Cellular Proteins and Their Fatty Acids in Health and Disease

Asim K. Duttaroy and Friedrich Spener (Eds.) Cellular Proteins and Their Fatty Acids in Health and Disease

Asim K. Duttaroy and Friedrich Spener (Eds.)

Cellular Proteins and Their Fatty Acids in Health and Disease

Editors: Professor Dr. Asim K. Duttaroy Institute for Nutrition Research University of Oslo POB 1046 Blindern N-0316 Oslo Norway Professor Dr. Friedrich Spener Institut für Biochemie Universität Münster Wilhelm-Klemm-Str. 2 48149 Münster Germany

n This book was carefully produced. Nevertheless, authors and publisher do not warrant the information contained therein to be free of errors. Readers are advised to keep in mind that statements, data, illustrations, procedural details or other items may inadvertently be inaccurate.

Library of Congress Card No.: applied for British Library Cataloguing-in-Publication Data A catalogue record for this book is available from the British Library. Bibliographic information published by Die Deutsche Bibliothek Die Deutsche Bibliothek lists this publication in the Deutsche Nationalbibliografie; detailed bibliographic data is available in the Internet at © 2003 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim All rights reserved (including those of translation in other languages). No part of this book may be reproduced in any form – by photoprinting, microfilm, or any other means – nor transmitted or translated into machine language without written permission from the publishers. Registered names, trademarks, etc. used in this book, even when not specifically marked as such, are not to be considered unprotected by law. Printed in the Federal Republic of Germany Printed on acid-free paper Composition K+V Fotosatz GmbH, Beerfelden Printing Druckhaus Darmstadt GmbH, Darmstadt Bookbinding Buchbinderei Schaumann, Darmstadt ISBN

3-527-30437-1

V

Preface Long-chain fatty acids, in addition to providing the cell with energy, are substrates for membrane biogenesis and act as signalling molecules. These fatty acids and their derivatives directly or indirectly regulate cellular processes such as differentiation, development and gene expression as well as the activities of enzymes, membrane receptors and ion channels. Dietary fats have profound effects on gene expression and fatty acid-activated transcription factors (nuclear receptors) may have a fundamental role in regulating energy balance through their sensing of fatty acid flux in metabolically active tissues. Because of the functional roles of these fatty acids and of their structural features and physico-chemical properties, it is important to understand the mechanisms that evolved for uptake and retention of these molecules. The picture emerging is that the cell has multiple binding proteins in the membranes as well as in the aqueous compartments that assure adequate uptake and intracellular movement of long-chain fatty acids and their regulatory action. This book covers the various aspects of intracellular binding proteins (FABPs, ACBP, SCP-2), such as structure-function, ligand specificity, delivery of ligands by membrane-protein and protein-protein interaction, as well as their expression and roles pertaining to nutrition, health, and disease. Regulation and expression of membrane fatty acid transporters such as FABPpm, FAT, FATP, and ABC transporters are treated in further chapters. In addition, transcription factors PPARs, RXRs, RARs, LXR, and HNF4 which bind fatty acids or their derivatives are also dealt with in depth. They play a central role in regulating the storage and catabolism of dietary fats and essentially all major metabolic paths of lipids appear to be under control of one or more genes regulated by these transcription factors. Their roles in inflammatory disorders, obesity, cancers, and atherosclerosis are also discussed. Since these transcription factors require fatty acids or their derivatives as ligands, FABPs may play important roles in transporting these ligands. Many leading investigators have contributed their most recent developments to this book. We believe that it will prove to be an invaluable reference text for both those familiar with and those new to the exciting, and ever changing world of cellular proteins whose common denominator is binding of fatty acids.

VI

Preface

Finally, we would like to express our thanks to all contributors to this book and to the reviewers for their competent advice. The untiring support and patience of Ines Chyla and the staff at Wiley-VCH is gratefully acknowledged. Oslo and Münster, February 2003

Asim K. Duttaroy, Oslo Friedrich Spener, Münster

VII

Contents Preface

V

List of Contributors

XIX

Part 1

The Molecular Basis of Protein-Lipid Interaction and Functional Consequences 1

1

Structure-Function of CD36 and Evidence for its Role in Facilitating Membrane Fatty Acid Transport 3

1.1 1.2 1.3 1.4 1.5 1.6 1.7 1.8 1.9 1.10 1.11 1.12 1.12.1 1.12.2 1.12.3 1.12.4 1.12.5 1.12.6 1.13 1.14

Chris T. Coburn and Nada A. Abumrad Introduction 3 Primary Structure 4 Ligand Binding Domains 6 Membrane Localization and Role in Cell Signaling 6 CD36 Gene Structure and Regulation 8 CD36 Deficiency 10 CD36 and Platelet Function 11 CD36 and Atherosclerosis 12 CD36 and Phagocytosis 13 CD36 and Angiogenesis 14 CD36 and Malaria 14 CD36 and Fatty Acid Transport 15 CD36 is Identified as a Mediator of FA Uptake 15 CD36, SHR, and Insulin Resistance 17 CD36 Transgenic and Knockout Mice Models 18 CD36-null Mice – the Fed Phenotype 18 CD36-null Mice – the Fasting Phenotype 22 CD36 and Insulin Responsiveness in the Mouse 23 Perspectives and Future Directions 24 References 25

VIII

Contents

2

Role and Function of FATPs in Fatty Acid Uptake

2.1 2.2 2.3 2.4 2.5 2.6 2.7

Jean E. Schaffer Introduction 31 Identification of Fatty Acid Transporter Proteins 32 Structure of FATPs 32 Function of FATPs 34 Regulation of FATP expression 35 Significance of FATPs 36 References 37

31

3

Function, Expression, and Regulation of Human ABC Transporters 39

3.1 3.2 3.3 3.3.1 3.3.2 3.3.3 3.3.4 3.3.5 3.3.6 3.4 3.4.1 3.4.2 3.4.3 3.4.4 3.4.5 3.4.6 3.5 3.5.1 3.5.2 3.5.3 3.6 3.7

Gerd Schmitz and Thomas Langmann Introduction 39 Structural Features of ATP Binding Cassette (ABC) Transporters 40 Overview of Human ABC Gene Subfamilies 41 The ABCA (ABC1) Subfamily 45 The ABCB (MDR/TAP) Subfamily 46 The ABCC (CFTR/MRP) Subfamily 48 The ABCD (ALD) Subfamily 50 The ABCE (OABP) and ABCF (GCN20) Subfamilies 51 The ABCG (White) Subfamily 51 Diseases and Phenotypes Caused by ABC Transporters 52 Familial HDL-deficiency and ABCA1 52 Retinal Degeneration and ABCA4 (ABCR) 54 Cystic Fibrosis (ABCC7/CFTR) 56 Multidrug Resistance (ABCB1/MDR1, ABCC1/MRP1, ABCG2) 57 Adrenoleukodystrophy (ABCD1/ALD) 58 Sulfonylurea Receptor (ABCC8/SUR) 59 Function and Regulation of ABC Transporters in Lipid Transport 60 ABCA1 in Macrophage Lipid Transport 61 ABCG1 and Other ABCG members in Sterol Homeostasis 64 ABC Transporters involved in Hepatobiliary Transport 67 Conclusions and Perspectives 70 References 70

4

4.1 4.2 4.2.1 4.2.2 4.3 4.3.1 4.3.2

Albumin Receptors – Structure and Function

79

Nigel J. Brunskill Introduction 79 The Search for an Albumin Receptor 80 The Endothelium–Albumin Relationship: Early Concepts 80 Identification of Receptors for Native and Modified Albumin in Endothelial Cells 81 Albumin Receptors in the Kidney 83 Glomerular Handling of Albumin 83 Binding and Uptake of Albumin in the Kidney Proximal Tubule

83

Contents

4.4 4.4.1 4.4.2 4.5 4.5.1 4.5.2 4.6 4.7 5

5.1 5.2 5.2.1 5.2.2 5.2.3 5.3 5.3.1 5.3.2 5.3.2.1 5.3.2.2 5.3.2.3 5.3.2.4 5.3.3 5.3.4 5.4 5.4.1 5.4.2 5.4.3 5.4.4 5.4.4.1 5.4.4.2 5.4.5 5.5 5.6

Megalin and Cubilin as Proximal Tubule Albumin Receptors 84 Megalin 84 Cubilin 86 Albumin as a Signaling Molecule – Implications for Albumin Receptor Function 87 LDLR Family as Signaling Receptors 88 Megalin as a Signaling Receptor 89 Summary 90 References 90 Intracellular Lipid Binding Proteins: Evolution, Structure, and Ligand Binding

95

Christian Lücke, Luis H. Gutiérrez-González, and James A. Hamilton Introduction 95 The Evolution of Lipid Binding Proteins 95 The Calycin Superfamily 95 The Intracellular Lipid Binding Proteins 96 The Phylogeny of iLBPs 98 Structural Characteristics of iLBPs 99 The Common Three-dimensional Fold 101 The iLBP Subfamilies 103 Subfamily I 103 Subfamily II 105 Subfamily III 106 Subfamily IV 106 Dynamic Properties of iLBPs 107 Mutagenesis Studies 108 Ligand Binding Assays 109 Microcalorimetry 109 The Lipidex Assay 110 Fluorescence-based Binding Assays 111 The ADIFAB Assay 111 Thermodynamic Analysis 112 Kinetic Analysis 112 Lipid Binding Preferences 113 Concluding Remarks 113 References 114

6

Fatty Acid Binding Proteins and Fatty Acid Transport

6.1 6.2 6.3 6.4 6.5

Judith Storch and Lindsay McDermott Introduction 119 Equilibrium Binding of Fatty Acids to FABPs 119 In vitro Fatty Acid Transfer Properties of FABPs 122 Transfection Studies of FABP Function 125 Cellular Fatty Acid Transport via FABP-Protein Interactions 126

119

IX

X

Contents

6.6 6.7 6.8 7

7.1 7.2 7.3 7.4 7.5 7.6 7.7 7.8

Insights into FABP Function from Null Mice 128 Perspectives 130 References 131 Structure and Function of SCP-x/SCP-2 135

Udo Seedorf Introduction 135 The SCP-2 Gene Family 136 Structure of SCP-2 139 Role of SCP-2/SCP-x in Peroxisomal Metabolism 142 SCP-2/SCP-x Deficiency Affects the Activity of the Peroxisome Proliferator Activated Receptor PPARa Impact of SCP-2/SCP-x on Cholesterol Metabolism 145 Acknowledgements 147 References 147

143

8

Structure, Function, and Phylogeny of Acyl-CoA Binding Protein

8.1 8.2 8.3 8.4 8.4.1 8.4.2 8.4.3 8.5 8.6 8.7 8.8 8.8.1 8.8.2 8.9 8.10 8.11 8.12 8.13

Susanne Mandrup, Nils J. Færgeman, and Jens Knudsen Introduction 151 The ACBP Family 152 ACBP Structure and Ligand Binding Specificity 156 Regulation of ACBP Expression 157 Genomic Organization in Mammals 157 Expression Pattern in Mammals 158 Transcriptional Regulation of the Mammalian ACBP Gene 159 Expression Profile in Other Eukaryotes 160 Subcellular Localization 161 Regulation of Long-chain Acyl CoA Concentrations in vivo 161 Functions of ACBP 163 Clues obtained from in vitro Studies 163 In vivo Functions in Mammals 165 Acyl-CoA esters, ACBP, and PPARs 165 ACBP in African trypanosomes (T. brucei) 166 Functions, and Lessons from Yeast 166 Conclusions and Future Directions 167 References 168

9

9.1 9.2 9.3 9.4 9.5

Structure and Function of PPARs and their Molecular Recognition of Fatty Acids 173

Colin N. A. Palmer PPARs as Nuclear Receptors 173 DNA Binding 174 PPARs as Fatty Acid and Drug Binding Receptors 176 Species Differences in Pharmacology 179 Co-activator/Co-repressor Interactions 180

151

Contents

9.6 9.7 9.8 10

10.1 10.2 10.3 10.4 10.5 10.6 10.7 10.8 10.9 10.10 10.11 10.12 10.13 11

11.1 11.2 11.3 11.4 11.5 11.5.1 11.5.2 11.5.3 11.5.4 11.5.5 11.6 11.7 11.8

Cross-talk with Inflammatory Signaling PPARs as Phosphoproteins 183 References 185

182

Structure and Function of Retinoid Receptors RAR and RXR 191

Alexander Mata de Urquiza and Thomas Perlmann Retinoids in Development 191 Retinoid Receptors Transduce Retinoic Acid Signals 193 Retinoid Receptors Belong to the Nuclear Hormone Receptor Family 194 Nuclear Receptors Share a Common Structure 194 The LBD and Ligand-dependent Transactivation 196 Cross-talk 198 Co-activators 198 Co-repressors 199 Nuclear Receptors from an Evolutionary Perspective 201 Fatty acids as Endogenous Ligands for RXR 201 Perspectives 202 Acknowledgements 203 References 203 Liver X Receptors (LXRs) – Important Regulators of Lipid Homeostasis

209

Lene K. Juvet and Hilde I. Nebb Introduction 209 Nuclear Hormone Receptors 209 The Liver X Receptors, LXRa and LXRb 210 The Cholesterol Sensor: LXR 211 Interplay between Cholesterol and Fatty Acid Metabolism 214 LXR and SREBP-1c Activation: a New Link between Cholesterol and Fatty Acid Regulation 214 Direct Regulation of Target Genes by LXRs in Lipid Metabolism 215 LXRs as Insulin Sensors in Liver 216 Fatty Acid Regulation of LXR 217 LXRs in Adipose Tissue 218 Summary 219 Acknowledgements 219 References 220

12

Acyl-CoA Ligands of HNF-4a and HNF-4a/PPARa Interplay

12.1 12.2 12.3 12.4

Rachel Hertz and Jacob Bar-Tana Transcriptional Activation by HNF-4a 225 Fatty Acyl-CoA Ligands of HNF-4a 226 Xenobiotic Ligands of HNF-4a 230 HNF-4a and its Ligands in Health and Disease 232

225

XI

XII

Contents

12.4.1 12.4.2 12.4.3 12.5 12.6

Blood Lipids 232 MODY-1 232 Blood Coagulation 233 Liver HNF-4a/PPARa Interplay in Rodents and Humans References 236

Part 2

Role for Proteins in Cellular Homeostasis

13

Fatty Acid Binding Proteins and their Roles in Transport of Long-chain Polyunsaturated Fatty Acids across the Feto-placental Unit 241

13.1 13.2 13.3 13.4 13.5 13.6

13.7

239

Asim K. Duttaroy Introduction 241 Fatty Acid Uptake in the Feto-placental Unit 242 Identification of Membrane-associated Fatty Acid Binding Protein in Human Placenta 243 Identification and Location of FAT/CD36 and FATP in Human Placental Membranes 246 Presence of Cytoplasmic Fatty Acid Binding Proteins (FABPs) in Human Placenta 247 Presence of Nuclear Transcription Factors that Bind Fatty Acids in Human Placenta: Interaction Between Fatty Acid Binding Proteins and PPARc 248 References 250

14

Fatty Acid Binding Proteins of the Brain

14.1 14.2 14.2.1 14.2.2 14.2.3 14.3 14.4 14.5 14.6

Yuji Owada and Hisatake Kondo Introduction 253 Expression of FABPs in Developing Rat Brain 254 Localization of H-FABP 254 Localization of E-FABP 258 Localization of B-FABP 261 Significance of FABP Expression in Brain 261 Perspective 263 Acknowledgements 263 References 264

15

15.1 15.2 15.3

233

253

Cross-talk between Intracellular Lipid Binding Proteins and Ligand Activated Nuclear Receptors – A Signaling Pathway for Fatty Acids 267

Christian Wolfrum and Friedrich Spener Introduction 267 Fatty Acid Activated Nuclear Receptors 268 Intracellular Lipid Binding Proteins 269

Contents

15.4 15.5 15.6 15.7 15.8 15.9 15.10 15.11 16

16.1 16.2 16.2.1 16.2.2 16.2.3 16.2.4 16.3 16.3.1 16.3.2 16.3.3 16.4 16.4.1 16.4.2 16.4.3 16.5 16.6 17

17.1 17.2 17.3 17.3.1 17.3.2 17.3.3 17.4 17.5 17.6

Regulation of Fatty Acid Activated Nuclear Receptor Activity by iLBPs 270 L-FABP 271 A-FABP and E-FABP 274 CRABP-II 276 Other Members of the FABP Family 277 Mechanism of iLBP Import into the Nucleus 278 Conclusions and Perspectives 279 References 281 Arachidonic Acid Binding Proteins in Human Neutrophils

285

Claus Kerkhoff and Olof Rådmark Cellular Functions of Arachidonic Acid 285 The Two Myeloid-related Proteins S100A8 and S100A9 285 S100A8 and S100A9 Belong to the S100 Family 285 S100A8 and S100A9 Expression is Primarily Restricted to Cells of Myeloid Lineage 287 S100A8/A9 Protein Complexes Bind Polyunsaturated Fatty Acids 289 Translocation of S100A8 and S100A9 is Accompanied with Arachidonic Acid Transport 291 Putative Intracellular Functions of S100A8/A9 292 5-Lipoxygenase (5-LO) and 5-Lipoxygenase Activating Protein (FLAP) 292 Cyclooxygenases (COX-1 and COX-2) 294 NADPH Oxidase Complex 295 Extracellular Role of the S100A8/A9–Arachidonic Acid Complex 297 Transcellular Arachidonic Acid Metabolism 297 Cellular Uptake of Long-chain Fatty Acids (LCFAs) 298 Participation of S100A8/A9 in the Arachidonic Acid Uptake 299 Conclusion and Future Perspectives 302 References 303 PPARs, Cell Differentiation, and Glucose Homeostasis

309

Stephen R. Farmer Introduction 309 Regulation of PPAR Activity 309 PPARs and Differentiation 311 PPARc 311 PPARc and Adipogenesis 312 PPARc and Transcriptional Control of the Pleiotropic Functions of the Adipocyte 315 PPARa 316 PPARd 317 PPARs and Control of Glucose Homeostasis: Therapies for Metabolic Syndrome and Type 2 Diabetes 318

XIII

XIV

Contents

17.6.1 17.6.2 17.7 17.8 17.9 18

18.1 18.2 18.2.1 18.2.2 18.2.3 18.2.4 18.3 18.4 18.5 18.5.1 18.6 18.6.1 18.6.2 18.6.3 18.6.4 18.7 18.8 19

19.1 19.2 19.2.1 19.2.2 19.2.3 19.3 19.4 19.4.1 19.4.2 19.4.3

PPARc 318 PPARa 321 Conclusion 322 Acknowledgements 323 References 323 Role of FABP in Cellular Phospholipid Metabolism

327

Chris A. Jolly and Eric J. Murphy Fatty Acid Targeting 327 Phospholipid Metabolism 328 Diacyl Phospholipid Classes 329 Potential Mechanisms for Diacyl Phospholipid Classes 331 Plasmalogen Classes 331 Potential Mechanisms for Plasmalogen Classes 333 Neutral Lipid Mass 334 Cellular Phospholipid Composition 334 Phospholipid Acyl Chain Composition 335 Potential Mechanisms for Fatty Acyl Chain Alterations 336 Phosphatidic Acid Biosynthesis 337 FABP Increases Phosphatidic Acid Biosynthesis 337 L-FABP Conformers and Phosphatidic Acid Biosynthesis 338 Potential Mechanisms for Stimulation of Phosphatidic Acid Biosynthesis 338 Biological Significance 339 Conclusions and Perspectives 340 References 340 Membrane-associated Fatty Acid Binding Proteins Regulate Fatty Acid Uptake by Cardiac and Skeletal Muscle 343

Jan F. C. Glatz, Joost J. F. P. Luiken, Ger J. van der Vusse, and Arend Bonen Introduction 343 Molecular Mechanism of Muscular Fatty Acid Uptake 344 Passive Diffusional and Protein-mediated Fatty Acid Uptake 344 Membrane-associated Fatty Acid Binding Proteins 346 Putative Mechanism of Cellular Fatty Acid Uptake 347 Expression of FABPs in Heart and Skeletal Muscles Compared 348 Regulation of Muscular Fatty Acid Uptake 350 Acute Changes in Muscle Fatty Acid Utilization and Membrane FABPs 350 Signaling Pathway for FAT/CD36 Translocation to and from the Sarcolemma 351 Chronic Changes in Muscle Fatty Acid Utilization and Membrane FABPs 352

Contents

19.5 19.6 19.7 19.8 19.9 20

20.1 20.2 20.2.1 20.2.2 20.2.2.1 20.2.2.2 20.2.2.3 20.2.2.4 20.2.3 20.2.4 20.2.5 20.3 20.3.1 20.3.2 20.4 20.5 21

21.1 21.2 21.3 21.4 21.4.1 21.4.2 21.5

Concerted Action of the Proteins Involved in Muscle Fatty Acid Uptake 353 Alterations in Fatty Acid Uptake and Membrane FABPs in Disease 354 Concluding Remarks 355 Acknowledgements 355 References 356 Intestinal Fat Absorption: Roles of Intracellular Lipid-Binding Proteins and Peroxisome Proliferator-Activated Receptors 359

Isabelle Niot and Philippe Besnard Introduction 359 Intestinal LCFA Absorption: A Complex Phenomenon 360 Can LCFA Uptake be a Rate-limiting Step for Intestinal Fat Absorption? 360 Why do Enterocytes Express Different Membrane LBP? 363 FABPpm/mAspAT: A Protein in Search of a Function 364 FATP4: A Plasma Membrane-associated ACS-like Protein? 365 Caveolin-1: An LBP and a Caveolae Marker 365 FAT/CD36: An Involvement in a Vesicular Trafficking of LCFA? 366 Do the Different Soluble FABPs Exert the Same Function? 368 ACBP: A Universal Long-chain Acyl CoA Transporter 372 An Integrative Working Model 372 Intestinal LCFA Absorption: A Phenomenon Putatively Adaptable to the Lipid Content of the Diet 374 PPAR and Coordinatd LBP Regulation 374 PPARb/d: A Nuclear Receptor Involved in the Regulation of Intestinal Absorptive Area 376 General Conclusion 377 References 378 Fatty Acid Binding Proteins as Metabolic Regulators

383

J. M. Stewart Introduction 383 Established Interactions between Carbohydrateand Fatty Acid-based Energy Production 384 The Involvement of FABP in Metabolism: Working Hypothesis 384 Criteria for Physiological Relevance of Metabolite Modulation of Fatty Acid Binding to FABP 385 Mammalian Liver FABP 386 Mammalian Heart/Muscle FABP 387 Potential of Formation of Schiff Bases: Non-enzymatic Glycation of FABPs 388

XV

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Contents

21.6

21.7 21.8 21.9 21.10 21.11

Theoretical Effects and Implications of Reciprocal Cross-talk: How much Fatty Acid Would be Available to Interact with Hexokinase? 389 Difference in Binding of Fatty Acids and Modulation between Different Types of FABP 391 Where Else to Look: Other Enzymes that are Influenced by Fatty Acids 391 Summary 392 Acknowledgements 393 References 394

22

Role of Lipid Binding Proteins in Disease

22.1 22.1.1 22.1.2 22.1.3 22.2

Aline Meirhaeghe and Philippe Amouyel Polymorphism in FATP1 Gene and Triglyceride Metabolism 397 Fatty Acid Metabolism 397 FATP1 Polymorphisms 398 FABP2 Polymorphisms 399 References 400

397

23

PPARs in Atherosclerosis

23.1 23.1.1 23.1.2 23.1.3 23.1.4 23.1.5 23.2 23.2.1 23.2.2 23.3 23.3.1 23.3.2 23.4 23.4.1 23.4.2 23.5 23.6 23.7

Jorge Plutzky Atherosclerosis 401 Introduction 401 Atherosclerosis as a Clinical Syndrome 402 Cellular Constituents of Atherosclerosis 403 Atherosclerosis as an Inflammatory Disorder 404 Atherosclerosis as a Metabolic Disorder 404 PPAR in the Vasculature 405 PPARs in Vascular Biology and Atherosclerosis 405 Examining Evidence for PPAR in Vascular Responses 406 PPARc in Vascular Biology and Atherosclerosis 407 In vitro Evidence 407 In vivo Evidence 408 PPARa in Vascular Biology and Atherosclerosis 409 In vitro Evidence 409 In vivo Evidence 411 PPARd in Vascular Biology and Atherosclerosis 413 Conclusion 413 References 414

401

Contents

24

24.1 24.2 24.3 24.4 24.4.1 24.4.2 24.4.3 24.5 24.5.1 24.5.2 24.5.3 24.6 24.7 24.8

PPARs: Nuclear Hormone Receptors Involved in the Control of Inflammation 419

Liliane Michalik, Nguan Soon Tan, Walter Wahli, and Béatrice Desvergne Introduction 419 PPAR Expression Profiles and Modulation by Cytokines 420 Fatty Acids and their Metabolites are PPAR Ligands 421 PPARs and the Control of the Inflammatory Response 423 Anti-inflammatory Properties of PPARa 423 PPARb and the Keratinocyte Response to Inflammation 425 PPARc is Involved in the Control of Inflammation 427 Are PPARs Good Targets for the Treatment of Inflammatory Disorders? 428 PPARs in Skin Inflammatory Disorders 428 PPARs and the Progression of Atherosclerosis 428 PPARc Regulates Intestinal Inflammation 431 Conclusion 431 Acknowledgements 432 References 433

25

PPARs and Cancer

25.1 25.2 25.3 25.3.1 25.3.2 25.3.3 25.3.4 25.4 25.4.1 25.4.2 25.4.3 25.5 25.5.1 25.5.2 25.5.3 25.6 25.7

J. H. Gill and Ruth A. Roberts Introduction 437 The PPAR Family 437 PPARa 438 Expression and Activation 438 PPARa and Cancer 439 Species Differences 439 PPARa as a Therapeutic Target? 440 PPARc 441 Expression and Activation 441 PPARc and Cancer 442 PPARc as a Therapeutic Target? 442 PPARb 443 Expression and Activation 443 PPARb and Cancer 443 PPARb as a Therapeutic Target? 444 Future Directions 444 References 445

Subject Index

449

437

XVII

XIX

List of Contributors Philippe Amouyel INSERM U508 Institut Pasteur de Lille 1 rue du professeur Calmette BP 245 59019 Lille Cedex France

A. K. Duttaroy Institute for Nutrition Research University of Oslo P.O. Box 1046 Blindern 0316 Oslo Norway

Arend Bonen Department of Kinesiology University of Waterloo Waterloo, ON N2L 3G1 Canada

Steve Farmer Boston University of School of Medicine Department of Biochemistry 715 Albany St. Boston, MA 02118 USA

Nigel J. Brunskill Department of Cell Physiology and Pharmacology Department of Nephrology University of Leicester Medical Sciences Building University Road Leicester, LE1 9HN UK

Jason H. Gill Molecular Pathology Cancer Research Unit University of Bradford All Saints Road Bradford BD7 1DP UK

Chris T. Coburn and Nada A. Abumrad Department of Physiology and Biophysics Stony Brook University Stony Brook, NY 11794-8661 USA

Jan F. C. Glatz, Joost J. F. P. Luiken and Ger J. van der Vusse Department of Physiology, CARIM Maastricht University P.O. Box 616 6200 MD Maastricht The Netherlands

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List of Contributors

Luis H. Gutiérréz-González Institut für Organische Chemie und Biochemie II Technische Universität München Lichtenbergstr. 4 85747 Garching Germany Rachel Hertz and Jacob Bar-Tana Department of Human Nutrition and Metabolism Hebrew University Medical School 91120 Jerusalem Israel Chris A. Jolly Division of Nutritional Sciences University of Texas at Austin Gearing Hall 117 Austin, TX 78712 USA Lene K. Juvet and Hilde I. Nebb Institute for Nutrition Research University of Oslo P.O. Box 1046 Blindern 0316 Oslo Norway Claus Kerkhoff Institut für Experimentelle Dermatologie Universität Münster Von Esmarch Straße 56 48149 Münster Germany Christian Lücke and James A. Hamilton Department of Physiology and Biophysics Boston University School of Medicine Boston, MA 02118 USA

Susanne Mandrup, Nils J. Færgeman and Jens Knudsen Department of Biochemistry and Molecular Biology University of Southern Denmark Campusvej 55 5230 Odense M Denmark Alexander Mata de Urquiza and Thomas Perlmann Ludwig Institute for Cancer Research Karolinska Institute P.O. Box 240 17177 Stockholm Sweden Lindsay McDermott Department of Chemistry University of Glasgow Glasgow G12 8QQ UK Aline Meirhaeghe Clinical Chemistry Addenbrooke’s Hospital – Level 4 Hills Road Cambridge CB2 2QR UK Liliane Michalik, Nguan Soon Tan, Walter Wahli and Béatrice Desvergne Institut de Biologie Animale Bâtiment de Biologie Université de Lausanne 1015 Lausanne Switzerland

List of Contributors

Eric J. Murphy Department of Pharmacology, Physiology and Therapeutics University of North Dakota 501 N. Columbia Rd., Room 3700 Grand Forks, ND 58203 USA

Olof Radmark Department of Medical Biochemistry and Biophysics Division of Physiological Chemistry II Karolinska Institutet 17177 Stockholm Sweden

Isabelle Niot and Philippe Bresnard Laboratoire de Physiologie de la Nutrition (ENSBANA) Université de Bourgogne 1, Esplanade Erasme 21000 Dijon France

Ruth A. Roberts Aventis Pharma SA Centre de Recherche de Paris 94400 Vitry sur Seine France

Yuji Owada and Hisatake Kondo Division of Histology Department of Cell Biology Graduate School of Medical Science Tokohu University 2-1 Seiryo-cho, Aoba-ku Sendai 981-8575 Japan Colin N. A. Palmer Biomedical Research Centre Nienewells Hospitals and Medical School University of Dundee Dundee DD1 9SY UK Jorge Plutzky Cardiovascular Division Department of Medicine Brigham and Women’s Hospital Harvard Medical School 221 Longwood Ave., LMRC 307 Boston, MA 02115 USA

Jean E. Schaffer Departments of Internal Medicine Molecular Biology and Pharmacology Washington University School of Medicine 660 South Euclid Ave Box 8086 St. Louis, MO 63110-1010 USA Gerd Schmitz and Thomas Langmann Institut für Klinische Chemie und Laboratoriumsmedizin Universität Regensburg Franz-Josef-Strauß-Allee 1 93042 Regensburg Germany Udo Seedorf Institut für Arterioskleroseforschung Domagkstr. 3 48149 Münster Germany Friedrich Spener Institut für Biochemie Universität Münster Wilhelm-Klemm-Str. 2 48149 Münster Germany

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List of Contributors

Jack M. Stewart Biochemistry Program Department of Biology Mount Allison University Sackville, NB E4L 1G7 Canada Judith Storch Department of Nutritional Sciences Rutgers University New Brunswick, NJ 08901 USA

Christian Wolfrum Rockefeller University 1230 Yorck Ave. New York, NY 10021 USA

Part 1

The Molecular Basis of Protein-Lipid Interaction and Functional Consequences

3

1

Structure-Function of CD36 and Evidence for its Role in Facilitating Membrane Fatty Acid Transport Chris T. Coburn and Nada A. Abumrad

1.1

Introduction

CD36 (also known as GPIV, GPIIIb, PAS IV, and FAT) is an integral membrane glycoprotein with a wide cellular distribution. It has been identified on the surface of megakaryocytes, erythroid precursors, platelets, monocytes, dendritic cells, adipocytes, myocytes, retinal and mammary epithelial cells, and endothelial cells of the microvasculature and small intestine. Depending upon the cellular context, CD36 may perform its primary role as a mediator of fatty acid (FA) uptake, a cell adhesion molecule, or a class B scavenger receptor. This remarkable versatility may reflect both the diverse tissue distribution of CD36 and its ability to interact with a wide variety of ligands. CD36 binds long-chain FA with high affinity and is a major facilitator of FA uptake in muscle and adipose tissues. As a receptor for the extracellular matrix proteins thrombospondin 1 (TSP-1) and collagens type I and IV, CD36 acts as an adhesion molecule modulating platelet aggregation and the cell–cell interactions important for recruitment and trafficking of monocytes to damaged tissues. In a pathological context, CD36 is the receptor in the microvasculature for the Plasmodium falciparum protein expressed on the surface of malaria-infected erythrocytes and as a result contributes to the virulence of this form of malaria. In macrophages and dendritic cells, CD36 is a scavenger receptor important to recognition and phagocytosis of apoptotic cells. On macrophages it is also the major receptor mediating binding and internalization of oxidized low-density lipoproteins (oxLDL), a role reflecting its ability to bind anionic phospholipids as well as lipids or proteins modified by lipid peroxidation. Because of the varied roles of CD36, a great deal has been discovered about this protein. With functions impacting on lipid metabolism, atherogenesis and thrombosis, inflammation, platelet function, the pathogenesis of malaria and even angiogenesis, further study of the physiology and molecular interactions of CD36 will no doubt continue to progress at a rapid pace. This chapter presents an overview of current knowledge with particular emphasis given to the role of CD36 in lipid metabolism and metabolic homeostasis.

4

1 Structure-Function of CD36 and Evidence for its Role in Facilitating Membrane Fatty Acid Transport

1.2

Primary Structure

CD36 is the founder member of a gene family of structurally related glycoproteins expressed at the cell surface and within lysosomes. Known members include, in addition to CD36, the high-density lipoprotein receptor SR-B1 (also CLA-1), Drosophila plasma membrane proteins emp and croquemort, the ubiquitously expressed lysosomal integral membrane protein LIMPII, and the recently identified amoeboid endolysosomal proteins LmpA, LmpB, and LmpC. All are believed to share a “hairpin” topology defined by two transmembrane domains with both termini in the cytoplasm (Fig. 1.1). In this configuration, the intervening amino acids localize either to the cell surface (CD36) or to the lumen of lysosomal vesicles (LIMPII). This arrangement is exceedingly rare among membrane proteins but has been experimentally confirmed for both CD36 and SR-B1 [1, 2]. Numerous studies have contributed to the development of a detailed model for the primary structure of CD36 (Fig. 1.1). The human cDNA predicts a sequence of 472 amino acids with an N-terminal signal peptide directing transcription to the endoplasmic reticulum (residues 1–30). Limited N-terminal sequencing of purified CD36 shows that the signal peptide is uncleaved but the initiating methio-

Cartoon of CD36 in the membrane, highlighting the major structural features. N-linked glycosylations are shown as triangles. Disulfide bonds are shown in green.

Fig. 1.1

1.2 Primary Structure

nine is removed. Residues 2–7 are in the cytoplasm while 8–30 form the N-terminal membrane-spanning domain. The second transmembrane domain (440–463) is near the C-terminus. An uninterrupted hydrophobic segment (186–204) is centrally located but not long enough to span the bilayer. This segment, which may form a hydrophobic pocket or may be associated with the outer leaflet, is not conserved in other known members of the CD36/LIMPII family. The cytoplasmic domain of CD36 consists of only 15 amino acids (6 at the Nterminus and 9 at the C-terminus). It contains four cysteines (N residues 3 and 7 and C residues 464 and 466), which were shown to undergo palmitoylation, confirming the membrane topology [1]. From examples of other acylated proteins, it can be speculated that palmitoylation of the cytoplasmic domain may play an important role in modulating interactions with other proteins and/or membrane localization. Since palmitoylation is reversible, a variable palmitoylation state may also serve to acutely regulate CD36 function. In this respect, in isolated rat adipocytes, insulin or energy depletion with 2,4-dinitrophenol was shown to rapidly increase CD36 palmitoylation by about 3- and 12-fold, respectively [3]. The extracellular domain of CD36 contains 10 potential glycosylation sites and glycosylation increases the apparent protein mass from 53 kDa (non-glycosylated) to between 78 and 88 kDa, depending on the tissue source. Extensive glycosylation is a characteristic of the CD36/LIMPII family of proteins perhaps affording protection in the protease-rich environments of lysosomes or at sites of inflammation and tissue injury. Indeed, CD36 was initially identified in platelets based on its resistance to protease digestion [4] and deglycosylation with endoglycosidase F yields a protein that is susceptible to a range of proteases [5]. A cursory examination of the primary structure of CD36 shows a natural division between the Nand C-terminal halves of the extracellular domain. The N-terminal half contains 7 of the 10 potential N-linked glycosylation sites as well as the internal hydrophobic domain, while the C-terminal half is proline-rich and contains all of the extracellular cysteines and subsequent interchain disulphide bonds. Whether this may translate into separate functional domains remains to be determined. In megakaryocytes and in CD36-transfected COS cells, Thr92, which fits within a protein kinase C consensus site, is constitutively phosphorylated during maturation of the protein in the Golgi apparatus [6]. The phosphorylation state of this residue appears to modulate the selectivity of CD36 on platelets for TSP-1 or collagen binding. A cAMP-dependent ectoprotein kinase A on the surface of platelets has also been shown to phosphorylated CD36 [7]. The phosphorylated residue was not determined but most likely occurs within a protein kinase A (PKA) consensus site around Ser237, though PKA phosphorylation of Thr92 cannot be ruled out. No functional change in CD36 activity has yet been attributed to this phosphorylation event.

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1 Structure-Function of CD36 and Evidence for its Role in Facilitating Membrane Fatty Acid Transport

1.3

Ligand Binding Domains

Monoclonal antibodies have been used to identify CD36 domains involved in ligand binding. Amino acids 155–183 define an immunodominant region shown to bind a number of anti-CD36 monoclonal antibodies, including the widely used OKM5 [8]. These antibodies block interactions of CD36 with TSP-1, OxLDL, malaria-infected red blood cells, apoptotic neutrophils, and phosphatidylserine [9–13]. Since antibody binding to CD36 might sterically hinder ligand interaction, synthetic and recombinant peptides have been used to more closely define the binding domains. These studies have shown that OxLDL and TSP-1 bind to sequences outside of the immunodominant domain. OxLDL binds with high affinity to CD36 amino acids 28–93 [14]. TSP-1 binds in a two-step process interacting first with CD36 residues 139–155. This induces a conformational change in TSP-1 unmasking a high-affinity site for amino acids 93–120 of CD36 [15]. Malaria-infected erythrocytes also bind within this region (residues 97–110) [6]. A possible site for long-chain FA binding in the extracellular domain of CD36 has been identified with an alignment comparing CD36 sequence with that of a representative member of the lipocalin family of cytosolic FA binding proteins [16]. Members of this family may exhibit as little as 20% sequence identity but share a common and distinct structural motif. The region comprising amino acids 127–279 of CD36 exhibits homology to human muscle FA binding protein (M-FABP) throughout 73% of its sequence, although identity is only 14.5%. Secondary structure predictions indicate this sequence may consist of a single a-helical region interposed between regions of sheets similar to the known structure of M-FABP and other lipocalin family members. It is also of interest that of the amino acids conserved throughout the lipocalin family, Arg126 and Tyr128 of MFABP, which interact with the FA carboxyl group and are necessary for FA binding, are conserved in this alignment (Arg272 and Tyr275 of CD36). It may be noteworthy that this region includes the hydrophobic domain of CD36 (186–204), thought to be membrane associated or to form a hydrophobic pocket.

1.4

Membrane Localization and Role in Cell Signaling

CD36 in many cells is associated with membrane microdomains rich in cholesterol and sphingolipid and known as rafts or caveolae. The long, largely saturated acyl chains of the sphingolipids favor tight packing with cholesterol and promote formation of small freely floating domains (hence rafts) within the membrane (reviewed in Ref. [17]). These detergent-resistant membranes (DRM), which can be biochemically isolated from the rest of the membrane by virtue of their insolubility in Triton X-100 at 4 8C, typically account for about 5% of the plasma membrane of mammalian cells. Although sphingolipid-rich rafts are mostly confined to the outer leaflet they are coupled to similar domains incorporating mono-un-

1.4 Membrane Localization and Role in Cell Signaling

saturated phospholipids within the cytoplasmic side. DRMs are enriched in and may promote clustering of receptors and signaling proteins such as transmembrane receptor kinases, the EGF and insulin receptors, protein kinases C and A, adenyl cyclase, intermediates of MAP kinase pathways (Ras, Raf, Sos, and Shc), heterotrimeric G proteins, and several Src family kinases. In many cells (especially adipocytes, myocytes, endothelial and epithelial cells but not platelets or monocytes) DRMs contain large amounts of one or more members of the caveolin family of proteins. Caveolins are the defining structural components of caveolae and promote formation of the typical 50–100 nm in diameter flask-shaped invaginations. Caveolae may be a specialized structural form of rafts and it is unknown whether rafts and caveolae with separate functions coexist within the membrane of some cells. CD36 lacks the caveolin scaffold recognition sequences present in many caveolae-sequestered proteins. However, recruitment of proteins to rafts and caveolae can also be accomplished by protein modification with attachment of closely spaced myristate and palmitate or dual palmitate chains that pack well into the ordered lipid environment. So the dual palmitoylation sites on each of the cytoplasmic tails of CD36 may provide a flexible signal to regulate its association with rafts or caveolae. The localization of CD36 in DRMs strongly suggests that it functions in cell signaling. However, this is not the only evidence in support for such a role. Association of CD36 with Src family kinases has been reported in resting platelets and endothelial cells [18, 19]. Kinases from this family, which are initially bound inactive to a membrane receptor, are activated following receptor oligomerization and subsequent transphosphorylation. The kinase may then dissociate from the receptor to interact with and phosphorylate downstream effectors. In accordance with this scheme, collagen binding as well as antibody-mediated clustering of CD36 have been shown to activate platelets concomitant with an increase in tyrosine phosphorylation [20, 21]. Immunoprecipitates of CD36 from resting platelets contain the Src family tyrosine kinases Fyn, Lyn, and Yes, whereas CD36/kinase associations are not detected following platelet activation with antiCD36 antibodies [18, 22]. Similarly, binding of TSP-1, which induces dimerization of CD36, has been shown in microvascular endothelial cells to activate the associated tyrosine kinase Fyn [23]. In monocytes, CD36-specific IgG antibodies induce an oxidative burst, while the Fab fragments, which cannot promote oligimerization, are unable to do so unless cross-linked by a secondary antibody [24]. It is noteworthy that a respiratory burst can be induced in neutrophils by inactivation of protein tyrosine phosphatases and can be inhibited by tyrosine kinase inhibitors [25, 26]. The role of CD36 in signal transduction after external stimuli may also apply to integrin-mediated signaling. Integrins comprise a large family of a b heterodimeric transmembrane proteins that function as receptors for cell adhesion molecules. The cytoplasmic domain of integrins connects to the cytoskeleton providing points of attachment, or focal adhesions, between actin filaments and components of the extracellular matrix. These adhesions are enriched in kinases and can relay signals to the cytoskeleton upon integrin binding, leading to the cytoskeletal rear-

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1 Structure-Function of CD36 and Evidence for its Role in Facilitating Membrane Fatty Acid Transport

rangements necessary for phagocytosis and cellular migration as well as for platelet and monocyte activation. Physical and/or functional associations between CD36 and integrins have been documented on the surface of platelets, macrophages, retinal pigment epithelial cells, and melanoma cell lines [27–31]. The integrins associated with CD36 function as receptors for either collagen or TSP-1 [32], so CD36 may function in concert with integrins to complement or stabilize cell interactions with these adhesion molecules enhancing their signaling efficiencies. For example, phagocytic uptake of apoptotic neutrophils by macrophages requires the coordinated functions of CD36 and the aVb3 integrin, possibly with TSP-1 bound to the surface of the apoptotic cell functioning as a molecular bridge [28].

1.5

CD36 Gene Structure and Regulation

The CD36 gene consists of 15 exons encompassing more than 32 kilobases on the q11.2 band of human chromosome 7 [33]. Both the N- and C-terminal transmembrane and cytoplasmic domains are encoded by single exons (exon III and part of exon XIV), while the extracellular domain is divided among 11 (exons IV–XIV). Recently, several mRNA transcripts arising from alternative splicing of coding exons in peripheral blood monocytes have been reported [34]. The protein products of these alternate forms have not yet been characterized, however. Sequence analysis of the proximal 5'-flanking region has identified a number of potential cis-regulatory elements [33, 35]. Of these, the most well characterized are response elements for transcription factors of the peroxisome proliferator activated receptor family (PPAR, nucleotides –272 to –260), the CCAAT/enhancer binding protein family (C/EBP, nucleotides –9 to –1), and the polyomavirus enhancer binding protein 2/core binding factor family (PEBP2/CBF, nucleotides – 103 to –98). The 5'-proximal promoter also contains a TATA box and a CAAT box appropriately situated with respect to the transcriptional start site. The functions of these transcription factors are consistent with the expression pattern of CD36. The C/EPB proteins play essential roles in the adipocyte and myeloid differentiation programs. The PEBP2/CBF proteins are likewise important regulators of myeloid-specific gene expression. PPAR transcription factors have regulatory roles in adipogenesis and FA metabolism and are present in all cell types expressing CD36. The peroxisome proliferator activated receptors (PPARa, PPARc, and PPARd are members of the nuclear receptor family of ligand-activated transcription factors, which include the retinoid, steroid, and thyroid hormone receptors. PPARs participate broadly in the transcriptional control of genes involved in lipid and carbohydrate metabolism (reviewed in Ref. [36]). Synthetic agonists for PPARa collectively known as fibrates, decrease serum triglyceride levels and are widely prescribed for the treatment of hypertriglyceridemia and compound lipidemia. Synthetic PPARc agonists, known as thiazolidinediones, or glitazones, act as insulin sensitizers and

1.5 CD36 Gene Structure and Regulation

are used for treatment of type II diabetes. The natural ligands for PPARs have not been firmly established. However, a number of long-chain FA and naturally occurring FA derivatives have been shown in vitro to stimulate PPAR-mediated transcription, suggesting that PPARs may act as FA sensors to allow modulation of gene expression according to FA supply. PPARs play complementary roles in lipid homeostasis. PPARa is expressed in tissues exhibiting high rates of FA catabolism such as heart, muscle, liver, kidney, and brown-adipose tissue [37]. In these tissues, PPARa plays an active role in regulating FA catabolism by modulating the expression of genes involved in FA uptake, esterification, mitochondrial import, and b-oxidation. PPARa is highly expressed in adipose tissue and is a key regulator of adipogenesis and insulin sensitivity [38]. It actively promotes lipid storage by inducing expression of genes involved in FA uptake and triglyceride synthesis. PPARd occurs at low levels in most cell types and emerging evidence indicates it too is involved in lipid metabolism and adipocyte differentiation [39, 40]. Long-chain FAs were also shown to activate this receptor [41]. Consistent with the role of CD36 in FA metabolism, all three receptor subtypes appear capable of transcriptionally regulating CD36 expression. CD36 is induced in cardiomyocytes by synthetic PPARa agonists, whereas cardiomyocytes from PPARa-deficient mice exhibit significantly reduced levels [42, 43]. In pre-adipocytes, CD36 mRNA is strongly induced by glitazones, long-chain FAs, and the non-metabolizable FA analog 2-bromopalmitate [44, 45]. When the PPARd transcription factor (also known as the fatty acid-activated receptor, or FAAR) was stably transfected into 3T3-C2 fibroblasts, the cells acquired the capacity to induce the expression of CD36 as well as adipocyte cytosolic FA binding protein in response to FA [45]. Transcriptional induction of these genes exhibited inducer specificities identical to those described in pre-adipocytes. CD36 expression and FA uptake appear to be closely linked to adipocyte differentiation. Pre-adipocytes can be induced to differentiate in vitro by the addition of mitogens and hormones such as insulin and glucocorticoids, leading to increased cAMP levels and subsequent expression of the C/EBP transcription factors. These transcription factors in turn mediate the expression of CD36 and PPARc. In a positive feedback loop, the increased CD36-facilitated FA uptake leads to further increases in PPARc-mediated CD36 expression. Expression of CD36 appears to ensure a ready supply of FA (or FA-derived) ligands necessary for PPARcmediated gene transcription and full progression of the differentiation program. Antisense expression of CD36 in 3T3-F442A preadipocytes resulted in a marked decrease in FA uptake and a complete block of insulin and triiodothyronine induced differentiation [46]. Increasing the amount of FA in the cell growth medium led to an induction of CD36 expression above levels of the antisense mRNA, an enhancement in FA uptake, and rescue of cell differentiation. PPAR activation of CD36 gene transcription may provide a link to the increased CD36 expression observed with pathologic states characterized by hyperlipidemia. CD36 expression is increased, for example, in animal models of genetic obesity and diabetes [47–52]. CD36 expression is also increased in mice fed a high fat diet

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1 Structure-Function of CD36 and Evidence for its Role in Facilitating Membrane Fatty Acid Transport

[51, 53]. In a recent in vivo experiment in humans, elevated plasma concentrations of FA were maintained for 5 hours in normal weight subjects by infusion of a commercial soybean oil emulsion with added heparin. Following the infusion, PPARc and CD36 mRNAs isolated from subcutaneous fat samples were increased 2-fold and greater than 7-fold, respectively, in comparison to pretreatment levels [54]. Similar results were obtained following lipid infusion in lean Zucker rats [55]. These findings suggest that elevated plasma FA may regulate the partitioning of calories to adipose tissue by inducing genes that promote fat cell differentiation, FA uptake, and triglyceride deposition.

1.6

CD36 Deficiency

CD36 was originally identified as a cluster designation (CD) marker for erythroid precursors, monocytes, and platelets. CD molecules are cell surface antigens defined by a panel of monoclonal antibodies raised against cells of hematopoietic origin. Because of its expression on platelets and circulating monocytes, CD36 deficiency in humans can be easily ascertained and has been divided into two subgroups. The type I CD36-deficient phenotype is distinguished by a lack of CD36 expression on the surface of both platelets and monocytes. The type II phenotype lacks expression on the surface of platelets but monocyte expression is near normal. There is little evidence in this phenotype for the existence of a platelet-specific silent allele [56]. Indeed, type II-deficient individuals may be found to be homozygous for the wild-type gene [57]. This finding suggests the possibility of deficiencies in other megakaryocyte proteins such as proteins involved in the intracellular processing or transport of CD36 to the membrane. CD36 deficiency has a prevalence of 3–11% in Asian populations, 5–18.5% in African populations, and less than 0.3% in Caucasians [58–60]. Incidences in the Japanese population of the type I and type II phenotypes are 1.0% and 5.8% respectively [56]. A number of mutations associated with CD36 deficiency have been identified. The most common mutation among the Japanese is a T for C substitution at cDNA nucleotide 478, resulting in a proline substitution for serine 90. This mutation has been reported to result in degradation of the immature protein in transfected cells [61]. In sub-Saharan Africa the most common mutation is a nonsense mutation resulting from the substitution of G for T at nucleotide position 1264. This mutation encodes a truncated protein lacking the C-terminal membrane spanning domain. CD36-deficient individuals appear healthy. However, a marked defect in myocardial uptake of long-chain fatty acids has been described [62] and the deficiency may be linked to some forms of cardiac hypertrophy [63]. A recent study with a limited number of subjects suggests an association between CD36 deficiency and blood lipid abnormalities with impaired insulin responsiveness [64]. This study, which used the sensitive euglycemic hyperinsulinemic clamp technique, documented in all cases abnormalities of glucose metabolism. However, conclusions

1.7 CD36 and Platelet Function

from this study were not supported by the findings of Yanai et al. [65] who reported that young CD36-deficient patients showed no sign of insulin resistance. Our data with CD36-null mice (Hajri et al., unpublished observations) are more consistent with the findings of Yanai et al. Our data suggest that the effect of CD36 deficiency on insulin responsiveness is strongly diet-dependent and consideration of this interaction could help reconcile some of the divergent effects in humans. Incidence of CD36 deficiency is high in many subpopulations known to be at high risk for diabetes type II. However, the role of CD36 deficiency in the etiology of diabetes type II in these subpopulations may prove difficult to define as a result of the complexities introduced by dietary influences and because of the polygenic nature of the disease. Of clinical importance, type I individuals may produce isoantibodies against CD36 following a transfusion or during pregnancy which can lead to refractoriness to blood group-matched platelet transfusions, post-transfusion purpura, or neonatal immune thrombocytopenia [66–71]. For this reason type I deficiency is sometimes referred to as the Naka-negative phenotype, where anti-Naka is a CD36 isoantibody formed following immunization with the wild-type protein. The identification of CD36 as the Naka isoantigen seen with type I deficiency suggests that CD36 is indeed absent from all cell types in these individuals.

1.7

CD36 and Platelet Function

When vascular injury occurs, binding to the exposed collagen and fibronectin of the subendothelial matrix induces platelet activation. This is associated with platelet degranulation and the release of coagulation factors and adhesive proteins such as TSP-1, which mediate further platelet binding and aggregation. CD36 is a major glycoprotein on the surface of platelets and an adhesive receptor for both collagen and TSP-1. As a result it is likely to play a role in both platelet activation and in secretion-dependent aggregation. The phosphorylation state of Thr92 has been suggested to modulate the activity of CD36 between these two roles [6]. CD36 in megakaryocytes is constitutively phosphorylated during maturation in the Golgi and before transfer to the plasma membrane. The phosphorylated protein on the surface of resting platelets exhibits high affinity for collagen and weak affinity for TSP-1. However, upon platelet activation, released phosphatases are thought to dephosphorylate CD36, increasing its affinity for TSP-1 while preventing its interaction with collagen. CD36 deficient individuals show no evidence of hemostatic abnormalities and platelets from these individuals respond normally to a variety of physiological agonists [72]. This is not surprising since the processes of platelet activation and adhesion are highly redundant, involving multiple structurally diverse adhesive ligands and receptors and defects in more than one component of these pathways are usually required for manifestation of hemostatic abnormalities.

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1.8

CD36 and Atherosclerosis

Atherosclerosis is a principal cause of death in the United States, Europe, and parts of Asia. Its complications include ischemic heart disease, myocardial infarction, and stroke. Atherosclerotic lesions are initiated in the arterial tunica intima by accumulation of lipid-filled macrophages, or foam cells, in the subendothelial space, giving the appearance of a fatty streak. In a second stage, proliferation of smooth muscle cells generates a plaque with an acellular lipid core derived from necrotic foam cells and a fibrous cap of smooth muscle cells and collagen. The final stage or complicated lesion shows evidence of thrombus formation with deposition of fibrin and platelets. The blood vessel has become narrowed to the point of occlusion by the luminal and subendothelial deposits. CD36 is implicated in the development and early progression of atherosclerotic lesions as a result of its function in binding and uptake of oxidatively modified low-density lipoproteins (oxLDL). According to the oxidative hypothesis, the majority of the lipid accumulated in the intima derives from uptake of these particles by CD36 and other scavenger receptors on macrophages [73]. LDL entrapped by extracellular matrix proteins in the subendothelial space of lesion-prone arterial sites is subject to oxidative modification by endothelial cells, smooth muscle cells, and resident macrophages. oxLDL is internalized by CD36 and other scavenger receptors on macrophages, which bypasses the feedback control exercised by the classical LDL receptor pathway and thus leads to the formation of lipid-filled “foam cells”. Diffusible products of LDL oxidation and others released by smooth muscle and endothelial cells exposed to oxLDL are chemotactic toward circulating monocytes. Further intimal accumulation of macrophages promotes more LDL oxidation and foam cell formation. The consequent rise in lipid peroxidation products ultimately leads to foam cell necrosis, and continuous irritation of the endothelial cell layer causes a host of other effects that may favor lesion progression. A number of studies point to CD36 as the dominant scavenger receptor on macrophages [74, 75], which could reflect its ability to bind even minimally oxidized LDL. More extensive oxidation leads to recognition by additional receptors, including the type A I/II scavenger receptors (SR-AI/II), SR-B1, macrosialin, and LOX-1 [76]. The fraction of uptake mediated by CD36 varies in ex vivo models depending upon the method used for LDL oxidation [77]. To better evaluate the contribution of CD36 to atherogenesis, Febbraio and colleagues crossed CD36-null mice into the atherosclerosis-prone apolipoprotein E (apoE)-null strain [78]. Despite a modest worsening of the pro-atherogenic serum lipid profile in the doublenull mice, there was a 70% reduction in aortic lesion size with high fat feeding as compared with the apoE-null mice. The specific modification on oxLDL that is recognized by CD36 has not been identified. LDL oxidation produces major alterations in both the lipid and protein components. These include extensive hydrolysis of phosphatidylcholine, increased density, increased negative charge, fragmentation of apoB, and derivatization of lysine groups by highly reactive lipid peroxidation products [79]. Current evidence

1.9 CD36 and Phagocytosis

suggests that CD36 may bind to an anionic lipid product or that lipid components are essential for recognition of the modified apoprotein [10].

1.9

CD36 and Phagocytosis

The phagocytic clearance of apoptotic cells serves to limit tissue injury by protecting against release of the potentially harmful contents of dying cells. Much of the work on the role of CD36 in this process has centered on its involvement in the phagocytosis of apoptotic neutrophils. Circulating neutrophils responding to chemotactic signals migrate to sites of inflammation where they phagocytose and destroy invading microbes. Perhaps because of their high content in lysosomes and granules with potent degradative enzymes, neutrophils are programmed from inception to undergo apoptosis with an average lifespan of about five days. If not phagocytosed, apoptotic neutrophils eventually disintegrate by secondary necrosis, releasing their histotoxic contents into the extracellular space and inciting further tissue damage. CD36 is involved in both the initial recognition and (through its cooperation with integrins) the engulfment of apoptotic cells [80]. Furthermore, signaling through CD36 may suppress the secretion of pro-inflammatory cytokines released by macrophages when they phagocytose and digest necrotic cells. The ability of dying cells to be phagocytosed without eliciting an inflammatory response is a hallmark of apoptosis. The mechanism by which macrophages recognize apoptotic cells is linked to alterations in their surface chemistry, which may vary depending on the cell type (reviewed in Ref. [81]). CD36 appears capable of recognizing a number of these modifications. Apoptotic neutrophils, for example, express anionic TSP-1 binding sites on their surface. CD36 also recognizes anionic phospholipids on the surface of many apoptotic cells. The anionic lipid phosphatidylserine (PS) is normally confined to the inner leaflet of the plasma membrane and often apoptosis leads to loss of membrane asymmetry and to PS exposure on the outer leaflet. Furthermore, much of this PS may be oxidatively modified by reactive oxygen species generated during apoptosis [82]. Indeed, epitopes on the surface of apoptotic cells cross-react with antibodies against oxLDL, making it likely that CD36 binds to oxidative modifications of apoptotic cell membranes [83]. Similar interactions may be involved in the CD36-mediated recognition and phagocytosis of photoreceptor rod outer segments by retinal pigment epithelial cells [29, 84]. Rod outer segments are photo-damaged membrane segments shed daily by photoreceptors. These segments must be phagocytosed and the essential FA docosahexaenoic acid (22:6n–3) they contain must be recycled back to photoreceptor cells for maintenance of normal vision.

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1.10

CD36 and Angiogenesis

Angiogenesis, or formation of new blood vessels, is necessary for both tissue development and wound healing. It is a highly regulated multistep process involving endothelial cell proliferation, migration, and tube formation. Recruitment of endothelial cells for angiogenesis is driven by growth factor inputs and is dependent upon appropriate cues from the extracellular matrix to support cell proliferation and migration. TSP-1, secreted in the extracellular matrix during injury or inflammation by platelets, fibroblasts, vascular smooth muscle cells, macrophages, or endothelial cells, generates a potent anti-angiogenic signal to limit neovascularization and vessel density. This angiostatic activity may reflect the ability of TSP-1 to induce apoptosis in microvascular endothelial cells, a process mediated by CD36 [23]. Binding of TSP-1 to CD36 on the cell surface induces receptor oligomerization with activation of the associated Fyn tyrosine kinase. This ultimately leads to programmed cell death through subsequent downstream activation of caspase-3 and p38 MAP kinase. Neovascularization may require a delicate balance between the opposing actions of growth factors and TSP-1. To modulate the activity of TSP-1, several growth factors may be capable of downregulating CD36 expression on endothelial cells. In isolated adipocytes, epidermal growth factor and platelet-derived growth factor can phosphorylate PPARc via the MAP kinase cascade, decreasing its transcriptional activity [85]. Activation of this pathway in macrophages by transforming growth factor b1 and b2 downregulates CD36 expression [86]. Although this has not been demonstrated to occur in microvascular endothelial cells, it is certainly a possibility. Microvascular endothelial cells express PPARc and PPARc agonists inhibit growth factor-induced endothelial cell proliferation and tube formation [87].

1.11

CD36 and Malaria

The World Health Organization recognizes malaria as one of the world’s primary health problems, causing more mortality than any other parasitic disease. More than 40% of the earth’s population is at risk for malaria and an estimated 300– 500 million clinical cases each year result in 1.1 to 2.7 million deaths [88]. Almost 90% of these deaths occur in sub-Saharan Africa, and young children are the most affected. Malaria is an acute and chronic protozoan infection of the red blood cell. Of the four species of malaria parasite affecting humans, Plasmodium falciparum is by far the most virulent and accounts for the majority of illnesses and fatalities. Virulence of P. falciparum results from its ability to evade reticuloendothelial filtration beds in the spleen, where parasite-infected erythrocytes are detained and phagocytosed. Expression of an antigenically variant receptor, P. falciparum erythrocyte membrane protein 1 (PfEMP-1) on the surface of the host cell mediates its adhe-

1.12 CD36 and Fatty Acid Transport

sion to the endothelium of post-capillary venules. As a result, infected red blood cells become absent from the circulation as they are sequestered in the vascular beds of major organs, leading to microcirculatory obstruction with severe tissue ischemia and metabolic dysfunction. A number of endothelial receptors for PfEMP-1 have been identified based on their ability to support adhesion of infected erythrocytes in vitro. However, under physiological flow conditions, adhesion is almost exclusive to CD36, accounting for greater than 90% of the parasite isolates tested [89, 90]. Owing to its expression on macrophages, the CD36/PfEMP-1 interaction may also be beneficial to the host. Binding of infected red blood cells to activated monocytes induces a respiratory burst [91]. The presence of redox-active ferrousprotoporphyrin derived from the catabolism of hemoglobin by the intracellular parasite makes the parasite particularly susceptible to oxidation. In addition, it has recently been shown that CD36 on the surface of macrophages mediates the nonopsonic phagocytosis of infected cells [92]. In this process clustering of CD36 within the membrane upon binding of the infected red blood cell leads to activation of a tyrosine kinase signaling cascade and phagocytosis. This role for CD36 in the innate immune response may explain, in part, why parasitized erythrocyte isolates from cases of non-severe malaria are more likely to show a higher avidity of binding to CD36 [93]. Strengthening of the innate immune response would be particularly beneficial to children as protective immunity to P. falciparum develops slowly, requiring recurrent prolonged and often clinically significant attacks. Interestingly, mutations in CD36 occur at an exceptionally high frequency (up to 18.5%) within populations of malaria endemic regions of sub-Saharan Africa. The most common is a nonsense mutation resulting from the substitution of G for T at nucleotide position 188 of exon 10 (corresponding to the cDNA nucleotide 1264) [60]. Individuals homozygous for this mutation show a complete lack of CD36 on platelets and monocytes. Heterozygosity for this mutation occurs with a prevalence of 17.5%, whereas only 1% are homozygous for the mutation [94]. Although there are conflicting data on the selective advantage that such mutations may offer with respect to malaria, it has been suggested that mutations in CD36 might exist as a balanced polymorphism, reducing parasite sequestration without significantly depressing the innate immune response [60, 94].

1.12

CD36 and Fatty Acid Transport 1.12.1

CD36 is Identified as a Mediator of FA Uptake

As a result of their hydrophobic nature, fatty acids readily partition into and diffuse across the lipid bilayer. The transport of FA across the plasma membrane may be viewed as the progression of three separate kinetic events: (1) partitioning of unbound FA from the aqueous phase into the outer leaflet of the membrane;

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1 Structure-Function of CD36 and Evidence for its Role in Facilitating Membrane Fatty Acid Transport

(2) diffusion, or flip-flop, to the inner leaflet; and (3) dissociation of the FA from the inner leaflet to the cytoplasm. Studies of the transport of long-chain FA across model membranes indicate that flip-flop is the rate-limiting step [95]. For the uptake of physiologically important long-chain fatty acids into giant unilaminar vesicles approaching cell size (³ 2000 Å) and membrane lipid composition, flip-flop times are between 1 and 10 seconds. Similar results are obtained with resealed erythrocyte ghosts in which transport rates of about 1 s–1 have been measured for oleate at 37 8C [96]. Given the unbound FA concentration in the blood and interstitium (5–11 nM), this intrinsic rate of transport may not be sufficient to meet the needs of tissues possessing a high metabolic capacity for FA such as adipose tissue, liver, or muscle. Consistent with this, extensive research has shown that protein-mediated FA transport occurs in parallel with passive diffusion and may predominate in these highly metabolic tissues. The link between CD36 and FA uptake was initially made in isolated adipocytes. Kinetic studies of the metabolism-independent uptake of FA into isolated rat adipocytes demonstrated the existence of a saturable and long-chain FA specific component, implicating the existence of a high-affinity carrier [97, 98]. The saturable component of uptake was sensitive to the common anion transport inhibitors phloretin and diisothiodisulfonic acid. The transport Km was determined to be within the range of physiological concentrations of unbound FA. A membrane protein was subsequently identified and isolated by specific labeling of adipocytes with tritiated membrane impermeable reactive FA analogs [99, 100]. Under the conditions used, labeling of this protein resulted in a 70% reduction in FA uptake with loss of the saturable component. The apparent involvement of this protein in the uptake of FA prompted its designation as a putative FA translocase (FAT). After limited N-terminal sequencing, the cDNA was isolated and shown to have 85% identity to human CD36 [101]. Consistent with a role for CD36 in FA uptake, CD36 purified from adipose tissue reversibly bound long-chain FA with high affinity and specificity [16]. In Ob17PY fibroblasts, stable transfection of CD36 was associated with an increase in the uptake of long-chain FA and with increased FA incorporation into phospholipids [102]. The increased uptake reflected appearance of a saturable, high affinity (Km*4 nM), and phloretin-sensitive component, and the magnitude of the increase generally correlated with the level of protein expression. The expression pattern and regulation of CD36 are consistent with its role in FA uptake. The distribution of CD36 favors tissues with a high metabolic capacity for FA such as adipose tissue, heart, and skeletal muscle [101]. Expression is also high in tissues exposed to large fluxes of FA such as microvascular (but not large vessel) endothelia, mammary secretory epithelia, enterocytes of the small intestine, and endothelial cells of the labyrinth zone of the chorioallantoic placenta [19, 53, 103, 104]. In the small intestine expression is highest in the jejunum, the site of greatest dietary FA absorption, and is upregulated by a high-fat diet. In muscle tissues, expression occurs with predominance in red oxidative fibers and is upregulated with chronic muscle stimulation concomitant with an increase in the

1.12 CD36 and Fatty Acid Transport

FA transport Vmax [105]. As recently shown by Bonen and colleagues, translocation of CD36 from an intracellular pool to the sarcolemma appears to mediate the contraction induced increase in FA uptake [106]. This mechanism would be analogous to the insulin-dependent regulation of glucose uptake by GLUT4 translocation in muscle and fat cells. 1.12.2

CD36, SHR, and Insulin Resistance

Until recently, evidence for an in vivo role for CD36 in FA metabolism was indirect and relied on the pattern of tissue distribution and on alterations in CD36 expression with metabolic or pathologic states. CD36 expression is increased, for example, in mice fed a high-fat diet and in animal models of genetic obesity and diabetes [47–52]. In 1999, Aitman et al. suggested, based on genetic linkage studies, that CD36 deficiency may underlie defects of FA metabolism and insulin responsiveness in the spontaneously hypertensive rat (SHR), which is a well-studied rodent model of human syndrome X [107]. Based on in vitro studies implicating CD36 in FA transport, it was proposed that the primary genetic defect in SHR might be compromised tissue FA utilization, which would contribute to the pathogenesis of insulin resistance by producing secondary alterations in basal glucose metabolism. An SHR congenic strain was generated by replacing a small region of chromosome 4 containing the deletion variant of CD36 with the corresponding wild-type segment from the normotensive Brown Norway rat [107]. Adipocytes isolated from the congenic strain showed significant rescue of insulin-stimulated glucose transport and of catecholamine-mediated lipolysis. More direct evidence for loss of CD36 function in FA uptake in the SHR was obtained recently with in vivo measurements of the uptake of a slowly metabolized FA analog [108]. FA uptake is significantly impaired in SHR heart, oxidative muscle, and adipose tissue while that of the glucose analog fluoro-2-deoxyglucose is greatly increased. The data confirmed that defective FA uptake is a primary factor behind some of the metabolic defects in the SHR. Supplementation of the diet with short-chain FA, which are not dependent on protein-facilitated transport for uptake, eliminated the compensatory increase in glucose uptake, the hyperinsulinemia and cardiac hypertrophy in the SHR. The linkage of CD36 deficiency to insulin resistance in the SHR has been questioned by the finding that the mutations in the CD36 gene, documented in the SHR colony established at the National Institutes of Health (SHR/NIH), were not present in the original strain kept in Japan (SHR/Izm). However, adipocytes from SHR/Izm exhibited the same defects in insulin responsiveness reported in SHR/ NIH [109]. To determine that the improved insulin responsiveness seen in the congenic SHR strain did not result from re-introduction of some other gene within the same chromosomal locus, a transgenic SHR/NIH line rescued for CD36 was generated [110]. Transgenic rescue of CD36 considerably improved serum FA levels, glucose tolerance, and insulin-stimulated glycogen deposition in muscle. The SHR/Izm and SHR/NIH strains diverged long before they were fully inbred

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1 Structure-Function of CD36 and Evidence for its Role in Facilitating Membrane Fatty Acid Transport

and differ throughout the entire genomes [111]. Thus the genetic basis for insulin resistance in the two strains may be different and allelic comparison to draw firm conclusions as to the role of CD36 in modulating the phenotype may not be justified. Regardless, data from both the congenic and transgenic rescue studies clearly show that defective CD36 could potentially be a quantitative trait locus for dyslipidemia, glucose intolerance, and insulin resistance. 1.12.3

CD36 Transgenic and Knockout Mice Models

Recent development of well-defined genetic models of CD36 deficiency and tissuespecific overexpression has added greatly to our understanding of the physiological role of CD36 as a mediator of FA uptake. Previous studies could not yield definitive data as a result of the complexity of the animal models used, which possessed polygenic aberrations. For example, in the SHR/NIH CD36 deficiency was linked to insulin resistance while in the obese Zucker rat, upregulation of CD36 in adipocytes was observed and preceded the development of insulin resistance [47]. In humans, studies used a limited number of subjects and were often complicated by a context of cardiac dysfunction. Transgenic mice with muscle-targeted CD36 overexpression were generated by insertion of a CD36 gene under control of the muscle creatine kinase promoter [112]. The metabolic phenotype of these mice is consistent with greater peripheral FA utilization. These mice have less body fat and lower serum FA, triglyceride, and cholesterol. Blood glucose is significantly increased, while insulin levels are similar in the fed state and higher in the fasted state. Soleus muscle from these mice displays an enhanced ability to oxidize FA in response to stimulation/contraction. Mice null for CD36 were shown to exhibit an increase in fasting serum FA, ketone bodies, triglyceride, and cholesterol [113]. The increase in cholesterol is mainly within the high-density lipoprotein fraction, whereas the increase in triacylglycerol reflects a rise in levels of very low density lipoproteins. Blood glucose is unchanged in the fed state but significantly decreased in the fasted state, as are insulin levels (Hajri et al., unpublished observations). The mice also have significantly less body fat and their adipocytes lack the high-affinity component of FA transport observed in cells isolated from wild-type controls [113]. 1.12.4

CD36-null Mice – the Fed Phenotype

The FA analog b-methyl 15-(p-iodophenyl) pentadecanoic acid (BMIPP, shown in Fig. 1.2) has been used as a metabolic tracer for studying FA uptake and utilization in vivo. Its usefulness has been demonstrated extensively in studies on both humans and laboratory animals [114]. Like native FA, tissue extraction of BMIPP from the blood equilibrates within 2–3 minutes and BMIPP is incorporated into phospholipids, diglycerides, or triglycerides [115, 116]. The stable iodination of

1.12 CD36 and Fatty Acid Transport Chemical structure of the FA analog [125I]BMIPP. The 3-methyl group of BMIPP inhibits b-oxidation, resulting in prolonged tissue retention without affecting its incorporation into complex lipids.

Fig. 1.2

BMIPP (terminal iodophenyl substitution) coupled with its prolonged tissue retention (inhibitory effect of the b-methyl group on b-oxidation) make it an ideal tracer for sensitive comparisons of tissue capacities for FA uptake in vivo. In Europe and Japan, BMIPP is commercially available and is used clinically to assess myocardial tissue viability in patients with advanced coronary artery disease by single photon emission computed tomography (SPECT). In normoxic myocardium long-chain FAs are the major energy source, accounting for 60–70% of ATP production. In ischemic myocardium, b-oxidation of FA is suppressed and, to compensate, glucose utilization is enhanced. A discordant decrease in BMIPP uptake relative to perfusion is therefore often seen in ischemic but viable myocardium and can be used to identify regions likely to respond positively to revascularization [117]. A similar approach was taken to investigate the metabolic phenotype of the CD36-null mouse. To evaluate the contribution of CD36 to FA uptake by various tissues, the biodistributions of [125I]BMIPP were compared between CD36-null mice and their wild-type counterparts [118]. In muscle it is well known that glucose transport is responsive to factors affecting the energy balance of the cell. Agents which inhibit lipid oxidation or decrease circulating levels of FA markedly enhance peripheral glucose utilization. Therefore, to determine if defects in FA uptake would perhaps be compensated for by an increase in glucose utilization, uptake of the non-metabolizable glucose analog [18F]fluoro-2-deoxyglucose (FDG, shown in Fig. 1.3) was also analyzed. FDG is a commonly used metabolic tracer. Like glucose, it is taken up by cells and phosphorylated by hexokinase, effectively trapping the label within the cell. The fluorine at the C-2 position inhibits further metabolism by blocking isomerization of the glucose 6-phosphate to fructose 6phosphate. Consistent with the pattern of CD36 expression, uptake of BMIPP was reduced in the fed state by 50–80% in heart, skeletal muscle, and adipose tissues of CD36null mice. In muscle tissues the magnitude of the defect increased with increasing oxidative capacity. For example, diaphragm muscle, which in the mouse is almost exclusively oxidative, exhibited a defect in BMIPP uptake nearly three times that of hip muscle, which is predominantly glycolytic. The defect in uptake was accompanied by a decreased incorporation of labeled FA into triglyceride but was not accompanied, in the fed state, by a compensatory increase in glucose uptake. The 50–60% reduction observed in BMIPP incorporation into triglycerides in CD36-null muscle and adipose tissues was associated with a 2- to 3-fold increase in labeled diglycerides. Identical results were obtained with [3H]palmitate in iso-

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1 Structure-Function of CD36 and Evidence for its Role in Facilitating Membrane Fatty Acid Transport

Chemical structure of the non-metabolizable glucose analog [18F]FDG (top panel). FDG is taken up by cells and phosphorylated by hexokinase. The fluorine at the C-2

Fig. 1.3

position blocks the next step in glycolysis (bottom panel), isomerization of glucose 6phosphate to fructose 6-phosphate.

lated adipocytes. The classical biosynthetic pathway for triglyceride is shown in Fig. 1.4. The apparent block in diglyceride to triglyceride conversion observed in these tissues occurred despite normal specific activities of the key enzymes longchain acyl-CoA synthetase and diacylglycerol acyltransferase (DGAT). Assuming that DGAT in these tissues was saturated with diglyceride (which accumulated), the data suggest that the decreased rate of triglyceride synthesis was determined by a deficit in the FA supply and a relatively low affinity of DGAT for long-chain acyl-CoA. Regulating FA esterification at the branch point between phospholipid and triglyceride synthesis makes sense physiologically since it would serve to first secure FA for pathways essential to the cell, namely b-oxidation and phospholipid synthesis. Only when the FA needs of these pathways are met, as reflected by a rise in long-chain acyl-CoA, would triglyceride synthesis in this model proceed optimally through DGAT. This would ensure that triglyceride deposition would not compete with b-oxidation when the FA supply is low and might in part explain why CD36deficient animals appear healthy under normal and non-metabolically challenged conditions.

1.12 CD36 and Fatty Acid Transport

The classical triacylglycerol biosynthetic pathway, showing steps that are altered by CD36 deficiency (highlighted by arrows). The enzyme diacylglycerol acyltransferase (DGAT) catalyzes step 5 at the bifurcation between triglyceride and phospholipid formation. (1) Acyl-CoA synthetase; (2) glycerol-3phosphate acyltransferase; (3) monoacylgly-

Fig. 1.4

cerol-3-phosphate acyltransferase; (4) phosphatidic acid phosphatase; (5) diacylglycerol acyltransferase. Broken arrows represent steps not shown. PC, phosphatidylcholine; PE, phosphatidylethanolamine; PS, phosphatidylserine; PI, phosphatidylinositol; PG, phosphatidylglycerol.

The observed decrease in triglyceride deposition in CD36-null muscle may have been due solely to a general lack of intracellular FA availability or may have been secondary to shunting of what FA was coming in towards oxidation. In this respect, it is interesting to note that the lipid pool distribution observed in CD36null heart in the fed state was similar to that observed in 16-hour fasted wild-type controls. Likewise, in hip muscle, the BMIPP lipid distribution from fed CD36null mice resembled the corresponding 6-hour fasted wild-type distribution (Coburn et al., unpublished observations). Whatever the mechanism, the altered lipid pool distribution observed in CD36-null muscle is consistent with the observed decrease in FA uptake.

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1 Structure-Function of CD36 and Evidence for its Role in Facilitating Membrane Fatty Acid Transport

1.12.5

CD36-null Mice – the Fasting Phenotype

Despite the significant defect in post-prandial FA uptake observed in muscle and adipose tissues of CD36-null mice, there is no compensatory increase in glucose utilization and serum FA and glucose levels are unchanged. However, significant changes are observed when the mice are fasted. In comparison to wild-type controls, fasting levels of plasma free FA and VLDL-triglyceride are significantly increased (2- and 1.4-fold, respectively) and those of plasma glucose [113] and insulin levels (Hajri et al., unpublished observations) are significantly decreased (by 25 and 30%, respectively) in CD36-null mice. To investigate the underlying mechanisms of this phenotype, differences in FA and glucose uptake between these mice in the basal and fasted states were evaluated with BMIPP and FDG (Coburn et al., unpublished observations). In fasted mice BMIPP uptake was significantly decreased in CD36-null adipose tissue, suggesting that lipolysis may be enhanced in comparison to controls. In CD36-null liver, both FDG and BMIPP uptake were significantly increased with fasting. The increase in FDG uptake is consistent with a previously documented autoregulatory mechanism whereby hepatic glucose output is inhibited under conditions of hyperlipidemia [119]. In highly oxidative cardiac and diaphragm muscles FDG uptake was dramatically increased (13- and 3-fold, respectively) with fasting, suggestive of a compensatory increase secondary to a decreased rate of FA oxidation. These findings are consistent with previous reports showing that agents such as methylpalmoxirate, which inhibits lipid oxidation, or nicotinic acid, which decreases circulating levels of FA, markedly enhance whole body glucose disposal [120–122]. In rats given methylpamloxirate, FDG uptake was found to be enhanced in heart and diaphragm by about the same extents seen here with no change observed in the white gastrocnemius, a glycolytic muscle [120]. Interestingly, a further study showed that a dramatic increase in the rate of glucose disposal following inhibition of FA oxidation in rats occurred only with fasting [121]. These observations have led us to propose the following model (shown in Fig. 1.5) to explain the CD36-null fasting phenotype. The increased fasting serum FA is likely a result of both increased mobilization from adipose tissue and decreased peripheral FA utilization. The elevation in serum FA increases flux and uptake of FA by the liver. This would increase triglyceride synthesis and incorporation into lipoproteins, as evidenced by the increase in fasting VLDL-triglyceride. FA oxidation is significantly decreased in CD36-null oxidative muscle but may be normal in glycolytic muscle due to the increased serum FA concentration and the lower oxidative capacity of this tissue. The presumed increase in hormone-sensitive lipase activity in adipose tissue may result from the fasting hypoinsulinemia observed in these mice. Circulating levels of cortisol as well as sympathetic activity to adipose depots are also likely to be increased, further stimulating lipolysis. The decrease in insulin secretion may initially occur as a result of the greatly increased glucose disposal by heart and highly oxidative skeletal muscles and may be compounded as the fast progresses and serum FA levels rise

1.12 CD36 and Fatty Acid Transport

Model of the CD36-null fasting phenotype. Small up/down arrows denote changes in comparison to fasted wild-type controls. In this model, the major defect is a dramatic increase in glucose uptake by oxidative muscles, compensating for the defect in FA oxidation. This occurs only with fasting and is likely induced by a drop in the energy charge of the cell subsequent to the initial drop in insulin. The resulting hypoglycemia causes a further decrease in insulin secretion and increased sympathetic activity, both of

Fig. 1.5

which would increase lipolysis in adipose tissue. The increased serum FA gives rise to increased FA uptake by liver and subsequently to increased VLDL triglyceride and ketone bodies. At some point the increase in FA uptake by liver induces an autoregulatory mechanism effectively clamping hepatic glucose output and thereby contributing to the hypoglycemia. This vicious cycle continues, escalating the hypoglycemia and dyslipidemia, as the fast progresses and is only partially compensated for by the rising ketone levels.

by a FA-induced clamping of hepatic glucose output. Although this model is likely to be amended as more data are obtained, it nevertheless illustrates the important regulatory role of CD36 in the homeostatic mechanisms controlling both substrate transfer between tissues and the balance of local substrate utilization. 1.12.6

CD36 and Insulin Responsiveness in the Mouse

As already noted, deficiency of FAT/CD36 has been genetically linked to insulin resistance in the SHR. In humans, incidence of CD36 deficiency ranges between 0.3 to 18.5% and is highest in subpopulations with high rates of diabetes type II. We examined insulin responsiveness of the CD36-null mouse, which would represent a good model of the CD36-deficient human in terms of the magnitude of the defect in FA uptake [118]. The CD36–/– mouse fed a chow diet rich in complex carbohydrates and low (5%) in fat was more insulin-sensitive than the wild-type

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1 Structure-Function of CD36 and Evidence for its Role in Facilitating Membrane Fatty Acid Transport

control. It cleared glucose faster, reflecting a several-fold enhancement in glucose utilization by muscle. In vitro, insulin responsiveness of glycogenesis by the null soleus was enhanced. However, when the chow diet was switched to one high in fructose, the CD36–/– but not the wild-type mouse developed marked glucose intolerance and hyperinsulinemia, reflecting decreased muscle glucose uptake. Both mice groups had glucose intolerance with feeding a high-fat diet but muscle insulin responsiveness was partially protected in the CD36-null mouse. In conclusion, CD36 deficiency in the mouse enhances insulin responsiveness on a high-starch/ low-fat diet. It predisposes to the insulin resistance induced by high fructose and, partially protects from that induced by high fat. If these data could be extrapolated to the human case, CD36 deficiency would constitute an important risk factor for the insulin resistance linked to high consumption of simple sugars. It is also possible, based on preliminary data from mice with muscle CD36 overexpression, that high levels of CD36 in muscle may impact susceptibility to glucose intolerance and diminished insulin responsiveness with consumption of high-fat diets.

1.13

Perspectives and Future Directions

The studies with the CD36-deficient and CD36-overexpressing mice models outlined in the previous section indicate that CD36 is essential for normal rates of FA uptake by muscle and adipose tissues in vivo. Studies have also identified several other FA transport proteins in these tissues, including FABPpm and FATP1 [123, 124]. It is unlikely that the functions of these proteins are entirely redundant. Otherwise, these tissues would be better able to compensate for the lack of CD36 expression. Furthermore, the expression patterns of these proteins only partially overlap with that of CD36. They are also found in highly metabolic tissues exhibiting little or no CD36 expression, such as liver and kidney. Consequently, these proteins are likely to play important and distinct roles in the maintenance of metabolic homeostasis. As more is learned about the various proteins that facilitate FA transport, our understanding of how balanced whole-body utilization of metabolic substrates is achieved, should greatly improve. The existence of a number of differentially regulated plasma membrane proteins capable of enhancing FA uptake likely provides an adaptive pathway both within the cell and between tissues to regulate FA transport and distribution to match changing cellular needs. Tissue and temporal regulation of transport is necessary in the course of the normal daily nutritional transitions and during altered metabolic states such as fasting and strenuous exercise to maintain a consistent and adequate supply of fuel to all organs of the body. Furthermore, to the extent that FA and glucose homeostasis are inextricably linked, these proteins are very likely integral to the local homeostatic mechanisms which determine the balance of substrate utilization according to substrate availability, hormonal status, and energy demand. In this context, determining the role

1.14 References

CD36 plays in the etiology of diet-induced diabetes type 2 and metabolic syndrome X in humans will undoubtedly be important since mutations in the CD36 gene are relatively frequent and with high incidence in subpopulations prone to these diseases. The demonstration of the metabolic phenotype of CD36-null mice has provided a broad overview of the role of CD36 in FA metabolism. While this has allowed us to answer a number of questions, it has suggested many others. A question of considerable interest, for example, is to what extent CD36 might play an essential role in vivo in supplying ligands for PPAR activation. PPARa-deficient mice exhibit a phenotype very similar to that of the CD36-null mouse with depressed myocardial FA uptake and b-oxidation [43]. A large decrease in CD36 expression in this mouse model is observed and its direct contribution to the phenotype is unknown. In addition, if CD36 expression is needed early in this program to supply PPARa with the ligands necessary for full expression of the oxidative genotype, it is possible that in the absence CD36 there is maintenance of some aspects of the fetal metabolic phenotype.

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2

Role and Function of FATPs in Fatty Acid Uptake Jean E. Schaffer

2.1

Introduction

In mammalian cells such as myocytes and adipocytes, long-chain fatty acid (LCFA) uptake is efficient and highly regulated. Free fatty acids (FFA) are generated by lipase-mediated hydrolysis of triglycerides in adipose stores or by hydrolysis of triglycerides from lipoprotein particles in plasma. Within serum, FFA are largely bound to albumin, resulting in low nanomolar concentrations of free unbound LCFAs under normal physiologic conditions [1]. Developmental and physiologic regulation of LCFA utilization in accordance with nutritional and hormonal signals suggests that discrete mechanisms exist for vectorial movement of LCFAs into/out of cells. Experimental evidence provides support for two classes of mechanisms of LCFA transport: non-protein-mediated and protein-mediated permeation. Rapid flip-flop of un-ionized LCFAs within the membrane does not require facilitation by membrane proteins and likely occurs under pathophysiologic conditions in which high unbound LCFA concentrations are observed [2, 3]. On the other hand, proteinmediated transport mechanisms are likely to be important for permeation of LCFA anions and for un-ionized LCFAs when present at physiologic (low nanomolar) concentrations [4, 5]. Precedence for the involvement of membrane proteins in LCFA transport in mammalian cells comes from studies of Escherichia coli. The outer membranebound fatty acid transport protein fadL and an inner membrane-associated acylcoenzyme A (CoA) synthetase (fadD) are required for LCFA import and utilization in this prokaryote [6–10]. Moreover, kinetic analyses of LCFA transport in mammalian cells using radiolabeled substrates show that transport is efficient, saturable at low unbound LCFA concentrations, specific for particular LCFAs with competition between substrates, and inhibited by prior protease treatment of the cell surface [5, 11–14].

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2.2

Identification of Fatty Acid Transporter Proteins

Initial efforts to identify plasma membrane proteins that facilitate LCFA transport in mammalian cells took advantage of the observation that many transport proteins bind their substrates specifically with high affinity. First, a plasma membrane fatty acid-binding protein (FABPpm) was purified by oleate-agarose affinity chromatography from hepatocytes, adipocytes, jejunal enterocytes and cardiac myocytes, and proposed to play a role in fatty acid import [15, 16]. This protein shares amino acid identity with mitochondrial aspartate aminotransferase (mAspAT) [17]. Subsequently, fatty acid translocase (FAT) was identified to play a role in LCFA import on the basis of its ability to bind a sulfosuccinimidyl LCFA derivative that inhibits LCFA import [18]. This 88 kDa plasma membrane glycoprotein is the murine adipose homolog of CD36 [19], a known scavenger receptor [20]. Expression of the cDNAs for either mAspAT [21] or CD36 [22] increases the saturable component of LCFA import into cells, consistent with a role for each of these proteins in mammalian cell LCFA transport. Subsequently, a functional, expression cloning strategy was used to isolate a cDNA encoding the first member of the fatty acid transport protein (FATP1) family. Based on the observation that adipocytes have efficient LCFA import, a 3T3-L1 adipocyte cDNA library was expressed in Cos7 cells, which were then screened for uptake of a fluorescent fatty acid analog by flow cytometric analysis. Cells with increased uptake were isolated, cDNAs recovered and amplified, and cDNAs were re-transfected in subsequent rounds of screening. Screening of 106 independent colonies from the library led to the isolation of multiple independent cDNAs encoding a novel protein, FATP1. Based on its isolation in this screen, FATP1 was proposed to function as a transporter for LCFAs. A second cDNA recovered in this screen encoded long-chain acyl-CoA synthetase 1 (ACS1). ACS1 catalyzes esterification of LCFAs (C14–C18) at the 1-carbon position with CoA [23], a required activation step for most anabolic and catabolic pathways in which LCFAs are used. The identification of ACS1 in the screen for proteins that increase LCFA uptake suggests that esterification of LCFAs is coupled with membrane transport in mammalian cells. As has been proposed for LCFA import in E. coli [6], ACS may facilitate import in mammalian cells through vectorial acylation. Consistent with this model, overexpression of FATP1 and of ACS1 in fibroblasts have synergistic effects on LCFA uptake [24]. Furthermore, ACS1 is an integral membrane protein that localizes to several cellular membranes of adipocytes, including the plasma membrane where it co-distributes with FATP1.

2.3

Structure of FATPs

FATP1 is a 646 amino acid protein that is expressed in cells and tissues with high-level fatty acid import for metabolism or storage [25]. Expression of FATP1 in mammalian cells increases import of radiolabeled and fluorescently labeled

2.3 Structure of FATPs

LCFAs and very long-chain fatty acids (VLCFAs), but not medium-chain substrates. FATP1 is a member of a large family of related proteins from diverse organisms including Saccharomyces cerevisiae, Caenorhabditis elegans, Mycobacterium tuberculosis, rats, mice, and humans [26]. In mice, different FATP isoforms (mmFATP1–5) have distinct tissue-specific distributions of expression. Northern analysis demonstrates that isoforms mmFATP1, 3, and 4 are widely expressed, whereas mmFATP2 and mmFATP5 are expressed in a more restricted pattern. Most tissues show expression of at least one isoform of FATP, and several, such as liver, heart and kidney, express multiple isoforms. On the basis of sequence conservation, the FATP protein family also includes proteins initially characterized as very long-chain acyl-CoA synthetase (VLACS) enzymes (see below), suggesting that fatty acid transport and esterification are evolutionarily and/or functionally linked. FATP1 is an integral membrane protein found at the plasma membrane as well as on internal cellular membranes in adipocytes that natively express the protein. It is not known whether the presence of FATP1 on intracellular membranes is due to specific or regulated targeting of the protein to those membranes or whether this simply reflects newly synthesized FATP1 protein within the secretory pathway. Compared with natively expressed protein, overexpressed FATP1 is less efficiently targeted to the plasma membrane [27]. Another FATP family member, FATP4, is natively localized at the brush border membrane on the apical side of enterocytes that face the intestinal lumen in the mouse and human gut [28]. This localization is consistent with a role for FATP4 in absorption of dietary LCFAs. By contrast, human VLACS, which contains a potential C-terminus peroxisome targeting sequence, co-localizes with a peroxisomal marker in HepG2 cells [29]. Experimental characterization of the topology of FATP1 demonstrates an unusual predicted structure for FATPs. The primary amino acid sequence of FATP1 has multiple hydrophobic domains, consistent with a membrane protein; however, the only region predicted to have significant a-helical structure is the N-terminal signal sequence. FATP1 has a long hydrophobic N-terminal region of 190 amino acids that contains three stretches of sequence, each independently capable of directing integral membrane association of reporter sequences [27]. The extreme Nterminus of FATP1 faces the extracellular/luminal space, residues 1–190 contain at least one transmembrane domain, and the C-terminus of FATP1 faces the cytosolic space. Residues 191–257 are not membrane associated, likely face the cytosol, and contain a motif (IYTSGTTGXPK) that is implicated in interactions with ATP [30]. By contrast, immunofluorescence studies of FATP2, using selective permeabilization conditions, indicate that the C- and N-termini of FATP2 are both oriented toward the peroxisome matrix [29, 31].

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2.4

Function of FATPs

Among the various FATP family members described to date, function in LCFA import has been demonstrated for murine isoforms mmFATP1, mmFATP2, mmFATP4, and mmFATP5; for the C. elegans ortholog ceFATPb; for the M. tuberculosis ortholog mtFATP; and for the S. cerevisiae ortholog fat1p [26, 28, 32]. In mammalian overexpression studies, the various FATP isoforms/orthologs have been expressed in cells with low basal fatty acid import. Uptake assays have been performed by brief incubations of cells (15–60 seconds) with low concentrations of radiolabeled or fluorescently labeled free LCFAs in buffered solutions containing albumin (BSA; fatty acid:BSA ratios of less than 5:1). Alternatively, in assays for FATP4 function, LCFAs have been solubilized by complexing in mixed micelles containing bile acids (taurocholate) and fatty acids (bile acid:fatty acid of 2:1 to 5:1) [28]. At the end of uptake assay incubations, cells have been washed at 4 8C with buffer containing phloretin to inhibit LCFA permeation (influx and efflux) and/or 0.1% BSA to remove cell surface-bound LCFAs. Depending on the level of expression of the particular FATP, increases in uptake from 2- to 100-fold have been observed in different cell backgrounds using transient and stable modes of expression of the different isoforms. Conversely, antisense depletion of FATP4 expression in primary cultures of enterocytes to 40% of basal levels specifically diminishes the rate of oleic acid uptake by enterocytes to 40% of basal levels [28]. These findings suggest that FATP4 accounts for most LCFA transport activity in isolated enterocytes. These findings are mirrored by studies in S. cerevisiae, in which knockout of fat1p significantly reduces the rate of oleic acid uptake [32]. The high degree of sequence conservation between VLACS enzymes and FATPs suggests that these proteins are not only evolutionarily related but also functionally related proteins. When rat liver VLACS was purified from rat liver homogenates on the basis of its enzymatic activity and subsequently cloned, this peroxisomal enzyme was found to have 40% sequence identity with mmFATP1 [33]. Moreover, there is 89% sequence identity between rat liver VLACS and mmFATP2, a murine FATP isoform identified on the basis of sequence similarity to FATP1. The high degree of sequence conservation raised the possibility that these sequences represent species and/or tissue specific orthologs of the same protein. In addition to sequence conservation between these proteins, overexpression of a number of FATP family members in mammalian cells results in increased cellular CoA synthetase activity. When overexpressed in Cos cells, FATP1 and FATP4 increase total cellular VLACS activity; whereas overexpression of FATP5 increases cholate CoA ligase activity [34–36]. Moreover, overexpressed FATP1 in Cos7 cells co-segregates with VLACS activity in crude cellular fractions [34]. On the other hand, mutations of conserved sequences appear to affect both uptake and esterification functions. The 11 amino acid motif IYTSGTTGXPK (FATP1 sequence 247–257) is conserved in a number of proteins that either bind ATP or catalyze reactions that proceed through adenylated intermediates, including acyl-CoA synthetases. Conservative substitutions in this motif result in a properly expressed and targeted plasma

2.5 Regulation of FATP expression

membrane protein with impaired ability to bind ATP and transport LCFAs compared with wild-type protein [30]. In addition, substitution of residues 249–254 or deletion of FATP1 amino acids 503–524 leads to synthesis of proteins of expected molecular mass and diminishes VLACS activity in cellular lysates [34], although it is unclear whether either of these mutated proteins is targeted in a fashion similar to wild-type FATP1 or whether either functions in LCFA import. Moreover, disruption of the yeast FATP1 ortholog fat1p yields a strain with impaired LCFA import and utilization [32], normal long-chain ACS activity [32, 37], diminished VLACS activity, and increased concentrations of free very long-chain fatty acids (VLCFAs) [37– 39]. Taken together, these studies suggest that FATP1 contributes to the observed LCFA transport function by catalyzing vectorial acylation. Several alternative interpretations of these data have been suggested. First, due to tight coupling of LCFA transport and esterification, FATP1 may closely associate with esterifying enzymes such as VLACS in a cell surface transport complex [40]. Second, FATP1 may be a bifunctional molecule with separable transport and esterification activities [39]. Third, changes in the magnitude of LCFA uptake due to changes in FATP1 expression may more indirectly affect esterification [40, 41]. For example, disruption or overexpression of a gene encoding a protein in a metabolic pathway may result in compensatory metabolic changes in the cells. Or the observed changes in VLACS activity may result from FATP-mediated uptake of LCFA ligands for peroxisome-proliferator activated receptors (PPARs) that in turn alter the level of expression of the peroxisomal enzyme, VLACS. Proponents of these alternative models cite discrepancies in substrate specificity of transport and esterification activities. FATP1 facilitates import of LCFAs and VLCFAs, whereas known VLACS enzymes have minimal activity toward long-chain substrates. Definitive assessment of putative enzymatic activity of FATP1 awaits studies in which purified FATP1 protein is assayed outside a context in which metabolic compensations are likely to occur.

2.5

Regulation of FATP expression

FATP1 mRNA expression is regulated by cis-acting elements in the 5' untranslated region of the FATP1 gene. PPARa and c agonists induce FATP1 mRNA in a tissue-specific manner and are associated with increases in LCFA transport [42, 43]. Because FATP1 mRNA is also induced by retinoid X receptor (RXR) agonists, it has been proposed that transcription of FATP1 is regulated by PPAR-RXR heterodimers [44]. These agents likely act at the functional cis-acting PPAR-response element located from –458 to –474 in the upstream region of the FATP1 gene [45]. On the other hand, FATP1 transcription is negatively regulated by insulin [46] through a cis-acting insulin response sequence present from –1347 to –1353 upstream of the FATP1 gene [47]. Abundance of FATP1 mRNA is increased in Zucker diabetic fatty rats in adipocytes and this finding correlates with increased FFA uptake [48]. These findings are all consistent with a role of FATP1 in regulated LCFA transport. Although some studies show no changes in FATP1 mRNA

35

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2 Role and Function of FATPs in Fatty Acid Uptake

or protein levels associated with physiologic or pathophysiologic changes in cellular LCFA utilization [49, 50], it is possible that post-translational modifications or alterations in subcellular localization serve as an important means of regulation of FATP1 function. Moreover, these studies do not examine potential changes in expression of other FATP family members.

2.6

Significance of FATPs

Whether FATP family members function as transporters for LCFAs remains to be established. Although functional studies suggest these proteins play a role in LCFA import in mammalian cells, several questions remain. First, the membrane topology for each of these proteins does not resemble those of polytopic membrane transporters for hydrophilic substrates. Many transporters are predicted to have transmembrane domains (4–12 per transporter) consisting of primarily a-helical structures of 17 or more amino acids that span the phospholipid bilayer and form a three-dimensional channel through which substrate passes. Second, gain and loss of function studies are potentially confounded by cellular metabolic compensations. Third, tight coupling of transport and esterification render measurements of transport alone difficult. Fourth, loss of function studies may be complicated by compensatory upregulation of expression of other highly related protein family members. Regardless of the exact mechanism of action of FATP family members, the importance of FATP proteins in LCFA transport is underscored by experiments in murine enterocytes in which antisense depletion of FATP4 protein significantly decreases LCFA import [28]. Similarly, disruption of the yeast Saccharomyces cerevisiae FATP1 homolog fat1p results in impaired LCFA uptake and impaired growth under conditions in which de novo LCFA biosynthesis is inhibited and LCFAs are supplied as the sole carbon source [32, 39]. There is great interest in the generation of mice in which the various FATP isoforms are disrupted, although to date there are no published data available on the knockout phenotypes. No human diseases have been identified as resulting from FATP1 mutations. However, an intronic polymorphism in the FATP1 gene is associated with increased plasma triglyceride levels in women [51]. It is as yet unclear whether this polymorphism is associated with altered levels of expression or function of FATP1 in tissues that would impact on total body fatty acid homeostasis. Because of the physiologic significance of regulated LCFA flux in vivo, understanding the mechanism of action of proteins implicated in LCFA uptake is an important goal. Vertebrate animals have evolved an ability to store excess nutrients in the form of esterified LCFAs in adipocytes and to remobilize these substrates in conditions of nutritional deprivation. Regulation of these processes implies regulated LCFA transport across the adipocyte membrane. On the other hand, cells such as cardiac and skeletal myocytes have limited capacity for de novo LCFA biosynthesis and storage, yet these cells rely on import of LCFAs to provide

2.7 References

metabolic substrates. Evidence is emerging that imbalance of LCFA uptake and utilization in cells with limited capacity for triglyceride storage results in lipotoxicity [52–55]. Cellular dysfunction and death from excess lipid accumulation may play a role in the pathogenesis of diabetes [56] and heart disease [57, 58]. Future studies of the mechanism of LCFA import and the manner in which it is regulated has the potential to aid in the development of novel therapeutic approaches for these common human diseases.

2.7

References 1 2

3 4 5 6

7 8 9

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13 14

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G. Richieri, Kleinfeld, A. J. Lipid Res. 1995, 36, 229–240. F. Kamp, Zakim, D., Zhang, F., Noy, N., Hamilton, J. Biochemistry 1995, 34, 11928–11937. J. A. Hamilton, Kamp, F. Diabetes 1999, 48, 2255–2269. A. M. Kleinfeld, Chu, P., Romero, C. Biochemistry 1997, 36, 14146–14158. D. D. Stump, Fan, X., and Berk, P. D. J. Lipid Res. 2001, 42, 509–520. K. Klein, Steinberg, R., Fiethen, B., Overath, P. Eur. J. Biochem. 1971, 19, 442–450. W. D. Nunn, Simons, R. W. Proc. Natl Acad. Sci. USA 1978, 75, 3377–3381. G. B. Kumar, Black, P. N. J. Biol. Chem. 1991, 268, 15469–15476. P. N. Black, Di Russo, C. C., Metzger, A. K., Heimert, T. L. J. Biol. Chem. 1992, 267(35), 25513–25520. P. N. Black, DiRusso, C. C. Biochim. Biophys. Acta 1994, 1210, 123–145. S. Mahadevan, Sauer, F. Arch. Biochem. Biophys. 1974, 164, 185–193. N. A. Abumrad, Perkins, R. C., Park, J. H., Park, C. R. J. Biol. Chem. 1981, 256, 9183–9191. N. A. Abumrad, Park, J. H., Park, C. R. J. Biol. Chem. 1984, 259, 8945–8953. W. Stremmel, Strohmeyer, G., Berk, P. D. Proc. Natl Acad. Sci. USA, 1986, 83, 3584–3588. B. J. Potter, Stump, D., Schwieterman, W., Sorrentino, D., Jacobs, L. N., Kiang, C. L., Rand, J. H., Berk, P. D. Biochem. Biophys. Res. Commun. 1987, 148, 1370–1376.

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29

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2 Role and Function of FATPs in Fatty Acid Uptake 30

31 32

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S. M. Stuhlsatz-Krouper, Bennett, N. E., Schaffer, J. E. J. Biol. Chem. 1998, 273, 28642–28650. B. T. Smith, Sengupta, T. K., Singh, I. Exp. Cell Res. 2000, 254, 309–320. N. J. Faergeman, DiRusso, C. C., Elberger, A., Knudsen, J., Black, P. N. J. Biol. Chem. 1997, 272, 8531–8538. A. Uchiyama, Aoyama, T., Kamijo, K., Uchida, Y., Kondo, N., Orii, T., Hashimoto, T. J. Biol. Chem. 1996, 271, 30360– 30365. N. Coe, Smith, A., Frohnert, B., Watkins, P., Bernlohr, D. J. Biol. Chem. 1999, 274, 36300–363004. T. Herrmann, Buchkremer, F., Gosch, I., Hall, A. M., Bernlohr, D. A., Stremmel, W. Gene 2001, 270, 31–40. S. J. Steinberg, Mihalik, S. J., Kim, D. G., Cuebas, D. A., Watkins, P. A. J. Biol. Chem. 2000, 275, 15605–15608. P. A. Watkins, Lu, J.-F., Steinberg, S. J., Gould, S. J., Smith, K. D., Braiterman, L. T. J. Biol. Chem. 1998, 273, 18210– 18219. J. Choi, Martin, C. J. Biol. Chem. 1999, 274, 4671–4683. C. DiRusso, Connell, E., Faergeman, N., Knuddsen, J., Hansen, J., Black, P. Eur. J. Biochem. 2000, 267, 4422–4433. A. Stahl, Gimeno, R. E., Tartaglia, L. A., Lodish, H. F. Trends Endocrinol. Metab. 2001, 12, 266–273. J. E. Schaffer. Am. J. Physiol. 2002, 282, E239–E246. G. Martin, Schoonjans, K., Lefebvre, A. M., Staels, B., Auwerx, J. J. Biol. Chem. 1997, 272, 28210–28217. K. Motojima, Passilly, P., Peters, J. M., Gonzalez, F. J., Latruffe, N. J. Biol. Chem. 1988, 273, 16710–16714. G. Martin, Poirier, H., Hennuyer, N., Crombie, D., Fruchart, J. C., Heyman, R. A., Besnard, P., Auwerx, J. J. Biol. Chem. 2000, 275, 12612–12618.

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39

3

Function, Expression, and Regulation of Human ABC Transporters Gerd Schmitz and Thomas Langmann

3.1

Introduction

Members of the ATP binding cassette (ABC) transporter superfamily, which are found in all three kingdoms of life, namely prokaryotes, archae bacteria and eukaryotes, represent one of the biggest protein families described so far. ABC transporter genes are highly conserved among different genomes and have been sustained throughout the evolutionary tree. They are usually multispan membrane proteins that mediate the active uptake or efflux of specific substrates across various biological membrane systems [1]. The development of these two different directions of transport, import or export, most likely occurred even before the differentiation of eukaryotes from prokaryotes [2]. In bacteria, these bidirectional transport functions either provide a mechanism for nutrition supply, as in the MalK transporter from Escherichia coli [3], or serve as a defense mechanism, allowing bacteria to protect themselves from their own or foreign toxins [4]. During evolution, eukaryotes have developed specialized ABC proteins as a type of early innate immune system, protecting cells from harmful substances. Thus in the human system several ABC proteins (MDRs, MRPs, ABCG2) are responsible for increased drug exclusion in compound-treated tumor cells, providing cellular mechanisms for the development of multidrug resistance [5]. ABC transporters have also received attention because mutations in these molecules are the cause of various inherited human diseases, including familial HDL deficiency (ABCA1) [6–8], some chorioretinal diseases (ABCR or ABCA4) [9], progressive familial intrahepatic cholestasis (PFIC) type II (PFICII: BSEP or ABCB11) and type III (PFICIII: MDR3 or ABCB4) [10–12], Dubin–Johnson syndrome (cMOAT or ABCC2) [13], pseudoxanthoma elasticum (MRP6 or ABCC6) [14], adrenoleukodystrophy (ALDR or ABCD2) [15], and b-sitosterolemia [16]. In addition, functional polymorphisms have been described in various ABC genes including ABCA1 [17], ABCA4 [18], and ABCG1 [19]. Most interestingly, a significant number of human ABC transporters are critically involved in bile acid, phospholipid, and sterol transport [20–23], whereas the expression of these ABC proteins is itself controlled by lipids. Therefore, it is obvious that ABC transporters are promising target molecules for the treatment of

40

3 Function, Expression, and Regulation of Human ABC Transporters

lipid disorders. In this chapter, we summarize the structural features of human ABC transporters, their role in human disorders and especially highlight the function and regulation of ABC proteins in cellular and total body lipid homeostasis.

3.2

Structural Features of ATP Binding Cassette (ABC) Transporters

A functional ABC transporter protein usually consists of two transmembrane domains (TMD) and two nucleotide binding domains (NBD) or ATP binding cassettes (ABC). The characteristic ABC domain is composed of two short, conserved peptides, the Walker A and Walker B motifs [24], which are required for ATP binding and which are also found in other ATP binding proteins [25] (Fig. 3.1). An additional element, the signature motif, is located between both Walker motifs and is characteristic for each ABC subfamily [26]. The TMD and ABC domains are either present in one polypeptide chain (full-size transporter) or in two polypeptides (half-size transporter) and several arrangements of the TMD and ABC motifs are found in human ABC proteins (Fig. 3.1). Among the full-size transporters domain structures such as (TMD-ABC)2 as well as TMD0-(TMD-ABC)2 (which contains an additional N-terminal series of five transmembrane spans) occur. (TMD-ABC)2 structures are represented in the

Diagram depicting domain arrangements of human ABC transporters. The ATP binding cassette (ABC) consists of Walker A and Walker B motifs, separated by the signature motif characteristic for each ABC transporter subfamily [24, 28]. The membrane spanning domains are depicted as barrels. (A) The TMD0-(TMD-ABC)2 structure of ABCC (MRP) family members is shown. In addition to the regular full-size type containing the (TMD-ABC)2 domain arrangement,

Fig. 3.1

this type displays an additional five transmembrane domains termed TMD0. (B) Prototype ABC transporter with the (TMD-ABC)2 structure. (C) Two alternative types of halfsize molecules, TMD-ABC and ABC-TMD. Only corresponding half-molecule organizations are able to form heterodimers. (D) The (ABC)2 type of molecules lacking transmembrane domains is unlikely to function as transporter.

3.3 Overview of Human ABC Gene Subfamilies

ABCA, ABCB, and ABCC families, whereas the TMD0-(TMD-ABC)2 arrangement is solely present in specific members of the ABCC subfamily (Tab. 3.1). The (ABC-TMD)2 is only found in yeast and not present in human ABC molecules. Half-size transporters can either occur in the TMD-ABC organization, as it is the case within the ABCD subfamily, or as ABC-TMD, which is found in the ABCG group of ABC proteins (Tab. 3.1). In both cases, creation of a functional transporter requires the assembly as a homodimer or heterodimer. Although the final destination of full-size transporters is the plasma membrane, these proteins are also found intracellularly as a result of vesicular trafficking processes. Also, the localization in intracellular membrane-bound vesicles, collectively named multivesicular bodies (MVBs), is conceivable [27]. Most half-size molecules are routed to intracellular membrane systems such as mitochondria, peroxisomes, the endoplasmic reticulum, and the Golgi compartment [28]. However, a member of the ABCG subfamily, ABCG2, has been localized to the plasma membrane [29]. In contrast to these membrane-spanning ABC transporters, proteins from the ABCE and ABCF subfamilies do not harbor TMD at all and contain a (ABC)2 domain structure (Fig. 3.1). As a consequence, they are not likely to be involved in any membrane transport function. Moreover, ABCE1 binds oligoadenylate, which is produced upon viral infections and seems to be a part of the innate immune system by controlling the RNase L pathway [30]. ABCF1 is associated with ribosomes and interacts with eukaryotic initiation factor 2 (eIF2) and thereby plays a key role in the initiation of mRNA translation [31]. The group of membrane-spanning ABC transporters can be split into two different sections depending on their mode of action. The active transporters or pumps, such as members of the ABCB (MDR/TAP) subfamily, couple the hydrolysis of ATP and the resulting free energy to movement of molecules across membranes against a chemical concentration gradient [32]. In contrast, recent work has identified several ABC proteins that show nucleotide binding and a subsequent conformational change but very low ATP hydrolysis. These molecules mainly function as transport facilitators and include ABCC7 (CFTR) [33], ABCC8 (SUR1), ABCC9 (SUR2) [34], and ABCA1 [35].

3.3

Overview of Human ABC Gene Subfamilies

A comprehensive description of the currently known human ABC transporters is given in Tab. 3.1. The list contains fully characterized ABC genes, as well as gene annotations derived from sequence information based on the analysis of the human genome [36, 37] and uses the proposed nomenclature of the Human Gene Nomenclature Committee (HUGO). The synonyms, the chromosomal location, the domain structure, and the tissue specificity and cellular location of each gene are itemized. Furthermore, the lipid-sensitive regulation and the known or putative function of human ABC transporters is mentioned. A short outline of each of the six known human ABC gene subfamilies is presented in the following paragraphs.

41

MTABC3 ABC 7 MAB C1

TAP1 TAP2 MDR 3

ABCB2 ABCB3 ABCB4 ABCB5 ABCB6 ABCB7 ABCB8 6p21 6p21 7q21.1 7p14 2q36 Xq12–q13 7q36

7p21

MDR1

9q34 16p13.3 1p22.1–p21 17q24 17q24 19p13.3

ABCB1

ABC2 ABC3 ABCR

ABCA2 ABCA3 ABCA4 ABCA5 ABCA6 ABCA7

9q31.1

17q24 17q24 17q24 2q34 7p11–q11

ABC1

ABCA1

Location

ABCA8 ABCA9 ABCA10 ABCA12 ABCA13

Alternative name

Gene

Tab. 3.1 Overview of human ABC gene subfamilies.

TMD-ABC TMD-ABC (TMD-ABC)2 (TMA-ABC)2 T MD-ABC TMD-ABC TMD-ABC

(TMD-ABC)2

(TMD- ABC)2 (TMD- ABC)2 (TMD-ABC)2 (TMD- ABC)2 ( TMD-ABC)2

(TMD-ABC)2 (TMD-AB C)2 (TMD-ABC)2 (TMD-ABC)2 (TMD-ABC)2 (TMD-ABC)2

(TMD-ABC)2

Domain structure

+

– – + – + – –

Ubiquitous, ER Ubiquitous, ER Liver, apical membrane Ubiquitous Mitochondria Mitochondria Mitochondria

– + – – –

+ + – + + +

+

Lipid regulated

Excretory organs, apical membrane

Ovary Heart Muscle, heart Stomach Low in all tissues

Brain Lung Photoreceptors Muscle, heart, testes Liver Spleen, thymus, PBMC

Macrophages, liver

Tissue expression and cellular location

Fe/S clusters Fe/S clusters Fe/S clusters

Phospholipids, PAF, aldosterone, cholesterol, amphiphiles, b-amyloid peptide Peptides Peptides Phosphatidylcholine

Phospholipids Sphingolipids (e.g. ceramide) and serine-phospholipids (e.g. PS)

Choline-phospholipids and cholesterol Estramustine, steroids Surfactant phospholipids N-Retinylidene-PE

Known or putative transported molecule

42

3 Function, Expression, and Regulation of Human ABC Transporters

MTABC2 BSEP

MRP1

MRP2

MRP3

MRP4

MRP5

MRP6

CFTR SUR1 SUR2 MRP7 MRP8 MRP9

ABCB9 ABCB10 ABCB11

ABCC1

ABCC2

ABCC3

ABCC4

ABCC5

ABCC6

ABCC7 ABCC8 ABCC9 ABCC10 ABCC11 ABCC12 7q31.2 11p15.1 12p12.1 6p21 16q11–q12 16q11–q12

16p13.1

3q27

13q32

17q21.3

10q24

16p13.1

12q 24 1q42 2q24

(TMD-ABC)2 TMD0-(TMD-ABC)2 TMD0-(TMD-ABC)2 TMD0-(TMD-ABC)2 (TMD-ABC)2 (TMD-ABC)2

TMD0-(TMD-ABC)2

(TMD-ABC)2

(TMD-ABC)2

TMD0-(TMD-ABC)2

TMD0-(TMD-ABC)2

TMD0-(TMD-ABC)2

TMD-ABC TMD-ABC (TMD-ABC)2

Exocrine tissue Pancreas Heart, muscle Low in all tissues Low in all tissues Low in all tissues

Kidney, liver

Ubiquitous

Prostate

Lung, intestine, liver, basolateral membrane

Liver

Lung, testes, PBMC

Heart, brain, lysosomes Mitochondria Liver, apical membrane

– – – – – –

–

+

+

–

+

+

+ – + GSH-, glucuronate-, sulfate-conjugates, GSSG, sphingolipids, LTC4, PGA1, PGA2, 17b-glucuronosyl estradiol GSH-, glucuronate-, sulfate-conjugates, bilirubin glucuronide, LTC4, 17b-glucuronosyl estradiol, taurolithocholate 3-sulfate, anionic drugs glucuronate-, sulfate-conjugates, 17b-glucuronosyl estradiol, taurolithocholate 3-sulfate Xenobiotics, nucleosides (ATP/ADP/AMP/adenosin, GTP/GDP) Xenobiotics, nucleosides (ATP/ADP/AMP/adenosin, GTP/GDP) Anionic cyclopentapeptides (e.g. BQ123) Chlorideions, ATP Sulfonylureas Sulfonylureas

Fe/S clusters Monovalent bile salts (e.g. TC)

3.3 Overview of Human ABC Gene Subfamilies 43

6p21.33

ABC-TMD ABC-TMD ABC-TMD ABC-TMD ABC-TMD

(ABC)2 (ABC)2

(ABC)2

(ABC)2

TM-ABC TM-ABC TM-ABC TM-ABC

Domain structure

Ubiquitous Placenta, intestine Liver Liver, instestine Liver, instestine

Ubiquitous Ubiquitous

Ubiquitous

+ – + + +

– –

–

–

+ + – +

Peroxisomes Peroxisomes Peroxisomes Peroxisomes Ovary, testes, spleen

Lipid regulated

Tissue expression and cellular location

long-chain long-chain long-chain long-chain

fatty fatty fatty fatty

acids acids acids acids

Plant and shellfish sterols Plant and shellfish sterols

Phospholipids, cholesterol Drug resistance

Translation elongation initiation factor 2

Oligoa denylate

Very Very Very Very

Known or putative transported molecule

Notes: The currently known 48 human ABC transporters from six different subfamilies and their typical features are listed. The proposal of the Human Genome Organization (HUGO) for the numbering of human ABC transporter genes has been used and the common names have been included additionally. The domain structure of ABC transporters has been adapted from Klein et al. [28] or by generation of hydrophobicity plots. An excellent regularly updated website established by Michael Müller (http://nutrigene.4t.com/humanabc.htm) provides supplementary information concerning database entries.

21q22.3 4q22 11q23 2p21 2p21

White MXR White2 White3 White 4

ABC50

ABCF1

4q31

ABCG1 ABCG2 ABCG4 ABCG5 ABCG8

OABP

ABCE1

Xq28 12q11–q12 1p22–p21 134q24.3

7q36 3q25

ALD ALDR PMP70 PMP69

ABCD1 ABCD2 ABCD3 ABCD4

Location

ABCF2 ABCF3

Alternative name

Gene

Tab. 3.1 (continued)

44

3 Function, Expression, and Regulation of Human ABC Transporters

3.3 Overview of Human ABC Gene Subfamilies

3.3.1

The ABCA (ABC1) Subfamily

The ABCA family contains solely full-size transporters (Tab. 3.1), and with ABCA1, ABCA4 (ABCR), and ABCA2 the largest proteins with 2261, 2273, and 2436 amino acids, respectively. Most of the ABCA proteins are expressed ubiquitously at low levels and also predominantly in specific tissues, such as ABCA1 in macrophages and ABCA4 (ABCR), which seems to be restricted to photoreceptor cells [9]. In contrast to all other ABC subgroups, the ABCA subfamily has no counterpart in yeast and appears for the first time in Caenorhabditis elegans [38]. Based on the genomic locations and phylogenetic analyses [39], two distinct divisions of ABCAs can be formed. The first group contains five genes located in a cluster on chromosome 17q24 (ABCA5, ABCA6, ABCA8, ABCA9, and ABCA10) and the second group consists of seven genes distributed over six different chromosomes (ABCA1, ABCA2, ABCA3, ABCA4, ABCA7, ABCA12, and ABCA13). Interestingly, the transcriptional control of at least seven ABCA members (Tab. 3.1) is controlled or influenced by lipids [40–45], indicating an important role of the whole ABCA subfamily in cellular lipid transport processes [23, 46]. ABCA1, the founding member of the family, is under extensive investigation and it is now widely accepted that its predominant role is associated to the regulation of cellular phospholipid and cholesterol release via an indirect mechanism, possibly by ATP-sensitive regulation of an as yet uncharacterized molecule [35]. In contrast, ABCA4 is an active retinoid–PE complex transporter which displays strong, lipid-activated ATPase activity [47–49] comparable to active pumps such as ABCB1 (MDR1). In addition to the high expression in neuronal tissues [50], ABCA2 is also present in liver, kidney, and macrophages [45, 51]. ABCA2 co-localizes with endosomal/lysosomal markers and contains a lipocalin signature motif, a feature found in a family of proteins linked to the transport of sterols including retinoids, steroids, lipids, and bilins [52]. Thus it is conceivable that ABCA2 sequesters lipids or lipid–steroid complexes via its lipocalin domain into endosomal/lysosomal vesicles, which could serve as a secretory pathway for these molecules [51]. This hypothesis is further supported by the lipid-sensitive induction of ABCA2 in human macrophages [45]. Although ubiquitously expressed, the ABCA7 protein is predominantly found in myelo-lymphatic tissues [43, 44] and a pivotal role in the development of hematopoietic cell lineages has been suggested [53]. Interestingly, there is recent evidence that ABCA7 may be involved in the transport of phosphatidylserine and ceramidespecies and thus be linked to apoptotic processes [54]. The ABCA3 protein is an integral part of the surfactant lamellar body membrane in lung alveolar type II cells [55]. Pulmonary surfactant is a complex mixture consisting of phospholipids, neutral lipids, and specific proteins. It is essential for normal lung function because it reduces surface tension at the air–liquid interface of alveolar spaces. Phospholipids comprise 80% of the mass of surfactant, of which 80–85% are phosphatidylcholines (PC). Among the PC molecular species, dipalmitoyl-PC (PC16:0/16:0) is the principle surface tension-lowering

45

46

3 Function, Expression, and Regulation of Human ABC Transporters

molecule, ranging from 40 to 60 mol% in adult mammals, whereas disaturated palmitoylmyristoyl-PC (PC16:0/14:0), together with the monounsaturated palmitoylpalmitoleoyl-PC (PC16:0/16:1) and palmitoyloleoyl-PC (PC16:0/18:1), comprise up to 38% of total PC [56]. Lung surfactant also contains four unique proteins: surfactant protein A (SP-A), SP-B, SP-C, and SP-D [57]. Lamellar bodies are enriched in SP-B and SP-C and it has been proposed that these hydrophobic proteins are secreted together with the phospholipids. SP-A and SP-D are secreted independently of lamellar bodies. The localization of ABCA3 in lamellar bodies of alveolar type II cells and the finding that raised ATP levels in bronchoalveolar lavage fluid are sufficient to stimulate surfactant secretion [58] implicate ABCA3 in the processing of pulmonary surfactant by transporting phospholipids and/or specific surfactant proteins for secretion. Since lamellar bodies are also important structures in other cells with barrier function such as keratinocytes in the skin and intestinal epithelial cells and because ABCA3 is expressed in other cells as well, a similar function in this cellular system could be envisioned [22, 59]. 3.3.2

The ABCB (MDR/TAP) Subfamily

The ABCB family is the only subgroup of human ABC transporters that contains full-size (ABCB1, ABCB4, ABCB5, and ABCB11) and half-size transporters (ABCB2, ABCB3, ABCB6–ABC10) (Tab. 3.1). ABCB1 (MDR1) is probably the best studied ABC transporter. It was the first human ABC protein to be cloned [60] and has the ability to mediate multidrug resistance in cancer cells. ABCB1 is localized to the apical membrane of polarized cells and the major sites of expression are found in the liver, the intestine, and the blood–brain barrier. One proposed physiological function of MDR1 is the protection of cells by exporting lipophilic cytotoxic drugs. In addition to ABCB4 (MDR3), which only translocates phosphatidylcholine (PC) across membranes [61], ABCB1 can transport a variety of lipids: PC analogs, phosphatidylethanolamine (PE), sphingomyelin (SM), cholesterol, and glucosylceramide (GlcCer) molecules, which carry a shortened fatty acid at the C2-position of the glycerol or sphingosine backbone [62]. Of particular interest is the finding that ABCB1 (MDR1)-overexpressing cells have elevated levels of cholesterol, GlcCer [63–65] and caveolin 1 [66], all of which are constituents of raft plasma membrane microdomains involved in pathways of lipid efflux from cells. However, these data need further confirmation. Since ABCB1 itself is localized in Triton X-100-insoluble caveolin/cholesterolrich domains [67] and because cholesterol can directly interact with the substrate binding site of ABCB1 [68], it has been suggested that the transport of cytotoxic drugs, which are mostly lipophilic, is coupled to the translocation of cholesterol and sphingolipids [69]. A recent report has indicated that ABCB1 is also involved in the secretion of platelet-activating factor (PAF) [70]. PAF (1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) is a potent bioactive lipid that is synthesized by a broad range of cells, including circulating infammatory cells, endothelial cells, and epithelial cells. It has

3.3 Overview of Human ABC Gene Subfamilies

a variety of biological effects including activation of inflammatory cells and is involved in many pathological conditions, such as angiogenesis in breast cancers, metastasis, shock, sepsis and multiple organ failure. Since PAF is a naturally occurring short-chain phosphocholine and because MDR1 recognizes short-chain analogs of PC and is expressed in many cell types, including epithelial cells, a model for direct translocation of PAF across the plasma membrane has been proposed. Once present on the cell surface, PAF interacts with the PAF receptor on a neighboring cell and elicits its signaling mechanisms. An unexpected role of ABCB1 in the immune response has been recently identified: mdr1a–/– mice kept under pathogen-free conditions develop spontaneous intestinal inflammation. It is thought that this type of colitis is due to a disturbance of the mucosal layer as a consequence of a defect in the membrane integrity of intestinal epithelial cells [71]. In this context, altered intestinal intraepithelial lymphocyte populations and a disturbed cytokine response has been documented in mdr1a–/– mice [72, 73]. ABCB1 has also been implicated in the efflux of brain b-amyloid protein, since pharmacological blockade of ABCB1 rapidly decreases extracellular levels of bamyloid secretion. Also, in vitro binding studies showed that addition of synthetic human -amyloid peptides to hamster mdr1-bearing vesicles resulted in saturable uptake of these peptides, suggesting that they interact directly with the transporter [74]. These results and the finding that apolipoprotein E (apoE) is also associated with b-amyloid peptides [75] implies that ABCB1 can co-transport apoE and b-amyloid and thereby may contribute to the etiology of Alzheimer’s disease. Two half-size members of the subfamily, ABCB2 (TAP1) and ABCB3 (TAP2), are transporters associated with antigen presentation (TAP) and form a functional heterodimer to transport peptides from the cytoplasm into the endoplasmic reticulum, from where the presentation of peptide antigens via major histocompatibility complex (MHC) I starts [76] (Fig. 3.2). A transient complex containing a class I heavy chain–b2 microglobulin (b2m) heterodimer is assembled onto the TAP molecule by numerous interactions with the ER chaperones calnexin, ERp57, calreticulin, and the specialized tetherin molecule, tapasin [77]. Most interestingly, virusinfected and malignant cells have developed strategies to escape immune surveillance by affecting TAP expression or function [78]. The immediate-early gene product ICP47 of herpes simplex virus type I binds to the cytoplasmic face of TAP and thereby blocks peptide entry, whereas the ER-resident human cytomegalovirus protein US6 inhibits TAP function by blocking the ER-luminal part of the transporter (Fig. 3.2) [79–81]. ABCB9, which is closely related to ABCB2 and ABCB3, has been mainly found in lysosomes [82]. Although ABCB9 has been proposed to be involved in TAP-dependent processes, its exact function is currently unknown. The remaining four ABCB proteins (ABCB6, ABCB7, ABCB8, and ABCB10) are all targeted to the inner mitochondrial membrane and play a role in cellular iron homeostasis by transporting iron–sulfur (Fe/S) cluster precursor proteins [82–85]. In this respect, a mutation in ABCB7, which is located on the Xchromosome, has been linked to X-linked sideroblastic anemia and ataxia (XLSA/ A) (see Tab. 3.2) [86].

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Proposed role of TAP proteins (ABCB2, ABCB3) in antigen presentation. Endogenous proteins are degraded in the ubiquitin-proteasome pathway. The peptides are transported into the ER lumen by a full-size complex composed of TAP1 and TAP2 [76]. The correct folding, assembly and loading of

Fig. 3.2

MHC I molecules is mediated by numerous accessory proteins including calnexin, calreticulin, ERp57, tapasin, and TAP [77]. Stable MHC I–peptide complexes leave the ER through the Golgi compartment to the cell surface for recognition by T-cell receptors.

3.3.3

The ABCC (CFTR/MRP) Subfamily

The ABCC subfamily comprises 12 full-size ABC proteins which perform such diverse functions as drug resistance, ion transport, nucleoside transport, and ion channel regulation (Tab. 3.2). A special subgroup within the ABCC family can be distinguished by the presence of a TMD0-(TMD-ABC)2 domain arrangement (Fig. 3.1A). Seven members display this special membrane topology (ABCC1, ABCC2, ABCC3, ABCC6, ABCC8, ABCC9, and ABCC10), whereas the other proteins in this subfamily exhibit the (TMD-ABC)2 structure. Although the TMD0 part is not required for transport activity, a linker region designated L0 is essential for proper ABCC1 function [87]. Among the (TMD-ABC)2 molecules, ABCC7 (CFTR) is characterized by an extraordinary domain structure: it contains a regulatory domain, which is controlled by cAMP/PKA-dependent phosphorylation and thereby enables ATP binding and hydrolysis at the nucleotide binding cassettes,

3.3 Overview of Human ABC Gene Subfamilies Tab. 3.2 Human ABC transporter genes and corresponding dieseases or phenotypes.

Gene

Alternative name

Disorder or phenotype

Reference

ABCA1 ABCA4

ABC1 ABCR

ABCB1

PGY1, MDR1

6–8 9 9 9 9 9 9 59, 70

ABCB2 ABCB3 ABCB4

TAP1 TAP2 MDR3

ABCB7 ABCB11

ABC7 SPGP, BSEP

Familial HDL deficiency, Tangier disease Stargadt macular dystrophy (STGD) Fundus flavimaculatus (FFM) Retinitis pigmentosa 19 (RP) Cone–rod dystrophy (CRD) Cone dystrophy (CD) Age-related macular degeneration (AMD) Multidrug resistance, inflammatory bowel disease (ulcerative colitis) Immune deficiency Immune deficiency Progressive familial intrahepatic cholestasis type 3 (PFIC-3) Intrahepatic cholestasis of pregnancy (ICP) X-linked sideroblastosis and anemia (XLSA/A) Progressive familial intrahepatic cholestasis type 2 (PFIC-2)

ABCC1 ABCC2 ABCC6 ABCC7 ABCC8

MRP1 MRP2 MRP6 CFTR SUR1

Multidrug resistance Dubin–Johnson Syndrome (DJS) Pseudoxanthoma elasticum (PXE) Cystic fibrosis (CF) Persistent hyperinsulinemic hypoglycemia of infancy (PHHI)

162 13 14 163 184

ABCD1 ABCD3

ALD PXMP1, PMP70

Adrenoleukodystrophy (ALD) Zellweger syndrome 2 (ZWS2)

15 104, 105

ABCG2 ABCG5 ABCG8

ABCP, MXR, BCRP White3 White 4

Multidrug resistance b-Sitosterolemia b-Sitosterolemia

117 16 16

76–78 76–78 10–12 10–12 83 11

which in turn control opening and closing of the chloride channel [88]. Mutations in ABCC7 (CFTR) cause cystic fibrosis by affecting numerous secretion processes. ABCC1, ABCC2, and ABCC3 are all able to transport anticancer drugs, whereby ABCC1 (MRP1) mainly transports glutathione-conjugated (GSH) molecules and therefore has been termed GS-X pump [89]. In addition to cancer drug resistance, the physiologic function of GS-X pumps is closely related with cellular detoxification, oxidative stress, and inflammation [90]. ABCC2 (MRP2), which is located in the apical membrane of polarized epithelial cells and particularly to the canalicular membrane of hepatocytes, appears to participate in the hepatobiliary secretion of organic anions and has therefore originally called canalicular multispecific organic anion transporter (cMOAT) [91, 92]. ABCC3 (MRP3) is also an organic ion transporter but prefers glucuronate conju-

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gates over GSH conjugates [93]. ABCC4, ABCC5, ABCC11, and ABCC12 are MRP-like proteins which lack the additional N-terminal domain, and ABCC4 and ABCC5 have been shown to function as cellular efflux pumps for nucleosides, including anti-human immunodeficiency virus drugs such as PMEA [94] and nucleotide analogs (e.g. 6-mercaptopurine and thioguanine) [95]. Although the physiological role as well as the potential participation in drug efflux of ABCC6 (MRP6) is still unclear [96], mutations in the gene have been detected in the connective tissue disorder pseudoxanthoma elasticum (PXE, Tab. 3.2) [97]. Since ABCC6 is highly expressed in liver and kidney cells, sites where PXE is not very pronounced, one hypothesis suggests that ABCC6 may transport or remove toxic metabolites which destroy connective tissue cells [98]. ABCC11 and ABCC12 are most closely related to the ABCC5 gene and are found tandemly duplicated on chromosome 16q12 (Tab. 3.1) [99]. Since ABCC11 and ABCC12 were mapped to a region harboring gene(s) for paroxysmal kinesigenic choreoathetosis, a disease characterized by recurrent, brief attacks of involuntary movements induced by sudden movements or startling, ABCC11 and ABCC12 represent positional candidates for this disorder [100, 101]. Interestingly, several paroxysmal neurological manifestations and idiopathic age-dependent seizures are known to be caused by ion channel-related genes [102]. The two remaining members of the ABCC subfamily ABCC8 (SUR1) and ABCC9 (SUR2) bind sulfonylurea with high affinity and interact with potassium inward rectifiers KIR6.1 and KIR6.2, to form a large octameric channel with the stoichiometry (SUR/KIR6.x)4 [103]. These heteromeric channels regulate insulin release in response to glucose metabolism and sulfonylureas are widely used to stimulate insulin secretion in type 2 diabetic patients because they close these ATP-sensitive potassium (KATP) channels in the pancreatic beta-cell membrane (see Fig. 3.5) [34]. 3.3.4

The ABCD (ALD) Subfamily

This subfamily is composed of four peroxisomal half-size ABC transporters with a TMD–ABC domain structure. They are involved in very long fatty acid (VFLA) transport. A variable pattern of homo- and heterodimerization for all ABCD members has been proposed [104–106] and mutations in ABCD1 and ABCD3 are associated with adrenoleukodystrophy (ALD) and Zellweger syndrome 2 (ZWS2), respectively (Tab. 3.2) [107, 108]. An interesting finding is the transcripitonal regulation of ABCD genes by lipids (Tab. 3.1). In this respect, recent reports have provided evidence that nuclear hormone receptor ligands, especially RXR ligands and PPAR ligands, induce the ABCD2 promoter [109, 110].

3.3 Overview of Human ABC Gene Subfamilies

3.3.5

The ABCE (OABP) and ABCF (GCN20) Subfamilies

This subfamily contains four half-size ABC transporters, which are ubiquitously expressed in human tissues and do not possess transmembrane domains. The ABCE1 gene encodes an oligoadenylate binding protein (OABP), which is only found in multicellular eukaryotes and seems to participate in innate immune defense [30]. Oligoadenylates, which are produced from virus-infected cells are activators of RNaseL, which in turn degrades cellular RNAs and thereby blocks protein synthesis in infected cells. ABCE1 binds these oligonucletides and thus inhibits RNAseL, which implies that ABCE1 is involved in the negative control of immune reactions. ABCF1, the human homolog of the yeast GCN20 gene, shares some interesting features with ABCE1. Thus, ABCF1 is involved in the control of protein synthesis and also in the control of the immune system. ABCF1 binds to the translation elongation initiation factor 2 (eIF2) and seems to modulate its phosphorylation state [111]. In addition, ABCF1 has been co-purifed with ribosomal components confirming its role in protein translation [31]. In another interesting study, Richard and colleagues identified ABCF1 as a TNF-induced transcript in synoviocytes. They suggest that this ABC protein could be part of inflammatory processes related to rheumatoid arthritis. Since functionally related genes tend to be clustered on chromosomes and because ABCF1 is located on chromosome 6p21.33 (Tab. 3.1) in close proximity to class I MHC, the proposition that ABCF1 mediates inflammatory processes is very likely. 3.3.6

The ABCG (White) Subfamily

The human white or ABCG subfamily consists of five fully cloned genes (ABCG1, ABCG2, ABCG4, ABCG5, and ABCG8) and one gene so far only found in rodents (ABCG3) [22]. The ABCGs are thought to dimerize to form active membrane transporters. Among the half-size molecules ABCG proteins have a peculiar domain organization characterized by a nucleotide binding domain (ATP binding cassette) at the N-terminus followed by six transmembrane-spanning domains (Tab. 3.1 and Fig. 3.1). The founding member of this group, ABCG1, was independently described by Chen et al. and Croop et al. as the human homolog of the Drosophila white gene [112, 113] and its genomic organization, including the promoter region, has been described recently [114, 115]. Earlier indications linked ABCG1 with the congenital recessive deafness (DFNB10) syndrome, based on its chromosomal localization on chromosome 21q22.3 [116]. However, a recent report [117] has excluded ABCG1 along with five other known genes as candidates for DFNB10. Also, conflicting data exist whether the G2457A polymorphism in the 3UTR of the ABCG1 mRNA is associated with mood and panic disorders and related to suicidal behavior [19, 118]. The most interesting report dealing with ABCG1 function came from a study by Klucken et al., which identified ABCG1 as a sterol-induced gene that participates

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in cholesterol and phospholipid efflux, especially in macrophages and foam cells [41]. The second well-known member of the ABCG subfamily ABCG2 has been identified by different approaches and is known under the names ABCP [119], BCRP [120], and MXR [121]. The protein has been shown to be amplified and overexpressed in human cancer cells and is capable of mediating drug resistance even in the absence of the classical MDR proteins ABCB1 (MDR1) and ABCC1 (MRP1) [121–123]. In contrast to most other half-size ABC transporters, the bulk of the ABCG2 protein has been localized to the plasma membrane, with a minor fractions found within intracellular membranes [29]. It was only a short time ago when two other ABCG transporters ABCG5 and ABCG8 had been identified and linked to the human disease b-sitosterolemia by two independent approaches [124–126]. The latest paper on an ABCG member has reported the cloning of the complete cDNA of ABCG4 and identifed this transporter as a sterol-sensitive gene [127].

3.4

Diseases and Phenotypes Caused by ABC Transporters

Eighteen out of 48 currently known human ABC proteins have been linked to human monogenetic disorders or cause special disease phenotypes (Tab. 3.2) [98]. Since ABC transporters represent a combination of enzymes and structural proteins, homozygous mutations cause severe human diseases, which are inherited in a recessive manner. As described below, these genetic diseases are found in five of the seven ABC subfamilies (Tab. 3.2). In addition, heterozygous mutations in ABC genes have been connected with susceptibility to complex, multigenic disorders. It is also worth mentioning that due to the pleiotropic functions of ABC transporters, the disease states affected by mutations in ABC transporter genes are just as complex and diverse as the cellular functions of these proteins. 3.4.1

Familial HDL-deficiency and ABCA1

The major clue that ABCA1 is involved in cellular cholesterol removal and lipid efflux was the identification of mutations in the human gene as the defect in familial HDL-deficiency syndromes such as classical Tangier disease (Tab. 3.2) [6–8]. The most striking feature of these patients is the almost complete absence of plasma HDL, low serum cholesterol levels, and a markedly reduced efflux of both cholesterol and phospholipids from cells, strongly supporting the idea that both lipids are co-transported [128, 129]. The lack of ABCA1 function in these patients has a major impact on plasma HDL levels and composition. Thus plasma HDL from TD patients is composed of small pre-b1-migrating HDL particles containing solely apoAI and phospholipids but lacking free cholesterol and apoAII [130, 131]. The low HDL levels seen in Tangier disease (TD) are mainly due to an enhanced catabolism of these HDL precursors [131–134]. In addition, the size of the HDL

3.4 Diseases and Phenotypes Caused by ABC Transporters

particle strongly correlates with the amount of cholesterol efflux and plasma HDL concentrations [135, 136]. In TD patients, neither cholesterol absorption nor metabolism is significantly affected, however, the concentration of LDL-cholesterol is only 40% of healthy controls and the particles are often enriched in triglycerides. The reduction in LDL levels is mainly caused by disturbance of the cholesterol ester transfer pathway resulting in changes of LDL composition and size [137]. Interestingly, obligate heterozygotes for TD mutations have approximately 50% of plasma HDL, but normal LDL levels [138]. Studying 13 different mutations in 77 heterozygous individuals, Clee et al. described a more than 3-fold risk of developing coronary artery disease in affected family members and earlier onset compared with unaffected members [139, 140]. However, these results seem to be biased towards the atherosclerotic phenotype, since the prevalence of splenomegaly is much higher in the European group of ABCA1 deficiency patients [46]. These authors also reported an age-dependent modification of the ABCA1 heterozygous phenotype [140]. In addition to the absence of plasma HDL, patients with genetic HDL-deficiency syndromes display accumulation of cholesteryl esters either in the cells of the reticulo-endothelial system (RES), leading to splenomegaly and enlargement of tonsils or lymph nodes, or in the vascular wall, leading to premature atherosclerosis [46]. This indicates differences in macrophage trafficking into tissues in the absence of ABCA1 which may be a reflection of the specific localization of mutations within the ABCA1 gene. In this context, it is of note that the pool size of CD14dimCD16+ monocytes is inversely correlated with plasma HDL-cholesterol levels [141] and the expression of ABCA1 is high in phagocytes [40] but low in antigen-presenting dendritic precursor cells (unpublished observation). These observations may provide clues for a potential interlink between ABCA1 function and the control of monocyte differentiation and phagocyte/dendritic cell lineage commitment. Accordingly, we have previously hypothesized that ABCA1 function regulates the differentiation, lineage commitment (phagocytic versus dendritic cells), and targeting of monocytes into the vascular wall of the RES [142]. This concept has been substantiated by recent work from our laboratory demonstrating accumulation of macrophages in liver and spleen in LDL receptor-deficient mouse chimeras that selectively lack ABCA1 in their blood cells [143]. The fact that the absence of ABCA1 from leukocytes is sufficient to induce aberrant monocyte recruitment into specific tissues identifies ABCA1 as a critical leukocyte factor in the control of monocyte targeting. In addition to phagocytes, dendritic cells have been shown to be increased in atherosclerotic lesions and have been implicated in T cell activation in atherogenesis [144]. Expression of ABCA1 appears to inhibit monocyte differentiation into macrophages and may thus shift the balance between phagocytic differentiation and dendritic cell differentiation towards the latter [145]. Taking into account that dendritic cells are capable of inducing primary immune responses, ABCA1 may function, through this mechanism, as a modulator of innate immunity in atherogenesis. An interesting clue as to how ABCA1 may be implicated in the control of monocyte/macrophage trafficking at the cellular level comes from the observation

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that apoAI-mediated lipid efflux in ABCA1-deficient cells is paralleled by the downregulation of the protein Cdc42 and filopodia formation [146]. Cdc42, like rho and rac, is a member of the family of small GTP binding proteins which are sequentially activated by extracellular stimuli in mammalian cells [147]. Cdc42 controls a wide range of cellular functions including cytoskeletal modulation, formation of filopodia and vesicular processing. Rho proteins are known to induce the formation of stress fibers and focal adhesions; rac proteins regulate formation of lamellipodia and membrane ruffles. It is thus tempting to speculate that ABCA1 modulates cellular mobility of monocytes/macrophages through this mechanism and thus may affect recruitment of monocytes into the vessel wall. This regulator function for filopodia formation and cytoskeletal reorganization may even extend to platelet aggregation, vascular smooth muscle cell migration, and endothelial cell integrity, since these cells have been shown to express ABCA1 [148]. 3.4.2

Retinal Degeneration and ABCA4 (ABCR)

In addition to ABCA1, the ABCA4 (ABCR) gene located on chromosome 1p21 (Tabs 3.1 and 3.2) is another example how several mutations in one ABC transporter gene can cause pleiotropic effects. Thus, many different clinical phenotypes, associated with various forms of eye degeneration, and the age of onset as well as disease severity are associated with distinct mutations in ABCA4 [9]. As summarized in Tab. 3.2, ABCA4 has been found to be a causal gene for a series of retinal diseases. As an effort of several laboratories in 1997 [149–151], mutations in ABCA4 have been identified in Stargadt disease (STGD), a juvenile-onset macular dystrophy characterized by rapid central visual impairment and progressive bilateral atrophy of the retinal pigment epithelium, as well as in the late-onset form termed fundus flavimaculatus. Although only 60% of the mutations in the ABCA4 gene of STGD have been determined, all segregated chromosomal regions in these patients have been mapped to a locus between chromosomes 1p13 and 1p22. In addition to the monogenic STGD, ABCA4 mutations have been described in the autosomal recessive diseases cone–rod dystrophy (CRD) [152, 153] and retinitis pigmentosa (RP) [152, 154–156], which are both genetically and clinically heterogeneous disorders. Cone–rod dystrophy mainly displays cone degeneration, whereas retinitis pigmentosa affects predominantly rod photoreceptors. Age-related macular degeneration (AMD), the leading cause of severe central visual impairment among the elderly, is the fourth disease state associated with ABCA4 dysfunction. The disease is also characterized by progressive accumulation of large quantities of lipofuscin with retinal pigment epithelial cells and delayed dark adaptation [157]. Athough AMD is strongly influenced by environmental factors such as smoking, heterozygous mutations in ABCA4 have been proposed to increase the susceptibility to develop AMD. Thus, the two most frequent AMD-associated ABCA4 variants D2177N and G1961E, increase the risk of developing AMD by approximately 3-fold and 5-fold, respectively [158, 159].

3.4 Diseases and Phenotypes Caused by ABC Transporters

Model for the role of ABCA4 (ABCR) in rod outer segments. Left panel: schematic drawing of a rod photoreceptor. Right panel: magnification of rod disc membranes. Rhodopsin is manufactured from opsin and 11-cis retinal in the Golgi of the rod inner segment and transported to rod outer segment discs. Upon light absorption the 11-cis form of retinal is converted to an all-trans form, which reacts with phosphatidylethanolamine (PE) to form the Schiff-base product N-retinylidene-PE Fig. 3.3

(N-RPE). ABCA4 is thought to flip N-RPE to the outer leaflet of the disc membrane. There, all-trans retinal is generated by hydrolsysis of N-RPE and subsequently reduced to all-trans retinol by retinol dehydrogenase prior to its delivery to the retinal pigment epithelial cells and re-esterification [62]. Under the effect of short-wave light or in ABCA4 deficiency, alltrans retinal accumulates, causing photooxidative damage and generation of toxic A2E (Nretinyl-N-retinylidene ethanolamine) [63].

In addition to the above described results based on the phenotypical analysis of ABCA4 mutations, data from in vitro studies and ABCA4 knockout mice have shed light on the transport function of ABCA4 in photoreceptor cells. Recombinant liposome-reconstituted ABCA4 displays all-trans-retinal-stimulated ATPase activity [47, 48, 160] and ABCA4 knockout mice exhibit an acute light-dependent accumulation of all-trans retinal within rod outer segments and a progressive lightdependent culmination of lipofuscin-derived A2E (N-retinyl-N-retinylidene ethanolamine) [161, 162]. Based on these data, a model for the function of ABCA4 in rod disc membranes has been proposed [162]. As summarized in Fig. 3.3, all-trans retinoids, which are released from rhodopsin by photobleaching, react with the primary amine of PE to form the condensation product N-retinylidene-PE (NRPE). ABCA4 is thought to flip N-RPE to the outer leaflet of the disc membrane, where all-trans retinal is generated by hydrolysis of N-RPE and subsequently re-

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duced to all-trans retinol by retinol dehydrogenase prior to its delivery to the retinal pigment epithelial cells and re-esterification [163]. 3.4.3

Cystic Fibrosis (ABCC7/CFTR)

Cystic fibrosis, caused by mutations in ABCC7 (CFTR) (Tab. 3.2) is one of the most frequent inherited diseases in Caucasian populations with a prevalence of 1:900 to 1:2500, whereas African and Asian individuals are affected to a much lesser extent. Interestingly, a three base pair deletion (DF508) accounts for 70–80% of the mutated alleles in northern European populations. The total number now comprises more than 1000 CFTR mutations (http://www.genet.sickkids.on.ca/cftr/). The spectrum of the disease severity is dependent on the residual function of ABCC7 [164, 165]. Patients with two affected alleles develop a severe disease with a disturbed exocrine function of the pancreas leading to nutritional deficiencies, bacterial lung infections, and a blockade of the vas deferens causing male infertility. In

Schematic diagram summarizing ABCC7 (CFTR) interactions in the plasma membrane. CFTR interacts with various proteins in the plasma membrane including syntaxin 1a, CAL (CFTR-associated ligand), and EBP50/NHERF [170–175]. Syntaxin 1a contains coiled-coil protein motifs which bind the N-terminal part of CFTR. Syntaxin 1a controls the vesicular trafficking of CFTR through the Golgi to the plasma membrane and thereby inhibits its channel function. CFTR also contains a PSD-95/Disc-large/ZO-1 (PDZ)-interacting motif at its C-terminus, which binds the PDZ-proteins CAL and EBP50/NHERF (ezrin, radixin, and moesin) binding phosphoprotein of 50 kDa/Na+/H+ exchanger regula-

Fig. 3.4

tory factor). CAL has one PDZ domain and two coiled-coil motifs, which organize CFTR into a cluster in the apical membrane. EBP50/NHERF binds to CFTR through its PDZ1 domain and thereby links the protein to the cytoskeleton via ezrin. Ezrin serves as a PKA-anchoring protein and facilitates cAMPdependent phosphorylation of the CFTR regulatory domain and channel activity. In addition to these direct protein–protein interactions, CFTR indirectly regulates several ion channels such as ROMK2, ENaC, CaCC, and ORCC. CaCC and ORCC are activated by Ca2+dependent purinergic receptors (P2Y2), which are in turn modulated by CFTR-dependent ATP release.

3.4 Diseases and Phenotypes Caused by ABC Transporters

contrast, patients with one partially functional allele retain residuary pancreatic function and have a milder disease phenotype [166]. Before ABCC7/CFTR was identified, it was known in the 1980s that the apical membrane of different epithelia displays a Cl– conductance, which could be activated by cAMP and which was defective in cystic fibrosis. With the identification of the ABCC7/CFTR gene in 1989 [167] and an impressing multitude of publications in the following years, it became more and more evident that ABC transporters are not exclusively ATP-driven pumps, but moreover can exhibit a regulatory and/or channel function. As depicted in Fig. 3.4, ABCC7/CFTR can act as a cAMP-regulated chloride channel as well as a regulator of outwardly rectifying chloride channels (ORCC) [88, 168]. In both cases targeting to the apical membrane of epithelial cells and activation of the regulatory (R) domain by PKA are a prerequisite for proper ABCC7 function. It is now widely recognized that ABCC7 interacts with various proteins in the plasma membrane [169]. Thus, the N-terminus of CFTR binds the coiled-coil protein syntaxin 1a and the C-terminal region of CFTR binds to PDZ domain proteins, a family of proteins containing a 80–90 amino acid motif that binds the Cterminus of a variety of ion channels and receptors [170]. At least three PDZ domain-containing proteins, NHE-RF or EBP50, CAP70, and CAL, bind to the CFTR C-terminus (Fig. 3.4) [170–174]. Because EBP50 associates with Ezrin, which itself binds the regulatory subunit of PKA and has a binding site for F-actin [175], it is likely that EBP50 anchors CFTR to the actin cytoskeleton at a site where it can be targeted by PKA. CAP70, a subapical protein, is able to bind two ABCC7 molecules simultaneously via PDZ3 and PDZ4 [173] and thus can mediate cross-linking of CFTR dimers and thereby enhance either direct or indirect (ORCC) chloride channel activity. Taken together, the N-terminus of ABCC7 is required for binding of syntaxin 1a and other components of the SNARE-dependent vesicular trafficking machinery, whereas the C-terminus of CFTR is necessary for cytoskeletal fixation via EBP50/Ezrin proteins. In addition, multimerization of CFTR molecules could be potentially achieved by CAP70-dependent linkage. 3.4.4

Multidrug Resistance (ABCB1/MDR1, ABCC1/MRP1, ABCG2)

When cells are exposed to toxic compounds, as is the case for tumor cells treated with chemotherapy, resistance against these drugs can occur by a variety of mechanisms. Among these are decreased cellular uptake, increased intracellular detoxification, modification of target proteins, and enhanced extrusion from cells. Although in most cases one compound initially causes these events, cells can become resistant to a variety of drugs with structural similarities to the initial compound. This multidrug resistance (MDR) is mainly caused by three drug efflux pumps, ABCB1 (MDR1), ABCC1 (MRP1), and ABCG2 (MXR, ABCP, BCRP) (Tabs 3.1 and 3.2). ABCB1, which was described in the mid 1970s by its ability to confer a multidrug resistant phenotype to cancer cells upon chemotherapy [176], is a highly promiscous

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3 Function, Expression, and Regulation of Human ABC Transporters

transporter of hydrophobic drugs, e.g. vinblastine, colchicine, VP16, adriamycin, and of such diverse substrates as lipids, steroids, xenobiotics, and peptides [177]. In small-cell lung carcinoma cells, which did not display high ABCB1 activity, overexpression of ABCC1 (MRP1) was identified [178]. Thereafter a similar substrate pattern for ABCC1 compared to ABCB1 was reported, including doxorubicin, daunorubicin, vincristine, and colchicine. However, in contrast to ABCB1, which has mainly organic cations as substrates, ABCC1 is able to transport organic and anionic compounds, the latter mainly in conjugated forms [20, 95]. Another ABC protein amplified and involved in MDR is ABCG2, which confers resistance to anthracyclin chemotherapeutic drugs such as mitoxantrone. These findings have been supported by in vitro studies: ABCG2-transfected drug-sensitive breast cancer cells are resistant to mitoxantrone, daunorubicin, and doxorubicin, and also display an enhanced rhodamine-123 efflux [120]. Interestingly, an R482G mutation in ABCG2 can significantly alter its substrate specificity and concomitantly change the drug-resistance phenotype [179].

3.4.5

Adrenoleukodystrophy (ABCD1/ALD)

The X-linked adrenoleukodystrophy (ALD) is an inherited peroxisomal disorder caused by mutations in ABCD1 (Tab. 3.2), resulting in progressive neurological dysfunction, occasionally associated with adrenal insufficiency [180]. ALD is characterized by the accumulation of unbranched saturated fatty acids with a chain length of 24–30 carbons, particularly hexacosanoate (C26), in the cholesterol esters of brain white matter and in adrenal cortex and in certain sphingolipids of brain [15, 107]. It took a long period of research for the causative defect to be identified and therapies for ALD to be developed; these include the famous approach developed by the Odone family of dietary treatment with oleic and erucic acids (glyceryl trierucate and trioleate oil), known as Lorenzo’s oil [181, 182]. Adrenoleukodystrophy belongs to a group of defects in peroxisomal b-oxidation [183]. The first step in the oxidation of very-long-chain fatty acids (VLCFA) involves their activation by conversion into CoA esters and the transport into peroxisomes [184]. The ABCD1 protein is thought to mediate this transport process [185] and evidence for this function comes from experiments using overexpression of human cDNAs encoding the ABCD1 protein and its closest relative, the ABCD2 (ALDR) protein. With this approach, Netik et al. could restore the impaired peroxisomal b-oxidation in fibroblasts of ALD patients [186]. The accumulation of very-long-chain fatty acids could also be prevented by overexpression of the ABCD2 protein in immortalized ALD cells. Moreover, the peroxisomal b-oxidation defect in the liver of ABCD1-deficient mice could be restored by stimulation of ABCD2 and ABCD4 gene expression through dietary treatment with the PPAR agonist fenofibrate [186]. These results implicate that a therapy of adrenoleukodystrophy might be possible by drug-induced overexpression or ectopic expression of ABCD genes.

3.4 Diseases and Phenotypes Caused by ABC Transporters

3.4.6

Sulfonylurea Receptor (ABCC8/SUR)

Familial persistent hyperinsulinemic hypoglycemia of infancy (PHHI) is characterized by unregulated insulin secretion from pancreatic beta cells. The defect has been localized to chromosome 11p15.1–p14, a chromosomal region containing the ABCC8 (SUR1) gene and the KCNJ11 (Kir6.2) gene [187]. Subsequently, causal mutations in the ABCC8 gene have been described in PHHI families [188] (Tab. 3.2); however, no defects in the KCNJ11 gene have found so far. As described earlier in this chapter (see Section 3.3.3), ABCC8 and KCNJ11 form together an inwardly rectifying potassium channel (Fig. 3.5). Under hyperglycemic

Fig. 3.5 Schematic model for KATP channelcontrolled insulin secretion from pancreatic bcells. Entry and metabolism of glucose into pancreatic b-cells leads to increased levels of intracellular ATP and concomitantly decreases ADP levels. The increase in the ATP/ADP ratio causes binding of ATP to the nucleotide binding domains of ABCC8 (SUR1) and to KIR6.2 [34]. Thereby, the KATP channel closes and the plasma membrane is depolarized. The opening of voltage-gated Ca2+ channels and voltage-dependent Na+ channels raises the intracellular Ca2+ concentration by Ca2+ influx and mobilization of intracellular Ca2+ stores, respectively. The increased level of intracellular Ca2+ stimulates the dephosphorylation of b2-

syntrophin and the dissociation of 2-syntrophin–utrophin–actin complexes from ICA 512 and secretory granules. Following dissociation of b2-syntrophin, ICA 512 is cleaved by Ca2+/ calmodulin (CaM)-activated calpain, resulting in the mobilization of secretory granules from the cytomatrix and exocytosis of insulin. The pancreatic KATP channels are also regulated by important therapeutic pharmacological agents, such as sulfonylureas and K+ channel openers. Sulfonylureas, widely used in the treatment of NIDDM, stimulate insulin secretion by closing the KATP channels, while K+ channel openers inhibit insulin secretion by opening the KATP channels [103].

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conditions the intracellular ATP/ADP ratio increases and thereby causes the ATPsensitive octameric K+-channel complex to close, which in turn depolarizes the beta-cell membrane. The subsequent opening of voltage-dependent calcium channels allows calcium influx and initiates insulin release via several successive steps including dephosphorylation of b2-syntrophin, dissociation of b2-syntrophin–utrophin–actin complexes from the islet cell autoantigen (ICA) 512, ICA 512 cleavage by l-calpain, and exocytosis of secretory granules. Interestingly, polymorphisms in calpain-10, a related protease also proposed to be involved in the processing of ICA512, affect insulin secretion and have been linked to type 2 diabetes [189, 190]. Hypoglycemia decreases the intracellular ATP/ADP ratio and causes opening of the ABCC8/KCNJ11 complex, which hyperpolarizes the plasma membrane and inhibits Ca2+ influx and thereby stops insulin secretion [191]. Mutations in ABCC8 lead to defective ATP-sensing of KCNJ11 channels and thereby to abnormal behavior in response to hypoglycemia, resulting in persistent insulin secretion despite low glucose levels. The knowledge of the function of ABCC8 has become very important in clinical practice, since sulfonylureas, drugs widely used as oral hypoglycemics to promote insulin secretion in the treatment of non-insulin-dependent diabetes mellitus (NIDDM), are high-affinity inhibitors of ABCC8 (SUR1) and ABCC9 (SUR2). In addition to the causal mutations in PHHI, polymorphisms in the ABCC8 gene have been associated with hyperinsulinemia and type 2 diabetes in Mexican Americans [192] and French Caucasians [193], respectively.

3.5

Function and Regulation of ABC Transporters in Lipid Transport

Due to the strong interest in the primary drug-transporting ABC proteins, other aspects of cellular functions, such as lipid homeostasis, of this large transporter superfamily has been for long time remained unknown and unappreciated. The first implication that ABC proteins could participate in lipid binding and/or transport came from mdr2 knockout mice, which displayed a complete absence of phospholipids from bile and as a consequence developed liver disease [194]. Subsequently, the translocation of phospholipids by the human homolog MDR3 (ABCB4) was demonstrated [61, 62, 195, 196]. Only a few years later, the identification of the sterol-responsiveness of ABCA1 [40] and of other ABC family members [41] had paved the way for the identification of the gene defect in HDL deficiency [6–8], which was a major clue in proving the importance of ABC transporters in macrophage cholesterol efflux. In a similar manner, the discovery of the genetic defect in b-sitosterolemia has identified ABCG5 and ABCG8 as proteins which extrude dietary sterols from intestinal epithelial cells and from the liver to the bile duct [124, 126]. As is clear from Tab. 3.1, a significant number of ABC transporters feature a lipid-sensitive regulation, which implies that in addition to the currently established ABC proteins, further members of this superfamily could have similar functional

3.5 Function and Regulation of ABC Transporters in Lipid Transport

properties. The following section will summarize the current knowledge of ABC transporters in lipid transport with a special emphasis on transport processes in macrophages, liver, and intestine, which reflect the major organ systems in sterol metabolism. 3.5.1

ABCA1 in Macrophage Lipid Transport

Several factors control the expression and activity of ABCA1. Induced cholesterol influx into macrophage cells has been shown to be a potent inducer of ABCA1 expression [40]. Since the cloning of the complete human and mouse ABCA1 genes, a number of transcriptional control elements acting via alternative promoters have been characterized [197–199] (Fig. 3.6). The ABCA1 upstream region contains a macrophage-specific promoter preceding exon 1. This sequence binds the repressors ZNF202 and USF1/2, as well as the activating factors Sp1/Sp3 and the oxysterol-induced RXR/LXR heterodimer [200, 201]. A second promoter located downstream of exon 1 has been recently implicated in the liver/steroidogenic expression of ABCA1 [198] (Fig. 3.6). The The LXR/RXR-responsive elements in promoter 1 triggers retinoic acid and oxysterol-dependent activation of the ABCA1 promoter and thereby confer the observed induction of ABCA1 during lipid loading of macrophages. The most likely endogenous ligand for LXRa and LXRb is 27-hydroxycholesterol, since CYP27-deficient cells are not able to upregulate ABCA1 in reponse to sterols and since overexpression of CYP27 activates LXR/RXR [202]. The earlier described LXR ligands 20(S)-hydroxycholesterol, 22(R)-hydroxycholesterol, and 24(S),25-epoxycholesterol are not present in cholesterol-loaded macrophages, rendering them unlikely to be natural ligands of LXR [202]. In contrast to LXR/RXR, the zinc finger transcription factor ZNF202 is a transcriptional repressor of ABCA1 gene expression, which also prevents the induction of the gene by oxysterols by recruiting the universal co-repressor KAP1 (KRAB domain-associated protein 1) [203]. Due to the strong upregulation of ABCA1 expression in response to oxysterols, LXR agonists have been proposed to be promising candidates for therapeutic activation of ABCA1 [199, 204–206] (Fig. 3.7). It stands to reason that especially under disease conditions such as NIDDM, where the cells have low glucose levels, low ATP levels, and associated low HDL-cholesterol levels, excessive mitochondrial energy production could induce mitochondrial exhaustion. This may ultimately result in cellular ATP shortage, a process that likely enhances the programmed cell death of lesion macrophages. Mitochondrial exhaustion may also inhibit mitochondrial 27-OH sterol synthesis and its export from the mitochondrion, a critical pathway for LXR activation in response to cellular cholesterol stress (Fig. 3.7) [202]. Since 27-OH sterol is the predominant oxysterol in macrophage-derived foam cells and atherosclerostic lesions [207], this mechanism may indeed be of pathophysiological significance in atherogenesis. In light of these complexities, treatment with LXR agonists bears the potential risk of inducing mitochondrial failure and pro-apoptotic effects and may thus negatively affect lesion formation.

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Diagram representing the human ABCA1 and ABCG1 gene promoters Upper panel: The ABCA1 upstream region contains two alternative promoters. Promoter 1 mainly directs macrophage-specific ABCA1 expression and contains binding sites for the transcription factors ZNF202, Sp1, Sp3, E-box binding factors, LXR/RXR, and TATA binding proteins. Promoter 2 is active in liver and steroidogenic tissues and contains putative binding motifs for HNF3, SREBP, LRH/SF1, LXR/RXR, C/EBPs, and a TATA box [198–202]. Lower panel: The ABCG1 gene contains at

Fig. 3.6

least three alternative promoters. Binding of ZNF202 to promoter 2 and binding of LXR/ RXR to promoter 3 has been determined experimentally. The functionality of promoter 1 has not been demonstrated so far. ZNF202, zinc finger transcription factor 202; Sp1, specificity protein 1; Sp3, specificity protein 3; LXR, liver X receptor; HNF3, hepatic nuclear factor 3; SREBP, sterol regulatory element binding protein; LRH, liver x receptor homolog; SF1, steroidogenic factor 1; NjFB, nuclear factor kappa B [203, 215].

ABCA1 appears to be localized on the plasma membrane and surface expression of ABCA1 is upregulated in macrophages by cholesterol loading [208]. Recent evidence indicates that ABCA1 and Cdc42 are associated with a Lubrol detergent-resistant raft subfraction, whereas ABCA1 is not detectable in Triton-resistant rafts [209, 210]. In addition, the fact that ABCA1 is detectable in the cytosol and Golgi compartment of unstimulated fibroblasts also raises the intriguing possibility that it is a mobile molecule that may shuttle between the plasma membrane and the Golgi compartment. Thus, ABCA1 could be a constituent of a vesicular transport route for lipids involving the Cdc42/N-WASP/Arp pathway (Fig. 3.7). Initial studies on the biologic role of ABCA1 supported the view that this transporter, like MDR1 and MDR3 [62], functions as a translocator of lipids between the inner and outer plasma membrane [211]. This was based on experiments showing an increase in cholesterol and phospholipid export under conditions of forced expression of ABCA1 and ABCA1-null mutant cells from TD individuals

3.5 Function and Regulation of ABC Transporters in Lipid Transport

Synopsis of ABC lipid transporters, cellular lipid trafficking pathways, and energydependent activation of ABCA1. The model view presented highlights the interdependence of ABCA1 function and the availability of ATP, thus emphasizing the requirement of mitochondrial integrity for the proper function of ABC transporters. The transcriptional activation of ABCA1 induced by oxidized sterols such as 27-OH cholesterol is shown. ACAT, acyl-CoA:cholesterol acyltransferase; ANT, adenine nucleotide translocator; Apaf-1, apoptotic protease-activating factor 1; PDH, pyruvate dehydrogenase; PKA, protein kinase A; UC, unesterified cholesterol; VDAC, voltage-dependent anion channel; ABC, ATP binding cas-

Fig. 3.7

sette transporter; ACS, acyl-CoA synthetase; CE, cholesteryl ester; DAG, diacylglycerol; FA, fatty acid; FABP, fatty acid binding protein; FATP, FA transfer protein; FA-CoA, fatty acid acyl-CoA; GlcCer, glucosylceramide; HE1, Niemann-Pick C2 protein; HSL, hormone-sensitive lipase; L, lysosome; LacCer, lactosylceramide; LB, lamellar body; LCAT, lecithin-cholesteryl acyltransferase; Lipo, lipoprotein; MVB, multi-vesicular body; NCEH, neutral cholesteryl ester hydrolase; NPC, Niemann-Pick C protein; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PL, phospholipid; PS, phosphatidylserine; SPM, sphingomyelin; TG, triglycerides [207–211].

that characteristically display the reverse scenario [128, 208]. However, recent work from our laboratories indicated that the ATP turnover of ABCA1 occurs at a very low rate, whereas nucleotide binding induces conformational changes [35]. Based on this information it is likely that ABCA1 acts rather as a facilitator of cholesterol/phospholipid export within the cellular lipid export machinery than exerts bona fide pump function [35]. It will be exciting to elucidate the exact molecular mechanisms by which ABCA1 mediates the export of lipid compounds from the cell.

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3.5.2

ABCG1 and Other ABCG Members in Sterol Homeostasis

Following its cloning in 1996 [112], it was four years before ABCG1 attracted great attention because of its striking similarities with ABCA1 in its expression pattern in monocytic cells. Using a differential display approach ABCG1 was identified as a target gene involved in macrophage lipid homeostasis [41]. Like ABCA1 [40], ABCG1 is upregulated during the differentiation process of monocytes into mature macrophages and is strongly induced by foam cell conversion of these macrophages under sterol loading conditions using acLDL. Conversely, cholesterol unloading conditions achieved by further incubation with HDL3, as the cholesterol acceptor results in the suppression of ABCG1 mRNA and protein expression [41]. In the mean time, these results have been confirmed by other groups as well [212–214]. The observed upregulation of ABCG1 is not restricted to acLDL but is also operative when using other types of modified LDL, such as oxidized LDL or enzymatically modified LDL, but not with free cholesterol or native LDL. Of special interest is the finding that ABCG1 regulation by lipids occurs exclusively in human or murine monomyeloid cells, such as primary human macrophages, THP-1 cells, RAW246.7 cells, peritoneal macrophages, and foam cells of atherosclerotic lesions. The sterol-sensitive induction seen in these cells is independent of pro-inflammatory stimuli and the oxidative state of the cell as treatment with TNFa or LPS has no impact on ABCG1 mRNA expression [213]. In addition to lipoprotein-derived lipids, some oxysterols and RXR-specific ligands upregulate ABCG1 expression via the LXR/RXR pathway. Evidence for a significant role of these nuclear receptors in ABCG1 induction comes from two different types of experiments. First, macrophages devoid of LXRa and LXRb fail to upregulate ABCG1 mRNA upon oxysterol treatment, and secondly, retroviral expression of LXR in RAW246.7 cells facilitates the induction of ABCG1 in response to LXRa and LXR ligands [213]. A first characterization of the ABCG1 promoter (promoter 2 in Fig. 3.6) demonstrated its functionality and elucidated the minimal region required for liver- and macrophage-specific expression of the gene [114]. Further reports have shown that the ABCG1 gene displays a highly complex transcriptional profile due to the existence of at least three independent promoters (Fig. 3.6). Whereas the activity of promoter 1 has not been proven so far [115], promoter 3 of ABCG1 has been shown to bind the transcription factors LXR/RXR and thereby mediate the sterol-dependent induction of the gene [215]. In addition to this activating, sterol-regulated pathway, an independent inhibitory mechanism involving the transcriptional repressor ZNF202 and promoter 2 of ABCG1 has been described [203]. ZNF202 regulates a number of genes involved in general lipid metabolism and in particular has been shown to bind to the apoE, ABCA1, and ABCG1 promoters and thereby to modulate cellular lipid efflux. Taken together, transcription from ABCA1 and ABCG1 genes seems to be dominated by sterol-dependent activating mechanisms involving LXR/RXR and by sterol-independent repressory mechanisms mediated by ZNF202.

3.5 Function and Regulation of ABC Transporters in Lipid Transport

Although the remarkable regulation of ABCG1 gene expression by cellular lipid components revealed its importance in macrophage lipid metabolism, direct evidence for a functional role in lipid trafficking came from an antisense strategy to block ABCG1 expression [41]. Specific antisense oligonucleotides which had no effect on ABCA1 levels caused a 32% and a 25% reduction in macrophage cholesterol and phospholipid efflux, respectively, thereby directly linking ABCG1 with cellular lipid trafficking (Fig. 3.7). Since the same ABCG1 antisense oligonucleotides lead also to a significant inhibition of apoE secretion, the pathways involving ABCG1 seem to be at least in part distinct from acceptor mediated lipid efflux. Also, the residual phospholipid and cholesterol efflux present in cells from patients with Tangier disease along with a compensatory upregulation of ABCG1 in these cells further supports a function of ABCG1 in intracellular mobilization of lipid stores [212]. First steps in the elucidation of the localization of ABCG1 showed that the protein is predominantly localized in intracellular compartments mainly associated with the ER and Golgi membranes [41, 206, 216]. The small fraction of ABCG1 surface staining detected in immunocytochemical analysis is presumably due to unspecific binding of polyclonal ABCG1 antibodies to the macrophage receptor, as a ABCG1–GFP fusion protein is absent from the plasma membrane [206]. There is still a lack of knowledge regarding the question whether ABCG1 functions as a heterodimer or homodimer. Both forms are conceivable for ABCG1 since both cases have been described within the subfamily, e.g. ABCG2 acts as homodimer, whereas ABCG5 and ABCG8 most likely cooperate as heterodimers. In addition to the above described lipid efflux pathways operative in macrophages and liver cells, two other members of the ABCG subfamily, namely ABCG5 and ABCG8 (Tabs 3.1 and 3.2), have been implicated recently in the efflux of dietary sterols from intestinal epithelial cells back into the gut lumen and from the liver to the bile duct (Fig. 3.9). The sterols in a normal western diet usually consist of cholesterol (250–500 mg) and non-cholesterol sterols (200–400 mg), mainly plant sterols like sitosterol and also sterols from fish. In healthy individuals approximately 50–60% of the cholesterol is absorbed and retained, whereas the retention of non-cholesterol sterols is less than 1% [217, 218]. These subtle mechanisms are disrupted in b-sitosterolemia or phytosterolemia or shellfishsterolemia, a rare autosomal recessive disorder first described by Bhattacharyya and Connor in 1974 [219]. The disease is characterized by enhanced trapping of cholesterol and other sterols, including plant and shellfish sterols, within the intestinal cells and the inability to concentrate these sterols in the bile. As a consequence affected individuals have strongly increased plasma levels of plant sterols e.g. b-sitosterol, campesterol, stigmasterol, avenosterol, and 5saturated stanols, whereas total sterol levels remain normal or are just moderately elevated [220, 221]. Another biochemical feature of b-sitosterolemia is a reduced cholesterol synthesis due to a lack of HMG-CoA reductase. Despite the almost normal total plasma sterol levels, the disease shares several clinical characteristics with homozygous familial hypercholesterinemia (FH). Patients display tendon and tuberous xanthomas at an early age, premature development of atherosclerosis, and coronary ar-

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tery disease. In some cases hemolytic episodes, hypersplenism, platelet abnormalities, arthralgiasis, and arthritis have been described [222]. In 1998 Patel et al. [223] managed to localize the b-sitosterolemia locus to chromosome 2p21 and a recent fine mapping allowed workers to narrow the gene within a 2 cM region between markers D2S2294 and Afm210ex9 [125]. Using a combination of positional cloning and genome database survey, Lee et al. [126] identified ABCG5, which was mutated in nine unrelated b-sitosterolemia patients. Almost at the same time, Berge et al. [124] used a microarray analysis to search for LXR-regulated genes and identified ABCG5. Since ABC transporters are often found in clusters, the group screened nearby regions and found a second new member of the ABCG subfamily, ABCG8, which displayed 61% sequence similarity and was also mutated in sitosterolemia patients. The fact that the translational start sites of both ABC transporter genes are separated by only 374 bp and arranged in a head-to-head orientation led to the assumption that ABCG5 and ABCG8 have a bi-directional promoter and share common regulatory elements [124], although no functional promoter data have been provided so far. The highest expression level of both transporters is found in liver and intestine and high-cholesterol diet feeding in mice induced the expression of both genes. These findings, together with the observed clinical and biochemical features of b-sitosterolemia patients, assume that ABCG5 and ABCG8 play an important role in reducing intestinal absorption and promote biliary excretion of sterols. To date, several mutations and a number of polymorphisms have been identified in ABCG5 and ABCG8 [124, 126, 224, 225]. Interestingly, sequence analysis of both genes showed that the majority of the analyzed patients were homozygous for a single mutation and that the total number of different mutations is very low. This strongly suggests that sitosterolemia has its origin in a limited number of founder individuals. Another striking finding is that mutations in b-sitosterolemia patients occur exclusively either in ABCG5 or ABCG8, but never in both genes together [225, 226]. The coordinate regulation of both genes and the finding that mutations in either gene cause b-sitosterolemia strongly suggest that the ABCG5 and ABCG8 proteins form a functional heterodimer. As depicted in Fig. 3.8, dietary sterols including cholesterol and plant sterols which enter the intestinal epithelial cells via micellar transport are released along the lysosomal route. b-Sitosterol and other plant sterols are directly transported back to the gut lumen by the heterodimeric ABCG5/ABCG8 complex in a sort of kick-back mechanism, which may also efflux cholesterol, thereby regulating total sterol absorption. The retained sterols are routed along the ACAT pathway in the ER and either stored as cholesteryl esters in lipid droplets or alternatively packed into chylomicrons for further transport back to the liver (Fig. 3.8). In the liver alternative processes are conceivable. The sterols are either transported to peripheral tissues by VLDL and LDL particles or converted to bile acids. Also, a direct track into the bile duct for excretion exists, possibly mediated by ABCG5 and ABCG8. In addition to ABCG5 and ABCG8, other ABC transporters including ABCG1 and ABCA1 may also participate in intestinal sterol absorption mechanisms. Data from ABCA1–/– mice strongly suggest that ABCA1 is involved in the absorption of cho-

3.5 Function and Regulation of ABC Transporters in Lipid Transport

Proposed role of ABC proteins in intestinal sterol metabolism. ABCG5, ABCG8, and ABCA1 are sterol-induced members of the ABC transporter family. ABCG5 and ABCG8, which are mutated in sitosterolemia, form a heterodimer to mediate the export of absorbed plant sterols and cholesterol into the gut lumen. In contrast, ABCA1 expression and function are required for the uptake of

Fig. 3.8

sterols into intestinal epithelial cells. Implications for the intracellular location and vesicular trafficking of these proteins are presented. Abbreviations not defined in text: CE, cholesteryl ester; DAG, diacylglyceride; DGAT, acylCoA: diacylglycerol transferase; HSP70, heat shock protein 70; L, lysosome; MAG, monoacylglyceride; Mic, micelle; MTP, microsomal transfer protein; Sit, sitosterol [22, 124–126].

lesterol and in the uptake of lipophilic vitamins [208, 227]. With this respect, it will be of special interest to determine in which membrane compartment, the apical or the basolateral part of intestinal epithelial cells, the ABCA1 molecule is located. 3.5.3

ABC Transporters involved in Hepatobiliary Transport

The formation of bile is an elementary physiological function of the liver, which involves numerous transport proteins located in the basolateral (sinusoidal) and apical (canalicular) membranes of hepatocytes (Fig. 3.9). Bile, which is composed of bile salts, phospholipids, cholesterol, bilirubin and many other small molecules, is necessary for the micellar absorption of lipids from the intestine as well as for the excretion of endogenous and xenobiotic compounds [228]. The first step in hepatobiliary transport, the uptake of compounds into liver cells is mediated by proteins of the solute carrier (SLC) superfamily [229]. Among these, the Na+/taur-

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Overview of lipid transport proteins in hepatocytes. Monovalent bile salts, such as taurocholate, are taken up into hepatocytes by the sodium-taurocholate co-transporting polypeptide (NTCP) [229]. The organic anion transporting polypeptides 1 and 2 (OATP1–2) are responsible for the charge-independent uptake of bulky organic compounds, including bile salts and other organic anions, uncharged cardiac glycosides, steroid hormones, and certain type 2 organic cations [230]. Small, type 1 organic cations are transported by the organic cation transporter OCT1. Several ABC proteins belonging to the ABCB (MDR) subfamily or ABCC (MRP) subfamily are expressed in liver [231]. ABCB1 (MDR1) is

Fig. 3.9

responsible for the excretion of bulky amphiphatic compounds into bile, whereas ABCB4 (MDR3) is a phosphatidylcholine translocase. Monovalent bile salts are secreted into the bile canaliculi by the bile salt export pump BSEP (ABCB11). ABCC2 (MRP2) functions as a multispecific organic anion transport protein in the canalicular membrane. ABCC1 (MRP1), expressed at very low levels in the basolateral membrane in normal hepatocytes, has a similar substrate specificity to MRP2. ABCC3 (MRP3) preferentially translocates conjugates with glucuronate or sulfate, whereas the physiological substrates for ABCC6 (MRP6) are unknown.

ocholate co-transporting peptide (NTCP), located in the basolateral membrane is responsible for the uptake of the majority of bile salts in hepatocytes. Small (type I) organic ions (e.g. choline, drugs, and monoamine neurotransmitters) are transported by the organic cation transporter 1 (OCT1), whereas bulky (type II) organic cations, glutathione conjugates, and some amount of bile acids are taken up the organic anion-transporting polypeptide (OATP1) [230]. The subsequent step in hepatobiliary transport, the translocation of compounds from hepatocytes into the bile, involves ABC transporters localized in the hepatocyte apical (canalicular) membrane [231]. These ABC proteins belong to the ABCB (MDR) and ABCC (MRP) subfamilies. Despite the low expression level of ABCB1

3.5 Function and Regulation of ABC Transporters in Lipid Transport

(MDR1) in normal human liver [232], data from Mdr1a/1b knockout mice, which are very sensitive to xenobiotics, neurotoxins, and chemotherapeutics, provide evidence that the major function of ABCB1 is the protection of hepatocytes against harmful substances by active translocation into the bile [233, 234]. It is now widely accepted that ABCB4 (MDR3), which is exclusively expressed in the liver apical membrane, is a bile canalicular phosphatidylcholine translocase (Fig. 3.9). This function has been confirmed by a series of experimental data: (1) mice with a target disruption of the Mdr2 gene, the mouse homolog of ABCB4 (MDR3), exhibit a complete absence of PC and strongly decreased levels of cholesterol from bile [194]; (2) transgenic expression of human MDR3 in these mice can fully restore PC secretion into the bile [235]; and (3) mutations in the human ABCB4 (MDR3) gene cause progressive familial intrahepatic cholestasis (PFIC) type 3 [10] (Tab. 3.2). The third member of the ABCB subfamily involved in hepatobiliary secretion is ABCB11 (SPGP). Gerloff et al. [236] have shown that membrane vesicles isolated from ABCB11-overexpressing Sf9 cells display ATP-dependent taurocholate uptake characteristics similar to those of liver canalicular membrane vesicles, and thus concluded that ABCB11 is the major, if not the only bile salt transporter of mammalian liver, hence the name bile salt export pump (BSEP). Further support for this proposition comes from the findings that the ABCB11 (BSEP) gene is mutated in patients with progressive intrahepatic cholestasis type 3 (PFIC3) [11], a syndrome characterized by very low levels of biliary bile salts and elevated concentrations of serum bile salts. In the ABCC (MRP) subfamily, at least four members have been shown to be expressed in liver cells [95]. In hepatocytes and other polarized epithelial cells, ABCC2 (MRP2) is localized and is highly expressed at the canalicular membrane. In contrast, ABCC1 (MRP1) present at the basolateral membrane domain, is expressed very low in normal liver. As listed in Tab. 3.1 and displayed in Fig. 3.9, physiological substrates for ABCC1 and ABCC2 comprise glutathione conjugates (e.g. leukotriene C4), estrogen- and bilirubin-glucuronides, taurolithocholate 3-sulfate, and glutathione disulfide (GSSG). However, due to the differences in the overall expression levels and because of greatly different transport kinetics, ABCC2 seems to be the major transporter of anionic conjugates. Likewise, hereditary defects of ABCC2 in humans cause the Dubin–Johnson syndrome, which is associated with defects in biliary secretion of amphiphilic anionic conjugates including bilirubin-glucuronides [237, 238]. Glucuronate- and sulfate-conjugates are also substrates for ABCC3 (MRP3), which has been localized to the basolateral membrane of hepatocytes [239]; however, in contrast to ABCC1 and ABCC2, glutathione conjugates are poor substrates for ABCC3. ABCC6 (MRP6), which has been localized to the lateral hepatocyte membrane [240], is capable of transporting the anionic cyclopentapeptide BQ123, an endothelin receptor antagonist; however, the physiological substrate for ABCC6 has not been elucidated so far.

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3.6

Conclusions and Perspectives

Although our knowledge of lipid-transporting ATP binding cassette transporters has grown substantially over the last few years, the detailed molecular mechanisms by which lipid compounds are transported across cellular membranes still await clarification. Analysis of the transcriptional and metabolic regulation, the intracellular localization and membrane domain association, the exact substrate specificity, and the functional activity of these proteins will provide helpful hints towards the understanding of working mechanisms of ABC lipid transporters. Based on their complex architecture it can be expected that ABC lipid transporters engage in multifaceted interactions with an array of yet to be identified effector molecules at specialized membrane compartments. The recent finding that ABCA1 is not an active pump but may rather function as a regulator similar to ABCC7 (CFTR) or ABCC8 (SUR1) supports this hypothesis. It will be a fascinating task to characterize the functional partners of ABC lipid transporters and to determine whether these include other ABC lipid transporters. In light of the now documented role of the prototypic cholesterol-responsive ABC molecules ABCA1 and ABCG1, it can be postulated that other ABC transporters which show a cholesterol-dependent regulation in macrophages, especially members of the ABCB and ABCC subfamilies play critical roles in macrophage lipid homeostasis.

3.7

References 1 2 3 4 5

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C. F. Higgins, Annu. Rev. Cell Biol. 1992, 8, 67–113. W. Saurin, M. Hofnung, E. Dassa, J. Mol. Evol. 1999, 48, 22–41. H. Nikaido, FEBS Lett. 1994, 346, 55– 58. J. Young, I. B. Holland, Biochim. Biophys. Acta 1999, 1461, 177–200. T. Litman, T. E. Druley, W. D. Stein, S. E. Bates, Cell Mol. Life Sci. 2001, 58, 931–959. M. Bodzioch, E. Orso, J. Klucken, T. Langmann, A. Bottcher, W. Diederich, W. Drobnik, S. Barlage, C. Buchler, O. M. Porsch, W. E. Kaminski, H. W. Hahmann, K. Oette, G. Rothe, C. Aslanidis, K. J. Lackner, G. Schmitz, Nature Genet. 1999, 22, 347– 351. A. Brooks-Wilson, M. Marcil, S. M. Clee, L. H. Zhang, K. Roomp, M. vanDam, L. Yu, C. Brewer, J. A. Collins,

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Albumin Receptors – Structure and Function Nigel J. Brunskill

4.1

Introduction

Human albumin, a 585 amino acid globular protein with a molecular weight of 66 000 Da, accounts for approximately 60% of the total protein in blood serum. The total body mass of albumin is about 300 g for a 70-kg individual, and between 40% and 50% of the albumin pool is normally found in the circulatory compartment [1, 2]. Thus the concentration of albumin in healthy human serum is around 40 g L–1 or *0.6 mM. Albumin has a variety of crucial functions in the body. As the most abundant blood protein it is the major contributor to the maintenance of oncotic pressure within the circulation and in the interstitial fluid, thereby strongly influencing the transendothelial flux of water and small solutes. In addition, through interactions with the endothelial glycocalyx, albumin contributes to a permselective barrier restricting the transendothelial passage of many molecules [3–7]. Accordingly, in many experimental models removal of albumin from the vascular perfusate significantly increases microvessel permeability [7–10]. Endothelial/albumin interactions may also reduce red cell and platelet adherence, and restrict binding of other plasma proteins [11, 12]. Albumin also acts as a carrier for drugs, amino acids, fatty acids, hormones, sterols, and bilirubin [13]. Many of these molecules are presented by albumin to various cells, an activity which involves its own transport across the endothelium. The preservation of normal albumin levels in the body is important for health, and abnormal circulating albumin concentrations are often observed in disease. Low plasma albumin levels are the hallmark of the nephrotic syndrome [14]. In many inflammatory illnesses albumin may be considered a negative acute-phase reactant [15]. Increased synthesis of acute-phase reactant proteins such as fibrinogen, C-reactive protein, a2-microglobulin, etc. are accompanied by suppressed synthesis of albumin leading to a decrease in its blood concentration [15]. Prolonged depression of blood albumin concentration is an adverse feature in disease, and in dialysis patients for example, reduced blood albumin concentrations are a powerful predictor of death [16, 17]. In nephrosis higher than normal concentrations of albumin prevail in the proximal tubule, and “albumin toxicity” has been invoked as a mediator of renal injury in such individuals [18, 19].

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Thus albumin plays a key role in the maintenance of normal physiology and homeostasis. Conversely in disease, altered or locally inappropriate albumin levels may contribute to the evolution of pathophysiology in cardiovascular, renal, and other conditions. It is not surprising therefore that the mechanism(s) of interaction between albumin, and the cells it comes into contact with, should have interested a diverse group of investigators over the last 30–40 years.

4.2

The Search for an Albumin Receptor 4.2.1

The Endothelium–Albumin Relationship: Early Concepts

Initial physiological studies indicated that albumin, and other macromolecules, crossed the endothelium via an hydraulic conductive pathway that could be simply modeled as pores or filtration slits assumed to be located at intracellular junctions [3, 8]. This represented the standard view of endothelial albumin transport until the late 1980s. At this time it became apparent from work performed in a number of laboratories that molecules such as insulin [20], low-density lipoprotein [21], and transferrin [22] could be transported across the endothelium by a receptor-mediated vesicular endocytic pathway. In the 1960s investigators interested in the clearance of metabolically altered albumin by the liver had examined the handling of formaldehyde-treated albumin (f-albumin) by liver cells in vivo and in vitro. These experiments revealed very rapid clearance of f-albumin by sinusoidal liver cells, which were able to take up and degrade this chemically modified albumin [23, 24]. A putative receptor for f-albumin was subsequently purified from rat liver by affinity chromatography and partially characterized. This receptor was found to be composed of two glycoprotein subunits with Mr of 53 000 and 30 000 respectively. The receptor exhibited a high affinity for f-albumin (KD 6.2 lg mL–1, or approximately 10–8 M) but not native albumin [25, 26]. Rapid and highly efficient hepatic uptake of albumin-bound long-chain fatty acids was suggested to be due primarily to a direct interaction of the albumin– fatty acid complex with hepatocytes. Saturable and reversible binding of [125I]albumin to isolated hepatocytes reinforced the belief that a putative albumin receptor may allow the interaction of albumin directly with cells [27]. The suggestion of receptor-mediated endocytosis of peptides and proteins by endothelial cells, and the presence of receptors for modified albumin in liver cells provoked groups in several laboratories to postulate the existence of receptors for native albumin in the endothelium.

4.2 The Search for an Albumin Receptor

4.2.2

Identification of Receptors for Native and Modified Albumin in Endothelial Cells

Evidence for endothelial receptors with affinity for native albumin was obtained from experiments where mouse organs were in situ perfused with gold–albumin (alb-au) complexes followed by perfusion fixation and electron microscopy of capillary endothelia [28]. Even at very early time points alb-au could be detected in membrane microdomains with the appearance of uncoated pits, and in vesicular structures towards the luminal front of the cell. The binding was saturable at low concentration and blocked by unlabeled native albumin. At later time points albau appeared to be discharged from the abluminal membrane into the subendothelial space. For the first time experimental evidence strongly indicated the possibility of specific endothelial binding sites for albumin and receptor-mediated endocytosis of albumin by these cells. Using cultured rat microvascular endothelial cells Schnitzer et al. demonstrated binding of native unmodified albumin that was specific, saturable, and reversible [5]. Thus albumin fulfilled many of the criteria required of a specific ligand binding to its receptor. Eventually, by lectin affinity chromatography and protease digestion of radioiodinated surface proteins, this group of investigators was able to purify a 60 kDa sialoglycoprotein (gp60) with albumin binding activity from microvascular endothelial cells in culture [29, 30]. This protein was found to be immunologically similar to glycophorins [31], a family of erythrocyte membrane proteins thought to have an important role in restricting non-specific low-affinity interactions at the cell surface and the prevention of non-specific hemagglutination [32]. Anti-glycophorin antisera were able to recognize gp60 without inhibiting albumin binding [31]. Working separately and using different methodologies, other workers also established the presence of albumin binding proteins in endothelial cells. Ghinea et al. identified two major proteins of 18 and 31 kDa (gp18 and gp30) on the surface of microvascular endothelial cells from lung and epididymal fat pad that possessed binding activity for alb-au and [125I]albumin. Binding was specific and of high affinity with KD *60 nM. Two minor albumin binding proteins were also found of 56 and 73 kDa, the 56 kDa protein being of similar size to, and possibly representing gp60 [33, 34]. Subsequent work revealed a wide tissue distribution for gp18 and gp30 and indicated a generally more avid interaction with chemically modified forms of albumin compared to native albumin [35–37]. This pattern of expression, along with their higher affinity for conformationally altered albumin, is consistent with a role for gp18 and gp30 in albumin catabolism and the widespread removal of senescent forms of albumin, an activity prevalent in a variety of organs [38]. Broadly, therefore, albumin receptors appeared to fall into two groups: those able to bind native albumin and mediate its transcytosis, and those which could bind chemically modified albumins ultimately destined for catabolism and lysosomal degradation. Although the identities of gp18 and gp30 require further study, more is known about gp60. This molecule is widely expressed on the surface of

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continuous endothelia in organs such as heart, lung, and skeletal muscle [39]. Accordingly the continuous endothelia in these organs bind albumin, and albumin can be detected within intracellular vesicular structures. Organs such as adrenal gland, pancreas, and duodenal mucosa possess a non-continuous or fenestrated endothelium. Fenestrated endothelium lacks gp60 and cannot absorb albumin into vesicular structures [39]. Presumably because fenestrated endothelia form a discontinuous barrier with large gaps, plasma molecules are allowed free access to the interstitium without the requirement for endothelial binding and transcytosis. Lately, further similarities between gp60 (christened albondin by one group of workers) [40] and other proteins have been recognized. Polyclonal antisera raised to purified gp60 cross-react with secreted protein, acidic and rich in cysteine (SPARC) [41]. SPARC is a secreted matricellular protein released from many cell types and is found primarily in tissues undergoing consistent turnover, or at sites of damage [42]. It is thought to regulate cellular differentiation and response of tissues to injury [42]. Binding of albumin to SPARC secreted by endothelial cells has been described [43]. Several antisera raised to purified SPARC also recognize gp60, and prevent albumin binding to gp60. However some anti-SPARC peptide antisera raised against N-terminal peptides fail to recognize gp60 and do not inhibit albumin binding [41, 44]. Thus the proteins SPARC and gp60 share common epitopes and are immunologically and functionally related. Whether the albumin binding properties of SPARC are important to its function is not clear. There seems little doubt that gp60 is a functional and important receptor for native albumin in endothelia. Antibodies made against gp60 inhibit albumin binding to both immobilized gp60 and endothelial cells; prevent internalization of albumin by endothelia in situ and in vitro; decrease capillary permeability; and greatly reduce transendothelial transport of albumin in vitro [40, 41]. Activation of gp60 by cross-linking results in 2- to 3-fold increase in albumin uptake and a corresponding increase in permeability to other macromolecules but not to water [45, 46]. Very interestingly, activation of gp60 by albumin binding or by receptor crosslinking results in phosphorylation of a number of cell proteins [42]. Notable amongst these are gp60 itself, caveolin-1, and the Src family tyrosine kinases pp60c-Src and Fyn. These intriguing results raise the possibility that gp60 can act as a signaling receptor, and that its ligand, albumin, may have intrinsic signaling properties. More work is required to clone and sequence this protein, to study the regulation of its expression, to establish the pharmacology of gp60–albumin interactions, and to clarify the signaling events precipitated by albumin–gp60 binding. Until this information is available the precise importance of gp60 in endothelial cell biology will remain obscure.

4.3 Albumin Receptors in the Kidney

4.3

Albumin Receptors in the Kidney

Endothelial cell biologists have not had a monopoly over research into albumin receptors. In the last 20 years renal physiologists and nephrologists have also had a major interest in the mechanisms of cellular binding and handling of albumin. Nephrological studies of albumin binding reflect widespread interest in the handling of glomerular filtered albumin by renal tubular cells, and the potential pathophysiological effects of the inappropriate albumin concentrations encountered in the kidney proximal tubule in nephrotic syndrome. 4.3.1

Glomerular Handling of Albumin

Renal plasma flow and subsequent glomerular filtration results in the passage of water, low molecular weight solute and some macromolecules, including albumin, into the tubular fluid. The quantity of intact albumin excreted in the urine of healthy subjects is however negligible. Clearly, therefore, filtered albumin must be removed from glomerular ultrafiltrate and processed by tubular cells. It is postulated that excess albumin interacting with the proximal tubule may contribute very significantly to the pathophysiology of chronic renal failure [18, 19]. The mechanism of renal handling and glomerular filtration of albumin is currently the subject of considerable controversy. The traditional view, and prevailing dogma, holds that glomerular permeability for albumin is low as a result of both the size and charge selectivity of the glomerular filtration barrier [47–50]. Tubular micropuncture studies in rodents support this concept with measured albumin concentrations of 10–135 mg L–1 in proximal tubular fluid [51–55], meaning approximately 8 g of albumin must be filtered in 24 hours. This view has recently been challenged by a new hypothesis. The new paradigm suggests that albumin is freely filtered by the glomerulus, and that large quantities of albumin enter the tubular fluid. An as yet unidentified high-capacity retrieval pathway for albumin in the proximal nephron is postulated to prevent the undesirable excretion of filtered albumin in this model [56, 57]. Whichever of these conflicting models eventually proves to be correct, however, tubular cell/albumin interactions represent an important component [58]. 4.3.2

Binding and Uptake of Albumin in the Kidney Proximal Tubule

A number of elegant morphological studies have established that albumin re-absorption occurs by endocytosis through a very extensive and prominent tubulovesicular network largely in the proximal tubule [59]. Early studies confirmed that albumin and other proteins were re-absorbed and catabolized by proximal tubular cells but contributed little mechanistic information [60, 61]. The classic studies of Park and Maack were the first to investigate the kinetics of albumin re-absorption

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in proximal tubule segments [62, 63]. Two uptake systems were identified, one of high affinity but low capacity, the other with low affinity but high capacity. These findings were subsequently reproduced in studies using cultured proximal tubule cells [64–67]. Identification of two uptake systems for albumin implied the presence of two binding sites. We examined binding of [125I]albumin to opossum kidney (OK) cells at 4 8C, and ligand blotting of OK cell lysates to identify albumin binding proteins [68]. The presence of two albumin binding sites was confirmed, one of high affinity (KD 154 ± 7 mg L–1) and one of much lower affinity (KD 8300 ± 1000 mg L–1), the KD of the more avid site being appropriate for a re-absorptive receptor given the measured concentration of albumin in proximal tubular fluid (see above). Competition studies showed that albumin binding was sensitive to a number of agents known to inhibit binding to scavenger receptors. Ligand blotting revealed the existence of albumin binding proteins with molecular weights of 14, 18, and 30 kDa, reminiscent of those found in endothelial cells [68]. Employing different methodologies, another group also found albumin binding proteins of similar character in rat kidney [69]. Thakkar et al. used an optical biosensor to study albumin binding proteins in LLCPK cells [70]. A single binding site/receptor was identified which was capable of binding albumin together with a variety of other proteins such as retinol binding protein, 1 microglobulin, cystatin C, and 2 microglobulin. The equilibrium binding constant KA for albumin was 8.0 ´ 104 M–1. Although these studies provided helpful information, the precise identity of the albumin receptor(s) remained unclear. Several groups had noted that megalin, a receptor for the endocytic uptake of various ligands, was localized along the endocytic pathway in proximal tubular cells [71–74]. With this observation in mind Cui et al. provided convincing evidence that megalin was responsible for albumin binding and endocytic uptake in rat proximal tubules [75]. Their findings indicated firstly that known megalin ligands significantly inhibited albumin uptake in the proximal tubule, and that secondly megalin-sepharose columns bound [125I]albumin [75]. The same investigators later demonstrated that megalin cooperates with a second protein, cubilin, to mediate albumin uptake in rats, dogs and OK cells [76, 77]. The significance of these observations is described below.

4.4

Megalin and Cubilin as Proximal Tubule Albumin Receptors 4.4.1

Megalin

Megalin is a giant membrane glycoprotein originally identified as the Heymann nephritis antigen in rats. Initial purification by gel electrophoresis suggested a molecular weight of 330 kDa and the protein became known as gp330 [78]. When cloned, originally by Saito et al. from rat [79] and then by Hjalm et al. from hu-

4.4 Megalin and Cubilin as Proximal Tubule Albumin Receptors

man kidney [80], the protein was found to be a single polypeptide of 4660 amino acids forming a glycoprotein with a molecular weight of 600 kDa. The protein became known as megalin on account of its size. Megalin has a single transmembrane domain, a large extracellular N-terminal domain, and a short intracellular C-terminal tail [79]. The extracellular portion contains 36 cysteine-rich ligand binding domains, 16 EGF precursor domains, and 40 YWTD repeats [81]. There are four clusters of ligand binding domains consisting of multiple complement type repeats [81]. Megalin belongs to the low-density lipoprotein receptor (LDL-R) family and possesses the seven characteristic features of this family: cell surface expression; extracellular ligand binding domain consisting of complement-type repeats; Ca2+-dependent ligand binding; recognition of receptor-associated protein and apolipoprotein E (apoE); epidermal growth factor precursor homology domain containing YWTD repeats; single membrane spanning region; and receptor-mediated endocytosis of multiple ligands [81, 82]. Like other members of the LDL-R family megalin binds a diverse range of ligands with high affinity. These include apoE [83, 84], lipoprotein lipase [85, 86], aprotinin [87], plasminogen activator inhibitor 1 [88], tissue plasminogen activator [89], receptor-associated protein (RAP) [89, 90], gentamicin [87], cubilin [91], and others. In general, ligands binding to LDL-R family members are destined for lysosomal breakdown, often with the release of crucial cellular nutrients. In accordance with its role as a re-absorptive receptor megalin expression is found on re-absorptive surfaces, particularly epithelia such as proximal tubule cells, glomerular podocytes, and choroid plexus [92, 93]. Megalin is also present in the visceral yolk sac, parathyroid hormone-secreting cells, type II pneumocytes and the small intestine [92–96]. This pattern of cellular expression obviously implicates megalin in the re-absorption of molecules from tubular fluid, intestinal fluid and in transport across the blood–brain barrier [92, 97]. Thus Cui et al. examined the ability of megalin to bind albumin in the proximal tubule [75]. Using a micropuncture approach these workers showed that the megalin ligands RAP and gentamicin inhibited albumin uptake in the rat proximal tubule. RAP is a chaperone protein for LDL-R family members. Present in the endoplasmic reticulum, RAP assists with correct folding, prevents premature ligand binding, and acts as a universal antagonist for the binding of all ligands to all the different LDL-R [98–101]. Albumin was also found to bind strongly to purified sepharosemegalin columns. This study was the first to demonstrate that megalin could bind albumin, although crucially the affinity of this interaction has not been determined. The importance of megalin as a proximal tubular albumin binding protein was, however, questioned by the study of megalin knockout mice. Almost all megalin–/– animals die in the immediate perinatal period due to severe malformation of the forebrain [102]. Only 2% of these animals survive to adulthood but those survivors exhibit low molecular weight proteinuria but not albuminuria [103].

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4.4.2

Cubilin

Cubilin is a 3600 amino acid protein with a non-glycosylated molecular mass of 400 kDa. Complete DNA sequences from human [104], rat [93], and dog [105] are available and reveal the absence of a transmembrane domain and little structural homology with other endocytic receptors. This protein was first identified as the target of teratogenic anti-renal brush border antibodies in rabbits and named gp280 based on an estimated molecular weight [106]. Cubilin was eventually found to be identical to the intrinsic factor cobalamin receptor [107], and other identified ligands include RAP, high-density lipoprotein and apolipoprotein AI [108, 109]. An interaction with megalin has also been described and it has been suggested that megalin may be a co-receptor involved in the endocytosis and trafficking of cubilin [93]. The sequence of cubilin predicts 27 bone morphogenic protein 1 (CUB) domains, most likely constituting the ligand binding domains. These regions are preceded by a stretch of 110 amino acids and eight EGF-type repeats. The N-terminal region possess an amphiphatic helix with resemblance to the ligand binding regions of apolipoproteins essential for anchoring of the protein to the external surface of the cell membrane [110]. Whilst expression of cubilin is more restricted than that of megalin it is found in small intestinal epithelium [111, 112] and placenta [113]. Renal proximal tubule cells express cubilin on their brush border, and in all components of the coated pit endocytic and recycling pathway, a distribution extremely similar to that of megalin [92]. Imerslund–Grasbeck syndrome an inherited vitamin B12-deficiency disease caused by the absence of functional cubilin is associated with proteinuria [114– 116]. A variety of cubilin mutations are responsible for this disease [117]. Inherited deficiency of cubilin in dogs is also associated not only with vitamin B12 malabsorption, but also with proteinuria [118]. Thus considerable circumstantial evidence makes cubilin an attractive candidate albumin receptor in the proximal tubule. Recent work confirms that: dogs with a functional cubilin defect fail to reabsorb albumin in their proximal tubules; cubilin binds albumin by affinity chromatography; albumin, cubilin and megalin co-localize in endocytic compartments in rat proximal tubules; low molecular weight albumin binding proteins identified in earlier studies [69] may be cubilin fragments; both megalin and cubilin are required for efficient albumin re-absorption by OK cells [76, 77]. A picture of albumin binding in the kidney proximal tubule is now beginning to emerge (Fig. 4.1). It seems that megalin and cubilin work in a cooperative and synergistic manner to mediate efficient albumin binding and re-absorption [117]. A highaffinity (KD *7 nM) Ca2+-dependent and RAP-inhibitable interaction exists between these two proteins [93], the two receptors share several ligands and have similar localization. Although megalin and cubilin contribute importantly to albumin binding and uptake in the proximal tubule, it is probable that other albumin receptors are present for the following reasons: megalin knockout mice show little or no albumi-

4.5 Albumin as a Signaling Molecule – Implications for Albumin Receptor Function

Megalin and cubulin as cooperative albumin receptors in the kidney proximal tubule. Albumin binds to both megalin and cubilin followed by internalization of megalin–albumin–cubilin complexes via internalization sequences in the cytoplasmic tail of megalin.

Fig. 4.1

It is probable that other intracellular proteins interact with the cytoplasmic tail of megalin (see text). Whether or not such interactions are regulated by agonist binding is not yet determined.

nuria; cubilin or megalin antibodies and antisense oligonucleotides reduce albumin uptake only by 20–30% in proximal tubule cells; RAP and intrinsic factor B12 complexes inhibit albumin uptake by only 40% and 50% respectively in OK cells [77]. Furthermore albumin binding and transcytosis in endothelia cannot be mediated by megalin or cubilin because these cells do not express them.

4.5

Albumin as a Signaling Molecule – Implications for Albumin Receptor Function

Albumin itself has traditionally been regarded as a benign or biologically inert molecule. Evidence in support of a role for albumin in cell signaling is now beginning to accumulate however. As described above, studies of gp60 in endothelial cells demonstrated albumin-evoked activation of tyrosine kinases [42]. Other workers have described a potentially receptor-mediated survival factor type effect of albumin in cultured endothelial cells [119]. Astrocytes manifest a marked and sustained reduction in intracellular Ca2+ on exposure to lipid free albumin [120]. In view of the potential pathophysiological effects of albumin in the kidney, work in several laboratories has investigated how albumin may alter cell phenotype by signaling in proximal tubular epithelia. Recent work in our laboratory exemplifies this approach. Using gene transfection and dominant negative mutants we have

87

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demonstrated that albumin stimulates activity of the type 1 phosphoinositol 3-kinase (PI3-kinase) p85/p110 and the ribosomal protein p70s6 kinase in PTEC [121, 122]. In vitro proximal tubular epithelial cells proliferate briskly [122, 123] when incubated with albumin. Our work has shown that albumin stimulates proliferation of proximal tubular cells via a kinase cascade involving PI3-kinase, pp70s6 kinase [122], and extracellular signal regulated p42/p44 MAP kinase (ERK) [124]. Appreciable stimulation of PI3-kinase occurs at albumin concentrations likely to be found in the proximal tubule in health. Therefore these data indicate that albumin signaling in proximal tubular cells may not only be involved in the pathogenesis of tubulo-interstitial disease but may also play a role in maintaining proximal tubular growth and homeostasis in health. Hyperplasia of proximal tubular cells has now been demonstrated in albuminuric rats with protein overload proteinuria [125] and also been confirmed in proteinuric humans [126]. Incubation of proximal tubular cells with albumin in vitro causes activation of the NFjB family of transcription factors [127, 128]. As a consequence of albumininduced NFjB activation, these cells in culture produce a variety of chemoattractants, such as MCP1 and RANTES in a dose-dependent manner [127, 128]. In vivo models of albuminuric renal disease demonstrate prominent interstitial accumulation of macrophages associated particularly with those proximal tubules displaying the most marked accumulation of intracellular protein [129]. These heavily protein overloaded tubules also demonstrate the greatest chemokine expression [129]. Therefore this evidence indicates that albumin may induce, via proximal tubular cells, a pro-inflammatory environment in the kidney. Thus albumin alters proximal tubular cell function and phenotype in the manner of a signaling molecule. These recent findings of albumin signaling obviously challenge the accepted dogma described above although the mechanisms of albumin-induced signal generation are unclear. 4.5.1

LDLR Family as Signaling Receptors

The enigmatic effects of albumin described above could be explained by signaling through an albumin receptor. Cubilin, being an entirely extracellular protein without any transmembrane or intracellular region is unlikely to be involved in signaling. Members of the LDL-R family such as megalin are much more likely candidates. The cytoplasmic tails of the LDL-R family share homologous sequences, particularly internalization related WxNPxY motifs [81]. Traditionally, members of this family have been regarded solely as endocytic receptors that serve to bind and internalize extracellular ligands prior to lysosomal breakdown. This paradigm has very recently been challenged [130]. In particular, current evidence suggests that certain non-endocytosis-related cytoplasmic adaptor proteins may interact with the cytoplasmic domains of certain members of the LDLR family. Using yeast two hybrid and co-precipitation approaches the neuronal adaptor proteins Disabed-1 (Dab1) and FE65 have been shown to bind to the cytoplasmic domains of LDL-R and LRP [131]. This interaction with Dab1 is felt to be

4.5 Albumin as a Signaling Molecule – Implications for Albumin Receptor Function

crucial for LDL-R family function in neuronal tissues. However neither of these proteins have a role in endocytosis, but rather function in signaling pathways involving tyrosine kinases and cytoskeletal components. Mice expressing mutant Dab1 display a profound brain phenotype indistinguishable from animals lacking the extracellular signaling molecule Reelin, or both VLDLR and apoER2 [132, 133]. Reelin binds with high affinity to apoER2 and VLDLR, and more weakly to LDL-R. Taken together these observations suggest that Reelin/apoER2/Dab1 act in a common signaling pathway. The cytoplasmic domain of ApoER2 also binds the JNK-interacting proteins JIP1 and JIP2, which act as molecular scaffolds for the JNK-signaling pathway [134]. Ligand binding to LRP has been shown to activate cAMP-dependent protein kinase via direct association with the GTP-binding protein Gsa [135]. The cellular consequences of such interactions remain speculative. 4.5.2

Megalin as a Signaling Receptor

Megalin possesses a longer cytoplasmic tail (209 amino acids in human, 213 in rat) than other LDL-R family members with unique sequence motifs [79, 80] (Fig. 4.2). In particular human megalin has three WxNPxY domains acting as coated pit internalization sequences [136] and/or phosphotyrosine interaction domains [137], four Src homology 3 (SH3) binding regions conforming to the XpUPpXP SH3 binding site consensus recognition motif [138], and one Src homology 2 (SH2) recognition motif for the p85 regulatory subunit of PI3-kinase [139]. In addition, there are multiple phosphorylation sites for protein kinase C, casein kinase II, and cAMP/cGMP-dependent protein kinase [80]. These findings suggest that megalin may have signaling or trafficking functions in addition to, or distinct from, those of other LDLR family members.

Distribution of human megalin cytoplasmic tail sequence motifs potentially involved in protein–protein interactions and signaling. NPxY domains numbered 1st–3rd.

Fig. 4.2

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Megalin knockout mice exemplify the crucial physiological role of this receptor. The great majority of these animals die in the perinatal period, displaying a severe developmental brain phenotype, holoprosencephaly, together with major abnormalities in PTEC structure [102]. Only 2% survive but with considerable proteinuria [103]. The severity of these phenotypic changes is difficult to explain on the basis of an endocytic defect alone. Indeed a variety of proteins have now been shown to interact with the cytoplasmic tail of megalin [140–142]. Of particular interest are Disabled-2 (Dab2), SEMCAP-1, JIP1, JIP2, PIP4,5-kinase homolog, and ANKRA. These proteins are involved in Ras and ERK signaling, GTP-binding protein signaling, JNK scaffold assembly, inositol metabolism, and probable Rafkinase binding [143] respectively. In kidney glomerular podocytes the megalin cytoplasmic tail binds the membrane associated guanylate kinase protein MAGI-1 [144], possibly facilitating the assembly of a signaling complex around megalin. The precise physiological relevance of the binding of these proteins to megalin remains to be determined.

4.6

Summary

Albumin is a ubiquitous protein which comes into contact with nearly every cell type. Not surprisingly many investigators have studied how albumin–cell interactions are regulated. Considerable progress has been made particularly in the study of endothelia and kidney proximal tubular cells. Further work is required to complete the identification of endothelial albumin receptors, and it is likely that receptors for albumin other than megalin and cubulin may be located in the kidney tubule. The concept of albumin as a signaling molecule is relatively new, and is poised to open up exciting avenues for further important research into structure and function in health and disease.

4.7

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Intracellular Lipid Binding Proteins: Evolution, Structure, and Ligand Binding Christian Lücke, Luis H. Gutiérrez-González, and James A. Hamilton

5.1

Introduction

With the isolation of the first “fatty acid binding protein” (FABP) nearly 30 years ago [1], a new family of intracellular lipid binding proteins (iLBPs) was discovered. Since then a large variety of 14–16-kDa iLBPs (127–137 residues) have been reported. These cytosolic proteins bind various amphiphilic molecules, such as fatty acids, bile acids or retinoids [2]. As a result of their interactions with these essential lipids, the iLBPs potentially have multiple tasks inside the cell. Aside from cellular uptake and transport of lipids, the iLBPs also likely play a role in the regulation of lipid metabolism [3, 4] (see also Chapters 13, 15, 20, and 21). However, the entire spectrum of possible functions of the different iLBP types is currently still being investigated by various biochemical, biophysical, and biological approaches [5, 6]. Here we will describe the structures of the iLBPs, as deduced from X-ray crystallography and high-resolution NMR spectroscopy, and discuss binding assays used to study protein:lipid interactions in iLBPs.

5.2

The Evolution of Lipid Binding Proteins 5.2.1

The Calycin Superfamily

The iLBPs belong to a larger superfamily of lipid binding proteins, the so-called calycins [7], that show a common up-and-down b-barrel fold [8]. Despite amino acid sequence homologies as low as *10%, the different branches of this superfamily are apparently genetically related [9]. Hence, the calycins consist of three main protein families: the avidins, the lipocalins, and the iLBPs. The avidins are biotin binding proteins from oviparous vertebrates. Their structure comprises eight antiparallel b-strands that form a b-barrel structure with an internal binding pocket [10]. A prokaryotic form, the streptavidin, has been isolated from Streptomyces [7].

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The lipocalins include a wide variety of extracellular lipid binding proteins such as the plasma retinol binding protein (RBP), b-lactoglobulin, a2-globulin, major urinary protein, insecticyanin, and the epididymal retinoic acid binding protein [11–16]. Bacterial forms have also been reported [17]. The overall fold of lipocalins is very similar to the avidins: the ligand binding pocket is located inside a b-barrel structure formed by eight antiparallel b-strands. In retinoid-binding lipocalins the hydrophobic end of the retinoid ligand is immersed into the protein cavity and the solvent-accessible polar headgroup emerges at the protein surface [5], in contrast to the intracellular retinoid binding proteins. The iLBPs developed from a single ancestral gene that connects them to the above-described extracellular lipid binding proteins [9]. iLBPs appear to be restricted to the animal kingdom, where they occur in various kinds of invertebrates as well as all vertebrate classes [18, 19]. Their structure consists of 10 antiparallel b-strands forming a b-barrel structure that encloses the lipid binding cavity [20]. 5.2.2

The Intracellular Lipid Binding Proteins

The iLBP genes in vertebrates consist of four exons and three introns with a comparable intron phase [2], even though the intron length can vary. This common gene structure suggests that present day iLBPs developed via gene duplication and branching from a single precursor gene. In the human genome, most iLBPs are distributed on different chromosomes [21]. Because of their different tissue distributions and lipid binding preferences, the various iLBP types were named according to either the tissue of first isolation or the primary ligand type. In total, 16 vertebrate iLBPs have been discovered up to now. They consist of nine FABP forms (adipocyte- (A-), brain- (B-), epidermal- (E-), heart- (H-), intestinal- (I-), liver- (L-), basic liver- (Lb-), myelin- (M-), and testis-type (T-) FABP), four cellular retinol binding proteins (CRBP-I through CRBP-IV), two cellular retinoic acid binding proteins (CRABP-I and CRABP-II), and an intestinal bile acid binding protein (I-BABP). (Note: The various iLBPs have also been designated in the literature as ALBP or aP2 (for A-FABP), BLBP (for B-FABP), KLBP or Mal-1 (for E-FABP), Z protein (for L-FABP), myelin P2 (for M-FABP), and TLBP (for TFABP). Also, I-BABP is usually referred to in the literature as “ileal lipid binding protein” (ILBP).) Amino acid sequence comparisons have shown that orthologous iLBPs (i.e. the same type from different organisms) display a much higher homology than paralogous iLBPs (i.e. different types from the same organism) [2]. Figure 5.1 shows a sequence comparison of the 14 human iLBPs presently known. The basic liver-type FABP (Lb-FABP), which is different from the mammalian L-FABP (approx. 40% sequence homology) [22], appears to have existed before the divergence of fish and tetrapods [23], but has not been detected in mammals to date; in fact, since no Lb-FABP gene sequence has been found in the human genome, the Lb-FABP gene may have been silenced and/or completely removed during the mammalian evolution [19]. A human T-FABP form has also not

5.2 The Evolution of Lipid Binding Proteins

Sequence comparison of the 14 presently known human iLBPs. Dashed lines separate different iLBP subfamilies. Residues with shaded background are identical in at least half of the protein sequences. The most important residues for ligand binding, as described in Section 5.3.2, are printed bold and in italics. The amino acid alignment was performed manually based on homologies in the primary, secondary, and tertiary structures. The numbering refers to the H-FABP

Fig. 5.1

sequence; the total number of residues is given at the end of each sequence. The sequences are taken from the SWISS-PROT database and have the following accession numbers: H-FABP (P05413), B-FABP (O15540), AFABP (P15090), M-FABP (P02689), E-FABP (Q01469), I-FABP (P12104), L-FABP (P07148), I-BABP (P51161), CRBP-I (P09455), CRBP-II (P50120), CRBP-III (P82980), CRBP-IV (Q96R05), CRABP-I (P29762), and CRABP-II (P29373).

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yet been reported. The 14 presently known human iLBP sequences exhibit about 20–75% sequence homology – with the CRABP sequences most strongly conserved. Orthologous iLBPs in mammals, on the other hand, show sequence homologies that generally range between 80% and 90%. iLBPs are also found in a variety of invertebrates, including roundworms (nematodes), flatworms (platyhelminthes), and insects (arthropods). The iLBP genes of invertebrates generally have only two introns [24]. Except for the tobacco hornworm (Manduca sexta L.) FABP, which is similar to the L-FABP [25], most invertebrate FABPs resemble the mammalian H-FABP form. In fact, about 18% of the total protein content in the flight muscle tissue of migratory locust (Locusta migratoria) is a homolog of H-FABP that apparently regulates the fatty acid metabolism as an energy source for sustained flight activity [26]. This seems to be also the case in vertebrates, as indicated by the unusually high cellular FABP levels in the flight muscle tissues of migratory birds [27, 28], which is dependent on a variety of factors such as the developmental stage of the animal, the migration season and physical activity. 5.2.3

The Phylogeny of iLBPs

Since the iLBPs of vertebrates and invertebrates show 25–47% sequence homologies as well as a highly conserved three-dimensional fold, these protein forms must have existed prior to the vertebrate/invertebrate split approximately 650 million years ago [18, 19, 29]. However, iLBPs are not found in yeast or plants, suggesting they evolved after the separation of the animal kingdom from plants and fungi about 1200–1000 million years ago. A phylogenetic tree of the 16 vertebrate iLBPs has been computed by Schaap and co-workers [19], as shown in Fig. 5.2. The various iLBP branches show different evolutionary rates (i.e. substitutions per amino acid residue per year) [19]. For example, the fastest amino acid substitution rate is displayed by I-BABP, and the slowest by CRABP-I. The I-BABP/LFABP group also has the largest evolutionary distance to the other iLBP subfamilies. The earliest iLBP gene duplication took place about 930 million years ago [19], along with the appearance of higher eukaryotes. It is possible that this development coincided with the demand for intracellular transport proteins that provide an efficient lipid transport between different compartments of an eukaryotic cell. In particular, the fast transport of fatty acids to mitochondria and other organelles, e.g. for immediate energy production, may have been essential for the further development of advanced eukaryotic life forms. The primordial iLBP gene possibly represented a universal binding protein for hydrophobic ligands, which over the years evolved into a multitude of different, better adapted amino acid sequences with specialized binding properties. Since most present-day iLBPs are able to bind fatty acids, it has been proposed that such binding was also a characteristic of the ancestral iLBP [19].

5.3 Structural Characteristics of iLBPs

Phylogenetic tree showing schematically the evolutionary development of the different iLBP types from vertebrates [19]. The gene duplication times are indicated at the branching points, wherever they could be estimated reliably. The tree has been rooted by

Fig. 5.2

including a lipocalin sequence (von Ebner’s gland protein) as an outside reference in the analysis. This figure was kindly provided by F. G. Schaap, G. J. van der Vusse, and J. F. C. Glatz, University of Maastricht, The Netherlands.

5.3

Structural Characteristics of iLBPs

High-resolution three-dimensional structures are now known for nearly all iLBPs. Originally, tertiary structure information was obtained mainly from X-ray structure analyses (Tab. 5.1). NMR studies first probed specific features of the iLBP structures that could be easily identified in the NMR spectra, either through isolated resonances or with the help of specific isotope labeling [61–69]. The development of stable magnets at higher fields (³ 500 MHz 1H Larmor frequency), of multidimensional heteronuclear NMR techniques, and of more advanced isotope-enrichment techniques for proteins allowed NMR spectroscopists to advance to sophisticated structure determi-

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5 Intracellular Lipid Binding Proteins: Evolution, Structure, and Ligand Binding Tab. 5.1 Crystal state structures of iLBPs (dashed lines separate different iLBP subfamilies).

ILBP

Source

Ligand

Resolution PDB ID (Å) code

CRABP-I

murine

CRABP-II

bovine human

trans-retinoic acid apo synthetic retinoid trans-retinoic acid synthetic retinoid synthetic retinoids

2.9 2.7 2.8 1.8 2.2 2.1 & 2.0

CRABP-II (R111M) human CRBP-I rat CRBP-II rat

apo trans-retinol apo trans-retinol CRBP-III human apo ––––––––––––––––––––––––––––––– L-FABP rat oleic acid ––––––––––––––––––––––––––––––– I-FABP rat heterogeneous palmitic acid apo apo oleic acid myristic acid I-FABP (R106Q) rat oleic acid I-FABP (V60N) rat apo A-FABP murine apo apo oleic acid stearic acid hexadecanesulfonic acid palmitic acid arachidonic acid ANS ––––––––––––––––––––––––––––––– A-FABP murine synthetic ligands (chem. modified) A-FABP murine apo (C1G/V32D/F57H) A-FABP murine synthetic ligand (C1G/V32D/F57H) (chem. modified) B-FABP human oleic acid docosahexaenoic acid E-FABP human heterogeneous H-FABP bovine heterogeneous human heterogeneous elaidic acid oleic acid stearic acid

References

1CBR 30 1CBI 31 2CBR 32 1CBS 30 30 1CBQ 2CBS & 32 3CBS 2.3 1XCA 33 2.1 1CRB 34 2.1 1OPA 35 1.9 1OPB 35 2.3 1GGL 36 –––––––––––––––––– 2.3 1LFO 37 –––––––––––––––––– 2.5 – 38 2.0 2IFB 39 1.96 1IFB 40 1.2 1IFC 41 1.75 – 42 1.5 1ICM 43 1.74 1ICN 43 2.1 1DC9 44 45 2.5 – 46 1.6 1LIB 46 1.6 1LID 46 1.6 1LIF 47 1.6 1LIC 47 1.6 1LIE 1ADL 48 1.6 2ANS 49 2.5 –––––––––––––––––– 2.4 & 2.4 1A18 & 50 1A2D 1.9 1AB0 51 2.7

1ACD

51

2.8 2.1 2.05 3.5 2.1 1.4 1.4 1.4

1FE3 1FDQ 1B56 – 2HMB 1HMR 1HMS 1HMT

52 52 53 54 55 56 56 56

5.3 Structural Characteristics of iLBPs Tab. 5.1 (continued)

ILBP

Source

Ligand

Resolution PDB ID (Å) code

M-FABP

bovine

FABP FABP

hornworm heterogeneous locust heterogeneous

References

heterogeneous 2.7 – 57 oleic acid 2.7 1PMP 34 ––––––––––––––––––––––––––––––––––––––––––––––––– Lb-FABP chicken heterogeneous 2.7 – 58 1.75 2.2

1MDC 1FTP

59 60

Tab. 5.2 Solution state structures of iLBPs (dashed lines separate different iLBP subfamilies).

iLBP

Source

CRABP-II CRABP-II (R111M) CRBP-I

human human rat

CRBP-II –––––––––– I-BABP

–––––––––– I-FABP

I-FABP (D17-SG) –––––––––– B-FABP E-FABP H-FABP

Ligand

PDB ID code

Reference

apo 1BLR 73 apo 1BM5 74 apo 1JBH 75 trans-retinol 1KGL 75 rat apo 1B4M 76 trans-retinol 1EII 77 ––––––––––––––––––––––––––––––––––––––– porcine apo 1EAL 78 glycocholic acid 1EIO 79 human taurocholic acid – 80 ––––––––––––––––––––––––––––––––––––––– rat palmitic acid 1URE 81 apo 1AEL 82 human heterogeneous 3IFB 83 rat heterogeneous 1A57 84 ––––––––––––––––––––––––––––––––––––––– human heterogeneous 1JJX 85 human stearic acid 1JJJ 86 bovine palmitic acid 1BWY 87 human heterogeneous 1G5W 88

nations approximately 10 years ago. Starting initially with the identification of secondary structure elements [70–72], complete tertiary structures of iLBPs were subsequently solved by high-resolution NMR spectroscopy as well (Tab. 5.2). 5.3.1

The Common Three-dimensional Fold

Despite the high degree of divergence of their amino acid sequences and binding properties, the overall protein fold has been conserved in all iLBP types. A representative example of the typical iLBP fold (i.e. H-FABP) is shown in Fig. 5.3. The

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5 Intracellular Lipid Binding Proteins: Evolution, Structure, and Ligand Binding Ribbon diagram representing the common iLBP fold. Ten antiparallel b-strands (A to J) form a b-barrel that is closed on one side by two short a-helices (aI and aII). An internal water-filled protein cavity defines the ligand binding site. The figure was produced with MOLSCRIPT [89] and Raster3D [90] using the atomic coordinates of human H-FABP.

Fig. 5.3

iLBPs consist of 10 antiparallel b-strands arranged in a + 1, + 1, + 1, . . . topology to form a b-sheet structure that wraps around to create a b-barrel fold [20]. Because of the clam-shell appearance, this fold has also been dubbed “b-clam” [38]. The bottom of the clam is sealed off by a number of hydrophobic side-chains clustered around the N-terminus, whereas the mouth of the clam is almost completely covered by a helix-turn-helix domain consisting of two short a-helices. There is a so-called “gap” in the b-sheet between b-strands D and E, where no hydrogenbonding network between the backbone atoms exists [34, 38, 46]. This gap produces an opening in the protein surface, but one that is not large enough to serve as a ligand entrance. Instead, a region located between the turns bC–bD and bE– bF as well as a-helix II has been postulated as the so-called “entry portal” for the various ligand molecules [39]. The interior of the protein structure contains a relatively large, water-filled cavity, where the respective ligand can bind – well shielded from the external solvent [39, 40]. Upon entering the binding pocket, the hydrophobic ligand displaces several, but not all of the internal waters, and occupies approximately a quarter to a half of the entire cavity space. The cavity is lined with both hydrophobic as well as hydrophilic side-chains, which interact with either the ligand or the internally bound water molecules to keep them in place. Hence, intricate networks of wellordered, hydrogen-bonded water molecules inside the protein cavity have been described for the holo forms of A-FABP, H-FABP, CRABP-II, and I-FABP [30, 41, 47, 56]. These internal water molecules apparently support the overall protein stability as well as the electrostatic interactions inside the binding pocket. Moreover, they may play a role during the ligand binding process, when they presumably enter or exit the protein cavity through a small opening in the gap between bstrands D and E [56, 91, 92]. The three-dimensional structures of iLBPs in the apo and holo form are generally highly comparable [30, 35, 41, 46, 75]. Only the solution state structures of IFABP [82] and CRBP-II [76] showed significantly different backbone conformations between the free and ligand-bound states: a-helix II is either partially or

5.3 Structural Characteristics of iLBPs

completely unfolded in the structures of the apo forms. However, this finding contradicts the X-ray data of I-FABP [41] and CRBP-II [35], where both apo and holo forms include a fully intact a-helix II. Such discrepancies between the three-dimensional structures in the crystal and in solution are rather unusual for iLBPs. In the case of apo I-FABP, missing resonance assignments and the consequent lack of NOE-derived distance constraints in that particular region might lead to the inference of a shortened a-helix II in solution. Similarly, the occurrence of multiple amino acid spin-systems in the portal region of apo CRBP-II could possibly mask the existence of a-helix II in solution. The solution state structures of CRBP-I in the ligand-free and retinol-bound forms [75], on the other hand, show no differences for a-helix II. Hence, the above-mentioned reports of a deformed ahelix II in solution should be viewed with caution, as missing NOEs do not necessarily imply a lack of structure. Instead, NOEs might be concealed by dynamic effects that cause, for example, line-broadening or heterogeneities of resonance signals in the NMR spectra. 5.3.2

The iLBP Subfamilies

Based on sequence homologies and binding properties, the mammalian iLBPs have been divided into four subfamilies [93]. Despite the common three-dimensional fold of the protein backbone, the iLBP subfamilies show characteristic differences in their ligand types and binding geometries. The structures of several protein:lipid complexes, representing all four subfamilies, are displayed in Fig. 5.4.

5.3.2.1 Subfamily I

Subfamily I contains all the retinoid binding iLBPs [5, 95], including four cellular retinol binding proteins (CRBP-I through CRBP-IV) as well as two cellular retinoic acid binding proteins (CRABP-I and CRABP-II). These intracellular retinoid binding proteins are specialized in binding vitamin A and its derivatives (i.e. retinol, retinal, and retinoic acid), which generally consist of three parts: (1) a b-ionone ring, (2) a polyisoprene chain, and (3) a polar headgroup in one of three possible oxidation states – carboxylate (retinoic acid), aldehyde (retinal), or alcohol (retinol). The CRBPs prefer retinol over retinal, whereas the CRABPs bind retinoic acid only. In the bound form (Fig. 5.4), the b-ionone ring is generally located near the ligand entrance below the helix-turn-helix domain, either in a cis (CRABP) or a trans (CRBP) conformation [30, 34, 35, 75, 77]. The polyisoprene chain extends through the protein cavity, usually in an all-trans conformation. The functional headgroup is always immersed into the binding pocket, where it interacts in a non-covalent fashion with different residues. In the CRABPs, the carboxylate group of the ligand is complexed via hydrogen bonds by the side-chains of Arg111, Arg132, and Tyr134, which correspond to the highly conserved residues Arg106, Arg126, and Tyr128 in the fatty acid binding proteins of iLBP subfamily

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5 Intracellular Lipid Binding Proteins: Evolution, Structure, and Ligand Binding

Graphic representation of several ligand complexes that are exemplary of the four mammalian iLBP subfamilies (I–IV). The protein backbone is shown as a tube (yellow) with the respective ligand displayed as spacefilling model inside the binding cavity. Subfamily I is represented by CRABP-I and CRBPI in complex with retinoic acid and retinol, respectively. Subfamily II consists of L-FABP (with two molecules of oleic acid) and I-BABP (with glycocholate). The I-FABP of subfamily III is shown in complex with one molecule of

Fig. 5.4

palmitic acid in a linear conformation. The subfamily IV proteins, such as E-FABP (fitted with palmitic acid), H-FABP (with oleic acid) and B-FABP (with DHA), generally bind one fatty acid molecule in a U-shaped conformation. The atomic coordinates were taken from the following entries in the PDB database: CRABP-I (1CBR), CRBP-I (1CRB), L-FABP (1LFO), I-BABP (1EIO), I-FABP (2IFB), HFABP (1HMS), E-FABP (1B56) und B-FABP (1FDQ). The figure panels were produced with GRASP [94].

5.3 Structural Characteristics of iLBPs

IV (Fig. 5.1). In the CRBPs, the retinol and retinal ligands are bound either to Gln108 (CRBP-I and CRBP-II) or His108 (CRBP-III and CRBP-IV) [36, 96], the residue corresponding to Arg111 in the CRABPs.

5.3.2.2 Subfamily II

Subfamily II consists of only two proteins, the intestinal bile acid binding protein (I-BABP) and the liver-type FABP (L-FABP). (Note: Lb-FABP might also belong to this iLBP subfamily, but has not been detected in mammals to date. Although its overall fold is similar to L-FABP, its ligand binding arrangement appears to be quite different. Chicken Lb-FABP for example binds only a single fatty acid, which adopts an U-shaped conformation in the central portion of the protein cavity [58].) These proteins interact with a wide selection of relatively bulky ligands in addition to fatty acids. I-BABP is the only iLBP that binds bile acids (both conjugated and unconjugated) [64], while L-FABP exhibits the largest variety of potential ligands, including fatty acids, acyl-CoA esters, eicosanoids, bile acids, cholesterol, lysophosphatidylcholine, bilirubin, and heme [97]. Furthermore, L-FABP is the only FABP that binds two long-chain fatty acids at the same time [98, 99]. The iLBPs of this subfamily lack several residues in the b-strands G and H because of deletions in their amino acid sequences [64], thus creating an additional opening in the protein surface that appears to serve as a second ligand entry portal. Moreover, the considerably higher backbone flexibility of I-BABP and L-FABP compared with other iLBPs [78] (see also Section 5.3.3), possibly accounts for their ability to bind more bulky groups such as the steroid moieties of bile acids or the porphyrin ring of hemes. In the L-FABP complex (Fig. 5.4) [37], the presumably high-affinity fatty acid ligand rests in a bent conformation at the bottom of the protein cavity, where the carboxylate group interacts via hydrogen bonds with the side-chains of Ser39, Arg122, and Ser124, which corresponds to Thr40, Arg126, and Tyr128 in the HFABP of subfamily IV (Fig. 5.1). The second, low-affinity fatty acid ligand stretches from the center of the first ligand molecule to the portal region, where its solvent-accessible carboxylate group emerges at the protein surface. Even though the corresponding residues Ser38 (or in some cases Thr38), Arg121, and Ser123 also exist in the I-BABPs, the fatty acid binding affinity of that iLBP type is rather low, presumably because of differences in the hydrophobic protein:lipid contacts inside the binding site. Instead, I-BABP preferentially binds bile acids and their derivatives with the steroid moiety immersed into the protein cavity and the solvent-accessible carboxylate tail at the protein surface (Fig. 5.4) [79]. In porcine I-BABP, the side-chains of the hydrophilic residues Tyr97, His99 (Gln99 in human IBABP), Glu110, and Arg121 in b-strands H, I, and J show contacts with the hydroxy groups on the polar face of the glycocholate ligand, but the complex formation appears to be defined primarily by van der Waals interactions between the non-polar face of the bile acid and the mostly hydrophobic residues in b-strands C, D, and E that line the binding cavity. In the case of human I-BABP, however, a slightly different orientation of the bound bile acid has been reported [80].

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5.3.2.3 Subfamily III

The sole member of subfamily III, the intestinal-type FABP (I-FABP), preferably binds a single long-chain fatty acid molecule. The ligand adopts a slightly curved conformation (Fig. 5.4) [39, 81], in a reversed orientation compared with the lowaffinity ligand in L-FABP. Its solvent-inaccessible carboxylate group is buried deep inside the protein cavity, where it forms hydrogen bonds with Arg106, which corresponds to Arg111 in the CRABPs, Gln108/His108 in the CRBPs, and Arg106 in the iLBPs of subfamily IV (Fig. 5.1). In addition, Trp82 and Gln115 also interact with the fatty acid via H-bonds. The convex face of the fatty acid ligand is lined by mostly hydrophobic side-chains, while ordered water molecules are clustered at the concave face as a sort of hydration shell – within van der Waals distances to the hydrocarbon chain of the lipid [42]. Finally, the hydrophobic tail of the fatty acid ligand interacts with the side-chain ring of Phe55, which also exists as Phe57 in most CRBPs as well as subfamily IV iLBPs and has been proposed to serve as an adjustable portal lid (see Section 5.3.4).

5.3.2.4 Subfamily IV

Subfamily IV contains the most diverse assortment of iLBPs, including the A-, B-, E-, H-, M-, and presumably T-FABPs. For T-FABP, however, neither the endogenous ligand nor the three-dimensional structure is known. All other members of this subfamily preferentially bind a single long-chain fatty acid in a U-shaped conformation [34, 46, 52, 53, 56, 87] and exhibit a short 310-helical loop as an additional secondary structure element at the N-terminus (Fig. 5.4). In the case of BFABP, however, it was shown that “very long-chain” polyunsaturated fatty acid ligands (> C20) adopt a helical conformation inside the binding pocket [52]. In addition, A-, E-, and M-FABP have been reported to bind retinoids or eicosanoids in vitro, and the same binding properties are predicted for T-FABP [19]. Finally, while most iLBPs contain maximally one or two cysteine residues without the formation of disulfide bridges, the E-FABP and T-FABP types display 5–6 and 4 conserved cysteines, respectively. (Note: Even though the mouse and rat B-FABPs both contain five conserved cysteine residues, only two of these are found in the chicken and human B-FABP forms and none in bovine B-FABP.) The only disulfide bridge established to date in the iLBP family has been found in E-FABP between Cys120 and Cys127 [53]. (Note: The same two cysteine residues also exist in MFABP (Cys117 and Cys124). Surprisingly though, despite biochemical evidence for a disulfide linkage, the S-S distance in the X-ray structure appears to be too large [34].) The positions of the cysteine residues in T-FABP suggest that an S-S bond is highly unlikely. In all structures of this subfamily, the carboxylate group of the fatty acid forms a network of hydrogen bonds with the highly conserved residues Arg106, Arg126, and Tyr128 (Fig. 5.1), while the hydrophobic tail interacts with Phe57 at the entry portal. The fatty acid chain is U-shaped, thus creating intramolecular non-polar contacts that are energetically favorable for binding. The upper side of the bent fatty acid ligand, facing a-helix I, is surrounded by mostly hydrophobic side-

5.3 Structural Characteristics of iLBPs

chains, while the lower side shows van der Waals interactions with a cluster of bound, well-ordered water molecules located in the central portion of the protein cavity [100]. This close lipid : water arrangement inside the cavity, with the internal water acting as a sort of hydration shell, is similar to that described above for IFABP, even though the conformation of the fatty acid ligand is entirely different. 5.3.3

Dynamic Properties of iLBPs

In spite of their common three-dimensional fold, iLBPs display distinct dynamic properties in solution, which have been revealed by recent studies employing NMR techniques, Fourier transform infrared spectroscopy (FT-IR), and molecular dynamics (MD) calculations. NMR relaxation and exchange data have provided important insights into the backbone dynamics of various iLBP molecules. Using 15N-, 13C- and 2H-NMR techniques, the relative mobilities of the protein (backbone as well as side-chains) and the ligand have been investigated [75–77, 86, 101–105]. The backbone dynamics are influenced by the presence of the ligand, which stabilizes the overall protein structure. In fact, multiple stable conformational states in the portal region have been observed for H-FABP in the presence of a mixture of endogenous fatty acid ligands [88]. Moreover, the backbone relaxation and hydrogen/deuterium exchange data strongly suggest that ligand binding properties such as affinity and specificity are related to the protein dynamics [78, 86]. Hence, the differences in the binding properties of certain iLBP types correlate with variations in their conformational stabilities, as indicated also by biochemical fluorescence-based denaturation studies [72, 106, 107]. Hydrogen/deuterium exchange data from FT-IR [108] and NMR experiments [70, 78, 86] for example clearly show that L-FABP and I-BABP, which can bind a variety of bulky ligand molecules, display faster exchange of backbone amide protons in the b-sheet structure than I-FABP, E-FABP, or H-FABP. The latter three proteins apparently developed a specialization for fatty acid binding that is possibly achieved by a combination of structural and dynamic aspects [68]. Interestingly, even evolutionary closely related iLBPs, such as E-FABP and H-FABP, exhibit marked differences in the exchange behaviour of their backbone amide protons [86]. More recently, histidine titration studies have provided strong evidence that the structural arrangement of water molecules inside the iLBP cavity has a major influence on the dynamic properties of these proteins [68]. The protein dynamics of some iLBPs have also been simulated by MD calculations [91, 92, 104, 109–118]. The earliest studies focused on comparisons of the apo and holo structures and confirmed the increased backbone stability in the presence of a bound ligand molecule. More recently, with the development of greater computing capabilities, it has become feasible to include a larger number of water molecules in the MD simulations. Consequently, the electrostatics, movement, residence times, and exchange pathways of water inside the fully-solvated protein cavity have been calculated for several iLBPs. In particular, the water flux through the gap between b-strands D and E was confirmed by MD calculations of

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I-FABP [91, 92]. Moreover, a “structural” water molecule (WAT135), which interconnects the backbone atoms of three residues located in the turn bD–bE as well as b-strand F, was found to be highly immobile not only in MD simulations of IFABP [92, 116]. NMR studies of H-FABP and I-FABP, employing heteronuclear cross-relaxation experiments [116, 119] or magnetic relaxation dispersion [120] to determine the residence times of long-lived water molecules within the picosecond-to-nanosecond timeframe, confirmed the presence of such a slow-exchanging water molecule at the position of WAT135 as an additional factor in the backbone stability. 5.3.4

Mutagenesis Studies

Arg126 (H-FABP numbering) is highly conserved in all iLBPs that bind ligands with a carboxylic acid headgroup. Site-directed mutagenesis has shown that substitution of Arg126 with Gln in A-FABP leads to a significant reduction in the binding affinity for fatty acids [121]. A comparable result was observed for a mutation of Arg106 in I-FABP [43]. Similarly, mutations of R111 and R131 in CRABP-I or R111 and R132 in CRABP-II decreased the respective binding affinities for retinoic acid [122, 123], whereas the replacement of Gln108 by Arg enabled CRBP-II to bind fatty acids [124]. Another important residue for ligand binding is Phe57 (H-FABP numbering), which is usually either conserved or replaced by other hydrophobic residues in most iLBPs. The phenyl ring of this residue has been postulated to act as a lid in the portal region of I-FABP [43], possibly controlling ligand entry and/or exit; however, the position of the phenylalanine side-chain has been found to vary considerably in other iLBPs [43, 46, 56]. Even though the mutation of this residue showed no notable effects on ligand binding in H-FABP and B-FABP [125, 126], it appears to influence both ligand binding as well as protein stability in A-FABP [127]. Single and multiple mutations in the portal region of A-FABP have furthermore hinted at changes in the association and dissociation rates of the ligand molecules without marked differences in the binding affinities [128]. Fluorescence studies with anthroyloxy-labeled fatty acids (see also Chapter 6) have suggested that positively-charged lysine residues on the protein surface could play a role in the ligand binding process [129, 130]. Substitutions of two lysine residues, Lys21 and Lys58, in the portal regions of A-FABP and H-FABP led to a significant decrease in the rates of transfer of fatty acids from the protein to model membranes. Hence, these side-chains have been postulated to govern the electrostatics that cause the proteins to interact either with the ionized fatty acid ligand directly or with the negatively-charged phospholipid membranes that hold the fatty acid. (Note: Similar data, suggesting an electrostatic interaction between the fatty acid and an arginine residue on the protein surface, has been obtained with a Arg56 mutant of I-FABP [131].) Based on a linear increase in the transfer rates of ligand with the concentration of phospholipid vesicles, it was proposed that AFABP, B-FABP, H-FABP, I-FABP, and CRBP-I release fatty acids by a transient

5.4 Ligand Binding Assays

collision-based mechanism, whereas an aqueous diffusion-controlled mechanism without direct membrane contact has been suggested for L-FABP and CRBP-II [132–135]. The helix-turn-helix domain closes the b-clam structure nearly completely. It is, however, not absolutely essential for the structural integrity of the protein. A genetically produced “helix-less” I-FABP mutant still shows the typical clam topology, even though it is less stable in guanidine hydrochloride [136]. With the a-helices missing and the opening to the binding pocket considerably enlarged [84], the association rate for oleic acid is still comparable to that of the wild-type protein. However, the dissociation rate of the helix-less mutant is increased by more than an order of magnitude, thus indicating that the helix-turn-helix domain might in fact play a role in the regulation of ligand binding [137].

5.4

Ligand Binding Assays

Significant differences in ligand binding, conformational stability, and surface properties between the various iLBPs suggest that these proteins carry out distinct functions in the cells and tissues where they occur. The factors determining the ligand binding affinities have been studied by several biochemical and biophysical methods. Some results of the ligand binding assays employed in recent years are discussed below with an emphasis on fatty acid:protein interactions, since most of the binding studies performed to date have focused on the different FABP types. 5.4.1

Microcalorimetry

The most direct approach to obtain thermodynamic data such as ligand binding constants is “isothermal titration calorimetry” (ITC), which measures the heat of interaction between protein and ligand in a series of small titration steps. This method, first applied to FABPs by Miller and Cistola [138], was used to distinguish between endo- and exothermic binding reactions and to identify affinity classes of different order of magnitude, such as the high- and low-affinity binding sites of L-FABP. However, rather few ITC results have been reported for other iLBPs [52, 139, 140], mainly because of the experimental limitation that most iLBP ligands exhibit very low solubilities in aqueous solution. The Kd values determined by ITC for mono- and polyunsaturated fatty acids are in the nanomolar range, in-between the values estimated from the Lipidex and the ADIFAB results (Tab. 5.3). Moreover, the thermodynamic data obtained for fatty acid binding to different FABP types generally show a primarily enthalpic contribution to the free energy of binding (DG8 *–30 to –40 kcal mol–1), comparable to the ADIFAB results described below.

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5 Intracellular Lipid Binding Proteins: Evolution, Structure, and Ligand Binding Tab. 5.3 Oleic acid binding affinities determined for different iLBP types using the ITC, Lipidex,

and ADIFAB methods. iLBP type

ITC

Lipidex a) Kd (nM)

ADIFAB b)

A-FABP B-FABP E-FABP H-FABP I-FABP L-FABP g) M-FABP CRABP-I i) CRBP-II i)

n. d. c) 46.7 ± 1.4 d) n.d. 470 ± 80 e) 250 ± 150 f) 6 ± 4 h) n. d. n. d. n. d.

1560 ± 250 380 ± 20 820 ± 20 440 ± 50 570 ± 40 890 ± 30 310 ± 30 n. d. n. d.

57 7 n. d. 4 35 15 5 360 2200

a) Mean values ± SD obtained for human FABPs at 25 8C (from Ref. [107]). b) Values obtained for human FABPs at 37 8C (from Ref. [141]). c) Not determined. d) Mean value ± SD obtained for human B-FABP at 30 8C (from Ref. [53]). e) Mean value ± SD obtained for locust H-FABP at 25 8C (from Ref. [139]). f) Mean value ± SD obtained for rat I-FABP at 37 8C (from Ref. [138]). g) Values refer to high-affinity binding site. h) Mean value ± SD obtained for human L-FABP at 37 8C (from Ref. [140]). i) Protein type from mouse.

5.4.2

The Lipidex Assay

The first binding assay for FABPs was based on the “Lipidex procedure” developed by Glatz and Veerkamp [142], which uses a lipophilic Sephadex material (hydroxyalkoxypropyl dextran) to sequester small hydrophobic molecules from aqueous solution. This material can therefore be employed to delipidate iLBPs after protein purification, in order to subsequently study protein:lipid complex formation with 14C-labeled fatty acids. The dissociation constants (Kd) thus obtained for various fatty acid:FABP complexes are in the micromolar range [99, 107, 143]. Since oleic acid is one of the most prevalent natural fatty acids, it is interesting to compare its binding affinities to different FABP types. The strongest binding of oleic acid is displayed by three FABPs of subfamily IV, B-FABP, H-FABP, and MFABP. The order of oleic acid binding affinities for all FABPs studied is: BFABP & H-FABP & M-FABP > I-FABP > E-FABP & L-FABP > A-FABP [107]. Moreover, orthologous H-FABPs bind oleic acid with Kd values between 0.2 and 0.45 lM [143], whereas the dissociation constants of the human paralogs generally range from 0.3 to 2.0 lM [107]. The Lipidex assay yields dissociation constants that are usually 1–2 orders of magnitude higher than the corresponding results derived from other methods (Tab. 5.3). Although relative binding affinities of different FABPs can be studied by this approach, the ADIFAB assay described below probably provides the more accurate Kd values.

5.4 Ligand Binding Assays

5.4.3

Fluorescence-based Binding Assays

A spectrophotometric analysis of ligand binding was easily established for the retinoid-binding iLBPs, since vitamin A and its derivatives are fluorophores. The Kd values for retinol and retinal binding to CRBPs were found to be in the nanomolar range. More precisely, the affinities for all-trans retinol are: CRBP-I (<10 nM), CRBP-II (*10 nM), CRBP-III (*60 nM), and CRBP-IV (109 nM) [36, 96]. Interestingly, retinoic acid binding to CRABPs is even stronger (Kd = 0.06 and 0.13 nM for CRABP-I and CRABP-II, respectively) [144]. A similar approach was chosen to analyze the binding affinities of bile acids and their derivatives to I-BABP. Since I-BABP contains only a single Trp residue located in the ligand binding site [79], tryptophan fluorescence measurements have been employed to demonstrate binding preferences in the order taurine-conjugated > glycine-conjugated > unconjugated bile acids [107]. At the same time, the positions of the hydroxy groups on the steroid moiety seem to define the bile acid affinities to I-BABP in the order deoxycholic acid (3a-,12a-dihydroxy) > cholic acid (3a-,7a-,12a-trihydroxy) > chenodeoxycholic acid (3a-,7a-dihydroxy). Since typical dietary fatty acids are not natural fluorophores, and since most iLBPs generally contain more than one Trp residue in the binding cavity, other approaches have been developed to study fatty acid binding to FABPs. Fluorescentlylabeled fatty acid derivatives and analogs (carrying for example anthroyloxy, dansyl, or NBD fluorophores) have been employed in various binding studies (see Section 5.3.4), but are expected to have different thermodynamic and kinetic properties because of their modified chemical structures. Hence, caution must be exercised when extrapolating such data to the physiological fatty acids. Finally, since several FABP types are able to bind ANS, an “ANS competition assay” that monitors fatty acid binding to I-FABP has been introduced [145, 146]. This method produced binding constants and other thermodynamic data comparable to the results obtained with the ADIFAB assay described in the following section [147]. 5.4.4

The ADIFAB Assay

The ADIFAB assay makes use of a chemically modified I-FABP. A fluorescent acrylodan molecule, which is covalently attached to the Lys27 side-chain of rat IFABP, is thereby employed to monitor any fluorescence-induced changes due to ligand binding. One major advantage of this procedure is that binding studies can be implemented under true equilibrium conditions. Moreover, in addition to obtaining binding constants, this method provides both kinetic and thermodynamic data such as the rate of ligand association (kon) and dissociation (koff) or changes in the heat capacity (DCP), enthalpy (DH8), entropy (TDS8) and free energy (DG8) of binding [131, 148–151].

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5.4.4.1 Thermodynamic Analysis

Analogous to the Lipidex assay data, B-FABP, H-FABP, and M-FABP showed the lowest dissociation constants for most fatty acids (Tab. 5.3) [141, 152, 153], with Kd values for oleic acid between 4 and 7 nM. No FABP type displayed a pronounced preference for any particular fatty acid, with the order of fatty acid binding affinities determined as B-FABP & H-FABP & M-FABP > L-FABP > I-FABP  CRABP-I  CRBP-II. In comparing fatty acids of identical chain length, the binding affinity decreases exponentially with an increasing number of double bonds. On the basis of the general trends of binding as measured by ADIFAB, Richieri and co-workers [147, 152] have postulated the so-called “solubility hypothesis”. According to this hypothesis, the affinity of a fatty acid for any FABP type is dependent on its solubility in the aqueous solvent, and the FABP cavity is considered as an organic, non-polar solvent environment. Such a mechanism would suggest mostly entropic contributions to the free energy of binding. Although the results obtained for I-FABP agree fairly well with this idea, the data for other FABP types show an additional release of free energy upon binding that deviates from the predictions of the solubility hypothesis [141]. In fact, for most FABP types the total free energy of binding (DG8 usually *–10 kcal mol–1) is largely enthalpic in nature and the fatty acid solubility is reflected as only a small entropic contribution (except for fatty acid binding to CRABP-I where the energetic distribution is the reverse) [141, 148]. Clearly, the distinct molecular architectures of the various FABP types render the ligand:protein interactions strongly dependent on the arrangement of both the protein side-chains as well as the water molecules present inside the lipid binding cavity [68]. Numerous site-specific variants of A-FABP and I-FABP at locations in the fatty acid binding site [151, 154] showed Kd values that ranged from about 200-fold larger to 30-fold smaller compared to the wild-type proteins, even though the changes in the enthalpy and entropy of binding were usually compensatory. Hence, the effects of amino acid substitutions on ligand binding cannot always be explained on the basis of the binding affinity alone; rather, understanding these effects may require additional knowledge of the thermodynamic contributions.

5.4.4.2 Kinetic Analysis

Rate constants for the interaction of different FABP types with fatty acids have been determined as a function of temperature by stopped-flow fluorometric measurements with ADIFAB [149]. In all cases, the fatty acid:FABP equilibrium was achieved within 2 s at 378C or within 20 s at 10 8C. For each fatty acid the off-rate constants (koff) varied about 10-fold among the different FABP types (*0.25– 5.0 M–1 s–1), whereas the kon values generally fluctuated less than 2-fold (*5– 10 ´ 107 M–1 s–1). H-FABP consistently showed the lowest koff rates of different iLBP types [149], which corresponds with its higher binding affinities. The order of koff values for oleic acid binding, for example, has been determined as HFABP < L-FABP (high-affinity site) < I-FABP < A-FABP. In addition, a series of IFABP mutants with substitutions in the ligand binding site, in the gap between

5.5 Concluding Remarks

b-strands D and E, or in the portal region displayed either slower or faster koff rates compared to the wild-type, but the kon values were never significantly higher than for the native protein [131]. A similar result had also been obtained with the helix-less I-FABP mutant [137] (see Section 5.3.4), which suggests that ligand binding is not so much determined by certain attractive forces at the protein surface, but rather by the protein:lipid interactions inside the binding pocket. 5.4.5

Lipid Binding Preferences

Although there are some discrepancies in the quantitative results obtained with the different lipid binding assays, certain binding preferences are commonly observed (compare, for example, references [107, 141, 152, 153]). The iLBP subfamily IV members B-, H-, and M-FABP generally exhibit the strongest fatty acid binding. Of the remaining FABPs, I-FABP prefers saturated over polyunsaturated fatty acids, possibly because of the linear binding conformation, while the preference for saturated fatty acids is less pronounced for A-, E-, and L-FABP. The latter three proteins show fatty acid binding affinities in the order L-FABP (high-affinity site) & E-FABP > A-FABP. Nevertheless, the Kd values of different FABPs for a specific fatty acid vary usually by no more than one order of magnitude. Hence the question arises as to why so many FABP/iLBP types have evolved. CRABP-I and CRABP-II, for example, are expressed in different cell types, even though their primary sequences are very similar (74% identity) and their binding affinities for retinoic acid differ only 2-fold [144]. Such intriguing questions remain to be answered.

5.5

Concluding Remarks

In the last 20 years of their nearly 1 billion year history, remarkable advances have been made in understanding the structural biology of iLBPs. There are not many other protein families for which dozens of complete tertiary structures are known. Nevertheless, although their structures and their in vitro binding properties are well described, the functional aspects of these proteins are still under investigation and debated. The tissue-specific distributions of iLBPs as well as their particular ligand binding preferences appear to imply distinct functional roles. Yet, even genetic experiments with different iLBP knockout mice (see Chapters 19 and 20) have not revealed essential functions, but rather showed the animals to be viable without any particular iLBP [5, 155]. One final interesting note about these proteins: since the b-barrel structure is consistently preserved among the family members, and since most members bind fatty acids, it might be assumed that nature has evolved this particular structural motif explicitly for binding of fatty acids. However, there are other fatty acid binding proteins for which high-resolution structures of the ligand complex exist, such

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as human serum albumin, maize non-specific lipid transfer protein, and peroxisome proliferator activated receptor d (PPARd), that all show a predominantly helical fold [156].

5.6

References 1

2 3 4

5 6 7 8 9 10

11

12

13

14

15 16

17

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21

22

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24

25

26

27

28

29

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6

Fatty Acid Binding Proteins and Fatty Acid Transport Judith Storch and Lindsay McDermott

6.1

Introduction

Long-chain fatty acids (FA) are required by cells as membrane phospholipid constituents, metabolic substrates, precursors for signaling molecules, and mediators of gene expression. They are in constant flux and need to enter and leave cells rapidly and, presumably, in a regulated manner. The relatively low aqueous solubility of fatty acids would strongly suggest that specific and efficient mechanisms must exist for their intracellular transport. High levels of fatty acid binding proteins (FABPs) are found within cells and although it has been shown that these proteins non-covalently bind fatty acids with high affinity, their true in vivo functions have remained elusive. This chapter focuses on recent findings assessing the transport function of FABPs, and on data supporting putative mechanisms by which FABPs may be involved in cellular FA uptake, efflux, and intracellular transport.

6.2

Equilibrium Binding of Fatty Acids to FABPs

There are 12 members of the mammalian FABP family, each with specific tissue expression and, with the exception of the retinol and retinoic acid binding proteins, each is named after the first tissue of isolation (Tab. 6.1). In the tissues where they are found, FABPs are typically expressed abundantly, at levels of 1–3% cytosolic protein [1] and, as their name suggests, are capable of non-covalently binding long-chain fatty acids with high affinity and a 1:1 molar stoichiometry. Ileal lipid binding protein (I-LBP) and liver fatty acid binding protein (L-FABP) prove exceptions to this rule: both can bind more bulky, hydrophobic ligands such as lysophospholipids, bile acids, eicosanoids, and some drugs [2, 3], I-LBP does not bind fatty acids [4], and L-FABP is capable of binding two fatty acids simultaneously [5]. A recent NMR spectroscopic study showed two distinct binding environments for these FA, and suggested that binding of the first fatty acid precedes and may facilitate binding of the second [6].

120

6 Fatty Acid Binding Proteins and Fatty Acid Transport Tab. 6.1 Members of the family of mammalian intracellular fatty acid-binding proteins.

Name

Occurrence

Reference

E-FABP (K-FABP)

Epidermis, adipose, mammary tissue, tongue epithelia, testis Heart muscle, cardiac and skeletal muscle, brain, mammary gland, kidney, adrenals, ovaries, testis Brain, central nervous system Peripheral nervous system Adipose, macrophages Small intestine Small intestine (distal) Liver, small intestine Brain, skin, testis Epidermis, adrenal Liver, kidney, testis, lung Small intestine

91

H-FABP B-FABP M-FABP A-FABP I-FABP I-LBP L-FABP CRABP-I CRABP-II CRBP-I CRBP-II

92 93 94 86, 95 96 97 98 99 100 99 101

E-FABP, epidermal FABP; K-FABP, keratinocyte FABP; H-FABP, heart FABP; B-FABP, brain FABP; M-FABP, myelin FABP; A-FABP, adipocyte FABP; I-FABP, intestinal FABP; I-LBP, ileal lipid binding protein; L-FABP, liver FABP; CRABP, cellular retinoic acid binding protein; CRBP, cellular retinal binding protein.

Equilibrium binding studies have been used in an attempt to elucidate the functional characteristics of each FABP type. Most recently the ligand binding specificity of eight human FABPs (heart, liver, intestine, adipocyte, myelin, epidermal, brain fatty acid binding protein, and ileal lipid binding protein) were directly compared by Zimmerman et al. [4] using the Lipidex assay. By determining the equilibrium distribution of FAs between the resin and FABP, binding affinities were measured and, with the exception of I-LBP, ranged from 0.2 to 4.0 lM. The results obtained showed that the proteins have a lower affinity for palmitic acid than for oleic and arachidonic acids. Contrary to these findings and using the ADIFAB (acrylodated intestinal fatty acid binding protein) method, Richieri and colleagues showed that human FABPs from brain, heart, intestine, liver, and myelin pertained little or no selectivity for a particular FA, and obtained Kd values ranging from 2 to 400 nM [7]. ADIFAB consists of intestinal fatty acid binding protein covalently modified with the fluorescent acrylodan group, which exhibits a marked red-shift in fluorescence emission maximum upon fatty acid binding, enabling unbound concentrations of FAs to be accurately measured [8]. A further study using isothermal titration calorimetry (ITC) to determine the binding of FAs to human L-FABP yielded a stearate Kd value approximately 100 times larger (weaker binding) than that determined by Richieri et al. [7], and concluded, again in contrast to Richieri et al. that L-FABP preferentially binds unsaturated relative to saturated FAs [9]. It is possible, however, that the poor solubility of stearate, as well as the high fatty acid concentrations necessary for the ITC injection method, resulted in a lower stearate concentration in the reaction vessel, thus producing lower values for heat change, and therefore uncertain Kd values.

6.2 Equilibrium Binding of Fatty Acids to FABPs The three-dimensional structure of a representative cytoplasmic fatty acid binding protein. The protein comprises a flattened bbarrel capped by two short a-helices. The helices and closely positioned b-turns are believed to behave as a portal for ligand entry and exit [12, 13].

Fig. 6.1

In spite of these discrepancies over absolute Kd values, it is clear from these and earlier in vitro binding experiments that FABPs bind long-chain saturated and unsaturated FA. Indeed, NMR and X-ray crystallographic structures of holo FABPs reveal the position of the bound FA within the individual protein structures. In some structures, including intestinal FABP (I-FABP) and adipocyte FABP (A-FABP), the fatty acid adopts a bent conformation [5, 10], while in others such as heart FABP (H-FABP), it adopts a U-shaped conformation [11, 12]. Notwithstanding a wide variance in primary sequence, all members of the FABP family consist of a b-barrel structure capped by two a-helices, the latter believed to behave as a portal for ligand entry and release (Fig. 6.1) [13]. This hypothesis was supported recently by fluorescence-based experiments comparing AFABP and a triple mutant (V32G, F57G, K58G), designed to enlarge the putative portal opening by reducing the size of portal amino acids [14]. By comparing analinonaphthalene sulfonic acid (ANS) and oleate binding affinities and ANS binding rates, it was found that enlargement of the A-FABP portal region increased ligand accessibility into the cavity with only modest effects on ligand binding affinity, suggesting that dynamic fluctuations in this region regulate cavity access. Indeed, the solution structure of apo I-FABP was shown to exhibit a higher degree of mobility in this portal area in comparison with that of the ligand-bound IFABP complex relative to other domains in these proteins [15]. In an attempt to better understand the mechanism by which FAs bind to and dissociate from the binding cavities of FABPs, Richieri et al. constructed 31 single amino acid mutants within the portal region and in the region of the gap between the bD- and bE-strands of I-FABP, and determined binding affinities and rate constants for FA binding [16]. Together with experiments examining these parameters as a function of ionic strength, it was suggested that the FA initially binds through an electrostatic interaction to Arg56 on the surface of the protein, before inserting into the binding cavity, with a reversal of these steps for the dissociation reaction.

121

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The structural and biochemical studies of FA binding to FABPs do not directly demonstrate a ligand transport function for the proteins, however they nevertheless are consistent with their participation in such processes. It has been suggested that upon binding to FABP, the FAs traverse the aqueous cytoplasm in a more energetically favorable manner owing to increased aqueous solubility of FAs, in that lateral diffusion of FABP-bound FAs proceeds more rapidly than that of membrane-bound FAs [36].

6.3

In vitro Fatty Acid Transfer Properties of FABPs

The mechanisms by which members of the FABP family transfer FAs to membranes has been examined in a series of in vitro experiments. These studies examined the rate of anthroyloxy-labeled fatty acid (AOFA) movement from FABPs to model acceptor vesicles containing a non-exchangeable fluorescence quencher, using a fluorescence resonance energy transfer assay [17–19]. Transfer of fluorescent FA analogs from adipocyte, heart, intestinal, epidermal, brain, and myelin FABPs (A-, H-, I-, E-, B-, and M-FABPs), and of retinol from cellular retinal binding protein I (CRBP-I), to membrane targets appears to involve collision of the holo protein with both zwitterionic and anionic membranes, with more effective collisional interactions occurring with the latter [20–25]. FA transfer rates were directly proportional to acceptor membrane phospholipid concentration, and were modulated by changes in the acceptor vesicle charge, implying that FABP and membrane come into direct physical contact, with ligand transfer occurring during the collision. Further studies suggested that positively charged (lysine) residues on the protein surface are involved in this ligand transfer process and are likely to participate in the formation of effective FABP–membrane complexes that involve electrostatic interactions [21, 22, 26, 27]. Site-directed mutagenesis of A-FABP and H-FABP demonstrated that lysines in the helical cap domain are important for establishing these ionic interactions [21, 27]. In particular, lysine residues on aI, aII, and the bCD-turn of both proteins, and possibly the bA-strand of A-FABP but not H-FABP, were shown to be directly involved in these charge–charge interactions. Indeed, by removing I-FABP’s two a-helices, fatty acid transfer no longer occurred via a collisional mechanism [24]. Furthermore, creation of chimeric proteins of A- and H-FABPs revealed that the aII-helix is important in determining the absolute fatty acid transfer rates, while the aI-helix appears to be particularly important in regulating protein sensitivity to the negative charge of membranes [28]. The FABP a-helical domain, part of the same region believed to enable FA entry and exit, is therefore extremely important not only for the direct interaction with membrane phospholipids during ligand transfer to membranes, but also in regulating FA transfer rates. The direct interaction of the FABPs with membranes was further assessed using fluorescence-based assays and direct physical measurements and, as suggested by the transfer experiments, the a-helical domain and surface lysine resi-

6.3 In vitro Fatty Acid Transfer Properties of FABPs

dues therein proved particularly important for membrane association. Pre-incubation of anionic vesicles with I-FABP prevented the subsequent binding of the peripheral membrane protein cytochrome c, suggesting that the I-FABP was membrane-bound. In contrast, helix-less I-FABP demonstrated 80% less efficiency in preventing cytochrome c binding than intact I-FABP [24]. Further direct support for I-FABP–membrane interactions was provided by surface pressure measurements, Brewster angle microscopy and infrared reflection-absorption spectroscopy (IRRAS), which revealed that I-FABP interacted with 1,2-dimyristoyl phosphatidic acid monolayers to a stronger extent than its helix-less variant. IRRAS studies also showed I-FABP to induce a stronger conformational ordering of the lipid acyl chains than helix-less I-FABP [29]. The interaction of A-FABP with vesicles was also directly measured using FTIR spectroscopy, and it was found that A-FABP interacts much more strongly with acidic than zwitterionic membranes, and that neutralization of A-FABP positive surface charges by acetylation considerably weakens its interactions with negatively charged vesicles [30]. As for I-FABP, these data supported observations gained from the cytochrome c binding assay whereby A-FABP, but not acetylated A-FABP, was able to prevent subsequent cytochrome c binding to model anionic membranes [22]. Notably, in all theses studies, the degree of membrane interaction correlated directly with the rate of fatty acid transfer, indicating that FABP–membrane interactions are functionally related to their fatty acid transport properties. While the primary mode of FABP–membrane interaction appears to involve the establishment of ion pairs between positive charges on the protein surface and negative membrane charges, recent mutagenesis studies have suggested that hydrophobic interactions between I-FABP helix II residues and membrane phospholipids may also play a role in establishing the collisional interactions that promote FA transfer [31]. Indeed, it has been noted that those FABPs displaying a collisional mechanism for fatty acid transfer, possess a conserved, solvent-exposed, bulky hydrophobic side-chain located on aII, namely a phenylalanine, leucine, isoleucine, or methionine [32]. L-FABP on the other hand, displaying a diffusional mechanism for fatty acid transfer, possesses a Glu in this position. The aforementioned results for I-FABP thus suggest that exposed hydrophobic residues could be involved in membrane association, thereby rendering a subsequent interaction with a receptor protein more efficient and permitting the exchange of ligand without its entry into an aqueous phase. L-FABP and CRBP-II are the only members of the FABP family that were found not to transfer FA, or retinol in the case of CRBP-II, via a collisional mechanism [17, 20]. L-FABP transfers FAs to membranes almost 50-fold slower than members of the family exhibiting a collisional FA transfer mechanism [17]. Despite overall structural similarity, this particular FABP appears to transfer its ligand via aqueous diffusion, a mechanism that does not involve direct protein– membrane contact. The rate of FA transfer from L-FABP to membranes was modulated by neither the concentration of acceptor membranes nor their composition, however, changes in the ionic strength of the buffer directly affected the transfer rates indicating that the transfer rate is regulated by aqueous solubility of the fatty acid. CRBP-II also displayed a diffusional mechanism, and indeed proved

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ineffective in preventing cytochrome c from binding to phospholipid vesicles [20]. Recently, however, Davies et al. were able to observe L-FABP binding to anionic phospholipid vesicles using the fluorescent probe DAUDA, an undecanoate (11:0) derivative [33]. Upon binding to FABP, the fluorescence maximum of DAUDA becomes blue shifted and exhibits a substantial increase in intensity [34]. By mixing a pre-formed DAUDA:L-FABP complex with anionic vesicles, an immediate decrease in DAUDA fluorescence occurred indicating a release of DAUDA from the protein. As this did not occur with zwitterionic vesicles, the results suggested that interaction of L-FABP with the anionic membrane interface induces a rapid conformational change, resulting in a reduced affinity of DAUDA for the protein. The nature of this interaction was suggested to involve both electrostatic and nonpolar forces. It is important to note, however, that these L-FABP-membrane interactions were observed at very low ionic strength assay conditions, but were not found at physiologic ionic strength. From these experiments it was hypothesized that those FABPs exhibiting a collisional mechanism for FA transfer are most likely to be involved in the targeted transfer of FAs, whereby the proteins interact either with specific membrane lipids and/or membrane protein domains to transfer their fatty acid ligand [17, 20, 22, 25]. Recently it was also discovered that transfer of anthroyloxylated fatty acids (AOFAs) from phospholipid membranes to I-FABP (in the opposite direction from that initially examined) also occurs via a collisional mechanism [35]. It is possible then that FABPs may utilize membrane–protein interactions not only for the acquisition of ligand, but also for their delivery. By displaying a diffusional mechanism for fatty acid transfer whereby no direct protein–membrane interaction occurs, it is likely that L-FABP and CRBP-II may function in the capacity of cytosolic fatty acid or retinol reservoirs [17, 20]. Nonetheless, just as the apparent membrane interactions may be surrogates for FABP–protein interactions, the absence of apparent L-FABP– or CRBP-II–membrane interactions does not preclude protein–protein interactions; however the nature of any such interactions is likely to be different from that of the “collisional” FABPs. An additional approach used to examine the transport function of the FABPs is fluorescence recovery after photobleaching (FRAP) for solution conditions. In such studies, the effective diffusion (Deff) of the fluorescent probe N-(7-nitro-2,1,3benzoxadiazol-4-yl)-stearate (NBDS) is evaluated in individual cells or in so-called “model cytosol”. Using this approach, it has been found that the rate of NBDS movement correlates directly with the intracellular level of L-FABP in HepG2 cells and hepatocytes [36–38], the level of I-FABP in embryonic stem cells [39], the total FABP level (L-FABP + I-FABP) in rat enterocytes isolated from different intestinal segments [38], and the L-FABP concentration in solutions prepared to resemble cytosol with intracellular membranes [40]. The mechanism for these effects likely involves FABPs acting to limit fatty acid partitioning into “immobile” membranes, thereby increasing the rate of movement of the fatty acid [36]. Additionally, specific effects of FABPs in cells were also suggested by results in which permeabilized HepG2 cells were used to generate various cytosolic compositions by incubation with different protein-containing solutions; in these studies, albumin was found

6.4 Transfection Studies of FABP Function

to be only 4-fold more effective than equal concentrations of L-FABP in increasing the Deff of NBDS, despite the fact that its fatty acid binding capacity is of far greater magnitude greater than that of L-FABP, as is its FA binding affinity [40].

6.4

Transfection Studies of FABP Function

To assess FABP function within a more physiological milieu, FABP genes have been transfected into model cell cultures and subsequent changes in FA uptake and metabolism examined. A series of experiments examining L-cell fibroblasts or embryonic stem cells transfected with L-FABP and/or I-FABP appeared to suggest a role for L-FABP in cellular FA uptake [41–47]; when NBDS [48] or the fluorescent fatty acid analog cis-parinaric acid (cPnA) were added to cells expressing L-FABP and compared with control cells, or cells expressing I-FABP, a 2-fold increase in fluorescence intensity was observed. However, care should be taken over the interpretation of these results given that the quantum yield for cPnA binding to L-FABP differs by a similar degree to that of cPnA binding to I-FABP [49]. More recently, nevertheless, Wolfrum et al. used peroxisome proliferators to increase L-FABP levels in HepG2 cells, and antisense L-FABP to decrease L-FABP mRNA expression, and the net oleate uptake was shown to correlate directly with the L-FABP content of the cells [50]. When I-FABP was expressed at 2-fold higher concentrations in L-cells, it was found that cPnA uptake was lower relative to cells with lesser I-FABP levels [46]. However, the decreased level of I-FABP expression in differentiated relative to undifferentiated embryonic stem cells was also correlated with a decrease in fatty acid uptake [41]. Results of experiments involving overexpression of [Ala54] and [Thr54]IFABP (two I-FABP forms created by a single base pair alteration in the human IFABP gene) in Caco-2 intestinal cells must also be viewed carefully. A 2-fold increase in net fatty acid uptake was obtained from [Thr54]I-FABP-transfected cells and approximately 5-fold more triacylglycerol was secreted into the basolateral medium relative to [Ala54]I-FABP-transfected cells [51]. In differentiated cells of both lines, the endogenous levels of L-FABP were decreased relative to control cells, although L-FABP levels nevertheless remained 2- to 3-fold higher than levels of IFABP [51]. Moreover, it has subsequently been demonstrated that, in contrast to earlier indications, Caco-2 cells do in fact express I-FABP [52, 53]. A recent publication, nevertheless, reported that parent Caco-2 cells as well as mock-transfected cells fail to express detectable levels of I-FABP mRNA or protein at any stage of differentiation [54]; upon transfecting cells with I-FABP, radiolabeled oleic acid was used to monitor fatty acid metabolism and it was deduced that I-FABP expression in intestinal cells leads to reduced triacylglycerol secretion. Clearly the precise function of IFABP in the enterocyte remains uncertain. Given the additional variable of high levels of expression of L-FABP in this cell type as well, an understanding of the functional properties of enterocyte FABPs, in particular, remains a challenge.

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CHO cells transfected with A-FABP showed a 1.5- to 2-fold increase in the net uptake of oleate [55] and, interestingly, expression of equivalent levels of a mutated form of A-FABP with reduced fatty acid binding failed to produce a change in uptake. In contrast, expression of A-FABP in L6 myoblasts did not alter the net fatty acid uptake [56], although the degree of differentiation in these cells differed from that of control cells [52]. Recently, electroporation of A-FABP into 3T3-L1 pre-adipocytes was used as an alternative technique for modifying the level of cellular FABP directly. Incorporation of A-FABP was found to result in an increase in the initial rate of palmitate uptake relative to that of control cells [57], supporting an A-FABP-mediated transport function. An early report suggested an increase in net fatty acid uptake in cells overexpressing H-FABP [58], whereas overexpression of H-FABP in L6 myoblasts was reported not to alter uptake [56]. Expression of CRBP-I in Caco-2 cells increased net retinol uptake by about 2-fold; in this clone a large decrease in endogenous Caco2 expression of CRBP-II was also found [59]. Thus, some studies have yielded conflicting results, although some have provided strong evidence for an intracellular FABP-mediated transport function. A general concern with stable transfections in cultured cell lines is that parallel alterations may be occurring due to clonal variability, and secondary changes due to the altered expression of a specific gene, in this case an FABP, may also occur. The latter issue is not as serious as the former, as it at least points to the potential involvement of the FABP in a cellular process, if not to its precise role at the molecular level. It would seem of great importance for transfection studies, especially, to demonstrate a dose-dependent functional response to FABP expression. Thus far, only the studies of Wolfrum et al. [50] have been so rigorously performed. The use of direct protein transfer techniques, including streptolysis, electroporation, and lipid- and peptide-based protein transfer reagents, avoid entirely the issue of clonal variability, and in large part the concern about secondary changes in cellular processes. Excepting the aforementioned electroporation studies, these approaches have not yet been widely applied to the FABPs.

6.5

Cellular Fatty Acid Transport via FABP-Protein Interactions

As noted above, in vitro studies suggested a potential for FABPs to act as targeting proteins, conveying their ligands to particular domains on organellar membranes, and/or to specific protein receptors. Recently a number of protein–protein interactions involving FABPs have indeed been discovered, suggesting that fatty acids may be transported around the cell in a regulated manner. Using yeast two-hybrid assays, an interaction between A-FABP and hormonesensitive lipase (HSL) was discovered and further confirmed with experiments including GST-pulldowns and co-immunoprecipitation of the HSL:A-FABP complex [60]. The A-FABP interaction domain of HSL was found to reside in the N-terminal portion of the protein, whereas the catalytic domain is known to be localized

6.5 Cellular Fatty Acid Transport via FABP-Protein Interactions Fatty acid binding proteins and fatty acid efflux. It is possible that upon hydrolysis of lipid by hormone sensitive lipase (HSL) or other lipases, the resulting fatty acid (FA) is bound by fatty acid binding protein (FABP) and transported through the cytoplasm to CD36/FAT, whereupon the fatty acid is off-loaded for efflux out of the cell. Adipocyte FABP-HSL [60] and heart FABPCD36 complexes have been identified [62].

Fig. 6.2

to the C-terminus. Recent studies showed that HSL residues His194 and Glu199 appear to be critical for interactions with A-FABP [61]. These results suggest that A-FABP may function to traffic fatty acids away from the triglyceride droplet after hydrolysis by HSL, thus promoting further lipolysis by diminishing end-product inhibition. It is still unclear exactly where A-FABP is taking the fatty acid. However, given the discovery of an association between H-FABP and the cytoplasmic domain of the putative transmembrane fatty acid transporter CD36/FAT in milkfat globule membranes, as determined by gel filtration and coimmunoprecipitation [62], it is possible that A-FABP transports its bound fatty acid to the plasma membrane and, via an interaction with CD36/FAT, promotes the efflux of the fatty acid out of the cell (Fig. 6.2). Alternatively, in the adipocyte or in other cell types, an FABP could transport its bound fatty acid to internal sites for re-esterification. Again using the yeast two-hybrid system an interaction between L-FABP and the lipid-activated transcription factor peroxisome proliferator activated receptor a (PPARa) was found [63]. This was further assessed by pull-down assays and immunoprecipitation, and was shown to be independent of ligand binding. PPARa is believed to be a nuclear target for fatty acids [64, 65] and initiates gene expression of enzymes involved in lipid metabolism [66, 67]. Such an association, therefore, suggests that L-FABP serves to directly traffic its fatty acid ligand, gained possibly from an interaction with CD36/FAT and/or other transmembrane transporters, to the nucleus and, thereby, directly functions in the regulation of gene expression (Fig. 6.3). Indeed, recent results indicate that A- and E-FABPs localize in the nuclei of 3T3-LI adipocytes as well as the cytoplasm, suggesting that these two FABPs may also exert their action at the level of the nucleus [68]. An interaction between the extracellular domain of CD36 and the S100A8/ S100A9–arachidonic acid complex has also been identified in endothelial cells [69], implying a role for this protein in transcellular eicosanoid metabolism. Given that the coordinate regulation of gene expression [70, 71] and similar abundance [72] of cytoplasmic FABPs and membrane FA transporters have been repeatedly demonstrated, it is highly possible that a concerted action for FA transport exists.

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Fatty acid binding proteins and gene regulation. Fatty acids (FA) taken up by CD36/FAT may then be bound in the intracellular space by fatty acid binding proteins (FABP) for transport through the cytoplasm to the nucleus. Upon entering the nucleus, the FABP:FA complex binds to PPAR, the FA becomes PPAR-bound, and gene expression is

Fig. 6.3

initiated. CD36 has been shown to play a role in fatty acid uptake [102], FABP–CD36 complexes have been identified [63], and an association between liver FABP and PPARa has been shown [58]. There is also evidence to suggest that adipocyte and epidermal FABPs may be partially localized to the nucleus [68].

6.6

Insights into FABP Function from Null Mice

Several mouse models null for different FABPs have been created by targeted gene disruption. These are providing important support for the transport functions of these proteins, as well as new insights into additional potential functions. Of the four FABP “knockouts” described thus far, the H-FABP-null mouse provides perhaps the clearest example of functional consequences. Mice lacking expression of H-FABP displayed a substantial decrease in long-chain fatty acid uptake into the heart [73]. Further studies in cardiac myocytes isolated from wildtype and H-FABP–/– animals demonstrated that defective fatty acid uptake and oxidation appears to be the underlying cause of the phenotype observed in the whole animal [74]. Interestingly, the CD36/FAT-null mouse displays a very similar FA uptake phenotype [75], again suggesting a concerted action by the two types of FA transport proteins in cellular FA trafficking. In the H-FABP-null animals as well as in the CD36 nulls, physiological compensation for the decreased FA uptake appears to occur by an increase in glucose uptake and oxidation in the heart [73, 74], and not by compensatory increases in other members of the FABP family. H-FABP is also expressed in the mammary gland, however the H-FABP–/– mice were reported to have no overt phenotype in this tissue [76]. Mice null for I-FABP also showed no apparent compensation with other FABPs. The I-FABP–/– mice developed hyperinsulinemia that was independent of

6.6 Insights into FABP Function from Null Mice

body weight gain, an unusual dissociation [77]. The I-FABP knockout mice gained more weight and had higher levels of serum triglycerides [77]. This could indicate an involvement of the protein in lipid absorption, metabolism, or secretion, which requires further investigation. An intriguing finding in this model was that the effects on weight and serum triglycerides were observed only in male mice [77]. This gender dependency suggests a previously unexplored interaction between IFABP and sex hormones. In contrast to the H-FABP and I-FABP nulls, mice null for the ap2 gene, which encodes A-FABP, showed a dramatic increase in expression of another FABP, keratinocyte FABP (K-FABP), in adipose tissue [78, 79], perhaps accounting for the absence of a dramatic phenotype in the animals. Initial investigations of low fatfed mice showed few differences between wild-type and A-FABP–/– animals, however feeding a high-fat diet resulted in lower levels of plasma insulin and reduced adipocyte mRNA levels of tumor necrosis factor a (TNFa) relative to wild-type mice; the absence of hyperinsulinemia appeared to occur despite the presence of high-fat diet-induced obesity [78]. Conversely, however, it was found that younger aP2–/– mice, despite maintaining lower glucose levels, did in fact develop hyperinsulinemia on a high-fat diet; the plasma insulin levels were directly correlated with the degree of adiposity in both wild-type and A-FABP–/– mice [79]. Further, adipocyte TNFa secretion was not reduced relative to wild-type mice [79]. These results indicate that a dissociation between the development of obesity and the development of hyperinsulinemia, a hallmark of obesity, is not apparent in the AFABP–/– mouse under all circumstances. The aP2–/– mice have also been reported to have modest decreases in lipolysis in some [80, 81] but not all [79] investigations, as well as a small increase in basal levels of de novo fatty acid synthesis [79]. Interestingly, the pancreatic insulin secretory response to b-adrenergic stimulation was suppressed in aP2–/– mice [80], and when aP2 deficiency was introduced into the genetically obese ob/ob mouse, animals lacking A-FABP showed decreased pancreatic insulin secretion as well as improved glucose tolerance [82]. Thus, despite the discrepancies and despite the fact that the mechanisms of the null phenotypic changes are not clear, the collective results indicate that further explorations of the role of A-FABP in fatty acid flux and systemic lipid and glucose metabolism are warranted. A recent report of dramatic increases in serum levels and adipose expression of a bone morphogenic protein in the A-FABP-null mice [83] may point to a heretofore unrecognized interaction between fatty acid metabolism and bone development. At this point, however, the nature of any such association remains to be explored. A potentially critical involvement for A-FABP in the development of diet-induced atherosclerosis has been recently revealed by studies of A-FABP–/– mice crossed with mice deficient in apoE, the latter being a well-established model of dietary atherosclerosis. ApoE–/– animals develop severe coronary arterial occlusion on a high-fat diet; in dramatic contrast, the A-FABP–/–/apoE–/– mice developed only trivial lesions, strongly indicating a role for A-FABP in the accumulation of lipid-rich foam cells in the arterial intima [84, 85]. As A-FABP is highly expressed not only in adipose tissue but also in macrophages [86], the results suggested that it plays a critical

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role in the development of hypercholesterolemia-induced atherosclerosis, likely at the level of the macrophage. Indeed, recent bone marrow transplantation studies showed that macrophage-expressed A-FABP, rather than adipocyte A-FABP, was likely to be primarily involved in the development of dietary atherosclerosis [84, 85]. K-FABP was also found to be expressed in macrophages, however unlike the compensatory upregulation of expression observed in adipose tissue of the ap2–/– mice, macrophage K-FABP expression remained unchanged [84]. The mechanisms for the apparently pro-atherosclerotic effects of macrophage A-FABP do not appear to be primarily related to cholesterol ester accumulation, as this was altered to a modest extent, or not at all, in macrophages from the A-FABP–/–/apoE–/– mice [84, 85]. Interestingly, levels of interleukin 6, interleukin 1b, macrophage inflammatory proteins 1a and 1b, and macrophage chemoattractant protein 1 were decreased in the apoE/A-FABP-deficient macrophages relative to apoE–/–, indicating a role for A-FABP in inflammatory cytokine and chemokine expression [84, 87]. Recently, the generation of a skin-type FABP (K- or E-FABP)-null mouse (EFABP–/–) was reported [88]. Here, too, it appears that compensatory changes in another FABP are found: mice lacking E-FABP showed increased expression of H-FABP. A dramatic phenotype was not observed in the E-FABP–/– mice, however changes in the rate although not the extent of transepidermal water loss were observed [88]. The mechanisms by which the FABPs may participate in the water barrier function of the skin are not yet known.

6.7

Perspectives

It has often been suggested that the expression of more than one type of FABP in a single tissue or even a single cell type, is a strong predictor that FABPs perform functions other than or in addition to bulk binding and transport. Recent studies of Widstrom et al., using displacement of the fluorescent probe ANS, showed that H-FABP can bind arachidonic and linoleic acid metabolites, which serve as components of cell signaling cascades [89]. If the FABPs do in fact act to target as well as bind FAs, as the data reviewed here suggest, then there must be specific signals for protein trafficking. An interesting feature of A-FABP is that Phe57 on the b-CD turn rotates by more than 908 into the binding cavity of the apoprotein relative to the holoprotein [90]. As discussed earlier, differences in apo-FABP versus holo-FABP tertiary structures are especially notable in the helix-turn-helix ‘portal’ domain [15]. Differences in the regulation of fluorescent FA transfer from I-FABP to membranes relative to transfer in the opposite direction further suggests differential trafficking properties of apo- and holoFABPs [35]. Thus, structural changes in apo- versus holo-FABPs might act to alter interactions with other cellular structures, prevent competition between apo- and holo-proteins for interaction with the same receptor, act as a signal for a ligand-bound protein, or change FABP affinity for a membrane. Evidently there is much to be learned about the precise mechanisms by which FABPs participate in the uptake, efflux, and intracellular transport and metabolism of their small hydrophobic ligands.

6.8 References

6.8

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3 4

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8

9

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Atshaves, B. P., Foxworth, W. B., Frolov, A., Roths, J. B., Kier, A. B., Oetama, B. K., Piedrahita, J. A., Schroeder, F. Am. J. Physiol. 1998, 274, C633–C644. Luxon, B. A., Milliano, M. T. Am. J. Physiol. 1997, 273, C859–C867. Atshaves, B. P., Foxworth, W. B., Frolov, A., Roths, J. B., Kier, A. B., Oetama, B. K., Piedrahita, J. A., Schroeder, F. Am. J. Physiol. 1998, 274, C633–C644. Jefferson, J. R., Powell, D. M., Rymaszewski, Z., Kukowska-Latallo, J., Lowe, J. B., Schroeder, F. J. Biol. Chem. 1990, 265, 11062–11068. Jefferson, J. R., Slotte, J. P., Nemecz, G., Pastuszyn, A., Scallen, T. J., Schroeder, F. J. Biol. Chem. 1991, 266, 5486–5496. Schroeder, F., Jefferson, J. R., Powell, D., Incerpi, S., Woodford, J. K., Colles, S. M., Myers-Payne, S., Emge, T., Hubbell, T., Moncecchi, D. Mol. Cell Biochem. 1993, 123, 73–83. Prows, D. R., Murphy, E. J., Schroeder, F. Lipids 1995, 30, 907–910. Prows, D. R., Schroeder, F. Arch. Biochem. Biophys. 1997, 340, 135–143. Murphy, E. J., Prows, D. R., Jefferson, J. R., Schroeder, F. Biochim. Biophys. Acta 1996, 1301, 191–198. Murphy, E. J. Am. J. Physiol. 1998, 275, G244–G249. Nemecz, G., Hubbell, T., Jefferson, J. R., Lowe, J. B., Schroeder, F. Arch. Biochem. Biophys. 1991, 286, 300–309. Wolfrum, C., Buhlmann, C., Rolf, B., Borchers, T., Spener, F. Biochim. Biophys. Acta 1999, 1437, 194–201. Baier, L. J., Bogardus, C., Sacchettini, J. C. J. Biol. Chem. 1996, 271, 10892–10896. Le Beyec, J., Delers, F., Jourdant, F., Schreider, C., Chambaz, J., Cardot, P., Pincon-Raymond, M. Exp. Cell Res. 1997, 236, 311–320. Darimont, C., Gradoux, N., Cumin, F., Baum, H. P., De Pover, A. Exp. Cell Res. 1998, 244, 441–447. Gedde-Dahl, A., Kulseth, M. A., Ranheim, T., Drevon, C. A., Rustan, A. C. Lipids 2002, 37, 61–68. Sha, R. S., Kane, C. D., Xu, Z., Banaszak, L. J., Bernlohr, D. A. J. Biol. Chem. 1993, 268, 7885–7892.

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Prinsen, C. F., Veerkamp, J. H. Biochem. J. 1998, 329(2), 265–273. Liou, H. L., Storch, J. FASEB J. 2001, 15, A1090. Claffey, K. P., Crisman, T. S., Ruiz-Opazo, N., Brecher, P. FASEB J. 1988, 2, A1783. Levin, M. S. J. Biol. Chem. 1993, 268, 8267–8276. Shen, W. J., Sridhar, K., Bernlohr, D. A., Kraemer, F. B. Proc. Natl Acad. Sci. USA 1999, 96, 5528–5532. Shen, W. J., Liang, Y., Hong, R., Patel, S., Natu, V., Sridhar, K., Jenkins, A., Bernlohr, D. A., Kraemer, F. B. J. Biol. Chem. 2001, 276, 49443–49448. Spitsberg, V. L., Matitashvili, E., Gorewit, R. C. Eur. J. Biochem. 1995, 230, 872–878. Wolfrum, C., Borrmann, C. M., Borchers, T., Spener, F. Proc. Natl Acad. Sci. USA 2001, 98, 2323–2328. Krey, G., Braissant, O., L’Horset, F., Kalkhoven, E., Perroud, M., Parker, M. G., Wahli, W. Mol. Endocrinol. 1997, 11, 779–791. Ellinghaus, P., Wolfrum, C., Assmann, G., Spener, F., Seedorf, U. J. Biol. Chem. 1999, 274, 2766–2772. Wolfrum, C., Ellinghaus, P., Fobker, M., Seedorf, U., Assmann, G., Borchers, T., Spener, F. J. Lipid Res. 1999, 40, 708–714. Issemann, I., Green, S. Nature 1990, 347, 645–650. Helledie, T., Antonius, M., Sorensen, R. V., Hertzel, A. V., Bernlohr, D. A., Kolvraa, S., Kristiansen, K., Mandrup, S. J. Lipid Res. 2000, 41, 1740–1751. Kerkhoff, C., Sorg, C., Tandon, N. N., Nacken, W. Biochemistry 2001, 40, 241– 248. van der Lee, K. A., Vork, M. M., De Vries, J. E., Willemsen, P. H., Glatz, J. F., Reneman, R. S., van der Vusse, G. J., Van Bilsen, M. J. Lipid Res. 2000, 41, 41–47. Van Nieuwenhoven, F. A., Willemsen, P. H., van der Vusse, G. J., Glatz, J. F. Int. J. Biochem. Cell Biol. 1999, 31, 489– 498. Pelsers, M. M., Lutgerink, J. T., Nieuwenhoven, F. A., Tandon, N. N., van

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Structure and Function of SCP-x/SCP-2 Udo Seedorf

7.1

Introduction

In most eukaryotic cells, the bulk of cholesterol is synthesized at the endoplasmic reticulum (ER), whereas almost 90% of the free, non-esterified fraction of this essential membrane lipid resides in the plasma membrane and the endocytic recycling compartment [1, 2]. Cholesterol is found mainly at the inner leaflet of the bilayer, where it limits membrane fluidity which is thought to stabilize the complex supramolecular structures that are formed between lipids, receptors, adaptor proteins, and the cytoskeleton at the cell surface. The cholesterol hydroxyl group forms a hydrogen bond with a phospholipid carbonyl oxygen atom, whereas the bulky steroid moiety and the flexible hydrocarbon tail are directed to the hydrophobic inner portion of the membrane. It has been proposed that cholesterol is not evenly distributed within the inner leaflet of the membrane, but that it is concentrated in cholesterol-rich microdomains called lipid rafts or caveolae [3, 4]. Lipid rafts are also rich in sphingomyelin and VIP21 caveolin, a 21–24 kDa integral membrane protein that binds cholesterol in a 1 : 1 molar ratio [3, 4–7]. The highly asymmetric distribution of cholesterol in cells makes it conceivable that intracellular trafficking of cholesterol requires target-specific transport mechanisms that mediate its translocation from the site of synthesis at the ER to the lipid rafts of the plasma membrane. Moreover, there must be some means that keep cholesterol from diffusing to the bilayer’s outer leaflet and that inhibit its lateral diffusion within the membrane. There is growing evidence from the literature that the Golgi apparatus also plays a key role in cholesterol trafficking (reviewed in Ref. [3]). Eukaryotes are characterized by endomembranes that are connected by vesicular transport along secretory and endocytic pathways. The compositional differences between the various cellular membranes are maintained by sorting events, and it has long been believed that sorting is based solely on protein–protein interactions. However, the central sorting station along the secretory pathway is the Golgi apparatus which is the site of synthesis of the sphingolipids. It is presumed that, as Golgi cisternae mature, ongoing sphingolipid synthesis attracts ER-derived cholesterol and drives a fluid–fluid lipid phase separation that segregates sphingolipids and sterols from

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7 Structure and Function of SCP-x/SCP-2

unsaturated glycerolipids into lateral domains. While sphingolipid domains move forward to the plasma membrane, unsaturated glycerolipids are retrieved by recycling vesicles budding from the sphingolipid-poor environment. Currently, it is not clear whether soluble cholesterol carrier proteins play a role in the process of target-specific intracellular cholesterol trafficking. The best-studied candidate for a soluble sterol carrier has been sterol carrier protein 2 (SCP-2), also known as the non-specific lipid transfer protein, purified almost 20 years ago on the basis of its ability to activate the enzymatic conversion of 7-dehydrocholesterol to cholesterol by liver microsomes in vitro [8]. Because SCP-2 promotes intermembrane exchange of a wide variety of sterols in vitro and its expression affects sterol trafficking in certain tissue culture systems and animal models, it has long been thought that the protein acts as a substrate carrier in various aspects of sterol metabolism [9–12]. On the other hand it appears noteworthy that purified SCP-2 binds most fatty acids and fatty acyl-CoAs with similar or even higher affinity than sterols [13]. Phytanoyl-CoA binding was shown to be ~10-fold better than binding of cholesterol as measured with a highly specific fluorescence resonance energy transfer (FRET) assay [14, 15]. Wouters et al. studied the fate of fluorescently labeled pre-SCP-2 (Cy3-pre-SCP-2) microinjected into BALB/c 3T3 fibroblasts [16]. The protein co-localized to a high degree with the immunofluorescence pattern for the peroxisomal enzyme acyl-CoA oxidase. Proteolytic removal of the C-terminal leucine of the peroxisomal targeting signal (AKL) abolished targeting of the labeled pre-SCP-2 to peroxisomes. Moreover, they investigated the association of SCP-2 with peroxisomal enzymes by measuring FRET between the microinjected Cy3-pre-SCP-2 and Cy5-labeled antibodies against the peroxisomal enzymes ACO, 3-ketoacyl-CoA thiolase, PBE, PMP70, and catalase. The data revealed a specific association of SCP-2 with acyl-CoA oxidase, 3-ketoacyl-CoA thiolase, and PBE in the peroxisomes. These studies showed a close association of SCP-2 with other essential components of the peroxisomal fatty acid b-oxidation system, which supports a role of SCP-2 in regulating peroxisomal b-oxidation (e.g. by facilitating the presentation of substrates and/or stabilization of the substrates or enzymes). The purpose of this review is to summarize our current knowledge about the structure of SCP-2 and discuss potential functions of the protein in vivo.

7.2

The SCP-2 Gene Family

The SCP-2 gene family includes four distinct members: SCP-2, SCP-x, D-PBE (MFE-2), and UNC-24/hSLP-1. Apart from SCP-2, which is expressed as an individual protein, all other homologs contain their SCP-2 domains at their C-termini (Fig. 7.1). Mammalian SCP-2 is synthesized as 143-amino-acid precursor which is processed presumably in peroxisomes to the 123-amino-acid mature SCP-2. The human SCP-2-encoding gene comprises 16 exons, which span ~100 kb on chromosome 1p32 [17–19]. Alternate transcription initiation regulates the expression of SCP-2 and a second gene product that consists of 547 amino acids (named ster-

7.2 The SCP-2 Gene Family

SCP-2/SCP-x

Domain structure of the currently known members of the SCP-2 gene family. Amino acid numbers of known processing sites are indicated by arrows. SCP-2, sterol carrier protein-2; SCP-x, sterol carrier protein-x; PBE, peroxisomal bifunctional enzyme; hSLP, human stomatin-like protein.

Fig. 7.1

ol carrier protein-x, SCP-x) [20]. SCP-x represents a fused protein consisting of a thiolase, extending from amino acids 1 to 404, and SCP-2 which is located at the C-terminus [21, 22]. The fused gene can be traced back to Drosophila melanogaster [23] (GenBank accession no. X97685), whereas two separated genes for SCP-2 and the thiolase are present in Caenorhabditis elegans and several yeast species [24, 25]. In addition, SCP-2 homologs could be identified in several bacterial species and the methanogenic archaeon Methanococcus jannaschii [26]. It is known from in vitro studies that SCP-x has lipid transfer activity similar to SCP-2 [27]. The substrate specificity of the SCP-x thiolase shows a preference for medium straight-chain acyl-CoA substrates, 2-methylbranched-chain fatty acylCoAs (such as 3-ketopristanoyl-CoA) and bile acid precursors (such as 3a,7a,12atrihydroxy-24-ketocholestanoyl-CoA) [28–30]. The last two substrates are not oxidized effectively in mitochondria but require peroxisomal b-oxidation, which coincides with the peroxisomal localization of SCP-x. The properties of the newly discovered SCP-x thiolase thus differ clearly from the long-known peroxisomal thiolase (called pTh1), that was identified by Hashimoto and co-workers almost 15 years ago [31]. Unlike SCP-x, pTh1 acts preferentially on the 3-keto derivatives of the straight very long-chain fatty acids (VLCFAs) that are metabolized in peroxisomal b-oxidation [31, 32]. Since the SCP-2 domain harbors the PTS1 signal it is required for the import of SCP-x into peroxisomes [14]. Once the protein has arrived in the peroxisome, SCP-x is subject to proteolytic cleavage yielding pTh2 and SCP-2. We found that only approximately half of the total SCP-x protein is processed whereas the other half remains in the form of the fused precursor [28]. It is known that processing does not affect the catalytic activity of the thiolase which exists in three distinct forms: homodimeric SCP-x, homodimeric pTh2, and heterodimeric SCP-x/pTh2 [33].

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The third member of the gene family consists of an 80-kDa precursor cloned originally as 17b-hydroxysteroid dehydrogenase type IV (17b-HSD4). This precursor comprises a C-terminal domain similar to SCP-2 [34], which is fused to a peptide with acyl-CoA 2-enoyl hydratase/3-hydroxyacyl-CoA dehydrogenase activity (also known as peroxisomal bifunctional enzyme, PBE) [35]. Processing occurs after import of the 80-kDa precursor into peroxisomes at the junction between the acyl-CoA 2-enoyl hydratase and 3-hydroxyacyl-CoA dehydrogenase domains [36]. The SCP-2-like domain is required for import of the 80-kDa precursor into peroxisomes and confers a similar intrinsic lipid transfer activity to the fusion protein as was demonstrated for SCP-x [37]. Since the precursor protein has a modular organization of three functionally distinct domains the protein was called multifunctional enzyme 2 (MFE-2). b-Oxidation of acyl-CoA species in mammalian peroxisomes can occur via either multifunctional enzyme type 1 (MFE-1) or type 2 (MFE-2), both of which catalyze the hydration of trans-2-enoyl-CoA and the dehydrogenation of 3-hydroxyacyl-CoA, but with opposite chiral specificity. The well-known multifunctional enzyme 1 (MFE1), which is also called L-bifunctional enzyme [38], converts trans-enoyl-CoA to their 3-keto derivatives via the l-hydroxy stereoisomer. In contrast, the new MFE-2 catalyzes the same transformation via the d-stereoisomer [39, 40]. Therefore, Hashimoto and co-workers have introduced the name D-bifunctional enzyme for MFE-2. The two MFE proteins differ in their substrate specificity. Whereas the MFE-2 catalyzes the formation of 3-ketoacyl-CoA intermediates from the CoA esters of bile acid precursors, very long straight-chain fatty acyl-CoAs and 2-methylbranched-chain fatty acyl-CoAs, the activity of MFE-1 is only high with the straight-chain substrates [39–42]. Convincing evidence showing that these findings are relevant also in vivo were obtained by van Grunsven et al. who identified a patient with isolated MFE-2 deficiency [43]. Besides a severe block in pristanic acid b-oxidation, the patient had a block in normal peroxisomal degradation of the cholesterol side-chain in bile acid synthesis. A surprising finding was that MFE-2 deficiency also affected peroxisomal b-oxidation of VLCFA-CoA. Although secondary causes cannot be excluded at present, this finding suggests strongly that MFE-2, together with MFE-1, may play an important role also in b-oxidation of straight VLCFA-CoA substrates. Thus, the newly discovered MFE-2 may act simultaneously on all three major substrates of peroxisomal b-oxidation (VLCFA, 2methyl branched-chain fatty acids, and bile acids) which suggests that MFE-2 may in fact be the more important enzyme in human peroxisomal b-oxidation than the better-known MFE-1. More recently, positional cloning and molecular characterization of the unc-24 gene of Caenorhabditis elegans led to the identification of a new member of the SCP-2 gene family [44]. The unc-24 gene is required for normal locomotion and interacts with genes that affect the worm’s response to volatile anesthetics. In C. elegans, unc-24 is genetically epistatic to unc-1, which is the primary determinant of anesthetic sensitivity. It is presumed that UNC-24 is required for the correct organization of lipid rafts in the plasma membrane of neurons [45]. The predicted gene product contains a domain similar to part of two ion channel regulators (the

7.3 Structure of SCP-2

erythrocyte integral membrane protein stomatin and the C. elegans neuronal protein MEC-2), juxtaposed to a domain similar to SCP-2. Sequence analysis suggested that the SCP-2-like domain of UNC-24 is tethered to the plasma membrane by the stomatin-like domain which may be regulatory [44]. Recently, cDNA clones encoding a human homolog of UNC-24 were isolated from a human cerebral cortex cDNA library [46]. The bipartite stomatin-like protein called hSLP-1 consists of 394 amino acids. The major stomatin-like part starts at the N-terminus whereas the SCP-2-like domain is located at the C-terminal end. The SLP-1 transcript is mainly expressed in the brain, with the highest levels in the frontal lobe, cerebral cortex, caudate nucleus, amygdala, temporal lobe, putamen, substantia nigra, and hippocampus.

7.3

Structure of SCP-2

Nuclear magnetic resonance (NMR) spectroscopy was used to determine the secondary structure and the three-dimensional polypeptide backbone fold of human SCP-2 [47]. Sequence-specific assignments were obtained for nearly all backbone 1 H and 15N resonances, as well as for about two-thirds of the side-chain 1H resonances, using uniform 15N-labeling of the protein combined with homonuclear two-dimensional 1H NMR and three-dimensional 15N-correlated 1H NMR. Three a-helices comprising the polypeptide segments of residues 9–22, 25–30, and 78– 84 were identified by sequential and medium-range nuclear Overhauser effects (NOE). The analysis of long-range backbone–backbone NOEs showed that hSCP-2 further contains a five-stranded b-sheet including the residues 33–41, 47–54, 60– 62, 71–76, and 100–102, which is a central feature of the molecular architecture. The first three strands are arranged in an antiparallel fashion, the polypeptide chain then crosses over this three-stranded sheet in a right-handed sense so that the fourth strand is added parallel to the first one. The fifth strand runs antiparallel to the fourth one, so that the overall topology is +1, +1, –3x, –1 (Fig. 7.2 A). Subsequently, a more refined NMR structure of the mature SCP-2 was presented together with NMR studies of nitroxide spin-labeled substrate binding [48]. Compared with the first study, an additional a-helix could be identified within the C-terminal segment of the protein. By use of the spin-labeled substrate 16-doxylstearic acid, fatty acid binding was mapped to a hydrophobic surface area formed by amino acid residues of the first and third helices, and the b-sheet. These structural elements are all located in the polypeptide segment 8–102, previously identified by site-directed mutagenesis to be crucial for the activity of SCP-2 [27]. The data supported that the lipid-binding site is covered by the C-terminal segment 105–123 which carries the PTS1 targeting signal. This implied that ligand binding could affect the topology of the PTS1 signal in relation to the protein structure. The crystal structure of the rabbit SCP-2 was determined at 1.8 Å resolution [49]. The structural topology showed an overall good agreement with the NMR data of the human SCP-2 (Fig. 7.2 B) [48]. The core of the protein was formed by

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7 Structure and Function of SCP-x/SCP-2

Structure of SCP-2. (A–C) Ribbon drawing of the structures of human and rat SCP-2 and the human MFE-2 SCP-2-like domain. (A) Polypeptide segment 8–116 of human SCP-2 as revealed by three-dimensional NMR [47]. (B) Crystal structure of the rabbit mature SCP-2 [48]. (C) Crystal structure of the

Fig. 7.2

human MFE-2 SCP-2-like domain [51]. The ligand bound to the protein is Triton X-100. (D) Space-filling model of rabbit SCP-2. The two shown orientations reveal the proposed exit and entrance of the tunnel (see gray arrows).

a five-stranded antiparallel b-sheet flanked by five instead of four helices. The putative binding site was predicted to represent a hydrophobic tunnel providing the environment for apolar ligands such as fatty acids and fatty acyl-coenzyme As. This tunnel was formed by the C-terminal segment (residues 114–123), together with part of the b-sheet and four a-helices (Fig. 7.2 D). It is interesting that some of the structurally well-characterized non-specific lipid transfer proteins from plants also have hydrophobic tunnel-like cavities, which have been identified as the binding sites for fatty acids and related apolar ligands. Despite the fact that plant non-specific lipid transfer proteins are smaller proteins than SCP-2, show no sequence homology to SCP-2, and are structurally unrelated, the cavities of these two classes of proteins are very similar with respect to size, shape, and hydrophobicity, suggesting a common functional role. Since these studies were all performed on the mature 123 amino acid protein, they did not allow predictions about the precise topology of the 20 amino acid leader sequence which is present in pre-SCP-2. From circular dichroism, mass spectroscopy, and antibody accessibility data, Schroeder et al. concluded that the pres-

7.3 Structure of SCP-2

ence of the pre-sequence had dramatic effects on the overall structure of SCP-2 [50]. In comparison to mature SCP-2, they found that pre-SCP-2 had 3-fold less ahelix, 7-fold more b-structure and 2-fold less binding of anti-SCP-2 antibodies. In addition, carboxypeptidase had a 6-fold higher reactivity towards pre-SCP-2 than towards the mature SCP-2. In addition, pre-SCP-2 did not enhance sterol transfer from plasma membranes. Nevertheless, the same concentration of guanidine hydrochloride was required for 50% unfolding and the ligand binding sites displayed the same high affinity for the binding of lipids. These data are in clear contrast to results published by Weber et al., who studied the pre-form of the rabbit SCP-2 protein with three-dimensional 15N-resolved NMR spectroscopy [51]. In spite of its low solubility in aqueous solution of only approximately 0.3 mM, these workers obtained sequential 15N and 1H backbone resonance assignments for 105 out of the 143 residues. From comparison of the sequential and medium-range NOEs in the two proteins, all regular secondary structures previously determined in mature human SCP-2 were also identified in pre-SCP-2 of the rabbit. Nearidentity of the backbone 15N and 1H chemical shifts and 1:1 correspondence of 24 long-range NOEs to backbone amide groups in the two proteins showed that the residues 21–143 adopted the same globular fold in pre-SCP-2 of the rabbit and mature human SCP-2. The conclusion was that the N-terminal 20-residue leader peptide of pre-SCP-2 is flexibly disordered in solution and does not observably affect the conformation of the polypeptide segment 21–143. These structural data correspond to functional results obtained by Seedorf et al., who showed almost identical sterol and phospholipid transfer activities for the mature SCP-2 and preSCP-2 of the rat [28]. Recently, Haapalainen et al. presented the structure of the SCP-2-like domain comprising amino acid residues 618–736 of the human MFE-2 at 1.75 Å resolution in complex with Triton X-100 [52]. The SCP-2-like domain of MFE-2 adopted an a/b-fold consisting of five b-strands and five a-helices. The overall architecture was similar to the rabbit and human SCP-2 structures (Fig. 7.2C). However, the structures differ in that the hydrophobic tunnel traverses the protein in MFE-2/ SCP-2. Interestingly, this tunnel was occupied by an ordered Triton X-100 molecule. The tunnel was large enough to accommodate molecules such as straightchain and branched-chain fatty acyl-CoAs and bile acid intermediates. In addition, relatively large empty apolar cavities were observed near the exit of the tunnel and between a-helices C and D. In addition, the C-terminal peroxisomal targeting signal was ordered in the structure and solvent-exposed, which is not the case with unliganded SCP-2. The structure of SCP-2 is consistent with that of a soluble lipid carrier and it may fulfill a role in peroxisomes similar to that of the acyl-CoA binding protein (ACBP) in the cytosol. One could imagine, for instance, that SCP-2 protects various CoA esters from undesired hydrolysis by peroxisomal thioesterases. On the other hand, it is also possible that the protein is part of a peroxisomal antioxidant mechanism protecting double-bonded fatty acyl-CoAs from superoxide-fostered peroxidation. Data from in vitro studies appear to support both concepts, and thus further in vivo studies are required in order to reach a definitive conclusion.

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7.4

Role of SCP-2/SCP-x in Peroxisomal Metabolism

Genetic approaches to the identification of a human inherited disorder that would result from SCP-2 mutations have not been successful so far. Therefore, we investigated the biological function of SCP-2/SCP-x by employing gene targeting in mice [14]. The null mice exhibited spontaneous peroxisome proliferation, hepatocarcinogenesis, and marked alteration of gene expression in the liver. The biochemical defect related to a severe block at the level of the thiolytic cleavage in pristanic acid boxidation (2-methyl-branched fatty acid, see Fig. 7.3 for a schematic illustration of peroxisomal b-oxidation). In addition, SCP-2/SCP-x deficiency also affected peroxisomal b-oxidation of phytanic acid (3,7,11,15-tetramethylhexadecanoic acid) [14]. Although secondary causes cannot be excluded at present, the data raised the possibility that the acyl-CoA binding function of SCP-2 may play a role in intra-peroxisomal delivery of phytanoyl-CoA to the phytanoyl-CoA a-hydroxylase enzyme. Conversely, no abnormalities were found for oxidation of VLCFA-CoA in fibroblasts, whereas a moderate decrease was found in primary hepatocytes. In agreement with the substrate specificity of pTh2, the null mice also had a block in normal peroxisomal degradation of the cholesterol side-chain in bile acid synthe-

Current view of the peroxisomal boxidation systems. Enzymes and carrier proteins which are part of the classical l-hydroxyspecific, peroxisome proliferator-inducible boxidation system are shown on the left: adrenoleukodystrophy protein (ALDP), acyl-CoA oxidase (AOX), l-specific peroxisomal bifunctional enzyme (L-PBE), peroxisomal thiolase 1 (pTHIOL-1). A second non-inducible d-hydroxy-specific b-oxidation system (right) consists of pristanoyl-CoA oxidase (POX), trihydroxycholestanoyl-CoA oxidase (THCAOX), d-spe-

Fig. 7.3

cific peroxisomal bifunctional enzyme (D-PBE, also called multifunctional enzyme 2, MFE-2), and sterol carrier protein-x (SCP-x, also called peroxisomal thiolase 2). Null mouse models which are associated with sustained PPARa activation are shown in gray boxes. Those without sustained PPARa activation are boxed with a broken line. Gene targeting of POX, THCAOX, and pTHIOL-1 has not yet been performed. The carrier systems required for peroxisomal import of BCFA and bile acid precursors are not known.

7.5 SCP-2/SCP-x Deficiency Affects

Role of the SCP-x thiolase in bile acid synthesis. 27-Hydroxylation occurs in mitochondria, oxidation to cholestenic acid is

Fig. 7.4

performed by cytosolic alcohol dehydrogenases.

sis [53]. The defect in bile acid synthesis is illustrated schematically in Fig. 7.4. Specific inhibition of b-oxidation at the thiolytic cleavage step in bile acid synthesis was supported by the finding of pronounced accumulation in bile and serum from the null mice of 3a,7a,12a-trihydroxy-27-nor-5b-cholestane-24-one (which is a known bile alcohol derivative of the cholic acid synthetic intermediate 3a,7a,12a-trihydroxy-24-keto-cholestanoyl-coenzyme A). Moreover, these mice had elevated concentrations of bile acids with shortened side-chains (i.e. 23-norcholic acid and 23-norchenodeoxycholic acid), which may be produced via a-rather than b-oxidation. These results demonstrated that the SCP-x thiolase is critical for peroxisomal b-oxidation of the steroid side-chain in conversion of cholesterol into bile acids.

7.5

SCP-2/SCP-x Deficiency Affects the Activity of the Peroxisome Proliferator Activated Receptor PPARa

Spontaneous peroxisome proliferation and marked alteration of gene expression in the liver are early events related to SCP-2/SCP-x deficiency in mice which may be attributed to sustained PPARa activation [54]. Similar effects were reported in acyl-CoA oxidase (AOX)-null mice. AOX catalyzes the first step of peroxisomal b-

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7 Structure and Function of SCP-x/SCP-2

oxidation of VLCFA, which converts fatty acyl-CoA to 2-trans-enoyl-CoA. Mice deficient in AOX exhibit increased levels of VLCFA, particularly after stress with VLCFA-enriched diets [55]. The block in peroxisomal b-oxidation of VLCFA is associated with steatohepatitis, increased hepatic H2O2 levels, and hepatocellular regeneration. Similar to the SCP-2/SCP-x-null mice, the liver of AOX-null mice displayed profound generalized spontaneous peroxisome proliferation and increased mRNA levels of genes that are regulated by PPARa. Hepatic adenomas and carcinomas developed in AOX-null mice by 15 months of age, probably due to sustained activation of PPARa [56]. These observations implicate putative substrates for peroxisomal b-oxidation as biological ligands for PPARa. Many of the pleiotropic effects that result from the AOX gene disruption resemble those that are present in SCP-2-null mice, also showing spontaneous peroxisome proliferation and evidence of chronic PPARa activation. Although steatohepatitis was virtually absent in the SCP-2/SCP-x-deficient strain, hepatocarcinomas and adenomas developed shortly after the age of one year. It could be shown that branchedchain fatty acid (BCFA) serum concentrations correlate well with expression of PPARa target genes in SCP-2-null mice [54]. Moreover, treatment with BCFA led to pleiotropic effects that could be mimicked with PPARa agonists bezafibrate and Wy 14,643 but not with the retinoid-X receptor, RXRa agonist 9-cis-retinoic acid. The findings were in line with binding of BCFA to a fused glutathione-S-transferase murine PPARa ligand binding domain with almost the same affinity as the strong artificial PPARa agonist Wy 14,643 and phytanic acid-induced expression activation of a peroxisome proliferator response element (PPRE)-driven reporter gene in vitro [54]. Taken together, the currently available data provide strong support for BCFA acting as signal involved in direct stimulation of PPARa. This is particularly noteworthy since direct binding or activation of rodent PPARa could so far not be demonstrated for other natural substrates of peroxisomal metabolism. In AOX-null mice, AOX deficiency imposes a block on VLCFA-CoA entering the b-oxidation pathway (Fig. 7.3). It is conceivable that unmetabolized VLCFACoA may function as biological ligands of PPARa/RXRa, leading to sustained transcriptional enhancement of genes with PPRE-containing promoters in this system. Long-chain acyl-CoAs were once considered to represent a metabolic message responsible for the induction of the b-oxidation system [57, 58]. This raises the question whether free fatty acids and unmetabolized synthetic peroxisome proliferators can act as direct ligands of PPARa in vivo or whether activation of this receptor is mediated by their CoA esters or downstream derivatives resulting from their b-oxidation. It is known that sulfur-substituted fatty acid derivatives and peroxisome proliferators of the fibrate class are activated to their esters with CoA. Although these cannot enter the b-oxidation spiral, they could still function efficiently as peroxisome proliferators in vivo, implying that b-oxidation is not essential to generate the PPARa agonists [58, 59]. On the other hand, progressive VLCFA accumulation in X-linked adrenoleukodystrophy (X-ALD), a peroxisomal disorder with impaired VLCFA metabolism associated with neurological abnormalities and death during childhood, does not lead to spontaneous peroxisome proliferation in liver parenchymal cells in X-ALD patients or in mouse models for

7.6 Impact of SCP-2/SCP-x on Cholesterol Metabolism

this disease, developed recently by inactivating the X-ALD gene [60–63]. Free VLCFA bind only weakly to recombinant PPARa [64, 65]. In addition, dietary lipid overload, leading to increased VLCFA levels, does not induce peroxisome proliferation [60–63]. These results imply that, under in vivo conditions, the free VLCFAs are not effective inducers of PPARa. The remarkable induction of spontaneous peroxisome proliferative response in AOX-null mice raises the possibility that the PPARa signal-transducing event is immediately distal to the acyl-CoA synthase catalyzed fatty acid activation step. However, several long-chain fatty acylCoAs neither bound to recombinant PPARa nor induced PPARa activation in vitro [64]. Thus, the factors that mediate peroxisome proliferation in AOX-null mice are not yet clear. One possibility is that PPARa activation is mediated by a still unknown PPARa ligand that is b-oxidized within the peroxisome [66]. Such an endogenous ligand of PPARa may potentially contribute to enhanced PPARa activation in the liver of AOX-null mice. Despite the fact that phytanic and pristanic acid can be regarded as a bona fide PPARa agonists in mice, the involvement of so far unknown endogenous ligands that accumulate along with the BCFAs in the SCP-2-null mice and signal PPARa activation cannot be ruled out at present. The data establish clearly that both genes, AOX and SCP-2/SCP-x, are required for efficient peroxisomal oxidation of certain fatty acids and at the same time they are key regulators of PPARa function in vivo. Thus, these mouse models may provide helpful clues in the search for so far unknown natural PPARa agonists and in screening for in vivo antagonists for this receptor.

7.6

Impact of SCP-2/SCP-x on Cholesterol Metabolism

Many studies have been published in which potential functions of SCP-2 in cholesterol metabolism were investigated using assays in vitro, but relatively little is known regarding the role of SCP-2 in intact cells. Moncecchi et al. transfected mouse L-cell fibroblasts with cDNAs encoding mouse pre-SCP-2 and SCP-2. Expression of pre-SCP-2, but not of SCP-2, enhanced the rate and extent of [3H]cholesterol uptake compared to control or mock-transfected cells slightly by 1.3-fold [67]. Puglielli et al. reported that the rapid transport of de novo synthesized cholesterol to the plasma membrane was reduced after treatment with SCP-2 antisense oligonucleotides of normal fibroblasts, which suggested that the major fraction of newly synthesized cholesterol may be transported to the plasma membrane via an SCP-2-dependent mechanism [11]. According to Baum et al., overexpression of SCP-2 in McA-RH7777 rat hepatoma cells enhances the rate of cholesterol cycling, which reduces the availability of cholesterol for cholesterol ester synthesis and alters the activity of a cellular cholesterol pool involved in regulating apolipoprotein AI-mediated high-density lipoprotein cholesterol secretion. The net result of these changes was a 46% increase in plasma membrane cholesterol content [12]. An interesting experiment was performed by Zanlungo et al. who used adenovirus-mediated SCP-2 gene transfer in order to obtain hepatic overexpres-

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sion of SCP-2 in C57BL/6 mice [68]. The procedure, which led to an 8-fold increase of SCP-2 protein levels in the liver, decreased HDL-cholesterol and increased LDL-cholesterol concentrations with no effect on VLDL-cholesterol. LDL receptor expression and cholesterol synthesis were repressed whereas hepatic cholesterol concentrations were increased. The latter finding would imply an altered hepatic cholesterol balance in which a net increase in hepatic cholesterol is established in the presence of downregulated supply of cholesterol to the liver via the LDL receptor pathway and repressed cholesterol de novo synthesis. Since the SRB1 cholesterol uptake pathway was not affected, one could consider decreased VLDL secretion or repression of bile acid synthesis. However, VLDL concentrations, CYP7A1 expression and fecal excretion of bile acids were unaffected, whereas biliary cholesterol concentrations and bile acid secretion rates were increased. The SCP-2-overexpressing mice had a higher intestinal cholesterol absorption rate than the mock transfected controls. Although this could have contributed to the observed rise of cholesterol in the liver, it remained unclear how hepatic overexpression of SCP-2 may be related to enhanced cholesterol absorption which occurs at the level of the intestine. Apart from a significant depletion of the hepatic cholesterol ester storage pools, no gross abnormalities of cholesterol metabolism were observed in SCP-2/SCP-xnull mice during the initial characterization [14]. Thus, the conclusion that SCP-2 is an important mediator of intracellular cholesterol trafficking could not be made. On the other hand, it was shown that the SCP-2/SCP-x-null mice have a specific defect in the peroxisomal steps of bile acid synthesis which was attributed to the absence of the SCP-x thiolase in these mice [53]. In order to understand the apparent discrepancy existing between a defect in bile acid synthesis and hepatic cholesterol depletion rather than accumulation, we characterized key regulators of hepatic cholesterol metabolism, intestinal cholesterol absorption and biliary lipid secretion in the SCP-2/SCP-x-null mouse [69]. Compared with chow-fed wild-type animals, SCP-2/SCP-x-null mice had higher bile flow and lower bile salt secretion rates, slightly decreased hepatic apolipoprotein expression, significantly increased HMG-CoA reductase expression, and at least 4-fold upregulation of CYP7A1. In addition, the bile salt pool size was reduced and intestinal cholesterol absorption was unaltered in SCP-2/SCP-x-null mice. When SCP-2/SCP-x-null mice were challenged with a lithogenic diet, a smaller increase of hepatic free cholesterol failed to suppress HMG-CoA reductase expression and biliary cholesterol secretion increased to a much smaller extent than phospholipid and bile salt secretion. Since it is known that CYP7A1 plays a central role in regulating the hepatic cholesterol balance [70], increased CYP7A1-mediated 7a-hydroxylation of cholesterol may be the most important reason for cholesterol depletion in the SCP-2/ SCP-x-null mouse liver. However, CYP7A1 induction does not result in a corresponding expansion of the bile acid pool size due to the defect in one of the final steps of the major bile acid synthetic pathway. Thus, it cannot be excluded at present that most of the cholesterol metabolism-related abnormalities that are present in this mouse model are secondary to the bile acid synthetic defect caused by absence of the thiolase rather than SCP-2.

7.8 References

7.7

Acknowledgements

The authors’ work was supported by grants from the Deutsche Forschungsgemeinschaft (grant Se 459/2 and Sonderforschungsbereich 556) and the Interdisziplinäres Zentrum für Klinische Forschung, IZKF (Project B9) of the Medical Faculty, University of Münster. 7.8

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Structure, Function, and Phylogeny of Acyl-CoA Binding Protein Susanne Mandrup *, Nils J. Færgeman *, and Jens Knudsen

8.1

Introduction

Long-chain fatty acyl-CoA (LCACoA) esters are increasingly being recognized as modulators of a wide range of cellular functions. Acyl-CoA binding protein (ACBP) a *10-kDa cytosolic protein, is named after its ability to bind C14–C20 LCACoA esters with high specificity and affinity (KD 1–10 nM). The protein was original named diazepam binding inhibitor (DBI) from its reported ability to inhibit diazepam binding to the GABA receptor [1], a function which could not be reproduced by other groups [2]. The protein, or the gene encoding the protein, has been found in all eukaryotic species, but it appears to be absent from prokaryotes with the exception of the bacteria Deinococcus radiodurans. The extremely high conservation of ACBP among all eukaryotic species and the fact that ACBP is expressed in all cells and tissues suggest that its function is associated with one or more basal cellular function(s) common to all cells. However, the precise biological function of ACBP is not known at present. A large body of in vitro experimental evidence indicates that ACBP is able to act as an intracellular acyl-CoA transporter and pool former. Recent work indicates that ACBP is required for protein sorting and vesicular trafficking in yeast [3]. A database search shows that ACBP, in addition to being a functional protein on its own, also occurs as a domain in a large number of proteins, including enzymes and potential regulatory proteins containing DNA binding motifs and/or ankyrin binding repeats and in the FERM motifs occurring in radixin, moesin, and receptor JAK kinases [4]. In this review we focus on the phylogenetic aspects and the reported functions of ACBP, which have been directly linked to its ability to bind acyl-CoA esters.

* These authors contributed equally to this work.

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8.2

The ACBP Family

The ACBP is an approximately 10-kDa protein consisting of 86–92 amino acid residues depending on the species. ACBP has a highly conserved sequence and has been found in all eukaryotic species examined, ranging from yeast and plants to reptiles, birds, and mammals. Mouse and rat ACBP have 97% identity at the amino acid level, while species as diverse as humans and Saccaromyces cerevisiae exhibit 48% identity. A BLAST database search in November 2001 with the human ACBP sequence revealed 43 basal ACBP sequences (82–92 residues) from 30 different species, published either as proteins or as gene sequences. In addition to the 43 basal ACBP sequences, the database search also identified 26 sequences where the ACBP sequence occurs as a domain in a larger protein. A phylogenetic analysis based on the sequence of the ACBP domain only (Fig. 8.1 and Tab. 8.1) shows that the basal 82–92-residue ACBP form has evolved in a large group of organisms from the yeast Schizosaccharomyces pombe to humans. The sequencing of the full genome of the nematode Caenorhabditis elegans, fruit fly (Drosophila melanogaster), Arabidopsis thaliana, and pufferfish allows a more detailed analysis of the number of ACBP genes. The fruit fly expresses one basal isoform grouping together with the ACBP isoforms from other species (Fig. 8.1). In addition to the general basal ACBP isoform, the fruit fly has evolved four additional basal isoforms (Fig. 8.1). Silkworm (Bombyx mori) also expresses two different basal ACBP forms (Fig. 8.1). In addition to the basal ACBP found in all tissues, some mammals, including rat, mouse, and cow, have evolved a testes-specific ACBP isoform, which is expressed in spermatozoa only [5] (Fig. 8.2). Interestingly, the human gene encoding this testes-specific form has undergone a frameshift and is no longer functional [6]. Ducks express two different 86-residue isoforms, a generally expressed ACBP and a brain-specific isoform [7]. Exactly how many different ACBP isoforms higher eukaryotes express are presently unknown. Silkworms also express two different basal ACBP isoforms. It is interesting that the ACBPs of the yeasts Saccharomyces monacensis and S. cerevisiae are only distantly related to that of Schizosaccharomyces pombe (Fig. 8.1). The plant ACBPs have evolved independently of the vertebrate and mammalian forms. Interestingly, this group also comprises a spider ACBP sequence. C. elegans expresses only one 86-residue basal isoform, however, this organism also expresses ACBP domain proteins with 114, 125, and 145 residues with a highly conserved ACBP domain. Whether these proteins should be regarded as basal ACBP isoforms or ACBP domain proteins with a different function is not known at present. The genomes of the pufferfish and A. thaliana express one and two ACBP basal isoform, respectively. It will be an interesting challenge for future research to elucidate the biological significance of having five very similar basal isoforms in fruit fly, only one in A. thaliana, and four isoforms of different sizes in C. elegans. The power of combining the advanced genetic tools available for C. elegans and fruit fly together with RNA-interference technology for fast and efficient gene knockdowns should make these two organisms ideal model systems to investigate the in vivo function of ACBPs.

8.2 The ACBP Family

Evolution of ACBP and ACBP domain proteins. The number attached to the protein name indicates predicted number of amino acid residues. ANK, ankyrin binding repeats; ECI, enoyl-CoA hydratase/isomerase; PECI, peroxisomal enoyl-CoA hydratase/

Fig. 8.1

isomerase ACBP, acyl-CoA binding protein; BolA, BolA-like protein. The Arabidopsis thaliana ACBP domain protein-containing kelch motifs is not included in this figure. Note that Trypanosoma brucei basal ACBP isoforms (bold) group together with ACBP domain proteins.

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8 Structure, Function, and Phylogeny of Acyl-CoA Binding Protein Tab. 8.1 Accession numbers for protein sequences used in Fig. 8.1.

Name

Accession no.

Name

Accession no.

Bovine_86_ Human_86_ Pig_86_ Dog_86_ White ear_marmoset_testis s_80_ Rat_86_ Mouse_86_ Armadillos_86_ Duck_86_ Chicken_86_ Turtle_86_ Carp_86_ Fruit_fly_86_ Fruit_fly_84a_ Fruit_fly_84b_ Fruit_fly_82a_ Fruit_fly_89_ Fruit_fly_82c_ Tobacco_hornworm_89_ Silkworm_90_ Silkworm_84_ Nematode_C. elegans_85_ Protozoan_P. falciparum_88_ Protozoan_T. brucei_92_ Digitalis_89_ Digitalis_91_ Cotton_88_ Rape_91_ Arabidopsis_91_ Castor bean_89_ Spider_F. agrestis_86_ Rice_93_

P07107 P07108 NP_065438 BAA34531 AAF79123

Pufferfish_86_ Pufferfish_87_ Bacteria_D. radiodurans_83_ Duck_brain_86_ Frog_brain_87_

JGI_35709 JGI_19661 F75551 P45882 P45883

P11030 NP_031856 P82934 S63594 S63595 S63593 AAC19408 P42281 AAF50608 AAF50610 AAF50367 AAF52610 AAF50609 P21428 AAF78042 AAF78043 O01805 PF00887 AAF75257 CAB56694 CAB56693 Q39779 Q39315 AAG31305 O04066 O22643 BAA92736

AAF79118 NP_067607 NP_067269 BAB15159 BAB14553 NP_006108 MGC2404 XP_017862 AB27836.1 AAB71197 NP_035998 BAB23735 P07106 AAF51185 AAF50834 AAF47423 AAC19408 NP_200159 T09886 T09017 AAD03482 AAF64540 T26494 Q20507 T16468 T19386 T24859

Yeast_ S. cerevisiae_86_ Yeast_S. monacensis_86_ Yeast_S. bayanus_86_ Yeast_S. pombe_86_

NP_011551 CAA69946 AAB31936 T39465

Cow_testis_86_ Rat_testis_86_ Mouse_testis_86_ Human_527_ Human_305_ Human_PECI_358_ Human4_Ank_281_ Human_407_ Mouse_Ank_281_ Mouse_444_ Mouse_PECI_358_ Mouse_504_ Cow_532_ Fruit_fly_323_ Fruit_fly_ANK_292_ Fruit_fly_262_ Carp_355_ Arabidopsis_ANK_338_ Arabidopsis_361_ Arabidopsis_ANK_353_ Arabidopsis_215_ Arabidopsis_Kelch_668 Nematode_C. elegans_114_ Nematode_C. elegans_124_ Nematode_C. elegans_145_ Nematode_C. elegans_265_ Nematode_C. elegans_ ANK_375_ Fungi_N. crassa_376_ Pufferfish_ANK_429_ Pufferfish_407_ Pufferfish_407_

T49431 JGI_6891 JGI_6573 JGI_6573

In addition to existing as an individual protein, the ACBP domain is also found as a conserved domain in larger multidomain proteins (Fig. 8.1). Whether these ACBP domains bind long-chain acyl-CoAs is presently unknown. The C. elegans, fruit fly, A. thaliana, and pufferfish genomes all encode ACBP domain proteins. A functional domain analysis using the DART program (NCBI, http:// www.ncbi.nlm.nih.gov/Structure/lexington/lexington.cgi? cmd=rps) divides the

8.2 The ACBP Family Fig. 8.2. Three-dimensional structure

of recombinant bovine ACBP in complex with palmitoyl-CoA seen from above (A) and from the side (B). The protein is shown in a ribbon presentation. The four a-helices are A1 of residues 3–15 (gray), A2 from residue 21– 36 (light blue), A3 from 51–62 (light yellow), and A4 from residue 65 to 84 (light green). The palmitoyl chain is shown in red and the CoA head group in green.

ACBP domain proteins into three groups. The first group contains both an ACBP domain and an enoyl-CoA isomerase domain. These proteins are all peroxisomal D2-D3-enoyl-CoA-isomerases (PECI) required for peroxisomal b-oxidation of unsaturated fatty acids [8] (Fig. 8.1). The function of the ACBP domain in these enzymes is unknown; it might either present the substrate for the isomerase or participate in the catalytical process. Preliminary data show that human PECI is active without the ACBP domain (our unpublished results), indicating that the ACBP domain is not part of the active site. The second group of proteins contains both an ACBP domain and one or more ankyrin binding repeats. These proteins are not only encoded by all four abovementioned genomes but also by the mouse and human genome. Ankyrin binding repeats are known to be involved in specific protein–protein interaction, and thereby target the ACBP–ankyrin repeat proteins to specific cellular sites The function of these proteins is presently unknown. In addition to the ACBP domain and the ankyrin domain, the human ACBP 282 contains a potential nuclear targeting signal sequence. The C. elegans ACBP 385 ankyrin repeat protein also contains a highly conserved BolA DNA binding domain in addition to the two other domains. This protein might represent an acyl-CoA regulated transcription factor, like the E. coli transcription factor FadR [9, 10].

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The third group of genes encodes potential ACBP domain proteins with 145– 504 residues, which contain no other known functional domains. The A. thaliana genome contains a gene encoding a hypothetical 668-residue protein containing three kelch domains. The kelch domain was first identified as the fruit fly ring channel protein [11], and has since been found in an extensive family of protein structures with diverse functions. A structure similarity search using the program Finding 3-D Similarities in Protein Structures (http://cl.sdsc.edu/) shows that a perfect matching ACBP domain also appears as a subdomain in the FERM domain present in ridixin, which plays a role in the formation of membrane-associated cytoskeleton by linking actin filaments and adhesion proteins [4].

8.3

ACBP Structure and Ligand Binding Specificity

The three-dimensional structures of bovine and Plasmodium falciparum ACBP (PfACBP) have been solved by both NMR and X-ray crystallography and by X-ray crystallography, respectively [12–14]. The bovine ACBP crystal and NMR structures are almost identical and overall very similar to the PfACBP crystal structure. The fold of the peptide backbone of bovine ACBP shows the protein as an updown-down-up four a-helix bundle with an overhand loop connecting helix A2 and A3 (Fig. 8.2). The bundle arrangement of ACBP is unique amongst known four-helix folds. The bundle arrangement is skewed, since helix A3 is disjoint to helix A1 and A4, resulting in just four helix–helix interfaces, instead of the usual six seen in the well-known supercoiled four-helix bundles. The structure of bovine ACBP is shaped as a relatively flat disc. Bovine ACBP, yeast ACBP, and ACBP from the plant Arabidopsis thaliana bind saturated and unsaturated C14–C22 acyl-CoA esters in a one-to-one binding mode with high specificity and affinity (KD = 1–15 nM) [15–18]. Free CoA binds with a KD of 2 lM, [19]. ACBP does not bind fatty acids, acyl carnitines, cholesterol, or nucleotides [16]. The structural basis for the high-affinity binding of LCACoA to ACBP was studied by solving the structure of palmitoyl-CoA in complex with bovine ACBP using NMR spectroscopy. The structures of apo and holo forms of ACBP are practically identical [20]. No distances in holo ACBP are significantly longer than the ones in apo ACBP, but the C-terminal of A4 is significantly closer to the C-termini of A2 and A3 and the area around Phe49. This suggests that binding of ligand induces a tightening of the structure of bovine ACBP. The ligand binding site of ACBP is divided distinctly into three subsites: one for the adenine ring, one for the 3'-phosphate, and one for the palmitoyl part of the ligand. The first two sites are similar in the bovine ACBP and PfACBP. The 3'phosphate group, which contributes with 40% of the total binding energy, interacts strongly with ACBP through a massive network of hydrogen bonds and salt bridges to Tyr28, Lys32, and Lys54. The aromatic ring of Tyr31, which stacks with the adenine ring, and which is structurally supported by the aromatic rings of Tyr73 and Phe5, forms the hydrophobic pocket.

8.4 Regulation of ACBP Expression

The x-end (C12–C16) of the palmitic acid acyl chain makes several non-polar interactions with residues in the cleft between helices A2 and A3, especially with the side-chains of Met24, Leu25, and Ala53. Comparison of the high-resolution Xray structures of the PfACBP and bovine ACBP crystal structures reveals a number of minor differences between the two molecules [14]. The insertion of two additional residues in the loop between a-helix 1 and a-helix 2 in PfACBP, together with the alterations Ala53Lys, Lys50Ile, and Asp21Asn, change the binding pocket and close the tunnel at the end of the acyl-chain, with the result that PfACBP exhibits a preference for a shorter ligand (C14). This modification in PfACBP chain length specificity might have occurred in order to insure the synthesis of massive amounts of the di-C14:0-GPI-anchored protein coat lining to protect this parasite. This indicates that minor changes in ligand preference might have major biological significance. In this connection it is interesting that the sequence differences Asn19Ser and Glu23Ala in the two A. thaliana ACBP isoforms change the preference for unsaturated fatty acids (our unpublished results). Thus, although the general overall pattern of chain length specificity looks similar for all ACBPs, the biological importance of small changes in ligand preference for optimum chain length and degree of saturation should not be overlooked.

8.4

Regulation of ACBP Expression 8.4.1

Genomic Organization in Mammals

So far, the genomic structures of the rat [21], the human [22], and the mouse (our unpublished results) ACBP genes are known. In addition, a number of processed pseudogenes have been characterized [21, 23]. Cumulatively, these data show that the mammalian ACBP gene has all the hallmarks of a typical housekeeping gene, i.e. a 5' CpG island, several transcriptional initiation sites, and processed pseudogenes [21]. The gene covers approximately 8 kb and is composed of four exons, which give rise to a transcript of 0.45 kb. An alternative transcript generated by insertion of an additional exon between exon 1 and 2 has been isolated from several human cell types. Using the ATG codon in exon 1, this alternative transcript has the potential to encode a protein of 67 amino acids, which, since the reading frame is shifted by insertion of the alternative exon, would be totally unrelated to ACBP except for the three first amino acids. Alternatively, using the downstream ATG codon in the alternative exon, a protein of 104 residues, which shares the last 84 residues with ACBP, can be encoded [22]. However, the existence of these proteins has not been demonstrated. Interestingly, although the sequence of the region encompassing the alternative exon is highly conserved between humans and rodents, we have found no evidence of alternative splicing either in rat or in mouse cells (our unpublished results). In addition, the reading frame in this alternative exon is not conserved between humans and rodents [23]. Thus, it appears

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that the high conservation of this region cannot be explained by evolutionary conservation of alternative splicing. 8.4.2

Expression Pattern in Mammals

In keeping with the housekeeping gene characteristics of the mammalian ACBP gene, ACBP appears to be ubiquitously expressed from early stages of mammalian embryogenesis [24] as well as in adult tissues (reviewed in Ref. [25]). However, the level of ACBP differs markedly among different cell types. High concentrations are found in steroid-producing cells (glomerulosa and fasciculate cells of adrenal cortex, leydig cells of testis) [26], keteratinocytes, and cells from sweat and sebaceous glands [27]. Lower concentrations are found in epithelial cells involved in water and electrolyte transport (intestinal mucosa, distal convoluted tubules of kidney) [26]. Interestingly, fruit fly ACBP is also highly expressed in potassiumtransporting cells in the urine-secreting Malpighian tubules [28]. Hepatocytes contain moderate amounts of ACBP, however, the total amount of ACBP in liver is relatively high due to the diffuse presence of ACBP in all hepatocytes. Also brain exhibits differential expression with highest expression in area postrema, the cerebellar cortex, and ependyma of the third ventricle [29]. The expression pattern described above suggests that ACBP expression is more linked to secretion, ion and water transport, and energy metabolism than lipid synthesis. The expression level of the mammalian ACBP is slightly affected by feeding status. Fasting rats for 24 hours resulted in a 33% decrease in liver ACBP levels [30] and in reduced ACBP mRNA level as well [31], whereas the level in heart and kidney was unaffected. High-fat diet for 48 hours increased liver ACBP levels by 36%. Hepatic levels of ACBP continued to increase and remained elevated with prolonged exposure to high fat (28 days). Heart ACBP did not respond to shortterm fat feeding but was increased after prolonged exposure [30]. Androgens, which stimulate growth of the human prostate cancer cell line LNCaP [32], also stimulate de novo fatty acid synthesis, cholesterol synthesis, and lipid accumulation and induce ACBP expression in this cell line [33]. Similarly, androgens induce ACBP expression in several accessory sex organs in the male rat [34]. ACBP expression is also significantly induced during in vitro differentiation of 3T3-L1 pre-adipocytes [35], a process that is accompanied by a marked triglyceride accumulation and de novo fatty acid synthesis. This could indicate that ACBP expression is linked to lipid synthesis. However, both growing LNCaP cells and differentiating 3T3-L1 cells undergo dramatic structural and functional changes, and it is therefore possible that other functions besides general lipid synthesis require increased expression of ACBP in these cells. Testis-specific ACBP (ELP) is highly expressed in the late haploid stage of male germ cell development only, with the first immunohistochemical staining being present in the elongated spermatid [5, 36]. During the elongation process and spermatozoa formation, the spermatid undergoes dramatic morphological changes. This could indicate a role for ELP in membrane remodeling. In primary

8.4 Regulation of ACBP Expression

cerebella, astroglial, and C-6 cells the highest expression of ACBP was observed in actively dividing cells [37]. Finally, it has been reported that acute stress induces increased ACBP content in selected areas of rat brain [38, 39]. 8.4.3

Transcriptional Regulation of the Mammalian ACBP Gene

The sterol regulatory element binding protein (SREBP-1)/adipocyte determination and differentiation factor 1 (ADD1), which has been shown to be involved in the coordinated induction of fatty acid synthesis and glycolysis by insulin in the liver [40] and in the androgen-induced lipogenic gene expression in LNCaP cells [41], is likely to play a key role in regulation of ACBP expression in liver. A functional sterol regulatory element (SRE) has been identified in the proximal promoter of both the human and rat ACBP gene [42] (our unpublished results) (Fig. 8.3). However, it still remains to be shown that this SRE is functional in a chromatin context. The activation of ACBP expression during adipocyte differentiation is likely to be mediated primarily by the peroxisome proliferator activated receptor c (PPARc) with SREBP-1 playing a more modulatory role. We have recently identified a peroxisome proliferator response element (PPRE) in intron 1 of the rat ACBP gene (Fig. 8.3). The element is functionally conserved in the human gene, it binds PPARc/RXR in the chromatin context and mediates inducibility by PPARc-specific ligands in adipocytes [43]. Interestingly, the element is located in a highly conserved region of in-

Model illustrating the transcriptional regulation of ACBP expression by PPAR/RXR and SREBP. Regulation of ACBP expression in response to feeding and fasting may be mediated via SREBP-1 homodimers binding to a SRE in the proximal promoter. The adjacent NFY binding site is necessary for the activation by SREBP-1. The ACBP gene is also regulated by PPAR/RXR heterodimers, which bind to a PPRE in intron 1 and mediates acti-

Fig. 8.3

vation by PPAR agonists. Thiazolidinediones (TZD) activate PPARc, whereas fibrates like clofibrate and bezafibrate activate PPARa. In addition, activation of ACBP expression during adipocyte differentiation is likely to be mediated by PPARc/RXR. Numbers refer to the position of the respective regulatory elements and the start of exon 2 relative to the ATG start codon in the rat gene.

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tron 1, suggesting that the presence of a PPRE and possibly other regulatory elements could explain the high conservation of the intronic region. Since ACBP expression in the liver is downregulated by fasting, a process during which most other PPARa target genes are induced [44, 45], ACBP does not appear to belong to the group of traditional PPARa target genes. However, ACBP expression is increased in rat liver by high fat feeding [30], and by various peroxisome proliferators [31, 46], which are known to induce liver mitochondrial and peroxisomal b-oxidation by virtue of their ability to activate PPAR. The failure to respond to the increased PPARa activity during fasting conditions indicates that the regulation by SREBP-1 and other transcriptional regulators overrides that of PPARa.

8.5

Expression Profile in Other Eukaryotes

Expression profiles of ACBP from other eukaryotes support the notion that ACBP could be involved in lipid metabolism, water and ion transport, energy metabolism, and membrane remodeling or all of these. Determination of ACBP expression profiles in different tissues of the fruit fly, using rabbit–rat anti-ACBP, show that the expression is high in cardia, in part of the potassium-secreting Malpigian tubules, in the fat body, and in the gametes of both sexes [28]. However, these results should be treated with caution because fruit flies express five short and three long ACBP isoforms (our unpublished data). Interestingly, the mRNA level of one of these isoforms, (gene product CG15829 with the potential to encode an 82-residue protein) is specifically induced for 1.5 hours after bacterial infection [47]. After 6 hours the mRNA level is still increased 3-fold and after 12 hours the mRNA level is back to normal. No change in mRNA level of this particular ACBP isoform is seen during fungal infection. This adds a new and interesting dimension to the possible function of ACBP. ACBP is expressed at high levels in the larval midgut of fruit fly [28], the tobacco horn worm (Manduca sexta) [48], and silkworm [49]. The larval midgut is involved in fatty acid absorption, and in the tobacco horn worm the expression in the midgut appears to be increased by feeding [50]. It is therefore tempting to speculate that ACBP plays a role in fatty acid absorption. However, in rat intestine high expression of ACBP is found in water and electrolyte-transporting cells (see above). The silkworm expresses at least two different ACBPs. The pheromone gland form (pg-ACBP) is expressed at very high levels in the pheromone gland, at low levels in most larval tissues, in adult fat body and in ovary. Midgut ACBP (mg-ACBP) is only expressed in the midgut of larvae and in the pheromone gland of the adult female [49]. The pheromone gland contains large lipid droplets containing a mixture of triglycerides also containing D10,12 hexadecadienoate, a precursor for the sex pheromone (E,Z)-10,12-hexadecadiene-1-ol (bombykol), which is synthesized from palmitoyl-CoA [49]. This would indicate a role of mg-ACBP in the synthesis of pheromones and pheromone precursors by ensuring the supply

8.7 Regulation of Long-chain Acyl CoA Concentrations in vivo

of palmitoyl-CoA and shuttling of acyl-CoA intermediates in the pheromone synthetic pathway. Thus, like in mammalian spermatozoa, it appears that specialized ACBP isoforms may have specific functions in specialized tissues, where they are highly expressed. In Brassica napus (oil seed rape) and in Arabidopsis thaliana, ACBP is expressed in a wide range of tissues at similar levels [51, 52]. One exception is developing seed where the ACBP content is increased to very high levels during seed development [52]. However, the amount of ACBP did not correlate with the rate of lipid metabolism in developing embryos or cotyledons of seedlings, although it was absent from dry seeds [51]. A recent study showed that ACBP was able to relieve product inhibition by acyl-CoA esters on fatty acid synthesis in isolated plastids from oil seed rape [51] and to increase the activity of glycerol-3-phosphate acyltransferase in vitro [53].

8.6

Subcellular Localization

Like other intracellular lipid-binding proteins, ACBP was originally thought to be confined to the cytosol. However, it has recently been shown using immunofluorescence and confocal microscopy as well as immunoelectron microscopy that a considerable amount of ACBP localizes to the nucleus in several cell lines [54, 55] and in liver [55]. Similarly, immunogold electron microscopy has shown that yeast ACBP, Acb1p, is evenly distributed over the cytosol and nuclei but is not detected over mitochondria and in peroxisomes (our unpublished results).

8.7

Regulation of Long-chain Acyl CoA Concentrations in vivo

In order to evaluate the physiological relevance of the regulatory effects of LCACoA esters, it is of great importance to consider the intracellular concentration of these esters. The total cellular concentration of LCACoA esters has been reported to be in the range of 5–160 lM, depending on the tissue and its metabolic state (Ref. [56] and references therein). The size of the intracellular pool of LCACoA esters is determined by the rates of fatty acyl-CoA synthesis and utilization. While acylCoA synthesis is largely determined by the rate of activation by acyl-CoA synthetases of either imported fatty acids, endogenously synthesized fatty acids, or fatty acids from lipolysis of cellular lipids, utilization is determined by the rate of degradation by b-oxidation, incorporation into cellular lipids, acyl-CoA hydrolysis, and protein acylation. While the total cellular concentrations of LCACoA esters have been determined (see Ref. [56] for a review), the concentration of free unbound LCACoA available for metabolism and regulatory purposes is not known. The Dr Jekyl and Mr Hyde character of LCACoA esters suggests that the concentration of LCACoA esters must be tightly controlled. In this regulatory

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scheme, intracellular acyl-CoA binding proteins and acyl-CoA hydrolases are assumed to play important roles. A number of reports suggest that the ratio of LCACoA to ACBP is close to one [31, 57]. Based on the reported in vitro binding affinity and the cellular level of ACBP it can be calculated that the unbound concentration of LCACoA would be in the low nanomolar range when the acyl-CoA/ACBP ratio is below one [56]. LCACoA esters can also bind to a number of other proteins in the cell including the high affinity-binding site on acyl-CoA synthetase and acyl-CoA-utilizing enzymes, or they can be compartmentalized in cellular organelles. A number of predicted ACBP domain proteins may also contribute to the regulation of the intracellular concentration of LCACoA esters. It is likely that these domain proteins bind LCACoA esters with similar affinity as ACBP, and thus exchange LCACoA esters with ACBP thereby creating local pools of acyl-CoA esters. Acyl-CoA hydrolases are also believed to contribute to the regulation of the size of the different acyl-CoA pools [58]. Acyl-CoA hydrolases are found in most subcellular compartments and include short-, medium-, and LCACoA hydrolases (see Ref. [56] and references therein) [59, 60]. Acyl-CoA hydrolases usually display Km values ranging from 0.1–6 lM for LCACoA esters [61–63]. Some acyl-CoA hydrolases have been shown to be induced by ingestion of hypolipidemic drugs (see Ref. [56] and references therein) [60, 64, 65], which also induce increased acylCoA levels and ACBP in rat liver [31, 66, 67]. It is very likely that acyl-CoA hydrolases could act as “scavengers” if the free LCACoA pool rises to unacceptable levels. This is substantiated by the observation that the psychrophilic Antarctic yeast Rhodoturula aurantiaca is not viable at nonpermissive temperatures due to inactivation of a LCACoA thioesterase and concomitant accumulation of myristoyl-CoA [68]. In this scenario it should be borne in mind that the presence of acyl-CoA hydrolases also ensures that the pool of coenzyme A is not exhausted. In yeast, a peroxisomal acyl-CoA hydrolase (Tes1p) is believed to act when acyl-CoA esters accumulate to prevent depletion of the peroxisomal CoA pool required for oxidation of fatty acids [59]. Likewise, the presence of acyl-CoA hydrolases in mitochondria prevents depletion of the mitochondrial CoA pool, ensuring mitochondrial b-oxidation, whereas the presence of acylCoA hydrolases in the nucleus prevents harmful levels of acyl-CoA esters and ensures CoA for the synthesis of acetyl-CoA for histone acetylation and ultimately transcriptional activation. Hence, acyl-CoA hydrolases not only prevent accumulation of LCACoA, but also function as a valve ensuring sufficient concentrations of CoA for CoA-utilizing systems. It is in this context that the function of ACBP should be seen. The presence of a high cellular acyl-CoA hydrolase activity requires either high-affinity acyl-CoA binding proteins to act as acyl-CoA pool formers and transporters or direct channeling of acyl-CoA from acyl-CoA synthetases to acyl-CoA-utilizing enzymes. This ensures low free acyl-CoA concentrations and plenty of acyl-CoA esters for metabolic and regulatory purposes. The fact that fatty acid synthesis occurs despite the fact that the Ki for inhibition of acetyl-CoA carboxylase is 5.5 nM, clearly indicates that the cytosolic free acyl-CoA concentration in this situation is at least below the 5 nM range.

8.8 Functions of ACBP

The dynamic range of this regulatory system in vitro and in vivo is not known. The concentration of circulating free unbound fatty acids is believed to be kept in the range of 1–10 nM by circulating albumin [69]. Whether an increase in circulating fatty acids is able to induce an increase in intracellular free LCACoA concentrations is not known. In this context it is interesting that fasting induces increased levels of circulating fatty acids, increased cellular levels of LCACoA, increased expression of cytosolic and mitochondrial acyl-CoA hydrolases, and decreased expression of ACBP (see above). ACBP efficiently protects acyl-CoA from hydrolysis by cellular acyl-CoA hydrolases [57, 70, 71]. This suggests that even the reduced level of ACBP, despite increased expression of acyl-CoA hydrolases during fasting, is able to support the increased flux of fatty acids through the acylCoA pools, without increasing the cytosolic and nuclear free acyl-CoA concentrations.

8.8

Functions of ACBP 8.8.1

Clues obtained from in vitro Studies

In vitro, ACBP has a strong attenuating effect on the inhibition by LCACoA esters of acetyl-CoA carboxylase and the mitochondrial adenine nucleotide translocase, and ACBP stimulates the mitochondrial long-chain acyl-CoA synthetase [72]. Bovine liver ACBP is able to extract acyl-CoA esters from multilamellar liposomes immobilized on one nitrocellulose membrane, transport the extracted acyl-CoA to a different membrane, and donate it to mitochondrial b-oxidation and to microsomal glycerolipid synthesis (Fig. 8.4) [72]. Carnitine palmitoyl-transferase 1 (CPT1) efficiently uses the acyl-CoA/ACBP complex as substrate [72–74], and kinetic studies indicate that CPT1 prefers the acyl-CoA/ACBP complex over free acyl-CoA as substrate [74]. This strongly suggests a role of ACBP in b-oxidation. ACBP efficiently protects acyl-CoA from hydrolysis by cellular acyl-CoA hydrolases [57, 70, 71], indicating a role of ACBP as acyl-CoA pool former. Overexpression of either bovine or yeast ACBP in Saccharomyces cerevisiae led to an increased intracellular acyl-CoA level, supporting the in vivo acyl-CoA pool-forming function of ACBP [18, 75]. In vitro ACBP stimulates incorporation of arachidonic acid from arachidonoylCoA into phospholipids by the acyl-CoA-lysophospholipid acyltransferase in red blood cells at low arachidonoyl-CoA concentrations [76]. However, the effect of ACBP on glycerolipid synthesis in vitro is less clear [70, 71] and depends on the ACBP and acyl-CoA used. The effect of ACBP on glycerol-3-phosphate acyltransferase activity depends on the concentration ratio of ACBP/acyl-CoA [51, 57]. At low substrate concentrations, ACBP inhibits phosphatidic acid synthesis. At higher acyl-CoA concentrations, ACBP initially stimulates phosphatidic acid synthesis, and after reaching an optimum phosphatidic acid synthesis drops at higher ACBP

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Model of proposed functions of ACBP. Fatty acids from exogenous sources or endogenously synthesized are activated to LCACoA and incorporated into triglycerides (TG) or phospholipids (PL) or bound to ACBP. ACBP has been shown to be able to

Fig. 8.4

protect LCACoA esters from hydrolysis by thioesterases (TE), to be involved in lipid remodeling and vesicular trafficking. In addition, ACBP may donate LCACoA to b-oxidation and other metabolic pathways and to regulation of gene expression.

concentrations. These data indicate that ACBP can relieve the inhibitory effect of acyl-CoAs on the enzyme activity and that increasing concentrations of ACBP efficiently compete with the enzyme for acyl-CoA esters. To what extent glycerol-3phosphate acyltrasferase can use the acyl-CoA/ACBP complex as substrate is not fully known. However, the fact that ACBP in 15-fold excess of acyl-CoA can desorb acyl-CoA from an immobilized phospholipid bilayer and subsequently transport and donate the bound acyl-CoA to glycerol-3-phosphate acyltransferase in microsomes immobilized on a different membrane, strongly indicates that ACBP can donate acyl-CoA to glycerol-3-phosphate acyltransferase [72]. The regulatory functions of the ACBP/acyl-CoA complex were investigated in experiments with the ryanodin receptor Ca2+ release channel from rabbit muscle terminal cisternae. This channel has been shown to be activated by palmitoyl-CoA in the micromolar range [77]. Addition of 6 lM palmitoyl-CoA in the presence of 6.6 lM bovine ACBP to the terminal cisternae did not affect Ca2+ release, but significantly reduced the rate of reuptake of an added Ca2+ pulse. However, preincubation of the terminal cisternae membranes with increasing concentrations of palmitoyl-CoA/ACBP complex strongly potentated caffeine-induced Ca2+ release. This

8.9 Acyl-CoA esters, ACBP, and PPARs

effect was proportional to the concentration of the complex and independent of the calculated concentration of unbound palmitoyl-CoA [78]. These results strongly indicate that the acyl-CoA/ACBP complex can either donate acyl-CoA directly to the ryanodine receptor or act as a regulator of the receptor itself. 8.8.2

In vivo Functions in Mammals

The high evolutionary conservation of ACBP as well as the ubiquitous expression in mammals suggests that ACBP is involved in basal cellular functions in eukaryotic cells. While in vitro studies clearly define multiple metabolic pathways in which ACBP could take part, it is still unknown to what extent ACBP actually plays a role in these pathways in vivo. Thus, although in vitro studies suggest a role of ACBP in esterification of fatty acids into glycerolipids, clones of the McARH 7777 rat hepatoma cell line overexpressing ACBP only showed a marginal increase in esterification compared with control cells [79]. However, great care should be taken in the interpretation of these results since they are based on a single cell line and on selection of individual clones. ACBP expression is significantly upregulated during adipocyte differentiation, and expression of high levels of ACBP antisense RNA in the 3T3-L1 pre-adipocyte cell line decreased endogenous ACBP levels, expression of the adipogenic transcription factors C/EBPa and PPARc, and of accumulation of triacylglycerides [80]. The fact that this inhibition could be relieved by addition of synthetic PPARc ligands made us speculate that ACBP might belong to the group of genes involved in the synthesis of endogenous PPARc ligands. However, at present it is not known why reduced ACBP expression blocks differentiation of 3T3-L1 cells. Further insight into the function of ACBP in this context will have to await targeted disruption of the ACBP gene in mice or construction cell lines where the expression of ACBP can be regulated in time.

8.9

Acyl-CoA esters, ACBP, and PPARs

Whereas fatty acids and fatty acid derivatives, in particular products of the cyclooxygenase and lipoxygenase pathways, are known to activate members of the PPAR family, acyl-CoA esters may be natural antagonists of the PPARs. In a recent study, several acyl-CoA esters were reported to bind directly to PPARa and PPARc and to inhibit the recruitment of co-activators in vitro [81]. In another study a non-hydrolyzable C16:0 acyl-CoA analog was shown to behave as a PPARa and PPARd antagonist in vitro by decreasing the interaction with the response element, increasing the interaction with co-repressors and decreasing the interaction with co-activators [82, 83]. Despite the convincing in vitro data it still remains to be shown that CoA esters can act as antagonists in vivo.

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To investigate how the level of ACBP affects the ability of fatty acids to activate the different PPARs we transiently overexpressed ACBP in CV-1 cells and measured the ability of the PPAR ligand, tetradecylthiaacetic acid (TTA), to activate the different PPARs. We found that increased ACBP expression inhibited the ability of TTA to activate all PPAR subtypes. The mechanism by which ACBP causes this effect is not clear. One possibility is that the increased acyl-CoA buffering capacity introduced by increased levels of ACBP allows an increased flux of fatty acids through the acyl-CoA pool and thereby increases the rate of TTA esterification and decreases the available concentration of TTA for PPAR activation.

8.10

ACBP in African trypanosomes (T. brucei)

Trypanosoma brucei expresses a slightly different ACBP with increased affinity for C10 to C14 acyl-CoA esters. T. brucei are shielded from their host’s defense by a coat of variant surface glycoproteins molecules, each attached to the plasma membrane by a glycosylphosphatidylinositol anchor. The glycosylphosphatidylinositol anchor undergoes a remodeling step from a di-palmitoyl to a di-myristoyl derivative during synthesis [84]. It has been demonstrated that T. brucei ACBP enhances the fatty acid remodeling of the GPI anchor in vitro [84]. Interestingly, ACBP was shown to be essential for T. brucei viability, using targeted homologous recombination [85].

8.11

Functions, and Lessons from Yeast

ACBP was originally identified on its ability to induce synthesis of medium-chain acyl-CoA esters by goat mammary gland fatty acid synthase in vitro [86]. Likewise, ACBP in the yeast Saccharomyces cerevisiae, Acb1p, has been shown to facilitate removal of newly synthesized acyl-CoA esters from the yeast fatty acid synthase in vitro [87], which is consistent with the observation that depletion of Acb1p in S. cerevisiae results in accumulation of C18:0-CoA and diminished levels of C14:0-CoA. Moreover, disruption of Acb1p resulted in reduced levels of unsaturated acyl-CoA esters like C16:1-CoA and C18:1-CoA, implying that Acb1p is involved in intracellular acyl-CoA pool formation. Despite changes in the acyl-CoA composition, depletion of Acb1p in S. cerevisiae does not affect general glycerolipid synthesis and glycerolipid turnover [3]. This indicates that Acb1p, which is 49% identical to human ACBP, is not required for general lipid synthesis in yeast. Fatty acid composition was only slightly affected, and only the level of C26:0 fatty acid was significantly reduced. However, mass spectrometric analysis of plasma membrane lipids revealed that the relative levels of the sphingolipids IPC and MIPC were 25–40% increased in Acb1p-depleted cells, and that the relative levels of lysophosphatidic acid (LPA), lysophosphatidylserine, and lysophosphatidylinositol were 1.7- to 2.2-fold in-

8.12 Conclusions and Future Directions

creased in plasma membranes from cells exhausted of Acb1p [3]. The increased level of LPA was caused by an increase in the unsaturated LPA species only and was accompanied by a large decrease in the content of C16:1/C18:1-PA. This could indicate that Acb1p is required for delivery of specific acyl-CoA esters for synthesis of particular phospholipid species in, for example, the plasma membrane. The observation that non-hydrolyzable LCACoA ester analogs inhibited homotypic vacuole fusion [88], budding and fusion of transport vesicles [89–91], as well as homotypic vacuole fusion [92], lends credence to the suggestion that acyl-CoAs are used for fatty acylation of either a protein and/or a lipid required in the fusion process. Acb1p-depleted yeast exhibits strongly perturbed plasma membrane structures, accumulation of 50–60 nm vesicles and autophagocytotic-like bodies [3]. Furthermore, the strain exhibits multilobed vacuoles, which are unable to undergo homotypic vacuole fusion in vitro (our unpublished results). These results strongly imply that ACBP exerts a function in vesicular trafficking most likely by donation of acyl-CoA to either protein or lipid acylation (Fig. 8.4).

8.12

Conclusions and Future Directions

Long-chain acyl-CoA esters are key players in many cellular functions, including lipid metabolism, membrane remodeling, protein acylation, pheromone synthesis, and cell signaling (Fig. 8.4). It is therefore to be expected that ACBP, due to its ability to bind acyl-CoA esters with very high affinity and specificity, participates in multiple cellular functions. The experimental evidence strongly indicates that ACBP is able to create an intracellular protected pool of acyl-CoA esters. Together with acyl-CoA thioesterases ACBP is likely to play a key role in regulating cytosolic free acyl-CoA concentrations and thereby also influence acyl-CoA-regulated functions. Very little is known about to what extent this pool is available for, or required for, general lipid synthesis. Investigations with yeast indicate that ACBP is not required for general glycerolipid synthesis in this organism. Whether the same is the case in higher eukaryotes is not known, and in this context it should be kept in mind that cytosolic thioesterases have not been identified in yeast. However, it would not make sense that an acyl-CoA synthesized by an ER-associated acyl-CoA synthetase should pass over cytosolic ACBP in order to be donated to the first enzyme in glycerolipid synthesis, a-glycerol-P-acyltransferase in the same membrane. Kinetic studies with CPT1 indicate that the acyl-CoA/ACBP is the preferred substrate for CPT1. This observation indirectly indicates a role for ACBP in b-oxidation, however, this suggestion does not correlate with the expression pattern, which groups the ACBP gene together with genes involved in lipid synthesis in mammals. Yeast data demonstrate that ACBP is involved in membrane remodeling and protein acylation and is required for normal vacuole fusion to occur. The large differences in ACBP expression level between different cell types in multicellular organisms indicate that ACBP, in addition to its general housekeep-

167

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ing functions, have acquired specialized functions in some cell types. In addition, the tissue-specific expression of five basal 82–92-residue ACBP isoforms in fruit fly further indicate that ACBP have acquired even more specialized functions by gene duplication and development of biochemically distinct isoforms. The advanced molecular genetic tools developed for this organism make fruit fly a powerful model system for studying ACBP function. Finally, the identification of multidomain proteins containing an ACBP domain fused to other domains, such as ankyrin binding repeats and DNA binding domains, indicates that these proteins could be components in hitherto unknown acyl-CoA-regulated signal pathways.

8.13

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Structure and Function of PPARs and their Molecular Recognition of Fatty Acids Colin N. A. Palmer

9.1

PPARs as Nuclear Receptors

Peroxisome proliferation is an adaptive response to lipid overload, where the size and number of peroxisomes are increased [1]. This provides a greater capacity for b-oxidation of fatty acids in the peroxisomes. This increase in peroxisomes is therefore accompanied by increases in the expression of enzymes involved in lipid oxidation and includes the regulation of peroxisomal and non-peroxisomal enzymes. One protein that is very highly induced during peroxisomal proliferation is the microsomal fatty acid x-hydoxylase (CYP4A) [2–4]. The dicarboxylic acids formed by this group of enzymes are thought to be preferentially targeted to the peroxisome for b-oxidation. The peroxisomal b-oxidation enzymes such as acylCoA oxidase, and the enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase are also induced during peroxisomal proliferation [5]. Peroxisome proliferation has largely been characterized as a pathological response to a wide range of structurally dissimilar compounds which are collectively known as peroxisome proliferators [6]. This group of compounds includes certain pesticides, plasticizers, adhesives, fuel additives, and the fibrate class of lipid-lowering drugs. Indeed most of the compounds classed as peroxisome proliferators have lipid-lowering effects in rodents, and this appears to be through the activation of hepatic lipid oxidation; however, this phenomenon is mainly studied as it results in liver cancer, and the vast bulk of the research into this phenomenon was performed in toxicology laboratories until the mid-to-late 1990s. Indeed it was in the toxicology laboratories of AstraZeneca in Macclesfield that Isseman and Green characterized the receptor for peroxisome proliferators [7]. Up until this point it was thought that these compounds worked through a non-receptormediated mechanism [8]. Several investigators had failed to characterize a receptor through classical biochemistry. In the late 1980s it was thought that the receptor for another group of xenobiotics, the planar aromatic hydrocarbons, was related to the receptors for the sex hormones. This was because the biochemical characterization of the mode of action of this receptor suggested a very similar mechanism of action [9]. The molecular cloning of the receptors for the sex hormones had revealed the presence of a superfamily of receptors with unknown

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function (orphan receptors). These receptors contained a recognizable DNA binding domain containing two conserved zinc fingers and a ligand binding domain that was rather less conserved. One approach to screening for novel ligands for orphan receptors was to make chimeric receptors using the DNA binding domain of the glucocorticoid receptor and the ligand binding domain of the orphan receptor. This would allow the detection of an “orphan” transcriptional signal through a reporter system based on the glucocorticoid response element. One such screen yielded a murine orphan receptor that was clearly activated by the diverse spectrum of compounds classed as peroxisome proliferators [7]. This receptor was named the peroxisome proliferator activated receptor (PPAR). Studies in Xenopus laevis revealed a subfamily of three receptors that were clearly similar to the mouse PPAR. The one which was most closely related to the mouse form was designated PPARa, with the other two forms being designated PPARb and PPARc [10]. Subsequent cloning has confirmed the conservation of this subfamily from frog to humans, and it has been shown that these receptors are expressed in an overlapping yet distinct range of tissues and cell types [11]. Most research has concentrated on the role of PPARa in the liver and PPARc in adipocytes [12–14]. The activation of these receptors by fatty acids was demonstrated [15–19], and detailed binding analysis of fatty acids to the human isoforms has been performed [20–24]. However, the nature of the endogenous ligands is still unclear and the relative role that different nutritionally derived fatty acids have in the activation of different PPAR isoforms has not been elucidated as yet. This chapter gives an account of what is known about the mechanisms by which PPARs activate gene transcription in response to fatty acid and drug signals.

9.2

DNA Binding

Shortly after the molecular characterization of PPARa, the region of the peroxisomal acyl-CoA oxidase gene promoter that was reponsible for the transcriptional activation by clofibrate was characterized using the rat hepatoma cell line H4IIE [25]. The region of DNA that conferred a response to clofibrate contained recognizable nuclear receptor binding motifs or “half-sites”, AGGTCA, and contained a repeat of two half-sites in the same direction (direct repeat) separated by one nucleotide. Several groups immediately synthesized oligonucleotides corresponding to this enhancer element and confirmed that it was indeed a functional PPAR response element in transactivation assays [19, 26]. Peroxisome proliferator response elements were then characterized in the genes encoding a host of fatty acid metabolizing enzymes such as CYP4A6 [27] and the enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase enzyme [28]. However, evidence of direct binding of PPAR to these elements was not provided in the initial reports. The reason for this was made clear when Steven Kliewer described how PPAR required a heterodimeric partner for binding to DNA. This heterodimeric partner

9.2 Binding

was the retinoid X receptor (RXR) [29]. This is a promiscuous partner that facilitates the binding of several nuclear receptors to DNA, including the thyroid and vitamin D receptors (TR, VDR). RXR is absolutely required for the binding of PPAR to DR1 motifs and this heterodimerization requires the ligand binding domain of both receptors. Deletion of the N-terminal AB domain of PPAR unmasks a monomeric DNA binding capability, where the truncated PPAR can bind to single half-sites as a monomer [30]. These studies revealed that the PPAR response elements not only involve the direct repeats but also involve recognition of sequences immediately 5' of the repeat. The 5' sequences correspond to the sequence AAACT [31–33]. In contrast, sequences 3' of the repeat have no effect on receptor binding. These 5' sequences are recognized by an a-helix adjacent to the zinc finger of PPAR known as the C-terminal extension (CTE) or “GRIP” box. This GRIP box has been extensively characterized in orphan receptors that bind as monomers such as ROR and ReverbA, and the CTE of PPAR is highly conserved with these monomeric receptors. A similar binding mechanism has been observed with the thyroid hormone receptor, where the GRIP box helix interacts with the sequences that separate the repeats [34]. TR has also been shown to bind DNA as a monomer, however PPAR is the only protein that has been demonstrated to have this activity completely inhibited by the AB domain. The additional binding interactions conferred by the CTE do not enhance the binding of PPAR to perfect direct repeats, rather they help to stabilize interactions with imperfect repeats. The use of imperfect repeats appears to generate specificity in signaling, in order to distinguish between a wide range of nuclear receptors such as ARP-1, COUP-TF, and HNF-4 [31, 35]. This is important, as these competing receptors can bind consensus DR1s very efficiently and are expressed in the same cell types as PPARs. Indeed, proteins such as ARP-1 and HNF-4 bind rather poorly to many natural peroxisome proliferator response elements (PPREs) due to divergence in the core repeat sequence, whereas PPAR binds very well to the natural PPREs with the help of additional interactions with the 5' flanking sequences. The results of the above analysis revealed that PPAR was unusual in the polarity of the heterodimeric binding to DNA, with PPAR on the 5' half-site and RXR on the 3' half-site [30–33, 36]. This is in contrast to the VDR/RXR, RAR/RXR, and TR/RXR heterodimers where RXR is bound to the 5' half-site. It appears that this difference in polarity forms the basis for rexinoid/peroxisome proliferator synergy in signaling, as RXR appears to be unresponsive to rexinoids in some cases where it is bound on the 5' half-sites [36]. The role of ligand binding in the binding of PPAR to DNA has also been explored. It is clear that using excess amounts of recombinant PPAR and RXR, heterodimerization, and DNA binding can occur in the absence of ligand. However, it has been demonstrated that in the presence of limiting concentrations of PPAR ligands will promote cooperative binding of PPAR and RXR to DNA [20, 37]. This finding suggests that the PPAR/RXR heterodimerization interface is modulated conformationally by the binding of ligand. It would be less likely that the ligand

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binding affects the conformation of the zinc finger DNA binding domain, but this has not been ruled out. This ligand-dependency of DNA binding by PPARs has not yet been demonstrated in vivo. Currently the most extensively characterized consensus for DNA binding has been generated for PPARa target genes. However it would appear that PPARd- and PPARc-selective target genes do exist, and some selectivity of binding to adipocyte-specific PPREs, such as ARE6 and 7, has been demonstrated [38]. However very little is known about the sequence selectivity for PPREs that occurs with the different members of the PPAR subfamily. One group has used forced evolution to generate a consensus for PPARd binding sites and has demonstrated selective activation of a synthetic consensus site by PPARd [39, 40]. However, these studies have not been confirmed with the mapping of natural PPARd-selective promoter elements.

9.3

PPARs as Fatty Acid and Drug Binding Receptors

For the first 5 years of the characterization of PPARs it was still unclear whether they were activated by direct binding of the various peroxisome proliferators, or whether the different compounds provoked the production of an endogenous ligand. It was not until 1995 that the group of Steven Kliewer at Glaxo Wellcome demonstrated that the insulin-sensitizing drug rosiglitazone bound directly to and activated PPARc [41]. This group utilized radiolabeled rosglitazone to determine a binding affinity of 50 nM to the ligand binding domain of PPARc that had been expressed in E. coli and purified. This was the first finding of a compound that was of a high enough affinity for PPARs to demonstrate specific binding using a radioligand. The same group then demonstrated that a natural prostanoid fatty acid 15-deoxy D12,14PGJ2 could displace rosiglitazone and was an agonist of PPARc [42]. This was the first direct demonstration that a fatty acid could bind to PPARs. They then used the same displacement assay to show that non-steroidal anti-inflammatory drugs also displaced rosiglitazone and acted as agonists [43]. It was not until 1997 that binding assays were designed to prove that peroxisome proliferators bound directly to PPARa [20, 23]. This again was facilitated by the development of a high-affinity radioligand for use in displacement studies [23]. These studies showed conclusively that PPs such as Wy14,463 bound directly to PPARa at concentrations required for the activation of PPARa. In 1998 a simple assay for the binding of ligands to PPARs was described. It was shown that the fatty acid cis-parinaric acid became fluorescent when bound to PPARc [24, 44]. Also this fatty acid has a distinct spectral peak at 319 nm that is shifted to 324 nm when bound to PPAR. Both of these properties can be used to monitor displacement of the CPA by test ligands. Using this procedure it was found that certain NSAIDs, including diclofenac, bind PPAR with a relatively high affinity. Indeed, diclofenac displays an IC50 of 700 nM in these assays and it was found that diclofenac is a partial agonist that can antagonize rosiglitazone action and inhibit adipogenesis [45]. This antagonistic NSAID has an affinity for PPARc similar to, or higher than, that of the

9.3 PPARs as Fatty Acid and Drug Binding Receptors

clinically used PPARc agonists pioglitazone and troglitazone. The fluorescent binding properties of this fatty acid have been used to monitor the effect of amino acid substitutions in the ligand binding domain. Mutation of glutamic acid 291 of the ligand binding domain of PPARc to glycine has been shown to reduce ligand binding with these assays [44]. In a similar fashion, it has been demonstrated that the trans-isoform of parinaric acid is a high-affinity ligand for PPARa [46]. The sophistication of our knowledge of the binding of fatty acids and drugs has increased greatly since the crystallization of the ligand binding domains of all three PPARs. Both GlaxoWellcome and AstraZeneca have been engaged in the crystallization of these molecules and they have published structures which include the binding modes of fatty acids, high-affinity agonists, and partial agonists [21, 47–52]. These structures have revealed the residues involved in binding to the different ligands and have shown common interactions with both drugs and fatty acids for the PPARs. One particular point of note is the role of carboxylic acid in the activation of PPARs. It has been shown that it is the carboxylic acid residue of eicosapentanoic acid that makes hydrogen bonds with the activation helix 12 (AF2 domain) of PPARd [21]. It is this interaction that appears to stabilize a “charge clamp” for the binding of co-activator proteins containing LXXLL motifs [47]. This interaction has been visualized with the structure of the PPARa ligand binding domain co-crystallized with a synthetic LXXLL motif from the steroid receptor coactivator 1 (SRC-1) protein. This regulatable interaction represents the molecular basis for transcriptional activation by PPARs. This arrangement is also seen for the binding of a dihydro-cinnamic acid derivative, AZ242, to PPARc and PPARc [50]. The thiazolidinediones are bound to PPARc with the TZD head group making interactions similar to those of the carboxylic acids, however the TZD head group is sterically hindered from binding to PPARa by residue Tyr314. The orientation of the agonist binding to PPARs is therefore rather predictable, although the interactions that occur in the larger ligand binding cavity between helixes 2', 3, 6, and 7 are much more variable. This region provides multiple hydrophobic interactions which result in different binding modes for long-chain fatty acids. This resulted in the crystallization of EPA in two conformations within the ligand binding domain of hPPARd i.e. in a tail-up or tail-down mode. This large cavity can bind ligands without interactions with the AF-2 domain. This was visualized with the structure of PPARG/GW0072 [53]. GW0072 is a high-affinity partial agonist and the lack of interactions with the AF-2 domain as seen in the co-crystal structure explain rather nicely the reason for the compound’s lack of agonist activity (Fig. 9.1). The large size of the PPAR ligand binding cavity has been used to explain the apparent promiscuity of this receptor – the PPAR has an internal volume of 1300 Å3 [21] compared with around 450 Å3 for the more conventional receptors, such as the estrogen receptor [54]. However, the size of this cavity argues against the role of free fatty acids/prostanoids as being endogenous ligands for these receptors. It is possible that the endogeous ligands for these receptors are larger fatty acyl-containing molecules, such as sphingolipids, inositol lipids, or acyl glycerols. These compounds have long been appreciated as biological messengers. Recently there have been reports of such molecules binding and activating

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Agonists and antagonists bound to PPARc. Shown are molecular visualizations of the ligand binding domain of PPARc with (A) the agonist, rosiglitazone, and (B) the partial antagonist, GW0072, bound inside the ligand binding cavity. It can be seen that the partial antagonist is buried deep in the molecule and has less interaction with the cavity between

Fig. 9.1

the transactivation domain helix 12 (blue ribbon) and helix 3 (purple ribbon). The ligands are displayed as ball and stick models with carbons (yellow), oxygen (red), nitrogen (blue) and sulfur (green). The visualizations are derived from the co-ordinates in the PDB entries 2PRG and 4PRG, using INSIGHT II for SGI (with the expert help of Dr Jack Flanagan).

PPARs; however, these have not produced a compelling argument for any individual endogenous ligand or biological rationale for their function [55–57]. Interestingly, fatty acyl-CoA esters appear to be potent antagonists of PPAR signaling [37]. Another interesting aspect of the ligand binding domain of PPARs is their activation by the highly unstable and reactive prostanoids 15-deoxy D12, 14PGJ2. These compounds are used routinely by large numbers of laboratories as specific PPARc ligands. PPAR-independent mechanisms of 15-deoxy D12, 14PGJ2 action have also been proposed. Unfortunately many of these studies are poorly interpreted. The investigators that used 15-deoxy D12, 14PGJ2 to probe PPAR function as opposed to other PPAR function have misinterpreted the original studies. It is quite clear that 15-deoxy D12, 14PGJ2 is quite a pan-PPAR agonist [20] and it appears that the original description of the specificity for PPARc was due to the nature of the chimeric receptors used [42]. The activity of 15-deoxy D12, 14PGJ2 on all three of the native receptors has been demonstrated and confirmed in our laboratory (unpublished observations). Comparisons of the action of 15-deoxy D12, 14PGJ2 and TZD drugs are particularly confused by this fact. The activation of other PPARs by 15deoxy D12, 14PGJ2, may also confound the experiments with cells that lack PPARc, as some other PPAR may substitute in these experiments. These considerations, along with the inevitable wide reactivity of these compounds, make many of the 15-deoxy D12, 14PGJ2 studies difficult to interpret. Indeed original studies into the anti-proliferative effects of D12(-PGJ2 noted that the prostanoid entered the nucleus and became covalently bound. The prostanoid could then be recovered under reducing conditions with DTT. 15-deoxy D12, 14PGJ2 has been found to modulate NFjB directly and indirectly by covalent modification of IjB [58–60]. It is possible, however, that NFjB is also indirectly inhibited by a covalently modified PPARc. Indeed, close examination of

9.4 Species Differences in Pharmacology A reactive cysteine is buried in the ligand docking site of PPARc. Shown is the topology of Cys285 in the crystal structure of rosiglitazone bound to the PPARc ligand binding domain. Cys285 is the only cysteine in the entire ligand binding domain of PPARc. This has been shown to react with irreversible antagonists such as GW9662. The color scheme and visualization methods are the same as Fig. 9.1 with the exception of the sulfur in Cys285 being shown in brown for contrast.

Fig. 9.2

the PPARc ligand binding domain reveals that there is only one cysteine in the entire ligand binding domain and that this cysteine is buried deep in the ligand binding pocket on the inner face of helix 3 (Fig. 9.2). This cysteine is the reactive target of an irreversible antagonist of PPARc that was discovered by Merck [61], and is also the target of the high-affinity antagonist from GSK, GW9662 [62–64]. We have found that 15-deoxy D12, 14PGJ2 activates PPAR reporter constructs in a non-saturable manner with an efficacy over 100 times that seen with high-affinity ligands such as rosiglitazone (unpublished data). We have also found that it is impossible to inhibit this activity with competitive antagonists such as diclofenac. It is therefore possible that 15-deoxy D12, 14PGJ2 may possibly be an irreversible super-activator of PPAR which would make the comparison of their function on PPARs with those activities seen with reversible agonists quite difficult. This hypothesis has still to be confirmed by analytical methods such as mass spectrometry, but is an exciting potential new avenue of PPAR research.

9.4

Species Differences in Pharmacology

The PPARs are closely conserved between mammalian species [> 95% mouse versus humans). But as with the total genome difference between these species it is the small number of differences that do occur that are important in giving the phenotypic difference between mice and humans. It is clear that mice and humans have very different lipid handling capabilities, and have very different diets. The mouse is a poor model for many PPAR-associated diseases, including heart disease, diabetes, and cancer; and the pharmacology of fatty acids and drugs available for the study of PPAR function is also quite different between the species. Indeed it would appear that some of the selectivity seen for polyunsaturated fatty acids (PUFAs) in mammalian PPARc is not apparent in the frog PPARc [22, 24]; however, it is when considering the synthetic compounds that species differences have been the most apparent. One of the most potent compounds reported

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for the frog PPARa is the synthetic triple-bonded mimetic of arachidonic acid, ETYA. This compound activates Xenopus PPARa with a very high potency, but does not activate mouse PPARa with the same potency. The species differences were isolated to specific residues of helix 3 [65]. This demonstrates that species differences in the ligand binding cavities of the PPARs do provide different pharmacology. This was also found to be the case for Wy14,643, which is a very efficacious and specific activator of mouse PPAR with an EC50 of around 400 nM. It does not activate other murine receptors until around 100 times that concentration. In contrast, the EC50 of Wy14,643 with the human receptor is around 5 lM with activation of PPARd at 35 lM and PPAR at 60 lM [66]. Many studies have been published using this compound at 100–250 lM in human cells and have claimed numerous biological activities for PPARa in human cells. These studies should be interpreted with great caution. A converse example of this occurs with the recent series of compounds described as specific ligands for PPARd [67]. These compounds are selective for human PPARd yet have very poor selectivity for mouse PPARd. In our studies into the role of PPARd in atherosclerosis, we have found that compound F has an affinity of 2 nM for human PPARd, 400 nM for PPARc, and > 5 lM for PPARa [68]. In contrast, the mouse PPARd receptor is activated very poorly by compound F and at concentrations very similar to that seen for mouse PPARc. We find that this compound is very effective in promoting lipid accumulation into primary human monocytes and human monocytic cells, whereas activators of PPARa and PPARc are inactive and even oppose lipid accumulation. This has led us to speculate that PPARd may promote certain aspects of atherosclerosis. However, it is clear that these compounds are not suitable for use in murine models of atherosclerosis. Recently, high-affinity ligands for PPARd that retain their specificity in murine models have been described [69], which should allow for the resolution of this issue. It is clear therefore, that the species-specific pharmacology of PPAR agonists must be taken into account in the proper design and interpretation of experiments using such compounds.

9.5

Co-activator/Co-repressor Interactions

As mentioned previously, the role of PPAR is to attract the transcriptional machinery to target genes in a ligand-dependant manner. The first point of contact is a family of proteins known as co-activators. These molecules are not an integral part of the polymerase complex, they have been characterized as part of the mediator complex, which modifies chromatin to an open configuration by acetylation and recruits the polymerase complex for initiation. Co-activators are large molecules that have the potential to bind many transcription factors at a gene target and these molecules probably integrate complex signals that occur in the regulatory regions of genes.

9.5 Co-activator/Co-repressor Interactions

The binding of co-activators such as SRC-1, PBP, and CBP/p300 utilizes a LXXLL motif to bind to the charge clamp formed by helix 12 of the PPAR ligand binding domain in the presence of ligand [47, 70]. Dominant-negative mutants of PPAR are generally defective in recruiting the co-activators. Experimentally, the deletion of helix 12 has been used to generate dominant-negative forms of PPAR for probing PPAR function in cells. In addition, there have been cases of naturally occurring dominant-negative mutations in PPARc. A few individuals with extreme insulin resistance have been characterized as being heterozygous for dominant negative point mutations in the ligand binding domain of PPARc [71]. Point mutations in the AF-2 domain have provided dominant-negative PPARc for experimental purposes, including a mutant form with Leu468 and Gln471 changed to Ala [72]. Both the point mutation and deleted AF-2 form of PPARc have been expressed in retrovirus and used to explore the role of PPARc in adipocyte function [72, 73]. One interesting finding with this mutant is that it does not repress the action of rosiglitazone on the uptake of glucose in adipocytes [74]. The authors speculate that this process may be mediated by the action of a specific coactivator PGC-1, which has been shown to bind the N-terminal portion of the ligand binding domain along with residues in the AF-2 helix 12. The mode of binding to the AF-2 domain appears to be distinct from that of SRC-1 [75–77] and it would appear that the binding interactions of individual co-activators are quite different [78]. It is predicted, therefore, that a dominant-negative form of PPARc with the AF-2 domain deleted would antagonize the effects of rosiglitazone on glucose uptake, although this remains to be tested. The action of PGC-1 has also been shown to mediate isoform-specific induction of the UCP-1 gene [79]. In vivo studies have shown that PPARa activators are inefficient inducers of UCP-1, yet PPARc activators are quite efficacious. It has been shown that this is due to the inability of PGC-1 to act as a co-activator for PPARa when phosphorylated by the p38 MAP kinase pathway. This phenomenon is not observed with PPARc signaling on the UCP-1 gene, thus resulting in a PPARcspecific response. It is clear that this specificity is totally dependant on the status of the p38 MAP kinase pathway in the target cells. Co-repressors in PPAR biology are less well characterized. The concept of a corepressor was developed from other nuclear receptors such as the thyroid hormone receptor (TR) [80]. These receptors have profound repressive qualities in the absence of ligand. They can silence quite constitutively active promoters. In contrast, many PPAR reporter systems are rather inactive without PPAR and introduction of PPAR can provide transcriptional activation without the apparent addition of ligand [81]. It has been shown, however, that dominant-negative forms of PPARc bind to co-repressors such as N-COR and SMRT and are defective for their ligand-dependant dissociation [71, 72]. It has also been shown that PPARc binds to co-repressors in solution, but not when bound to DNA [82]. The binding domain for the co-repressors has recently been visualized by X-ray crystallography to residues in helix 4 and 12 of PPARa, and is mediated by overlapping yet distinct residues compared with those involved in co-activator binding in other nuclear receptors [48]. These findings have not clarified the role of co-repressors in PPARc

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biology, but they do suggest an intriguing possibility that co-repressors may be involved in the biology of non-DNA bound PPARc. In contrast, it has been shown that PPARd does possess quite powerful repressive activities. It appears that unliganded PPARd can repress the action of PPARa and PPARc on the activation of common target genes [83]. This has mainly been demonstrated at the in vitro level using consensus PPRE-driven reporter constructs, however the biological relevance of this has yet to be demonstrated. Interaction with co-activators and co-repressors would suggest the potential for nongenomic activities; however, the biology of non-genomic actions of PPARs is, as yet, poorly defined. Nevertheless, as will be discussed in the next section, it may constitute a major part of the function of PPARs.

9.6

Cross-talk with Inflammatory Signaling

One of the most exciting aspects of PPAR biology is their potential to modulate inflammatory processes. This is also, however, the area that it is almost impossible to interpret from the current literature. This is due to the existence of many discordant studies and the use of rather poorly selective compounds (or the poor use of reasonably selective compounds). It has been proposed that PPARc mediates the anti-inflammatory actions of the x-3 fish oils such as eicosapentaenoic acid (EPA) [84], however the pharmacology of the thiazolidinedione drugs in vitro and in vivo has been contradictory and does not completely support this hypothesis [58, 85–87]. Of course there is a very good precedent for the ability of nuclear receptors to have anti-inflammatory activity: the glucocorticoid receptor (GR) is the archetypal anti-inflammatory mediator and there are many parallels between the proposed anti-inflammatory mechanisms for PPAR and those of GR. Both have been shown to mediate the repression of pro-inflammatory genes such as COX-2, iNOS, TNFa, and IL-6 in various cell types, including macrophages, smooth muscle cells, and colonic epithelia [86, 88, 89]. These repressions do not appear to occur via the binding of the nuclear receptor to the regulatory elements of these genes, rather the nuclear receptors appear to inhibit the activation of transcription factors such as AP-1 and NFjB. The GR positively modulates the transcription of anti-inflammatory mediators such as IjB and this requires classical binding to a glucocorticoid response element (GRE); however it is clear that GR has many properties that do not require DNA binding. Indeed, although the complete GR gene deletion in mice is lethal, if the DNA binding activity is just abolished by “knockin” point mutations, then the lethality is avoided [90]. It appears that the functions of the GR that are important for viability do not require the DNA binding function of this receptor. In these mice classical GRE-mediated responses do not exist, however, many processes that require gene repression, such as T cell apoptosis, are still functional. In a similar vein it may be possible that many functions of PPARs are unrelated to the classical PPAR/RXR heterodimer binding to PPREs and may rely on

9.7 PPARs as Phosphoproteins

“off the DNA” processes. One example of this is the repression of cyclin D by PPARc ligands [91–93]. In this case, it has been proposed that liganded PPARc sequesters CBP/p300, thus limiting the transcriptional potential of the cyclin D gene [91]. As mentioned previously, the unliganded PPAR in solution may bind to co-repressors and thus may influence the expression of genes that do not contain PPAR binding elements. This may be of importance in pathological conditions such as cancer and inflammation where PPARs may become overexpressed. We have found that this is indeed the case and it appears that overexpression of PPARc in cell lines can increase the expression of cyclin D and result in increased proliferation (unpublished data). This would provide a rationale for the finding that PPARc is overexpressed in tumors and that ligand binding-defective mutations can arise somatically in tumors [94]. Our findings provide evidence for a physiological consequence of the overexpression of such mutant proteins in cancer, as they would be predicted to provide a growth advantage over cells producing small amounts of wild-type protein. In a similar fashion it is therefore clear that the role for PPARs is likely to be complex and will require the use of rigorous pharmacology and genetic tools to determine specific roles for PPAR subtypes.

9.7

PPARs as Phosphoproteins

Fatty acids are known to modulate many aspects of cellular signaling, including the activation of membrane receptors and subsequent activation of intracellular kinases. It was therefore thought for a period that ligands may activate PPARs indirectly by phosphorylation. This concept has been replaced by the knowledge that fatty acids bind PPARs directly, but it is still apparent that PPARs are phosphoproteins [95, 96] and that their signaling potential can be modulated by the action of kinases. The most characterized phosphorylation of PPAR is at Ser112 of PPARc2. This residue is phosphorylated by both the classical ERK pathway and the stress-activated JNK pathway [97–101]. The result of this phosphorylation is the inhibition of ligand binding and transcriptional activation of the receptor. A mutant PPARc with an aspartic acid substitution to mimic the phosphorylated serine has a 10-fold decrease in its affinity for ligands [97]. This was quite unexpected as the residue is in the AB domain portion of the receptor, rather close to the zinc finger DNA binding domain, and on the other side of the DNA binding domain from the ligand binding domain. This suggested that the AB domain may wrap round and physically affect the shape of the ligand binding domain. This has a precedent in the androgen receptor, where the AB domain has a profound effect on the ligand-dependent transcriptional activity of the ligand binding domain [102]. The phosphorylation of Ser112 has been presented as having two different physiological consequences. The first of these is in cancer cells, where the RAS pathway is highly active. It appears that the cells are quite insensitive to growth inhibi-

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tion by PPARc ligands such as rosiglitazone. However, when these cells are treated with inhibitors of the MAP kinase pathway they become sensitive to much lower concentrations of PPARc ligands [103, 104]. Another situation where this has been shown to have a functional consequence is in the differentiation of adipocytes. Insulin is required for the full differentiation of adipocytes, however mutation of Ser112 to alanine results in faster adipogenesis in the presence of insulin [97]. This has been presented as a feedback mechanism in adipogenesis, where high levels of insulin-induced MAP kinase activation will self-limit the adipogenesis program. This concept was reinforced by the discovery of four extremely obese individuals who had single base pair changes in their PPARc gene that inactivated this phosphorylation site [105]. However, these mutations have not been found in any other populations. The finding that phosphorylation may inactivate PPAR signaling may provide insights into complex gene regulation by PPARs. It is clear that PPARs can be transcriptionally activated by fatty acids, however the concentrations required to do so do not reflect their binding affinity for PPARs. In particular PPARc is poorly activated by arachidonic acid, even at 100 lM. The binding affinity for PPARc for arachidonic acid is around 1 lM and for PGJ2 6–8 lM [21, 24]. Activation of transcription of a PPRE can be detected with concentrations of PGJ2 at around 1 lM, and this may be accounted for by direct modification of PPARc, but why is arachidonic acid so poor at activating PPARc? In addition to metabolic reasons, one potential mechanism may be the activation of PKC or another signal transduction pathway by arachidonic acid, which may in turn inactivate PPARc and prevent binding to arachidonic acid. Other fatty acids are less prone to such activation and may proceed to activate the PPAR directly. This concept may help explain the efficacy of 9-hydroxyoctadecadienoic acid (9-HODE) in the activation of PPARc. This fatty acid has a poor affinity for PPARc when compared with linoleic acid, yet is much more efficacious in transactivation assays [106]. Another example of this complexity is seen in the products of 15-lipoxygenase such as 13-hydroxyeicosatetraenoic acid (13-HETE), which have been shown to promote phosphorylation and downregulation of PPARc activity, and yet can also be direct activating ligands [40]. It therefore appears that PPARs represent a point of integration of fatty acid signals both at the membrane and in the nucleus, rather than a purely direct response to the physical binding of fatty acids. The AB domains of PPARs are rich in proline/serine repeats and appear to be prime targets for phosphorylation, and it would appear that phosphorylation may modulate PPAR turnover and degradation. PPARc is degraded through a proteasomal pathway and this appears to be promoted by ligand binding [107]. In contrast, ligand binding has been proposed to stabilize PPARa [108]. Proteosomal degradation is known to be regulated by phosphorylation and again Ser112 is implicated in this regulation [109]. In addition, the PPARc2-specific AB domain contains a consensus PEST sequence, which is known to target degradation upon phosphorylation. However, PPARc2 appears to have a longer half-life than PPARc1 [109]. This amino acid sequence also appears to be required for efficient adipogenesis as revealed by sophisticated genetic analysis including the “knockdown” of PPAR ac-

9.8 References

tivity in mice [110], although the mechanism by which the PPARc2 AB domain mediates adipogenesis remains to be elucidated. PPARd also appears to be regulated by phosphorylation, as has been observed with activation of the protein kinase A (PKA) pathway by forskolin [96]. This PKA activation is via the AB domain of PPARd, but is quite weak and appears to act solely as a sensitization to ligand activation. In contrast, PPARd is strongly activated by potent activators of kinase signaling such as TNFa and phorbol ester, however it would appear that this may be due to generation of endogenous ligands by these pathways, rather than phosphorylation of the PPAR protein [111]. Further work is required to clarify the role of phosphorylation in the activation of PPAR activity by inflammatory mediators.

9.8

References 1 2 3 4

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Structure and Function of Retinoid Receptors RAR and RXR Alexander Mata de Urquiza and Thomas Perlmann

10.1

Retinoids in Development

Vitamin A and its biologically active metabolites, the retinoids, play essential roles in embryonic development, differentiation, and maintenance of homeostasis in the adult organism [1–3]. Adult animals suffering from vitamin A deficiency (VAD) display a number of abnormalities, including impaired vision, fertility, immune response, and epithelial differentiation. Furthermore, altering the levels of retinoic acid (RA) during embryogenesis leads to phenotypical malformations affecting for example cranofacial, CNS, limb, and heart development (reviewed in Refs [4] and [5]), underscoring the importance of correct vitamin A levels during gestation. Some of these abnormalities are thought to arise due to dysregulation of Hox gene expression. Hox genes encode a family of homeobox-containing transcription factors that play crucial roles in informing cells of their position along the anterio-posterior axis (reviewed in Refs [3] and [5]). The overlapping expression domains of these genes along the anterio-posterior axis are thought to specify the positional identity of cells along this axis, thereby enabling them to adopt a correct developmental fate. Interestingly, several Hox genes contain RA response elements (RAREs; see below) in their promoters, indicating that retinoids are involved in regulating their expression. Accordingly, embryos that develop in the absence of RA or in the presence of excess RA, display altered Hox gene expression [5, 6]. The distribution of retinoids in vivo has been analyzed using transgenic mice that express a retinoic acid-inducible reporter gene [7–14]. The results suggest that the brachial and lumbar regions of the developing spinal cord are “hot spots” of RA synthesis. In addition, reporter gene expression has been detected in the developing forebrain, forelimbs, and optic cup, as well as at the midbrain/hindbrain boundary [7, 9–11]. A better understanding of how this highly localized synthesis of RA is achieved in tissues has been gained with the cloning of enzymes responsible for RA production and degradation. Dietary all-trans retinol (atROL) is converted to all-trans retinal (atRAL), a reaction catalyzed by one of various ROL dehydrogenases (ROLDH). In the following step, atRAL is converted into atRA by a RAL dehydrogenase (RALDH) (reviewed in Refs [15] and [16]) (see Fig. 10.1 A). 9cRA has been suggested to form by spontaneous isomerization from the all-trans

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Fig. 10.1 (A) Metabolic steps leading from retinol to retinoic acid and one of its oxidation products. The initial step is rate-limiting, and is the only reversible step in this process. 9-cis Retinoic acid (see B) is presumably formed via spontaneous isomerization from

the all-trans form. (B) Structures of three natural RXR agonists, including 9-cis retinoic acid (left), the fatty acid docosahexaenoic acid (middle), and the chlorophyll metabolite phytanic acid (right) (see text for details).

form. Although atRA and 9cRA are the two best-characterized retinoids, other bioactive forms also exist in vivo, for example 3,4-didehydroRA and 4-oxo-RA [17, 18]. 4-oxo-RA is one of several breakdown products of atRA in catabolic reactions catalyzed by a family of cytochrome P450 enzymes, the CYP26 family [19–21], involved in attenuating the RA signal (see Fig. 10.1 A). In early development, most embryonal RA is thought to be synthesized by RALDH2 [22–26]. Intriguingly, a comparison of the expression patterns of RALDH2 and CYP26 suggests that both enzymes are present in complementary and non-overlapping domains, thereby creating areas differing in their RA levels [19, 24, 25]. CYP26 expression is restricted to the anterior- and posterior-most structures of the embryo, including the presumptive head and tailbud regions, keeping RA levels low. RALDH2 on the other hand is expressed in more central areas of the embryo, from the posterior end of the developing hindbrain to the anterior regions of the developing tail, and is responsible for RA synthesis in this region. Targeted disruption of the CYP26 gene gives rise to embryos where anterior brain structures are transformed to more posterior ones, resembling the morphogenetic defects generated by excess RA administration [5, 27, 28]. Conversely, disruption of the RALDH2 gene produces embryos with phenotypes indicating an

10.2 Retinoid Receptors Transduce Retinoic Acid Signals

expansion of anterior brain structures, reminiscent of the defects generated by vitamin A deficiency [6, 29, 30]. Taken together, these results suggest that disturbances in the graded synthesis of RA leads to posteriorization (excess RA) or anteriorization (shortage of RA) of structures in the developing head, largely due to misexpression of Hox genes. In an elegant study, Koide and co-workers show that active repression of RA target genes needs to be maintained for correct development of anterior regions [31]. This suggests that the mere absence of RA in anterior tissues is not sufficient for normal development, but that active repression of RA-responsive genes per se is essential for correct anterior patterning. Interestingly, retinoid receptors are directly involved in this repression by a mechanism that will be further discussed below (see “co-repressors” below). Within cells, cellular retinol binding proteins I and II (CRBP-I and -II) and cellular retinoic acid binding proteins I and II (CRABP-I and -II), act as cytoplasmic carriers for ROL and RA, respectively. Based on expression studies, it was thought that CRBPs function in protecting retinol from the cellular environment and presenting ROL to RA synthesizing enzymes. In contrast, CRABPs were suggested to be important in sequestering and promoting breakdown of excess RA in embryonic regions sensitive to the teratogenic effects of retinoids (see, for example, Refs [32–34]). However, animals carrying null mutations in both CRABP-I and -II are indistinguishable from wild-type littermates, suggesting that CRABPs are dispensable for normal embryonal development and adult physiology [35]. On the other hand, although not essential for embryonal development, adult mice lacking CRBP-I show decreased liver retinyl ester storage and predisposition to vitamin A deficiency [36], suggesting an important role for CBRP-I during vitamin A-limiting conditions. Interestingly, two CRBP-III genes have recently been identified [37, 38], showing partially overlapping patterns of expression, and it will be interesting to see what roles these proteins might play in vivo.

10.2

Retinoid Receptors Transduce Retinoic Acid Signals

The cloning and characterization of the nuclear hormone receptors (NRs) that bind and transduce the retinoid signal represent landmarks in our understanding of retinoid physiology (see references in Ref. [39]). Two families of retinoid receptors exist, the retinoic acid receptors (RARs) and the retinoid X receptors (RXRs), each present in three isotypes, a, b, and c (reviewed in Refs [39–41]). In addition, each RAR and RXR isotype exists in several isoforms (for example RARa1 and a2) due to differential promoter usage. Expression studies of RARa, b, and c show that RARa is ubiquitously expressed, while RARb and RARc show a more temporal and spatial restriction, often in a non-overlapping fashion [32–34, 42, 43]. RXRs are similarly differentially expressed both during development and in adult animals [42–45]. RXRb is expressed in a general fashion, while RXRa is abundant in tissues associated with lipid metabolism. RXRc expression is highly restricted to a

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few distinct areas, including the developing striatum and spinal motor neurons (where it is co-expressed with RARb). A strong effort has been made during the last few years to understand the specific functions of the different retinoid receptors during development. Despite the unique distributions of the different receptors, genetic ablation studies have revealed a surprising redundancy in function among the different members of each receptor subtype. The results of single and double RAR or RXR mutants, as well as compound RAR/RXR mutants, have been extensively reviewed [41, 46]. Taken together, the results suggest that RARs and RXRs are important for correct regulation of numerous developmental processes. Although many known VAD phenotypes are not apparent in single RAR–/– mice, simultaneous ablation of two RAR genes recapitulates most aspects of VAD. Additionally, compound mutants of RXRa and RARa, b, or c together reproduce almost the entire VAD phenotype spectrum, supporting the notion that RAR/RXR heterodimers are the active units for retinoid signaling in vivo (summarized in Ref. [46]). Interestingly, a number of malformations not described in VAD studies are also observed, either suggesting roles for unliganded receptors or reflecting difficulties in achieving complete VAD by dietary deprivation.

10.3

Retinoid Receptors Belong to the Nuclear Hormone Receptor Family

NRs comprise a large and evolutionary well conserved family of transcription factors found in organisms as diverse as nematodes, flies, and mammals (reviewed in Refs [47–49]). NRs are thought to function as ligand-activated transcription factors, exerting widely different biological functions by regulating target gene expression positively and/or negatively, and include the receptors for certain small, lipophilic molecules. RARs bind both all-trans and 9-cis retinoic acids, whereas RXRs only bind 9-cRA (see Ref. [50] and references therein). Retinoid receptors activate transcription by recognizing and binding consensus sequences known as RA response elements (RAREs) in the promoters of target genes (see below). RAR binds DNA as a heterodimer with RXR, while RXR also has the ability to bind DNA as a homodimer. Additionally, RXR forms heterodimers with a number of other NRs, including the receptors for thyroid hormone (TR) and vitamin D3 (VDR) [50], thereby coupling retinoid signaling to a multitude of other cellular signaling pathways.

10.4

Nuclear Receptors Share a Common Structure

As mentioned above, retinoid receptors belong to the NR family of transcription factors. With only a few exceptions, all NRs share a common structure of functionally separable domains, including an N-terminal domain (NTD), a central DNA binding domain (DBD), and a C-terminal ligand binding domain (LBD)

10.4 Nuclear Receptors Share a Common Structure

Fig. 10.2 Nuclear receptor domains and functions. (A) NRs consist of defined domains, with variable degrees of conservation within the NR superfamily. The NTD, which is the most variable, has a ligand-independent transactivation function (AF-1) shown to be important in basal transcription by some receptors. The more conserved DBD and the LBD are responsible for DNA and ligand binding, respectively. In addition, both domains play important roles in dimerization. A ligand-dependent transactivation function (AF-2) is local-

ized to the LBD. NTD, N-terminal domain; DBD, DNA binding domain; LBD, ligand binding domain; AF-1, activation function 1; AF-2; activation function 2. (B) Structures of the RXR DBD homodimeric complex (left) and of the RXR–RAR DBD heterodimeric complex (right) on DR1 DNA response elements. Contacts between receptor partners form over the minor groove of the DNA helix, with additional protein–DNA contacts stabilizing the complex. DR1, direct repeat 1. Modified from Ref. [52].

(Fig. 10.2 A). The highly conserved DBD is responsible for recognizing and binding to specific DNA sequences in the promoters of target genes, and is also involved in dimerization between receptors. The LBD, besides binding ligand, plays an essential role in the initial interaction between receptor heterodimers, as well as in ligand-dependent transactivation. The LBD also harbors a ligand-dependent activation function (AF-2), mediating ligand-dependent interactions with so-called co-activators (see below). The NTD is less conserved between different NRs, both in amino acid composition and length, and contains a ligand-independent activation function (AF1), important in the basal transcriptional activity of some receptors. A hallmark of the NR family is the well-conserved DNA binding region. This cysteine-rich zinc binding domain, has been shown to contain structural features important for correct DNA target sequence recognition and binding, as well as dimerization (Fig. 10.2B) (see Ref. [50] and references therein). Most NRs bind socalled hormone response elements (HREs), arranged as one or two half-sites of the consensus nucleotide sequence 5'-AGGTCA-3'. Half-sites can be arranged as direct- (DR), inverted- (IR), or everted repeats (ER), with a varying number of nucleotides separating the repeats. Studies of receptor dimers bound to DNA have

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shown that RXR and its heterodimer partners bind response elements arranged as two direct repeats spaced by one to five nucleotides (DR1 to DR5) ([51]; reviewed in Ref. [52]). Depending on the spacing between the two repeats, different RXR heterodimers will bind and activate transcription. It seems that both heterodimer partners cooperate to ensure correct binding specificity and affinity by making partner-specific protein contacts that stabilize the complex only on a correctly spaced DR motif. Additionally, RXR has the ability to switch its polarity on DR elements, binding either the upstream or the downstream half-site [52]. Importantly, the RXR heterodimer partner not only influences the response element of choice, but also the ability of RXR to become activated by ligand. For example, heterodimers between RXR and the peroxisome proliferator activated receptors (PPARs) are induced by both PPAR and RXR ligands, therefore said to be permissive to RXR activation. RAR–RXR heterodimers, on the other hand, are non-permissive in that they require binding of RAR ligands in order to become responsive to RXR ligands [53–55]. It has been suggested that the initial agonistinduced interaction between RAR and co-activators (see below) is necessary to induce the correct structural rearrangements that allow RXR to become receptive to its ligand [56]. However, several groups have obtained results that are not easily explained by this model. For example, RAR–RXR heterodimers can become responsive to an RXR ligand even after addition of an RAR antagonist, a situation when co-activators are not recruited by RAR [57, 58]. It is therefore still somewhat unclear why some heterodimers are permissive while others are not. An additional dimerization interface important for the initial interaction between NRs is found in the neighboring ligand binding domain (see below). This region has been shown to mediate cooperative binding on all three classes of DNA repeats (direct, inverted, and everted) [59, 60]. In receptor heterodimers, the second dimerization region formed within the DBD restricts receptors to direct repeat elements.

10.5

The LBD and Ligand-dependent Transactivation

The crystal structure of the LBD of several NRs has been solved, including unliganded (apo) and liganded (holo) RXRa, as well as holo RARc [61, 62] (reviewed in Ref. [63]). The results reveal a common fold, consisting of 12 a-helices (H1– H12) and one b-turn, arranged in a three-layered antiparallel “sandwich” with a hydrophobic core (Fig. 10.3 A). The center of this sandwich contains the ligand binding pocket, lined mostly by hydrophobic and polar residues. As mentioned, the LBD contains the ligand-dependent activation function AF-2, and residues critical for its function have been mapped to helix 12 (the AF-2 core) [64]. Crystallographic studies have provided a model that accounts for the structural transitions involved in ligand activation of NRs (reviewed in Refs [63, 65]). In this so-called “mouse-trap” model, the initial interaction between ligand and LBD leads to structural changes that trap the ligand within the LBD in an induced fit mechanism. These transitions are subsequently followed by a repositioning of he-

10.5 The LBD and Ligand-dependent Transactivation

Fig. 10.3 Schematic drawing of the LBD structure of apo RARa (A), holo RARc bound to all-trans retinoic acid (atRA) (B), and antagonist-bound estrogen receptor (ER) a (C). atRA is shown in “stick” form in the center of the LBD in (B), and the ER antagonist, raloxi-

fene, is depicted as a bent cylinder in (C). Note the different positions of helix 12 (shown in black) in each situation. The a-helices of the LBD are represented as numbered rods. Modified from Ref. [65].

lices 3 and 4, which together with helix 11 now move to form a hydrophobic cleft on the surface of the LBD. The most profound conformational change involves helix 12 (H12) itself (Fig. 10.3). In the absence of ligand, H12 protrudes from the LBD and is exposed to solvent, whereas in the holo-receptor, it rotates and folds back towards the LBD, thereby compacting its structure. In its final position, H12 seals the pocket, trapping the ligand inside (compare Fig. 10.3 A and B). In addition, in this new conformation, H12 has a major role in positioning so-called coactivator proteins in the hydrophobic cleft formed by residues on helices 3, 4, and 11, a process important in transcriptional activation (discussed below). Once transcription of target genes has been initiated, the hormone response needs to be attenuated in order to control the transcriptional output. Cells can regulate the activity of proteins involved in transcriptional activation by affecting their stability. Several reports show that retinoid receptors are targeted for ubiquitin-mediated degradation upon ligand binding [66–69]. Although somewhat conflicting, the results suggest that only active receptors are degraded. Interestingly, a component of the proteasome that mediates the degradation of ubiquitinated proteins, SUG1, has also been shown to function as a co-activator for certain NRs, including RAR [70], by interacting with the AF-2 helix. This could provide a functional link between ligandinduced transcriptional activation and subsequent degradation of NRs.

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10.6

Cross-talk

In addition to acting as ligand-dependent transcription factors, some NRs also become activated or inactivated in the absence of ligand, for example by phosphorylation of the receptor itself or of its co-regulatory proteins. This is especially the case for the steroid hormone receptors (reviewed in Ref. [71]), but also the activity of retinoid receptors can be modulated through phosphorylation, both in the presence and absence of ligand (see, for example, Refs [72–74]). Phosphorylation of a serine residue in the RAR DBD has for example been shown to lower the interaction between RAR and RXR, thereby decreasing transcriptional activation, and could be a way of regulating the activity of receptors in vivo [75]. On the other hand, phosphorylation of serine residues in the N-terminal AF-1 of RAR has been shown to have a positive effect on ligand-independent transcription [74, 76]. Perhaps the best established example of cross-talk is the inhibition of AP-1 transcriptional activity by several NRs including retinoid receptors. These modulatory activities are believed to be critical for the common antiproliferative effects of retinoids and other NR ligands (reviewed in Ref. [77]).

10.7

Co-activators

NRs require accessory factors, so-called co-activators, in order for ligand-dependent activation of target genes to occur. The first co-activators to be described interacted with the estrogen receptor (ER) in the presence of ligand [78]. One such protein, RIP160 (for receptor interacting protein 160), later shown to be identical to SRC-1 (steroid receptor co-activator-1) [79], interacted with several NRs in a hormone-dependent manner. Since then, an ever-increasing number of co-activators have been cloned and characterized (reviewed in Ref. [80]). The best-characterized co-activators belong to one of three classes: the p160 family, including SRC-1/N-CoA1, GRIP-1/ TIF2, and ACTR/pCIP/RAC3/AIB-1; the homologous CBP and p300 co-activators; and the recently isolated TRAP/DRIP complexes (see Ref. [81] for abbreviations and references) (Fig. 10.4). Members of the p160 family as well as CBP/p300 and p/CAF (CBP associated factor) show intrinsic histone acetyltransferase (HAT) activity [82–86], suggesting that co-activators may play direct roles in chromatin remodeling at promoters by acetylating histone proteins [87]. The TRAP/DRIP complexes were isolated by their ability to interact with ligand-bound TR and VDR, respectively. They form large multiprotein complexes that share common subunits [88, 89], and are thought to play an important role as bridging molecules between DNA-bound NRs and the basal transcription machinery [90]. Co-activators interact with NRs via one or several leucine-rich a-helices, also known as NR boxes, with the consensus sequence LxxLL (where L corresponds to leucine and x is any amino acid residue) [91, 92]. Structural and functional studies indicate that the co-activator LxxLL helix is accommodated along the hydropho-

10.8 Co-repressors

bic cleft on the surface of the receptor LBD that forms upon ligand binding [93– 96]. Two strictly conserved amino acid residues on the receptor form a “charge clamp” that correctly places the co-activator LxxLL motif on the LBD, leading to transcriptional activation of the receptor. In the crystal structures of ER and RAR bound to antagonists, the AF-2 helix of the receptor is not repositioned correctly as in the ligand-bound receptors. Instead, it is translocated to overlap the co-activator interaction site, thereby preventing co-activator binding [96–99]. This in turn would facilitate the recruitment of another group of regulatory factors, corepressors, explaining the molecular mechanism behind antagonistic repression of NRs. The development of novel techniques for studying protein–chromatin interactions at specific promoters has yielded exciting new insights explaining the process of transcriptional initiation (reviewed in Ref. [100]). The results suggest that ligand-bound receptors continuously cycle on and off target promoters, transiently interacting with response elements on DNA, recruiting cofactors and RNA polymerase II to initiate transcription, and subsequently dissociating from DNA again [101]. Furthermore, it seems that cofactors with HAT activity not only acetylate histones at the promoter itself, but also further away on the DNA template to allow better access of proteins to promoter DNA. The exact sequence of events involved in transcriptional initiation is however unclear, and both a stepwise or concerted recruitment of p160 and TRAP/DRIP co-activators to the initiation complex has been suggested [102, 103] (see Fig. 10.4).

10.8

Co-repressors

Certain NRs, such as RAR and TR, repress basal transcription in the absence of ligand by binding the promoters of target genes, a process known as silencing [104]. The molecular mechanisms behind this phenomenon involves two related corepressor proteins, N-CoR (nuclear receptor co-repressor) and SMRT (silencing mediator of retinoid and thyroid hormone receptor), which both interact with RAR and TR in a ligand-independent manner [105, 106]. It has recently been shown that corepressors bind a region on the surface of the receptor LBD that overlaps the co-activator-interacting site. N-CoR and SMRT contain a-helical structures similar in sequence to the LxxLL helix of co-activators, with the consensus LxxI/HIxxxI/L (where L is leucine, I is isoleucine, H is histidine, and x is any amino acid residue) [107–109]. Thus, due to sequence similarities, corepressors are able to bind the same region on the NR LBD as co-activators, thereby masking the co-activator site and repressing transcription. Structural transitions in the LBD that occur upon ligand binding move the corepressor and allows AF-2 helix repositioning, which further displaces the corepressor. The extended corepressor motif no longer fits in the cavity due to steric hindrance by the AF-2 helix, and instead the co-activator gains entry to the site. Analogous to co-activators forming large multiprotein complexes, N-CoR and SMRT also interact with other proteins to repress transcription. Histone deace-

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Fig. 10.4 The different steps and proteins involved in NR transcriptional activation. DNAbound NRs (here exemplified as an RAR–RXR heterodimer) inhibit transcriptional activation by recruiting corepressors with histone deacetylase activity, keeping promoter DNA packed into histones, i.e. in a silent form. Upon exposure to ligand, co-repressors are released and co-activators of the p160 family and CBP/ p300 are recruited, either before or together with complexes like the TRAP/DRIP proteins.

The ATP-dependent activity of the SWI/SNF complex, initially acts to unwind DNA at the promoter. Histone acetylation by co-activators allows stronger interaction between the NRs and DNA, ultimately leading to recruitment of RNA Pol II and other accessory factors. These proteins recognize and bind DNA sequences of the TATA box at the transcriptional initiation site. RNA Pol II is finally released from the promoter and initiates gene transcription. See text for abbreviations and further details.

tylases (HDACs 1 and 2) are bridged to unliganded NRs via N-CoR/SMRT and mSin3 co-repressors, thereby mediating silencing (reviewed in Ref. [80]) (Fig. 10.4). Deacetylation of core histones by HDACs is recognized as a mechanism for keeping chromosome domains transcriptionally silent, and would explain how NRs mediate repression.

10.10 Fatty acids as Endogenous Ligands for RXR

10.9

Nuclear Receptors from an Evolutionary Perspective

Based upon the homologies within the superfamily, NRs have been divided into six subfamilies [110]. The ability to bind ligand and the structure of the ligand in several cases seems unrelated to which subfamily the respective receptors belong. For example, RAR and TR, which bind two unrelated ligands, are nonetheless more related in sequence than, for example, RAR and RXR, which both bind retinoic acid isomers, suggesting that ligand-binding ability is independent from the evolutionary origin [111]. The fact that orphan receptors are present in all subfamilies, whereas liganded receptors are not, further suggests that the ancestral receptor did not have a ligand. Interestingly, several recent reports have revealed the unexpected presence of fatty acids/lipids in the ligand-binding pockets of several NR crystals, including RXR, the Drosophila RXR ortholog Ultraspiracle, and retinoic acid-related orphan receptor b (RORb) [99, 112–114]. Apparently, these LBDs bind lipids derived from the bacterial strains used to express the proteins, and it seems likely that such binding is a prerequisite for a stable LBD conformation. These findings suggest an intriguing model explaining how the ligand-activated receptors of today have evolved. Accordingly, the primordial NR may have used a ubiquitous lipid as a structural element/cofactor to stabilize the LBD in its active state. In this view, the primordial “lipid binding domain” was permissive to those evolutionary adaptations necessary for a regulated domain to evolve, i.e. a structure that is regulated by bona fide ligand interactions. Ligand identity can thus be viewed as the result of an independent, convergent evolutionary process that took place in several different receptors, unrelated to their evolutionary origin within the NR superfamily.

10.10

Fatty acids as Endogenous Ligands for RXR

The signaling status of RXR in vivo is still a matter of debate, as its proposed natural ligand, 9-cis RA, has proved difficult to identify in mammalian tissue [115]. Nonetheless, several reports show that simultaneous addition of both RXR- and RAR-specific ligands often leads to synergistic biological effects (see, for example, Refs [57, 116, 117]). Therefore, it seems likely that ligand-induced activation of RXR does occur, a conclusion that has been corroborated by experiments in transgenic mice [14, 118]. Recent experiments have identified novel endogenous RXR ligands and expanded the perspectives on RXR functions in vivo. Interestingly, recent data suggests that the chlorophyll metabolite phytanic acid can bind and activate RXR [119, 120] (Fig. 10.1 B). Although phytanic acid is a low-affinity ligand and high serum levels would be required to activate RXR, significant activation might occur in patients with certain metabolic disorders such as Refsum’s disease [119, 120]. Docosahexaenoic acid (DHA or C22 : 6 cis 4, 7, 10, 13, 16, 19) (Fig. 10.1 B), a long-chain polyunsaturated fatty acid (PUFA), is an RXR ligand, does not activate

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RAR, TR, or VDR, and promotes the interaction between RXR and the co-activator SRC-1 [121]. Interestingly, other PUFAs including docosatetraenoic (C22 : 4 cis 7, 10, 13, 16), arachidonic (C20 : 4 cis 5, 8, 11, 14) and oleic (C18 : 1 cis 9) acids, also activate RXR, although DHA is more potent [121]. Unlike 9cRA, which binds and activates RXR with high affinity, DHA is a low-affinity ligand for this receptor, reaching half-maximal activation at a concentration of about 40–50 lM (our unpublished observations). However, while 9cRA has been difficult to identify in vivo, DHA, which accumulates in the mammalian CNS during late gestation and early postnatal development, constitutes between 30 and 50% of total membranebound fatty acids in the postnatal brain (reviewed in Refs [122] and [123]). Therefore, it seems likely that sufficiently high intracellular concentrations of free DHA may exist in neurons. Little is known about the mechanisms of DHA release from its phospholipid compartment, although some reports implicate phospholipases A2 and C in this process (see, for example, Ref. [124]). This mobilization could potentially supply enough free DHA to compensate for its rather low affinity towards RXR. Deficiencies in DHA have been shown to cause neurological abnormalities, impaired learning abilities and growth retardation (see, for example, Refs [125] and [126]). Memory deficits have recently been shown to improve upon either RA or DHA treatment, suggesting functional overlap between the two pathways [127–129]. Interestingly, the analyses of gene targeted mice lacking one or several genes encoding RXR isotypes have demonstrated overlapping functions with DHA, for example in the process of memory formation [130], vision [131], and reproduction [132]. Moreover, several RXR heterodimerization partners, such as PPARs, liver X receptors and farnesoid X receptor, contribute to energy and nutritional homeostasis in response to their respective ligands (reviewed in Ref. [133]). DHA could thus play an important modulatory role in these processes by binding and influencing the RXR subunit of such heterodimers. Structural analysis of the RXR LBD bound to DHA shows that the receptor LBD adopts the canonical conformation of a ligand bound NR, with the activation function AF-2 helix (H12) packed towards the hydrophobic groove formed on the surface of the protein [134]. The highly flexible fatty acid molecule is optimally accommodated to the ligand cavity of RXR, occupying 80% of the pocket (72% for 9cRA) and making ligand–protein contacts similar to those of 9cRA.

10.11

Perspectives

A few issues stand out as particularly likely to attract researchers’ attention over the next coming years. For example, although structural studies have provided valuable information as to how nuclear receptors in general, and retinoid receptors in particular, interact with each other, with DNA, and with co-regulatory proteins, most such studies have been performed on isolated domains of the receptors. Notably, crystal structures of receptors bound to DNA have been produced using

10.13 References

only the DNA binding domain. Similarly, ligand and co-activator binding studies have focused on the ligand binding domain. Undertaking similar studies using full-length receptors will undoubtedly provide additional and valuable data on how nuclear receptors perform their essential roles in vivo. Moreover, while the importance of retinoid receptors during embryonal development has been subject to detailed analysis, their roles in adult homeostasis are less clear. Gene ablation studies suggest that RXRa is the preferred partner of RARs in development in vivo [46]. Mice lacking the RXRa gene die before birth, a fact that has hampered the detailed analysis of its roles in adult homeostasis. Several studies have now employed tissue-specific knockout techniques to learn more about this important receptor (see, for example, Refs [135–137]), and recent data suggest that RXR ligands have potent effects on pathways involving other heterodimer partners than RAR [138]. Despite much work, little is known about retinoid receptor target genes, something that new approaches such as microarray experiments will help to address. It will be interesting to understand if isotype-specific retinoid receptor ligands induce the expression of different sets of downstream genes, perhaps by affecting the interaction between receptor and a certain co-activator or co-repressor. Perhaps the biggest mystery in retinoid action concerns the issue of specificity, i.e. how a simple small molecule such as retinoic acid can evoke such pleiotropic responses in development and adult physiology. Clearly, depending on cellular context the retinoid signal can be interpreted in many different ways. Reaching an understanding of how the diverse mechanisms discussed in this chapter can be differentially integrated to achieve the appropriate cellular responses should remain a major challenge in future retinoid receptor research.

10.12

Acknowledgements

This work was supported by the Göran Gustafsson Foundation.

10.13

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Liver X Receptors (LXRs) – Important Regulators of Lipid Homeostasis Lene K. Juvet and Hilde I. Nebb

11.1

Introduction

Several physiological processes are regulated by nuclear receptors. The role of liver X receptors (LXRs) as the body’s key sensing apparatus for maintaining cholesterol homeostasis has been elucidated over the past 4 years. LXRs regulate cholesterol catabolism, storage, absorption, and transport through the transcriptional control of key target genes involved in these processes. This work has been facilitated by characterization of the phenotype of LXR knockout animals, and use of this mouse model to identify LXR target genes. LXRa and LXRb were considered to be orphan nuclear receptors until the endogenous cholesterol metabolites oxysterols were identified as their specific ligands. LXRs require heterodimerization with the retinoid X receptor (RXR) to be transcriptionally active. Together they bind to DNA in conjunction with a variety of cofactors to an LXR response element preferentially consisting of a direct repeat of the hormone receptor response element half-site spaced by four nucleotides. Several LXR target genes in lipid homeostasis have been identified recently. In addition crosstalk between LXR and other transcription factors in lipid metabolism occurs, as well as hormonal signaling pathways. In this review, we focus on recent progress in understanding the physiological functions of LXRs as lipid regulators.

11.2

Nuclear Hormone Receptors

Nuclear hormone receptors belong to a superfamily of transcription factors mediating the transcriptional activity of endogenous or exogenous ligands [1–5]. This family contains approximately 50 members in humans [6] and includes the classical steroid hormone receptor family and the thyroid/retinoid/vitamin D receptor families. A growing number of proteins that possess the structural features of nuclear hormone receptors, but lack known ligands, have been identified. These are called orphan nuclear receptors. Identifying ligands for this class of receptors has accelerated the physiological characterization of the pathways they control.

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The protein structures are common for the members of this superfamily of transcription factors, and contain three specific functional domains. They usually include a poorly understood N-terminal transcriptional activation domain (AF-1), a central DNA binding domain (DBD) consisting of two highly conserved zinc fingers, and a C-terminal ligand binding domain (LBD) separated by a variable hinge region from the DBD. In addition the LBD contains an AF-2 domain involved in dimerization and transactivation properties of nuclear receptors. In general, in the presence of their ligands nuclear hormone receptors alter the transcriptional rate of specific genes. Upon ligand binding, nuclear receptors undergo a conformational change, releasing associated co-repressor proteins and promoting interaction with co-activators that allow the receptor to communicate with the general transcription machinery to regulate target gene expression [7, 8]. These target genes are regulated by selective interactions between the conserved DNA binding domain and their cognate response elements [3, 5].

11.3

The Liver X Receptors, LXRa and LXRb

LXRa (NR1H3 (Nuclear Receptors Nomenclature Committee, 1999), also described as RLD-1) was first isolated from rat liver and later from human liver [9, 10]. LXRa is expressed to a high extent in liver, with lower levels present in kidney, intestine, spleen, and adipose tissue [11]. The tissue specificity indicates that LXR might play a physiological role in lipid homeostasis. The main metabolic pathways are restricted (although not completely) to defined tissues such as triglyceride storage in adipose tissue; fatty acid oxidation in liver, kidney, and muscles; and lipoprotein synthesis in liver and intestine mucosa. The LXRa subtype, LXRb (NR1H2, also described as OR-1/UR/NER/RIP15) [12–15] is more ubiquitously expressed [11], but so far little is known about its function. The two subforms are highly related and share ~78% amino acid sequence identity in both DNA and ligand binding domains. LXRs require heterodimerization with the retinoid X receptor (RXR) to be transcriptionally active [10, 16]. LXRs are permissive RXR partners, as the heterodimer complex is activated by specific ligand for both receptors [16]. If both RXR and LXR ligands are present, a synergistic activity is obtained. The RXR/LXR heterodimer binds preferentially to a DNA sequence consisting of two conserved direct repeats of hexanucleotide motif (AGGTCA) separated by four bases, now commonly referred to as an LXR responsive element (LXRE) of the DR4 type [10, 12]. A major breakthrough in understanding the biological function of the LXRs was the identification of naturally occurring oxygenated cholesterol derivatives (oxysterols) as ligands for both receptors. This was achieved by screening organic extracts and natural compound libraries [17, 18]. Among oxysterols, 22(R)-hydroxycholesterol, 24(S)-25-epoxycholesterol, and 24(S)-hydroxycholesterol are the most potent oxysterols that bind and activate LXR, whereas the 22(S)-isomer showed no activation [17]. Other oxysterols that were potent activators of LXR were 20(S)-hy-

11.4 The Cholesterol Sensor: LXR Tab. 11.1 LXR target genes and hormone response elements (LXRE).

Target gene

LXRE

Tissue

Reference

Cyp7A1 CETP ABCG1 (LXRE1) (LXRE2) ABCG5 ABCG8 ABCA1 ApoE SREBP-1c

TGGTCA ctca AGTTCA GGGTCA ttgt CGGGCA

Liver Liver

18 98

TGGTCA ctca AGTTCA AGTTTA taat AGTTCA not identified not identified AGGTTA ctat CGGTCA GGGTCA ctgg CGGTCA GGGTT A ctgg CGGTCA

Macrophages, CNS

LXRa LPL FAS TNFa ACC ApoCI/apoCII/ap oCIV

AGGTTA ctgc TGGTCA TGGTCA ccac CGGTCA GGGTTA ctgc CGGTCA GGGCTA tgga AGTCGA GGGTTA cctc GGGTCA GGGTCA ctgg CGGTCA

34, 35, 97 34 Liver, intestine 47 Liver, intestine 47 Macrophages, intestine, CNS 36, 47, 97 Macrophages, adipose tissue 42, 43 Liver, adipose tissue, intestine, 30, 97 CNS Macrophages 44–46 Macrophages, liver 63 Macrophages, liver 60 Macrophages 99 Not reported 100 Macrophages 43

droxycholesterol, 20,22-dihydroxycholesterol, and 4b-hydroxycholesterol. Oxysterols that activated LXR, but with lower potency, included 26-hydroxycholesterol, 25-hydroxycholesterol, and 7a-hydroxycholesterol [19–20]. A binding assay based on scintillation proximity technology demonstrated that the most potent oxysterols bind with Kd values of 0.1–0.4 lM [18]. Extensive structure–activity relationship studies on LXR ligands have been performed and resulted in several synthetic LXR agonists, including a potent, high-affinity (Kd = 50 nM), non-steroidal ligand called T0901317 [21, 22]. This finding, together with the identification of cholesterol 7a-hydroxylase (CYP7A1) as a target gene of LXR (Tab. 11.1) [18] led to the suggestion that LXRs are part of a new hormone signaling pathway that plays an important role in the regulation of cholesterol homeostasis [17, 19]. CYP7A1 is the first and rate-limiting enzyme in conversion of cholesterol to bile acids [23–25]. Table 11.1 lists the LXR target genes that will be discussed during this review.

11.4

The Cholesterol Sensor: LXR

Cholesterol exerts essential physiological functions as an important constituent of cell membranes and as intermediates in crucial biosynthetic pathways such as synthesis of steroid hormones and bile acids. Cholesterol balance is achieved by equilibrium between dietary and biliary cholesterol absorption, cellular de novo

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11 Liver X Receptors (LXRs) – Important Regulators of Lipid Homeostasis

synthesis from acetyl coenzyme A, and hepatic catabolism into bile acids. The liver is considered as the principal cholesterol biosynthetic organ, and it produces up to 50% of newly generated cholesterol for export into the bloodstream and for intrahepatic storage as cholesterol esters. However, nearly all cells in the body contain the enzymatic machinery to synthesize cholesterol from acetyl-CoA. Thus, cholesterol is not an essential nutrient (i. e. the body is capable of synthesizing amounts adequate to meet its needs). However, significant amounts of cholesterol are still obtained by dietary intake (varies widely from 0.1 g for individuals on lowcholesterol diets to nearly 1 g on unrestricted diets) [26, 27]. Conversion of cholesterol to bile acids in the liver is the most important pathway for elimination of cholesterol from the body. A dysregulation of the input and output pathways leads to gallstone formation and hyperlipidemia, which may lead to metabolic disorders such as atherosclerosis. The first report demonstrating the importance of LXRa for maintenance of cholesterol homeostasis came with results from studies using LXRa-deficient mice. Peet et al. [28] demonstrated that LXRa plays a role in the cholesterol elimination process. The LXRa-knockout mice were reported to appear identical to wild-type littermates with regard to morphology, histology, and parameters such as serum and hepatic cholesterol levels and lipoprotein profiles when the animals were fed a standard chow diet (<0.02% cholesterol). However, striking differences between wild-type mice and LXRa–/– mice were observed when fed a diet enriched in cholesterol (2%). The wild-type mice exhibited an increase in liver CYP7A1 mRNA levels of 3- to 6-fold. This change was accompanied by an increased bile acid pool size and subsequent fecal excretion of bile acids. These effects ultimately resulted in an increased bile acid/cholesterol output to maintain body cholesterol homeostasis. LXRa–/– mice fail to make these changes in bile acid status and as a consequence these animals accumulate large amounts of liver cholesterol. Interestingly, LXRb–/–mice maintain their resistance to dietary cholesterol [29], in contrast to LXRa–/– mice where LXR is unable to compensate for the loss of LXR. However, the LXRa/b double knockout mouse shows a more severe liver phenotype than the LXRa–/– mouse upon cholesterol ingestion [28–30]. Because the process of cholesterol catabolism is liver-specific, other tissues in the body must deal with elevated cholesterol by effluxing the excess cholesterol back into the serum, where it is transported to the liver by reverse cholesterol transport. This process is achieved through a number of membrane ATP-binding cassette (ABC) transporters that deliver cholesterol to high-density lipoproteins (HDLs) which serve as the primary serum transporter of cholesterol back into the liver. This process is especially important in cells like enterocytes and macrophages, since they can be exposed to large levels of sterols due to unsaturable uptake of free cholesterol from diet and serum. Several recent studies have defined a pathway for cholesterol efflux from lipidloaded cells. ABCA1 and ABCG1, two members of the ABC family of transporter proteins are highly induced in macrophages loaded with cholesterol [31–33]. In macrophages, activation of the RXR–LXR heterodimer by either naturally occurring oxysterols or RXR/LXR agonists stimulates transcription of ABCA1 and

11.4 The Cholesterol Sensor: LXR

ABCG1 (Tab. 11.1) [33–36]. ABCA1 is the protein mutated in Tangier disease, a rare autosomal recessive disorder characterized by extremely low circulating levels of HDL, premature coronary heart disease, and accumulation of cholesterol in macrophages [37]. This condition derives from the inability to transfer cholesterol and phospholipid to apolipoprotein acceptors. Expression of ABCA1 is also upregulated by oxidized low-density lipoproteins (oxLDL). Another receptor, peroxisome proliferator activated receptor c (PPARc), is involved in uptake of oxLDL by the scavenger receptor CD36 in macrophages [38]. In addition to lipid uptake, PPARc is also involved in cholesterol efflux. Ligand activation of PPARc by oxLDL leads to primary induction of LXRa and secondary induction of ABCA1 [39]. In contrast to ABCA1, there is much less information on the function of ABCG1. The results of studies with cultured cells treated with antisense oligonucleotides to ABCG1 suggest that this protein may be involved in controlling the efflux of cellular cholesterol to HDL and/or secretion of apoE [32, 40]. ABCA1 is thought to mediate the active efflux of cholesterol and phospholipids to apolipoproteins (apo) acceptors, most importantly apoA1, the major apolipoprotein of HDL [41]. ApoE is also a possible acceptor for effluxing phospholipids and cholesterol. ApoE expression is increased following activation of LXR both in macrophages and in adipocytes (Tab. 11.1) (42, 43). Moreover, the ability of oxysterols and synthetic ligands to regulate apoE expression in adipose tissue and peritoneal macrophages is reduced in LXRa–/– or LXRb–/– mice and abolished in double knockouts. These findings support a central role for LXR signaling pathways in the control of macrophage cholesterol efflux through the coordinate regulation of apoE, ABCA1, and ABCG1 expression (Tab. 11.1). Interestingly, LXR induces the LXRa gene itself in human macrophages (but not murine) by a process that is dependent on a distally localized LXRE (Tab 11.1) [44–46]. OxLDL, oxysterols, and synthetic LXR ligands all induce the expression of LXR mRNA in human monocyte-derived macrophages. Autoregulation of the LXRa gene is suggested to be an important regulator of this lipid-inducible efflux pathway in human macrophages. Along this line, LXRs might prevent the over-accumulation of sterols in the macrophages by the induction of multiple ABC transporters and acceptor proteins involved in this pathway. In the small intestine increased dietary and/or secreted biliary cholesterol activates LXR as in macrophages and increases transcription of at least three ABC transporters, ABCA1, ABCG5, and ABCG8 [47]. In enterocytes, these transporters are hypothesized to increase cholesterol efflux into the intestinal lumen and thereby prevent net sterol absorption.

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11.5

Interplay between Cholesterol and Fatty Acid Metabolism 11.5.1

LXR and SREBP-1c Activation: a New Link between Cholesterol and Fatty Acid Regulation

Three sterol regulatory element binding proteins (SREBP-1a, SREBP-1c, and SREBP-2) with overlapping functions control lipid synthesis in liver and other tissues of mammals [48]. SREBP-1a and SREBP-1c are derived from a single gene through the use of alternate promoters that give rise to alternate first exons, thereby changing the N-terminal sequences of the proteins [48, 49]. SREBP-2 is derived from a different gene. In contrast to the other members of the basic helix-loop-helix zipper family, SREBPs are synthesized as inactive precursors bound to endoplasmic reticulum membrane, where they form complexes with SREBP cleavageactivating protein (SCAP) [50]. SCAP escorts the SREBPs to the Golgi complex where they upon request are cleaved sequentially, thereby liberating the N-terminal domain so that it can enter the nucleus to activate transcription [51]. To gain insight into the distinct roles of each SREBP isoform in vivo, transgenic mice that overexpress truncated, active nuclear forms of human SREBP-1a, 1c, or 2 in the liver have been produced and characterized [52–54]. These transgenic animals show different patterns of increased hepatic synthesis and accumulation of cholesterol and/or fatty acids. SREBP-1c was found to be more selective in activating lipogenic enzyme genes, which are involved in energy storage through synthesis of fatty acids and triglycerides (see Ref. [48] and reviewed in Ref. [54]), whereas SREBP-2 was more specific for controlling cholesterol biosynthesis. Overexpression of nuclear SREBP-1a profoundly activated both cholesterol and fatty acid synthetic genes [55]. Several observations indicate a link between LXR and SREBP-1c in cholesterol and fatty acid metabolism [28–30]. The LXRa–/– mice, as well as mice homozygous for a knockout of both isoforms of LXRa/b–/–, differed from the wild-type mice in a downregulation of liver mRNA levels for SREBP-1c [28–30]. Furthermore, the mRNA levels of several lipogenic enzyme genes such as fatty acid synthase (FAS), acyl-CoA carboxylase (ACC), stearoyl-CoA desaturase-1 (SCD-1) in liver were also downregulated in LXRa knockout mice and barely detectable in LXR/ double knockout mice [28–30]. In normal mice, the level of SREBP-1c mRNA was increased when the animals were treated with T0901317, a non-sterol synthetic ligand for LXR [30]. This response did not occur in LXRa/bdouble knockout mice. The mRNA level of SREBP-1a and SREBP-2 was not affected by these treatments, indicating that the LXR induction is specific for the SREBP-1c promoter. One of the pharmacological responses in animals treated with LXR agonists is the significant increase in serum triglycerides [22]. Furthermore, recent observations have shown that the basal transcription level of the SREBP-1c gene in cultured rat hepatoma cells depends on the synthesis of an endogenous sterol that activates LXR [56]. When synthesis of this sterol is blocked by an inhibitor of

11.5 Interplay between Cholesterol and Fatty Acid Metabolism

3-hydroxy-3-methylglutaryl (HMG)-CoA reductase, the level of SREBP-1c mRNA falls, and it is restored when the cells are supplied with the LXR ligands T0901317 and 22(R)-hydroxycholesterol [56]. Along this line, Repa et al. [30] (Tab. 11.1) identified an LXRE in the mouse SREBP-1c promoter in a cell-based reporter assay together this reveals a possible regulatory interplay between cholesterol and fatty acid metabolism. The notion that fatty acid and cholesterol metabolism is tightly linked through a cross-talk between LXR and SREBP-1c is further supported by the downregulation of SCD-1 in LXR knockouts [28–30]. SCD-1 is a target gene of SREBP-1c [52, 57], which is responsible for the D9-cis desaturation of stearoyl-CoA and palmitoylCoA, converting them to oleoyl-CoA and palmitoleoyl-CoA, respectively. The role of SCD-1 in cholesterol homeostasis was recently shown in SCD-1–/– mice. These mice showed an impaired production of oleic acid and a corresponding diminished capacity to esterify cholesterol for hepatic storage and packaging into very low-density lipoproteins (VLDLs) for export to other tissues [58]. The benefit of upregulating SCD-1 is to increase oleoyl-CoA; the preferred substrate for acyl-CoA cholesterol acyltransferase (ACAT)-mediated cholesterol esterification for storage under high cholesterol conditions [59]. Under conditions of high cholesterol, LXR may then indirectly promote the esterification of free cholesterol to protect the cell from its harmful effects. Furthermore, a coordinated increased fatty acid synthesis by induction of FAS will yield the phospholipids required for lipoprotein transport of excess cholesterol, and for maintaining plasma membrane integrity by providing the correct phospholipid/cholesterol ratio. From a physiological viewpoint the identification of SREBP-1c as a direct target of LXR is important to promote lipid synthesis to coordinate a homeostatic balance between fatty acids and sterols to ameliorate the effects of high free cholesterol levels in liver [30]. 11.5.2

Direct Regulation of Target Genes by LXRs in Lipid Metabolism

The findings that a cross-regulation between cholesterol and lipid metabolism exists in liver, prompted several research groups to investigate other lipid-regulating genes as potential targets of LXRa and LXRb action, which further expands the role of the LXRs as key regulators of lipid metabolism. Recently, both a direct and indirect regulation of FAS gene expression by LXRs was demonstrated [60]. FAS is a central enzyme in the pathway of de novo lipogenesis, catalyzing all the steps in the conversion of malonyl-CoA to palmitate. A downregulation of FAS expression in the liver of LXRa–/– and LXRa/b–/–, in addition to increased plasma triglyceride levels in mice administered with the synthetic LXR ligand, T0901317, supported further the regulatory role of LXRs in the control of lipogenesis [28–30]. Previously, SREBPs were shown to regulate FAS expression through direct interaction with the FAS promoter at multiple sites [61, 62]. Until now, the effects of LXR activation on FAS have been proposed to be entirely secondary to induction of SREBP-1c. According to this notion, a high-affinity binding site for LXR was identified in the FAS promoter [60]. These results indicate that LXR signaling pathways modulate FAS expression

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Fig. 11.1 LXR regulated pathways. LXR target genes are depicted in yellow boxes. HDL-Ch, HDL-cholesterol. Modified from Ref. [101].

through distinct but complementary mechanisms and suggest that the FAS gene may be a critical target in the control of the lipogenesis by LXRs [60]. A direct LXR regulation was recently also demonstrated for the expression of lipoprotein lipase (LPL) [63]. Mice fed diets containing high cholesterol or an LXRselective agonist exhibited a significant increase in LPL expression in the liver and macrophages, but not in adipose tissue or muscle. This cholesterol-induced LPL gene expression is directly regulated by LXR through LXR binding to a DR4 LXR response element in the intronic region between exons 1 and 2 of the LPL gene. LPL is a key enzyme for lipoprotein metabolism, responsible for hydrolysis of triglycerides in circulating lipoproteins, releasing free fatty acids to peripheral tissues. Figure 11.1 shows an overview of the LXR regulated pathways discussed. 11.5.3

LXRs as Insulin Sensors in Liver

Insulin is probably the most important hormonal factor influencing lipogenesis. It is known that both glucose and insulin are required for the production of fatty acids via the induction of hepatic lipogenic enzymes. The main role of insulin is to maintain the blood glucose concentration nearly constant in the face of large fluctuations in the dietary intake of glucose. Different groups have shown that SREBP-1c is upregulated by insulin in vivo and in primary hepatocyte cultures [64–66]. These observations raised a possibility that SREBP-1c could be a metabolic mediator of insulin action in the liver [67]. However, there are reports indicating that SREBP-1c is not the only transcription factor involved. In refed SREBP-1c–/– mice some hepatic lipogenic enzyme genes were completely abol-

11.5 Interplay between Cholesterol and Fatty Acid Metabolism

ished, whereas others were only partially suppressed, indicating that other factors in addition to SREBP-1c might be involved in the refeeding response [68]. A recent study has also demonstrated a diminished hepatic response to fasting/refeeding and LXR agonists in mice with selective deficiency of SREBP-1c [69]. Furthermore, the rapid effect of insulin on gene transcription of genes such as glucokinase does not require an increased amount of the mature form of SREBP-1c in the nucleus, suggesting that additional actions of insulin are [70, 71]. We recently demonstrated that insulin induces LXR mRNA levels in liver. This leads to an increase in the transcription of LXR target genes, such as SREBP-1 as well as many genes encoding enzymes in fatty acid and cholesterol biosynthesis [72]. The insulin regulation of these genes was abolished in LXRa/b double knockout mice, providing strong evidence that LXRs play a central role as insulin sensors in hepatic lipid homeostasis. This observation indicates a cross-regulation between LXR and SREBP-1c in response to insulin. At present, the consequence of the altered upregulation of both these genes in LXRa/b double knockout mice compared to wild-type animals is not fully understood, and further study will be required to determine if there are other metabolic defects in these mice under different dietary conditions. However, when LXRa–/– and LXRb–/– mice are subjected to fasting/refeeding regimen, SREBP-1c mRNA levels are similarly affected in both mouse strains compared to wild-type mice. The mechanisms by which insulin stimulates the transcriptional activity of the LXRs are presently unknown. However, regulation of transcriptional activity of LXR by insulin could be through divergent insulin signaling pathways such as the MAP kinase and phosphatidylinositol 3-kinase (PI3-kinase) pathways [70, 73–75]. Furthermore, several cis-acting elements that mediate the effect of insulin on gene transcription have recently been defined. These are referred to as insulin response sequences or elements (IRSs/IRE) (reviewed in Ref. [76]). In vitro studies have clearly established the importance of the upstream stimulatory factors (USFs) in the regulation of the fatty acid synthase promoter by insulin through insulin response elements called E boxes of CANNTG sequence [77]. Further studies are needed to clarify the mechanisms by which insulin stimulates the transcriptional activity of LXR in liver. We have earlier shown a PPARa-dependent fatty acid upregulation of LXRa mRNA and protein [78], and PPAR has previously been shown to be phosphorylated in response to insulin, resulting in stimulation of basal as well as ligand-dependent transcriptional activity of PPARa [79, 80]. PPARa could therefore be an upstream factor mediating the insulin effect on LXRa. 11.5.4

Fatty Acid Regulation of LXR

A cross-talk between cholesterol and fatty acid metabolism mediated by LXRa and PPARa was recently demonstrated in rat and mouse liver, where fatty acids (unsaturated being more effective than saturated fatty acids) transcriptionally upregulated LXRa (but not LXRb) in a PPARa-dependent manner [78]. PPARa is a member of the nuclear hormone receptor superfamily. PPARs have a pivotal role in

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regulation of intermediary metabolism, both in liver and adipose tissue, where they control the expression of several genes associated with fatty acid metabolism [81–85]. Conversely, fatty acids and their metabolites are reported to be natural ligands for the different PPARs [83, 86–88], and they might also control the expression of these receptors, as demonstrated for PPARa [89]. The upregulation of LXRa via PPARa seems to be a direct induction through a peroxisome proliferator response element (PPRE) located –722 bp in the LXRa 5'-flanking region (unpublished data). Since this regulation was observed both in cultured primary hepatocytes as well as in the intact animal, this regulation might have a direct physiological relevance in the control of lipid metabolism. Classically it was believed that fatty acid and cholesterol metabolism occurred along distinct biochemical pathways with relatively little interaction. However, LXRa itself has been demonstrated to affect the expression of various genes involved in fatty acid metabolism, such as stearoyl-CoA desaturase, FAS, ACC, and SREBP-1c [28–30]. The regulation of the cholesterol sensor LXRa by fatty acids indicates another important point of cross-talk between these two chemically distinct classes of lipids, i.e. fatty acids and cholesterol. This cross-regulation leads us to hypothesize that when the organism is challenged with an increased lipid load, usually composed of both fatty acids and cholesterol, an integrated response is mounted allowing it to handle this challenge [78]. Furthermore, recent data demonstrate that unsaturated fatty acids inhibit transcription of the SREBP-1c gene by antagonizing ligand-dependent activation of the LXR in cultured rat hepatoma and human HEK-293 cells and in cell-free assays that reflect LXR activation [90, 91]. This might partially explain the long-known ability of dietary unsaturated fatty acids to lower the levels of mRNA for SREBP-1c [92–94], and thereby decrease the synthesis and secretion of fatty acids and triglycerides in livers of humans and other animals. 11.5.5

LXRs in Adipose Tissue

Additional observations strengthen that LXR are fatty acid responsive and under a variety of dietary conditions they control lipid homeostasis in cross-talk with other transcription factors. Recent data in our group clearly show that LXRa is a PPARc target gene in adipose tissue (unpublished data). LXRa is expressed during 3T3L1 adipocyte differentiation after induction of PPARc. In contrast, LXRb is expressed already in pre-adipocytes. LXRa is expressed approximately two days after PPARc during adipocyte differentiation, while another well-established PPARc target gene, aFABP, appears at around the same time as PPAR. A similar cross-regulation between LXRa and PPARc also occurs in human primary adipocytes and in obese Zucker rats. This tight cross-regulation between PPARc and LXRa may reflect an important physiological interplay between these transcription factors in adipocytes, with a possible role for LXRa in controlling lipid homeostasis also in adipose tissue. It is well known that adipocytes play a central role in energy balance, both as a reservoir, storing and releasing fuel, and as endocrine cells, secret-

11.7 Acknowledgements

ing factors that regulate whole body energy metabolism [95]. Understanding this cell type is becoming increasingly important because of the rising incidence of obesity and its associated disorder, type II diabetes. The function of LXRa in adipocytes is unknown, however, adipocytes contain the largest pool of non-esterified cholesterol in the body [96], and this transcription factor may be important for cholesterol regulation in adipocytes.

11.6

Summary

Natural ligands for LXRs were identified less than 4 years ago. A rapid increase in our understanding of these receptors has recently occurred as a result of studies on mice in which the genes encoding LXRa and LXRb have been deleted. The data from these studies strongly implicates LXRs as cholesterol sensors and our understanding of the hepatic response to dietary cholesterol has increased. Thus, intracellular and extracelluar cholesterol levels are tightly maintained within a narrow concentration range by an intricate control mechanism orchestrated by a number of transcription factors. These recent findings also highlight the complexity of cellular cholesterol regulatory mechanisms and a cross-regulation between cholesterol and fatty acid metabolism. The observation that SREBP-1c is a target for LXRs suggests that LXRs may be involved in the control of lipogenesis. Recent work has also implicated LXRa as a PPAR target gene in different cell types, such as macrophages (PPARc), adipocytes (PPARc) and liver (PPARa). These studies suggest that, under a variety of dietary conditions, the control of lipid homeostasis is dependent upon cross-talk between these transcription factors. This clearly demonstrates that metabolic regulation cannot be discussed in the context of a single metabolite or transcription factor. Taken together, LXRs functions as essential regulatory components not only in cholesterol homeostasis but also in triglyceride metabolism.

11.7

Acknowledgements

We thank Professor Jan-Åke Gustafsson and PhD student Stine M. Ulven for critical reading of the manuscript.

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tafsson, J. A., Nebb, H. I. Mol. Endocrinol. 2000, 14, 741–752. Shalev, A., Siegrist-Kaiser, C. A., Yen, P. M., Wahli, W., Burger, A. G., Chin, W. W., Meier, C. A. Endocrinology 1996, 137, 4499–4502. Juge-Aubry, C. E., Hammar, E., SiegristKaiser, C., Pernin, A., Takeshita, A., Chin, W. W., Burger, A. G., Meier C. A. J. Biol. Chem. 1999, 274, 10505–10. Sørensen, H. N., Treuter, E., Gustafsson, J.-Å. Vitam. Horm. 1998, 54, 121– 166. Schoonjans, K., Staels, B., Auwerx, J. J. Lipid Res. 1996, 37, 907–925. Schoonjans, K., Martin, G., Staels, B., Auwerx, J. Curr. Opin. Lipidol. 1997, 8, 159–166. Brun, R. P., Spiegelman, B. M. J. Endocrinol. 1997, 155, 217–218. Desvergne, B., Wahli, W. In: Baurerle, P. ed. Inducible Gene Expression. Boston: Birkhauser, 1994, vol. 1, pp. 142–176. Gottlicher, M., Widmark, E., Li, Q., Gustafsson, J. A. Proc. Natl Acad. Sci. USA 1992, 89, 4653–57. Forman, B. M, Chen, J., Evans, R. M. Ann. NY Acad. Sci. 1996, 804, 266–275. Willson, T. M., Lehmann, J. M., Kliewer, S. A. Ann. N Y Acad. Sci. 1996, 804, 276–283. Steineger, H. H., Sorensen, H. N., Tugwood, J. D., Skrede, S., Spydevold, O., Gautvik, K. M. Eur. J. Biochem. 1994, 225, 967–974. Ou, J., Tu, H., Shan, B., Luk, A., DeBose-Boyd, R. A., Bashmakov, Y., Goldstein, J. L., Brown, M. S. Proc. Natl Acad. Sci. USA 2001, 98, 6027–32. Yoshikawa, T., Shimano, H., Yahagi, N., Ide, T., Amemiya-Kudo, M., Matsuzaka, T., Nakakuki, M., Tomita, S., Okazaki, H., Tamura, Y., Ohashi, K., Takahashi, A., Sone, H., Osuga, Ji. J., Gotoda, T., Ishibashi, S., Yamada, N. J. Biol. Chem. 2002, 277, 1705–11. Hannah, V. C., Ou, J., Luong, A., Goldstein, J. L., Brown, M. S. J. Biol. Chem. 2001, 276, 4365–72. Mater, M. K., Thelen, A. P., Pan, D. A., Jump, D. B. J. Biol. Chem. 1999, 274, 32725–32.

11.8 References 94

Xu, J., Teran-Garcia, M., Park, J. H., Nakamura, M. T., Clarke, S. D. J. Biol. Chem. 2001, 276, 9800–07. 95 Spiegelman, B. M., Flier, J. S. Cell 2001, 104, 531–543. 96 Krause, B. R., Hartman, A. D. J. Lipid Res. 1984, 25, 97–110. 97 Whitney, K. D., Watson, M. A., Collins, J. L., Benson, W. G., Stone, T. M., Numerick, M. J., Tippin, T. K., Wilson,

98 99 100 101

J. G., Winegar, D. A., Kliewer, S. A. Mol. Endocrinol. 2002, 16, 1378–85. Luo, Y., Tall, A. R. J. Clin. Invest. 2000, 105, 513–520. Landis, M. S., Patel, H. V., Capone, J. P. J. Biol. Chem. 2002, 277, 4713–21. Zhang, Y., Yin, L., Hillgartner, F. B. J. Biol. Chem. 2001, 276, 974–983. Lu, T. T., Repa, J. J., Mangelsdorf, D. J. J. Biol. Chem. 2001, 276, 37735–38.

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Acyl-CoA Ligands of HNF-4a and HNF-4a/PPARa Interplay Rachel Hertz and Jacob Bar-Tana

12.1

Transcriptional Activation by HNF-4a

HNF-4a is a member of the superfamily of nuclear receptors (reviewed in Ref. [1]). The spliced variants HNF-4a1, a2 and a3 differ in their C-termini and are expressed in liver, intestine, pancreas, and kidney as products of the HNF-4a gene at the human chromosome 20 (reviewed in Ref. [2]). HNF-4a2 is the predominant HNF-4a isoform in liver. HNF-4c is encoded by the HNF-4c gene at the human chromosome 8 and is not expressed in liver [3, 4]. The HNF-4b isoform has so far been identified in Xenopus only [5]. As a member of the superfamily of nuclear receptors, HNF-4a consists of a highly conserved DNA binding domain (DBD) linked through a hinge region to a ligand binding domain (LBD) [2]. The N-terminus consists of an acidic AF-1 domain (amino acids 1–24) having autonomous transcriptional activity [6]. The DBD (amino acids 50–116) consists of two folded zinc-finger motifs which bind to respective response elements/enhancers in the promoters of HNF-4a responsive genes. The LBD (amino acids 132–370) harbors a ligand binding site, dimerization domain, and hydrophobic AF-2 transactivation domain (amino acids 360–370) [7], and is C-flanked by an F-domain (amino acids 370–455) which may suppress transactivation by intramolecular tethering to the AF-2 domain [8, 9]. HNF-4a response elements consist of direct repeat hexamers 5'-RG(G/T)TCA separated by one nucleotide (DR-1) [2]. HNF-4a binds to its response element as homodimer [10] and its binding may be competed by PPAR/RXR, RAR/RXR, COUP-TFI (EAR-3), or COUP-TFII (ARP-1). Preference is determined by nucleotides 1, 2, and 4 of the core motif as well as by the promoter context [11]. The binding affinity of HNF-4a to DNA and its nuclear localization may be affected by its phosphorylation [12–14]. In analogy with other nuclear receptors, transactivation by HNF-4a is mediated by protein co-activators of the CBP/P-300, SRC-1, PGC-1, and Grip-1 families which bind to the AF-2 activation domain [15–18]. Transcriptional co-activation by HNF-4a co-activators having an intrinsic acetyltransferase activity is accounted for by acetylation of nucleosomal histones as well as of HNF-4a itself [19]. Transcriptional modulation by HNF-4a binding to its DR1 response elements is further complemented by its direct interaction with other

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transcription factors (e.g. HNF-1 [20], COUP [21], SP-1 [22], SMAD [23], HIF-1 [24], and SHP [25]). HNF-4a is required for the expression of many traits of the hepatic, enteric, and pancreatic differentiated state [26–29]. Disruption of the murine HNF-4a by homologous recombination results in embryo death due to failure of visceral endoderm to differentiate [30]. HNF-4a responsive genes [2, 26, 27, 31–36] encode some transcription factors (e.g. HNF-1a, PXR); enzymes and proteins involved in fatty acids, lipoproteins, and lipid metabolism (apoAI, AII, B, CII, CIII, Lp(a), microsomal triglyceride transfer protein (MTP), mitochondrial fatty acyl-CoA dehydrogenases, fatty acid binding protein (FABP)); carbohydrate metabolism (insulin, GLUT2, glucose-6-phosphatase, PEPCK, pyruvate kinase, aldolase B, glyceraldehyde-3-P dehydrogenase); amino acid and protein metabolism (ornithine transcarbamylase, tyrosine aminotransferase, phenylalanine hydroxylase, anti–trypsin 1); P450 enzymes (steroid 15a-hydroxylase (CYP2A4), fatty acyl x-hydroxylase (CYP4A6), cholesterol-7a-hydroxylase (CYP7A1), drug-metabolizing P450 enzymes (CYP3a4–6)); hematopoiesis (erythropoietin, transferrin); blood coagulation (factors VII, IX, and X, fibrinogen) and others (e.g. cellular retinol binding protein, transthyretin). Since HNF-4a activates the transcription of some nuclear receptors (e.g. HNF-1a) and may further directly interact with other transcription factors (e.g. HNF-1a), the above list of HNF-4a responsive genes may include some which are transcriptionally affected by HNF-4a indirectly.

12.2

Fatty Acyl-CoA Ligands of HNF-4a

Until recently, HNF-4a was considered to be an orphan receptor. Various longchain saturated or unsaturated fatty acyl-coenzyme A (CoA) thioesters longer than C12 have now been reported by us to specifically bind to the full length HNF-4a or its LBD recombinants [37, 38]. Binding of long-chain fatty acyl-CoAs has been verified by direct fluorescence methods, based on quenching of the HNF-4a(LBD) tryptophan (Trp) fluorescence by bound ligand, or the increase in fluorescence yield of a fluorophore (e.g. parinaroyl-CoA) bound to the hydrophobic ligand pocket of HNF-4a(LBD) [38]. Trp fluorescence quenching by added fatty acyl-CoAs (e.g. palmitoyl-, stearoyl-, lineoyl-, arachidonoyl-CoAs) has indicated a single binding site with Kd values in the 2.0–4.0 nM range (Fig. 12.1). Binding of fatty acyl-CoAs is specific but not exclusive, as the binding affinities of the free acids are significantly lower (Kd values of 421–742 nM) than of the respective fatty acyl-CoAs. The spectral overlap of HNF-4a(LBD) Trp fluorescence emission with parinaroyl-CoA absorption/exitation spectrum provides for fluorescence resonance energy transfer (FRET) analysis of the intermolecular distance between HNF-4a(LBD) Trp and bound acyl-CoA [38]. FRET analysis has yielded an intermolecular distance of £ 42 Å between the interacting ligand and protein, thus pointing to direct molecular interaction rather than non-specific coaggregation. These data refute claims made by Bogan et al. [39] for a restricted ligand binding pocket of HNF-4a de-

12.2 Fatty Acyl-CoA Ligands of HNF-4a

Fig. 12.1 Binding of long-chain fatty acylCoAs to HNF-4a(LBD). Binding was measured by Trp fluorescence quenching induced by titration of 170 nM rHNF-4a(LBD)(132– 455) with palmitoyl-CoA (A), stearoyl-CoA (B),

linoleoyl-CoA (C), and arachidonoyl-CoA (D). Linearization of the respective hyperbolic plots (inset) results in a single exponential, and yields dissociation constants in the range of 2–4 nM [38].

duced by computer modeling of HNF-4a(LBD) based on its apparent homology with the progesterone receptor and RXR LBDs. Binding affinities of fatty acyl-CoAs determined by HNF-4a trp fluorescence quenching are three orders of magnitude higher than those previously derived by radiolabeled acyl-CoAs competition assays [37]. This difference conforms with other examples where radioligand competition assays typically yield Kd values that are 2–3 orders of magnitude higher than those determined by direct methods [40– 42], due to the inherently required separation step between bound and free ligand in the radioligand assay. The recently determined Kd values of 2–4 nM are in the range of dissociation constants of other acyl-CoA binding proteins [40–42] as well as in the range of estimated nuclear fatty acyl-CoAs [43], thus conforming with the proposed role of long-chain fatty acyl-CoAs as endogenous ligands of HNF-4a [37]. Binding of long-chain fatty acyl-CoAs was verified as well for HNF-4c (Hertz et al., unpublished results). Binding of fatty acyl-CoAs resulted in HNF-4a conformational changes as deduced by circular dichroism or by protection of HNF-4a from limited proteolysis [38] (Fig. 12.2). In line with binding data, protection of HNF-4a from limited pro-

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Fig. 12.2 Conformational changes induced in HNF-4a(LBD) by ligand binding. (A) Limited proteolysis of [35S]methionine-labeled rHNF4a by chymotrypsin was measured in the absence (lane 2) or presence of 50 lM C16:1-CoA (lane 3), 100 lM C16:1-CoA (lane 4), 50 lM C16:1 (lane 5), 100 lM C16:1 (lane 6), 50 lM C14:0-CoA (lane 7), 100 lM C14:0-CoA (lane 8),

50 lM C14:0 (lane 9), and 100 lM C14:0 (lane 10). Lane 1 – undigested protein. Ligand-protected HNF-4a fragment(s) is marked by an arrow. (B) The far UV CD-spectrum of 2 lM rHNF-4a (LBD) was measured in the absence (filled circles) and in the presence of 50 nM arachidonoyl-CoA (empty squares) or palmitoyl-CoA (empty triangles).

teolysis required significantly higher concentrations of the free fatty acids than the respective fatty acyl-CoAs. The extent and nature of conformational changes induced by long-chain fatty acyl-CoAs were dependent on chain length and degree of saturation of respective acyl-CoA ligands. The transcriptional activity of HNF-4a was affected by long-chain fatty acyl-CoA thioesters, as function of their chain length and degree of unsaturation. Thus, fatty acids of C14–C16 activate, while long-chain (x-3) polyunsaturated fatty acids (PUFAs) (e.g. 20:5, 22:6) suppress HNF-4a transcriptional activity in cells transfected with the full-length HNF-4a and a reporter plasmid enhanced by the C3P response element of the apoCIII promoter [37], or promoted by the apoCIII promoter (–854/–1) (Fig. 12.3). The specific requirement for intracellular fatty acylCoAs in transfected cells incubated in the presence of the respective free acids was verified by shifting the dose–response curve to the left or to the right by transfecting the cells with a fatty acyl-CoA synthase or esterase, respectively [37]. Direct modulation of the transcriptional activity of HNF-4a by fatty acyl-CoA ligands was further verified in in vitro transcription assays consisting of the fulllength HNF-4a recombinant and an apoCIII(C3P)-enhanced template [37]. Activation/inhibition of HNF-4a transcriptional activity by acyl-CoAs is due to modulating the binding affinity of HNF-4a to its DNA cognate enhancer [37, 44], and/or shifting the equilibrium between active HNF-4a dimers and inactive oligomers [37], and/or modulating its transactivation capacity as verified in transfection studies using the GAL-HNF-4a(LBD) chimera (Fig. 12.4). Since each of these tran-

12.2 Fatty Acyl-CoA Ligands of HNF-4a

Fig. 12.3 Modulation of HNF-4a transcriptional activity by long-chain fatty acids in transient transfection. The effects of fatty acids proligands of fatty acyl-CoA agonists and antagonists of rHNF-4a were measured in Cos-7 cells transfected with a reporter plasmid consisting of the apoCIII promoter (–854/–1) upstream of CAT and an expression plasmid for

the full-length HNF-4a. Transfected cells were cultured in serum-free medium containing 200 lM C14:0, 150 lM C20:5, or 200 lM C22:6 complexed to 60 lM albumin. Fold induction refers to CAT activity in cells transfected with pSG5-HNF-4a as compared with cells transfected with pSG5.

Fig. 12.4 Modulation of GAL-4-HNF-4a(LBD) transcriptional activity by HNF-4a agonists in transient transfection. Modulation of transactivation by fatty acids proligands of fatty acylCoA agonists of HNF-4a was measured in Cos-7 cells transfected with a reporter plasmid consisting of UAS upstream of CAT and

an expression plasmid for the GAL-4-hHNF4a(LBD)(132–410) chimera. Transfected cells were cultured in serum-free medium containing 150 lM C14:0 or 150 lM C16:1 complexed to 60 lM albumin. Fold induction refers to CAT activity in cells incubated in the absence of added ligands.

scriptional parameters may be independently modulated by HNF-4a ligands, the resultant effect of a ligand on HNF-4a transcriptional activity may depend on the conditions prevailing within a given cell type and which may determine whether the transcriptional activity of HNF-4a is dominated by its dimerization, DNA binding or transactivation.

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The high affinity of HNF-4a for its acyl-CoA ligands may indicate that the apparent constitutivity of wild-type HNF-4a in transfection studies may reflect its saturation with an endogenous agonist ligand. The ubiquitous availability of fatty acylCoAs and their reported nuclear concentration in the range of 1–10 nM conform with this view. Similarly, the apparent constitutivity of wild-type HNF-4a in in vitro transcription assays [37] may perhaps reflect the availability and binding of an endogenous agonist ligand in cells expressing the wild-type recombinant [Hertz et al., in preparation]. This view implies that partial agonists of HNF-4a may still present as antagonists, if displacing an endogenous bound agonist of higher capacity. Transcriptional modulation by dietary fatty acids mediated by their respective acyl-CoAs requires that the acyl-CoAs profile of a concerned cell type should reflect the composition of dietary fatty acids. However, since fatty acyl-CoAs are intermediates in metabolic pathways leading from the respective free acids to esterified or oxidized end-products, the putative relationship between dietary long-chain fatty acids and their respective intracellular acyl-CoAs is not redundant and must be carefully analyzed. A novel ESI/MS/MS method now established by us offers a reliable, sensitive, and high-resolution method for characterizing and quantitating long-chain fatty and xenobiotic acyl-CoAs [45]. Using this method, the composition of liver fatty acyl-CoAs was indeed found to reflect the composition of dietary fat thus allowing for liver transcriptional events exerted by dietary fatty acids and transduced by their respective acyl-CoAs.

12.3

Xenobiotic Ligands of HNF-4a

Peroxisome proliferators (PPs), like fibrate drugs (e.g. clofibrate, bezafibrate, gemfibrozil, ciprofibrate, fenofibrate) or substituted long-chain fatty acids (e.g. b,b'-tetramethylhexadecane dioic acid (Medica 16), tetradecylthioacetic acid (TTA)), are potent hypolipidemics in rodents and humans (reviewed in Refs [46–48]). Since all consist of amphipathic carboxylates which may serve as substrates for the acylCoA synthase(s) [49, 50] (reviewed in Ref. [51]), and since lipoproteins assembly and plasma clearance are controlled by proteins encoded by HNF-4a responsive genes [32], the hypolipidemic activity of PPs may be accounted for by targeting HNF-4a. Indeed, acyl-CoA thioesters of some hypolipidemic PPs (e.g. fibrate drugs, Medica homologs) have recently been reported by us to bind to the fulllength HNF-4a or its LBD recombinant proteins yielding Kd values of * 3 and * 30 nM for Medica 16-CoA and bezafibroyl-CoA, respectively [52, 38]. Binding was specific for the respective acyl-CoAs whereas binding of the respective xenobiotic free acids required significantly higher concentrations (34 and 57 nM for Medica 16 and bezafibrate, respectively). Similarly, protection of HNF-4a from limited proteolysis required significantly higher concentrations of hypolipidemic PP free acids than the respective acyl-CoAs [52]. The transcriptional activity of HNF-4a is potently suppressed by hypolipidemic PPs in cells co-transfected with an expression vector for HNF-4a and a reporter

12.3 Xenobiotic Ligands of HNF-4a Fig. 12.5 Suppression of HNF-4a transcriptional activity by hypolipidemic PPs in transient transfection. Suppression of the transcriptional activity of HNF-4a by hypolipidemic PP proligands of HNF-4a acyl-CoA antagonists was measured in Cos-7 cells transfected with a reporter plasmid consisting of the apo (C3P)3 enhancer of the apoCIII promoter upstream of the thymidine kinase promoter-CAT and an expression plasmid for the full-length HNF-4a. Transfected cells were cultured in Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum and increasing concentrations of Medica 16 (n), Medica 18 (s), nafenopin (^), bezafibrate (·), or Cl-DICA(HOOC-CCl2-(CH2)10- CCl2COOH) (t). The a-chlorine atoms of Cl-DICA interfere with its CoA-thioesterification by the fatty acyl-CoA synthase. 100% activity amounts to 2.4- ± 2.8-fold activation in cells transfected with pSG5-HNFa as compared with pSG5 [52].

plasmid fused to a HNF-4a cognate enhancer (Fig. 12.5) [52]. The specific requirement for the respective intracellular acyl-CoAs of hypolipidemic PPs was confirmed in transfection assays by shifting the dose–response curve to the left or to the right upon co-transfecting the cells with fatty acyl-CoA synthase, or inhibiting the endogenous synthase by triacsin C, respectively [52]. The specific requirement for the respective acyl-CoAs of hypolipidemic PPs for suppressing the activity of HNF-4a was further corroborated by lack of effect of amphipathic carboxylates which, due to a structural constraint, do not serve as substrates for the acyl-CoA synthase (Fig. 12.5). Hence, suppression of HNF-4a activity by hypolipidemic PPs is limited by their intracellular conversion to their respective acyl-CoAs. The putative suppression of HNF-4a transcriptional activity by high concentrations of the free acid form of hypolipidemic PPs still remains to be investigated. The content of liver xenobiotic acyl-CoAs following treatment with hypolipidemic PPs in vivo was verified by ESI/MS/MS [52, 45]. Liver xenobiotic acyl-CoAs in rats fed with hypolipidemic PP (e.g. fibrate drugs, Medica homologs) exceeded by 2-fold total liver fatty acyl-CoAs with concomitant increase in its nuclear content, thus becoming the dominant liver acyl-CoA species. Hence, modulation of transcription of liver HNF-4 responsive genes by hypolipidemic drugs may be accounted for by their respective acyl-CoAs.

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12.4

HNF-4a and its Ligands in Health and Disease 12.4.1

Blood Lipids

Some proteins encoded by HNF-4a responsive genes may play a dominant role in the onset and progression of various forms of dyslipoproteinemia leading to atherogenesis and coronary heart disease. Specifically, increased plasma apoB levels and decreased plasma apoAI may result in increased plasma LDL-cholesterol/ HDL-cholesterol ratio while overexpression of apoCIII, MTP, and apoAII may lead to hypertriglyceridemia. Since the synthesis of apolipoproteins is regulated at the transcriptional level [reviewed in Ref. [32]), their expression as modulated by the transcriptional activity of HNF-4a and related transcription factors may be crucial in maintaining the homeostasis of plasma cholesterol and triglycerides. The agonistic/antagonistic/neutral effects of fatty acyl-CoA ligands of HNF-4a conform with the reported in vivo effects of dietary fatty acids on blood lipids. Thus, increase in plasma very low-density lipoproteins (VLDLs) induced by dietary fatty acids of C12–C16 and their decrease induced by (x-3)PUFAs [53] are in line with the effect of the respective acyl-CoAs in activating (C12:0–C16:0 fatty acylCoAs) or inhibiting ((x-3)PUFA-CoAs) HNF-4a-controlled transcription of liver genes encoding proteins controlling lipoproteins production (e.g. MTP, apoB) and their plasma clearance (e.g. apoCIII) [37]. Hence, HNF-4a may serve as target for modulating blood lipids by fatty acid nutrients. Similarly, HNF-4a may serve as target for modulating blood lipids by hypolipidemic fibrate drugs or substituted long-chain dioic acids. Suppression of HNF-4a transcriptional activity by hypolipidemic drugs [52] may account for both, inhibition of VLDL production due to suppression of apoB and/or MTP as well as activation of clearance of plasma triglycerides-rich lipoproteins (VLDLs and chylomicrons) due to suppression of apoCIII. The increase in HDL-cholesterol induced by hypolipidemic PPs in humans follows the paradigm of inverse relationship between plasma triglycerides and plasma HDL-cholesterol (reviewed in Ref. [54]). 12.4.2

MODY-1

Mutations in the human HNF-4a gene may result in maturity-onset diabetes of the young (MODY)-1 [55] (reviewed in Ref. [56]). MODY-1 is associated with HNF4a nonsense or frameshift mutations yielding truncated function-less proteins (e.g. Q268X, R154X), as well as missense mutations in the hinge region (e.g. R127W), ligand binding domain (e.g. V255M, E276Q) or the F-domain (e.g. V391I) which result in variable loss of transcriptional activity. Failure to activate transcription by missense MODY-1 mutants has been ascribed to impaired recruitment of co-activators [57]. Also, mutations in the HNF-4a binding site of the HNF-1a gene promoter may result in MODY-3 due to decreased expression of HNF-1a-responsive genes involved in glucose-stimulated insulin secretion [58].

12.5 Liver HNF-4a/PPARa Interplay in Rodents and Humans

MODY-1 is characterized by impaired insulin secretion in line with the direct (HNF-4a/DR-1-mediated) [59] and indirect (HNF-1a-mediated) [34] modes of transcriptional activation of the insulin gene promoter by HNF-4a. Furthermore, MODY-1 patients [33, 35] are hypolipidemic, in line with the profile of liver HNF4a-responsive genes coding for apolipoproteins and their assembling proteins. Thus, serum levels of apoAII, apoCIII, and triglycerides are reduced in MODY-1 subjects independently of their hyperglycemia [33]. Similarly, plasma triglycerides, apoB100, apoAII, and apoC contents are reduced in conditional hepatic HNF-4a knockout mice, while apoAI, apoE, and apoB48 remain unaffected [31]. The hypotriglyceridemic–hypocholesterolemic profile induced by HNF-4a hapo–insufficiency or hepatic HNFa disruption is essentially similar to that induced by respective hypolipidemic PPs, thus indicating that mutational or pharmacological suppression of hepatic HNF-4a may both result in lowering blood lipids. Missense mutants of the ligand binding domain of HNF-4a are defective in their transactivation capacity due to defective binding of HNF-4a agonist ligands (Hertz et al., submitted). The mutants are rescued by excess agonist ligands, reaching activities in the wild-type range. These findings point to means for rescuing selected MODY-1 patients by HNF-4a agonist nutrients or drugs. 12.4.3

Blood Coagulation

Mutations in the HNF-4a DNA binding sites of factors VII and IX gene promoters result in hemophilia [60, 61].

12.5

Liver HNF-4a/PPARa Interplay in Rodents and Humans

HNF-4a and PPARa are highly expressed in rodent liver and share some apparent common features. Thus, PPARa binds to DR-1 response elements essentially homologous to those which bind HNF-4a. However, in contrast to HNF-4a, which binds as homodimer, binding of PPARa to cognate enhancers requires the heterodimer PPARa/RXR where PPARa and RXRa interact with the upstream and downstream core hexamers of a given enhancer, respectively (reviewed in Ref. [62]). Also, in an apparent analogy with HNF-4a, PPARa is activated by natural long-chain unsaturated, branched chain or saturated fatty acids in this decreasing order as well as by xenobiotic hypolipidemic PPs (e.g. fibrate drugs, Medica homologs) [62]. However, in contrast to HNF-4a, PPARa activation requires the free acid form of PPARa agonists [50] while being inhibited by the respective acyl-CoAs [63, 64]. Since both nuclear receptors are highly expressed in liver, are modulated by natural and xenobiotic amphipathic carboxylates or their CoA-thioesters, respectively, and may compete for the same or similar response elements of respective gene promoters, the contribution made by each to the physiological or pharmacological effect induced by a given carboxylic nutrient or drug may depend on their mutual hepatic abun-

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dance and on the availability of their respective ligands and transcriptional co-activators. Specifically, the role of each in mediating the hypolipidemic effect induced by hypolipidemic PPs is of crucial importance since PPARa activation results in rodents in liver hypertrophy, hyperplasia, and peroxisome proliferation culminating in non-genotoxic hepatocarcinogenesis [65, 66]. The hypolipidemic effect exerted by hypolipidemic PPs in rodents is indeed accounted for by PPARa activation since none is induced in PPARa knockout mice [67], and hypolipidemic PPs which fail to be endogenously thioesterified to their respective acyl-CoA thioesters are still effective in rodents [52]. The hypolipidemic activity induced in rodents by PPARa activation by its agonists is due to both, activation of clearance of plasma triglycerides-rich lipoproteins as well as inhibition of liver VLDL production. Activation of clearance by PP-activated PPARa in rodents is due to suppression of apoCIII expression by hypolipidemic PPs. ApoCIII suppression is accounted for by HNF-4a displacement from the apoCIII gene promoter by binding of PP-activated PPARa to the HNF-4a response element, while PPARa binding is transcriptionally non-productive [68]. Suppression of liver VLDL production by PP-activated PPARa is accounted for by diverting long-chain fatty acids from esterification to lipids into oxidation, as a result of PPARa induction of genes coding for enzymes of b-, a-, and x-oxidation (reviewed in Ref. [48]). Hypolipidemia induced in rodents by the direct suppression of HNF-4a transcriptional activity by the respective acyl-CoAs of hypolipidemic PP is still questionable [52]. Its verification in PPARa knockout mice is problematic since the expression of liver acyl-CoA synthase is controlled by PPARa [69], thus limiting the availability of respective PP-CoA ligands of HNF-4a in PPARa knockouts. In contrast to rodents, the human liver is essentially non-responsive to PPARa (reviewed in Refs [70–72]). Thus, the pleiotropic effect induced by PPARa agonists in murine hepatocytes is not induced in human liver cells, human liver transplants or humans treated with hypolipidemic PPs. Lack of response of the human liver to PPARa activation is due to the low content of liver hPPARa as compared with rodents [73, 74], non-functional splice variants of liver hPPARa which do not respond to PPARa agonists [73, 74], and lack of PPARa co-activator in the human liver (M. Bronner, submitted). These quantitative and qualitative human characteristics as contrasted with rodents may all cooperate in essentially nullifying PPARa transcriptional activity in human liver cells even under conditions where hPPARa content is increased to its level in rodent liver cells [75, 76]. However, in spite of lack of response of the human liver to PPARa activation, hypolipidemic PPs are very effective as hypolipidemic agents in humans. Hence, the hypolipidemic activity of PPs in humans due to transcriptional suppression of liver HNF-4a-responsive genes (e.g. apoCIII, MTP, apoB, and others) must be independent of PPARa, but accounted for by direct suppression of HNF-4a transcriptional activity by the respective acyl-CoAs of hypolipidemic PPs. Indeed, HNF-4a responsive genes which control the composition and clearance of plasma lipoproteins (e.g. apoCIII, apoAI), are robustly suppressed in HepG2 cells by hypolipidemic PPs which yield the respective CoA-thioesters, but not by PPs which due to structural constraints fail to be endogenously CoA-thioesterified [52]. The

12.5 Liver HNF-4a/PPARa Interplay in Rodents and Humans

Fig. 12.6 Suppression of HNF-4a responsive genes by carboxylic effectors of PPARa and HNF-4a. Suppression of HNF-4a-responsive genes may be mediated directly or indirectly by suppression of HNF-4a transcriptional activity or by displacement of HNF-4a from its cognate enhancer by activated PPARa/RXR under conditions where PPARa/RXR is transcriptionally non-productive, respectively. The PPARa-dependent indirect pathway requires the free acid form (RCOOH) of PPARa car

boxylic agonists (e.g. hypolipidemic PPs, PUFA). The HNF-4a direct pathway requires the acyl-CoA thioesters (RCO-SCoA) of HNF4a carboxylic antagonists (e.g. hypolipidemic PP-CoA, PUFA-CoA). In cell types where both PPARa and HNF-4a are functional, the two pathways may synergize suppression of HNF-4a responsive genes. However, only the direct HNF-4a pathway is active in the human liver.

latter also fail to exert hypolipidemia in humans in contrast to their hypolipidemic efficacy in rodents (J. Bar-Tana, unpublished results). Hence, suppression of liver HNF-4a responsive genes by hypolipidemic PPs in humans and rodents is transduced by two diverging pathways, namely a direct one active in the human liver independently of PPARa, and consisting of HNF-4a suppression by its acylCoA antagonists, and an indirect one inactive in the human liver but active in the rodent liver and consisting of HNF-4a displacement by PPARa activated by its free acid agonists (Fig. 12.6). Direct suppression of HNF-4a transcriptional activity offers an hypolipidemic mode of action of PPs in humans independent of liver PPARa and its proliferative–carcinogenic activity.

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12.6

References 1

2

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11 12 13

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D. J. Mangelsdorf, C. Thummel, M. Beato, P. Herrlich, G. Schutz, K. Umesono, B. Blumberg, P. Kastner, M. Mark, P. Chambon et al. Cell 1995, 83, 835–839. F. M. Sladek, In: Tronche, F., Yaniv, M. eds, Liver Gene Expression. Austin, Texas: R. G. Landes Co., 1994, pp. 207–230. T. Drewes, S. Senkel, B. Holewa, G. U. Ryffel, Mol. Cell. Biol. 1996, 16, 925– 931. S. Taraviras, T. Mantamadiotis, T. Dong-Si, A. Mincheva, P. Lichter, T. Drewes, G. U. Ryffel, A. P. Monaghan, G. Schutz, Biochim. Biophys. Acta 2000, 1490, 21–32. B. Holewa, D. Zapp, T. Drewes, S. Senkel, G. U. Ryffel, Mol. Cell. Biol. 1997, 17, 687–694. E. Kistanova, H. Dell, P. Tsantili, E. Falvey, C. Cladaras, M. HadzopoulouCladaras, Biochem. J. 2001, 356, 635– 642. M. Hadzopoulou-Cladaras, E. Kistanova, C. Evagelopoulou, S. Y. Zeng, C. Cladaras, J. A. A. Ladias, J. Biol. Chem. 1997, 272, 539–550. V. P. Iyemere, N. H. Davies, G. G. Brownlee, Nucleic Acids Res. 1998, 26, 2098–2104. F. M. Sladek, M. D. Ruse, L. Nepomuceno, S. M. Huang, M. R. Stallcup, Mol. Cell. Biol. 1999, 19, 6509–22. G. Jiang, L. Nepomuceno, K. Hopkins, F. M. Sladek, 1995, Mol. Cell Biol. 15, 5131–43. H. Nakshatri, P. Bhat-Nakshatri, Nucleic Acids Res. 1998, 26, 2491–2499. B. Viollet, A. Kahn, M. Raymondjean, Mol. Cell Biol. 1997, 17, 4208–4219. G. Jiang, L. Nepomuceno, Q. Yang, F. M. Sladek, Arch. Biochem. Biophys. 1997, 340, 1–9. E. Ktistaki, N. T. Ktistakis, E. Papadogeorgaki, I. Talianidis, Proc. Natl Acad. Sci. USA 1995, 92, 9876–80. E. Yoshida, S. Aratani, H. Itou, M. Miyagishi, M. Takiguchi, T. Osumu, K. Murakami, A. Fukamizu, Biochem.

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H. Wang, P. Maechler, P. A. Antinozzi, K. A. Hagenfeldt, C. B. Wollheim, J. Biol. Chem. 2000, 275, 35953–59. M. Stoffel, S. A. Duncan, Proc. Natl Acad. Sci. USA 1997, 94, 13209–14. R. Jover, R. Bort, M. J. Gomez-Lechon, J. V. Castell, Hepatology 2001, 33, 668– 675. R. Hertz, J. Magenheim, I. Berman, J. Bar-Tana, Nature 1998, 392, 512–516. A. D. Petresen, R. Hertz, J. Bar-Tana, F. Schroeder, A. B. Kier, J. Biol. Chem. 2002, 277, 23988–99. A. Bogan, Q. Dallas-Yang, M. D. Ruse, Jr., Y. Maeda G. Jieng, L. Nepomuceno, T. Scanlon, F. E. Cohen, F. M. Sladek, J. Mol. Biol. 2000, 302, 831–851. M. J. McArthur, B. P. Atshaves, A. Frolov, W. D. Foxworth, A. B. Kier, F. Schroeder, J. Lipid Res. 1999, 40, 1371–1383. J. Knudsen, M. V. Jensen, J. K. Hansen, N. J. Faergeman, T. B. Neergaard, B. Gaigg, Mol. Cell Biochem. 1999, 192, 95–103. R. E. Gossett, A. A. Frolov, J. B. Roths, W. D. Behnke, A. B. Kier, F. Schroeder, Lipids 1996, 31, 895–918. M. Elholm, A. Garras, S. Neve, D. Tornehave, T. B. Lund, J. Skorve, T. Flatmark, K. Kristiansen, R. K. Berge, J. Lipid Res. 2000, 41, 538–545. F. Rajas, A. Gautier, I. Bady, S. Montano, G. Mithieux, J. Biol. Chem. 2002, 277, 15736–44. B. Kalderon, V. Sheena, S. Shachrur, R. Hertz, J. Bar-Tana, J. Lipid Res. 2002, 43, 1125–32. J. C. Fruchart, H. B. Brewer, E. Leitersdorf, Am. J. Cardiol. 1998, 81, 912–917. O. Faergeman, Curr. Opin. Lipidol. 2000, 11, 609–614. J. Bremer, Prog. Lipid Res. 2001, 40, 231– 268. M. Bronfman, M. N. Morales, L. Amigo, A. Orellana, L. Nunez, L. Cardenas, P. C. Hidalgo, Biochem. J. 1992, 284, 289–295. R. Hertz, I. Berman, J. Bar-Tana, Eur. J. Biochem. 1994, 221, 611–615. P. A. Watkins, Prog. Lipid Res. 1997, 36, 55–83.

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Part 2

Role for Proteins in Cellular Homeostasis

241

13

Fatty Acid Binding Proteins and their Roles in Transport of Long-chain Polyunsaturated Fatty Acids across the Feto-placental Unit Asim K. Duttaroy

13.1

Introduction

Fatty acids serve as a metabolic energy source, as building blocks for membrane lipids, and as cellular signaling molecules such as eicosanoids [1–6]. In fact, essential fatty acids (EFAs) and their long-chain polyunsaturated fatty acid (LCPUFA) derivatives are of critical importance in cell growth and development [2–6]. They are also increasingly being recognized as important intracellular mediators of gene expression [7]. Because of the fundamental role of EFA and LCPUFA as structural elements and functional modulators, it was hypothesized that the EFA/ LCPUFA status of tissue or cells is an important determinant of health and disease [2–6]. The multiple roles of fatty acids suggest that careful regulation of all aspects of their disposition, including cellular uptake and subsequent intracellular transport is critical for the maintenance of cellular integrity. However, the very property that makes long-chain fatty acids well suited to be components of membranes (i.e. their acyl chain hydrophobicity) complicates for the organism the task of transporting them from sites of intestinal absorption, hepatic synthesis, and lipolysis to sites of utilization. The insolubility of fatty acids in aqueous environment requires the use of specific trafficking mechanisms to deliver fatty acids for cellular needs [8]. The uptake of fatty acids was long considered to be a passive process, involving partitioning of the fatty acid molecule into the lipid bilayer of the plasma membrane, however studies from various laboratories have clearly demonstrated the presence of different fatty acid binding proteins both in the cytosol as well as in the cell membranes, and their involvement in the uptake and intracellular transport of these water-insoluble molecules [9–12]. In this chapter, I will discuss fatty acid binding/transport proteins and their important roles in fatty acid uptake and metabolism in the feto-placental unit.

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13.2

Fatty Acid Uptake in the Feto-placental Unit

During fetal development the deposit of LCPUFAs in the fetus is rapid during growth, and it is suggested that a failure to accomplish a specific component of brain growth due to inadequacy of LCPUFAs in critical membrane lipids may lead to irrevocable damage [13]. Fetal brain and retina are very rich in arachidonic acid, 20 : 4n-6 (ARA) and docosahexaenoic acid, 22 : 6n-3 (DHA), and a sufficient supply of these fatty acids during the last trimester of pregnancy and the neonatal period is of great importance [13–15]. Many studies have shown that the levels of LCPUFA are higher in the fetal than the maternal circulation [13, 16, 17]. At birth, linoleic acid, 18:2n-6 (LA) represents about 10% of the total fatty acids in cord plasma compared to 30% in maternal plasma, but surprisingly, ARA concentration in cord plasma is twice (*10%) that observed in the mother (*5%). Similarly, a-linolenic acid, 18 : 2n-3 (ALA) concentration in the new-born (0.6%) is half that in the mother (0.3%), whereas DHA concentration is double (3% versus 1.5%) [13, 16, 17]. This situation in which the relative plasma concentration of the n-3 and n-6 LCPUFAs (mainly DHA and ARA) exceeds that of their precursors (ALA and LA) is specific to the new-born and is never observed in adult. It is obviously an extremely favorable situation for the development of the new-born, especially at a time when high quantities of ARA and particularly of DHA are needed by the brain and retina. Placental transport of LCPUFAs from the maternal plasma is in practice crucial for fetal growth and development because fetal synthesis of LCPUFAs is thought to be very low [13–14, 18]. Moreover, human placental tissue lacks both the D6 and D5 desaturase activities [13, 18], therefore any LCPUFA in the fetal circulation must primarily be derived from the maternal plasma. However, three very important questions arise: (i) what are the underlying mechanisms leading to this situation, at birth? (ii) how does the situation evolve throughout gestation? and (iii) how are the n-3 and n-6 LCPUFAs (DHA and ARA) preferentially transferred? In the materno-fetal unit, free fatty acids (FFAs) are the main class of naturally occurring lipids transferred across the placenta, irrespective of species or of the maternal source from they originate in the maternal circulation are the major source of fatty acids for transport across the placenta [13, 19]. Maternal lipoprotein lipase must be active to facilitate placental uptake of FFAs from circulating triacylglycerol but not from chylomicrons [13, 14]. Recently, we have shown that placental microvillous membranes (MVM) exhibit two distinct triacylglycerol hydrolase activities: a minor activity at pH 8.0 and second major activity at pH 6.0 [20, 21]. Triacylglycerol hydrolase activity in MVM at pH 8.0 appeared to be lipoprotein lipase (identified by important criteria such as serum stimulation and salt inhibition) whereas the activity at pH 6.0 was unique as it was almost abolished by serum but was not affected by high NaCl concentrations. Therefore, it is possible that the inhibitory effect of serum on triacylglycerol hydrolase activity at pH 6.0 in MVM may not allow it to act on maternal lipoproteins but only on intracellular

13.3 Identification of Membrane-associated Fatty Acid Binding Protein in Human Placenta

triacylglycerol stores in placenta. If it is true then this membrane-bound triacylglycerol hydrolase (optimum pH 6.0) may play an important role in releasing FFAs by hydrolyzing the placental stores of triacylglycerol. This unique triacylglycerol hydrolase activity at pH 6.0 therefore may be involved in the packaging and/or release of FFAs from the placenta, whereas lipoprotein lipase which is mostly present in placental macrophages may be responsible for hydrolysis of maternal plasma lipoproteins as suggested by Bonet et al. [22]. The uptake of FFAs was long considered to be a passive process, involving partitioning of the fatty acids molecule into the lipid bilayer of the plasma membrane. There are now increasing reports providing evidence of the involvement of several membrane-associated fatty acid binding/transport proteins in the uptake of FFAs into a variety of mammalian tissues: hepatocytes, adipocytes, cardiomyocytes, and jejunal mucosal cells [23, 24]. Although LCFAs move very rapidly across artificial protein-free lipid bilayers, fatty acid binding or transport proteins may be required in biological membranes to promote cellular uptake of LCFAs from a complex environment [25, 26]. Several membrane proteins have been suggested to mediate FFA uptake into cells, such as plasma membrane fatty acid binding protein (FABPpm, 40 kDa) [24, 27], fatty acid translocase (FAT, 88 kDa) or CD36 [28] and fatty acids transport protein (FATP, 63 kDa) (for details see relevant chapters). Most of the tissues such as intestine, adipose, liver, etc. have more than one membrane fatty acid transporter, but the physiological implications are yet to be understood [29].

13.3

Identification of Membrane-associated Fatty Acid Binding Protein in Human Placenta

The placenta is composed of highly specialized trophoblast cells, which arise from the embryo and differentiate to perform specialized functions. These include invasion of the uterine wall, nutrient and waste transport, metabolism, evasion of the maternal immune system, and cytokine and hormone production. While passive diffusion does occur for some nutrient transfer, the fetal requirement for these nutrients is so great that passive diffuison alone is not adequate. Therefore specific nutrient carriers or transport proteins are located in the placenta and act to facilitate transfer and meet the increased nutrient demands of the fetus during gestation. The mechanisms involved in preferential uptake of maternal LCPUFAs by the placenta were first examined in our lab by determining the fatty acid binding characteristics of human placental membranes [30]. The binding of EFAs/LCPUFAs to human placental membranes was highly reversible. In addition, oleic acid, 18 : 1n-9 (OA) binding was inhibited very strongly by LCPUFAs (ARA, c-linolenic acid, 18 : 3n-6, and eicosapentaenoic acid, 20:5n-3, EPA), followed by the relevant parent EFA (LA and ALA). The lack of strong inhibition of the binding of EFAs/ LCPUFAs to placental membranes by OA suggests the existence of stronger affinities for EFAs/LCPUFAs compared with OA [30]. EPA and its eicosanoid metabo-

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13 Fatty Acid Binding Proteins and their Roles in Transport of Long-chain Polyunsaturated Fatty Acids

lites are growth inhibitory as they reduce the availability of ARA and its metabolites by competing at cyclooxygenase/lipoxygenase and desaturation/elongation pathways of EFA metabolism. However, EPA has very little inhibitory effect on ARA binding to human placental membranes [30]. In contrast to EPA, its parent fatty acid, ALA inhibited the binding of both ARA and LA strongly. Competition experiments also suggested that the binding sites in human placental membranes have heterogeneous binding affinities for fatty acids. Binding sites had a strong preference for LCPUFA: the order of competition was AA>>>LA>ALA>>>>OA [30]. Evidence for the involvement of membrane protein in fatty acid uptake came from the trypsin-treated placental membranes which showed a decrease in specific [14C]fatty acid binding compared with that of untreated membranes [30]. The presence of FABPpm both in sheep and in human placental membranes was demonstrated [31, 32]. A 40-kDa protein which binds only long-chain fatty acids was isolated and purified from human placental membranes using established procedures [24]. The apparent molecular mass of the protein was determined by gel permeation chromatography and by SDS-polyacrylamide gel electrophoresis (PAGE). The pI value and the amino acid composition of human placental membrane fatty acid binding protein are different from those of hepatic or gut FABPpm [32]. In addition, unlike ubiquitous FABPpm [24], placental FABPpm (pFABPpm) does not have glutamic-oxaloacetic transaminase (GOT:l-aspartate:2-oxaloglutarate:aminotransferase, EC 2.6.1.1) activity [33]. Therefore, despite similar size and membrane location (both are peripherally membrane-bound protein), pFABPpm and ubiquitous FABPpm differ both in structure and function. Pre-incubation of placental membranes with polyclonal antiserum against pFABPpm inhibited the binding of fatty acids but the degree of inhibition varied depending on the type of fatty acid [30]. Antibody-mediated inhibition was much more acute for the LCPUFA binding than the OA binding, suggesting again the heterogeneity of fatty acid binding activity of p-FABPpm and that this may be preferentially involved in the placental LCPUFA uptake. Fatty acid binding activity, PAGE radiobinding assay, and Western blot analysis of microvillous and basal membranes of the human placenta clearly demonstrated that the p-FABPpm is exclusively located in the microvillous membranes of the human placenta [34]. To further elucidate the mechanisms of preferential transfer of maternal plasma LCPUFA by human placenta, direct binding of the purified p-FABPpm with fatty acids was investigated [35]. p-FABPpm bound only 14.58% of the total fatty acids when the protein was incubated with fatty acid mixture with the exact composition and concentration as the maternal plasma FFA pool during the last trimester of pregnancy. Almost 98% of the ARA and 87% of DHA present in the mixture bound to the protein, which was much higher than that for OA and LA (21% and 13% respectively) [35, 36]. Binding of palmitic and a-linolenic acid was nil or insignificant. In contrast, under identical experimental conditions, human serum albumin (HSA) bound 36% of the total fatty acids but did not show any preference for particular fatty acids [35, 36]. Radiolabeled fatty acid binding revealed that pFABPpm had higher affinities (Kd) and binding capacities (Bmax) for LCPUFA

13.3 Identification of Membrane-associated Fatty Acid Binding Protein in Human Placenta

compared with other fatty acids. The apparent Bmax values for oleic acid, linoleic acid, arachidonic acid, and docosahexaenoic acid were 2.0 ± 0.14, 2.1 ± 0.17, 3.5 ± 0.11, 4.0 ± 0.10 mol per mol of p-FABPpm, whereas the apparent Kd values were 1 ± 0.07, 0.73 ± 0.04, 0.45 ± 0.03 and 0.4 ± 0.02 lM, respectively [37]. In the case of HSA, the Kd and Bmax values for all fatty acids were around 1 lM and 5 mol per mol of protein, respectively. This data provides direct evidence for the role of p-FABPpm in preferential sequestration of maternal LCPUFAs by the placenta for transport to the fetus [36]. In order to elucidate further the mechanisms by which fatty acids are taken up by the placenta, the uptake of OA, LA, ARA, and DHA by cultured human placental choriocarcinoma (BeWo) cells was examined [33, 35, 36]. BeWo cells display placental differentiation markers including the production of placental-specific proteins, and has been used extensively to study lipoprotein metabolism. Fatty acid uptake by BeWo cells was temperature-dependent and exhibited saturable kinetics. OA was taken up least and DHA most by these cells [33]. Moreover, competitive studies of fatty acid uptake by BeWo cells also indicated preferential uptake compared with OA in the order of DHA, AA, and LA. When fatty acid uptake was compared between BeWo cells and HepG2 cells, EFAs/LCPUFAs were taken up by BeWo cells preferentially over OA, however, no such discrimination was observed in HepG2 cells (Tab. 13.1). Western blot analyses demonstrated the presence of p-FABPpm in BeWo cells which was absent from HepG2 cells, confirming our previous observation that the placental FABPpm is different from the hepatic protein [33]. Furthermore, pretreatment of BeWo cells with anti-p-FABPpm antibodies inhibited most of the uptake of DHA (64%) and AA (68%), whereas OA uptake was inhibited only 32% compared with the controls treated with pre-immune serum (Tab. 13.2). The order of fatty acid uptake inhibition by the antibodies was DHA > AA >>> ALA > LA >>> OA, indicating that the p-FABPpm is involved in preferential

Tab. 13.1 Uptake of oleic and linoleic acids by BeWo and HepG2 cells a)

Radiolabeled fatty acid + unlabeled fatty acid

Fatty acid uptake (nmol mg–1 protein) BeWo cells

[3H]Oleic acid,18 : 1n-9 [3H]Oleic acid,18 : 1n-9 + linoleic acid,18:2n-6 (–10-fold) [14C]Linoleic acid,18 : 2n-6 [14C]Linoleic acid, 18 : 2n-6 + oleic acid18 : 1n-9 (–10-fold) [14C]Linoleicacid,18 : 2n-6 + a-linolenic acid, 18 : 3n-3 (–10-fold)

5.56 ± 0.16 1.41 ± 0.15 b) 6.72 ± 0.50 5.40 ± 0.30 1.14 ± 0.09 d)

HepG2 cells 22.89 ± 0.34 8.67 ± 0.48 c) 27.78 ± 1.59 16.83 ± 1.89 b) 17.49 ± 0.36 b)

a) Competition between [3H]Oleic acid and [14C]linoleic acid for uptake by BeWo and HepG2 cells was carried out by incubating these cells with radiolabeled fatty acid in the presence and absence of a 10-fold excess of unlabeled fatty acid. Data represent the mean ± SEM of three separate experiments in which triplicate determinations were performed. b) P > 0.05; c) P < 0.005; d) P < 0.001.

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13 Fatty Acid Binding Proteins and their Roles in Transport of Long-chain Polyunsaturated Fatty Acids Tab. 13.2 Effect of anti-human p-FABPpm antibody on fatty acid uptake by BeWo cells a)

Fatty acid

% Inhibition of fatty acid uptake by the anti-p-FABPpm antibody

Oleic acid, 18 : 1n-9 Linoleic acid, 18 : 2n-6 Arachidonic acid, 20 : 4n-6 Docosahexaenoic acid, 22 : 6n-3

32 ± 3.7 50 ± 2.4 b) 68 ± 4.5 b) 64 ± 3.3 b)

a) Cultured BeWo cells pre-treated with p-FABPpm antiserum were compared with a control preparation with pre-immune serum. b) P < 0.005 (significantly different from antibody inhibition of oleic acid uptake). Data represent the mean ± SD of three separate experiments in which triplicate determinations were performed.

uptake of LCPUFA by these cells similar to what was observed with human placental membranes [30]. These results clearly demonstrate that the p-FABPpm may be involved in the preferential uptake of LCPUFAs by these cells. Studies on the distribution of radiolabeled fatty acids in the cellular lipids of BeWo cells showed that DHA was incorporated mainly in the triacylglycerol fraction, followed by the phospholipid fraction, whereas for AA the reverse was true [38]. Almost 60% of the total DHA taken up by the cells was esterified into triacylglycerol whereas 37% was in phospholipid fractions. One of the main functions of the placenta is to deliver DHA into the fetal circulation, therefore, the triacylglycerol form may favor the transport of DHA to the fetal circulation.

13.4

Identification and Location of FAT/CD36 and FATP in Human Placental Membranes

In addition to p-FABPpm, the presence of other two fatty acid transporters, FAT/ CD36 and FATP, were demonstrated in human placenta using pure trophoblast cells and placental microvillous and basal membrane preparations [39]. FAT and FATP are present in both the placental membranes, microvillous and basal membranes, whereas p-FABPpm is only present in microvillous membranes [39]. Although the presence of multiple membrane fatty acid transporters in different tissues has been reported [40] their complex interactions in the uptake of FFAs are yet to be understood. Studies in other tissues suggested that these membrane proteins alone or in tandem may be involved in effective uptake of FFAs [40]. Unlike p-FABPpm, FAT/CD36 or FATP does not have any preference for LCPUFAs [38]. Therefore, location of FAT and FATP on the both sides of the bipolar placental cells may allow bidirectional flow of all types of FFA (non-essential, EFA, and LCPUFA) across the placenta, whereas the exclusive location of pFABPpm on the maternal side may favor the unidirectional flow of maternal plas-

13.5 Presence of Cytoplasmic Fatty Acid Binding Proteins (FABPs) in Human Placenta

ma LCPUFAs to the fetus by virtue of its preference for these fatty acids. However, further work is necessary to understand the mechanistic roles of these membrane proteins in trophoblasts which may provide insight into lipid transport and metabolism in the placenta.

13.5

Presence of Cytoplasmic Fatty Acid Binding Proteins (FABPs) in Human Placenta

Once taken up in the cell, the intracellular transport of FFAs is also thought to be mediated by FABPs within the cytosol. Human placenta contains both liver type (L-) and heart type (H-) FABPs [39]. As there are many differences between LFABP and H-FABP [41], the expression of these two proteins in the placental trophoblasts may be related to differential fatty acids transport and metabolism to meet demands for feto-placental growth and development [33]. The stoichiometry of binding is 1 mol fatty acid per mole protein for all FABP types except for LFABP which binds 1–2 mol FFA [41]. H-FABP only binds FFAs, whereas L-FABP binds heterogeneous ligands [41]. L-FABP has been implicated in cell growth and regulation by virtue of its binding to various growth stimulatory and inhibitory eicosanoids as well as selenium [14, 26, 42]. Prostaglandin (PG)E1 and heme bind L-FABP with higher affinity than fatty acids, as do the lipoxygenase metabolites of arachidonic acid [41]. The cyclooxygenase metabolites PGE2, TxB2, and LTB4 do not bind L-FABP, whereas the cyclopentenone prostaglandins (PGA1, PGA2, PGJ2, and D12-PGJ2) bind more avidly than oleic acid to L-FABP. PGD2, PGE2, and PGF2a are poor competitors and PGE1 is intermediate. L-FABP is probably responsible for transporting these cyclopentenone prostaglandins to the nuclear membranes. Recently, it has been demonstrated that L-FABP transports ligands to PPAR through protein–protein interaction, and thus may play an important roles in gene expression [43]. No such role has been shown for H-FABP as yet. The differential effects of FABPs on fatty acid uptake and esterifcation were demonstrated using transfected mouse L-cell fibroblasts. L-FABP increases fatty acid uptake and targets this fatty acid for esterification into phospholipids, while I-FABP does not increase fatty acid uptake but preferentially stimulates esterification into triacylglycerol [44]. It is possible that these proteins have different roles in terms of intracellular trafficking of fatty acids in syncytiotrophoblast. The LFABP and I-FABP genes exhibit distinct patterns of tissue specific and developmental regulation [45]. In developing heart, H-FABP mRNA was detected at the earliest time point in rat (19-day gestation period) [46]. However, very little is known about the regulation of expression of L-FABP and H-FABP in human placenta syncytiotrophoblasts. The different types of placental FABPs may be involved in the channelling of specific fatty acids to b-oxidation, in the synthesis of structural and storage lipid in the placenta, in the transfer to fetal circulation, or in placental growth and regulation.

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13.6

Presence of Nuclear Transcription Factors that Bind Fatty Acids in Human Placenta: Interaction Between Fatty Acid Binding Proteins and PPARc

Hypolipidemic drugs and fatty acids induce expression of L-FABP via peroxisome proliferator activated receptor a (PPARa) in liver [47]. Recent evidence has shown that membrane fatty acid transport proteins (FAT, FATP) are also modulated in a tissue specific manner by PPARs [48, 49], indicating that these fatty acid binding/ transport proteins are the target genes for PPARs. So far, three types of PPARs (PPARa, PPARb, and PPARc) have been reported in mammalian systems. PPARc is associated primarily with lipolytic effects and is known to regulate enzymes involved in adipose tissue differentiation and lipid storage and metabolism [50]. Both PPARc and RXRa are expressed in human placenta, and these two nuclear receptors cooperate to induce the synthesis of human choriogonadotropin (hCG), a hormone essential for human pregnancy [51]. The critical role of PPARc was earlier demonstrated in mice deficient in PPARc, which exhibit abnormal placental development and trophoblastic differentiation [52]. PPARc and RXRa ligands increase hCGb transcript levels, and the hCG promoter was suggested to contain binding sites for PPARc/RXRa heterodimers [51]. In view of the general changes in placental trophic hormone production, this heterodimer may thus favor placental differentiation and maintenance. Therefore, stimulation of differentiation may occur through direct induction of placental hormone production triggered by the ligandinduced activation of PPAR/RXR heterodimers, rather than through an increase in the levels of these receptors. Placental hormones would then induce and sustain mature placental function via a feed-forward amplification loop. The tissue-specific regulation of FAT/CD36 and FATP expression by PPARs has been demonstrated, with hepatic expression of FAT, and FATP under the control of PPARa, whereas in adipose tissue these proteins are under the control of PPARc [50, 53]. In contrast, it appears that FABPpm expression is not under the control of PPARa and PPARc [50, 53]. However, no such information is available with regard to the human placental fatty acid transport/binding proteins. Several naturally occurring ligands for PPARc have been identified, including fatty acids, oxidized LDL derivatives, and prostaglandin metabolites 15-deoxy-D12, 14 prostaglandin J2 (15-deoxy-D12, 14PGJ2) [50]. The potential natural ligands involved in activating PPAR/RXR heterodimers in the placenta are not known yet, however it is possible that these ligands may activate expression of fatty acid binding/transport proteins in human placenta. Expression of p-FABPpm permits placenta to sequestrate ARA and DHA [30], which may also act as ligands for PPAR and RXR. The presence of FAT, FATP, L-FABP, and H-FABP in placental trophoblasts [30, 36] may help to deliver the ligands (fatty acid and eicosanoids) to PPARc for gene expression. Therefore, it is conceivable that natural ligands for PPAR are either synthesized or taken up by the trophoblast, enabling PPAR/RXR heterodimer activation and subsequent hCG production. In addition to the PPAR family (PPARa, b, c1, and c2), several other transcription factors have been identified as targets for fatty acids regulation, including

13.6 Presence of Nuclear Transcription Factors that Bind Fatty Acids

HNFa, SREBP-1c, LXRa, and b, RXRa, and NFjB (please see relevant chapters). In addition to PPARc, the placenta has also sterol regulatory element binding proteins (SREBPs), transcription factors that regulate cholesterol and, in response to sterols, provide important insights into the regulation of lipid synthesis [54, 55]. SREBP-1 and -2 are separate gene products of approximately 125 kDa located in the membranes of the endoplasmic reticulum and nuclear envelope. The gene for SREBP-1 has two different transcription start sites that generate two mRNAs and proteins, SREBP-1a and -1c. SREBP-1 and -2 are both detected in placenta [54, 55]. Recent studies have demonstrated that SREBPs also regulate the transcription of ACC and FAS, key enzymes for fatty acid synthesis [56–58]. Insulin is also known to act via SREBP-1c via augmenting the nuclear content of SREBP-1c, while LCPUFAs suppress the nuclear content through a post-transcriptional mechanism [54, 55]. The simultaneous regulation of enzymes in both cholesterol and fatty acid synthesis pathways may allow for coordinate regulation of the synthesis of both lipid species in the placenta. Liver X receptor (LXR) is the newest twist to this story and we are at an early stage in understanding this pathway. LXRs play a major role in lipo-

Fig. 13.1 Schematic diagram of the putative roles of fatty acid binding/transporter proteins in placental fatty acid uptake and metabolism. The location of FAT and FATP on both sides of the placental membranes indicates

control of FFA flux in these cells, whereas the location of p-FABPpm exclusively on the maternal side of the placenta shows that it allows unidirectional transport of LCPUFAs from the maternal side to the fetal side.

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13 Fatty Acid Binding Proteins and their Roles in Transport of Long-chain Polyunsaturated Fatty Acids

genesis through the regulation of the gene encoding SREBP-1c [59, 60]. However, in hepatic cell lines, it appears that n-3 LCPUFA can interfere with LXR action [61, 62]. This network controls lipogenesis and triglyceride synthesis. While it appears that n3 PUFA oppose insulin action, it must be remembered that insulin induces SREBP1c gene transcription, while n-3 PUFA controls SREBP-1c by enhancing the turnover of the mRNA. n-3 PUFAs regulate hepatic glucose/lipid homeostasis by controlling the activity and/or abundance of separate transcription factors. Together, these effects may determine the shift in metabolism from fatty acid synthesis and storage to fatty acid oxidation in human placenta. Figure 13.1 summarizes the role and expression of fatty acid binding proteins in human placenta. Future studies should explore the relationships between these nuclear transcription factors and fatty acid binding/transport proteins to better understand the role of fatty acids in feto-placental growth and development.

13.7

References 1 2 3 4 5 6 7 8

9

10

11 12

13

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Fatty Acid Binding Proteins of the Brain Yuji Owada and Hisatake Kondo

14.1

Introduction

Fatty acids are important as substrates for energy supply, as building blocks of membrane lipids, as signaling molecules and as precursors for lipid mediators. Since fatty acids are hydrophobic molecules, they are solubulized and transported by specific intracellular lipid binding proteins, the low molecular mass polypeptides of 14–15 kDa termed fatty acid binding proteins (FABPs). They were originally named according to the tissue of their first isolation, such as heart (H-), epidermal (E-), and brain (B-) type [1–4], however, they show much wider tissue distribution than first thought [5–7]. Several roles have been assigned to these proteins: (i) control of cellular uptake of fatty acids and their subsequent utilization, (ii) intracellular compartmentation of fatty acids, (iii) modulation of activity of enzymes involved in fatty acid metabolism, (iv) protection of cellular enzymes and membranes from detergent effects of fatty acids, and (v) carriers of signaling fatty acids [8] (see also Chapters 5, 6, 13, and 15). A 12-kDa protein fraction capable of binding 14C-labeled oleic acid was partially purified from rat brain, and it stimulated synaptosomal Na-dependent amino acid uptake, which is highly sensitive to fatty acids, by sequestration of the inhibitory fatty acids [9]. Another study revealed a developmental dependence of H-FABP mRNA expression from low levels in brains of late fetal through suckling rats, to a subsequent 2- to 3-fold increase in adult brain, the latter being only 10% of that found in adult heart [10]. Furthermore, presence of several FABPs homologous to H-FABP was suggested in bovine brain because microheterogeneity of the amino acid sequence was observed; reportedly these proteins amounted to 0.4% of total soluble protein from bovine brain [11]. In retrospect, one may argue these proteins to be a mixture of several FABP types. Indeed, later studies revealed two further FABP types to be expressed in the brain. The one was termed psoriasis-associated (PA)-, cutaneous type (C)-, or epidermal-type (E)-FABP, because it was isolated from cultured human psoriatic keratinocytes, and from normal epidermis of human and rat skin [2, 12–14]. The other was termed brain-type (B)-FABP or brain lipid binding protein (BLBP) because it was identified for the first time in the brain of rodents [3, 15–17]. Further-

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more, it was found that H- and B-FABP represented 0.01% and 0.1% of brain total cytosolic protein, respectively [18]. Although H-, E-, and B-FABP all bind long-chain fatty acids, differences in preferences for fatty acid binding were noted as follows: H-FABP for n-6 polyunsaturated fatty acids, E-FABP for saturated fatty acids and B-FABP more for n-3 polyunsaturated fatty acids [19]. Interestingly, it has been shown that fatty acid binding activity of rat brain, examined by binding radiolabeled palmitic acid, remained at similar levels in white and gray matters up to postnatal day 40, but then increased with age in white matter only. This would indicate that fatty acid binding activity could be related to glial cells and related structures [20] and thus could be representative for FABP expression in these cells. Therefore, for understanding fatty acid binding in these tissues and, more generally, the functional significance of FABPs in neurons and glia, it is crucial to know the spatio-temporal localization of the three FABP types expressed in brain cells. Being aware that these cells have the highest heterogeneity in structure and function among various organs, we address in this review the detailed localization of H-, E-, and B- FABP in the developing and adult rodent brain and discuss the functional significance of these proteins for neural cells.

14.2

Expression of FABPs in Developing Rat Brain 14.2.1

Localization of H-FABP

A histological study based on in situ hybridization reported the temporal localization of mRNAs for H-, E-, and B-FABP in the developing rat brain [21] (Figs 14.1 and 14.2, Tab. 14.1). Interestingly, significant expression of H-FABP mRNA in embryonic brains is not observed, in contrast to the situation in heart (Fig. 14.1 B). After birth, H-FABP mRNA expression in brain becomes gradually positive with postnatal age, but is confined to the gray matter, the region in which neuronal cell bodies accumulate, throughout the postnatal course (Figs 14.1 C–F and 14.2 A, D, G). These histochemical data are in accord with the Northern blot data for whole rat brain reported earlier and addressed in the Introduction [10]. In the adult stage of rat brain, in situ hybridization revealed distinct H-FABP expression in the olfactory mitral cell layer, weak in the glomerular layer, minute expression in the granule cell layer, if at all (Fig. 14.2 A). The cerebral neocortex expresses this mRNA weakly to moderately through layers II–VI, and expression in the hippocampal neuronal layers is also evident (Fig. 14.2 D). In the cerebellum, H-FABP mRNA expression is evident in the Purkinje cells as well as the granule cells (Fig. 14.2 G). Small cells in the molecular layer are expression-positive. Although the expression level is very low in the striatum, hypothalamus, and midbrain, much higher expression is discerned discretely over individual neuronal somata in the thalamus and various brainstem nuclei, such as trigeminal or facial

14.2 Expression of FABPs in Developing Rat Brain

Fig. 14.1 Overview of mRNA expression for H-, E-, and B-FABP in the parasagittal sections of developing rat brain examined by in situ hybridization. Note distinct expression of E- and B-FABP in embryonic brain, while HFABP expression is seen in the heart only (rectangle in B). Postnatally E- and B-FABP expression decreases markedly after P21,

whereas H-FABP expression increases gradually during postnatal period. Cb, cerebellum; Cx, cerebral cortex; Cp, caudate putamen; Di, diencephalon; He, heart. Magnifications approximately ´15 (A, G, and M), ´20 (B, H, and N), ´6 (C, D, I, J, O, and P), ´4 (E, F, K, L, Q, and R).

nerve nucleus. H-FABP mRNA was not detected in the white matter, the ependymal cell layer, and the glia limitans delineating the external surface of rat brain [21]. By immunoblot analysis [22] levels of H-FABP in whole mouse brain were nearly undetectable until late embryonic stages, after which levels increased until early postnatal stages and a little bit lower in the adult brain. Also, by the criterion of immunohistochemistry, H-FABP expression becomes gradually positive in rat brain after birth, where it is confined to the gray matter throughout the postnatal course. In the adult brain, almost all neurons are weakly immunoreactive for H-FABP throughout the entire brain. The immunoreactivity is mainly local-

255

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Fig. 14.2 Higher magnification photomicrographs showing mRNA expression for H-, E-, and B-FABP in the parasagittal sections of olfactory bulb (A–C), hippocampus (D–F) and cerebellum (G–I) of adult rat brain examined by in situ hybridization. Expression of H-FABP is observed in gray matter only, which of BFABP mostly in white matter. In contrast, EFABP is observed in gray and white matter.

Arrows and asterisks in G, H, I show Purkinje cell layer and white matter of cerebellum, respectively. CC, corpus callosum; Cx, cerebral cortex; De, dentate gyrus; F, fimbria; gl, glomerular layer; IG, internal granular layer; Mi, mitral cell layer; N, olfactory nerve fiber layer; Hi, hippocampus. Magnifications approximately ´20 (A–F), ´15 (G–I).

ized in the cellular nucleus, resulting in the appearance of numerous small round profiles of the weakly immunoreactive structures and no distinct features in the uni- or multipolar processes in the gray matter (Fig. 14.3). The choroid plexuses are immunoreactive at levels higher than that of most neurons, in which the immunoreactivity is more evident in the nucleus than the cytoplasm. H-FABP was not detected in cells of the white matter, the glia limitans and the ependyma. In essence, the pattern of immunoreactivity accorded with that of mRNA expression alluded to before.

14.2 Expression of FABPs in Developing Rat Brain Tab. 14.1 Summary of mRNA expression levels of H-, E- and B-FABP in various brain regions of rats of embryonic day 18, postanatal day 7 and 35.

E 18 Cerebral cortex Ventricular germinal zone Mantle zone P7 Olfactory bulb Granule cell layer Mitral cell layer Glomerular layer Nerve fiber layer Cerebral cortex Hippocampus CA1–CA3 Dentate gyrus Caudate putamen Thalamus Hypothalamus Corpus callosum Cerebellum External granule cell layer Internal granule cell layer Purkinje cell Bergmann glia Brainstem Cranial nerve nuclei P 35 Olfactury bulb Granule cell layer Mitral cell layer Glomerular layer Nerve fiber layer Cerebral cortex Hippocampus CA1–CA3 Dentate gyrus Caudate putamen Thalamus Hypothalamus Corpus callosum

H-FABP

E-FABP

B-FABP

n.d. n.d.

+ ++

+++ n.d.

+ (N) + (N) + (N) n.d.

+ + + +

n.d. n.d. n.d. ++ (G)

+ (N) + (N)

++ (N, G) ++ (N, G)

n.d. ++ (G)

++ (N) + (N) n.d.

++ (N, G) ++ (N, G) ++ (G)

++ (G) ++ (G) ++ (G)

n.d. + (N) + n.d.

++ (N) ++ (N) + +

n.d. n.d. n.d. ++

+ (N)

+ (N, G)

n.d.

+ (N) + (N) + (N) n.d. + (N)

+ + + + +

(N) (N) (N) (G) (N, G)

n.d. n.d. n.d. + (G) n.d.

+ (N) + (N) + (N) ++ (N) + (N) n.d.

+ + + + + +

(N, G) (N, G) (N, G) (N, G) (N, G) (G)

n.d. + (G) n.d. n.d. n.d. + (G)

(N) (N) (N) (G)

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258

14 Fatty Acid Binding Proteins of the Brain Tab. 14.1 (continued).

Cerebellum Granule cell layer Molecular layer Purkinje cell Bergmann glia Brainstem Cranial nerve nuclei

H-FABP

E-FABP

B-FABP

+ (N) + (N) + n.d.

n.d. n.d. + +

n.d. n.d. n.d. +

+ (N)

+ (N, G)

n.d.

Expression levels are estimated by visual comparison in emulsion-coated brain sections for each FABP type. +++, intense; ++, moderate; +, weak; n.d., not detected. (N) and (G) respresent expression in neuron and glial cells, respectively.

Fig. 14.3 A photomicrograph showing HFABP protein expression in the adult rat cerebral cortex examined by immunohistochemistry. H-FABP expression is mainly localized in the cellular nuclei of neurons, resulting in appearance of numerous small round profiles (indicated by arrows). Magnification approximately ´100.

14.2.2

Localization of E-FABP

E-FABP mRNA is already expressed in rat brain at mid-term embryonic stage as evidenced by Northern blotting. After highest expression levels are attained at late embryonic stage, E-FABP mRNA levels decrease postnatally to become weak in the adult brain [21]. With regard to spatial regions of rat brain the gene encoding E-FABP is expressed at moderate levels in the mantle zone and in the ventricular germinal zone throughout the neuraxis at the embryonic stage (Fig. 14.1 G). The expression in the latter gradually decreases and only a thin line of expression signals is detected postnatally. In contrast, in the external granule cell layer, another germinal zone of early postnatal cerebellum, E-FABP mRNA is clearly visible by in situ hybridization (Fig. 14.1 I–L). The gray matter throughout the early postnatal brain expresses the gene at weak to moderate levels, although the expression in the cerebral cortex is already positive before birth. Among various gray matter regions, the expression is more evident in the cerebral cortex and the hippocampus. While

14.2 Expression of FABPs in Developing Rat Brain

the signals in the gray matter decrease in intensity gradually with postnatal age, those in the white matter increase to substantial levels in the adult stage (Fig. 14.2 E, H). As a result, in the adult stage, the expression is positive in both neurons and glia, however, with varying intensity in various brain regions [21]. Western blot analysis by De Leon and his group [23, 24] revealed expression of E-FABP (termed “DA11” by the authors) at high levels in prenatal and neonatal rat brain, a decrease with age, and faint occurrence in adult rat brains. The spatial and temporal occurrence of E-FABP in developing rat brain regions is as follows: it is weakly seen in the innermost layer of the germinal zone throughout the neuraxis, but clearly in the endothelial cells of many small blood vessels within the brain parenchyma. E-FABP is also present in most cells of glia limitans/pia-glia, the outermost thin layer of the brain, and in epithelial cells lining the choroid plexuses. However, the immunoreactivity is close to background levels in most cells of the mantle zone at mid-term embryonic stage, which is in contrast to the moderate levels of mRNA expression. One may hypothesize that this is due to negative post-transcriptional regulation. In late embryonic stage before birth E-FABP is weakly discernible in the cerebral mantle zone, where immunoreactive neurons appear as spindles with processes extending vertical to the cortical surface. E-FABP is also present in many glial cells and their processes in the outermost (olfactory nerve) layer of the olfactory bulb. The immunoreactivity of small vascular endothelium within the brain and spinal cord parenchyma is negative at latest embryonic stages. At early postnatal stages, E-FABP immunoreactivity remains evident in the olfactory nerve fiber layer and the pia-glia. Some of the E-FABP positive pia-glial cells in the dorsal brain surface extend thin immunoreactive processes into the brain parenchyma perpendicular to the brain surface. However, in contrast to the clear detection of E-FABP mRNA in the gray matter of the entire brain, the immunoreactivity is close to detection levels in most of the gray matter regions, except for some weak reactions in pyramidal cells and their vertical processes in the cerebral cortex (Fig. 14.4 A). In the hippocampus E-FABP-positive cells with short radiating processes (presumably dentate radial glial cells) are arranged in a line along the inner rim of the dentate granule cell layer, while no clear immunoreactivity is detected in the pyramidal cell layer. Numerous immunoreactive cells of glial morphology are seen scattered in the gray matter (Fig. 14.4 B), even more distinct in the white matter such as the hippocampal fimbria and the corpus callosum. The exact identification of immunoreactive glial cells by co-immunostaining with type-specific glial markers has to be done. In the cerebellum, the immunoreactivity in the external granule cells is weak in contrast to E-FABP mRNA levels. E-FABP is not detected in internal granule cells or Purkinje cells. On the other hand, E-FABP expression is weakly detected in cell bodies of Bergmann’s glial cell in the Purkinje cell layer and their vertical processes in the molecular layer. EFABP is also detected clearly in scattered glial cells having radiating processes in the internal granule cell layer and more numerously in the cerebellar medulla. At the adult stage, the intensity of E-FABP expression decreases in both the white and gray matter regions, particularly in the latter.

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14 Fatty Acid Binding Proteins of the Brain Fig. 14.4 Photomicrographs showing E-FABP protein expression in the P14 rat cerebral cortex examined by immunohistochemistry. E-FABP expression is observed in some pyramidal cells and their vertical processes in the cerebral cortex (A). Numerous E-FABP immunoreactive cells of glial morphology are seen scattered in the gray matter (B), E-FABP is also positive in the pia glia indicated by arrows. Magnifications approximately ´100.

Fig. 14.5 Expression of b-galactosidase in the cerebral cortex of brain from P14 E-FABP knockout mouse as examined by X-gal histochemistry. Note that positive nuclear stainings (blue color), indicating endogenous E-FABP promoter activity, are observed not only in neurons (arrows) but also in glial cells adjacent to neurons (arrowheads). The section was counter-stained with Eosin. Magnification approximately ´200.

Liu et al. [24] reported that E-FABP mRNA and protein expression in the rat brain was distinctly observed in prenatal and early postnatal neurons not in glial cells. In order to confirm E-FABP expression in glial cells, we applied gene targeting technology which allowed us to detect endogenous E-FABP promoter activity with high sensitivity by the expression of reporter protein, b-galactosidase [25]. In b-galactosidase histochemistry, reporter protein expression was clearly observed in glial cells as well as in neurons (Fig. 14.5) of postnatal brain and its distribution in developing mouse brain was basically correlated with our findings [26].

14.3 Significance of FABP Expression in Brain

14.2.3

Localization of B-FABP

Northern blot analysis of mRNAs from rodent brains [3, 16, 21] revealed that the gene encoding B-FABP is substantially expressed at mid-term embryonic stages. With progressing differentiation of the embryonic brain, B-FABP gene expression decreases gradually and becomes very weak in adult brains. In situ hybridization histochemistry [3, 4, 16, 21] revealed that in embryonic rodent brain B-FABP mRNA is very intensely expressed in the ventricular and subventricular zones of the entire brain and in the spinal cord (Fig. 14.1 M, N). At neonatal stages, the expression in the germinal zones remains most intense, and it is strongly positive in both the gray and white matter throughout the neonatal brain (Fig. 14.1 O). Among the brain regions, the expression is more evident in the olfactory periglomerular and nerve fiber layers, hippocampal neuronal layers, and the cerebellar Purkinje cell layer. No positive expression is seen in the cerebellar external granule cell layer. The expression decreases markedly throughout the entire brain upon reaching the adult stage (Fig. 14.1 P–R) with a few, yet distinct exceptions. B-FABP mRNA clearly is expressed in the olfactory nerve fiber layer (Fig. 14.2 C), the dentate granule cell layer (Fig. 14.2 F), the cerebellar Bergmann glia in the Purkinje cell layer (Fig. 14.2 I), and the ependymal cells lining the ventricular surfaces of the adult brain. In immunohistochemical studies for localization of B-FABP in mouse embryonic brain [3, 16], many cells in the ventricular germinal zone of the brain and the spinal cord are intensely immunoreactive for B-FABP. As the processes of these immunoreactive cells run perpendicular to the brain surface, this immunoreactivity can be ascribed to radial glial cells in embryonic brains. Throughout the postnatal development to the adult stage, B-FABP is detected in numerous glial cells, presumptive astrocytes, of the white matter, in radial glial cells spanning the granule cell layer of the dentate gyrus, and in Bergmann glial cells located adjacent to the cerebellar Purkinje cells. Unlike H- or E-FABP, B-FABP is not detected in the neurons of the gray matter.

14.3

Significance of FABP Expression in Brain

The occurrence of the three FABP-types in brain can be summarized as follows: H-FABP is expressed in neurons of the mature brain. E-FABP is distinctly expressed in both neurons and glia in pre- and early postnatal brain and it continues to be expressed in mature glia, presumptive astrocytes. However, weak signals for E-FABP were obtained by in situ hybridization in mature neurons. BFABP is strongly expressed in radial glial cells in pre- and perinatal brain but in the adult stage only weakly in mature glia. Such spatial and temporal differences of their occurrence in brain may reflect their distinct roles in modulating neural cell function. An interesting observation was reported by Pu et al. [27], who found

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H-FABP to be highly concentrated in synaptosomes from total brain of 4 months old mice but decreased thereafter. The same was observed for B-FABP, yet at lower levels as compared with H-FABP. The occurrence of B-FABP in neuronal synaptosomes of brain is at variance with data from immunohistochemistry and in situ hybridization, which revealed B-FABP to occur only in glia [3, 16, 21]. Although a contamination of synaptosomal fractions by glial proteins could be possible, a localization of B-FABP restricted to nerve ending terminals remains to be elucidated by immunoelectronmicroscopy. With regard to the very high expression levels of the gene encoding B-FABP in the germinal epithelium and radial glia at pre- and early postnatal stages, in vitro data of Feng et al. [16] are noteworthy. Administration of anti B-FABP antibody to mixed cultures of cerebellar neurons and glia inhibited the formation of glial fascicles and migration of neurons along them on the one hand, and on the other hand, B-FABP was found in the conditioned culture medium. The authors suggested that B-FABP was released to serve extracellularly as a migrational cue for immature neurons. A precedent for extracellular FABP action has been reported earlier. The possibility of a so-called “mammary-derived growth inhibitor” (MDGI), later shown to be identical to H-FABP [28], being released from the cell and subsequently acting on a local cell population has been inferred from the finding that proliferation of mammary stromal cells was inhibited [29]. It was also reported that a “mammary-derived growth inhibitor-related gene” (MRG), which is identical to human B-FABP [30], suppressed cell proliferation and tumor growth in human breast cancer cells [31]. Furthermore, overexpression of B-FABP in MDA-MB-231 human breast cancer cells induced changes in cellular morphology, and addtional treatment of the cells with docosahexaenoic acid (DHA), a strong ligand of B-FABP, resulted in growth inhibition proportional to B-FABP expression levels [32]. Taken together, B-FABP may be involved in the differentiation of brain cells, by directing the physiological action of long-chain fatty acids towards control of the cell cycles. The precise mechanism remains to be elucidated, however. In view of the regulatory potential of FABPs on the cell cycle and cellular differentiation, changes in the expression of the genes for H-, E-, and B-FABP, respectively, have been examined in the hypoglossal nucleus of adult rats after nerve crush [33]. This is a good model for axonal regeneration and induces a variety of proteins related to cell cycle regulation as well as to morphological and biochemical changes, such as increased incorporation of long-chain fatty acid into the hypoglossal nucleus [34]. Indeed, a marked increase of expression levels was observed for E-FABP, but not for H- or B-FABP in the hypoglossal neuronal somata. Induction of E-FABP expression following peripheral nerve injury was also reported for rat dorsal root ganglia after sciatic nerve crush [35]. Taking into account the transient expression of EFABP in immature neurons of the cerebrum at early postnatal stages, an increase of E-FABP expression in regenerating neurons is understandable. Outside the brain, a differentiation-dependent FABP expression in vitro has been described for a mouse fibroblast cell line (3T3-L1), which upon differentiation from pre-adipocyte to adipocyte produces huge amount of adipocyte (A)-type FABP [36]. There has been evidence that A-FABP is the target of the insulin receptor tyrosine kinase in 3T3-L1

14.5 Acknowledgements

cells, which phosphorylates A-FABP at Tyr19 [37]. Furthermore, H-FABP was also shown to be phosphorylated on its Tyr19 upon insulin stimulation [38]. An equivalent tyrosine residue as a potential target for phosphorylation has been identified in E-FABP, suggesting that E-FABP might serve as a signaling molecule in tyrosine kinase signaling during nerve regeneration. Very recently it was reported that gene regulation by long-chain fatty acids in hepatocytes is affected by interaction of FABP (actually liver-type FABP) with a nuclear receptor, i.e. peroxisome proliferator activated receptor a (PPARa) [39, 40]. To this end, localization of L-FABP in the nuclei of hepatocytes was reported already in 1989 [41]. In addition, H-FABP has been found in nuclei of myocytes [42], E-FABP in nuclei of 3T3L-1 adipocytes and in CV-1 cells after transfection [43] and B-FABP in nuclei of cultured radial glia [16]. It is thought that FABPs are involved in transactivation of fatty acid-activated nuclear receptors in nuclei of neural cells, considering that H-FABP was found in the nuclei of neurons, but proof has to be provided yet. In rat brain capillary endothelial cells E-FABP was found at embryonic stages but not in the adult stage, in contrast to capillary endothelial cells of other adult tissues, where either H-FABP or E-FABP or both are expressed [5, 44]. These adult tissues with FABP-containing endothelium require high levels of blood-derived fatty acids for energy production. However, it is generally known that the mature brain oxidizes fatty acids poorly, which may explain the absence of FABPs in capillary endothelial cells of the adult brain. Thus, at the mid-term embryonic stage, where E-FABP ceases to be expressed, a change in fatty acid metabolism in brain endothelium or brain itself may occur.

14.4

Perspective

The generation of gene knockout mice for a given protein molecule is a powerful tool today to gain functional insight for the target molecule. However, in view of the presence of various FABP-types on the one hand and their heterogeneous spatio-temporal expression patterns on the other hand, it can be assumed that compensatory expression of FABPs may be active in the brain in vivo. This must actually be the case as no obvious phenotypes related to neural function have so far been reported for H- and EFABP knockout mouse, respectively [25, 26, 45]. The phenotype analysis of B-FABP knockout mouse is now under way. It is certainly crucial to analyze these knockout mice in great detail, or to generate knockout mice targeting double or triple genes for H-, E-, B-FABPs, or to target conditionally a gene in a specific brain area.

14.5

Acknowledgements

We thank Professor Friedrich Spener, University of Muenster, for helpful discussions. This work was supported by a grant from Hitomi Foundation.

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14.6

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3

4 5 6

7

8

9

10

11

12

13

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16 17

S. Tweedie, Y. Edwards, Nucleic Acids Res. 1989, 17, 4374. G. Siegenthaler, R. Hotz, D. Chatellard-Gruaz, S. Jaconi, J. H. Saurat, Biochem. Biophys. Res. Commun. 1993, 190, 482–487. A. Kurtz, A. Zimmer, F. Schnütgen, G. Brüning, F. Spener, T. Müller, Development 1994, 120, 2637–49. Y. Owada, H. Kondo, Neurosci. Res. Suppl. 1994, 19, S92. M. Watanabe, T. Ono, H. Kondo, J. Anat. 1991, 174, 81–95. J. H. Veerkamp, R. J. Paulussen, R. A. Peeters, R. G. Maatman, H. T. van Moerkerk, T. H. van Kuppevelt, Mol. Cell. Biochem. 1990, 98, 11–18. R. Watanabe, H. Fujii, A. Yamamoto, T. Hashimoto, K. Kameda, M. Ito, T. Ono, J. Dermatol. Sci. 1997, 16, 17–22. J. F. Glatz, T. Börchers, F. Spener, G. J. van der Vusse, Prostaglandins Leukot. Essent. Fatty Acids 1995, 52, 121–127. N. M. Bass, E. Raghupathy, D. E. Rhoads, J. A. Manning, R. K. Ockner, Biochemistry 1984, 23, 6539–6544. R. O. Heuckeroth, E. H. Birkenmeier, M. S. Levin, J. I. Gordon, J. Biol. Chem. 1987, 262, 9709–17. F. Schoentgen, G. Pignede, L. M. Bonanno, P. Jollès, Eur. J. Biochem. 1989, 185, 35–40. P. Madsen, H. H. Rasmussen, H. Leffers, B. Honore, J. E. Celis, J. Invest. Dermatol. 1992, 99, 299–305. N. Y. Schurer, N. M. Bass, S. Jin, J. A. Manning, S. Pillai, M. L. Williams, J. Invest. Dermatol. 1993, 100, 82–86. R. Watanabe, H. Fujii, S. Odani, J. Sakakibara, A. Yamamoto, M. Ito, T. Ono, Biochem. Biophys. Res. Commun. 1994, 200, 253–259. S. G. Kuhar, L. Feng, S. Vidan, M. E. Ross, M. E. Hatten, N. Heintz, Development 1993, 117, 97–104. L. Feng, M. E. Hatten, N. Heintz, Neuron 1994, 12, 895–908. E. Bennett, K. L. Stenvers, P. K. Lund, B. Popko, J. Neurochem. 1994, 63, 1616– 24.

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S. C. Myers-Payne, T. Hubbell, L. Pu, F. Schnütgen, T. Börchers, W. G. Wood, F. Spener, F. Schroeder, J. Neurochem. 1996, 66, 1648–56. T. Hanhoff, C. Lücke, F. Spener, Mol. Cell. Biochem. 2002, in press. E. R. Galarza De Bo, F. M. Atlasovich, M. R. Ermacora, J. H. Torea, J. M. Pasquini, J. A. Santome, E. F. Soto, Neurochem. Int. 1992, 21, 237–241. Y. Owada, T. Yoshimoto, H. Kondo, J. Chem. Neuroanat. 1996, 12, 113–122. P. A. Sellner, W. Chu, J. F. Glatz, N. E. Berman, Brain Res. Dev. Brain Res. 1995, 89, 33–46. Y. Liu, C. A. Molina, A. A. Welcher, L. D. Longo, M. De Leon, J. Neurosci. Res. 1997, 48, 551–562. Y. Liu, L. D. Longo, M. De Leon, Brain Res. 2000, 852, 16–27. Y. Owada, H. Takano, H. Yamanaka, H. Kobayashi, Y. Sugitani, Y. Tomioka, I. Suzuki, R. Suzuki, T. Terui, M. Mizugaki, H. Tagami, T. Noda, H. Kondo, J. Invest. Dermatol. 2002, 118, 430–435. Y. Owada, I. Suzuki, T. Noda, H. Kondo, Mol. Cell. Biochem. 2002, in press. L. Pu, U. Igbavboa, W. G. Wood, J. B. Roths, A. B. Kier, F. Spener, F. Schroeder, Mol. Cell. Biochem. 1999, 198, 69–78. B. Specht, N. Bartetzko, C. Hohoff, H. Kuhl, R. Franke, T. Börchers, F. Spener, J. Biol. Chem. 1996, 271, 19943– 49. Y. Yang, E. Spitzer, N. Kenney, W. Zschiesche, M. Li, A. Kromminga, T. Müller, F. Spener, A. Lezius, J. H. Veerkamp, J. Cell Biol. 1994, 127, 1097– 1109. C. Hohoff, F. Spener, Cancer Res. 1998, 58, 4015–17. Y. E. Shi, J. Ni, G. Xiao, Y. E. Liu, A. Fuchs, G. Yu, J. Su, J. M. Cosgrove, L. Xing, M. Zhang, J. Li, B. B. Aggarwal, A. Meager, R. Gentz, Cancer Res. 1997, 57, 3084–91. M. Wang, Y. E. Liu, J. Ni, B. Aygun, I. D. Goldberg, Y. E. Shi, Cancer Res. 2000, 60, 6482–87.

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Y. Owada, A. Utsunomiya, T. Yoshimoto, H. Kondo, Neurosci. Lett. 1997, 223, 25–28. S. Yamazaki, J. G. Noronha, J. M. Bell, S. I. Rapoport, Brain Res. 1989, 477, 19– 28. M. De Leon, A. A. Welcher, R. H. Nahin, Y. Liu, M. A. Ruda, E. M. Shooter, C. A. Molina, J. Neurosci. Res. 1996, 44, 283–292. D. A. Bernlohr, C. W. Angus, M. D. Lane, M. A. Bolanowski, T. J. Kelly, Jr., Proc. Natl Acad. Sci. USA 1984, 81, 5468–5472. M. Bernier, D. M. Laird, M. D. Lane, Proc. Natl Acad. Sci. USA 1987, 84, 1844–48. S. U. Nielsen, F. Spener, J. Lipid Res. 1993, 34, 1355–1366. P. Ellinghaus, C. Wolfrum, G. Assmann, F. Spener, U. Seedorf, J. Biol. Chem. 1999, 274, 2766–72.

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C. Wolfrum, C. M. Borrmann, T. Börchers, F. Spener, Proc. Natl Acad. Sci. USA 2001, 98, 2323–28. U. Bordewick, M. Heese, T. Börchers, H. Robenek, F. Spener, Biol. Chem. Hoppe-Seyler. 1989, 370, 229–238. T. Börchers, C. Unterberg, H. Rüdel, H. Robenek, F. Spener, Biochim. Biophys. Acta 1989, 1002, 54–61. T. Helledie, M. Antonius, R. V. Sorensen, A. V. Hertzel, D. A. Bernlohr, S. Kolvraa, K. Kristiansen, S. Mandrup, J. Lipid Res. 2000, 41, 1740–1751. I. Masouye, G. Hagens, T. H. Van Kuppevelt, P. Madsen, J. H. Saurat, J. H. Veerkamp, M. S. Pepper, G. Siegenthaler, Circ. Res. 1997, 81, 297–303. B. Binas, H. Danneberg, J. McWhir, L. Mullins, A. J. Clark, FASEB J. 1999, 13, 805–812.

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Cross-talk between Intracellular Lipid Binding Proteins and Ligand Activated Nuclear Receptors – A Signaling Pathway for Fatty Acids Christian Wolfrum and Friedrich Spener

15.1

Introduction

Signal transduction at the cellular level refers to the movement of signals from outside the cell to the inside. The movement of signals can be simple, like that associated with receptor molecules of the acetylcholine class. Complex signal transduction involves the coupling of ligand–receptor interactions to intracellular events. These events include phosphorylations by tyrosine kinases and/or serine/ threonine kinases or ligand activated gene expression. The eventual outcome is an alteration in cellular activity and changes in the program of genes expressed within the responding cells. Signal transducing receptors can be divided into four general classes: · Receptors that penetrate the plasma membrane and have intrinsic enzymatic activity, including tyrosine kinases (e.g. receptors for PDGF, insulin, EGF, and FGF), tyrosine phosphatases (e.g. CD45 protein of T cells and macrophages), guanylate cyclases and serine/threonine kinases (e.g. activin and TGF receptors). Receptors with intrinsic tyrosine kinase activity either are capable of autophosphorylation as well as phosphorylation of other substrates or are coupled to intracellular tyrosine kinases by direct protein–protein interactions. · Receptors that are coupled, inside the cell, to GTP binding and hydrolyzing proteins (termed G-proteins). Receptors of this class all have a structure of seven transmembrane spanning domains and are also known as serpentine receptors. Examples are the adrenergic receptors, odorant receptors, and certain hormone receptors (e.g. for glucagon, angiotensin, and vasopressin). · Receptors that are found intracellularly and upon ligand binding migrate to the nucleus where the ligand–receptor complex directly affects gene transcription. · Intracellular ligand activated receptors which are mainly localized in the nuclear compartment and whose cellular localization is independent of ligand-bound status. Signaling pathways pertaining to the latter receptors will be the focus of this article.

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15 Cross-talk between Intracellular Lipid Binding Proteins and Ligand Activated Nuclear Receptors

Nutritional factors, such as carbohydrates, fatty acids, sphingosine derivatives, and sterols, are important modulators of cell function [1–5]. Fatty acids, which have been shown to be important regulators of cell-specific expression patterns, activate nuclear receptors of the family of peroxisome proliferator activated receptors (PPARs) [6–11]. These are members of the superfamily of nuclear hormone receptors which, upon activation, regulate target gene expression by binding in the form of a heteromeric complex with retinoic acid X receptors (RXRs) to peroxisome proliferator responsive elements (PPREs) (see Chapter 9). The ligand for RXRs is 9-cis retinoic acid [12]. Related nuclear hormone receptors are the retinoic acid receptors (RARs) which bind both 9-cis and all-trans retinoic acid and regulate target gene expression, also as heterodimers with RXR, via an all-trans retinoic acid receptor responsive element (RARE) (see Chapter 10). The majority of intracellular binding sites for activators of PPARs and RARs, i.e. straight- and branched-chain fatty acids in general, are provided by members of the family of intracellular lipid binding proteins (iLBPs). Among this family are the fatty acid binding proteins (FABPs), the cellular retinoic acid binding proteins (CRABPs), and cellular retinoid binding proteins (CRBPs) as well as the intestinal bile acid binding protein (I-BABP). Given the low solubility of fatty acids, the question arises how they reach their designated nuclear receptor target. A specific transport system for the fatty acids might be furnished by members of the iLBP family. Thus, this chapter will address the signal transduction pathways for fatty acids involving an iLBP and a nuclear receptor and furthermore will indicate other possible mechanisms which are focus of current research.

15.2

Fatty Acid Activated Nuclear Receptors

Two different subgroups of nuclear receptors exist which are activated by fatty acids, namely PPARs and RAR/RXRs. PPARs are expressed in vertebrate species and three distinct subtypes have been identified to date, namely PPARa, PPARb or d (also called NUC-I or FAAR), and PPARc (see Chapter 9). Similar to PPARs, RARs and RXRs are encoded by three distinct genes each resulting in a, b, and c isoforms. Each of these receptor subtypes includes several isoforms (e.g. b1, b2, and b4) formed by different splicing and usage of alternative promoters (see Chapter 9). PPARs, RARs, and RXRs exhibit a common structural motif consisting of four main domains shared by all members of the nuclear hormone receptor family: The N-terminal A/B domain containing the activation function 1 (AF-1), the highly conserved C domain which functions as DNA binding domain (DBD) with two zinc-finger motives, the D domain which acts as a hinge domain linking the DBD to the C-terminal E/F ligand binding domain (LBD) containing the activation function 2 (AF-2) [13, 14]. The two AF sites furnish distinct co-activator binding regions. The AF-1 domain acts in ligand-independent, the AF-2 domain in ligand-dependent fashion [15].

15.3 Intracellular Lipid Binding Proteins

PPAR subtypes reveal a distinct distribution pattern, with PPARa being expressed most abundantly in liver and to a lesser amount in kidney, heart, and brown adipose tissue [16, 17]. PPARb is the most widely distributed member of the family and is expressed in nearly every tissue [16], whereas PPARc expression is severely restricted [16, 17]. Two isoforms of this receptor have been identified to date, the c1 isoform which is mainly expressed in white and brown adipose tissue and to a much lesser extent in other tissues such as in kidney, heart, and macrophages [18, 19], and the c2 isoform which is expressed in white adipose tissue only [20]. PPARs are activated by a variety of fatty acids and different xenobiotics [6–11, 21]. In contrast RAR is selectively activated by 9-cis and all-trans isomers of retinoic acid. RXRs were thought to be selectively activated by 9-cis retinoic acid, however recent reports demonstrated that both docosahexaenoic acid [22] and phytanic acid [23] can also bind to and activate RXRa. Both PPARs and RARs activate target gene expression as heterodimers with RXRs, by binding to either conserved PPREs or RAREs, respectively, in the promoters of responsive genes [12, 24]. PPREs were found in various genes, and shown to be functional in genes involved in fatty acid degradation, storage and transport (e.g. fatty acid binding proteins) as well as carbohydrate metabolism (see Chapter 9). RAREs, in contrast, are found in the promoters of genes involved in embryonic development, i.e. members of the hepatocyte nuclear factor family [25] (see Chapter 10).

15.3

Intracellular Lipid Binding Proteins

iLBPs form a family of 14–15 kDa proteins which are involved in various intracellular processes, i.e. intracellular transport and storage of fatty acids, modulation of enzyme activity, differentiation and growth regulation, and signal transduction (reviewed in Ref. [26]). To date 13 members of this family have been identified, which can be divided into four subfamilies according to sequence homology and ligand binding (Fig. 15.1): (i) The intracellular retinoid binding proteins CRABPs and CRBPs; (ii) liver (L-)FABP, which accommodates two fatty acids [27, 28] and I-BABP; (iii) intestinal (I-)FABP, which binds a single fatty acid in a linear conformation [29], and (iv) FABPs with the fatty acid bound in a highly bent or Ushaped conformation [30–33] (brain (B-), adipocyte (A-), epidermal (E-), and heart (H-) FABPs belong to the this group). For discussions of iLBP structure and ligand binding see Chapter 5. Most members of the iLBP family show a restricted tissue expression pattern. L-FABP is expressed in hepatocytes, enterocytes, kidney, and lung [34–37], while IBABP and I-FABP expression is restricted to the intestinal cells [38, 37]. A-FABP is expressed in adipocytes, lipofibroblasts and interestingly in stimulated macrophages [39, 40], B-FABP is expressed in glial cells [41], while H-FABP and EFABP show a more widely spread expression pattern as reviewed in Ref. [26]. CRABPs exhibit different patterns of expression across cell and developmental

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Fig. 15.1 Phylogenetic analysis of the iLBP family. By phylogenetic analysis using GeneBee-Net [78] the iLBP family can be divided into four subfamilies, consisting of (i) CRABPI, II and CRBP-I, II; (ii) L-FABP, I-BABP; (iii)

I-FABP; (iv) M-, A-, H-, T-, B-, E-FABP. The phylogenetic relationship of iLBPs is reflected by their ligand selectivity and the conformation of the ligand in the bound state (see text).

stages. Independent of species analyzed CRABP-I is expressed almost ubiquitously in the adult, while CRABP-II is only expressed in skin fibroblasts, uterus, ovary, and the choroid plexus (for review see Ref. [42]). L-FABP expression is enhanced by PPARa in hepatocytes in response to fatty acid stimuli via a functional PPRE in its promoter [43]. Similarly, A-FABP is upregulated by PPARa during adipocyte differentiation [44]. Experimental evidence exists that CRABP-II, but not CRABP-I is regulated by retinoic acid via RAR [45].

15.4

Regulation of Fatty Acid Activated Nuclear Receptor Activity by iLBPs

The hypothesis that iLBPs can function as modulators of fatty acid activated nuclear receptor activity is strengthened by the fact that several iLBP types have been detected in the nuclear compartment [46–51]. Since iLBPs and fatty acid acti-

15.5 L-FABP

vated nuclear receptors are expressed cell specifically one can envisage that distinct partners are responsible for fatty acid transport and translation of their signals. Indeed, several iLBPs became known to influence fatty acid activated nuclear receptor activity, though with controversial results. This will be discussed below.

15.5

L-FABP

Based on the common ligand spectrum of PPARs and L-FABP, their common cellspecific expression, and the fact that they are found in the nucleus, both proteins form a likely pair for an involvement in signal transduction. Based on this circumstantial evidence our group analyzed the role of L-FABP in PPARa activation. Cell clones of HepG2 cell cultures, stably expressing antisense L-FABP mRNA that reduced intracellular L-FABP down to 18% of untransfected HepG2 cells, were used in a reporter gene-based assay. The expression of the reporter gene was controlled by an idealized PPRE [52]. After treatment of the cells with PPARa agonists the amount of reporter gene expressed as well as the amount of L-FABP was measured and correlated. A positive linear correlation was found between FABP concentration and PPARa activation by natural ligands (Fig. 15.2 A) and xenobiotics (Fig. 15.2 B). The steepest slope was obtained for the potent xenobiotic activator of PPARa, Wy14,643, which is also a ligand of L-FABP, the shallowest for stearic acid indicative for a weak activation potential; incidentally the latter acid also is bound with low affinity by L-FABP. Furthermore, extrapolation of the fitted graphs to zero L-FABP concentration would indicate a complete loss of PPARa activation and implies LFABP to be a mandatory ligand/agonist transporter in the hepatocyte-derived HepG2 cells. From the physiological point of view the concentration of L-FABP in rat liver was reported to be around 70 nmol g–1 while the concentrations of unesterified fatty acids ranges from 50 to 100 nmol g–1 [53], suggesting that L-FABP is able to bind most of the unesterified fatty acids and to transport them either to their places of metabolic utilization or to PPARa activation. The regulation of PPARc activity by L-FABP was analyzed by the same experimental design. PPARc activation by ciglitazone, a thiazolidinedione, decreased 2fold in HepG2 cells which contained 2 times less L-FABP. This finding was not unexpected, as L-FABP binds BRL49653 [27] which is also a member of this class of xenobiotics known to be activators of PPARc (see Chapter 9). The mechanism pertaining to this modulation of PPAR activity by L-FABP cannot be deduced from these experiments. One evident mechanistic possibility is that L-FABP translocates to the nucleus and interacts directly with PPARs for ligand/agonist transfer. By employing immuno-coprecipitation from nuclear lysates and an in vitro pulldown assay we demonstrated direct PPARa/L-FABP interaction and could extend these findings via mammalian two hybrid assay in Cos-7 cells to all three PPAR isoforms (Fig. 15.3 A). To address the ligand dependency of PPARa interaction, in vitro pulldown assays were also carried out with L-FABP saturated with oleic acid or Wy14,643. In either case no differences in interaction

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Fig. 15.2 Transactivation of human PPARa is dependent on L-FABP concentration. Antisense L-FABP mRNA HepG2 cells [79], were transfected with the expression vector for human PPARa, pSV-b-Gal and the CAT-reporter gene vector under the control of an idealized PPRE. Each data point represents a single clone with a total of eight clones with differing L-FABP values being measured. (A) Cells were treated for 24 h with 200 lM stearic acid

·

( ), 200 lM linoleic acid (s) and 100 lM phytanic acid (n). (B) Cells treated for 24 h with 200 lM bezafibrate ( ), 50 lM ETYA (s) and 200 lM Wy14,643 (n). b-Gal, CAT, and L-FABP concentrations were determined by ELISAs, DMSO control was set as 1. Note the different scale of ordinates. Each data point represents the mean of six independent experiments ± SD. From Ref. [52] with permission.

·

were observed in comparison to interaction with apo L-FABP [52]. Similarly, in the two-hybrid assay administration or omission of linoleic acid or Wy14,643 to Cos-7 cells in the case of PPARa and of ciglitazon in the case of PPARc, did not reveal differences in the interaction of L-FABP with either receptor [52]. Since apo and holo L-FABP interact with PPARs, apo L-FABP would compete with holo LFABP for this interaction and would decrease the efficacy of ligand transfer. Lawrence et al., however, reported that only holo L-FABP interacted with the nuclear membrane, suggesting a selective transfer of holo L-FABP to the nuclear compart-

15.5 L-FABP Fig. 15.3 Two-hybrid interaction of murine (A) L-FABP and (B) AFABP with murine nuclear receptors. Interaction was measured with the mammalian two-hybrid system in Cos-7 cells, employing a fusion protein of the GAL4DNA binding domain with either PPARa, b, c1, c2, or RXRa and a fusion protein of the VP16 activation domain with L-FABP or AFABP. Unspecific interaction was quantified by testing the fusion protein of the GAL4-DNA binding domain with p53 or the fusion protein of the VP16 activation domain with the SV40-T antigen. Positive and negative controls were used according to the supplier’s manual. CAT and b-Gal expression was measured by ELISAs. Each column represents the mean of six independent experiments ± SD.

ment [48]. This would minimize interference by apo L-FABP and thus enhance the rate of ligand transfer. The structural motifs in the nuclear receptor and in the ligand/agonist transporter responsible for the protein–protein contacts observed have not been identified so far. It has been demonstrated previously that the AF-2 site of PPARs interacts with the LXXLL motif of various co-activators in a ligand/agonist-dependent manner [15]. This motif is not found in L-FABP, however. Since this binding protein does not function as a basal transcription factor either, a different kind of interaction must take place. Other proteins like the recently identified PPARc co-activator 2 (PGC-2) interact specifically with the AF-1 site of PPARc [54] independent of the presence of a ligand/agonist. Moreover, even an enzyme like peroxisomal bifunctional enzyme (PBE) interacts with the AF-1 site, in this case of PPARa [55]. In these protein–protein contacts the LXXLL motif cannot be involved because it is not present in these nuclear co-activators. Our current hypothesis is that L-FABP interacts with the AF-1 site of PPARs to facilitate ligand transfer from the former to the latter. This view is strengthened by the finding that phosphorylation of the A/B domain containing the AF-1 site leads to a reduction in ligand binding [56]. All this, of course, needs experimental verification.

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The question whether PPARs are able to displace ligands from L-FABP can be answered by inspecting respective dissociation constants for ligand binding. A competitive fluorescence assay carried out in our laboratory revealed that PPARa is a high-affinity protein for peroxisome proliferators (in the 10 nM range) and a medium-affinity protein for fatty acids (in the 100 nM range) [6]. Isothermal titration calorimetry showed that L-FABP binds peroxisome proliferators with low affinities (around 1 lM range), but fatty acids with high affinity (around 10 nM) in the first and low affinity in the second binding site (100–500 nM) [27]. Thus PPARa would be able to displace peroxisome proliferators from both binding sites of L-FABP, while fatty acids would preferentially be drawn from the second binding site. From these data it is clear that L-FABP modulates either PPARa or PPARc activity and also most likely PPARb activity. The physiological significance of activation of the latter two nuclear receptor isoforms is questionable, because in cells where L-FABP is expressed, i.e. those in liver, intestine, and kidney, PPARa is the predominant subtype.

15.6

A-FABP and E-FABP

Adipose tissue is a major organ involved in lipid homeostasis. Since in adipocytes PPARc is predominantly expressed, fatty acids exert their effect at least partly via this nuclear receptor. Two FABPs, namely A-FABP and E-FABP, are expressed in adipocytes and thus could convey the signaling fatty acid to PPARc. Three papers have been published on this question with conflicting results. In all cases an approach based on transient transfection of either A-FABP or E-FABP into a cell system which does not express the endogenous protein was taken. In two cases the authors reported an increase in PPAR activity, while in the third a decrease in PPARc activity was observed. In the first paper it was demonstrated that A-FABP, when transfected into CV-1 monkey kidney fibroblasts, enhances PPARc activity in the absence of exogenous ligand stimuli [57]. In the second paper the observation was published that cotransfection of A-FABP or E-FABP with PPARc into the CV-1 cells led to a decrease in PPARc activity with or without stimulation by tetradecylthioacetic acid (TTA) [49]. The third group reported that cotransfection of A-FABP and E-FABP with PPARc and PPARb, respectively, into Cos-7 cells led to an increased activity of the nuclear receptor activity, which in the case of PPARc was specific for A-FABP and in the case of PPARb was specific for E-FABP [58]. The differences observed in these independent studies are, in our opinion, at least partially reflected by the experimental approach. First, all three studies employed a different reporter gene, the first study using the A-FABP promoter, the second and the third one an artificial reporter consisting of three idealized PPREs. Second, in the first and third study no RXRa-expressing plasmid was cotransfected, while in the second study all experiments were performed with cotransfected RXRa. In addition it is conceivable that transient transfection with

15.6 A-FABP and E-FABP

binding protein–DNA concentrations that are too high would artificially increase A-FABP and E-FABP concentrations to the effect that these proteins would eventually act as a sink for intracellular ligands. Conflicting results are reported on the interaction of A-FABP and E-FABP with different PPAR isoforms. Data of a recent mammalian two-hybrid study by us revealed a protein–protein interaction between A-FABP and PPARc (Fig. 15.3 B) [59]. In this system the binding strength of A-FABP was equal for the c1 and c2 isoforms and ligand independent, as was found for interaction of L-FABP with these PPARc subtypes [59]. In the other study employing a GST-pulldown assay [58] it was demonstrated that A-FABP selectively interacts with PPARc while E-FABP interacts only with PPARb. In both cases the interaction was weak in the absence of endogenous ligand and increased dramatically when either the PPARc-specific agonist troglitazone or the PPARb-specific agonist L165041 was added. The discrepancies between the two studies are most likely due to the different experimental approaches taken. First, one can argue that in a cellular environment provided by the mammalian two-hybrid assay endogenous ligands like fatty acids are in abundance and thus the interaction cannot be increased further by the addition of exogenous ligand. Second, the GST-pulldown assay employed only the ligand binding domain of the respective PPAR subtype, while in the mammalian two-hybrid system the full-length protein was used. Thus, at the moment it cannot be stated with certainty whether the interaction of A-FABP/E-FABP with PPAR isoforms is ligand dependent. Yet, even circumstantial evidence resulting from the generation of the PPARc+/– and the A-FABP–/– mice cannot conclusively answer whether A-FABP influences PPAR activity in a positive or negative manner. For a phenotypic analysis of this animal model one has to understand first how an increase or a decrease of PPARc activity contributes to development of insulin resistance. Until the generation of the PPARc+/– mouse (PPARc–/– animals die during embryonic development) it was generally assumed on the one hand that an increase of PPARc activity protects from insulin resistance. This assumption was based on findings that treatment with thiazolidinediones, potent activators of PPARc, leads to a decreased insulin resistance [60]. On the other hand, the PPARc+/– mouse, with decreased PPARc activity, is also protected from insulin resistance [61]. Does a decrease as well as an increase in PPARc activity provide protection from insulin resistance? Interestingly, A-FABP–/– mice similar to PPARc+/– mice did not develop insulin resistance when fed a high-fat diet [62]. Furthermore, when A-FABP–/– mice were crossed into the ob/ob background the severe phenotype of insulin resistance usually observed in these animals was abrogated [63]. Based on what is known about the effect of PPARc activity on insulin resistance one could speculate that in the absence of A-FABP, the positive regulator, less ligand is transported to PPARc for its activation, and consequently the concomitant decrease in PPARc activity leads to a protection from insulin resistance. In view of the finding that activation of PPARc by thiazolidinediones protects from insulin resistance, A-FABP in contrast might function as a negative regulator of PPARc activity by sequestra-

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Fig. 15.4 Dependence of the rate of transfer of all-trans retinoic acid (RA) from CRABP to RARa on the concentration of the latter. Holo CRABP-I or holo CRABP-II (1 mM) were mixed with RARa at various concentrations. Transfer of RA from the binding protein to the receptor was monitored by measuring the time-dependent decrease in RA fluorescence (kex = 360 nm, kem = 470 nm) following mixing

of holo CRABP with RARa. Data were fitted to a first-order reaction equation to obtain reaction rate constants. Apparent half-lives of the transfer reactions are shown as a function of the concentration of RARa. Data are presented as mean of independent experiments (n = 3–6), except [RARa] = 21 mM where n = 2. From Ref. [64] with permission.

tion of the ligand. Taken together, the question whether A-FABP regulates PPARc in a positive or negative manner cannot be answered to date. Moreover, possible compensatory effects of another member of the iLBP family, namely E-FABP, which exhibits a dramatically increased expression in A-FABP–/– animals [62], have not been considered up to now.

15.7

CRABP-II

A signal transduction pathway identical to that mediated by L-FABP/PPARa and c, has been found for CRABP-II and RARa. As for FABPs, it has been proposed that CRABP-II serves to solubilize and protect its branched-chain ligand in the aqueous cytosol, and to transport it between cellular compartments, including the nucleus. In support of this notion CRABP-II was detected not only in the cytosol, but also in nuclei of different cell types [50] to serve as a shuttle of all-trans retinoic acid to RARs [64]. By measuring the rate of dissociation of retinoic acid from CRABP-I and CRABP-II as a function of RARa concentration in a reconstituted system, different dissociation kinetics were observed for CRABP-I and CRABP-II. While RARa did not affect the rate of retinoic acid dissociation from CRABP-I it did influence the dissociation of retinoic acid from CRABP-II, suggesting that transport occurs by direct collisional transfer (Fig. 15.4) [64]. In an other study it

15.8 Other Members of the FABP Family

was demonstrated that CRABP-II and RARa interact in a ligand-independent fashion [65]. Residues in CRABP-II responsible for interaction with RARa were mapped to the entrance of the ligand binding pocket [66]. Finally overexpression of CRABP-II resulted in a marked stimulation of transcription of a reporter gene driven by RARE [64, 65]. From the physiological point of view the expression of CRABP-II is associated with cells that convert relatively large amounts of precursor to all-trans retinoic acid on demand; this suggests that cells with an increased requirement for alltrans retinoic acid require upregulation of both the synthesis of the signaling molecule and the expression of CRABP-II, allowing for rapid delivery of all-trans retinoic acid to the nuclear receptor. In mammary carcinoma cell lines CRABP-II expression is correlated with resistance to all-trans retinoic acid-induced growth arrest [67]. Expression of a CRABP-II antisense construct in SCC cells results in reduced sensitivity to all-trans retinoic acid-induced cell cycle arrest [68]. Together with the finding that the CRABP-II knockout mouse exhibits defects in limb development [69] these results demonstrate that CRABP-II modulates the effect of RAR and thereby affects growth regulation and morphogenesis in response to retinoic acid.

15.8

Other Members of the FABP Family

Binding assays with the mammalian two-hybrid system and by immuno-coprecipitation showed that, apart from L-, A- and E-FABP, no other FABP-type of the iLBP family interacts by protein–protein contacts with any PPAR isoform [52, 58, 59]. A likely candidate within the iLBP family for participation in signal transduction according to the protein–protein contact mechanism is I-BABP. This protein is phylogenetically and structurally closest related to L-FABP (see Chapter 5), but in contrast binds bile acid instead of fatty acid and has been implicated in bile acid transport. It can be envisaged that I-BABP plays a role in transporting bile acids to their nuclear receptor, the farnesoid X receptor (FXR), for activation of the latter. Respective experiments are in progress. The direct interaction of CRABP-II with RARa, without involvement of other proteins, was demonstrated in a reconstituted system [64]. L-FABP interacts with PPAR also directly [52]. Beyond these examples positive modulation of PPAR/agonist-regulated gene expression by iLBPs via protein–protein contacts is also conceivable through participation of third proteins, which help in ligand/agonist transfer from iLBP to nuclear receptor. In a report recently published is was stated that L-FABP interacts with at least three nuclear proteins [48] as determined by Western blotting. Older data exist in the literature on the presence of H-FABP within euchromatic nuclear regions of epithelial cells and on its direct association with chromatin [70]. The latter protein, a subfamily iv iLBP, is a case in point. Also known as “mammary-derived growth inhibitor” (MDGI), H-FABP inhibited growth either from “outside” (by addition to the media) [71] or from “inside” (by

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stable transfection) the cell [72] (reviewed in Ref. [26]). How H-FABP decreases growth of these cells is still not clear. One possible mechanism pertaining to “inside” action is increase of apoptosis triggered by H-FABP and PPARc in these cells. It has been reported by several independent groups that PPARc activation leads to an increase of apoptosis in several cell lines [73] and overexpression of HFABP could stimulate this effect and thereby decrease cell growth. No hypothesis is available for the “outside” action of H-FABP, furthermore, the role of the ligands/agonists also need consideration. Another possible signal transduction pathway can be envisaged due to similarities of ligand binding. It was recently demonstrated that RXRa binds and can be activated by docosahexaenoic acid [22]. Since RXRa as well as docosahexaenoic acid play an important role during brain development it seems indeed reasonable that the latter can activate RXRa. Of all iLBPs, B-FABP shows the highest binding affinity for n-3 fatty acids in general and docosahexaenoic acid in particular [30]. Since B-FABP is expressed in brain tissue it could be possible that it acts as a shuttle for docosahexaenoic acid to RXRa for activation of the latter. B-FABP does not interact directly with RXRa [59], so a third protein might be involved. The “sink” function of iLBPs in the artificial CV-1 monkey kidney fibroblast system, as reported for A- and E-FABP [49], would result in a negative modulation of PPAR/agonist-regulated gene expression. In principle, this mechanism is applicable to all iLBPs and would mean an inverse relationship between intracellular iLPB concentration and PPAR activation. The physiological relevance of such a mechanism has not yet been demonstrated.

15.9

Mechanism of iLBP Import into the Nucleus

The compartment of interaction of iLBPs and fatty acid activated nuclear receptors has not been identified so far. One the one hand the interaction could take place in the cytosol, where nuclear receptors would sequestrate ligands from iLBPs and subsequently translocate to the nucleus to induce gene transcription. On the other hand, the interaction could take place in the nucleus, which is strengthened by the fact that several iLBPs have been detected in the nuclear compartment. Pertaining to the latter findings the question remains how iLBPs enter the nucleus. With respect to their size with or without ligand, iLBPs might diffuse freely through the nuclear pores. This hypothesis is strengthened by the finding that the ratio of nuclear to cytosolic L-FABP is the same in control mice as in mice treated with bezafibrate [47], a known inducer of cytosolic L-FABP expression. Although on a protein-based scale the L-FABP concentration in the nucleus is 100-fold lower than that in the cytosol, concentrations related to volume may be different. Alternatively, protein modification might furnish a recognition signal for entering the nucleus. In L-FABP, for example, the carboxylate group of the second fatty acid bound protrudes from the binding pocket [74] and may furnish a negative

15.10 Conclusions and Perspectives

charge on the protein‘s surface for nuclear targeting. By employing L-FABP modified with fluorescein in a fluorescein filtration assay, it was shown that L-FABP with a negatively charged ligand (fatty acid, peroxisome proliferator) bound directly to nuclei of rat hepatocytes in a ligand-dependent manner [48]. Covalent modification of L-FABP may also furnish targeting signals, in line with this are modifications of L-FABP by cysteinylation and glutathionylation [75]. A-FABP and E-FABP, when recombinantly expressed in CV-1 or Cos-1 cells, translocate to the nucleus [49, 58]. In these cases the fatty acid bound to the proteins cannot furnish a recognition signal because its carboxy group is buried deeply inside the binding pocket [31]. Modification of A-FABP by phosphorylation at Tyr19 was reported upon insulin stimulation of adipocytes [76] and of H-FABP in mammary gland cells [31]; this could furnish an additional signal for nuclear import. Presently, all this is speculation and passive diffusion from the cytosol to the nucleus of either iLBP is a possibility as well. CRABP-II was localized in cytosol and nuclei of different cell lines, as well as in mouse embryonic tissue by Western blotting and immunohistochemistry with specific antibodies [50]. Since this protein does not have a nuclear localization signal, it is most likely that it enters the nuclear compartment by passive diffusion, yet unknown factors contributing to this nuclear localization cannot be ruled out.

15.10

Conclusions and Perspectives

Signaling by fatty acids involves on the one hand the signal itself, the intracellular transport protein, the nuclear receptor target, and the genes regulated in response to the signal. The signal transduction pathways described in this chapter are based on iLBP interaction with nuclear receptors (Fig. 15.5), but this may not be the only mechanism one can envision for fatty acid signaling. First, protein–protein contacts of iLBPs with nuclear receptors need not be a prerequisite in all cases, other proteins could function as auxiliary interaction partners. Second, iLBPs might function as negative regulators of nuclear receptors when present intracellularly in high concentrations, as may be deduced from one of the studies on A-FABP and E-FABP action. Third, phosphorylation of iLBPs, as reported in the case of A-FABP and H-FABP, could also be a novel form of signal transduction, where fatty acid binding is converted into a recognition signal by chemical modification. One has to keep in mind that the scheme shown in Fig. 15.5 summarizes, in a sequential manner, five steps that have been elucidated independently: (i) Binding of ligand by iLBP, (ii) interaction of iLBP with nuclear receptor, (iii) binding of ligand-loaded receptor to responsive element, (iv) transactivation of nuclear receptor, and (v) induction of target genes. Thus, iLBP-dependent target gene expression in vivo awaits validation. One approach to the achievement of this goal is application of a pertinent iLBP knockout mouse.

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Fig. 15.5 Scheme for iLBP-modulated nuclear receptor activation by fatty acids and drugs (solid arrows for the path of the ligands/agonists). iLBPs function as intracellular transporters of ligands/agonists to the nucleus for activation of the receptor, the latter being their ultimate target. The ligand/agonist activated receptors in turn regulate the expres-

sion of some iLBPs as well as of genes involved in degradation or storage of fatty acids and in embryonic development (dashed arrows). Nuclear receptors (PPAR, RAR); ACO, acyl-CoA oxidase; CPT-I, carnitine palmitoyl transferase I; hLPL, hormone-sensitive lipoprotein lipase; HNF, hepatocyte nuclear factor.

L-FABP and CRABP-II have been clearly identified as co-activators of nuclear receptor transactivation induced by fatty acids [52, 77]; more members of the iLBP family may follow. The focus on the design of drugs affecting gene regulation thus far is on ligand activated receptors, but the question can now be addressed whether the transport system is able to perform the proper shuttle function for drugs. Furthermore, iLBPs themselves could serve as novel therapeutical targets in an approach to modulate nuclear receptor activity.

15.11 References

15.11

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Lim, R. Godbout, F. Spener, J. C. Sacchettini, J. Biol. Chem. 2000, 275, 27045–54. A. Reese-Wagoner, J. Thompson, L. Banaszak, Biochim. Biophys. Acta 1999, 1441, 106–116. C. Hohoff, T. Börchers, B. Rüstow, F. Spener, H. van Tilbeurgh, Biochemistry 1999, 38, 12229–39. A. Müller-Fahrnow, U. Egner, T. A. Jones, H. Rüdel, F. Spener, W. Saenger, Eur. J. Biochem. 1991, 199, 271–276. F. Guthmann, C. Hohoff, H. Fechner, P. Humbert, T. Börchers, F. Spener, B. Rüstow, Eur. J. Biochem. 1998, 253, 430– 436. R. K. Ockner, J. A. Manning, J. P. Kane, J. Biol. Chem. 1982, 257, 7872–78. R. G. Maatman, E. M. van de Westerlo, T. H. van Kuppevelt, J. H. Veerkamp, Biochem. J. 1992, 288, 285–290. H. Poirier, I. Niot, P. Degrace, M. C. Monnot, A. Bernard, P. Besnard, Am. J. Physiol. 1997, 273, G289–G295. R. P. Green, S. M. Cohn, J. C. Sacchettini, K. E. Jackson, J. I. Gordon, DNA Cell. Biol. 1992, 11, 31–41. D. A. Bernlohr, T. L. Doering, T. J. Kelly, Jr., M. D. Lane, Biochem. Biophys. Res. Commun. 1985, 132, 850–855. M. D. Layne, A. Patel, Y. H. Chen, V. I. Rebel, I. M. Carvajal, A. Pellacani, B. Ith, D. Zhao, B. M. Schreiber, S. F. Yet, M. E. Lee, J. Storch, M. A. Perrella, FASEB J. 2001, 15, 2733–35. A. Kurtz, A. Zimmer, F. Schnütgen, G. Brüning, F. Spener, T. Müller, Development 1994, 120, 2637–49. M. Donovan, B. Olofsson, A. L. Gustafson, L. Dencker, U. Eriksson, J. Steroid Biochem. Mol. Biol. 1995, 53, 459– 465. I. Issemann, R. Prince, J. Tugwood, S. Green, Biochem. Soc. Trans. 1992, 20, 824–827. P. Tontonoz, E. Hu, B. M. Spiegelman, Curr. Opin. Genet. Dev. 1995, 5, 571–576. A. ström, A. Tavakkol, U. Pettersson, M. Cromie, J. T. Elder, J. J. Voorhees, J. Biol. Chem. 1991, 266, 17662–66. U. Bordewick, M. Heese, T. Börchers, H. Robenek, F. Spener, Biol. Chem. Hoppe-Seyler 1989, 370, 229–238.

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J. Gorski, M. Zendzian-Piotrowska, C. Wolfrum, A. Nawrocki, F. Spener, Mol. Cell. Biochem. 2000, 214, 57–62. J. W. Lawrence, D. J. Kroll, P. I. Eacho, J. Lipid Res. 2000, 41, 1390–1401. T. Helledie, M. Antonius, R. V. Sørensen, A. V. Hertzel, D. A. Bernlohr, S. Kolvraa, K. Kristiansen, S. Mandrup, J. Lipid Res. 2000, 41, 1740– 51. M. P. Gaub, Y. Lutz, N. B. Ghyselinck, I. Scheuer, V. Pfister, P. Chambon, C. Rochette-Egly, J. Histochem. Cytochem. 1998, 46, 1103–11. T. Börchers, C. Unterberg, H. Rüdel, H. Robenek, F. Spener, Biochim. Biophys. Acta 1989, 1002, 54–61. C. Wolfrum, C. M. Borrmann, T. Börchers, F. Spener, Proc. Natl Acad. Sci. USA 2001, 98, 2323–28. N. M. Bass, Mol. Cell. Biochem. 1993, 123, 191–202. G. Castillo, R. P. Brun, J. K. Rosenfield, S. Hauser, C. W. Park, A. E. Troy, M. E. Wright, B. M. Spiegelman, EMBO J. 1999, 18, 3676–87. C. E. Juge-Aubry, S. Kuenzli, J. C. Sanchez, D. Hochstrasser, C. A. Meier, Biochem. J. 2001, 353, 253–258. D. Shao, S. M. Rangwala, S. T. Bailey, S. L. Krakow, M. J. Reginato, M. A. Lazar, Nature 1998, 396, 377–380. A. V. Hertzel, D. A. Bernlohr, Mol. Cell. Biochem. 1998, 188, 33–39. N. S. Tan, N. S. Shaw, N. Vinckenbosch, P. Liu, R. Yasmin, B. Desvergne, W. Wahli, N. Noy, Mol. Cell. Biol. 2002, 22, 5114–27. B. Bleck, Ph.D.-Thesis, University of Münster, 2001. D. E. Moller, D. A. Greene, Adv. Protein Chem. 2001, 56, 181–212. T. Yamauchi, J. Kamon, H. Waki, K. Murakami, K. Motojima, K. Komeda, T. Ide, N. Kubota, Y. Terauchi, K. Tobe, H. Miki, A. Tsuchida, Y. Akanuma, R. Nagai, S. Kimura, T. Kadowaki, J. Biol. Chem. 2001, 276, 41245–54. G. S. Hotamisligil, R. S. Johnson, R. J. Distel, R. Ellis, V. E. Papaioannou, B. M. Spiegelman, Science 1996, 274, 1377–79.

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K. T. Uysal, L. Scheja, S. M. Wiesbrock, S. Bonner-Weir, G. S. Hotamisligil, Endocrinology 2000, 141, 3388–96. D. Dong, S. E. Ruuska, D. J. Levinthal, N. Noy, J. Biol. Chem. 1999, 274, 23695– 98. L. Delva, J. N. Bastie, C. Rochette-Egly, R. Kraiba, N. Balitrand, G. Despouy, P. Chambon, C. Chomienne, Mol. Cell. Biol. 1999, 19, 7158–67. A. Budhu, R. Gillilan, N. Noy, J. Mol. Biol. 2001, 305, 939–949. Y. Jing, S. Waxman, R. Mira-y-Lopez, Cancer Res. 1997, 57, 1668–72. H. P. Vo, D. L. Crowe, Anticancer Res. 1998, 18, 217–224. D. Fawcett, P. Pasceri, R. Fraser, M. Colbert, J. Rossant, V. Giguère, Development 1995, 121, 671–679. T. Müller, A. Kurtz, F. Vogel, H. Breter, F. Schneider, U. Angstrom, M. Mieth, F. D. Böhmer, R. Grosse, J. Cell Physiol. 1989, 138, 415–423. Y. Yang, E. Spitzer, N. Kenney, W. Zschiesche, M. Li, A. Kromminga, F. Müller, F. Spener, A. Lezius, J. H. Veerkamp, G. H. Smith, D. S. Salomon, R. Grosse, J. Cell. Biol. 1994, 127, 1097– 1109.

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C. Buhlmann, T. Börchers, M. Pollak, F. Spener, Mol. Cell. Biochem. 1999, 199, 41–48. E. Elstner, C. Müller, K. Koshizuka, E. A. Williamson, D. Park, H. Asou, P. Shintaku, J. W. Said, D. Heber, H. P. Koeffler, Proc. Natl Acad. Sci. USA 1998, 95, 8806–11. J. Thompson, N. Winter, D. Terwey, J. Bratt, L. Banaszak, J. Biol. Chem. 1997, 272, 7140–50. P. Dörmann, T. Börchers, U. Korf, P. Højrup, P. Roepstorff, F. Spener, J. Biol. Chem. 1993, 268, 16286–92. M. K. Buelt, L. L. Shekels, B. W. Jarvis, D. A. Bernlohr, J. Biol. Chem. 1991, 266, 12266–71. N. Noy, Cochrane Database Syst. Rev. 2000, 4, 481–495. L. I. Brodskii, V. V. Ivanov, L. Kalaidzidis Ia, A. M. Leontovich, V. K. Nikolaev, S. I. Feranchuk, V. A. Drachev, Biokhimiia 1995, 60, 1221–30. C. Wolfrum, C. Buhlmann, B. Rolf, T. Börchers, F. Spener, Biochim. Biophys. Acta 1999, 1437, 194–201.

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Arachidonic Acid Binding Proteins in Human Neutrophils Claus Kerkhoff and Olof Rådmark

16.1

Cellular Functions of Arachidonic Acid

The polyunsaturated fatty acid arachidonic acid (Ara) plays a major role in the immune response of polymorphonuclear neutrophils (PMNs). Here, arachidonic acid serves as a substrate for either cyclooxygenase or lipoxygenase in the generation of prostaglandins and leukotrienes, respectively. Prostaglandins and leukotrienes represent biologically active lipid mediators that act as paracrine and autocrine regulators through a family of transmembrane receptors. They regulate many cell functions and play crucial roles in a variety of physiological and pathophysiological processes, including chemotaxis, leukocyte adhesion, and various immune and inflammatory functions (for review see Refs [1–4]). In addition, arachidonic acid has been implicated in several important roles of neutrophil function. They include stimulation of the respiratory burst [5, 6], degranulation of intact PMNs [7–9], activation of protein kinase C [10, 11], regulation of intracellular Ca2+ levels [12, 13], and modulation of the H+ pump associated with NADPH oxidase activity [14, 15].

16.2

The Two Myeloid-related Proteins S100A8 and S100A9 16.2.1

S100A8 and S100A9 Belong to the S100 Family

The two proteins S100A8 and S100A9, which are abundant in monocytes and neutrophils, belong to the S100 family of calcium-binding proteins (for review see Refs [16–19]). Most of the genes encoding the human S100 proteins are clustered on chromosome 1q21 [20]. S100A8 and S100A9 are composed of two distinct helix-loop-helix motifs (EFhand) flanked by hydrophobic regions at either terminus and separated by a central hinge region (Fig. 16.1). The C-terminal EF-hand has a higher affinity for Ca2+ and encompasses 12 amino acids, whereas the N-terminal Ca2+-binding loop is formed by 14 amino acids. S100A8 has a significant Glu33 ? Asp33 substitution at position

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Fig. 16.1 Structural domains within S100A8 and S100A9. Amino acid sequences of human S100A8 and S100A9 were obtained from the SwissProt protein sequence database, and the alignment was performed with the help of LALIGN. Identical residues are indicated by “:”, and homologous residues are indicated by “.”. Both proteins share 28.3% identity in a

92-amino-acid overlap, and a 72.8% homology, respectively. Helical elements are predicted with the help of NNPREDICT, and they are indicated by blank boxes. The calcium binding loop I (12-amino-acid EF-hand) and II (14- amino-acid EF-hand) are indicated by gray boxes; the zinc binding motif is indicated by dark gray boxes.

14 of the calcium-binding loop in the N-terminal pseudo EF-hand. Since this residue is known to play an important role in calcium binding and is highly conserved within this position in S100 proteins [21], it is concluded that the N-terminal EF-hand of S100A8 is not likely to bind calcium with appreciable affinity. In addition to the binding of Ca2+, S100 proteins have been shown to bind other bivalent cations, such as Zn2+ and Cu2+. The binding motif for these bivalent cations is still in debate (for review see Ref. [22]) [23–25]. The zinc binding to the S100A8/A9 protein complex is assumed to play a role in inhibiting the growth of microorganisms that initially escape the microbicidal activity of intact neutrophils at sites of infection. The function appears to depend on the ability of S100A8/A9 to sequester zinc efficiently enough that free concentrations of Zn2+ fall below the low levels needed by most microorganisms [26–28]. Indeed, a truncated form of S100A9 lacking the consecutive His residues was less active in the Candida growth inhibition assay than was the untruncated form when combined with S100A8 [29]. S100A8 and S100A9 show a strong tendency to form heterodimers as well as higher order species in accordance with the general consensus that S100 protein function depends on heteromer formation. The formation of these protein complexes is not calcium-dependent as demonstrated by various studies [30, 31]. The S100A8/A9 protein complex is also formed in the absence of calcium. However, in addition to heterodimers a protein complex with an Mr of 48 kDa is analyzed in the presence of calcium. The 48-kDa protein is assumed to represent the tetrameric protein complex consisting of two molecules S100A8 and two molecules S100A9 [31]. There is evidence that the binding of calcium to S100 dimers induces conformational changes within the protein complexes (for review see Refs. [17, 20, 33]).

16.2 The Two Myeloid-related Proteins S100A8 and S100A9

To date there are no crystal structures of the S100A8/A9 protein complex available. However, there is sufficient sequence and structural similarity to other S100 proteins to suppose that the heterodimer is formed in a similar way to those of other S100 proteins. It is established that S100 dimers are stabilized by hydrophobic contacts between helices IV (and partly the helices I) of the subunits, which are antiparallel. The EF-hand loops I and II and the hinge regions are positioned on the outer surfaces of the dimer after calcium binding. The calcium-induced conformational changes lead to the exposure of hydrophobic surfaces that may allow interaction with target proteins. A model for target protein binding to other calcium-activated S100 dimers has been proposed by Groves et al. [32]. The role of C-terminal domain, namely helix IV, has been confirmed by the two-hybrid system and site-directed mutagenesis [34]. 16.2.2

S100A8 and S100A9 Expression is Primarily Restricted to Cells of Myeloid Lineage

Like other S100 proteins, S100A8 and S100A9 are expressed in a tissue/cell-specific manner. Their expression appears to be restricted to a specific stage of myeloid differentiation, because both proteins are expressed in circulating neutrophils and monocytes, but not in mature tissue macrophages. They are absent in lymphocytes. In peripheral blood monocytes their expression is downregulated during maturation to macrophages [35, 36]. It has been shown that the first activated or recruited phagocytes which migrate to inflammatory lesions express S100A8 and S100A9 [35]. This subset of phagocytes is present in acute but absent in chronic inflammatory disorders. Monocytes, which express the membrane-bound S100A8/A9 heterodimer, release high amounts of TNFa and IL-1b in contrast to their S100A8/A9 surface-negative counterparts. Thus, S100A8 and S100A9 are widely used as marker proteins for activated or recruited phagocytes. Patients suffering from inflammatory disorders, such as chronic bronchitis, cystic fibrosis, and rheumatoid arthritis, had elevated plasma levels of S100A8/A9. The demonstration that S100A8/A9 is specifically secreted from human monocytes upon protein kinase C (PKC) activation may indicate an extracellular role for S100A8/A9 [37]. In addition, the proteins are also expressed in the epidermis under chronic inflammatory conditions, such as psoriasis and malignant disorders [38–40]. These findings, together with the presence of enhanced S100A8/A9 levels in the sera of patients suffering from a number of inflammatory disorders [41– 43], have led to the assumption that S100A8 and S100A9 affect leukocyte trafficking and display a propagating role in inflammatory responses. The molecular mechanisms underlying the cell type-specific expression of S100A9 are unknown. The human S100A9 gene has been cloned and sequenced, and an upstream 1-kb fragment of its promoter was shown to drive the gene expression in myeloid cells [36]. A number of distinct regulatory regions upstream of the transcription initiation site have been demonstrated to either activate or repress promoter activity in a differentiation and tissue/cell-specific manner [44–49]. Recently, we identified a 27-bp region within the 1-kb proximal region that plays an important role in

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Fig. 16.2 Protein binding to the MRE and monocytic differentiation. (A) EMSA was performed with nuclear extracts from cultured human peripheral blood monocytes and the 32 P-labeled MRE probe. Freshly isolated monocytes showed two shifts in EMSA and the lower shift diminished with further differentiation of the monocytes to macrophages.

(B) After 1, 3, and 7 days, RNA was isolated from the cultured monocytes and Northern blot analysis was performed using digoxygenin-labeled S100A9 cDNA probe. (C) The ethidium bromide-stained 28S and 18S ribosomal RNA markers indicate equal loading of the gel.

the regulation of the S100A9 protein expression in myeloid and non-myeloid cells as shown by a detailed deletion analysis (Kerkhoff, unpublished observations). This novel regulatory region, termed MRP14 (S100A9) regulatory element (MRE), exhibits no putative binding sites for known transcription factors as analyzed by a detailed computer search using the “Transfec Matrix Table” [50]. EMSA analysis revealed that two nuclear protein complexes with different electrophoretic mobilities were specifically bound to MRE. They are referred to as MRE-binding complex A (MbcA) and MbcB, respectively. The formation of these two nuclear complexes strongly correlated with the S100A9 gene expression in a cell type-specific, activation- and differentiation-dependent manner. · Nuclear extracts prepared from S100A9-positive cells, such as MonoMac-6 and monocytes, resulted in the formation of MbcB, whereas in nuclear extract of S100A9-negative cells, such as lymphocytes, the lymphoid cell line Raji, and non-hematopoietic cells (i.e. epithelial cells, fibroblasts, and keratinocytes) the formation of MbcA was found. · During the maturation of human blood monocytes to macrophages the S100A9 gene expression is downregulated [35, 36]. In accordance with this, EMSA analysis of nuclear extracts of monocytes cultured for different time periods revealed that the formation of complex MbcB was decreased during cultivation, while the formation of complex MbcA was induced at day 3 (Fig. 16.2). The downregulation of S100A9 mRNA transcripts was confirmed by Northern blot analysis.

16.2 The Two Myeloid-related Proteins S100A8 and S100A9

· The activation of blood monocytes by either a calcium ionophore or the phorbol ester PMA also leads to a reduced level of S100A9 mRNA transcripts [37, 45]. As demonstrated by EMSA, MbcB was present in nuclear extracts of non-stimulated cells, whereas nuclear extracts of stimulated cells resulted in the formation of a complex with mobility similar to that of MbcA. Northern blot analysis confirmed that short-term incubation with these agents indeed resulted in the downregulation of S100A9 mRNA transcripts. Due to our findings, the complex MbcB is assumed to drive S100A9 gene expression, whereas MbcA might play a negative regulatory role. However, the nature of the nuclear proteins binding to the MRE is not clear at present. The elucidation of these DNA–protein complexes is currently under investigation. 16.2.3

S100A8/A9 Protein Complexes Bind Polyunsaturated Fatty Acids

Two groups have shown independently that the S100A8/A9 protein complex specifically binds fatty acids in a Ca2+-dependent manner [51, 52]. Detailed studies using purified S100A8 and S100A9 of human neutrophils revealed that S100A8/ A9 specifically bound Ara in a calcium-dependent manner. The estimated calcium concentration required to induce fatty acid binding was within the physiological range. The fact that the individual components of the protein complex were unable to bind fatty acids either in the absence or in the presence of calcium, leads to the assumption that docking of the two subunits creates an asymmetric fatty acid binding site located at the interface between the subunits. The identification of the Ara binding site is currently under investigation. Preliminary results indicate that the unique C-tail of S100A9 is involved in the binding of the ligand (Sopalla, unpublished observations). The protein complex which was able to bind Ara consisted of equal moles S100A9 and S100A8, and the binding of calcium to each EF-hand within the protein complex was a prerequisite for Ara binding [30, 53]. Binding studies revealed that Ara binding was saturable and had specific binding characteristics. A KD of 0.2 lM and a stoichiometry of 0.4 mol fatty acid per mol S100A8/A9 heterodimer was derived from Scatchard analysis [30]. Competition studies clearly indicated that specifically polyunsaturated fatty acids were bound by the protein complex in a saturable and reversible manner, whereas saturated fatty acids, such as palmitic and stearic acids, and the monounsaturated oleic acid were poor competitors [30]. The S100A8/A9 protein complex showed the highest specificity towards arachidonic acid (Tab. 16.1). It is of interest to note that the arachidonic trifluoromethylketon (AACOCF3) does not compete for AA binding to S100A8/A9 although it exhibits structural similarity to Ara. In addition, the S100A8/A9 complex did not show any affinity for Ara-derived eicosanoids. These assays therefore give insights into the nature of the fatty acid binding site. The affinity was significantly influenced by (i) the polar heads, (ii) the number of the double bonds, and (iii) their position within the fatty acid molecule. The specificity towards polyunsaturated fatty acids as well as the reversibility

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16 Arachidonic Acid Binding Proteins in Human Neutrophils Tab. 16.1 IC50 values of various competitors determined by displacement experiments.

Competitor

IC50 [lM]

ETYA EPTA Oleic acid c-linolenic acid a-linolenic acid Arachidonic acid Trien EDA ETA 5-HETE 5-OxoETE

no competition no competition no competition 5.0 15.0 0.9 1.1 1.1 3.1 100.0 no competition

1 nmol of wild-type S100A8/A9 was incubated with 1 lM [3H]AA and increasing concentrations of the non-labeled competitors (range 0–100 lM) in the presence of 5 mM CaCl2. IC50 is the concentration of competitor that leads to a 50% displacement of the bound ligand [3H]AA.

of the binding to the protein complex excluded the possibility that merely a solvation of insoluble fatty acid calcium salts occurred. These results also argue against a coordination of the fatty acid by one of the calcium ions of the S100A8/A9 complex. In contrast to our results, Siegenthaler et al. [52] found that S100A8/A9 shows specificity towards various fatty acids. This finding may be due to the fact that they used purified proteins from human keratinocytes for their binding and competition studies. Investigation of the Ara binding capacity in the cytosol of human neutrophils revealed that S100A8/A9 represented the sole Ara binding protein. This finding was corroborated by the facts that (i) there was only a low capacity for Ara binding in the neutrophilic cytosol in the absence of calcium; (ii) the Ara binding capacity in the cytosol was increased in a protein concentration-dependent manner after the addition of calcium which induces the formation of S100A8/A9 protein complexes; and (iii) the Ara binding capacity in the cytosol was depleted by immunoprecipitation with the S100A8/A9 protein complex-specific monoclonal antibody 27E10 [30]. Furthermore, the conclusion was supported by the finding that members of the fatty acid binding protein (FABP) family are not expressed in human neutrophils [54]. The finding that the S100A8/A9 protein complex represents the exclusive Ara binding capacity in the neutrophilic cytosol together with their unusual abundance in human neutrophilic cytosol [55] clearly indicate that S100A8/A9 complexes play an important role in the arachidonic acid metabolism of neutrophils. The calcium-induced binding of Ara points to a role of S100A8/A9 in the mobilization, metabolization, or release of Ara.

16.2 The Two Myeloid-related Proteins S100A8 and S100A9

16.2.4

Translocation of S100A8 and S100A9 is Accompanied with Arachidonic Acid Transport

S100A8 and S100A9 are predominantly localized in the cytoplasm. Upon elevation of the intracellular calcium level they are translocated from the cytosol to cytoskeleton and to plasma membrane. At a later time point, they appear as non-covalently associated S100A8/A9 heterodimers on the surface of monocytes [56, 57]. The mechanism by which the S100A8/A9 heterodimer penetrates the plasma membrane, and how the S100A8/A9 protein complex is anchored into the cell membrane, remains unclear since both proteins lack a transmembrane region. Upon the activation of protein kinase C S100A8/A9 heterodimers are released from human monocytes by a novel secretion pathway which is energy-consuming and depends on an intact microtubule network [37]. Moreover, the secretion of S100A8 and S100A9 does not necessarily require S100A8/A9 cell surface expression. Therefore, it is suggested that both surface expression and S100A8/A9 heterodimer secretion may represent two different, independent pathways. Interestingly, both translocation pathways are accompanied with the transport of arachidonic acid (Fig. 16.3): (i) Elevation of intracellular calcium levels in human neutrophils by opsonized zymosan resulted in a decrease of cytosolic S100A8/A9 levels and a parallel increase in membranes. In parallel, arachidonic acid was transported from the cytosol to the membrane, suggesting that S100A8/ A9–Ara complexes might play an important role in FA uptake and/or release [54]. (ii) In detailed cell-stimulation experiments with neutrophil-like HL-60 cells both the release of Ara and the secretion of S100A8/A9 heterodimers was investigated [30]. Arachidonic acid release was induced by either an increase of the intracellular calcium level by the calcium ionophore A23187 or an activation of protein kinases by the phorbol ester PMA [58–60]. S100A8/A9 was secreted from neutrophil-like HL-60 cells by PMA, whereas the calcium ionophore A23187 was ineffective in inducing the S100A8/A9 secretion. Immunoprecipitation experiments of

Fig. 16.3 Schematic presentation of S100A8/ A9–Ara complex translocation. S100A8 and S100A9 are predominantly localized in the cytoplasm. Upon elevation of the intracellular calcium level they are translocated from the cytosol to cytoskeleton and to plasma membrane. Upon the activation of protein kinase

C S100A8/A9 heterodimers are specifically secreted from monocytes and neutrophils. Both translocation to cellular structures and secretion are suggested to represent two different and independent pathways, and they are accompanied by Ara transport [30, 54].

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the cell supernatants revealed that (a) the secreted S100A8/A9 protein complex bound fatty acids and (b) the majority of the simultaneously released Ara was bound by the protein complex. Furthermore, the time courses of both Ara release and S100A8/A9 secretion were found to be similar. In contrast, the stimulation with A23187 led to a rapid release of Ara, but not to secretion of S100A8/A9. Consequently, no significant amounts of radiolabeled fatty acids were determined in the immunoprecipitate. Still, the functional consequences of Ara release either in the absence of S100A8/A9 secretion (as induced by calcium ionophore) or in parallel to S100A8/A9 secretion (as induced by PMA) remain unclear. The calcium-induced binding of polyunsaturated fatty acids to the S100A8/A9 protein complex makes it a good candidate for mediating effects of polyunsaturated fatty acids in a calcium-dependent way. It is likely that in resting neutrophils (as well as other inflammatory cells) only relatively low concentrations of cellular Ara are allowed to accumulate. The intracellular concentration of Ara is under the control of various acyltransferases which rapidly incorporate the fatty acid into particular glycerolipids followed by a slower remodeling into other glycerolipids (for review see Ref. [61]). The mobilization of esterified Ara from cellular sources and the subsequent metabolism of this Ara into oxygen-containing metabolites (eicosanoids) is the key regulatory event in most inflammatory cells. Its mobilization is induced via phospholipase(s) by an increase of calcium or by protein phosphorylation through MAP kinases (for review see Refs [62–64]). However, the mechanism by which arachidonic acid as well as eicosanoids, once biosynthesized, leave the producer cell to reach their target cells is still poorly understood. In addition, arachidonic acid is rapidly liberated from cellular phospholipids but the activities of the Ara-metabolizing enzymes are detectable within hours after stimulation [65–67]. Both PMA and A23187 led to a time-dependent increase in the level of immunodetectable COX-2. In parallel, the enhanced calcium level also induces S100A8/A9 protein complex formation and its Ara binding capacity. Thus, it could be envisioned that the Ara/S100A8/A9 complex may function as an intermediate reservoir. At a later time point, the complex might pass the ligand either to Ara-metabolizing enzymes, thereby initiating a delayed eicosanoid formation, or to Ara-dependent enzymes which are then promoted by their co-factor supply.

16.3

Putative Intracellular Functions of S100A8/A9 16.3.1

5-Lipoxygenase (5-LO) and 5-Lipoxygenase Activating Protein (FLAP)

The initial steps of leukotriene biosynthesis involve cytosolic phospholipase A2 (cPLA2), 5-lipoxygenase activating protein (FLAP), and 5-lipoxygenase (5-LO) (for reviews see Refs [68–70]). Upon cell activation leading to leukotriene (LT) biosynthesis, 5-LO as well as cytosolic phospholipase A2 (cPLA2) migrate to the nu-

16.3 Putative Intracellular Functions of S100A8/A9

clear membrane, this site has been recognized as a LT biosynthetic metabolon [71]. The clustering of cPLA2, 5-LO, and permanently membrane-bound FLAP seems logical since membrane phospholipids are the source of arachidonate. FLAP is thought to function as an arachidonic acid transfer protein. Thus, FLAP binds to a 125I-labeled photo affinity analog of arachidonic acid [72], and arachidonic acid and other cis-unsaturated fatty acids compete with FLAP inhibitors (BAY X1005, MK-886) with regard to binding to FLAP [73, 74]. FLAP is crucial for conversion of endogenous substrate by 5-LO, and in a study of transfected Sf9 cells FLAP also stimulated the utilization of exogenous arachidonic acid [70]. More recently, FLAP was found to greatly (190-fold) stimulate 5-LO utilization of another exogenous substrate (12(S)-HETE) in the 5-LO catalyzed conversion to 5(S),12(S)DHETE [75]. The sequence of FLAP is quite similar (31% identity) to microsomal LTC4 synthase (also an integral nuclear envelope protein); both proteins are regarded as members of the MAPEG family (membrane-associated proteins in eicosanoid and glutathione metabolism) [76]. As S100A8/A9, FLAP is a rather small protein (162 residues), and when the sequences were compared using Bestfit and Gap (GCG Wisconsin package) no obvious similarities appeared. Calcium ionophore is an efficient stimuli of cellular LT production, and it is well established that Ca2+ is a determinant of 5-LO activity in the cell under physiological conditions. Ca2+ probably has several functions in this process, including to bind to 5-LO, leading to increased hydrophobicity and membrane association and thus enzyme activity. The EC50 for Ca2+ activation of purified 5-LO is quite low (1–2 lM), and full activation is reached at 4–10 lM [77, 78]. 5-LO binds Ca2+ in a reversible manner [79], a Kd close to 6 M was determined by equilibrium dialysis and the stoichiometry of maximum binding averaged around two Ca2+ per 5-LO. Peculiarly, both the Kd of 6 lM and the Ca2+ concentration required for activation in vitro are considerably higher than the intracellular Ca2+ concentration estimated for stimulation of 5-LO in polymorphonuclear leukocytes (PMNLs) (approximately 200 nM) [80]. One could speculate that factors in the intracellular milieu (other proteins?) could adjust the Ca2+ affinity. We recently described that the putative N-terminal b-barrel of 5-LO resembles a C2 domain and binds Ca2+, leading to Ca2+ stimulation of enzyme activity [81], similar to cPLA2 and other proteins containing Ca2+ binding C2 domains [82, 83]. Membrane association is important for 5-LO activity; in vitro phosphatidylcholine was crucial for both basal and Ca2+-stimulated 5-LO activity in vitro [84]. Indeed, the N-terminal C2-like domain of 5-LO was also found important for translocation to the nuclear membrane [85]. Other Ca2+ effects contributing to cellular 5-LO activity are probably related to 5-LO translocation. These could involve kinase activity, the PKC inhibitor calphostin C reduced 5-LO product formation from endogenous substrate in PMNLs stimulated with ionophore A23187 [86]. 5-LO itself, but also other proteins (the translocation machinery?) are probable kinase substrates of importance for cellular 5-LO activity. 5-LO can be phosphorylated outside its C2-like domain, by mitogen-activated protein kinase activated protein kinase 2 (MAPKAP kinase 2), a MAPKAP kinase motif is present in 5-LO leading to phosphorylation at Ser271 [87]. These MAP-

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KAP kinases are phosphorylated and activated by p38 mitogen activated protein kinase (p38 MAP kinase) which exists in several isoforms, generally activated by cell stress or treatment of cells with inflammatory cytokines [88, 89]. Active 5-LO kinases (apparently MAPKAP kinases 2 and 3) were present in stimulated MonoMac 6 cells and PMNLs, but not in unstimulated cells. The cell stress inducer sodium arsenite was the most efficient MAPKAP stimulus in human PMNL, and this agent upregulated (3- to 4-fold) 5-LO activity in PMNLs also receiving arachidonic acid and platelet activating factor [87]. More recently, also other stress stimuli (osmotic stress, heat shock) were found to activate p38 MAPK and stimulate 5LO activity in human PMNL, and interestingly sodium arsenite and osmotic stress were effective also after chelation of Ca2+ [90]. This implies that phosphorylation can lead to activation of 5-LO in PMNLs in absence of increased Ca2+. Accordingly, the p38 MAPK inhibitor SB203580 blocked cell stress-induced 5-LO activity more efficiently, as compared to 5-LO activity induced by A23187 or thapsigargin. Phosphorylation may have effects on 5-LO subcellular localization in the cell, for example by unmasking an NLS, or by modifying interactions with other proteins. Of the several protein interaction partners identified for 5-LO, three (growth factor bound receptor protein 2 (Grb2), actin, and coactosin-like protein [91, 92]) relate to the actin cytoskeleton. However, precise functions of these interactions remain unclear. Other mechanisms for how phosphorylation could affect 5-LO activity can be envisioned. For cPLA2 it was concluded that the C2 domain, together with another region of the protein (subject to phosphorylation), both contributed to membrane binding and thus activity [93, 94]. It is possible that similar mechanisms are operative for 5-LO. S100A8/A9 protein complexes are abundant in monocytes and neutrophils, in which 5-LO products (LTB4 and LTC4) are the major eicosanoids produced. As mentioned above, the nuclear envelope is regarded as a metabolon for LT production; here Ara is released by the action of cPLA2 and further metabolized by 5-LO. For both cPLA2 and 5-LO, increased intracellular Ca2+ leads to association with the nuclear membrane and enzyme activity. At the same time, Ca2+ activates S100A8/A9 in the cytosol to bind Ara. Thus, in addition to the Ara reservoir suggested above another possible task for S100A8/A9 could be to bind spuriously liberated Ara, to prevent stray LT production by 5-LO in the cytosol. Cells containing cytosolic 5-LO vigorously produce LTs from exogenous Ara in the absence of translocation to the nuclear envelope [86]. A mechanism to prevent formation of 5-LO products at the wrong place in the wrong time would appear to be useful for the cell. 16.3.2

Cyclooxygenases (COX-1 and COX-2)

The other eicosanoid-generating enzyme represents the prostaglandin endoperoxide synthase, also referred to as cyclooxygenase (COX) (for review see Ref. [95]). COX catalyzes a cyclooxygenase (bis-oxygenase) reaction in which Ara is converted to prostaglandin G2 (PGG2) and a peroxidase reaction in which PGG2 undergoes

16.3 Putative Intracellular Functions of S100A8/A9

a two-electron reduction to prostaglandin H2 (PGH2), the common precursor to all prostanoids, including PGD2, PGE2, PGF2a, prostacyclin (PGI2), and thromboxane. Two different COX enzymes have been identified in cells. The first one, COX-1, is constitutively expressed by most cells types, whereas the other one, COX-2, is generally induced after cell activation. In most tissues the COX-1 isoenzyme appears to support the levels of prostanoid biosynthesis for maintaining organ and tissue homeostasis. COX-2 is thought to be involved predominantly in the inflammatory response. Both COX-1 and COX-2 are approximately 60% identical in sequence and are highly homologous in both active site regions. Both isoforms bind arachidonic acid prior to its metabolization [96]. The existence of three distinct pathways for prostaglandin production has been shown by E. Dennis and colleagues (for review see Ref. [97]). The first one, referred to as the “primed immediate phase”, takes place in minutes and is elicited by the Ca2+-mobilizing agonist platelet activating factor (PAF) but requires the cells to be exposed first to lipopolysaccharide (LPS) for 1 h. The second pathway or “delayed phase” is elicited by LPS for periods of time spanning several hours and takes place in the absence of Ca2+ elevations. In both of these routes the cytosolic phospholipase (cPLA2) appears to behave primarily as an initiator of the response, whereas the secretory PLA2 (sPLA2) plays an augmentative role by providing most of the Ara to be converted to prostaglandins via COX-2. The third pathway or “immediate response” is elicited by zymosan for longer times, takes place in the absence of LPS priming, and utilizes only the cPLA2 to affect the Ara release. In zymosan-stimulated cells cPLA2 couples directly to COX-2 for prostaglandin production, whereas in the other two pathways the bulk of the prostaglandins are produced by a sPLA2/COX-2 coupling mechanism. Interestingly, the model of the “primed immediate phase” requires a yet unknown cytosolic factor that mediates the activating effect of cPLA2-derived Ara on the sPLA2 [98]. PAF activation of the LPS-primed macrophages triggers the “fast” activation of the cPLA2 intracellularly. The elevated levels of intracellular Ara specifically lead to activation of sPLA2 at the outer leaflet of the plasma membrane. The Ara liberated by the sPLA2 can be taken up and utilized by COX-2 to generate prostaglandins such as PGE2. Whether the Ara–S100A8/A9 complex represents the accessory system remains to be elucidated. 16.3.3

NADPH Oxidase Complex

The O–2 generating NADPH oxidase is a multicomponent enzyme abundant in phagocytic cells, especially in neutrophils. It consists of a membrane-bound heterodimeric flavocytochrome b and cytosolic proteins, p47phox (phox for phagocyte oxidase), p67phox, p40phox, and a small G-protein, either Rac1 or Rac2. The enzyme is dormant in resting cells. Activation of the oxidase correlates with the migration of the cytosolic factors to the membrane and their binding to flavocytochrome b. It has been shown that arachidonic acid potentiates NADPH oxidase activation [5, 6, 14].

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Fig. 16.4 Schematic presentation of the intracellular Ara metabolism within human neutrophils. Arachidonic acids (AA) are taken up by fatty acid transporters (FAT). Acyl-CoA synthetases promote efficient and unidirectional FA transfer from the bilayer to metabolic sites, thereby preventing back flux. At normal physiological conditions arachidonic acids are predominantly incorporated into phospholipids (PL) by lysophospholipid:acyl-CoA acyltransferases (LAT). This pathway has high affinity towards unsaturated fatty acids but low capacity. When cells are incubated with high concentrations of unsaturated fatty acids their incorporation into triglycerides (TAG) is achieved that is catalyzed by the acylCoA:diacylglyceride acyltransferase (ADGAT). This accumulation within TAG appears to be

a mechanism by which cells can store the unsaturated fatty acid moiety. This pathway has low affinity but high capacity. At a later time point arachidonic acids are released from the TAG and incorportated into PL. Arachidonic acid-containing phospholipids are the substrates of the CoA-independent transacylases (CoA-IT) which catalyze the synthesis of ether-linked phospholipids. The mobilization of esterified arachidonic acids from PL by phospholipase A2 (PLA2) and their subsequent metabolism into eicosanoids by either lipoxygenases (LO) or cyclooxygenases (COX) is the key regulatory event in most inflammatory cells. The liberated arachidonic acids also play a role as a co-factor in the activation of NADPH oxidase.

Recently, Doussiere et al. [99] have shown that S100A8/A9 enhances the activation of NADPH oxidase in a cell-free system. Due to its high concentration in the cytosol of phagocytic cells (up to 45%) and selective arachidonic acid-binding capacity, the authors speculated that the S100A8/A9 protein complex might be used to store significant amounts of arachidonic acid, which could be implied at the onset of activation. In addition of being a storage protein for arachidonic acid, the S100A8/A9 complex might serve as a scaffold protein for the cytosolic factors of oxidase activation and help them to migrate to the membrane-bound flavocytochrome b in activated neutrophils. In this manner, the S100A8/A9 complex (p7/ p23 complex of bovine neutrophils) could be considered as a novel component of the NADPH oxidase activation complex in neutrophils. Figure 16.4 shows a schematic presentation of the intracellular Ara metabolism within human neutrophils.

16.4 Extracellular Role of the S100A8/A9–Arachidonic Acid Complex

16.4

Extracellular Role of the S100A8/A9–Arachidonic Acid Complex 16.4.1

Transcellular Arachidonic Acid Metabolism

Experiments with physiological stimuli, such as the chemoattractants fMLP and C5a [100], indicate that S100A8/A9 is secreted from inflammatory cells targeted to sites of inflammation by a range of environmental cues. The presence of S100A8/ A9 complexes at sites of acute and chronic inflammation has long been noted. For example, the assessment of serum levels of S100A8/A9 has been suggested to track disease activity in patients suffering from inflammatory disorders, such as chronic bronchitis, cystic fibrosis, and rheumatoid arthritis [41–43]. Despite the fact that S100A8 and S100A9 lack signal sequences, there is definitive evidence that these proteins are specifically secreted into the extracellular space [37]. Here, the S100A8/A9–Ara complex may have an extracellular role, probably as a vehicle to move arachidonic acid to bystander cells. Then Ara is taken up by bystander cells to be metabolized to eicosanoids, representing a particular transcellular pathway for eicosanoid metabolism. Interestingly, the physiological stimuli that induce both S100A8/A9 secretion and Ara release also promote release of oxygen species formed via the NADPH oxidase complex [101]. The respiratory burst produces superoxide anion (up to 40 lmol per 106 cells) corresponding to a maximal blood concentration of ~200 lM, which dismutates to hydrogen peroxide, and is converted by myeloperoxidase to the powerful two-electron (non-radical) oxidant hypochlorous acid (HOCl) that can modify proteins and lipids and kill invading pathogens [102, 103]. Although S100A8 is effectively oxidized in vitro by low amounts of reagent HOCl [104], the oxidation did not affect the arachidonic acid binding capacity of the S100A8/A9 protein complex [30]. Thus, binding of arachidonic acid to the complex may prevent the oxidation of the fatty acid. The arachidonic acid transport by S100A8/A9 might be limited by the concentration gradient of zinc between the bloodstream and the inflammatory lesion. The Ara binding capacity of the S100A8/A9 protein complex, which has been induced by the binding of calcium, is reversed by the binding of zinc. The concentration of zinc necessary to prevent Ara binding to S100A8/A9 is within the physiological range [53]. This finding may represent a mechanism by which the extracellular function is restricted to local environments. It is worthwhile mentioning that different structural changes are induced by either calcium or zinc, allowing interactions with different target proteins as well as different cell surface binding sites. Further investigations have to identify target proteins binding to S100 proteins in a Ca2+-independent but Zn2+- and Cu2+-dependent manner. In addition, they have to pinpoint the binding sites specific for S100A8 and S100A9 under the consideration of the ion bound to the protein complex. Adhesion of neutrophils to vascular endothelial cells is a characteristic feature of inflammation involving numerous adhesion proteins that regulate their trans-

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migration across the vascular endothelial barrier. The microenvironment at the interface between adherent neutrophils and endothelial cells represents a strategic site for exchange, transcellular synthesis, and metabolism of lipid mediators (for review see Refs [1–4]). Transcellular metabolism of eicosanoid intermediates produced by one cell type in close contact with another may amplify the formation of a given compound or result in products that neither of the participating cell types can generate alone (for review see Refs [105, 106]). For example, arachidonate derived from one cell is oxidized to leukotriene A4 (LTA4) by another and this can then be exported for conversion to LTB4 or cysteinyl leukotrienes (cys-LTs) by yet another. Neutrophils do not contain LTC4 synthase but are known to cooperate with endothelial cells or platelets (which do have this enzyme) to generate cys-LTs [107, 108]. In addition, several reports indicate that endothelial cells and neutrophils utilize both endogenous and exogenous arachidonic acid for eicosanoid biosynthesis [109, 110]. Thus, the liberation of arachidonate from cellular membranes as well as uptake of exogenous arachidonate increases the level of this otherwise rate-limiting substrate. At present, little is known about the membrane proteins involved in arachidonate uptake. The expression of one putative fatty acid transporter, the plasma membrane-bound fatty acid binding protein (FABPpm), has been demonstrated on the surface of endothelial cells [111], and the participation of a yet unknown membrane protein in the free fatty acid (FFA) uptake by neutrophils was found by Krischer et al. [112]. 16.4.2

Celluar Uptake of Long-chain Fatty Acids (LCFAs)

The precise mechanism of long-chain fatty acid (LCFA) uptake is not well understood. The concept of LCFA uptake consists of two components: a high-affinity, low-capacity protein-mediated transport process and a low-affinity, high-capacity diffusional event. Although fatty acids are hydrophobic and capable of transferring passively across the membrane lipid bilayer when present in high concentrations, a large body of evidence supports the presence of a protein-facilitated carrier system that operates at low substrate concentrations. The LCFA uptake has been shown to exhibit saturability, specificity [112–115], and sensitivity towards protein modifiers [116–118]. Because extracellular fatty acids are in equilibrium with albumin, passive as well as carrier-mediated LCFA uptake may co-exist and contribute differentially to net uptake depending on LCFA supply and cell types. However, at normal physiological conditions the majority of LCFA is supposed to be taken up by the protein-facilitated system. There are five candidate proteins which may mediate the LCFA uptake in mammalian cells: the plasma membrane-bound fatty acid binding protein (FABPpm) [119, 120], the 56-kDa renal FABP [121], the 22-kDa plasma membrane protein caveolin [115], the fatty acid transport protein (FATP) [122], and the fatty acid translocase (FAT)/CD36 [123, 124]. These putative fatty acid transporters are expressed in a wide variety of tissues, except the 56-kDa renal FABP (for review see Ref.

16.4 Extracellular Role of the S100A8/A9–Arachidonic Acid Complex

[125]). In general, the expression of the putative fatty acid transporters correlates strongly with tissue active in fatty acid metabolism. They are expressed to the greatest extent in tissues exhibiting high levels of plasma membrane fatty acid flux such as brain, skeletal muscle, heart, adipose, and liver, whereas expression levels in other tissue types is considerably lower. Other common features of these proteins are their upregulation during adipocyte differentiation and their transcription control by a new class of transcription factors, the peroxisome proliferator activated receptors [126, 127]. Furthermore, the protein candidates are altered with metabolic conditions relevant to FA metabolism. For many cell types it is not clear what the physiological significance of multiple transporters might imply. However, information related to the contribution of each protein to FA turnover in vivo is just beginning to emerge. 16.4.3

Participation of S100A8/A9 in the Arachidonic Acid Uptake

Recently, we demonstrated that (i) the uptake of exogenous arachidonic acid by human umbilical vein endothelial cells (HUVECs) is predominately mediated by the fatty acid translocase, FAT/CD36, and (ii) the S100A8/A9–arachidonic acid complexes directly interact with FAT/CD36, thereby accelerating the dissociation of the complex, which in turn would facilitate the uptake of fatty acids by the cells [128]. The Ara uptake by HUVECs in the presence of S100A8/A9 protein complexes exhibited many characteristic properties of a protein-mediated transport process: (i) the fatty acid uptake was shown to be temperature- and energy-dependent; (ii) the kinetic studies revealed evidence that the FA uptake was dependent on an equilibrium between the free fatty acid concentration and the fatty acidbinding proteins; (iii) the Ara uptake was inhibited by protein modifiers indicating that membrane proteins are involved in arachidonate transport. The participation of FAT/CD36 in Ara transport by HUVECs is well documented by several findings: HUVECs express the mRNA of FAT/CD36, and the FAT/CD36-specific inhibitor sulfo-N-succinimidyl oleate (SSO) abrogated the initial uptake of arachidonate. FAT/CD36 is localized in raft-like membrane domains. These microdomains are involved in numerous cellular functions including endocytosis, for which the formation of coated pits and clathrin-coated vesicles is not essential. Potassium depletion inhibits the formation of coated pits and clathrin-coated vesicles but does not affect the uptake of exogenous arachidonate by HUVECs. It is likely from several studies that the cytoskeleton is involved in different steps of both the receptor-mediated endocytosis and the non-clathrin-coated pitsmediated endocytosis [129–132]. For example, the microtubules are involved into endocytosis by promoting the recycling of membrane proteins. Microtubule disruption by colchicine or nocadazole perturbs the delivery of recycling proteins to the cell surface and the internalized proteins can no longer be effectively recycled back to the cell surface in the absence of polymerized microtubules. Actin filaments also play a role in the intracellular movement of vesicles and organelles via accessing proteins such as myosin ATPase. It has been proposed that whereas mi-

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crotubules are responsible for “long-range” transport processes in the cell, actin filaments are involved in the final steps of access of vesicles to the inner leaflet of the plasma membrane. However, in our studies both agents displaying antimicrotubular activity, such as colchicine, democolcine, or nocadazole, as well as agents perturbating the actin filament, such as cytochalasin D, did not affect the S100A8/A9-facilitated Ara uptake into HUVECs. Thus, our study clearly indicates that the influx of arachidonate is not dependent on coated pits and clathrin-coated vesicles and is consistent with the localization envisaged for the CD36-related receptor proteins. This conclusion agrees with a hypothetical model for FA transport proposed by Abumrad and colleagues [125]. They presented a model in which CD36, one of the fatty acid transporter candidates, facilitates Ara transport by the formation of a multimeric pore or channel. Interactions between CD36 and intracellular FA binding proteins or key metabolite enzymes, such as FA-acyl-CoA synthetase, could be envisioned to promote efficient and unidirectional FA transfer from the bilayer to metabolic sites, thereby preventing back flux. The model is sustained by Thorne et al. [133], who reported that FAT/CD36 forms covalently associated dimers and multimers. The expression of another putative fatty acid transporter, FABPpm, was previously demonstrated at the surface of endothelial cells [111]. The FABPpm-facilitated oleate uptake by hepatocytes was shown to be sodium-dependent. The uptake was diminished when Li+, K+, choline, or sucrose were substituted for Na+ in the incubation medium, and was reduced by 46% by ouabain, an inhibitor of Na+/K+-ATPase [119]. In addition, the Na+-dependent FA uptake was found to be stimulated by valinomycin [134]. However, in our studies the pre-incubation with ouabain was without significant effect on the initial arachidonic acid uptake. Therefore, these data give additional evidence that FABPpm did not represent the membrane protein facilitating Ara uptake by HUVECs. On the other hand, the uptake of arachidonate in the presence of S100A8/A9 complexes had some properties that differed from the reported mechanism of fatty acid transport. Abumrad and colleagues demonstrated that free fatty acid but not the BSA-bound form is the ligand for CD36. However, our kinetic studies revealed evidence for an interaction between FAT/CD36 and S100A8/A9 because although the calculated Vmax values were in the similar range the Km values were quite different, indicative of competitive inhibition. In addition, the fatty acid transport was significantly higher in the presence of S100A8/A9 than in the presence of BSA. The protein–protein interaction assay revealed evidence that S100A8/A9 directly interacts with CD36. Therefore, we suggest that the interaction of the S100A8/A9–Ara complex with CD36 may accelerate the dissociation of the complex, which in turn would facilitate the uptake of fatty acids by the cells. The uptake experiments using radiolabeled S100A8/A9 exclude the possibility that S100A8/A9 itself is taken up by the cells. Thus, the fatty acid must be transferred from the complex to CD36 prior to its transport. However, we cannot for certain exclude the alternative that S100A8/A9–Ara forms an equilibrium and that the free Ara is then transported. Figure 16.5 summarizes these results.

16.4 Extracellular Role of the S100A8/A9–Arachidonic Acid Complex

Fig. 16.5 Schematic presentation of Ara (AA) uptake by HUVECs mediated by S100A8/A9– Ara complexes. Under normal physiological conditions the majority of long-chain fatty acids (LCFAs) are taken up by a protein-facilitated system. The LCFA uptake exhibits saturability, specificity, and sensitivity towards protein modifiers. The free fatty acid but not the

protein-bound form is the ligand for the protein-mediated transport process. However, there is evidence that S100A8/A9–Ara complexes directly interact with CD36, thereby accelerating the dissociation of the complex, which in turn would facilitate the uptake of fatty acids by the cells [30]. SSO, sulfo-N-succinimidyl oleate.

CD36 is a 78–88-kDa cell surface glycoprotein present on monocyte/macrophages, platelets, adipose tissue, and microvascular endothelial cells. One of the striking features of CD36 is the wide and overlapping ligand binding specificity. CD36 binds a variety of ligands, including thrombospondin [135], collagen [136], Plasmodium falciparum-infected erythrocytes [137, 138], oxidized low-density lipoprotein (oxLDL) [139], anionic phospholipids, apoptotic cells, and fatty acids [124, 140]. The functional significance of many of these receptor–ligand interactions is unknown. Studies with isolated and cultured cells have provided evidence that FAT/CD36 functions as a putative transporter of long-chain fatty acids. The participation of FAT/CD36 in FA uptake and metabolism in vivo has recently been shown by Coburn et al. [141]. They presented data that the FA transport is reduced in heart, skeletal muscle, and adipose tissues of CD36 knockout mice. Strong evidence for CD36 as fatty acid transporter was also established by transfection experiments with Cos-7 cells using the pEF.BOS–CD36 expression vector [128]. The calculated kinetic constants were in accordance with the observation that CD36 protein expression was very low in Cos-7 cells, whereas the cells transfected with pEF.BOS–CD36 vector expressed higher levels of CD36. Endothelial cells actively regulate their environment by secreting humoral substances, including endothelin-1 and a variety of eicosanoids that have local actions. Under inflammatory conditions endothelial cells respond to potential harmful conditions with the appropriate, adaptive changes in function and mediator release. A considerable body of evidence supports altered eicosanoid synthesis in profile and quantity upon cell activation. Endothelial cell-derived eicosanoids have been shown to regulate smooth muscle contractility, thrombocyte aggregation, and to modulate the adhesion of monocytes and neutrophils to endothelium. The hall-

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mark of S100A8/A9 complexes is their accumulation at inflammatory loci as a consequence of specific secretion from activated leukocytes [37, 42, 43, 100]. These S100A8/A9 complexes have been shown to carry arachidonic acid [30] and are recognized by FAT/CD36 on endothelial cells as shown in the present study. Furthermore, endothelial cells utilize both endogenous and exogenous Ara for transcellular production of eicosanoids [109]. Together with the identification of FAT/CD36 as a major FA transporter on HUVECs, our results give strong evidence that the secreted S100A8/A9–Ara complex serves as a transport protein to move Ara to its target cells. Ara is then taken up by bystander cells to be metabolized to eicosanoids representing a particular transcellular pathway for Ara metabolism. This mechanism points to an important role in the initiation and regulation of the inflammatory response.

16.5

Conclusion and Future Perspectives

Polymorphonuclear neutrophils perform as part of the immune response several host-defense functions, such as phagocytosis of invading microorganisms and cell debris, release of proteolytic enzymes, generation of reactive oxygen metabolites, and release of a number of arachidonic acid-derived eicosanoids. Here, arachidonic acid-binding proteins may play an important role in the regulation of the appropriate, adaptive change in function and mediator release as response to potential harmful conditions in the extracellular environment. Of the arachidonic acidmetabolizing enzymes lipoxygenase might play the more profound role in eicosanoid generation since the primary arachidonic acid metabolite generated in human neutrophils is leukotriene B4 (LTB4), a powerful chemoattractant for neutrophils acting at less than nanomolar concentrations. LTB4 also promotes adherence of neutrophils to the endothelium, induces neutrophil aggregation and upregulates the expression of Mac-1. The conversion of arachidonic acid into prostaglandins does not represent a major route of arachidonic acid metabolism in neutrophils, however, the cells may produce substantial quantities of prostaglandin E2 (PGE2) and thromboxane B2 (TXB2) under some circumstances. The S100A8/A9 protein complex, representing the exclusive arachidonic acidbinding capacity in the human neutrophil cytosol, is suggested to have a dual function: (i) The S100A8/A9–Ara complex may function as an intermediate reservoir for arachidonate. At a later time point, the complex might pass the ligand either to Ara-metabolizing enzymes, thereby initiating a delayed eicosanoid formation, or to Ara-dependent enzymes which are then promoted by their co-factor supply. Whether the S100A8/A9–Ara complex might interact with either lipoxygenase or cyclooxygenase has to be investigated. (ii) S100A8/A9–Ara complexes are specifically secreted from activated human neutrophils and they are recognized by FAT/CD36 on endothelial cells. Together with the identification of FAT/ CD36 as the major FA transporter on HUVECs, it has been speculated that the secreted S100A8/A9 complex may serve as a transport protein to move Ara to its

16.6 References

target cells. Arachidonic acid is then taken up by bystander cells to be metabolized to eicosanoids representing a particular transcellular pathway for Ara metabolism. FAT/CD36 has been suggested to be involved into immune/inflammatory responses. Therefore, it can be envisioned that there is a close relationship between FAT/CD36 receptor and the ligand S100A8/A9 released from activated leukocytes at inflammatory loci. Their interaction might propagate host response by perpetuating recruitment and activation of cellular effectors. The identification of FAT/CD36 as a receptor of S100A8/A9–Ara complexes may be of general interest. Besides their expression in myeloid cells, the other tissue type found to express S100A8 and S100A9 is mucosal epithelium and involved epidermis in conditions such as psoriasis and malignant disorders. It is established that the epidermis requires essential fatty acids and arachidonic acid in order to synthesize cellular membranes and the extracellular lipid lamellar membranes in the stratum corneum. It has been shown that mRNA for FABPpm, FATP, and CD36/FAT were present in the epidermis, and disruption of the barrier resulted in an increase in CD36 mRNA, whereas the mRNA levels of the other fatty acid transporters were not altered [142]. Whether the purpose in FAT/ CD36 as well as S100A8/A9 upregulation is to enhance fatty acid transport remains to be elucidated.

16.6

References 1 2 3 4

5

6 7

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PPARs, Cell Differentiation, and Glucose Homeostasis Stephen R. Farmer

17.1

Introduction

The peroxisome proliferator activated receptors (PPARs) are lipid binding proteins that belong to the nuclear hormone receptor (NHR) superfamily of transcription factors; members of the PPAR family include PPARa, PPARd (also referred to as PPARb, NUC-1, and FAAR), and PPARc. As is the case with most NHRs, PPARs require association with a ligand to induce their transcriptional activity. The large group of PPAR ligands includes a variety of fatty acids and their derivatives, as well as several synthetic compounds that have been developed as pharmacological agents to target specific PPARs. A principal function of the PPARs is to act as regulators of lipid homeostasis by sensing changes in lipid metabolism through direct interaction with various fatty acids or their derivatives, and thereby to alter the expression of genes that control metabolic processes. PPARs have also been shown to participate in events that regulate the growth and differentiation of many cell types during the development of various tissues. This is particularly true of PPARc, which has been shown to regulate pre-adipocyte differentiation during the development of both white and brown adipose tissue. This chapter will review the regulatory role of the PPARs during the transcriptional control of genes associated with the differentiation and function of adipocytes, with a discussion of PPARs as targets of therapeutic agents that combat disorders associated with glucose homeostasis.

17.2

Regulation of PPAR Activity

The transcriptional activity of PPARs depends on several structural features contained within the individual molecules [1]. The DNA binding domain (DBD) is the most conserved domain among all members of the nuclear hormone receptor superfamily that facilitates docking of the PPARs to specific DNA sequences referred to as PPAR response elements (PPREs) in the promoters and enhancers of target genes. PPREs consist of two copies of a core hexanucleotide motif AGGT-

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CA arranged as direct repeats and separated by a either a single nucleotide or two nucleotides, giving rise to direct repeat elements DR-1 or DR-2, respectively [1, 2]. PPARs bind as heterodimers to their respective PPRE in which the heterodimeric partner is a member of the retinoid X receptor family (RXRs) [3]. Since all three PPARs (a, b, and c) can associate with RXR to bind to closely related core regulatory elements, the mechanism by which each PPAR selects a specific PPRE in a particular target gene is of question. It appears that 5'-flanking sequences of a PPRE, at least in part, contribute to this selective binding process; however, additional mechanisms likely exist. The PPAR/RXR heterodimer can associate with a variety of co-activators and co-repressors through direct protein–protein interactions involving specific transactivation domains, thereby imparting a more complex level of specificity. The ability of these domains to associate with co-activators and promote transcription is dependent upon the binding of an appropriate ligand to the ligand binding domain within the PPAR molecule. Like the PPARs, RXR also associates with its ligand, 9-cis-retinoic acid, and in so doing contributes to the transcriptional activity of the PPAR/RXR heterodimer. Several long-chain polyunsaturated fatty acids (PUFAs) and their derivatives have the ability to bind PPARs with varying affinities, and in some cases can act as functional ligands to enhance transcriptional activity [1, 4, 5]. This is particularly true of the eicosinoids, which are a class of lipids derived from PUFAs by the enzymatic action of lipoxygenases or cyclooxygenases that gives rise to leukotrienes, hydroxyeicosatetraenoic acids (HETEs), hydroxyoctadenoic acids (HODEs), or prostaglandins. In fact, some of these eicosinoids are high-affinity ligands for specific PPARs; for instance, 15-deoxy-d12,14-prostaglandin J2 and 13S-HODE are PPARc ligands [6–8], whereas 8S-HETE preferentially activates PPARa [1, 4, 9]. Furthermore, there is a growing list of synthetic compounds currently being developed as therapeutic agents to activate PPARs in order to effect specific physiologic processes. These drugs include hypolipidemic agents such as gemfibrozil and fenofibrate that are potent PPARa ligands and members of the thiazolidinedione family of insulin sensitizers that activate PPARc, which include troglitazone, pioglitazone, and rosiglitazone [10]. Therefore, specific PPAR ligands are being used as important tools to identify PPARc and PPARa target genes, and to determine how activation of these genes effects various biologic processes both in vitro and in the animal. For example, activation of PPARc with the thiazolidinediones induces many genes associated with the mature adipocyte phenotype and function, while activation of PPARa induces genes coding for proteins involved in b-oxidation. A similar analysis of PPARd target genes has lagged behind that of the other PPARs because a comparable potent PPARd ligand has only recently become available to the research community.

17.3 PPARs and Differentiation

17.3

PPARs and Differentiation 17.3.1

PPARc

PPARc exists as two isoforms, c1 and c2, generated by alternative splicing of at least three different transcripts from the same gene [11, 12]. Both isoforms share almost all the same exon sequences, except c2 contains an additional 30 amino acids at the N-terminus [11, 13]. The mouse and human PPARc genes consist of over 100 kb and contain at least nine exons [11, 14]. Coding exons 1–6 are conserved between c1 and c2 and transcription of these is driven by two upstream promoters, P1 and P2. Transcription from the P1 promoter generates a large nuclear precursor mRNA that contains two additional untranslated c1-specific exons, A1 and A2, that are spliced onto the 5' end of exons 1–6. The c2-specific exon, B1, is transcribed from the P2 promoter and is also spliced onto the 5' end of exons 1–6 to generate an mRNA transcript in which both exon B1 and exons 1–6 are translated, giving rise to the longer c2 isoform. The P1 promoter is at least 63 kb upstream of the P2 promoter and, consequently, it is very likely that each promoter contains specific regulatory DNA elements that direct transcription in a tissue-specific and differentiation-dependent manner. In fact, transcription from the PPARc gene has been detected in many different tissues in which the c1 isoform is the predominant transcript [15]. In contrast, transcription from the P2 promoter is adipose tissue-selective, giving rise to abundant expression of the PPARc2 isoform [13]. This observation raises the question of whether there is a unique role for PPARc2 that is not shared by PPARc1. In fact, a recent report suggests that PPARc2 alone is the master regulator of adipogenesis [16]. As mentioned above, PPARc, predominantly the c1 isoform, is expressed in many different cell types including adipocytes, hepatocytes, macrophages, and lining epithelial cells of the colon, bladder, prostate, and breast. It is generally considered that the role of PPARc in these unrelated tissues is to regulate energy metabolism; however, recent in vitro studies suggest that it may also participate in fundamental processes that regulate growth and differentiation. This is most elegantly illustrated from studies that have identified PPARc as a “master regulator” of adipogenesis. A role for PPARc during development has been difficult to assess since deficiency of the PPARc gene results in embryonic lethality due to a defect in the placenta [17, 18]. These observations suggest that PPARc is important for the development and/or function of placental tissue. In fact, it appears from one set of studies that PPARc is required for epithelial differentiation of trophoblastic tissue, which is critical for proper placental vascularization. These defects in the placenta appear to lead to myocardial thinning and embryonic death at E10.0 [17]. Adopting alternative methods of generating PPARc-null mice that circumvent the placental defects allowed investigators to define a role for PPARc in adipose tissue development. One approach involved supplementing PPARc-null embryos with wild-type placentas via aggregation with tetraploid embryos. Tetraploid-rescued

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mutant surviving to term exhibited lipodystophy (reduced fat mass), fatty liver and multiple hemorrhages [17]. Another approach was to generate chimeric mice from wild-type and PPARc–/– embryonic stem (ES) cells. The adipose tissues in these mice were composed of adipocytes derived exclusively from the wild-type ES cells [19]. Heterogeneous PPARc-deficient mouse embryos survived to birth, and developed into adult mice with reduced fat mass due to the presence of smaller adipocytes [18]. The requirement of PPARc for adipose tissue development is further supported by the reduced ability of PPARc-deficient embryonic stem cells or mouse embryo fibroblasts to differentiate into adipocytes in vitro under potent adipogenic conditions [18, 19]. Collectively, the results from these loss-of-function studies provide strong evidence supporting an obligatory role for PPARc in adipocyte differentiation, both in vitro and in vivo. 17.3.2

PPARc and Adipogenesis

The differentiation of pre-adipocytes into adipocytes is regulated by a network of transcription factors that interact to orchestrate the expression of many hundreds of proteins [20, 21]. At the center of this network is PPARc (Fig. 17.1), which is considered to function as a “master regulator” of adipogenesis. In fact, ectopic expression of PPARc in non-adipogenic fibroblasts under appropriate hormonal conditions induces their conversion into adipocytes [22]. This event involves the PPARc-associated transcription of a series of target genes that encode proteins in-

Fig. 17.1 PPARc plays a central role in the cascade of transcriptional events mediating pre-adipocyte differentiation. Exposure of preadipocytes to adipogenic hormones (glucocorticoids, insulin, and inducers of intracellular cAMP) induce C/EBPb, C/EBPd, and SREBP-1c (ADD1) expression. These

transcription factors activate both PPARc and C/EBPa, which induce each other’s expression as well as the many genes involved in the development of fat cells. PPARa is required for differentiation, while C/EBPa plays a critical role in converting immature adipocytes into mature insulin-responsive adipocytes.

17.3 PPARs and Differentiation

volved in several different biological processes. As expected, these include many enzymes and other proteins that regulate lipid and glucose metabolism in the mature fat cell such as the adipose-specific fatty acid binding protein (aP2), fatty acid transport protein (FATP), phosphoenolpyruvate kinase (PEPCK) and the insulindependent glucose transporter (GLUT4) [13, 23–25]. Not only does the adipocyte function as a storage depot for energy-rich lipids, but it is also an endocrine cell, secreting proteins that act at distance sites [26, 27]. One of the most prominent hormones secreted by mature adipocytes is leptin [28], whose expression is also regulated by PPARc [29, 30]. In addition to its role as a regulator of adipocyte differentiation, studies have demonstrated a role for PPARc in suppressing the growth of pre-adipocytes during their differentiation into mature, growth-arrested adipocytes [31, 32]. The expression of PPARc during adipogenesis is primarily regulated at the level of transcription, but there are recent reports of post-transcriptional mechanisms that can influence the abundance of PPARc in the mature fat cell [33, 34]. Prior to differentiation, trace amounts of PPARc1 protein are observed in pre-adipocytes. Exposure of confluent pre-adipocytes to the adipogenic inducers results in an extensive induction of both PPARc1 and PPARc2 expression within hours, which is preceded by a regulated expression of two members of the CCAAT/enhancer binding protein (C/EBP) family of basic leucine zipper transcription factors: C/EBPb and C/EBPd. In fact, early expression of these C/EBPs appears to play a direct role in activating PPARc expression during adipogenesis, since their ectopic expression in non-adipogenic fibroblasts induces transcription of the PPARc2 gene through C/EBP regulatory elements within its promoter [35–37]. Factors other than the C/EBPs have also been proposed to stimulate PPARc expression and facilitate terminal differentiation. Support for this notion stems from observations in mice deficient in both C/EBPb and C/EBPd that still express PPARc and are capable of developing adipose tissue, albeit to a significantly lower extent when compared with control litter mates [38]. One of these factors is the adipocyte determination and differentiation factor 1 (ADD1) [39], which is a member of the basic helix-loop-helix family of sterol regulatory element binding proteins (SREBPs) [40]. SREBPs are known to regulate cholesterol metabolism in the liver and other tissues [41], but it appears that ADD-1 (SREBP1c) also contributes to the transcriptional induction of the PPARc gene by binding SREBP response elements in its promoter [42]. Yet other factors acting early during adipogenesis may include glucocorticoid receptors based on our studies which demonstrate that glucocorticoids can induce both PPARc1 and PPARc2 gene expression in the presence or absence of the C/EBPs [36, 43]. These positive mechanisms are certainly not the only means by which PPARc gene expression is activated during adipogenesis. Recent investigations demonstrate a role for the suppression of inhibitory factors that maintain pre-adipocytes in an undifferentiated state. Most notable among these negative factors are members of the GATA and TCF/LEF families of transcription factors. In the former case, studies demonstrate that constitutive expression of GATA 2 or GATA 3 in pre-adipocytes blocks adipogenesis in part by inhibiting PPARc expression

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through interaction of these proteins with GATA response elements in the PPARc2 gene [44]. The anti-adipogenic activity of TCF/LEF factors, on the other hand, is accomplished by inducing the secretion of Wnt proteins (i.e. Wnt 10b) in pre-adipocytes that stimulate the Wnt signaling pathway [45]. In fact, ectopic expression of Wnt in pre-adipocytes blocks adipogenesis by inhibiting PPARc gene expression, the precise mechanism of which is not known. Once expressed, PPARc is considered to play a prominent role in regulating gene expression of proteins necessary for the development of mature adipocytes. As mentioned previously, an additional role for PPARc is to suppress pre-adipocyte growth during adipogenesis. In fact, the early phase of adipogenesis involves a burst of proliferation in response to exposure of confluent pre-adipocytes to hormonal inducers. Concurrent with this event, coined the “clonal expansion phase”, mechanisms responsible for inducing PPARc expression are occurring, resulting in abundant production of both PPARc1 and PPARc2 [32]. Studies suggest that activation of these PPARs during clonal expansion serve to arrest pre-adipocyte growth by inducing at least three different growth-suppression processes. Specifically, Spiegelman and collaborators demonstrated that activation of PPARc2 in proliferating fibroblasts and pre-adipocytes leads to decreased binding of E2F/DP heterodimers to their target genes, possibly through the PPARc-associated downregulation of the protein phosphatase PP2A, thereby preventing the cells from entering S phase [31]. Other studies have shown that exposing cells to PPARc ligands can inhibit cyclin D1 expression and retinoblastoma protein (RB) phosphorylation, both events that would also result in decreased E2F activity [46, 47]. We have demonstrated that PPARc induces the expression of the tumor suppressors p18INK4 and p21CIP1, which inhibit the cyclin-dependent kinases, also resulting in an attenuation of E2F activity [32]. To date only a limited number of genes expressed during the terminal stages of adipogenesis have been shown to contain PPREs. It is likely, therefore, that PPARc induces the expression of many adipogenic genes through the activation of intermediary trans-acting factors. In this regard, our recent investigations suggest that PPARc can directly trans-activate the C/EBPa gene and may induce C/ EBPa expression during adipogenesis [43]. In fact, PPARc and C/EBPa can crossregulate each other in order to maintain their high level of expression required for orchestrating the pleiotropic functions of the adipocyte [48]. Additional intermediary factors may include the STAT family of transcription factors, specifically STAT1, STAT5a, and STAT5b, whose differentiation-dependent induction during adipogenesis is regulated by PPARc [49]. Although complete adipocyte differentiation in mice requires STAT5 [50], the precise function of these transcription factors during adipogenesis has yet to be determined. It is clear that PPARc is an indispensable component of the network of factors regulating adipogenesis, therefore, production of its ligand may contribute significantly to the differentiation of pre-adipocytes. In fact, one of the reasons that various 3T3 fibroblasts are unable to differentiate into mature adipocytes following their exposure to adipogenic inducers may result from their inability to produce PPARc ligands. For example, ectopic expression of PPARc in non-adipogenic fi-

17.3 PPARs and Differentiation

broblasts requires a potent PPARc ligand such as a thiazolidinedione to activate the adipogenic program, and induce the expression of C/EBPa [51]. If, however, both PPARc and C/EBPa are co-expressed in these fibroblasts, differentiation ensues in a ligand-independent manner suggesting that C/EBPa activates a pathway that leads to PPARc ligand production [22]. Moreover, recent studies in our laboratory suggest that C/EBPb may also participate in this process, since ectopic expression of a dominant negative isoform of C/EBPb (LIP) renders 3T3-L1 pre-adipocytes ligand-dependent for their conversion into mature adipocytes [43]. Whether or not these C/EBPs directly transactivate the genes which code for enzymes controlling ligand synthesis is not known, however, one might postulate the involvement of a possible intermediary factor, ADD-1(SREBP-1c). In fact, ectopic expression of ADD-1 in non-adipogenic fibroblasts results in the secretion of lipids that are able to activate PPARc in in vitro assays [52]. 17.3.3

PPARc and Transcriptional Control of the Pleiotropic Functions of the Adipocyte

Until very recently, the results from both in vivo and in vitro investigations positioned PPARc and C/EBPa as co-master regulators of adipogenesis. For instance, mice deficient in either transcription factor are compromised with regard to their ability to develop adipose tissue [17, 53, 54]. In addition, ectopic expression of either C/EBPa or PPARc in non-adipogenic fibroblasts can induce adipogenesis [22, 55] and enhance the expression of one by the ectopic expression of the other [48]. It is possible, therefore, that either of these factors may be capable of acting independently to promote fat cell differentiation. Since both C/EBPa and PPARc can cross-regulate each other’s expression, definitive analysis of their independent roles requires the use of both C/EBPa- and PPARc-deficient cell lines. Earlier studies had shown that mouse embryonic fibroblasts lacking C/EBPa have reduced adipogenic potential; nevertheless, ectopic expression of PPARc in these cells with exposure to a potent exogenous ligand could restore their ability to accumulate lipid and express markers of the mature adipocyte phenotype [48]. The converse experiment with PPARc–/– mouse embryo fibroblasts had not been performed until recently because of the difficulty in generating donor PPARc-deficient embryos as discussed above. This problem was circumvented by applying an elegant series of transgenic and in vitro manipulations to generate mouse embryo fibroblasts that are heterozygous null at the PPARc locus (+/–) with the single + allele containing a floxed exon 2 (loxP sites flanking exon 2 of PPARc). Adenovirus-mediated expression of Cre recombinase in these cells resulted in disruption of the loxP allele with subsequent deletion of exon 2, with a concomitant production of PPARc–/– fibroblasts. These cells were incapable of undergoing differentiation following exposure to the adipogenic inducers including a potent PPARc ligand. More importantly, ectopic expression of C/EBPa could not rescue adipogenesis in these PPARc-deficient fibroblasts [56]. This observation, together with that made in C/EBPa-deficient fibroblasts in which ectopic PPARc drives differentiation, provides evidence to suggest that there is a unified pathway of transcrip-

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tional events that leads to the development of adipocytes. Accordingly, a model based on these data identifies PPARc as the direct regulator of adipogenesis, while C/EBPa provides additional functions that are dependent on PPARc activity. Some of these functions of C/EBPa include maintaining PPARc expression through its ability to transactivate the PPARc gene and establishing the mature adipocyte phenotype. An example of this latter function is demonstrated in studies addressing the mechanisms responsible for expression of the 3-adrenergic receptor and the insulin-dependent glucose transport system. Specifically, activation of PPARc alone in C/EBPa–/– fibroblasts under potent adipogenic conditions induces the formation of lipid droplets and the expression of several genes associated with adipocyte differentiation. Nevertheless, these engineered cells do not express the b3 adrenergic receptor, nor do they respond to insulin with respect to glucose uptake [48, 51, 57]. This defect can be overcome, however, by co-expressing C/EBPa together with PPARc in the C/EBPa–/– fibroblasts, thereby implicating a positive cooperative role for C/EBPa in conjunction with PPARc during terminal adipocyte differentiation (Fig. 17.1).

17.4

PPARa

The mouse PPARa gene spans at least 30 kb of DNA corresponding to eight exons, six of which are translated into a single polypeptide [58]. Transcription of the gene occurs relatively late in rat and mouse development (E13.5) in the same tissues where it is expressed in the adult animal [15, 59, 60]. Transgenic mice lacking PPARa are viable and fertile and exhibit no detectable gross phenotypic defects, suggesting that this PPAR does not play a critical role in development [61]. Furthermore, there are no studies suggesting that PPARa is a master regulator of any differentiation program; instead, it plays an important role in regulating gene expression in various differentiated cell types [1, 62]. Most notably, PPARa regulates lipid and glucose metabolism through its transcriptional activation of genes encoding components of these metabolic processes. In the adult rat, PPARa is abundantly expressed in liver, brown fat, heart, skeletal muscle, intestine, and pancreatic islets, as well as several other tissues [15]. This pattern of expression correlates closely with high levels of mitochondrial and peroxisomal boxidation of fatty acids in these tissues. In fact, PPARa derived its name from the fact that compounds known to induce peroxisome proliferation in rodent livers are also activators of PPARa [63, 64]. The fact that PPARa-null mice do not exhibit peroxisome proliferation in response to peroxisome proliferators, such as clofibrate, demonstrates that PPARa is a principal regulator of this process in mice [61]. Since its initial characterization, many investigations have shown that PPARa can upregulate the expression of key enzymes involved in several aspects of lipid metabolism in different cell types. For example, target genes of PPARa encode enzymes of the b-oxidation pathway in peroxisomes and mitochondria, including acyl-CoA oxidase (ACO) and medium-chain acyl-CoA dehydrogenase (MCAD) [65–

17.5 PPARd

67]. Support for an important role of PPARa in regulating a constitutive level of mitochondrial b-oxidation has come from studies in the PPARa-null mouse, which expresses significantly lower levels of at least seven mitochondrial enzymes in their livers compared with wild-type animals [68]. Other processes regulated by PPARa include fatty acid uptake and synthesis as revealed by the observation that fatty acid binding proteins (L-FABP) and the lipogenic malic enzyme genes are all upregulated in response to activators of PPARa [69–71]. Whether PPARa plays a role in regulating adipogenesis is still unclear. Brown adipose tissue expresses abundant amounts of PPARa and PPARc, but there is a paucity of information on its possible involvement in regulating adipogenic gene expression in brown adipocytes. Activation of PPARa in non-adipogenic fibroblasts can induce their conversion into lipid-laden cells that express some markers of the mature adipocyte [72]. Notwithstanding these observations, however, it is generally considered that PPARc is the principal regulator of adipogenesis, at least in white fat. This notion is further supported by the fact that PPARa-null mice contain a normal amount of adipose tissue; yet, PPARa may regulate genes expressed during the terminal stages of the differentiation process. For instance, studies have shown that PPARc regulates GLUT4 expression during adipogenesis in white fat cells [25]. It is possible, therefore, that PPARa provides this activity in other insulin-responsive cells that do not express PPARc, notably cardiac and skeletal muscle.

17.5

PPARd

Despite the fact that PPARd is expressed in many different cell types, there is relatively little known about its role in regulating gene expression. As previously mentioned, this is in part due to the lack of a specific PPARd agonist. Nevertheless, significant insight into the role of this transcription factor in regulating various physiologic processes has come from studying the PPARd-null mouse [73, 74]. Furthermore, following the development of a potent synthetic agonist, studies in obese Rhesus monkeys have demonstrated that PPARd can induce reverse cholesterol transport and lower triglyceride levels [75]. These data support the notion that PPARd also acts as a physiologic regulator of lipid homeostasis. Further analysis of the PPARd-null mouse provides evidence that PPARd may also be involved in regulating processes beyond lipid homeostasis, including early development, brain function and epidermal cell proliferation [73, 74]. With regard to lipid metabolism, PPARd-null mice display a dramatic reduction in fat mass, that is uniformly expressed in all types of adipose tissue, including gonadal, mesenteric, brown, and subcutaneous stores [74]. This defect, however, does not appear to result from an intrinsic function of PPARd within the fat cell, but instead seems to stem from a response of adipose tissue to an exogenous effector. Specifically, mice with an adipose-selective deficiency in PPARd expression have been shown to contain similar amounts of fat as their wild-type littermates [74]. It is important to

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note, however, that disruption of the PPARd gene in these adipose-selective knockouts relied on the expression of Cre recombinase driven by the adipose-selective fatty acid binding protein (aP2) gene promoter. Since aP2, however, is only expressed during the terminal stages of adipogenesis, it is likely that the floxed PPARd gene is deleted at this late stage; thereby, having already performed its function during the early stages of differentiation before its inactivation by the Cre enzyme. Accordingly, disruption of the gene would appear to have no effect on the development of adipose tissue. Such a possibility is consistent with observations made in vitro suggesting a role for PPARd in regulating PPARc expression during the early phase of adipogenesis [76–78]. Regardless, of whether this PPAR plays a role in regulating adipogenesis, studies in the PPARd-null mouse clearly demonstrate that PPARd, together with PPARa and PPARc, is involved in regulating lipid homeostasis.

17.6

PPARs and Control of Glucose Homeostasis: Therapies for Metabolic Syndrome and Type 2 Diabetes 17.6.1

PPARc

The discovery that the thiazolidinedione class of insulin-sensitizing drugs (TZDs) are also ligands for PPARc, supported the notion that this important regulator of adipose tissue differentiation and function is also a central regulator of glucose homeostasis in the body. The precise mechanism by which PPARc promotes insulin sensitivity and lowers blood glucose and lipid levels in type 2 diabetics is still unclear, although recent studies in mice suggest that the TZDs act through adipose tissue [79]. To this end, Reitman and co-workers have performed studies in the A-ZIP/F1 transgenic mouse, which lack white adipose tissue (WAT). The AZIP/F1 phenotype strikingly resembles humans with severe lipoatrophic diabetes, including a lack of fat, insulin resistance, hyperglycemia, hyperlipidemia, and fatty liver. Administration of rosiglitazone or troglitazone to these animals does not decrease blood glucose or insulin levels as observed in wild-type animals, suggesting that WAT is required for the anti-diabetic effects of TZDs. Treatment of these mice with TZDs does, however, lower circulating triglycerides and increase total body fatty acid oxidation, indicating that this property of the thiazolidinediones occurs in tissues other than white adipose tissue. An earlier study by Burant et al. [80], however, came to the opposite conclusion stating that troglitazone did not require adipose tissue to sensitize hyperglycemic mice to insulin. These investigators used a different mouse model in which the adipose tissue had been destroyed by the fat-specific expression of diphtheria toxin A. It appears, therefore, that additional investigations are needed, using other mouse models, before we can unequivocally conclude that TZDs act exclusively through adipose tissue to sensitize glucose intolerant animals to insulin.

17.5 PPARd

It is generally considered, however, that this process does involve PPARc since novel ligands designed to bind PPARc with high affinity are also potent insulin sensitizers in vivo [81]. Furthermore, mutations in the PPARc gene have been discovered in a few patients with severe insulin resistance where the resulting mutated PPARc proteins behave as dominant negative inhibitors of wild-type PPARc in in vitro assays [82]. These data strongly support the notion that PPARc regulates basal insulin sensitivity in humans. There are several possible mechanisms by which TZD activation of PPARc in adipose tissue might sensitize the organism to insulin [83]. Activation of PPARc by TZD has been shown to induce components of the insulin signaling and glucose transport system [84]. Furthermore, studies by Saltiel and co-workers demonstrate that TZD-associated activation of PPARc directly stimulates transcription of the CAP gene through PPAR response elements [85, 86]. CAP (c-Cbl-associating protein) is a multifunctional signaling protein that interacts with c-Cbl, and in so doing facilitates its tyrosine phosphorylation by insulin, a process considered to be involved in insulin-dependent glucose uptake in fat cells [87]. Our studies have also shown that activation of PPARc in engineered fibroblasts induces the insulindependent glucose transporter 4 (GLUT4) during adipogenesis [25], yet the precise mechanism remains elusive since the GLUT4 promoter does not appear to contain PPREs [88, 89]. Whether the PPARc-associated induction of glucose transport in adipose tissue can account for the anti-diabetic activity of the TZDs is of question, however, since fat only contributes to 10–20% of post-prandial glucose clearance. The major site for insulin-responsive glucose uptake is skeletal muscle, but it is unlikely that TZDs act directly on this tissue based on the studies discussed previously in the A-ZIP/F1 mice, and on the fact that skeletal muscle cells contain exceedingly low amounts of PPARc. It is now generally believed that adipose tissue is indirectly signaling to other insulin-responsive tissues by a process that is regulated by TZDs through the action of PPARc (Fig. 17.2). Another feature associated with type 2 diabetes is hyperlipidemia as quantified by an increased accumulation of triglycerides in muscle and liver, and elevated levels of free fatty acids in the circulation. There is growing evidence suggesting a link between these elevated lipid levels and insulin resistance in muscle and liver [90, 91]. It follows that enhanced lipid uptake in fat through the TZD-associated activation of PPARc should promote storage of triglycerides in adipose tissue. Adipose-specific induction of GLUT4 by PPARc may be involved in this process, since selective disruption of the GLUT4 gene in adipose tissue affects glucose uptake in both muscle and liver [92]. This phenomenon might be due to the fact that storage of fatty acids such as triglycerides in adipocytes requires the production of glycerol from glucose. The TZDs may also act by repressing the secretion of cytokines such as TNFa and IL-6 from adipose tissue, which have been implicated in the development of insulin resistance that precedes type 2 diabetes [93– 95]. Similarly, other secreted molecules may be affected by the activation of PPARc. One such recently discovered molecule is resistin, which is secreted from adipocytes and is proposed to promote systemic insulin resistance [96–98]. Circulating levels of resistin are increased in diet-induced and genetic forms of obesity,

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Fig. 17.2 PPARa and PPARc regulate glucose homeostasis in rodents. Modulating PPARa or PPARc activity in the tissues of various mouse models of obesity by either treatment with appropriate PPAR ligands or transgenic

manipulation of the corresponding genes affects glucose and lipid metabolism within insulin-responsive tissues as illustrated by the arrows. See text for details.

and decreased by the anti-diabetic drug rosiglitazone. Moreover, treatment of normal mice with recombinant resistin impairs glucose tolerance and insulin action. Based on these data, resistin is a hormone that potentially links obesity to diabetes; furthermore, it may serve as a possible target of the TZDs. Another adipocyte-derived hormone that may also play a role in glucose homeostasis and respond to TZDs is adiponectin. Studies have shown that decreased expression of adiponectin correlates with insulin resistance in mouse models of altered insulin sensitivity, and its administration to obese mice decreases insulin resistance by decreasing triglyceride content in muscle and liver. Moreover, insulin resistance in lipoatrophic mice was completely reversed by the combination of physiologic doses of adiponectin and leptin, but only partially by either molecule alone. It is possible, therefore, that decreased adiponectin results in the development of insulin resistance in mouse models of both obesity and lipoatrophy. More importantly, PPARc appears to enhance adiponectin expression in mature adipocytes [99]. These data also indicate that the replenishment of adiponectin or its activation by PPARc ligands might provide a novel treatment modality for insulin resistance and type 2 diabetes [100–103]. The role of PPARc in regulating glucose homeostasis appears to be more complex. A series of recent studies demonstrate that a moderate reduction of PPARc

17.5 PPARd

activity observed in heterozygous PPARc-deficient mice prevents the insulin resistance and obesity resulting from a high-fat diet [104]. In addition, studies show that treatment of mice fed a high-fat diet with both RXR and PPARc antagonists decreases triglyceride (TG) content in white adipose tissue, skeletal muscle, and liver. These inhibitors also potentiate leptin’s effects and increased fatty acid combustion and energy dissipation, thereby ameliorating diet-induced obesity and insulin resistance [105]. These data suggest that appropriate functional antagonism of PPARc/RXR activity may be a therapeutic approach to combat obesity-related type 2 diabetes. 17.6.2

PPARa

Recent investigations suggest that PPARa may also play a central role in regulating glucose homeostasis, and, consequently, its ligands may serve as therapeutic agents to treat type 2 diabetes (Fig. 17.2). This is demonstrated by activation of PPARa with highly specific ligands in two models of obesity-linked insulin resistance: diet-induced and genetic, which corrected the elevated blood glucose and insulin concentrations by sensitizing the animals to insulin and, thereby, improving glucose utilization [106]. Furthermore, this activity of PPARa also resulted in a significant decrease in adipose tissue mass by a mechanism independent of food intake and leptin gene expression. It is of interest that the effect of PPARa ligands is in sharp contrast to that mediated by PPARc ligands, which result in increased fat mass and inhibition of leptin gene expression. The insulin-sensitizing action of the PPARa ligands is likely due in part to the lipid-lowering effect of activating PPARa target genes, particularly those associated with b-oxidation of fatty acids. In fact, one study demonstrates that treatment of insulin-resistant, high-fatfed rats with the PPARa agonist, Wy14,643, improves muscle insulin action proportional to the extent to which activation of PPARa reduces the accumulation of lipid in the muscle [107]. Consistent with the notion that PPARa plays a role in glucose homeostasis is the observation that PPARa-null mice develop severe hypoglycemia when fasted compared with wild-type animals [108, 109]. Recent investigations suggest, however, that the role it plays is more complex and may depend on many physiologic factors. For instance, when PPARa-null mice are challenged with a high-fat diet, their adipose tissue depot increases, but they are protected from developing insulin resistance [110]. These observations, however, contradict those obtained in the studies demonstrating the insulin-sensitizing activity of PPARa as discussed above [106]. In addition, overexpression of PPARa in the hearts of transgenic mice leads to a metabolic phenotype strikingly similar to that of the diabetic heart in which myocardial fatty acid oxidation rates are increased while glucose uptake and oxidation are decreased [111]. Such conflicting observations may stem from the pleiotropic effects of PPARa in regulating different metabolic processes in insulin-responsive tissues. It is possible that activating PPARa in select tissues (i.e. liver) and eliminating PPARa gene expression from all tissues may have a similar

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effect on glucose homeostasis but through different mechanisms. For instance, its specific ligands may be acting as insulin-sensitizers by increasing b-oxidation in the livers of obese rodents [1]. This would result in a flux of fatty acids from peripheral tissues to the liver, thereby decreasing the delivery of triglycerides to skeletal muscle and adipose tissue. Since insulin resistance in skeletal muscle of obese animals is thought to be a result of the accumulation of lipids and fatty acid metabolites [91], directing lipids to the liver should sensitize the muscle to insulin. On the other hand, PPARa expressed in cardiac and skeletal muscle may be contributing to the insulin resistance observed in these tissues in response to a high-fat diet by the generation of fatty acid metabolites. It follows that the absence of PPARa in these tissues should sensitize muscle to insulin. It is clear, therefore, that we need to understand significantly more about the role of PPARa in regulating glucose homeostasis before its ligands are used as therapeutic agents to treat diabetes.

17.7

Conclusion

During the last several years, there have been significant advances in our understanding of the roles of the PPARs in regulating gene expression. The fact that these fatty acid binding proteins are targets of two major groups of therapeutic agents, the hypolipodemic fibrates and the thiazolidinedione family of insulin sensitizers, has contributed to this advancement. In addition, establishment of various transgenic mouse models has facilitated the identification of the PPARs as regulators of lipid and glucose homeostasis and as key components of transcriptional processes that determine the differentiation of specific cell types during development. This latter property of the PPARs is illustrated most clearly by the critical involvement of PPARc in regulating the differentiation of pre-adipocytes into adipocytes. In fact, very recent observations position PPARc as a master regulator of adipogenesis, which directs a unified pathway governing the differentiation of mesenchymal stem cells into mature fat cells within adipose tissue. PPARc in adipocytes of obese diabetic individuals is also considered a target of the thiazolidinedione class of insulin sensitizers such as rosiglitazone (AvandiaTM) and pioglitazone (ActosTM). This fact alone has generated tremendous interest in understanding the mechanisms by which excess fat tissue results in diabetes and how activating PPARc within this tissue sensitizes muscle to insulin. Much of the interest is focused within the pharmaceutical industry in an attempt to identify new targets of therapeutic agents that suppress the negative effects of fat on other insulin-responsive tissues such as muscle and liver. In this regard, several novel peptides secreted from fat cells, most notably resistin and adiponectin have recently been identified as potential regulators of insulin responsiveness in liver and muscle. Other investigations suggest that the level of circulating fatty acids and triglycerides may also contribute to insulin resistance associated with obesity. In fact, one of the effects of treating diabetic individuals with

17.9 References

TZDs is to lower the level of these blood lipids at the expense of increasing the accumulation of triglycerides within adipose tissue. To circumvent this unwanted side effect of the TZDs, industrial researchers are considering other drug targets that may regulate glucose homoestasis without increasing fat mass. PPARa may be one such target since treatment of obese animals with PPARa agonists corrects the associated insulin resistance by burning excess lipids rather than storing them within fat tissue. Studies implicating PPARa in regulating glucose homeostasis may be more complex and depend on many factors including the nutritional status of the individual. Clearly, there are many unanswered questions concerning the role of the PPARs in regulating energy metabolism and gene expression. Nevertheless, the intensity of the research effort focused on developing novel therapeutic agents to combat obesity, diabetes and their related disorders will address several of these questions.

17.8

Acknowledgements

I am grateful to Dr Deepa Prusty for her critical reading of the manuscript and valuable suggestions. I also acknowledge the National Institutes of Health for the following grants: DK51586 and DK58825.

17.9

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Role of FABP in Cellular Phospholipid Metabolism Chris A. Jolly and Eric J. Murphy

18.1

Fatty Acid Targeting

The fatty acid binding proteins (FABP) are a superfamily of proteins that have overlapping sequence homology, considerable substrate specificity, similarity in structural motifs, and yet are immunologically distinct from each other. FABP tissue distribution is broad, but generally focused in tissues that process a large quantity of fatty acids for one reason or another. For instance, heart FABP is found in the heart, where it appears to facilitate the uptake and utilization of fatty acids for b-oxidation [1, 2]. Both liver and intestinal FABP exist in the enterocyte, where these FABP are thought to be involved in fatty acid uptake and trafficking by the enterocyte [3, 4]. One important, yet unresolved, question is what is the role of these multiple FABP within the cellular milieu? Further, do these proteins have multiple physiological roles within cells; including fatty acid uptake, trafficking, modulation of phospholipid biosynthesis, of b-oxidation, of peroxisome function, and of gene expression? Some obvious answers lie in the role FABP appear to have in fatty acid uptake [5–8]. However, despite similar binding affinities in vitro, the different FABP do not increase fatty acid uptake equally when expressed in L-cells [8, 9]. This, in part, could be the result of different mechanisms used to bind and presumably extract fatty acids from the membrane [10–14]. Expression of these proteins stimulates the cytoplasmic diffusion of fluorescent fatty acids [7, 12], consistent with a role in fatty acid trafficking. However, do these proteins impact fatty acid targeting for esterification into distinct lipid pools? If FABP affect phospholipid metabolism, it would suggest that their expression would directly impact fatty acid targeting to distinct lipid pools. When intestinal (I-) and liver (L-)FABP are expressed separately in L-cell fibroblasts, but at the same level [8], there is not only a differential effect on fatty acid uptake [6, 8, 9], but also on fatty acid targeting to specific cellular lipid pools [6, 8, 9]. The incubation of L-cells expressing these FABP with [3H]oleic acid ([3H]-18:1) followed by separation of the major lipid classes is used to assess fatty acid targeting in the presence of I-FABP and L-FABP. Similar to the uptake results, where L-FABP but not I-FABP stimulated an increase in fatty acid uptake [7, 8], there is

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a differential targeting of fatty acid by each FABP [5, 6, 8, 9]. I-FABP targets the [3H]-18:1 predominantly for esterification into cholesteryl ester and triacylglycerol pools [8, 9]. Although there is a net decrease in the amount of [3H]-18:1 targeted to the total phospholipid pool in I-FABP-expressing L-cells, there is an increase in the amount of radiotracer targeted to the ethanolamine glycerophospholipid (EtnGpl) pool and a decrease in the radiotracer targeted to the choline glycerophospholipid (ChoGpl) pool [9]. L-FABP on the other hand targets [3H]-18:1 for esterification into both phospholipid and neutral lipid pools [6, 8]. Interestingly, the majority of the radiotracer targeted for esterification into the neutral lipids is found in the cholesteryl ester pool [6]. In contrast to I-FABP expression, L-FABP expression increases the net radioactivity found in the total phospholipid pool, with an increase in targeting to the ChoGpl and phosphatidylserine (PtdSer) pools, and to a lesser extent the sphingomyelin (CerPCho) pool [6]. Others have shown that I-FABP expression in Caco-2 cells increases the amount of triglyceride produced [15, 16], similar to the results discussed above for I-FABP expression in L-cells. Collectively, these studies demonstrate that FABP expression in cellular systems can alter fatty acid targeting in a manner that is dependent upon the type of FABP expressed. Hence, in cells such as the enterocyte, where both I- and LFABP are expressed, these two FABP may have completely different functions based on a selectivity inherent to each FABP. Certainly the potential to target fatty acid for esterification into specific pools is intriguing, although mechanism(s) underlying this selectivity are not fully understood.

18.2

Phospholipid Metabolism

The differential effects of I- and L-FABP on fatty acid uptake and targeting [6–9] suggest that expression of these proteins may impact L-cell steady-state phospholipid mass, giving some insight into the effects of these proteins on overall phospholipid metabolic changes. Indeed, FABP expression increases total phospholipid mass (Fig. 18.1) and the details of this increase were recently reported [17]. LFABP expression significantly increases the amount of total phospholipid in Lcells, increasing the mass from 266 ± 53 nmol mg–1 protein to 452 ± 26 nmol mg–1 protein in L-FABP-expressing cells [17]. I-FABP expression also increases the phospholipid content, increasing the total phospholipid mass to 343 ± 23 from control levels stated above [17]. Hence, there is a net increase in the phospholipid mass of 1.3-fold for I-FABP-expressing L-cells and a net increase in the total phospholipid mass of 1.7-fold for L-FABP-expressing L-cells compared with control. The lower magnitude of the effect by I-FABP relative to L-FABP is consistent with the fatty acid targeting data described in Section 18.1. However, the question remains, does FABP expression alter specific phospholipid class mass?

18.2 Phospholipid Metabolism

Fig. 18.1 Total cellular phospholipid content of L-cells expressing either I-FABP or L-FABP with values expressed as mean ± SD, n = 4. *Indicates significance from control cells with a P < 0.05. **Indicates significance from IFABP-expressing cells with a P < 0.05. Total

phospholipid content was assessed by assaying for lipid phosphorus and the total protein was determined using a modified dye-binding assay. Statistical analysis was done using a one-way ANOVA with Tukey’s post-test. Data are from Ref. [17] and used with permission.

18.2.1

Diacyl Phospholipid Classes

I- and L-FABP expression in L-cells differentially stimulates an increase in individual phospholipid mass (Fig. 18.2). L-FABP expression results in a net increase in the steady-state phospholipid mass of all major phospholipids, while I-FABP expression increases the mass of only two major phospholipid classes [17]. Expression of either I- or L-FABP increases the content of PtdSer and ChoGpl, but the magnitude of the increase is significantly different [17]. L-FABP increases PtdSer mass 5.6-fold, while I-FABP increases the mass 3.1-fold (Fig. 18.2). Although the 3.1-fold increase in PtdSer mass in I-FABP-expressing cells is significant from control, the nearly 2-fold difference compared to the PtdSer mass in L-FABP-expressing cells is intriguing and suggests a potential difference in the ability of these two different FABP to enhance specific Kennedy pathway biosynthetic steps. A less robust differential effect, yet still a significant effect, is observed in the steady-state ChoGpl mass, where I-FABP increases the mass 1.3-fold compared with a 1.5-fold increase in L-FABP-expressing L-cells (Fig. 18.2). While the differential effects observed could be related to expression of I- and L-FABP at different levels in the L-cells, this is not the case, as L-FABP accounted for 0.4% of the total cytosolic protein compared with I-FABP, accounting for 0.35% of the total cytosolic protein (Fig. 18.3) [8]. However, this differential effect could potentially be caused by the difference in the number of fatty acid binding sites in these two

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Fig. 18.2 Individual phospholipid levels of Lcells expressing either I-FABP or L-FABP with values expressed as mean ± SD, n = 4. *Indicates significance from control cells with a P <0.05. **Indicates significance from I-FABPexpressing cells with a P <0.05. Individual phospholipid were separated by high-performance liquid chromatography and mass determined by assaying lipid phosphorus. Total

protein was determined using a modified dyebinding assay. EtnGpl, ethanolamine glycerophospholipids; PtdIns, phosphatidylinositol; PtdSer, phosphatidylserine; ChoGpl, choline glycerophospholipids; CerPCho, sphingomyelin. Statistical analysis was done using a oneway ANOVA with Tukey’s post-test. Data are from Ref. [17] and used with permission.

Fig. 18.3 I-FABP and L-FABP content of Lcells stably transfected with the cDNA encoding for each protein. Values are expressed as

percentage of total cytosolic protein and were determined by Western blot. Data are from Ref. [8] and used with permission.

18.2 Phospholipid Metabolism

FABP. Although there are two fatty binding sites on L-FABP compared with one site on I-FABP, it is difficult to ascertain how this difference alone could account for the large differences observed between I- and L-FABP expression on steadystate phospholipid mass as well as fatty acid uptake and targeting. This point is important when one notes the magnitude of the increases observed for the various phospholipid fractions in L-FABP-expressing L-cells. For example, phosphatidylinositol (PtdIns) mass is increased 2.6-fold and EtnGpl mass increased 1.4-fold in L-FABP expressing L-cells compared to I-FABP expressing Lcells and control L-cells [17]. CerPCho mass is increased 1.7-fold in L-FABP-expressing cells compared with I-FABP-expressing and control L-cells. Thus, all of the major phospholipid classes are increased in L-FABP-expressing L-cells, suggesting an overall increase in the phospholipid biosynthetic capacity of the cells. The limited effect of I-FABP suggests that these two FABP increase phospholipid biosynthesis through different mechanism(s). However, it is also quite possible that I-FABP and L-FABP both stimulate a common mechanism and that L-FABP, but not I-FABP, also stimulates one or more biosynthetic pathways accounting for the observed effects of L-FABP on the steady-state phospholipid mass. 18.2.2

Potential Mechanisms for Diacyl Phospholipid Classes

Several mechanisms may account for the observed increase in phospholipid synthesis. The general elevation in phospholipid levels found in L-FABP-expressing L-cells may be the result of increased phosphatidic acid (PtdOH) biosynthesis, which is consistent with the observed L-FABP-induced stimulation of PtdOH synthesis in vitro [18–21]. This will be discussed in much greater detail below. However, this does appear to be plausible since PtdOH is the central and key intermediate for the Kennedy pathway [22, 23]. The difference in the magnitude of stimulation of PtdOH biosynthesis by I- and L-FABP [21] could account for the differential effect demonstrated herein. Further, L-FABP, but not I-FABP, are known to be localized in the endoplasmic reticulum [24]. It is also quite possible that L-FABP may affect other enzymes in the Kennedy pathway, in particular the portion of the pathway involved in PtdSer and PtdIns synthesis. As demonstrated above, these two phospholipids are selectively elevated by L-FABP expression, suggesting that the effect of L-FABP expression extends beyond a simple increase in PtdOH synthesis and may involve a number of lipid synthetic enzymes located in the endoplasmic reticulum. 18.2.3

Plasmalogen Classes

Both expression of I-FABP and that of L-FABP in L-cells increase the steady-state mass of plasmalogens, suggesting that expression of FABP in general may alter plasmalogen biosynthesis. Plasmalogens contain an ether linkage at the sn-1 position and a double bond between the a and b carbons of the fatty alcohol.

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Although plasmalogens are enriched in the kidney, heart, and brain, all mammalian cells contain significant amounts of these ether lipids. Recent evidence demonstrates that these fatty acids are rapidly turned over in the brain [25], suggesting a role in signal transduction. This potential role in brain is supported by a number of different heart lipid-mediated signaling pathways known to hydrolyze plasmalogens [26–28]. Thus, the observed increase in plasmalogen mass in FABPexpressing L-cells is quite intriguing (Fig. 18.4). In L-cells, L-FABP expression stimulates a significant 1.8-fold increase in ethanolamine plasmalogen (PlsEtn), while I-FABP expression only increases PlsEtn 1.3-fold as compared with control [17]. Expression of either FABP increases the level of choline plasmalogen (PlsCho), yet in a manner similar to that found for PlsEtn. Interestingly, although the magnitude of the increase in PlsEtn and PlsCho mass is different between I- and L-FABP-expressing L-cells, with L-FABP expression having a more robust effect, the net effect of these FABP on the ratio of PlsEtn to PlsCho mass are identical (Fig. 18.5). However, the important observation is that in I- and L-FABP-expressing L-cells this ratio is 1.32 ± 0.25 and 1.35 ± 0.24, respectively, whereas in control cells the ratio is 1.75 ± 0.21. Hence, FABP expression enhances the ability of L-cells to convert PlsEtn to PlsCho. This observation is important in that PlsCho is thought to have a role in lipid-mediated

Fig. 18.4 Plasmalogen content of L-cells expressing either I-FABP or L-FABP with values expressed as mean ± SD, n = 4. *Indicates significance from control cells with a P <0.05. **Indicates significance from I-FABP-expressing cells with a P <0.05. Phospholipids were separated by high-performance liquid chromatography and the EtnGpl and ChoGpl fractions subjected to mild acidic hydrolysis and

the samples re-separated to isolate the acid stable and acid labile fractions. The acid labile fraction represents the plasmalogens. PlsEtn, ethanolamine plasmalogen; PlsCho, choline plasmalogen. Statistical analysis was done using a one-way ANOVA with Tukey’s post-test. Data are from Ref. [17] and used with permission.

18.2 Phospholipid Metabolism

Fig. 18.5 The ratio of PlsCho to PlsEtn was determined using the mass values for individual cultures from the data in Fig. 18.4. Values represent means ± SD, n = 4. *Indicates significance from control cells with a P < 0.05.

**Indicates significance from I-FABP-expressing cells with a P < 0.05. Statistical analysis was done using a one-way ANOVA with Tukey’s post-test. Data are from Ref. [17] and used with permission.

signal transduction in heart [26–28] and brain [29, 30, 25]. The role of heart FABP and brain FABP on this process is not known. Thus, FABP expression not only enhances phospholipid biosynthesis, but also may stimulate an increase in the synthesis of the plasmalogen subclasses. The mechanisms underlying these effects are not known, but certainly an important topic of on-going studies. 18.2.4

Potential Mechanisms for Plasmalogen Classes

Several different mechanisms may account for the observed increase in plasmalogen biosynthesis. It is important to note that plasmalogen biosynthesis requires both peroxisomal [31, 32] and microsomal [33–35] steps. It is unclear the extent to which FABP are found associated with peroxisomes, although microsomal localization has been documented for L-FABP [24]. Formation of the 1-O-alkyl moiety occurs in the peroxisome [31, 32], while the desaturation of the 1-O-alkyl moiety occurs in the microsome [33–35]. Hence, stimulation of the microsomal desaturation of the ether lipid precursor is consistent with the endoplasmic reticulum localization of L-FABP [24]. However, as discussed below, L-FABP increases the proportion of 22:6 n-3 in L-cell phospholipids. This is an important observation in that 22:6 n-3 synthesis is peroxisomal-dependent [36], suggesting an upregulation of peroxisomal enzyme activity. This is also consistent with the known upregulation of L-FABP expression by peroxisome proliferator activated receptors (PPAR) a

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and d using a number of different systems [37–40]. Further, it has been reported that L-FABP itself is able to stimulate an increase in peroxisome proliferator activated receptor activity, suggesting a potential partial regulation of peroxisome proliferation [41]. Hence, it is quite plausible that the increase observed in plasmalogen synthesis is related to an increase in peroxisomal enzyme activity. Alternatively, because the final steps for plasmalogen biosynthesis are microsomal [33–35], I- and L-FABP expression may increase desaturase activity, promoting an increase in the flux through the synthetic pathway [25]. Such a mechanism is consistent with the known stimulation of Kennedy pathway enzymes [18–21]. Regardless of which mechanism is operative, there is a selective enhancement of the conversion of PlsEtn to PlsCho. It is well accepted that PlsEtn is the precursor for PlsCho [25, 42, 43], however the mechanism by which this conversion occurs is not fully understood. Hence, it is also plausible that FABP expression enhances not only peroxisome activity, but also increases the flux of PlsEtn to PlsCho, thereby resulting in a significant shift in the PlsEtn to PlsCho mass ratio.

18.3

Neutral Lipid Mass

Consistent with the targeting of fatty acids to the triglyceride and cholesteryl ester pools in I-FABP-expressing L-cells, there is a net statistically significant increase in the amount of cholesteryl ester and triglyceride in I-FABP-expressing L-cells relative to control [9]. Cholesteryl ester mass is increased nearly 1.3-fold and triglyceride mass is increased 1.6-fold in I-FABP-expressing L-cells compared with control. A similar effect of L-FABP is not known. Hence, consistent with the fatty acid targeting data, I-FABP expression increases the steady-state levels of cholesteryl esters and triglycerides in L-cells.

18.4

Cellular Phospholipid Composition

While phospholipid levels are predictive of an overall increase in phospholipid synthetic capacity of the cell, phospholipid compositional data yields information regarding synthesis of individual classes relative to each other. Hence, the large impact of L-FABP expression on L-cell phospholipid synthesis needs to be addressed by also examining compositional data. For I-FABP expression, there are no significant differences in lipid composition (mol%) compared with control [17]. However, L-FABP expression alters the composition of three phospholipid classes [17]. Both PtdSer and PtdIns are found to comprise a greater proportion of the phospholipid composition, suggesting that L-FABP expression enhances the synthesis of these two phospholipids relative to the other major classes. A decrease in the proportion of ChoGpl is observed, despite the 1.5-fold increase in mass. In essence, the increase in mass is proportionally smaller than that observed for

18.5 Phospholipid Acyl Chain Composition

Fig. 18.6 The mole percentage of individual glycerophospholipid class was calculated using the mass values for individual cultures from data in Fig. 18.4. Values represent means ± SD, n = 4. *Indicates significance from control cells with a P < 0.05. **Indicates

significance from I-FABP-expressing cells with a P < 0.05. Statistical analysis was done using a one-way ANOVA with Tukey’s post-test. Data are from Ref. [17] and used with permission.

PtdSer and PtdIns, suggesting that the synthesis of these phospholipids is upregulated by L-FABP expression relative to ChoGpl. No other significant differences are observed in L-cells expressing L-FABP relative to control. Thus, there once again appears to be a differential effect of FABP expression on phospholipid biosynthesis, suggesting that each FABP may have its own unique properties that alter lipid synthesis in a number of different ways. For plasmalogens, expression of either protein significantly increases the proportion of PlsCho in L-cells [17]. However, only L-FABP expression increases the proportion of PlsEtn, suggesting an actual upregulation of PlsEtn synthesis in LFABP-expressing cells (Fig. 18.6). Expression of either FABP enhances the apparent conversion of PlsEtn to PlsCho, consistent with the mass data demonstrating a large increase in PlsCho. Hence, FABP expression, by an unknown mechanism, increases the synthesis of plasmalogens.

18.5

Phospholipid Acyl Chain Composition

Because FABP expression alters fatty acid targeting and enhances phospholipid biosynthesis, there is a strong potential that phospholipid acyl chain composition may also be altered by FABP expression. I- and L-FABP expression in L-cells re-

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sults in an overall increase in polyunsaturated fatty acid (PUFA) content [17]. Interestingly, L-FABP expression increases the content of 22:6 n-3 in EtnGpl, ChoGpl, and PtdSer. This is consistent with the potential increase in peroxisomal enzyme activity as discussed in detail above. Furthermore, expression of I- and LFABP increases the content of 20:4 n-6 in ChoGpl and PtdSer. This is very interesting as 20:4 n-6 is a fatty acid well known to be involved in lipid-mediated signal transduction and is the precursor of the 2-series of prostaglandins and the 4series of leukotrienes. Hence, both L-FABP and I-FABP expression alter L-cell phospholipid acyl chain composition, although L-FABP expression appears to have a greater impact on acyl chain composition relative to I-FABP expression. In general, L-FABP expression increases PUFA, predominantly 20:4 n-6 and 22:6 n-3, and decreases the amount of esterified monounsaturated fatty acids (MUFA) found in the phospholipids. Changes in these two parameters results in a decrease in the unsaturated/saturated fatty acid index and an increase in the PUFA/ saturated fatty acid index. In general, I-FABP expression decreases MUFA and causes a number of changes in PUFA, resulting in a decrease in the unsaturated/ saturated fatty acid index. 18.5.1

Potential Mechanisms for Fatty Acyl Chain Alterations

Similar to synthesis parameters demonstrated herein, there is a differential effect of I- and L-FABP expression on phospholipid acyl chain composition. In part, these changes suggest that the individual FABP may impart a degree of substrate selectivity for fatty acid esterification. Because I- and L-FABP bind both fatty acids and fatty acyl-CoA with a high affinity, these proteins may facilitate interactions of these substrates with CoA-dependent and CoA-independent acyltransferases [44]. Further, within the cell L-FABP may exhibit preferential binding for PUFA over MUFA, similar to what has been observed in vitro [45]. This alone could account for the differential effects of I- and L-FABP on PUFA composition in phospholipids isolated from cells expressing these proteins. Collectively, the elevation in phospholipids pertinent to signal transduction as well as the elevation in the content of 20:4 n-6, suggests that L-FABP expression may very well impact the cellular function of L-cells expressing these proteins. Further work needs to be done to extend these studies into cells that are more physiologically relevant and into animal models. However, the potential that expression of FABP may regulate lipid-mediated signal transduction through modulating the overall lipid metabolism of the cell is an important point that needs further consideration.

18.6 Phosphatidic Acid Biosynthesis

18.6

Phosphatidic Acid Biosynthesis

FABP are known to alter phospholipid levels, suggesting an increase in phospholipid synthesis de novo. Phospholipids are synthesized de novo by the sequential acylation of glycerol-3-phosphate to lysophosphatidic acid (lysoPtdOH) via glycerol-3-phosphate acyltransferase (GPAT) then to phosphatidic acid (PtdOH) via lysophosphatidic acid acyltransferase (LAT) in the presence of acyl-CoA [46]. Not only is PtdOH the precursor for cellular phospholipids and triglycerides, but both lysoPtdOH and PtdOH are important intracellular signaling molecules [47, 48]. Fatty acid asymmetry may also be related to selection to the sn-1 or sn-2 position during synthesis de novo. In phospholipids, the sn-1 position predominantly contains saturated fatty acids and the sn-2 position predominantly contains unsaturated fatty acids. Several reports have shown that this occurs because the mitochondrial GPAT demonstrates a preference for saturated acyl-CoA while the microsomal GPAT shows no preference between saturated or unsaturated acyl-CoA. Thus, it is thought that lysoPtdOH is produced in the mitochondria and then transported to the microsome for acylation via LAT to PtdOH. FABP have been proposed to serve as the shuttle for acyl-CoA and lysoPtdOH between the mitochondria and endoplasmic reticulum in cells. ACBP may also shuttle acyl-CoA between mitochondrial and microsomal membranes, which may help direct acyl-CoA utilization for phospholipid biosynthesis or b-oxidation [49]. Similarly, L-FABP can extract PtdOH and lysoPtdOH from mitochondrial membranes suggesting that LFABP may help to transport lysoPtdOH and PtdOH to the microsome for use in phospholipid biosynthesis [46, 50, 51]. However, recent evidence now suggests that the acyl-CoA specificity of the mitochondrial GPAT is due to the presence of a protein that binds acyl-CoA and not the enzyme itself. Several observations provide evidence for this supposition. First, the study demonstrating that mitochondrial GPAT prefers saturated acylCoA was performed in the presence of albumin, which binds acyl-CoA [52]. Second, measuring mitochondrial GPAT activity in the absence of albumin indicates that GPAT has no preference between palmitoyl- or oleoyl-CoA (manuscript in preparation). Third, including albumin or ACBP in the reaction mixture results in the mitochondrial GPAT showing at least a 5-fold preference for palmitoyl-CoA over oleoyl-CoA (manuscript in preparation). Thus, these data suggest that FABP may play an important role in phospholipid fatty acid asymmetry found in cellular membranes through its ability to bind substrate and enhance the preference of the mitochondrial GPAT for saturated fatty acids. 18.6.1

FABP Increases Phosphatidic Acid Biosynthesis

Recent evidence using isolated, gel-purified microsomal membranes from rat liver have directly compared the ability of L-FABP [18], I-FABP [19, 21], and ACBP [53] to stimulate PtdOH biosynthesis in vitro. L-FABP and ACBP acted similarly by

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stimulating PtdOH biosynthesis 10-fold in the presence of unsaturated acyl-CoA (oleoyl-CoA and arachidonoyl-CoA). In contrast, both L-FABP and ACBP suppressed PtdOH biosynthesis in the presence of palmitoyl-CoA [53]. Neither LFABP or ACBP significantly influenced LAT activity. This suggests that L-FABP and ACBP are increasing microsomal PtdOH biosynthesis by altering GPAT activity, but not LAT activity. This is important because the rate-limiting step of PtdOH biosynthesis is the GPAT step, since LAT activity is 10-fold higher in microsomes compared to GPAT. I-FABP can also stimulate microsomal PtdOH biosynthesis in the presence of oleoyl-CoA although I-FABP is only half as effective when compared to L-FABP and ACBP [19]. 18.6.2

L-FABP Conformers and Phosphatidic Acid Biosynthesis

L-FABP isolated from rat liver appears to exist as two isoforms [54, 55]. There are no differences in amino acid composition between the isoforms [55], however, there are significant changes in their three-dimensional structure, suggesting that these two isoforms are essentially conformers [56]. The isoforms are referred to here as isoforms I and II [55]. Recent experiments have shown that isoform I can stimulate microsomal GPAT activity in the presence of oleoyl-CoA 7-fold but has no effect on palmitoyl-CoA utilization. In contrast, isoform II stimulates microsomal GPAT activity in the presence of palmitoyl-CoA 4-fold but does not significantly impact oleoyl-CoA incorporation into PtdOH [18]. The mechanism may be related to selective acyl-CoA binding to the isoforms since isoform I shows a slight preference for unsaturated fatty acid and acyl-CoA in relation to isoform II [56]. Inhibiting acyl-CoA hydrolysis does not appear to be involved in the mechanism since only isoform II was effective at preventing the hydrolysis of both palmitoyl- and oleoyl-CoA relative to isoform I [18]. These data indicate that not only are the types of FABP that are present in tissues important, but also the potential existence of FABP isoforms may add an additional level of complexity to modulating phospholipid metabolism. 18.6.3

Potential Mechanisms for Stimulation of Phosphatidic Acid Biosynthesis

The physiologic significance of ACBP reducing palmitate incorporation into PtdOH biosynthesis may be that palmitate is diverted toward b-oxidation. Both carnitine palmitoylacyltransferase (CPT) and GPAT are located on the mitochondria and may compete for palmitate [57] thus ACBP may divert palmitoyl-CoA towards b-oxidation. This suggestion is supported by studies in vitro showing that CPT activity in mitochondria is increased by ACBP and transfecting ACBP into a hepatocyte cell line increases b-oxidation in the presence of exogenous palmitate [58]. The mechanism by which ACBP increases CPT activity is not known, however, current evidence suggests that CPT prefers ACBP-bound acyl-CoA rather than free acyl-CoA. ACBP was shown to have a thousand-fold higher binding af-

18.6 Phosphatidic Acid Biosynthesis

finity for acyl-CoA compared to CPT and that CPT activity correlates linearly with increasing amount of ACBP-bound acyl-CoA but not free acyl-CoA [59]. Most studies examining the influence of ACBP on PtdOH biosynthesis and CPT activity in vitro have used purified recombinant ACBP from bacteria. This is important because it has been shown that native ACBP is at least twice as effective at stimulating microsomal PtdOH biosynthesis and inhibiting acyl-CoA hydrolysis when compared to recombinant ACBP [60]. Therefore, the influence of ACBP on PtdOH biosynthesis seen in vitro may be more dramatic when the native ACBP is present. The mechanism by which FABP stimulate GPAT activity is not known. The results described above regarding ACBP and the fact that L-FABP equally stimulates microsomal GPAT activity cannot be completely explained by preventing acyl-CoA hydrolysis, thereby resulting in an elevation of acyl-CoA levels. ACBP, but not LFABP, was effective at suppressing acyl-CoA hydrolase activity using palmitoylCoA and oleoyl-CoA as substrates [53]. It is tempting to speculate that a scenario may exist similar to that shown for ACBP enhancement of CPT activity. For FABP, the GPAT enzyme may show a preference for FABP-bound acyl-CoA over free acyl-CoA. This would suggest a more direct mechanism of FABP influence via interaction with the enzyme instead of simply increasing the soluble levels of acyl-CoA via membrane extraction. 18.6.4

Biological Significance

In addition to PtdOH acid biosynthesis, FABP may also play a role in altering phospholipid fatty acid composition via attenuation of deacylation/reacylation cycles of pre-existing phospholipids. For example, ACBP has been shown to increase arachidonic acid incorporation into phosphatidylcholine (Pt d Cho) in red blood cell membranes, indicating a role for ACBP in membrane Pt d Cho fatty acid remodeling [61]. Alternatively, FABP may increase the transacylation of fatty acids into pre-existing phospholipids. L-FABP isoform I was found to increase the transacylation of oleate and palmitate into phosphatidylethanolamine while isoform II had no effect. In contrast, isoform II significantly incorporated oleate and palmitate into phosphatidylserine and sphingomyelin while isoform I had no effect [18]. These data indicate that FABP may have an important role in fatty acid remodeling of select phospholipids. Taken together, the data in cell lines overexpressing FABP are in agreement with the data in isolated membranes, showing that FABP stimulate phospholipid biosynthesis and can influence phospholipid fatty acid composition. New results discussed here also indicate that FABP may have a novel role in influencing the acyl-CoA preference of mitochondrial GPAT and potentially the fatty acid and acylCoA preferences of other enzymes as well. Furthermore, the data in isolated membranes reveal unique functions of individual FABP, suggesting they are not simply general non-specific carriers of fatty acids and acyl-CoA.

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18.7

Conclusions and Perspectives

FABP expression can alter a number of important lipid parameters in cells, including fatty acid targeting, phospholipid content, neutral lipid content, and phospholipid acyl chain composition. The mechanism(s) involved in these processes are not fully understood and in many cases are based upon observed steady-state lipid parameters. Certainly, the enhancement of PtdOH biosynthesis is a major contributing factor, however this contribution alone does not explain all of the results reported herein. Several important points need to be made. First, not every FABP has the same functional role within cells. In fact, results presented herein demonstrate quite the opposite, that two FABP with similar binding affinities for fatty acids have dramatically different effects on cellular lipid metabolism. Second, binding data from experiments in vitro do not represent protein function, but rather convey simply the protein binding affinities for a number of different substrates. Third, that more studies using knockout strategies are required to further elucidate the role of FABP in cellular phospholipid biosynthesis. Using a variety of techniques to study fatty acid uptake, targeting, and metabolism in vivo, the physiological role of FABP in phospholipid metabolism described herein in L-cell fibroblast expression system can be further substantiated using more biologically relevant models. 18.8

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Membrane-associated Fatty Acid Binding Proteins Regulate Fatty Acid Uptake by Cardiac and Skeletal Muscle Jan F. C. Glatz, Joost J. F. P. Luiken, Ger J. van der Vusse, and Arend Bonen

19.1

Introduction

Long-chain fatty acids (LCFAs) are important compounds for both heart and skeletal muscle cells. Apart from serving as constituents of membrane phospholipids and their involvement in protein modification and in regulation of transcription, LCFAs are a main substrate for energy production [1–4]. Under normal circumstances LCFAs provide approximately 70% of cardiac ATP production, and in skeletal muscle, although their contribution depends on the muscle fiber type and the degree of exercise, they provide up to 50%, with glucose being the other main substrate [2–5]. However, in a number of pathological states there are marked alterations in the contribution of LCFAs to muscular energy production. For instance, during development of cardiac hypertrophy and its progression towards cardiac failure, there is a gradual decrease in cardiac LCFA utilization, while in contrast, the diabetic heart is unable to take up sufficient amounts of glucose and, therefore, relies almost completely on LCFAs for energy production [2, 3, 6]. Strikingly, in both conditions these shifts in substrate preference are paralleled by a diminished performance of the heart. In the case of diabetes this has been related to an increased storage of LCFAs in triacylglycerols, which occurs in both heart and skeletal muscle [3, 6, 7]. These combined findings suggest that proper functioning of heart and skeletal muscle requires appropriate tuning of the uptake, storage, and utilization of metabolic substrates, notably LCFAs and glucose, with respect to both substrate preference and the amounts used. The latter aspect also relates to the ability to respond flexibly to changes in energy demand, such as occur, for instance, during the transition from a resting to a contracting skeletal muscle. On their way from the microvascular compartment to cardiac or skeletal muscle myocytes, LCFAs first have to pass the capillary endothelium. As outlined in more detail elsewhere [3], in muscle, unlike other tissues such as liver, the interendothelial clefts do not allow the passage of the albumin–LCFA complex at a rate that could explain observed LCFA uptake rates. The corollary is that LCFAs could be released from albumin and then traffic through the capillary endothelial cells to bind to albumin present in the interstitial compartment.

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In recent years much progress has been made with respect to our understanding of the first step of LCFA utilization by myocytes, i.e. LCFA uptake. Compelling evidence is now available that LCFA can enter myocytes either by passive diffusion through the lipid bilayer or by protein-mediated transmembrane transport [8–13] and that the latter represents a site of control, allowing changes in the presence and/or activity of these membrane proteins to regulate LCFA uptake. In this chapter we discuss novel data indicating that the three membrane-associated fatty acid binding proteins (FABPs) identified to date facilitate transmembrane transport of LCFAs in muscle, and that one of these, namely fatty acid translocase (FAT/CD36), is also involved in the acute regulation of LCFA uptake. Moreover, alterations in heart and skeletal muscle LCFA utilization, as observed in cardiac hypertrophy/failure and in diabetes, appear to be related to abnormalities in the presence or functioning of FAT/CD36. The focus of this chapter is on cardiac and skeletal myocytes; transendothelial transport of LCFAs, which putatively also involves membrane-associated FABPs [14], will not be addressed (for further information see Ref. [3]).

19.2

Molecular Mechanism of Muscular Fatty Acid Uptake 19.2.1

Passive Diffusional and Protein-mediated Fatty Acid Uptake

Transmembrane transport of LCFAs by a diffusional mechanism has been clearly demonstrated both in studies with model membrane systems and in studies with isolated cells (reviewed in Refs [8, 15–17]). LCFAs cross the phospholipid bilayer or cell membrane in the un-ionized, i.e. protonated, form, and this apparent flipflop mechanism is supposed to occur with sufficient rapidity to explain observed rates of cellular LCFA uptake or release [8]. On the other hand, there is now ample evidence that membrane-associated proteins markedly facilitate LCFA uptake. For example, in studies with isolated rat cardiac myocytes we observed that the rate of uptake of radiolabeled palmitate (90 lM, complexed to 300 lM albumin, i.e. physiological conditions) decreased to 20–30% of the initial rate both in the presence of phloretin, a non-selective inhibitor of membrane transport processes, and after trypsin pre-treatment of the cells, which hydrolyzes (the extracellular part of) membrane-associated proteins (Fig. 19.1). However, when studied in the presence of etomoxir, an inhibitor of mitochondrial b-oxidation, palmitate uptake was also markedly decreased (Fig. 19.1) [18]. These findings indicate that cellular uptake and metabolism of LCFAs are tightly coupled processes and illustrate the difficulty of interpreting these data with respect to the involvement of membrane proteins in the LCFA uptake process. Indeed, in order to properly investigate LCFA uptake it is important to use experimental models in which the uptake process across the plasma membrane is divorced from LCFA metabolism.

19.2 Molecular Mechanism of Muscular Fatty Acid Uptake

Fig. 19.1 Studies of palmitate uptake by isolated adult rat cardiac myocytes (solid bars) and by rat heart giant sarcolemmal vesicles (shaded bars) to explore the involvement of membrane-associated proteins in the fatty acid uptake process. Cells or vesicles were pretreated with 0.25% (w/v) trypsin, 0.4 mM sulfo-N-succinimidyloleate (SSO), or the

metabolic inhibitor etomoxir (10 lM), or incubated in the presence of 0.4 mM phloretin, and the uptake of 14C-labeled palmitate (90 lM) complexed to albumin (300 lM) was monitored at 378C for 3 min (myocytes) or 15 s (vesicles). Values are expressed relative to control and represent means ± SD for 3–5 experiments. Data are from Refs [18, 22, 71].

To overcome this issue we have employed so-called giant sarcolemmal vesicles in our studies on heart and muscle LCFA uptake. These vesicles are prepared by incubation of thinly sliced muscle strips in a buffer containing collagenase and a high concentration of KCl [19–21]. The vesicles are formed by budding, are oriented right-side out, and are spherical with a diameter of 5–25 lm (Fig. 19.2). Giant vesicles thus obtained do not contain cellular organelles like mitochondria, but do contain water-soluble cytoplasmic constituents such as cytoplasmic FABP (FABPc), which can act as an intravesicular acceptor for LCFAs (see Section 19.2.3). Thus, palmitate taken up by such vesicles could be completely recovered as unmetabolized LCFAs [21, 22]. In the presence of phloretin as well as after trypsin pre-treatment, vesicular palmitate uptake was decreased to 30–40% of the rate without these inhibitors (Fig. 19.1). In addition, vesicular palmitate transport was inhibited by excess oleate as well as by sulfo-N-succinimidyl-oleate (SSO), a LCFA derivative that covalently modifies fatty acid binding proteins by virtue of its highly reactive sulfo-N-succinimidyl moiety and that does not penetrate biological membranes [23]. Together, these findings suggest that there is competition among LCFAs for the transport protein(s). Finally, etomoxir did not affect vesicular palmitate uptake (Fig. 19.1). Since metabolism is absent in the vesicles, the conclusion can now be drawn that in the presence of a physiological LCFA concentration muscular LCFA uptake takes place mainly by a protein-mediated uptake process, assisted by passive diffusion.

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346

A

B Fig. 19.2 Comparison of photomicrographs of two model systems employed for fatty acid uptake studies: (A) isolated adult rat cardiac mycoytes and (B) rat heart giant sarcolemmal

vesicles. The average size of heart giant vesicles (about 15 lm in diameter) is of similar magnitude as that of small cardiac myocytes. Bar = 50 lm.

19.2.2

Membrane-associated Fatty Acid Binding Proteins

At present three membrane proteins with the ability to non-covalently bind LCFAs have been identified. These proteins are 43-kDa plasma membrane FABP (FABPpm), a family of some five 60-kDa fatty acid transport proteins (FATP1–5), and 88-kDa fatty acid translocase (FAT/CD36) (reviewed in Refs [10, 24, 25]). Both FABPpm and FATP have been detected in virtually all tissues examined, but FAT/ CD36 shows a more restricted expression, as in several species and strains it is absent in liver and brain [26]. However, the three proteins are simultaneously expressed in heart and skeletal muscles, making striated muscle types useful tissues for the study of their putative involvement in LCFA uptake. Evidence is available from transfection studies with cells in culture that in the presence of FABPpm, FATP, or FAT/CD36 the rate of cellular LCFA uptake is markedly increased [27–29]. Moreover, muscle-specific overexpression of FAT/CD36 enhanced LCFA oxidation by contracting muscle and reduced plasma LCFA and triacylglycerols [30]. However, the exact manner in which each of these three proteins facilitates transmembrane translocation of LCFAs is not yet known, partly because their membrane topology is still poorly understood. Thus, FABPpm, which is a peripheral membrane protein [31], may exert its action solely by trapping LCFAs, after which the LCFAs cross the membrane by passive diffusion. FATP1 was recently found to have one transmembrane and multiple membrane-associated domains [32]. FATP1–5 also possess acyl-CoA synthetase activity, particularly for very longchain LCFAs [33–35], so that metabolic trapping might explain their facilitation of LCFA uptake. FAT/CD36 is a highly glycosylated, integral membrane protein that is believed to have only two transmembrane-spanning regions [24], making it unlike-

19.2 Molecular Mechanism of Muscular Fatty Acid Uptake

ly that it facilitates LCFA transport by channelling LCFAs through a pore in the membrane. In view of this notion it has been suggested that FAT/CD36 operates mainly through trapping LCFAs to the plasma membrane [10, 24], increasing the LCFA concentration near the sarcolemma, thus promoting LCFA diffusion through the cellular membrane. 19.2.3

Putative Mechanism of Cellular Fatty Acid Uptake

Taken together, the above findings may suggest that passive diffusion and proteinmediated LCFA uptake co-exist in such manner that the membrane-associated FABPs function in the trapping of LCFAs from extracellular donors and their release to intracellular targets, and that the actual transmembrane translocation step occurs by passive diffusion of LCFAs through the lipid bilayer (Fig. 19.3). This

Fig. 19.3 Schematic presentation of the cellular uptake and utilization of long-chain fatty acids (LCFAs or FAs) illustrating the presumed roles of various lipid binding proteins in this process. Following their dissociation from plasma albumin, the transmembrane translocation of LCFA most likely takes place either by passive diffusion through the lipid bilayer, or is facilitated by membrane-associated proteins, or by a combination of both. This includes FABPpm acting as scavenger and FAT/CD36 acting as scavenger and/or transporter of LCFAs. FATP most likely is involved in fatty acyl-CoA synthesis. Intracellularly, LCFAs are bound by cytoplasmic FABP and, after activa-

tion to fatty acyl-CoA, by acyl-CoA binding protein (ACBP) [72]. LCFA uptake can be modulated by recycling of FAT/CD36 between the plasma membrane and an endosomal compartment. Alterations in redistribution of FAT/CD36 can be mediated by insulin, following the binding of this hormone to its receptor and involving PI3-kinase, or by muscle contraction, which may activate a yet unidentified protein kinase (PKX). FABPpm, plasma membrane fatty acid binding protein; FATP, fatty acid transport protein; FAT, fatty acid translocase (CD36); FABPc, cytoplasmic fatty acid binding protein; ACBP, acyl-CoA binding protein; IR, insulin receptor; PKX, protein kinase X.

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unified concept would be in agreement with virtually all experimental findings made so far on this controversial topic [10]. The release of LCFAs from extracellular donors, in particular albumin, may also be facilitated by albumin binding proteins, i.e. glycoproteins gp18, gp31, and gp60, localized on the luminal membrane of endothelial cells and on the plasma membrane of parenchymal cells, all of which have a high affinity for albumin [36–38]. These proteins initially have been postulated to function in the endocytosis and endothelial transcytosis of albumin [39], but may also facilitate albumin–LCFA dissociation [40, 41] albeit presumably in a permissive and not a regulatory function with respect to transmembrane LCFA trafficking [18]. Inside cells, LCFAs are bound to FABPc, which acts as an acceptor protein and is viewed as an intracellular counterpart of plasma albumin [10, 42, 43]. The role of FABPc as intracellular LCFA acceptor and cytoplasmic carrier protein has now clearly been established. For instance, studies in vivo [44] and with cardiac myocytes isolated from mice lacking the heart-type FABPc gene [45] show that the rates of LCFA uptake and oxidation are markedly (approximately 50%) reduced, while there is a compensatory upregulation of glucose uptake and oxidation. These data demonstrate that, while FABPc strictly is not indispensable, it is significant for adequate cardiac LCFA utilization, especially in the case of an increased energy demand.

19.3

Expression of FABPs in Heart and Skeletal Muscles Compared

Insight into the functioning of the three membrane-associated FABPs, and their relation to (heart-type) FABPc, has also been obtained by comparison of their expression levels in rat heart and red (oxidative) and white (glycolytic) skeletal muscles. On the mRNA level the expressions of FABPpm, FATP1, FAT/CD36, and FABPc were relatively high in heart, somewhat lower in red skeletal muscle (m. soleus), and lowest in white skeletal muscle (m. extensor digitorum longus) (Fig. 19.4) [26]. These differences are in keeping with similar differences in LCFA uptake rate as measured in giant sarcolemmal vesicles [22] and with the known differences in LCFA oxidative capacities among the three muscle types [21, 46], and support the view that all the proteins are involved in muscle LCFA uptake. However, comparing the protein amounts of these FABPs disclosed marked differences. Immunochemical assessment of the total tissue protein level and of the amounts of the membrane proteins present on the sarcolemma, i.e. their functional pools, revealed that levels of FABPpm, FAT/CD36, and FABPc declined from heart to red muscle to white muscle, whereas for FATP an inverse relationship was found (Fig. 19.4) [22, 47]. Thus, while the FATP content was 8- to 10-fold lower in heart vesicle membranes than in muscle vesicle membranes, there was a 4- to 6-fold greater maximal LCFA uptake rate by heart giant vesicles. This finding suggests that FATP is not involved in (regulation of) the bulk uptake of LCFA by heart and muscle, although it cannot be excluded that it fulfills a permissive role or that it needs to be activated upon a change

19.3 Expression of FABPs in Heart and Skeletal Muscles Compared

A

B

C Fig. 19.4 Relative expression in adult rat heart, red skeletal muscle, and white skeletal muscle of four proteins implicated in longchain fatty acid uptake, i.e. the membrane-associated proteins FABPpm, FATP, and FAT/ CD36, and the cytoplasmic protein FABPc. (A) Expression on the mRNA level, examined by Northern blot analysis in heart, m. soleus (red skeletal muscle), and m. extensor digitorum longus (white skeletal muscle) of Wistar rats. (B) Expression on the total tissue protein level, as measured by quantitative Western blotting in heart, red skeletal muscle (m. soleus and red portion of m. gastrocnemius), and white skeletal muscle (white portion of m. gastrocnemius) of male Sprague–Dawley rats (FABPpm, FATP, and FAT/CD36), or as measured by immunoassay (ELISA) in heart, m. soleus, and m. extensor digitorum longus

of Wistar rats (FABPc). (C) Relative protein amounts present in the sarcolemma, as measured by quantitative Western blotting in giant sarcolemmal vesicle membranes obtained from heart and from red and white skeletal muscles, using the same tissues as for the total tissue protein level of these membrane proteins (B). The graph in the lower right corner shows the relation between sarcolemmal FAT/CD36 (arbitrary units) and the palmitate uptake rate (in pmol s–1 per mg membrane protein) as measured in giant vesicles obtained from the three muscle types. In each bar graph the highest value was arbitrarily set at 100%. Data are obtained from Refs [22, 26, 46], or are unpublished observations (total tissue protein levels of the membraneassociated proteins).

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in phosphorylation state. In this respect, rat FATP is known to contain a number of potential phosphorylation sites [48]. As already noted, others have recently provided evidence that FATP is a plasma membrane-localized (very) long-chain fatty acyl-CoA synthetase rather than a transporter, and that the previously observed FATPmediated increase of cellular LCFA uptake [28] is due to the enhancement of intracellular LCFA activation [34, 35].

19.4

Regulation of Muscular Fatty Acid Uptake 19.4.1

Acute Changes in Muscle Fatty Acid Utilization and Membrane FABPs

It is well established that muscle LCFA utilization rates can change acutely, i.e. within minutes, for instance during the transition from a resting to contracting skeletal muscle. It is also known that these LCFAs predominantly originate from extracellular sources, with mobilization of intracellularly stored LCFAs (triacylglycerols) contributing additonally to the increased rate of utilization [49]. If such acute changes in LCFA uptake are mediated, at least in part, by membrane FABPs, a mechanism must exist to acutely regulate the sarcolemmal presence and/or activity of these proteins. It is conceivable that such a mechanism could resemble the manner by which glucose transport is acutely regulated. When the muscular need for glucose increases, for instance as induced by contraction or by insulin, the membrane glucose transporter GLUT4 is translocated from intracellular sites to the plasma membrane [50]. Guided by this reasoning we have examined a possible analogy between the acute regulation of muscle glucose and LCFA uptake, and studied changes in LCFA uptake into skeletal and cardiac muscle following a contraction-induced increase in energetic need. LCFA uptake was studied using giant vesicles prepared from rat lower leg muscles after short-term (30 min) electrical stimulation via the sciatic nerve. Vesicles prepared from the resting contralateral leg muscles served as control. Uptake of palmitate was 1.4-fold higher in giant vesicles from contracting muscles than in those from controls (Fig. 19.5) [51]. In parallel, the plasma membrane FAT/CD36 content was also 1.4-fold higher in contracting muscles, while the sarcolemmal FABPpm content was unaltered. Importantly, these contraction-induced effects could be completely blunted in the presence of sulfo-N-succinimidyl-oleate (SSO), a specific inhibitor of FAT/CD36 [22]. In additon, when after termination of contraction, the muscles were allowed to recover for up to 30 min both palmitate uptake and vesicular FAT/CD36 content had decreased to similar values as found in the control muscles [51]. These short-term concomitant changes in vesicular LCFA uptake rate and FAT/ CD36 content suggest that in response to increased muscle contraction, FAT/CD36 is recruited from (an) intracellular store(s) to be associated with the sarcolemma, thereby permitting a higher LCFA uptake rate, and that upon recovery of the muscle the protein is internalized and the rate of LCFA uptake similarly decreased. Further

19.4 Regulation of Muscular Fatty Acid Uptake

A

Fig. 19.5 Translocation of FAT/CD36 from intracellular pools to the sarcolemma upon short-term electrical stimulation of adult rat hindlimb muscles. Muscles were stimulated via the sciatic nerve to contract for 30 min at 40 tetani per min, while the contralateral muscles from the same animal served as non-contracting controls. (A) Palmitate uptake rate, measured in giant sarcolemmal vesicles pre-

B

pared from these muscles, was 65% higher in the stimulated compared to control muscles. (B) Distribution of FAT/CD36 in resting and contracting muscles. After density gradient fractionation of the muscles into separate surface and intracellular compartments, FAT/ CD36 was visualized and quantified by Western blot analysis. Adapted from Ref. [51].

proof for this mechanism was obtained by subcellular fractionation of the control and contracting muscles and assessment by immunoblot of the presence of FAT/ CD36 in both the sarcolemmal and an intracellular (endosomal) fraction, and the redistribution of this protein in response to contraction [51]. The contraction-induced redistribution of FAT/CD36 then was also confirmed in the heart. For these studies we employed isolated rat cardiac myocytes which were electrically stimulated in vitro using a newly developed device [52]. With contracting myocytes initial palmitate uptake was 1.5-fold higher than with quiescent myocytes. This contraction-induced increase, just as in skeletal muscle, could be blocked by sulfo-N-succinimidyl-palmitate (SSP), suggesting that the contractioninduced increase in LCFA uptake is mediated by FAT/CD36 [52]. Whether FABPpm is also subject to contraction-induced recycling between an intracellular pool and the plasma membrane is not yet clear (see Ref. [53]). 19.4.2

Signaling Pathway for FAT/CD36 Translocation to and from the Sarcolemma

Besides contraction, muscle metabolism also is affected by hormones, in particular insulin. Exposure of skeletal muscle [54, 55] and heart to insulin markedly increases the rate of LCFA uptake and esterification. In studies in which rat hindlimbs were perfused in the absence or presence of insulin we recently observed

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that the resulting increase in LCFA uptake rate was accompanied by a translocation of FAT/CD36 from an intracellular depot to the sarcolemma [56]. More recently, similar observations were made in isolated rat cardiac myocytes (J. J. F. P. Luiken, unpublished observations). In further studies we have obtained evidence that the effects of muscle contraction and insulin on both LCFA uptake and FAT/CD36 translocation to the sarcolemma are additive (unpublished observations). This finding suggests that there are (at least) two separate intracellular pools from which FAT/CD36 can be recruited, one being sensitive to contraction and the other to insulin, or, alternatively, that there is a single depot from which FAT/CD36 can be mobilized following two independent signal transduction cascades (Fig. 19.3). This mechanism, again, resembles the well-documented manner by which muscle contraction and insulin can independently translocate the glucose transporter GLUT4 from intracellular stores to the sarcolemma [50]. The effects of insulin on FAT/CD36 translocation are mediated via the signaling protein phosphatidylinositol 3-kinase (PI3-kinase), in both muscle [56] and cardiac myocytes (J. J. F. P. Luiken, unpublished observations). This became evident from the inhibition of the insulin-inducible LCFA uptake into cardiac myocytes and skeletal muscles, when the activity of PI3-kinase was inhibited by wortmannin or LY 294002 [56]. The signaling enzymes involved in the contraction-mediated FAT/CD36 translocation have not yet been unraveled. 19.4.3

Chronic Changes in Muscle Fatty Acid Utilization and Membrane FABPs

The direction and rate of LCFA movement across the plasma membrane are determined by the transmembrane gradient of LCFAs [42, 57] and, therefore, depend on the plasma supply of LCFAs and the metabolic state of the cell. However, given the fact that both membrane-associated and cytoplasmic FABPs increase the rate of LCFA transport across the membrane, long-term alterations in the presence and/or activity of these FABPs would also have an impact on the actual (mean) rate of transport. In line with this notion are the observations that chronic changes in tissue LCFA utilization, such as induced by exercise training, nutrition, and pharmacological manipulations, are paralleled by concomitant changes in the tissue content of membrane as well as cytoplasmic FABPs (reviewed in Refs [10, 42, 43]). For instance, FAT/CD36 expression is upregulated with chronic (i.e. 7 days) stimulation of skeletal muscle concomitant with an increase in the maximal rate of LCFA transport [58]. Conversely, changes in cellular content or functioning of FABPs, such as experimentally induced by genetic manipulations (transgenic animals), lead to parallel limitations of the rate of LCFA uptake and utilization. For instance, mice with a disrupted gene encoding FAT/CD36 showed a 50–80% decreased in vivo uptake rate of iodonated LCFA analogs [59]. The factors that regulate chronic adjustments in the transcription of these proteins in heart and skeletal muscle are not yet fully elucidated. Interestingly, LCFAs themselves are capable of controlling transcriptional activity of so-called metabolic genes, including those involved in uptake, transport, and metabolic con-

19.5 Concerted Action of the Proteins Involved in Muscle Fatty Acid Uptake

version of LCFAs [60]. Studies with cultured rat neonatal cardiomyocytes showed that exposure of the cells to physiological levels of LCFAs in the surrounding medium induced approximately 3-fold increases in the mRNA levels of FAT/CD36, FABPc, and fatty acyl-CoA synthetase, together with a similar increase in the rate of cellular LCFA uptake and oxidation [61]. These data suggest that LCFAs, or their derivatives, can control their own rate of cellular utilization through fine-tuning of gene expression. Most likely, members of the family of peroxisome proliferator activated receptors (PPARs), in particular PPARa, mediate LCFA-induced modulation of gene expression [60].

19.5

Concerted Action of the Proteins Involved in Muscle Fatty Acid Uptake

Several investigators have suggested that (some of) the membrane-associated FABPs and FABPc might interact during the LCFA uptake process so as to efficiently channel these ligands from their extracellular binding sites up to their intracellular binding by FABPc [24, 41, 42]. Such a mechanism is feasible especially because it would guarantee that the cell and cellular components are optimally protected from the potentially adverse effects of LCFAs [62]. A number of observations provide evidence for such putative cooperation among the various proteins during the LCFA uptake process. First, in studies with heart and muscle giant sarcolemmal vesicles it was found that the effects of SSO, a specific inhibitor for FAT/CD36, and of anti-FABPpm antibodies on vesicular LCFA uptake were not additive. That is, addition of both compounds simultaneously, using concentrations at which they independently exerted a partial inhibition of LCFA uptake, did not inhibit LCFA uptake further than their individual degree of action [22]. This demonstrates that the facilatory effect of each of these proteins on LCFA uptake is dependent on the proper functioning of the other protein, possibly because the two proteins are in close vicinity of each other. Second, Spitsberg and co-workers [63] showed by co-immunoprecipitation experiments that in rat lactating mammary gland there is a protein–protein interaction between FAT/CD36 and heart-type FABPc (in their study referred to as mammaryderived growth inhibitor, which was later documented to be identical with HFABPc [64]). Third, the so-called a-helical domain of the FABPc molecule, which serves as a “lid” for the cavity in which the ligand is bound, was found to interact directly with an artificial phospholipid membrane [65], suggesting that this is the site of interaction between FABPc and a biological membrane. Such interaction is considered to dramatically facilitate the desorption of LCFAs from the membrane inner leaflet, a step that is generally believed to be rate-limiting in the overall process of cellular LCFA uptake [8, 15]. A simultaneous interaction of the FABPc molecule with both the phospholipid membrane (a-helical domain) and FAT/ CD36 (other part of the FABPc molecule) is not excluded. It would be of interest to examine whether the three membrane FABPs are located together in distinctive areas of the membrane so as to exert their putative

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concerted action. Using heart giant vesicles, we recently demonstrated a co-localization of FAT/CD36 and caveolin-3 [66]. Caveolin-3 is a scaffolding protein abundantly expressed in small invaginations of the plasma membrane called caveolae, which are proposed to serve as lipid delivery vehicles for subcellular organelles [11]. Whether FABPpm and/or FATP are also preferentially present in these membrane domains has not yet been studied.

19.6

Alterations in Fatty Acid Uptake and Membrane FABPs in Disease

For two major diseases that affect heart and/or skeletal muscle LCFA uptake and utilization, i.e. cardiac hypertrophy and diabetes, there are indications that the characteristic changes in the rate of muscular LCFA uptake are related to concomitant changes in the presence of membrane FABPs. In humans, the shift in myocardial substrate utilization seen during the development of hypertrophy, notably a decrease in the use of LCFAs together with an increase in that of glucose [3], has been associated with a mutation or deficiency in FAT/CD36 [67, 68]. The fact that FAT/CD36 knockout mice show impaired myocardial LCFA uptake [59] and develop cardiac hypertrophy [69] underscores the notion that FAT/CD36-mediated LCFA uptake may represent an important control site for myocardial LCFA utilization, and that FAT/CD36 deficiency and defective myocardial LCFA uptake are causally linked. In diabetes mellitus myocardial as well as skeletal muscle glucose utilization are markedly reduced and compensated for by an increase in LCFA utilization. In streptozotocin-induced diabetic rats this is accompanied by a slight increase in the expression of the genes encoding for FABPpm, FATP, FAT/CD36, and FABPc [26], while at the protein level there is a 2- to 3-fold increase in muscle content of FAT/ CD36, and a 1.3- to 1.8-fold increase in that of FABPc [47]. In view of the abovementioned observation that insulin induces FAT/CD36 translocation in muscle, it was of interest to investigate whether the cellular distribution of FAT/CD36 is also affected in the diabetic state. For this, we studied the obese Zucker rat, a well-established model for obesity and type 2 diabetes [70]. LCFA uptake into giant vesicles prepared from heart and skeletal muscle was about 1.8-fold higher in obese animals than in lean littermates [53]. Interestingly, these differences could not be associated with changes in FAT/CD36 mRNA nor tissue protein content, but in both tissues there was an increased abundance of FAT/CD36 at the sarcolemma (1.6-fold in heart, 1.8-fold in muscle) [53]. Thus, it appears that in heart and muscle of obese Zucker rats the total cellular pool of FAT/CD36 is similar to that found in lean animals, but a larger proportion of the protein is permanently relocated to the cell surface at the expense of the intracellular storage compartment, resulting in higher LCFA uptake rates. This altered relocation could be the result of either an increased mobilization of FAT/CD36 or an impairment in the rate of endocytosis. In any case, the machinery regulating the subcellular distribution of FAT/CD36 might play a pivotal role in the etiology of obesity and type 2 diabetes.

19.8 Acknowledgements

19.7

Concluding Remarks

Both membrane-associated and cytoplasmic FABPs play central roles in the uptake and intracellular transport of LCFA by heart and skeletal muscle. Their physiological significance most likely involves (i) an elevation of the LCFA transport capacity, and (ii) a careful control of the cellular handling of LCFA, thus performing a dual function of permissive transport to certain sites and sequestration from others. The emerging evidence for a concerted action of the various proteins involved further emphasizes this notion. The recent observations that LCFA uptake by heart and muscle is subject to short-term regulation involving the translocation of the membrane protein FAT/ CD36 from an intracellular depot to the plasma membrane (Fig. 19.3) indicates that the cellular FABPs not only facilitate but also regulate cellular LCFA metabolism. On the basis of these findings and the above-mentioned preliminary evidence for a concerted action among the various proteins involved it is conceivable that following its translocation to the sarcolemma FAT/CD36 associates with FABPpm to facilitate the LCFA uptake process. In this way FAT/CD36 would serve a regulatory and FABPpm a permissive role in muscular LCFA uptake. In accordance with such mechanism is our recent observation that LCFA uptake by giant vesicles from heart, skeletal muscle, liver, and adipose tissue correlates with the vesicular content of FAT/CD36 but not with that of FABPpm [66]. This hypothesis does not exclude, however, that in specific organs and/or under certain conditions FABPpm (as well as FATP) would also be subject to translocation from intracellular stores to the plasma membrane (see Ref. [53]). Finally, the significance of the membrane-associated FABPs is also clear from studies of several diseases. For instance, the notion that the altered rates of LCFA metabolism observed in obese Zucker rats could be associated with changes in cellular FAT/CD36 distribution rather than affecting the total tissue content of this protein [53] elicit the hypothesis that malfunctioning of the protein-mediated LCFA uptake process may be a critical factor in the pathogenesis of insulin resistance, and perhaps of other metabolic diseases in which lipid metabolism is altered. Future studies should be directed towards further unraveling the mechanism and regulation of cellular LCFA uptake, especially the signaling cascade(s) involved in the cellular redistribution of membrane transporters.

19.8

Acknowledgements

The authors thank J. Willems for his help in preparing the illustrations. Work in the author’s laboratories was supported by the Netherlands Heart Foundation (grant D98.012), the Canadian Institutes of Health Research, and the Ontario Heart and Stroke Foundation. Joost J. F. P. Luiken is a Dekker post-doctoral fellow of the Netherlands Heart Foundation.

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19.9

References 1

2

3

4

5 6

7

8 9 10 11 12 13 14

15 16 17 18

Van der Vusse, G. J., Glatz, J. F. C., Stam, J. C. G., Reneman, R. S. Physiol. Rev. 1992, 72, 881–940. Lopaschuk, G. D., Belke, D. D., Gamble, J., Itoi, T., Schonekess, B. O. Biochim. Biophys. Acta 1994, 1213, 263–276. Van der Vusse, G. J., Van Bilsen, M., Glatz, J. F. C. Cardiovasc. Res. 2000, 45, 279–293. Van der Vusse, G. J., Reneman, R. S. In: L. B. Rowell, J. T. Shepherd, eds. Handbook of Physiology. Integration of Motor, Circulatory, Respiratory, Metabolic Control during Exercise. American Physiology Society, 1996, pp. 952–994. Wolffe, R. R. Adv. Exp. Med. Biol. 1998, 441, 147–156. Stanley, W. C., Lopaschuk, G. D., McCormack, J. G. Cardiovasc. Res. 1997, 34, 25–33. Pan, D. A., Lillioja, S., Kriketos, A. D., Milner, M. R., Baur, L. A., Bogardus, C., Jenkins, A. B., Storlien, L. H. Diabetes 1997, 46, 983–988. Hamilton, J. A., Kamp, F. Diabetes 1999, 48, 2255–2269. Berk, P. D., Stump, D. D. Mol. Cell. Biochem. 1999, 192, 17–31. Glatz, J. F. C., Storch, J. Curr. Opinion Lipidol. 2001, 12, 267–274. Stremmel, W., Pohl, L., Ring, A., Herrmann, T. Lipids 2001, 36, 981–989. Dutta-Roy, A. K. Cell. Mol. Life Sci. 2000, 57, 1360–1372. Schaffer, J. E. Am. J. Physiol. 2002, 282, E239–E246. Goresky, C. A., Stremmel, W., Rose, C. P., Guirguis, S., Schwab, A. J., Diede, H. E., Ibrahim, E. Circ. Res. 1994, 74, 1015–1026. Hamilton, J. A. J. Lipid Res. 1998, 39, 467–481. Kleinfeld, A. M. J. Membr. Biol. 2000, 175, 79–86. Zakim, D. J. Membr. Biol. 2000, 176, 101–109. Luiken, J. J. F. P., Van Nieuwenhoven, F. A., America, G., Van der Vusse, G. J., Glatz, J. F. C. J. Lipid Res. 1997, 38, 745– 758.

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Juel, C. Biochim. Biophys. Acta 1991, 1065, 15–20. Ploug, T., Wojtaszewski, J., Kristiansen, S., Hespel, P., Galbo, H., Richter, E. A. Am. J. Physiol. 1993, 264, E270– E278. Bonen, A., Luiken, J. J., Liu, S., Dyck, D. J., Kiens, B., Kristiansen, S., Turcotte, L. P., Van der Vusse, G. J., Glatz, J. F. C. Am. J. Physiol. 1998, 275, E471– E478. Luiken, J. J. F. P., Turcotte, L. P., Bonen, A. J. Lipid Res. 1999, 40, 1007– 1016. Harmon, C. M., Luce, P., Beth, A. H., Abumrad, N. A. J. Membr. Biol. 1991, 121, 261–268. Abumrad, N., Coburn, C., Ibrahimi, A. Biochim. Biophys. Acta 1999, 1441, 4–13. Luiken, J. J. F. P., Schaap, F. G., van Nieuwenhoven, F. A., Van der Vusse, G. J., Bonen, A., Glatz, J. F. C. Lipids 1999, 34, S169–S175. Van Nieuwenhoven, F. A., Willemsen, P. H. M., Van der Vusse, G. J., Glatz, J. F. C. Int. J. Biochem. Cell Biol. 1991, 31, 489–498. Isola, L. M., Zhou, S. L., Kiang, C. L., Stump, D. D., Bradbury, M. W., Berk, P. D. Proc. Natl Acad. Sci. USA 1995, 92, 9866–9870. Schaffer, J. E., Lodish, H. F. Cell 1994, 79, 427–436. Ibrahimi, A., Sfeir, Z., Magharaie, H., Amri, E. Z., Grimaldi, P., Abumrad, N. A. Proc. Natl Acad. Sci. USA 1996, 93, 2646–2651. Ibrahimi, A., Bonen, A., Blinn, W. D., Hajri, T., Li, X., Zhong, K., Cameron, R., Abumrad, N. A. J. Biol. Chem. 1999, 274, 26761–26766. Stump, D. D., Zhou, S. L., Berk, P. D. Am. J. Physiol. 1993, 265, G894–G902. Lewis, S. E., Listenberger, L. L., Ory, D. S., Schaffer, J. E. J. Biol. Chem. 2001, 276, 37042–50. Watkins, P. A., Pevsner, J., Steinberg, S. J. Prostaglandins Leukot. Essent. Fatty Acids 1999, 60, 323–328.

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Okamoto, F., Tanaka, T., Sohmiya, K., Kawamura, K. Jpn Circ. J. 1998, 62, 499– 504. 69 Hahn, R. T., Febbraio, M., Bella, J. N., Silverstein, R. L. Circulation 1998, 98, I – 236 (abstract). 70 Halaas, J. L., Gajiwala, K. S., Maffei, M., Cohen, S. L., Chait, B. T., Rabinowitz, D., Lallone, R. L., Burley, S. K.,

Friedman, J. M. Science 1995, 269, 543– 546. 71 Glatz, J. F. C., Luiken, J. J. F. P., Bonen, A. J. Mol. Neurosci. 2001, 16, 123–132. 72 Knudsen, J., Jensen, M. V., Hansen, J. K., Faergeman, N. J., Neergaard, T. B., Gaigg, B. Mol. Cell. Biochem. 1999, 192, 95–103.

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Intestinal Fat Absorption: Roles of Intracellular Lipid-Binding Proteins and Peroxisome Proliferator-Activated Receptors Isabelle Niot and Philippe Besnard

20.1

Introduction

Lipids represent more than 40% of the daily caloric intake in the Western diet, although nutritional advice is 10% lower. This high fat supply, associated with a qualitative imbalance (excess of saturated fatty acids, high polyunsaturated fatty acids x-6/x-3 ratio) greatly contributes to the prevalence of obesity in the population, and the appearance of many diseases (e.g. atherosclerosis, non-insulin-dependent diabetes, breast and colon cancers), of which the human and social costs are dramatic. Although the small intestine is responsible for fat supply, its involvement in the etiology of these pathologies has been neglected. The fact that the cellular and molecular aspects of fat absorption are not fully elucidated may explain this paradox. Moreover, the small intestine was for many years considered only to be a selective barrier. Over the two last decades, a set of membrane and soluble proteins exhibiting a high affinity for long-chain fatty acids (LCFA) have been found in the intestinal absorptive cells. Even if the respective intestinal functions of these lipid-binding proteins (LBP) are not yet fully determined, their abundance and diversity indicate that the mechanism of fat absorption is more complex than initially thought. Therefore, an alteration in the LBP expression levels could influence the intestinal fat absorption capacity. The fact that several LBP are shown to be upregulated by fatty acids (FA) in other lipid-utilizing tissues (e.g. adipose tissue, muscle, and liver) is in keeping with this assumption. Unfortunately, an extrapolation to the small intestine of mechanistic models found in other organs remains hazardous since the gut exhibits peculiar environmental and cellular features. Indeed, the specific microclimate lining the intestinal mucosa creates a favorable environment for efficient lipid absorption. This characteristic is especially critical during the post-prandial period when the fat supply increases dramatically. Moreover, the frequent renewal of its mucosa predisposes the small intestine to nutrient-mediated short-term adaptations (reviewed in Ref. [1]). According to these new insights, the small intestine might become a target organ for pharmacological and nutritional manipulations to correct metabolic disorders secondary to chronic high fat intakes. In the present chapter, the involve-

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ment of LBP in the cellular and molecular mechanisms responsible for fat absorption will be discussed as well as their putative roles in intestinal versatility. 20.2

Intestinal LCFA Absorption: A Complex Phenomenon

Triglycerides (TG), mainly composed of LCFA (C > 16), constitute the major form of dietary lipids. Digestion of TG, which occurs essentially in the duodeno-jejunal lumen through the action of a colipase-dependent pancreatic lipase, releases 2monoglycerides and LCFA. In contrast to other energetic nutrients, LCFA show a limited solubility in aqueous solutions. To overcome this limitation, they are successively dispersed in mixed micelles in the intestinal lumen, bound to LBP in the absorptive cells (i.e. enterocytes), and transported into lipoproteins in the lymph (Fig. 20.1). 20.2.1

Can LCFA Uptake be a Rate-limiting Step for Intestinal Fat Absorption?

The mechanisms by which LCFA are transferred into the enterocyte are still a matter of debate. Three levels of complexity (membrane, cellular, and organ levels) bringing complementary informations must be considered to fully answer this crucial question. Simplified models, such as protein-free phospholipid vesicles, have been largely used to study in vitro the trans-bilayer movement of native or fluorescent FA. This transport can be viewed as occurring in three successive steps: (i) adsorption of LCFA on the membrane surface, (ii) “flip-flop” movement from the external hemileaflet of the phospholipid bilayer to the internal hemileaflet, and (iii) desorption from the bilayer into the inner of the vesicle. The adsorption step is thermodynamically favorable [2] and extremely fast. Similarly, desorption seems to be a spontaneous phenomenon, at least in a membrane model [2, 3]. The most controversial step is the trans-bilayer (flip-flop) transport. To determine the time course of native LCFA movement through the phospholipid bilayer, Kleinfeld and co-workers have used the fluorescent probe ADIFAB trapped within lipid vesicles [4]. Times found for FA trans-bilayer transfer were within a 70 ms to 10 s range, according to the FA type, vesicle size, and temperature. Therefore, these authors concluded that the flip-flop step is rate-limiting. By extrapolation, they suggest that passive diffusion alone might be insufficient to support the metabolic activity of cells known to have high requirements for LCFA, such as cardiomyocytes. Contradictory findings have been reported by Kamp and collaborators [5, 6]. Using the pH-sensitive fluorophore pyranin, they have shown that the presence of protonated FA in the medium induced a short-term decrease in the pH in the internal compartment. This finding is consistent with a rapid movement of protonated LCFA across the lipid bilayer, followed by their ionization in the vesicle and diffusion of protons to pyranin. A flip-flop rate shorter than 20 ms independent of

20.2 Intestinal LCFA Absorption: A Complex Phenomenon

Fig. 20.1 Lipid binding proteins involved in intestinal long-chain fatty acid absorption. The main steps of FA absorption are depicted: (1) LCFA uptake by both passive diffusion and protein-mediated transport, (2) trafficking of LCFAs and long-chain acyl-CoA, (3) lipoprotein formation and exocytosis into

lymph. LBP, lipid binding protein; FA, fatty acid; LCFA–, ionized fatty acids; LCFAH, protonated fatty acids; LCA, long-chain acyl-CoA; TG, triglycerides; PL, phospholipids; CE, cholesterolesters; ACS, acyl-CoA synthetases; ER, endoplasmic reticulum; VLDL, very low-density lipoproteins.

FA chain length was reported. By contrast, the transmembrane movement of negatively charged LCFA was several orders of magnitude slower. These last data strongly suggest that LCFA can rapidly cross the cell surface by passive diffusion when they are protonated. According to this conclusion, LCFA were found to distribute across model membranes [7], and plasma membrane from intact 3T3-L1 adipocytes [8] in response to imposed pH gradients.

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Nevertheless, although the membrane models are useful for studying step by step the flux of free FA across a phospholipid bilayer in a controlled environment, the results obtained are not easily transposable to a biological system. Kinetic studies realized in cultured cells exhibiting a rapid LCFA influx (i.e. hepatocytes, cardiomyocytes, adipocytes, and enterocytes) support the existence of a proteinmediated transport system in parallel to the passive diffusion mechanism, at least, at low LCFA concentrations. In isolated rodent enterocytes and in the human enterocyte-like Caco-2 cell line, transport of LCFA in both apical and basolateral membranes is saturable [9, 10]. Moreover, competitive inhibition by structurally related LCFA has been reported in Caco-2 cells [11]. The identification of several unrelated proteins exhibiting an affinity for LCFA in the brush border membrane (BBM) of the enterocyte correlates quite well with the implication of a proteinmediated process in the LCFA influx in the absorptive intestinal cells. However, the data obtained from protein-free phospholipid (PL) vesicles and cultured cells must be interpreted in the context of the natural organ environment to become physiologically relevant. In vivo, the first step of the fat absorption is the shuttling of LCFA from the luminal water phase to the vicinity of BBM of enterocytes by diffusion through the unstirred water layer (Fig. 20.1). With a thickness from 50 to 500 lm [12], the unstirred water layer is a low renewal area produced by the trapping of water molecules in a glycoprotein network composed of the mucus and the glycocalyx lining the intestinal epithelium. This is a low pH compartment generated by the presence of an efficient Na+/H+ antiport exchange system located in the BBM [13, 14]. The disruption of the Na+/H+ exchanger by amiloride decreases, in a dose-dependent manner, oleic acid uptake by rat jejunal sheets or rabbit BBM vesicles [15], demonstrating the importance of this acidic microclimate in FA absorption. At the surface of the BBM, LCFA are protonated as soon as the local pH becomes lower than their pKa. This phenomenon, by reducing the LCFA solubility in micelles, induces the release of protonated LCFA near the BBM [16], and likely facilitates their cellular permeation by passive diffusion (Fig. 20.1). Indeed, the un-ionized FA have a greater membrane permeation than their corresponding ionized species [5, 6]. Such an intestinal specificity explains why the kinetic characteristics found in other FA-utilizing organs (e.g. adipose tissue, cardiac and skeletal muscles, liver), suggesting that the predominance of protein-mediated transport in LCFA uptake cannot be extrapolated to the small intestine. The same restriction must be applied to intestinal cells in culture, which do not reproduce this complex extracellular microclimate. The physiological role of enterocytes being to allow an efficient absorption of nutrients, this specific extracellular environment is essential, especially during the post-prandial period, when the absorptive cells are subjected to a dramatic increase in the lipid load. In this case, the predominance of passive diffusion might explain why FA uptake cannot become rate-limiting for intestinal fat absorption. Such an unsaturable transport system would explain why the fecal fat loss is usually below 5% in healthy humans in spite of the high fat content found especially in the Western diet.

20.2 Intestinal LCFA Absorption: A Complex Phenomenon

20.2.2

Why do Enterocytes Express Different Membrane LBP?

The above conclusions raise an important question: Why is it in the physiological interests of the small intestine to express a set of membrane proteins exhibiting a high affinity for LCFA? Four unrelated LBP have been identified in the BBM of the enterocyte: plasma membrane fatty acid binding-protein (FABPpm) [17], fatty acid transport protein 4 (FATP4) [18], caveolin-1 (Cav1) [19], and fatty acid transporter (FAT/CD36) [20] (Tab. 20.1). In the gut, any research on the physiological function of a protein must take into account its expression along two spatial axes. Indeed, the small intestine is an heterogeneous organ characterized by a variable genotypic expression along the gastro-colic and crypt-to-villus axes at the origin of a functional specialization. For instance, the proximal part of the gut (i.e. the duodeno-jejunum) is known to be the main site for the absorption of energetic nutrients, while the ileum is the exclusive intestinal segment where bile acids and vitamin B12 are actively reabsorbed. Similarly, phenotypic changes occur as cells differentiate along the cryptto-villus axis. For this reason, only the enterocytes located in the upper two-thirds Tab. 20.1 Plasma membrane lipid binding-proteins found in the brush border membrane of the enterocyte.

LBPs

Structure

Tissue expression

Ligand(s)

Main characteristics

Plasma membrane fatty acid-binding protein (FABPpm/AspAT)

43-kDa peripheral protein associated with plasma membrane

Small intestine, adipose tissue, muscles, liver, placenta

LCFA LPC CS

Identical to mitochondrial aspartate-aminotransferase (AspAT)

Caveolin-1 (Cav-1)

22-kDa hairpinlike protein anchored in the plasma membrane by a unique sequence

Small intestine, adipose tissue, heart

LCFA CS

Marker of caveolae. Putative interaction with various signaling molecules

Fatty acid transporter (FAT/CD 36)

53–88-kDa hairpin-like protein with two membrane domains located near the Nand C-terminus tails

Small intestine, adipose tissue, muscles, heart

LCFA

Found in caveolae. Putatively homo/ heterodimerized, and associated with Src kinases

Fatty acid transport protein 4 (FATP4)

63-kDa protein with a short Nterminus extracellular sequence

Small intestine, liver, brain, kidneys

LCFA

Contains an ATP binding domain.

LCFA, long-chain fatty acids; LPC, lyso-phosphatidylcholine; CS, cholesterol.

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of the villi are considered to be fully absorptive cells. Spatial changes in the absorptive area also occur in the intestinal epithelium. Indeed, it is well known that the number and the height of villi are greater in the proximal than in the distal part of the small intestine [21]. This intestinal architecture helps to explain why the main site of fat absorption in healthy humans or animals subjected to a standard dietary lipid load is located in the upper site of jejunal villi.

20.2.2.1 FABPpm/mAspAT: A Protein in Search of a Function

The plasma membrane fatty acid binding protein (FABPpm) is a 43-kDa peripheralassociated membrane protein expressed in organs with high lipid requirements [22]. It is thought to adhere on the plasma membrane through a specific N-terminal peptide [23]. Immunofluorescence studies realized on rat intestine have shown that FABPpm is expressed in the jejunum and to a lesser extent in the ileum. FABPpm protein is found in the BBM and in the lateral cell border of enterocytes located both in the villi and crypts [17]. Binding studies revealed that FABPpm binds various LCFA with an apparent dissociation constant (Kd) of 80 nM [9]. The protein also exhibits a high binding affinity for lysophosphatidylcholine, monoglycerides, and cholesterol [9]. FABPpm is identical to mitochondrial aspartate-aminotransferase (mAspAT), an enzyme found in the cytoplasm and mitochondrial matrix, in which it catalyzes the transamination reaction linking the urea and Krebs cycles [24]. FABPpm/mAspAT isolated from plasma membranes lacks the leader sequence with which pre-mAspAT is initially synthetized. Therefore, the different subcellular targeting of the enzyme (i.e. mitochondria, cytoplasm, or plasma membrane) could be explained by a post-translational maturation [25]. Overexpression of the mAspAT expression vector in 3T3 fibroblasts triggers the appearance of an FABPpm-like protein in the cell surface and induces an increase in the cellular FA uptake [25]. According to these data, preincubation of jejunal explants with a mono-specific FABPpm antibody results in a partial, but significant, inhibition of [3H]oleate uptake, suggesting that FABPpm also plays a role in the intestinal absorption of LCFA [9]. Nevertheless, large amounts of antiserum were required to achieve such an inhibition. Moreover, conflicting reports regarding the ability of FABPpm to induce LCFA transport in Xenopus laevis oocytes raise a doubt about the physiological role of this protein in cellular LCFA uptake [25, 26]. Data on the regulation of FABPpm/mAspAT expression in the small intestine are presently lacking. Even if results obtained during fasting in red skeletal muscles [27], diabetes in adipocytes from Zucker rats [28], or ethanol load in human hepatoma HepG2 cells [29] are consistent with an involvement of FABPpm/mAspAT in FA metabolism, the precise role of this protein in intestinal fat absorption remains unclear.

20.2 Intestinal LCFA Absorption: A Complex Phenomenon

20.2.2.2 FATP4: A Plasma Membrane-associated ACS-like Protein?

Fatty acid transport proteins (FATP) are 63-kDa proteins first identified in 3T3-L1 pre-adipocytes by expression cloning strategy on the basis of their ability to facilitate LCFA uptake [18]. As the glucose transporter family, five and six different isoforms of FATP have been found in the rodent and human, respectively [30]. Each isoform exhibits a specific pattern of tissue expression [30]. For example, FATP5 and FATP2 are the major isoforms expressed in the liver [31], while FATP1 is essentially found in adipose tissue and FATP4 in the small intestine [32]. In this last organ, FATP4 expression level is especially high in the jejunum and the ileum, as compared to the duodenum [32], but lacking in the colon. The expression of FATP4 is sustained in the BBM of mature enterocytes located in the top of villi, while it is low or lacking in undifferentiated cryptic cells [32]. By reference to the only known predictive structure (FATP1), it appears that this LBP is essentially oriented toward the cytosol with only a short N-terminus extracellular sequence [31]. Since this segment does not contain any putative LCFA binding site, a role for FATP as FA pool formers at the surface of cells is unlikely. The mechanism by which FATP facilitate cellular FA uptake is not yet fully understood. It is thought to take place through an AMP-dependent mechanism [33]. The high amino acid sequence identity between FATP and acyl-CoA synthetases (ACS) suggests that these proteins might be plasma membrane ACS. In agreement with this observation, it was demonstrated that membrane extracts of Cos-1 cells transfected with a murine FATP4 expression vector exhibit elevated ACS activity, preferentially for very LCFA [34]. By contrast, murine FATP1 cellular overexpression enhances uptake of LCFA without any significant change in ACS activity [35]. Presently, it is unclear if FATP are either ACS-associated LBP proteins or plasma membrane ACS. The importance of the FATP4 expression level in intestinal LCFA uptake is supported by the fact that the induced depletion of FATP4 protein by an antisense strategy leads to a significant decrease in LFCA uptake in isolated enterocytes [32]. However, the physiological importance of FATP4 remains to be determined in vivo.

20.2.2.3 Caveolin-1: An LBP and a Caveolae Marker

Caveolins (Cav) are 21–24-kDa proteins mainly found in specific non-clathrincoated surface invaginations of the plasma membrane called caveolae. Three distinct caveolin isotypes have been identified. Cav-1 is found associated with Cav-2 in stable hetero-oligomers [36]. Therefore, they have nearly identical tissue distributions. They are especially expressed in well-differentiated cells such as enterocytes, while Cav-3 is mainly found in the muscle [37]. Cav-1 is an hairpin-like protein facing the cytosol and anchored in the plasma membrane through a unique peptidic sequence [38]. Since Cav-1 plays a crucial role in caveolae biogenesis, it is considered to be a specific marker of these subcellular structures [39]. Caveolins seem to exert pleiotropic functions. They are implicated in cellular trafficking, more particularly in the targeted delivery of specific molecules (e.g. cholesterol) to

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organelles (especially endoplasmic reticulum and Golgi apparatus) [40]. A role in signal transduction is also strongly suggested by the demonstration of direct interactions of Cav-1 with resident caveolar signaling molecules (G-protein subunits, Ha-Ras, Src tyrosine kinases or epidermal growth factor receptor) [41]. Since Cav1 also exhibits a binding affinity for LCFA [42, 43], it must be considered as a putative LBP. Cav-1 expression has been reported in both human intestine biopsies and in well-differentiated enterocyte-like Caco-2 cells [44], in which it appears to be mostly confined to the apical membrane. However, the implication of Cav-1 in LCFA uptake and trafficking remains to be established in the enterocyte. Nevertheless, such a possibility is supported by the recent demonstration of an accumulation of Cav-1 in the membrane surrounding lipid droplets in fibroblasts subjected to a LCFA load [45]. Moreover, Cav-1-null mice exhibit a hypertriglyceridemia phenotype which becomes especially dramatic during the post-prandial period. Analysis of the lipoprotein profile of fed Cav-1–/– mice reveals a strong rise in the fraction corresponding to chylomicrons and VLDL [46], suggesting that Cav-1 protein plays a role in the metabolic fate of LCFA in the enterocyte.

20.2.2.4 FAT/CD36: An Involvement in a Vesicular Trafficking of LCFA?

The fatty acid transporter (FAT) is a 88-kDa transmembrane glycoprotein identified, then cloned, in rat adipocytes by labeling with sulfo-N-succinimidyl derivatives of LCFA under conditions where LCFA uptake was significantly inhibited (see Chapter 1). It is found in various tissues characterized by a high FA metabolism, including the small intestine [20, 47, 48]. Rat FAT is homologous to the human scavenger receptor CD36 highly expressed in platelets, monocytes/macrophages, and endothelial cells [47]. Despite a large spectrum of binding specificity, FAT/CD36 is considered to be an LBP since it can bind ionized LCFA with an affinity in the nanomolar range and a stoichiometry of 3 mol FA by 1 mol protein [49, 50]. Amino acid sequence analysis of FAT/CD36 predicts two transmembrane domains located near the N- and C-terminal tails resulting in a hairspin configuration with a large extracellular hydrophobic domain [47]. A dual palmitoylation might target FAT/CD36 into caveolae, where it has been shown to be co-localized with Cav-1 [51–53]. Concordant data strongly suggest that FAT/CD36 plays a significant role in FA uptake by adipose tissue and muscles (reviewed in Ref. [54]). In the small intestine, its expression level (mRNA and protein) is especially high in the major site of fat absorption, i.e. duodeno-jejunum [48, 55]. Immunocytochemical studies in humans and rat demonstrate that FAT/CD36 is strictly localized in the BBM of well-differentiated enterocytes [48, 55] (Fig. 20.2 A). Intestinal FAT/CD36 gene expression has been shown to parallel the lipid contents of the diet. Indeed, jejunal mRNA levels are significantly upregulated when rats are chronically subjected to a high-fat diet [48] and downregulated when they are fed a low-fat chow [56]. This positive correlation, as well as its location, supports an implication of FAT/CD36 in the intestinal absorption of dietary fat. How-

20.2 Intestinal LCFA Absorption: A Complex Phenomenon Fig. 20.2 Lipid-mediated disappearance of FAT/CD36 from the apical surface of the intestinal epithelium in the rat. Immunolocalization of FAT/CD36 in the rat jejunum. (A) 48 h fasted rats, (B) 6 h after refeeding a standard laboratory chow containing 3% lipid in mass, (C) 6 h after refeeding an alipidic meal. Magnification ´400.

ever, the involvement of FAT/CD36 as an efficient FA transporter in the small intestine during the post-prandial period (i.e. high fat supply) remains questionable because its Kd value is in the nanomolar range. Since FAT/CD36 is found in caveolae, it might concentrate ionized LCFA in the cholesterol/sphingolipid-rich segments of the intestinal plasma membrane, leading to a selective FA uptake by a vesicular transport. In good agreement with this theory, we recently found that the re-feeding of starved rats with a standard laboratory chow containing 3% lipids in mass leads to a short-term disappearance of FAT/CD36 protein from the BBM of enterocytes (Fig. 20.2 B). Since this phenomenon is associated with a rise in the intracellular FAT/CD36 content (data not shown), we propose the existence of a dietary-mediated internalization of FAT/ CD36 in the small intestine. Interestingly, this translocation appears to be strictly lipid-dependent, since it is not reproduced when animals were re-fed an alipidic

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meal (Fig. 20.2C). This new in vivo observation might explain why a decrease in Vmax was obtained in isolated enterocytes from rats previously subjected to an intra-duodenal oleate load [57]. Intracellular movement of FAT/CD36 has recently been reported in other cell types. In muscles, FAT/CD36 is able to translocate rapidly from an intracellular pool to the sarcolemna during contraction, leading to an increase in the cellular FA uptake [58]. This membrane recruitment is an insulin- and a leptin-dependent phenomenon [59, 60]. Likewise, a translocation from cytosol to plasma membrane has also been demonstrated in type II pneumocytes in response to an increase in cellular cholesterol, providing a short-term regulation of FA uptake [61]. Taken together, these new data indicate that FAT/CD36 likely contributes to a physiological adaptive response of skeletal and cardiac muscles to environmental challenges (e.g. acute exercise). In the small intestine, the physiological impact of intracellular FAT/CD36 movement remains to be understood. The lack of a dramatic intestinal phenotype in FAT/CD36-null mice seems to preclude a fundamental role of this LBP in intestinal absorption, at least in the standard dietary condition. In brief, in the small intestine, LCFA permeation involves both a passive diffusion and a protein-mediated process. However, the relative contribution of passive versus facilitated process must to be determined and the simultaneous expression of four unrelated plasma membrane LBP remains intriguing. Their specific structural features and expression patterns along the small intestine suggest subtle in vivo specializations which might be highlighted by the use of genetically transformed mice in a near future. 20.2.3

Do the Different Soluble FABPs Exert the Same Function?

Once in the enterocyte, LCFA are either reversibly bound to soluble fatty acid binding proteins (FABP) or esterified in long-chain acyl-CoA esters (LCA) (Fig. 20.1). The FABP belong to a multigenic family of 14–15-kDa soluble proteins exhibiting a high affinity for various hydrophobic molecules (LCFA, bile acids or retinoids) [62]. Two different FABP are abundantly expressed in the small intestine: the intestinal type (I-FABP), which is strictly confined to this organ, and the liver type (L-FABP), also found in the liver and kidney. The members of the FABP family exhibit a similar tertiary structure that consists of two a-helices (aI, aII) and 10 antiparallel b-strands (bA–bJ) organized in two almost orthogonal b-sheets forming an hydrophobic pocket. [63]. A “portal region” consisting of the a-helices connected to the bC/bD- and bE/bF-strands allows the entry and the exit of LCFA [63]. The respective functions of these two soluble FABP in the small intestine is not yet fully established. It is likely that they may exert both common and specific physiological roles in this organ. From different transfection studies carried out either in cell lines in which I-FABP or L-FABP are lacking, such as the fibroblastic L-cells [64] and pluripotent mouse embryonic stem ES cells [65], or in which

20.2 Intestinal LCFA Absorption: A Complex Phenomenon

only one FABP type is constitutively expressed, such as hBRIE 380i (+I-FABP/–LFABP) [66] and HepG2 (+L-FABP/–I-FABP) [67], it can be concluded that LCFA uptake and diffusion at least in transfected cells are increased by both I-FABP and L-FABP. By contrast, convergent data indicate that I-FABP targets LCFA preferentially towards the TG pathway [66, 68–72], while L-FABP is rather involved in PL formation [73]. Comparison of I-FABP and L-FABP localization along the small intestine, binding properties, and gene regulation also favor a functional specialization. Even if these two FABP are co-expressed in the gut, subtle differences in the pattern of expression along the small intestine has been found in the mouse. Indeed, I-FABP mRNA levels increase from the duodenum to reach the highest levels in the proximal ileum, while the highest L-FABP expression occurs in the proximal jejunum then progressively falls to an undetectable level in the distal ileum (Fig. 20.3) [74]. Comparison of the binding properties (stoichiometry, specificity, and affinity) also reveals strong differences. Indeed, I-FABP only binds LCFA with a ratio of one FA for one protein. By contrast, L-FABP can bind two LCFA as well as a large number of bulky hydrophobic molecules including bile acids, heme, various xenobiotics, and carcinogens, but generally with a ratio of 1 for 1 (Fig. 20.3). The binding affinities of these two FABP have been determined accurately by Richieri and collaborators for LCFA [75]. L-FABP and I-FABP have similar Kd values for saturated LCFA; by contrast, I-FABP exhibits a lower avidity for unsaturated FA than L-FABP (Fig. 20.3). Finally, I-FABP and L-FABP genes appear to be under the control of different regulatory pathways since L-FABP gene expression is transcriptionally upregulated by LCFA [76], while a direct FA-mediated modulation of I-FABP gene expression has never been highlighted as well in vivo as in vitro [77–79]. By contrast, in enterocyte-like hBRIE 380 cells, the peptide YY (PYY) specifically induces I-FABP mRNA levels [80]. This gut regulatory peptide is secreted mainly by ileal endocrine cells, especially when dietary fat reach this distal part of the gut, for instance after a dietary overload [81]. The fact that PYY can act as a paracrine agent [82] might explain why I-FABP induction in rats subjected to a high fat diet occurs only in the ileum [83]. More recently, a downregulation of I-FABP expression by the epidermal growth factor (EGF) has been reported in highly differentiated enterocyte-like Caco-2 cells. This growth factor, found in the intestinal lumen, contributes to re-epithelialization after intestinal mucosa injury. It is noteworthy that EGF does not affect LFABP expression [71]. The highlighting of a human polymorphism in the gene coding for I-FABP (FABP2) has greatly contributed to a better understanding of its physiological importance in TG-rich lipoprotein synthesis. One base substitution in codon 54 of the FABP2 gene leads to the change of an Ala to a Thr. Initially found in the Pima Indians, this substitution is associated with a high TG plasma level, an insulin resistance [68], and an increase in the body mass index [84]. The fact that the muted Thr54-I-FABP exhibits a 2-fold greater affinity for LCFA than the wild Ala54-protein may explain these metabolic disturbances [68]. Indeed, a greater avidity of the mutant I-FABP for LCFA could lead to an increase in both cellular

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Fig. 20.3 Main differences in the distribution, regulation, and binding characteristics of soluble fatty acid binding proteins (FABP) in the small intestine. I-FABP and L-FABP mRNA distribution along the cephalo-caudal axis of the small intestine as determined in Swiss mice. The small intestine was divided into 10 equal segments of 5 ± 0.3 cm from the pylorus

to the ileocecal valvula, and the mucosa of each segment was scraped off. Steady-state levels of FABP mRNA were determined by Northern blotting. Curves were derived from three independent experiments (nine mice per data).The curves showing the modification of the binding affinity of FABP as a function of FA unsaturation are derived from Ref. [75].

FA uptake and TG-rich lipoprotein synthesis [68]. In accordance with this assumption, a dramatic rise in LCFA transport and TG secretion is found in Thr54-IFABP-transfected Caco-2 cells compared with cells transfected with the wild isoform [69]. Similar data have been recently reported in the human jejunal organ culture model in which the Thr54-encoding allele appear to be associated with a dramatic rise in TG synthesis and chylomicron output [70]. It is noteworthy that these metabolic disturbances occur without modification of I-FABP and L-FABP levels and in the absence of a neuroendocrine influence. Taken together, these data strongly suggest that I-FABP is involved in the target-

20.2 Intestinal LCFA Absorption: A Complex Phenomenon

ing of dietary LCFA towards the endoplasmic reticulum, where they participate to the synthesis of TG-rich lipoproteins. The fact that an EGF-mediated inhibition of I-FABP is associated with a decrease in [14C]palmitate uptake, TG synthesis, and secretion in Caco-2 cells is in good agreement with this assumption [71]. If compelling evidence demonstrates the I-FABP involvement in TG-rich lipoprotein synthesis, an implication of L-FABP in this pathway is not excluded, as it is suggested by the lack of dramatic phenotype found in I-FABP-null mice [85]. Such a redundant function might be physiologically useful especially during a high fat supply. In the intestinal cells, LCFA are mainly bound to L-FABP (predicted ratio of binding of FA to L-FABP/I-FABP = 3.3) [72], therefore it is likely that L-FABP can also exert buffering action to protect the cell against the harmful effect of an excess of free FA. The fact that L-FABP gene expression is modulated by the lipid content of the diet is in keeping with this assumption. This positive regulatory loop might be essential for the maintenance of the functional integrity of the intestinal mucosa, which is absolutely required for an efficient FA absorption. A part of these functional differences can be explained by structural specificities. Using a resonance energy transfer assay, Hsu and Storch have demonstrated that the transfer of fluorescent anthroyloxy-FA (AOFA) from I-FABP or L-FABP to acceptor membrane vesicles occurred by different molecular mechanisms [86]. For I-FABP, the transfer of AOFA requires a direct collisional interaction with the PL bilayer, while the FA exchanges between L-FABP and membranes are carried out by aqueous diffusion. Collisional exchanges occur by ionic interactions between a few amino acid residues of the helicoidal domain of I-FABP with anionic PL of membranes. Indeed, in helix-less mutant I-FABP obtained by site-directed mutagenesis, the collisional exchanges are totally suppressed. In these conditions, the transfer of the AOFA occurs by aqueous diffusion, as for L-FABP [87]. It is thought that the collision with a target membrane yields a conformational change in the flexible region of I-FABP backbone constituted by the distal half of the aII helix and the bC–bD turns, producing the “hinged opening” of the portal domain and the release of FA [88]. This dynamic domain seems to play a critical role in the binding characteristics of I-FABP. Therefore, a single modification of its amino acid sequence can have significant physiological consequences. A good illustration of this is that a punctual mutation in the portal domain such as the Ala54Thr mutation deeply affects the binding affinity of I-FABP, leading to a dramatic alteration in lipid metabolism. The fact that LCFA delivered on the apical side of the enterocyte are preferentially bound to I-FABP [72] ensures their dynamic transfer towards a cellular compartment devoted to TG synthesis. In summary, although I-FABP and L-FABP are related proteins with a comparable tertiary structure, the weak homology of peptidic sequences (less than 30% in the rat) leads to important physico-chemical differences. The collisional transfer of ligands from I-FABP to acceptor membranes may provide an efficient mechanism for LCFA desorption from the apical membrane of enterocytes then targeting to specific organelles, such as endoplasmic reticulum, facilitating the synthesis of TG-rich lipoproteins. Therefore, I-FABP, the expression of which is restricted to the small intestine, might be preferentially involved in the supply of

371

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20 Intestinal Fat Absorption: Roles of Intracellular Lipid-Binding Proteins

dietary lipids to the organism. L-FABP, through the control of the intracellular unesterified FA level not only may protect the cell against the detergent effects of free FA, but also may play a role in gene regulation of FA target genes, since LFABP is also found in the nucleus where it is thought to modulate the activity of transcription factors [89]. 20.2.4

ACBP: A Universal Long-chain Acyl CoA Transporter

Thio-esterification of LCFA in long chain Acyl-CoA (LCA) is an obligatory step initiating FA metabolism. It is catalyzed by a set of membrane-associated ACS (Fig. 20.1). The newly synthesized LCA are bound to a specific carrier protein, the acyl-CoA-binding protein (ACBP). This LBP is a ubiquitous 10-kDa soluble LCA transporter conserved from yeast to mammal (see Chapter 8). The 86-amino-acid residues of ACBP are folded in four a-helices, forming a boomerang structure [90, 91]. The acyl chain of the LCA is buried in a hydrophobic groove of the binding pocket, in which it is totally protected from the aqueous solvent by its acyl-CoA head [92, 93]. ACBP binds both medium- and long-chain LCA with a stoichiometry of 1:1 and an affinity in the nanomolar range [93, 94]. In the small intestine from rats and mice, ACBP is co-expressed with FABP in absorptive cells [95]. In rat, similar ACBP mRNA levels are found along the cephalo-caudal axis of the small intestine (I. Niot, P. Besnard, unpublished data), in contrast to what is observed for L-FABP and IFABP. The precise function of ACBP in this organ has not yet been determined, however, ACBP could contribute to lipoprotein synthesis since it has been reported to regulate the microsomal LCA disposal for TG and PL synthesis [92]. The fact that an abrogation of ACBP gene expression in pre-adipocytes leads to a drastic reduction in TG accumulation [96] is in keeping with this hypothesis. The detection of ACBP and LCA in the nucleus of different cells [97] suggests a possible interference of ACBP expression and/or LCA disposal in fundamental regulatory pathways [96]. 20.2.5

An Integrative Working Model

This overview, completed with theoretical considerations, allows us to propose the following working model (Fig. 20.4). During the post-prandial period, the high FA concentration found in the intestinal lumen and the specific low pH microenvironment lining the BBM produce a massive influx of protonated LCFA into the enterocyte, mainly by passive diffusion. This favorable concentration gradient is maintained by a range of cellular LBP (FATP4, I-FABP, L-FABP, and ACBP). Indeed, the flip-flop transfer of LCFA through the apical membrane of the enterocyte is facilitated by the presence of high concentrations of soluble FABP which promote the membrane desorption of FA by either a collisional process (I-FABP) or aqueous diffusion (L-FABP). The plasma membrane-associated ACS-like activity of FATP4, which generates LCA, also contributes to the reduction of cytoplasmic LCFA concentration. Moreover, it creates an unidirectional flux of LCFA, since LCA are unable to

20.2 Intestinal LCFA Absorption: A Complex Phenomenon

Fig. 20.4 Intestinal absorption of long-chain fatty acids: a working model. For a detailed explanation see the text. LCFA, long-chain fatty acids; LCA, long-chain acyl-CoA; TG, triglycerides; PL, phospholipids; CE, cholesterol esters; FATP4, fatty acid transport protein 4;

FAT/CD36, fatty acid transporter; Cav-1, caveolin 1; I-FABP, intestinal fatty acid binding protein; L-FABP, liver fatty acid binding protein; ACBP, acyl-CoA binding protein; ACS, acyl-CoA synthetases .

cross the plasma membrane. This vectorial transport might be reinforced by LCFA metabolization supported by soluble LBP (L-FABP, I-FABP, and ACBP), which are thought to facilitate LCFA/LCA trafficking and targeting. Alternatively or complementary to the diffusional process, FAT/CD36 and Cav-1, which are known to be caveolae components, might be implicated in a LCFA vesicular transport system. Such a high-capacity transport system might either provide LCFA to the endoplasmic reticulum to form TG or might constitute a LCFA or TG waiting pool for eventual lipoprotein synthesis, which is known to be the rate-limiting step in fat absorption [98]. Nevertheless, the existence of this last route, suggested by the FA-mediated disappearance of FAT/CD36 from BBM, remains to be fully demonstrated in the small intestine. In this model, the concomitant expression of membrane and soluble LBP facilitates the fat delivery to the organism. This concept suggests that LBP can be subjected to coordinated regulation of their expression.

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20.3

Intestinal LCFA Absorption: A Phenomenon Putatively Adaptable to the Lipid Content of the Diet

An adaptation of the small intestine to changes in the lipid content of the diet might greatly contributes to the metabolic efficiency of the fat absorption. Compelling evidence indicates that the FA-mediated regulation of both gene expression and modulation of the absorptive area might govern this intestinal versatility. 20.3.1

PPAR and Coordinated LBP Regulation

It is now well established that dietary lipids can regulate the transcription rate of genes implicated in their own metabolism. Some of these transcriptional effects are mediated through LCFA binding and activation of nuclear receptor termed peroxisome proliferator activated receptors (PPAR). Three different PPAR subtypes are known: PPARa, which is mainly found in the liver, promotes LCFA catabolism [99]; PPARc is expressed predominantly in the adipose tissue, where it regulates adipocyte differentiation and lipid storage [100, 101] and in the colon [102]; and PPARb/d is more ubiquitous since it is found in all tissues characterized by a high lipid metabolism [103]. This large distribution may explain why the function of PPARb/d remains elusive. Nevertheless, the dramatic phenotype displayed by PPARb/d-null mice (i.e. placental defect, altered myelination and epidermal cell proliferation, drop in fat stores) demonstrates that this nuclear receptor exerts crucial functions in various tissues [104]. PPARb/d appears especially implicated in lipid metabolism since its gene is known to induce the proliferation and differentiation of pre-adipocytes [105, 106] and to increase reverse cholesterol transport [107]. In rodent small intestine, PPARb/d is predominantly expressed, followed by PPARa and PPARc respectively [108]. Moreover, PPARb/d exhibits a similar expression throughout the cephalo-caudal axis, while the expression levels of PPARa and PPARc decrease progressively from the duodenum to the ileum [102, 103] (Tab. 20.2). Therefore, the three PPAR isotypes are co-localized in the main site of lipid absorption (i.e. jejunal mucosa). The physiological interest of this co-expression is not yet determined. It might constitute a redundant or an autoregulatory system if PPAR are simultaneously expressed in the same cells. First, PPARa is not absolutely required in the gut to reproduce FA-mediated induction of L-FABP gene expression, in contrast to what is found in the liver, where PPARb/d and PPARc are barely expressed [76]. Secondly, PPARb/d has been recently reported to act as an intrinsic transcriptional repressor for PPARa [109]. Nevertheless, a coordinated FA-mediated upregulation of intestinal LBP could be envisioned since FATP1 [101], FAT/CD36 [110], L-FABP [76], and ACBP [111] promoters contain functional PPAR responsive elements (PPRE) (Tab. 20.2). According to this hypothesis, an induction of FAT/CD36 mRNA levels occurred in jejunum from rats subjected to a chronic high fat diet [48]. Similarly, it has been shown that a fat-enriched diet or a direct lipid infusion

+++

+++ ++

++

++++ +

PPARa

PPARb/d PPARc

+++ +/–

+

– ud

Induction

Induction ud

–

FAT/CD36 L-FABP, FATP1 ACBP

LBPs with functional PPRE

The levels of expression determined by Northern blotting in male rodents fed a standard laboratory chow. (+/–), barely detectable; (+), weak expression; (++), moderate expression; (+++), high expression; (++++), very high expression; ud, undetectable. I. Niot, P. Besnard (unpublished data).

+++ +

++

Post-prandial period

Fasting

Ileum

Duodenum Jejunum

Nutritional regulation

Localization along the cephalo-caudal axis

Relative expression levels

PPAR isoforms

Tab. 20.2 Main characteristics of PPARs in the small intestine.

20.3 Intestinal LCFA Absorption: A Phenomenon Putatively Adaptable to the Lipid Content 375

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20 Intestinal Fat Absorption: Roles of Intracellular Lipid-Binding Proteins

in the intestinal lumen induced L-FABP gene expression [74]. A combination of experiments using both PPARa-null mice and a specific PPARb/d agonist has demonstrated that the L-FABP gene can be a PPARb/d target gene in this organ. This suggests that PPARb/d which is the major intestinal PPAR isotype, could contribute to metabolic adaptation of the small intestine to changes in the lipid content of the diet [74]. The fact that PPARb/d gene expression is specifically induced during the post-prandial period in contrast to PPARa is in keeping with this putative function (Tab. 20.2). The construction of animal models displaying gut-specific alterations of PPARb/d activity, by overexpression of either the native PPARb/d protein or a dominant-negative mutant, are required to determine the contribution of this PPAR isotype to fat absorption. PPARa or PPARb/d activation is dependent on the intracellular bio-availability LCFA or metabolic derivatives. Since L-FABP exhibits a high binding affinity for both LCFA and eicosanoids, a change in its protein level can indirectly influence PPAR activation, and consequently, the expression of target genes, including the L-FABP gene itself. L-FABP might exert a more active role in gene regulation through a direct protein–protein interaction with PPAR [89]. Since ACBP as well as L-FABP localize to the nucleus [97] and LCA function as PPAR antagonists at least in vitro [112], an involvement of this LBP in gene regulation can also been envisioned. 20.3.2

PPARb/d: A Nuclear Receptor Involved in the Regulation of Intestinal Absorptive Area

The intestinal epithelium undergoes rapid and continuous renewal throughout its life. In healthy adult humans, more than 17 millions of cells are born each day and then die, leading to a total renewal of the mucosa in 3 days. This means that proliferation, differentiation, and death must be coordinated to maintain a proper cellular census. These programs are executed in anatomically well-defined units consisting of invaginated flask-shaped crypts of Liberkühn and evaginated fingerlike villi. The crypts are the proliferative units of the intestine. They contains a population of multipotent stem cells anchored around the crypt basis. These cells are able to give rise to two types of long-lived daughter cells, one being committed to produce enterocytes, the other yielding mucus-producing goblets cells and possibly the endocrine and Paneth cells [113]. Progressive differentiation of enterocytes takes place during their migration along the villus. Therefore, modifications in the proliferation rate of the mucosa lead to rapid changes in the absorptive capacity of the small intestine. Several physiological as well as pathological situations (e.g. gestation/lactation or insulin-dependent diabetes) lead to hyperphagia, responsible for intestinal hypertrophy [114]. Conversely, atrophy of the intestinal mucosa occurs during starvation [115]. It has been shown recently that dietary lipids are responsible for enterocyte proliferation through a neuroendocrine pathway involving both the entero-endocrine hormone glucagon-like-peptide 2 and enteric nerves [113, 116]. The involvement

20.4 General Conclusion

of PPARb/d in FA-mediated changes in absorptive areas is also likely. Indeed, a tumor-promoting activity has been proposed for PPARb/d. This was evidenced by the findings that PPARb/d (i) is activated when the transcriptional complex b-catenin/TCF-4 is formed secondary to a mutation of the tumor suppressor protein adenomatous polyposis coli (APC) [117] and (ii) is consistently highly expressed in many colorectal cancers in which APC is mutated [117], while PPARb/d gene disruption abolishes the tumorigenicity of tumoral human colonocytes [118]. Taken together these data indicate that PPARb/d plays a key role in intestinal cell proliferation, especially during the neoplastic process. They also raise the question of PPARb/d involvement in the cellular proliferation of normal intestinal mucosa. In brief, dietary lipids, probably through the activation of PPAR, are able to upregulate genes encoding for proteins implicated in the intestinal uptake and metabolic fate of LCFA, such as LBP, and might contribute to the modulation of the absorptive area as an adaptative response of the small intestine to its environmental challenges.

20.4

General Conclusion

Intestinal LCFA uptake is a dual phenomenon involving diffusion and proteinmediated transport. The specific extracellular microenvironment lining the intestinal mucosa mainly promotes LCFA permeation by passive diffusion. Such a feature is unique and crucial to maintain efficient fat absorption during the postprandial period. The efficiency of fat absorption is likely reinforced by the simultaneous expression of several membrane and soluble LBP, which, all together but differently, favor cellular LCFA influx into the enterocyte. They might also constitute a local high-affinity system absolutely required for the maintenance of lipoprotein synthesis, when the lipid concentration is low in the intestinal lumen (during inter-prandial periods, when fasting, or in the distal ileum). Nevertheless, the respective function(s) of these LBP remain(s) to be established in vivo. The small intestine also exhibits a great versatility secondary to the FA-mediated regulation of genes (including LBP) and the absorptive area, which might contribute to the adaptation of its absorptive capacity to the lipid content of the diet. Although this intestinal feature constitutes a vital advantage when the organism is subjected to seasonal changes in dietary supplies, conversely, it becomes a risk factor in situations of constant plethora. Better knowledge of the cellular and molecular mechanisms underlying intestinal fat absorption might lead to new therapeutic strategies in order to correct disorders in lipid balance in the near future.

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20.5

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14

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Fatty Acid Binding Proteins as Metabolic Regulators J. M. Stewart

21.1

Introduction

It is clear from many reviews and chapters of this book that fatty acid binding proteins (FABPs) have functions arising from their ability to bind long-chain fatty acids (FAs) and to interact with either the membranous or aqueous fraction of cytoplasm (reviewed in Refs [1–4]). The potential of interactions between FABPs and other proteins has been supported by evidence that FABPs can alter enzyme kinetics (e.g. [5, 6]), interact directly with both hormone-sensitive lipase [7] and CD36 [8], and with peroxisome proliferator activated receptors (PPAR). PPARa and PPARc interact with FABPs, are transported into the nucleus, and modulate, among other things, genes coding for enzymes of FA metabolism (reviewed in Refs [9, 10]). Studies with mice in which the gene for heart FABP was ablated reflected not only the role of the protein in upregulating genes of FA metabolism, but also the compensatory activation of the glycolytic system and apparent reduction of the usual controls of glycolysis [11, 12]. FABPs can also bind and transport signaling lipids such as bilirubin, prostaglandin E1, and lipoxygenase metabolites of arachidonic acid [13, 14], although some signaling lipids such as sleep-inducing FA amides of the central nervous system do not bind to brain FABPs [15]. Further, FABPs may protect polyunsaturated fatty acids from peroxidation through preferential binding of them [16] while greater partitioning of oxidation products of polyunsaturated fatty acids onto FABPs when compared with unilamellar vesicles [17] may indicate protection of membranes from these monohydroxy fatty acids. Thus, FABPs may be involved in intracellular signaling, cell development, and membrane protection and it appears that they may be more than intracellular ferries of poorly soluble hydrophobes. This chapter develops the idea there is an interaction between carbohydrate and fatty acid energy metabolism that involves FABPs. Evidence is presented to support the contention that interactions between non-lipid metabolites and FABPs can modulate the partitioning of long-chain FAs into FABP binding sites. Additionally, experimental results showing the potential for reciprocal interaction between FAs and enzymes of glycolysis will be presented. Finally, new data will be

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21 Fatty Acid Binding Proteins as Metabolic Regulators

tendered indicating that different FABP types (muscle and liver) respond differently to non-lipid metabolites. The possible cross-talk between the two fuel streams in energy metabolism suggested is centred on substrate cycles involving hexokinase-glucose 6-phosphatase and phosphofructokinase-fructose 1,6-phosphatase in muscle and, in liver, glucokinase-glucose 6-phosphatase.

21.2

Established Interactions between Carbohydrateand Fatty Acid-based Energy Production

The exchange of rudimentary status information between carbohydrate and FA catabolisms by substrate level control and inhibition/activation of participating enzymes is well established. The reciprocal interaction between FA levels and glucose use, now referred to as the Randle cycle [18, 19] suggests that modulation of glycolytic flux depends upon the mitochondrial production of citrate which, when transported to the cytoplasm, inhibits the major flux generator of this pathway, phosphofructokinase-I (PFK-I). The other major effect of FA is to activate a pyruvate dehydrogenase kinase with concomitant phosphorylation and decrease in pyruvate dehydrogenase activity and an overall decrease in “glucose oxidation” by reducing the production of acetyl-CoA from pyruvate. The control of mitochondrial b-oxidation is vested in gating entry of FAs into the mitochondrial matrix through coordinated actions of carnitine palmitoyltransferase I (CPT I), palmitoylcarnitine translocase, and matrical carnitine palmitoyltransferase II (reviewed in Ref. [20]). A powerful inhibitor of CPT I is malonylCoA, the first committed metabolite in FA synthesis and itself a product of cytoplasmic citrate. CPT I may also be under phosphorylation control in liver: phosphorylation by cAMP-protein kinase increases the CPT I activity by 30–80% [21]. Fuel sensing mediated by malonyl-CoA in muscle has recently been reviewed [22].

21.3

The Involvement of FABP in Metabolism: Working Hypothesis

An additional level of lipidic control of glucose phosphorylation in muscle has been reported: CoA esters of long-chain FAs (in rat and human skeletal muscle) [23] and long-chain FAs (in bovine heart) [24] inhibit hexokinase I. There are many older reports that long-chain FAs inhibit a number of enzymes but uncertainty arises from those reports since unrealistically large FA concentrations were employed and detergental effects cannot be ruled out. One careful kinetic study from that pre-FABP era was the inhibitory effect of palmitate on muscle PFK-I (Ki = 25 lM, [25]) although the physical binding of FAs to PFK-I has not been studied. Thus, if FAs can influence enzymes of glycolysis (and elsewhere, see below), an involvement of FABP in the process is strongly implicated. Other areas

21.4 Criteria for Physiological Relevance of Metabolite Modulation of Fatty Acid Binding to FABP

where long-chain FAs have influence, such as the ADP/ATP translocase-porin-hexokinase complex of mitochondria [26] might also be examined productively. If FABPs modulate FA metabolism above the level of the genetic machinery, to what signals do they respond? Clearly any productive modulation must act in response to the instantaneous status of the cell/tissue. Some years ago we suggested that the metabolic status of the cell might be part of the signaling mechanism influencing FA binding characteristics of FABP [27]. Starting with this idea we began to search for metabolites or conditions within the cell that might influence the FABP operation through modulation of formation of the protein–FA complex. We hypothesized that the metabolic status of the cell could be signaled to FA transport through modulation of FA binding to FABP. This would probably be an allosteric modulation (in the strictest sense of the term: not the fatty acid binding site) since competitive interaction, such as observed with ferri- and ferroheme [28], would be a binary situation and not optimally adaptive. It must be pointed out that, in a strictly thermodynamic sense (i.e. mass action) the effect would result from repartitioning effects. Although it might seem unusual that a small, monomeric protein with a relatively small surface areas, such as FABP, would display “allosteric” properties usually “reserved” for large, multimeric enzyme systems, such regulation has been demonstrated for small proteins: cytochrome c has a saturable binding site for ATP that regulates the electron transport system and hence oxygen consumption and ATP production [29]. If FABPs are involved in the productive modulation of lipid metabolism and in targeting of FABPs for example, to the mitochondria [2, 30] there must be an exchange of information between the binding of FAs and the status of the cell.

21.4

Criteria for Physiological Relevance of Metabolite Modulation of Fatty Acid Binding to FABP

In the search for possible modulators of FABPs we compared the degree of binding of reporter ligands (radiolabeled or fluorescent) in the presence and absence of physiological concentrations of metabolites [31]. The assay conditions are particularly important since FA binding is effected by ionic strength, pH [2, 27, 32] and by temperature [33]. Statistically significant changes in binding to FABP must occur at metabolite levels that are within physiological concentration ranges. We examined concentrations of potential modulators found in unstressed tissue since metabolic flux is curtailed compared to the maximal flux attainable. A second level criteria that we are now beginning to explore is that the equilibrium dissociation constant (the apparent Kd) of any modulator must also fall within physiological concentration ranges to be part of a normal regulatory network. Finally, interactions that influence FA binding should be consistent with what is known about particular metabolic pathways and physiological states.

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21.4.1

Mammalian Liver FABP

As an initial test of the working hypothesis we examined the effect of glycolytic metabolites on the binding of both 14C-labeled oleate and the fluorescent probe cis-parinarate (cPnA) to rat L-FABP [27] at 37 8C. To do this we set the concentration of reporter molecules at the value of the Kd of the ligand where binding is most sensitive to change. After establishing the FABP–FA complex we challenged it with test metabolites. We reported that, of the early glycolytic intermediates, the most effective modulators of the ability of L-FABP to bind FAs were glucose and glucose-6-phosphate (G6P). Both of these metabolites increase the ability of FABP to bind long-chain FAs 30% and 40% for 6 mM glucose and 0.25 mM G6P respectively. An alternative binding assay [34] using the fluorescent probe cPnA provided the same result. In examining the effect of increasing concentrations of these two metabolites on oleate and cPnA binding, the concentrations required to produce a 50% increase in the observed FA binding change were 6 mM and 0.2 mM respectively, within the normal physiological range of glucose and G6P in rat liver. As part of this study we also examined glucose-1-phosphate (G1P), phosphate ion, fructose-6-phosphate (F6P), fructose-1,6-bisphosphate (F-1,6-P2) and fructose-2,6bisphosphate (F-2,6-P2), phosphoenol pyruvate, ATP, ADP, AMP and cAMP, NAD+ and NADH, NADP+ and NADPH and acetyl-CoA, none of which had an effect on FA binding to rat L-FABP (presented at the 4th International Conference on Lipid Binding Proteins, Maastricht, June, 2001). A recent study [2] included an examination of the effect of glucose and G6P on [1-14C]oleate binding to human H-FABP and L-FABP. While the paper states there was no effect of glucose or G6P on oleate binding as compared to the control, and their results for the muscle-type FABP support this contention (as do new results with bovine H-FABP below), there is a statistically significant increase (albeit small 7–8%) in binding to L-FABP when these two glycolytic intermediates were included in their assays. The differences in the size of effects of glucose and G6P on human L-FABP as compared to rat protein may reflect differences in the two proteins or may be an artifact of inadvertent increases in pH due to the temperature sensitivity of the Tris buffer used (dpKa/dT = –0.028 per degree) [35] in the Lipidex assay, presuming that was the assay used. We extended this study to L-FABP of another rodent, the ground squirrel (Spermophilus richardsonii) (unpublished results). In this system, glucose and G6P also increased the binding of cPnA to the FABP at 37 8C compared to the control by 20.6 ± 0.5% and 41.4 ± 2.2% respectively (n = 4; P < 0.05) when examined under the same conditions as reported before for rat [27] and using cis-parinarate as a fluorescent probe. In summary, Glc and G6P increased the ability of L-FABP to bind FAs in three animal systems and to different degrees. Since the number of ligands bound to the protein was not likely to increase (2 : 1 binding stoichiometry for L-FABP) the effect was probably centred on changes in the Kd for the ligand.

21.4 Criteria for Physiological Relevance of Metabolite Modulation of Fatty Acid Binding to FABP

Because of different experimental protocols and few reports of work with pure proteins, it is difficult to find a general metabolic scenario into which the L-FABP work can be fitted. However, in a recent study of the effect of oleate on the glycolytic/gluconeogenic and glycogen systems of fasted rat hepatocytes, Gustafson et al. [36] showed, in the presence of S4048, an inhibitor of endoplasmic reticulum G6P translocase, oleate increased intracellular G6P 3-fold and glycogen production 1.5-fold. They showed further that oleate did not have an inhibitory effect on glucokinase but stimulated G6P phosphatase flux (part of the putative hexokinase/G6P phosphatase substrate cycle) and decreased overall lactate production. The increase in G6P was attributed to increased gluconeogenesis. Thus it appears that long-chain fatty acids, while interacting with early glycolytic enzymes, foster gluconeogenesis in liver. Presumably, then, an increase in G6P or glucose could increase the partitioning of FAs to the FABP, and behave as a communicative link to lipid status. Other studies in our laboratory with rat L-FABP have examined the role of the ketone body, 3-hydroxybutyrate, where, at normal physiological levels (0.14 mM), there was only a small increase in the binding of FA (< 10%). At levels mirroring diabetic or fasting/starvation hepatic levels (about 7 mM) there was a 45 ± 5% increase in FA binding. The effect was saturable with a Ki (3-hydroxybutyrate) of 4.9 mM, which is not physiological under euglycemic or non-starvation conditions. Increased FA binding with elevated 3-hydroxybutyrate (when carbohydrate supply is curtailed as in diabetes or fasting/starvation) could reverse a tendency to gluconeogenesis and modify delivery of available FAs. 21.4.2

Mammalian Heart/Muscle FABP

For heart tissue, which has different hexokinase (HK) isoforms and a different FABP type from liver, the situation appears equally complex. We showed [24] that bovine heart hexokinase (type I) is inhibited by long-chain fatty acids in a manner that correlates positively with chain length and degree of saturation, but activated by FAs containing ³ 12 carbons. Also, hexokinase has a saturable binding site for cPnA and oleate with Kd values of 3 lM and 1.3 mM respectively (by fluorescence competition assay) while the FAs that activated HK did not bind to this site. We can now report work on the FABP purified from bovine heart using binding protocols the same as reported for rat liver FABP [27] and for muscle-type FABP from the spadefoot toad [37]. Figure 21.1 shows the effects of a number of early glycolytic intermediates on FA binding to the protein. Bovine H-FABP was insensitive to glucose and G6P, as reported by Veerkamp et al. [2] for human H-FABP and toad M-FABP [37]. There was no significant effect on the binding of FAs by physiological concentrations of F6P but there was significant inhibition of FA binding by F16P2. G1P and Pi did not have any effect on the binding of the fluorescent probe. In contrast to mammalian L-FABP, FA binding by bovine M-FABP was unchanged by 3-hydroxybutyrate. Unlike hepatic tissue, muscle and heart tissue do not actively produce glucose or ketone bodies. Heart and red skeletal muscles import carbon resources (carbohy-

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Fig. 21.1 The effect of various metabolites on the binding of cis-parinaric acid (1 lM) to bovine heart FABP compared with the control (no non-lipid metabolite). The assays were carried out at 37 8C, in 50 mM sodium phosphate buffer pH 7 as in Refs [27, 37]. The assays contained 20 lg of pure proteins per mL. the concentrations of metabolites not indi-

cated were G6P, 0.5 mM; F6P, 0.1 mM; F1,6P2, 0.03 mM; 3-hydroxybutyrate (ButAc), 0.1 mM; acetoacetate (AcAc), 0.02 mM. The asterisk indicates statistically significant differences from the control at the 95% confidence limit. The experiments were replicated 3 to 5 times with the data representing the means and the error bars the standard deviation.

drates, fatty acids, and ketone bodies) in response to energy needs and are predominantly aerobic. This essentially unidirectional resource flow is reflected in the poise of muscle pathways where the major control points are PFK-I, pyruvate dehydrogenase, and mitochondrial import of FAs. As discussed above, both long-chain FAs and their CoA derivatives inhibit glucose phosphorylation in heart and skeletal muscle. The focus of the interactions in muscle tissue appears to be PFK-I. Long-chain FAs inhibit muscle PFK-I (Ki = 25 lM) and F-1,6-P2 (at 0.03 mM) inhibits FA binding, establishing the possibility of a reciprocal interaction. Elevated FA could decrease glycolytic flux through actions on both HK and PFK-I, while decreased F-1,6-P2 could lift resting state inhibition of FA binding to M-FABP.

21.5

Potential of Formation of Schiff Bases: Non-enzymatic Glycation of FABPs

Carbohydrates that undergo ring opening and formation of a carbonyl group can form Schiff bases with primary amines, such as lysine residues that are prevalent in FABP and which appear involved in the collisional mechanism proposed for muscle-type FABPs [1]. Since many of the glycolytic intermediates that can ring open to produce the carbonyl influence binding of FAs to FABPs (G6P, glucose, F-1,6-P2) while G1P and F-2,6-P2 (which are locked in the closed hemiacetal and hemiketal rings) do not affect FA binding, there is a possibility that formation of

21.6 Theoretical Effects and Implications of Reciprocal Cross-talk

Fig. 21.2 The time course of glycation of bovine serum albumin and assorted FABPs (30 lg mL–1) with d-glucose analog NBDG (80 lM) in 10 mM sodium phosphate buffer, pH 7.0 and 1% (w/v) sodium azide at 25 8C. Over the time course as proteins (after sepa-

ration from small molecules with size exclusion) became glycated they became fluorescently tagged (473 nm excitation, 532 nm emission). A fluorescence of 0.5 represented 1 nmol fluorescent tag.

Schiff base may cause altered sequestration of FAs. To examine this possibility, we incubated a number of purified FABPs and bovine serum albumin with fluorescently tagged glucose (2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose; 2-NBDG) at room temperature and in 50 mM sodium phosphate, pH 7.0. We monitored the proteins for indications of covalent fluorescent tagging that would result from Schiff base formation. Periodically, aliquots of the incubation mixtures were removed and 2-NBDG was separated from the protein with a small size-exclusion column (Sephadex G-25). The protein fraction (appearing in the column void volume) was examined spectrofluorometrically to detect fluorescent tagging (excitation at 473 nm, emission at 532 nm; Molecular Probes, Inc.). Figure 21.2 shows the effect of incubating a number of FABPs and BSA with the fluorescent tag for 3 days. Only after 48 hours of incubation was there fluorescent tagging of the protein at detectable levels. The slowness of the non-enzymatic glycations we observed was consistent with non-enzymic glycation rates of human serum albumin (days) using other fluorometric methods [38]. Thus, it is unlikely that Schiff base formation was significant during the time required for making the measurements of the effects of metabolites on FA binding (minutes).

21.6

Theoretical Effects and Implications of Reciprocal Cross-talk: How much Fatty Acid Would be Available to Interact with Hexokinase?

The similarity of Kd values of HK and FABP for oleate and cPnA leads to an important question surrounding such mutual interactions. Given the large cellular concentrations of FABP, is there enough unbound FA to interact with HK (assuming it was in a compartment accessible to HK)? To model the amount of unbound FA in the presence of two different binding proteins requires the concentration of both proteins, the total FA concentration, and the equilibrium dissocia-

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Fig. 21.3 (A) The degree of saturation of a muscle-type FABP and hexokinase versus the amount of unbound fatty acid (oleate) in the model system. The saturation curve of hexokinase (lower curve) was generated using the concentration remaining after FABP equilibrated its binding site with ligand. The hexokinase binding curve was magnified by 10 ´ to

be observable at this scale. (B) The residual concentration of unbound fatty acid at various initial fatty acid concentrations in the presence of FABP, of FABP plus hexokinase. The upper line indicates the effect on the amount of unbound fatty acids if binding strength of FABP was decreased by a factor of two (i.e. Kd increased by 2-fold).

tion constants of participants to be known. The inctracellular concentration of FABP has been reported as 85 lM [39] while some estimates provide ranges of 200–400 lM [38]. An intermediary value of 250 lM muscle-type FABP was used in the following model. Determining the molar concentration of HK in heart tissue is more difficult since it is not directly reported in the literature. We can estimate from the maximal tissue activity and the measured kcat of the enzyme a reasonable enzyme concentration since Vmax = kcat[E]T, where [E]T is the total enzyme concentration and Vmax represents the maximal tissue activity. The kcat of type I HK is 64 s–1 [41] while the maximal activity in mammalian heart has been reported as 8 lmol min–1 g–1 tissue in porcine heart (cytoplasmic plus mitochondrial bound) [42]. Finally, the estimate must convert the tissue weight-based activities to concentrations: the assumption of 75% water content will suffice. Thus with Vmax = 8 lmol min–1 g–1, kcat = 64 s–1 we can estimate a minimum [E]T = 3 lM. Using a Kd of 1 lM for FABP and 1.3 lM for HK we can model equilibrium concentrations of unbound FA and degree of saturation of both protein systems at different initial FA concentrations. Figure 21.3 A and B shows the result of this very simple equilibrium model.

21.8 Where Else to Look: Other Enzymes that are Influenced by Fatty Acids

The amount of “free FA” in the simple model in which FABP is present is enough to saturate HK, as indicated by Fig. 21.3 A that shows the binding isotherms of FABP at various free FA concentrations, and the binding curve of HK using the FA remaining unbound in the presence of FABP. Figure 21.3 B shows the estimated concentration of unbound FA in the presence of FABP, of FABP plus HK, and the effect on the amount of unbound FA when the Kd value of the FABP for oleate is doubled, as is seen in toad muscle FABP in the presence of F1,6-P2 [37]. Thus, even with FABP present providing very low concentrations of unbound FAs, unbound FA could be sequestered on HK.

21.7

Difference in Binding of Fatty Acids and Modulation between Different Types of FABP

The data presented suggest that mammalian L-FABP and muscle types of FABP respond very differently to non-lipid metabolites. While the liver types increase the degree of FA binding in the presence of glucose and G6P, with an activation of binding also elicited by ketone bodies, muscle type does not respond to glucose or G6P, is inhibited by F-1,6-P2 and inhibited by ketone body in the form of 3-hydroxybutyrate. If FABPs are involved in fuel selection, the different metabolic poise of these tissues would demand that they operate differently. The differential response may offer some part of an explanation of the presence of different FABPs in different tissues and perhaps multiple forms in some tissues.

21.8

Where Else to Look: Other Enzymes that are Influenced by Fatty Acids

The influence of long-chain FAs on enzyme kinetics, specifically hexokinase, and PFK-I have been noted above. FAs also have an effect on the myoglobin system [43] and bind strongly to cytochrome c [44]. As part of an examination of potential sites of interference during hyperlipidemia we examined a battery of enzymes to see if elevated FA concentrations altered their reaction kinetics. We examined the enzymes of glycolysis (HK, PFK, PK, lactate dehydrogenase, aldolase, triosephosphate isomerase, pentose phosphate cycle (G6P dehydrogenase), the citric acid cycle (citrate synthase, malate dehydrogenase and a-ketoglutarate dehydrogenase), and b-oxidation (CPT-I). Additionally we examined the effects of fatty acids on the enzymes that degrade reactive oxygen species (catalase, superoxide dismutase). Table 21.1 shows the results of incubation of enzymes of these metabolic systems with oleate. In these studies, all enzymes were obtained from commercial sources and assays were established that provided maximal activities [45, 46]. We compared the maximal enzyme activities over a range of FA concentrations with the highest being 0.2 mM. The clearest conclusion from this survey is that most enzymes are not sensitive to concentrations of FAs. Those enzymes that are sensitive to FAs are those that

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21 Fatty Acid Binding Proteins as Metabolic Regulators Tab. 21.1 The effect of oleate on activity of selected mammalian enzymes.

Enzyme

Enzyme rate (lmol min–1 mg–1 protein) No olete

0.2 mM oleate

Glycolysis Hexokinase (bovine heart) Phosphofructokinase-I (rabbit muscle) Pyruvate kinase (rabbit muscle) Lactate dehydrogenase (rabbit muscle)

2.74 ± 0.10 (3) 18.6 ± 0.9 (3) 9.8 ± 1.6 (6) 1.8 ± 0.1 (5)

0.55 + 0.03 (3) 0.32 + 0.07 (3) 10.2 + 1.6 (3) 1.7 + 0.01 (5)

Citric acid cycle Citrate synthase (porcine heart)

54.8 ± 2.5 (3)

51.3 ± 3.1 (3)

Pentose phosphate Glucose-6-phosphate dehydrogenase (yeast) Glucose-6-phosphate dehydrogenase (bovine adrenals) Electron transport Ferrocytochrome c auto-oxidation a) Reactive oxygen Catalase (bovine liver) Superoxide dismutase (bovine erythrocyte) l-DOPA oxidation No SOD 10 mg SOD

26.6 ± 0.7 (3) 0.67 ± 0.04 (6)

0.050 ± 0.005 (4) 11.2 ± 0.4 (6)

0.250 ± 0.002 (3) 0.13 ± 0.1 (3)

0.20 ± 0.04 (3) 0.55 ± 0.2 0 (6)

0.100 ± 0.015 (4) 11.4 ± 0.3 (5)

0.260 ± 0.005 (3) 0.14 ± 0.01 (3)

a) Measured with Hansatech DW2/2 oxygen electrode at pH 7, 37 8C, in 50 mM Na phosphate buffer with initial [cytochrome c] = 0.25 mM. Units are mmol O2 min–1 mg–1. The enzymes were commercial preparations and were delipidated with Lipidex before assay. The animal source/tissue is listing in parentheses behind the enzyme name. All assays were done at 37 8C (except where noted). The numberof replicates are listed after values in parentheses. The values are means ± standard deviation. The reported activities were measured under conditions that produced maximal enzyme activities [44, 45]. Mean activities that were significantly different (P < 0.05) are in bold-face. Fatty acid solutions were prepared as in Refs [27, 37].

have controlling functions on various metabolic pathways and indicate that there is a closer connection between lipid and carbohydrate metabolism than has generally been appreciated.

21.9

Summary

The binding of FA to FABP can be modulated by non-lipid metabolites. Figure 21.4 summarizes one model (M-FABP/PFK-I) that forms the basis of present endeavours. There is a differential response of L- and M-FABP to metabolites. These differential responses may offer insight into why there are different FABP types expressed in the same tissue. While the ability of FAs to bind to cytoplasmic en-

21.10 Acknowledgements

Fig. 21.4 A model of the interaction scheme between early glycolysis and FABP in mammalian muscle or heart. The bracketed negative signs indicate inhibition of either enzyme activity or binding to FABP.

zymes may alter the concentrations of unbound FAs (probably sequestered in membranes) the reverse, where FABPs can influence enzyme substrates, must be evaluated. A consequence of FABP interactions with glycolytic intermediates may also influence the concentration of available substrate in the case of hexokinase and concentrations of F-1,6-P2 in the case of PFK-I. Significant fractions of cytoplasmic glycolytic substrates could be associated with FABPs.

21.10

Acknowledgements

Much thanks to John A. Blakely, my long-time research associate, for discussions, reading of the manuscript and technical expertise and to my colleague Kenneth B. Storey, Carleton University for stimulating discussions. The work of a number of students has been essential in this work, notably, Steven Wiseman, Jaime F. Claude, Stephen J. Crozier, Danny Jardine, Dr Monica Henry, and Philip Karpowicz. The National Sciences and Engineering Research Council of Canada, the Heart and Stroke Foundation of New Brunswick and Mount Allison University must be acknowledged for the funding that allowed the research to proceed.

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21.11

References 1 2

3 4 5

6 7

8

9 10

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12

13

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15

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17 18

J. Storch, A. E. A. Thumser, Biochim. Biophys. Acta 2000, 1486, 28–44. J. H. Veerkamp, T. B. van Moerkerk, A. W. Zimmerman, Eur. J. Biochem. 2000, 267, 5959–66. A. E. Thumser, J. Tsai, J. Storch, J. Mol. Neurosci. 2001, 16, 143–150. H-L. Liou, J. Storch, Biochemistry 2001, 40, 6475–85. C. A. Jolly, T. Hubbell, W. D. Behnke, F. Schroeder, Arch. Biochem. Biophys. 1997, 341, 112–121. D. Mukhopadhyay, M. Mukherjea, Ind. J. Biochem. Biophys. 1998, 35, 296–302. W-J. Shen, K. Sridhar, D. A. Bernlohr, F. B. Kraemer, Proc. Natl Acad. Sci. USA 1999, 96, 5528–32. V. L. Spitsberg, E. Matitashvili, R. C. Gorewit, Eur. J. Biochem. 1995, 230, 872–878. C. Qi, Y. Zhu, J. K. Reddy, Cell Biochem. Biophys. 2000, 32, 187–204. C. Wolfrum, C. M. Borrmann, T. Borchers, F. Spener, Proc. Natl Acad. Sci. USA 2001, 98, 2323–28. B. Binas, H. Danneber, J. McWhir, L. Mullins, A. J. Clark, FASEB J. 1999, 13, 805–812. F. G. Schaap, B. Binas, H. Danneberg, G. J. van der Vusse, J. F. Glatz, Circ. Res. 1999, 85, 329–337. A. K. Dutta-Roy, N. Gopalswamy, D. V. Trulzsch, Eur. J. Biochem. 1987, 162, 615–619. J. F. Glatz, M. M. Vork, C. P. Cistola DP, G. J. van der Vusse, Prostaglandins Leukot. Essent. Fatty Acids 1993, 48, 33– 41. J. M. Stewart, J. A. Blakely, N. M. Boudreau, K. B. Storey, J. Thermal. Biol. 2001, 27, 309–315. B. A. Ek, D. P. Cistola, J. A. hamilton, T. L. Kaduce, A. A. Spector, Biochim. Biophys. Acta 17, 75–85. B. A. Ek-von Mentzer, G. Zhang, J. A. Hamilton, J. Biol. Chem. 276, 15575–80. P. J. Randle, D. A. Priestman, S. C. Mistry, A. Halsall, J. Cell Biochem. 1994, 55(Suppl), 1–11.

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J. M. Stewart, J. F. Claude, J. A. Mac Donald, K. B. Storey, Comp. Biochem. Physiol. B 2000, 125, 347–357. H. Zoellner, J. Y. Hou, T. Hochgrebe, A. Pljak, M. W. Duncan, J. Golding, T. Henderson, G. Lynch, Biochem. Biophys. Res. Commun. 2001, 284, 83–89. M. M. Vork, J. F. C. Glatz, G. J. van der Vusse, J. Theor. Biol. 1993, 160, 207–222. R. M. Kaikaus, N. M. Bass, R. K. Ockner, R. K. Experientia 1990, 46, 617–630. T-Y. Fang, O. Alechina, A. E. Aleshin, H. J. Fromm, R. B. Honzatko, J. Biol. Chem. 1998, 273, 19548–53.

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B. Font, C. Vial, D. C. Gautheron, FEBS Lett. 1975, 56, 24–29. J. M. Stewart, Biochem. Cell. Biol. 1990, 68, 1096–1102. J. M. Stewart, J. A. Blakely, M. D. Johnson, Biochem. Cell. Biol. 2000, 78, 675– 681. J. M. Stewart, R. C. Carlin, J. A. MacDonald, S. C. van Iderstine, Invert. Reprod. Dev. 1994, 25, 73–82. J. M. Stewart, M. E. Brass, R. C. Carlin, H. Black, Can. J. Zool. 1993, 70, 720– 724.

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Role of Lipid Binding Proteins in Disease Aline Meirhaeghe and Philippe Amouyel

22.1

Polymorphism in FATP1 Gene and Triglyceride Metabolism 22.1.1

Fatty Acid Metabolism

Long-chain fatty acids (LCFAs) are an important source of energy for most organisms (pro- and eukaryotes). Cells metabolize and/or store intracellular LCFAs, depending on cell type and energy requirement. They can synthesize LCFAs or use plasma LCFAs released from the hydrolysis of dietary triglycerides (TGs). To understand the relationships between LCFAs, triglyceride metabolism, and FATP1, it is essential to explore how the organism processes the dietary fatty acids. In developed Western countries, human dietary lipids are mainly di- and triglycerides and account for *40% of caloric intake [1]. The pancreatic lipases in the small intestine break down triglycerides into three free fatty acids (FFAs) and one glycerol molecule [2]. The monoglycerol and fatty acid components are then absorbed by the enterocytes. Short- and medium-chain FAs (C < 12) pass out of the enterocyte without esterification. Bound to albumin, these small FAs circulate as non-esterified fatty acids (NEFAs) to the liver and other tissues via the hepatic portal system. Fatty acids with a carbon chain length greater than 12 atoms are re-esterified into TG molecules in the smooth endoplasmic reticulum of the enterocyte via the monoacylglycerol pathway. They are packaged with proteins (mainly apoB48) and phospholipids to form chylomicrons. These chylomicrons, which contain *90% TG, enter the lymphatic system and then the circulation at the thoracic duct, causing post-prandial lipemia. The removal of the TG occurs on the luminal side of capillary endothelial cells in adipose tissue, skeletal, and cardiac muscle, and in the liver through the action of the lipoprotein lipase (LPL) [3]. FFAs are then liberated and taken up into these specific tissues. In adipose tissue, the FFAs are re-esterified and stored as TG. The FFAs in muscle or liver enter the mitochondrial and peroxisomal b-oxidation pathways to generate energy (ATP) [4]. The existence of an LCFA transport system in these key tissues is therefore essential to facilitate the rapid and controlled transport of

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22 Role of Lipid Binding Proteins in Disease

LCFAs to adjust the metabolic requirements of the body and to cope with an increase in circulating LCFAs in the post-prandial state and in pathological conditions such as diabetes. 22.1.2

FATP1 Polymorphisms

The role of FATP1 is still poorly understood. In vitro, it acts as a transporter of LCFA but it is not established if it has the same function in vivo. The discovery of single nucleotide polymorphisms (SNPs) in a gene and the analysis of their impacts in various human populations are always very useful to approach the biological function of the protein encoded by that gene. Therefore, we searched for genetic polymorphisms in the human FATP1 gene by the single-stranded conformation polymorphism technique (SSCP) to better understand the physiological role of FATP1 in humans. The FATP1 gene is located on chromosome 19p13.1. It spans more than 13 kb and contains 12 exons [5] (see Fig. 22.1). We screened the whole coding sequence as well as the exon–intron boundaries, except for exon 1 and the promoter which were not yet cloned at that time. No polymorphisms in the coding sequence could be detected. However, we discovered three very close polymorphisms in introns 8 and 9. Due to their proximity, we decided to study the impact of the A/G substitution in intron 8 only in a large sample representative of the population of northern France [6]. This population is composed of 1195 individuals (601 men and 594 women) aged 35–64 years living in the urban community of Lille and randomly recruited from electoral rolls. In total, 1144 individuals have been genotyped for the A/G polymorphism. The frequency was 60% for the A allele (wild-type) and 40% for the G allele. This frequency was similar to that of individuals affected by type II diabetes (n = 47). We selected a subset of the population, including only individuals not taking medical treatment to avoid interference between drugs and biological variables (n = 838). We looked for associations between the genetic polymorphism and anthropometrical variables such as body weight, body mass index (BMI), waist-to-hip ratio or biological variables: circulating lipids (total-, HDL-, LDL-cholesterol, triglycerides), insulin, glucose, and leptin levels. The high frequency of the G allele allowed us to compare the three groups of genotypes (AA, AG, and GG). No difference between these groups could be detected regarding the anthropometric variables. However, we observed that plasma triglyceride levels were higher in individuals with the GG genotype (1.12 mmol L–1, n = 139) compared with AA individuals (1.07 mmol L–1, n = 301), the individuals carrying the AG genotype being intermediate (1.10 mmol L–1, n = 391) suggesting an allele-dependent dose association. We then stratified the population by gender to see if the effect of the polymorphism on triglycerides was homogeneous in men and women. As expected, plasma triglyceride concentrations were higher in men (1.73 ± 2.50 mmol L–1) than in women (1.10 ± 0.89 mmol L–1). In women, we detected a significant difference in the levels of triglycerides between the categories of genotypes. Indeed, the triglyceride concentration was 1.00, 0.95, and 0.90 mmol L–1 in women carrying the

22.1 Polymorphism in FATP1 Gene and Triglyceride Metabolism

Fig. 22.1 Gene organization of the human FATP1 gene. Exons are represented by black rectangles and introns by a solid line.

GG, GA, or AA genotype respectively. In men, the concentrations of triglycerides between the three groups of genotypes were not statistically different but a trend toward higher plasma triglyceride levels in men with the AG or GG genotypes (1.28 mmol L–1) compared with men with the AA genotype (1.23 mmol L–1) could be detected. By regression analysis, we were able to show that age, BMI, alcohol consumption, smoking status, and the A/G polymorphism explained 29% of the plasma triglyceride level variance in women. Although the plasma triglyceride concentrations in carriers of the rare allele of the polymorphism are still in the normal range, the observed association suggests an involvement of FATP1 in triglyceride metabolism and potentially in cardiovascular diseases. By promoting LCFAs uptake in adipose tissue, liver and muscle, FATP1 seems therefore able to regulate triglyceride metabolism in an indirect manner. Because the A/G polymorphism is located in the middle of intron 8, it is highly unlikely it has any functional incidence on the FATP1 mRNA or protein. This polymorphism is therefore probably in linkage disequilibrium with another functional mutation located elsewhere in the FATP1 gene, maybe in its 5' regulatory region not yet explored. 22.1.3

FABP2 Polymorphisms

It is worth noting that our results display similarities with a polymorphism in the FABP2 gene. This gene encodes the intestinal fatty acid binding protein (I-FABP), which is a small cytosolic protein involved in intracellular fatty acid transfer and metabolism, which is only expressed in intestinal enterocytes. There is a polymorphism at codon 54 causing the substitution of the alanine by a threonine. This polymorphism has been extensively studied in various population studies and in vitro. The Thr54 allele is associated with insulin resistance and enhanced fat oxidation rates among non-diabetic Pima Indians [7]. It is also associated with insulin resistance independently of body composition, habitual physical activity levels, or hormone replacement therapy (HRT) status in post-menopausal white women [8]. The Ala54Thr polymorphism modifies the qualitative properties of FABP2. Indeed, in vitro the Thr54-containing protein displays a 2-fold greater affinity for LCFAs than the Ala protein does [7]. This greater affinity has been suggested to

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22 Role of Lipid Binding Proteins in Disease

increase the absorption and processing of fatty acids; this could in turn be responsible for the greater fat oxidation rate and impaired insulin action reported in various studies. The Thr54 allele is also associated with an increased synthesis of esterified triglycerides, of de novo ApoB synthesis and of chylomicron output in a human jejunal organ culture model [9]. The Thr54 allele may facilitate the cytoplasmic attachment of fatty acids and their fast release to the endoplasmic reticulum where they undergo esterification to triglycerides, resulting in both ApoB protection from proteolytic degradation and enhanced chylomicron formation. If the polymorphism modifies the absorption of dietary fatty acids, it could also alter the post-prandial response. The Thr54 allele is associated with increased postprandial lipidemia. Indeed the rise in plasma triglyceride concentrations after a fat meal is enhanced in subjects carrying the Thr54 allele. The difference is seen in both chylomicron and VLDL-triglyceride particles. A strong positive correlation usually exists between the magnitude of the post-prandial triglyceride response and fasting triglyceride levels [10]. Indeed, the Thr54 allele is associated with increased plasma triglyceride levels in Aboriginal Canadians [11] and in Finnish populations [12]. The post-prandial triglyceride response correlates with fasting triglycerides in Ala54Ala but not in Thr54Thr subjects. Conversely, the Thr54Thr subjects show a strong correlation between triglycerides and insulin response [13]. However, it is not clear whether the triglyceride variation is due to increased lipid oxidation and impaired insulin action or whether this is a result of some other alteration caused by this mutation.

22.2

References 1 2 3 4 5

6

7

J. H. Weisburger, J. Am. Diet. Assoc. 1997, 97, S16–S23. C. Chapus, M. Rovery, L. Sarda, R. Verger, Biochimie 1988, 70, 1223–34. R. O. Scow, E. J. Blanchette-Mackie, Mol. Cell. Biochem. 1992, 116, 181–191. M. S. Rao, J. K. Reddy, Semin. Liver Dis. 2001, 21, 43–55 G. Martin, M. Nemoto, L. Gelman, S. Geffroy, J. Najib, J. C. Fruchart, P. Roevens, B. de Martinville, S. Deeb, J. Auwerx, Genomics 2000, 66, 296–304. A. Meirhaeghe, G. Martin, M. Nemoto, S. Deeb, D. Cottel, J. Auwerx, P. Amouyel, N. Helbecque, Arterioscler. Thromb. Vasc. Biol. 2000, 20, 1330–34. L. J. Baier, J. C. Sacchettini, W. C. Knowler, J. Eads, G. Paolisso, P. A. Tataranni, H. Mochizuki, P. H. Bennett, C. Bogardus, M. Prochazka, J. Clin. Invest. 1995, 95, 1281–87.

8

9

10 11

12

13

M. D. Brown, A. R. Shuldiner, R. E. Ferrell, E. P. Weiss, M. T. Korytkowski, J. M. Zmuda, S. D. McCole, G. E. Moore, J. M. Hagberg, Metabolism 2001, 50, 1102–05. E. Levy, D. Menard, E. Delvin, S. Stan, G. Mitchell, M. Lambert, E. Ziv, J. C. Feoli-Fonseca, E. Seidman, J. Biol. Chem. 2001, 276, 39679–84. H. M. Roche, M. J. Gibney, Prog. Lipid Res. 1995, 34, 249–266 R. A. Hegele, P. W. Connelly, A. J. Hanley, F. Sun, S. B. Harris, B. Zinman, Arterioscler. Thromb. Vasc. Biol. 1997, 17, 1060–66. R. Sipilainen, M. Uusitupa, S. Heikkinen, A. Rissanen, M. Laakso, J. Clin. Endocrinol. Metab. 1997, 82, 2629–32. J. J. Agren, R. Valve, H. Vidgren, M. Laakso, M. Uusitupa, Arterioscler. Thromb. Vasc. Biol. 1998, 18, 1606–10.

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PPARs in Atherosclerosis Jorge Plutzky

23.1

Atherosclerosis 23.1.1

Introduction

Atherosclerosis and its protean manifestations – including myocardial infarction, cerebrovascular event, peripheral vascular disease – remains a major cause of morbidity and mortality worldwide [1]. Although a common scourge of Western society, the exportation of atherosclerosis to developing countries reveals obvious and worrisome trends. This immense burden of disease has been countered with extensive efforts to better understand, prevent, and treat atherosclerosis and its complications. Over the years, this has led to an evolution of thinking regarding atherosclerosis [2, 3]. A century ago, atherosclerosis was considered primarily a degenerative disease of the elderly. Over time, it became apparent that atherosclerosis is a clinical entity manifest in a much broader demographic segment of the population. Early observations focused on intra-luminal arterial obstructions as being at the center of an imbalance between arterial supply and tissue demand that precipitated cardiac ischemia and infarction. Although true to some extent for “demand ischemia”, this view of a gradual “hardening of the arteries” gave way to understanding atherosclerotic plaque as a more dynamic lesion [4]. Critical to this was recognition of thrombus formation as an integral element in both the propagation of lesions as well as their complications [5, 6]. Most of acute cardiovascular events derive from plaque rupture and the ensuing occlusive thrombus that results [7]. More recently, the field has focused on two dominant issues regarding the nature of atherosclerotic plaque. The first is atherosclerosis as a chronic inflammatory disease [1]. The second is attention to how metabolic pathways and disorders fundamentally and pervasively influence vascular responses and the atherosclerotic process [8]. Both of these trends – inflammation and metabolism as critical components of the atherosclerotic process – underlie the explosive interest in the role of peroxisome proliferator activated receptors (PPARs) in vascular biology and atherosclerosis [9, 10]. We will review the current concepts in atherosclerosis itself before delving into the existing evidence implicating PPARs and their ligands as

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possible mediators in these responses, looking in turn at PPARa, PPARc, and PPARd (also known as PPARb). 23.1.2

Atherosclerosis as a Clinical Syndrome

Atherosclerosis can no longer be considered as a single pathologic process accounting for its various clinical manifestations. Two common scenarios highlight this spectrum of disease. The first is the 45-year-old man who presents with sudden death and is found at autopsy to have ruptured a single severe atherosclerotic plaque in his left anterior descending artery, with a subsequent rapid cascade of occlusive thrombus, acute cardiac ischemia, ventricular fibrillation, and death. Contrast this to the octagenarian who has a mild infarct as a complication of hip surgery and is found to have diffuse mild three-vessel disease. Both patients have atherosclerosis, but the manifestations are quite divergent. Similarly, distinctions can be made between atherosclerosis in the coronary arteries, cerebrovasculature, aorta, and femoral vessels. The presence of biomechanical factors, such as shear stress and hemodynamics, which alter cellular responses including gene expression is one of many likely contributors to such differences [11]. Here we will focus largely on coronary atherosclerosis. Atherosclerosis develops through a variety of recognized stages, from its earliest manifestation as a fatty streak through raised lesions to evolution into complicated advanced lesions [6]. An early seminal event in atherosclerosis is the entry of inflammatory cells like monocytes (MO) and their in situ development into macrophages (M}) and then foam cells as they take up lipid [1]. This arterial entry of MO/M} involves a series of specific cellular interactions heavily dependent on the expression of adhesion molecules on the luminal surface of endothelial cells (EC) [12]. Late-stage complicated lesions are characterized by a large necrotic and highly thrombogenic lipid core, separated from the circulation by the fibrous cap, a reactive response produced primarily by vascular smooth muscle cells (VSMCs) and consisting of collagen and other extracellular matrix materials [10]. Data from pathologic studies as well as thrombolytic trials, in which the culprit lesion leading to myocardial infarction (MI) could be seen by comparing coronary angiograms pre- and post-thrombolysis, revealed that most MIs occurred not in the most stenotic lesions but rather in those with more modest stenoses, in the 50–70% range [13, 14]. Through such work, the fissuring of the fibrous cap, known as plaque rupture, and the exposure of the circulation to the thrombogenic lipid core result in the occlusive thrombosis inducing most MIs. These observations have focused attention on the nature of the fibrous cap, what maintains it, and the forces and players involved with plaque rupture versus plaque stabilization [7]. One group of proteins highly implicated in plaque destabilization are the matrix metalloproteinases (MMPs) [15]. MMPs represent a large, complex, family of highly regulated matrix-degrading enzymes thought to be especially active in the shoulder regions of ruptured plaques. Another mechanism contributing to lesion formation is the superficial erosion of ECs.

23.1 Atherosclerosis

23.1.3

Cellular Constituents of Atherosclerosis

The complex interplay of factors and forces described above clearly involves unique and critical roles for different cellular constituents of the arterial wall in both normal vessel function and pathologic atherosclerotic responses. One fundamental shift in perspective over the past decade has been in understanding the endothelium as not a simple passive conduit but rather a dynamic organ involved in metacrine, paracrine, and endocrine function [16, 17]. In their critical position lining blood vessels, ECs represent the interface between circulating elements and the ultimate tissue response, serving as a transducer of those reactions. Endothelial changes like adhesion molecule expression are not only among the earliest cellular responses contributing to atherosclerosis, endothelial dysfunction also occurs as an early manifestation of clinical atherosclerosis [18]. Centrally involved in such changes, and a salient example of endothelial roles in vascular responses, is endothelial nitric oxide production, an endogenously produced substance synthesized by endothelial nitric oxide synthetase, that induces arterial relaxation through effects on VSMCs [19, 20]. The studies establishing the dependency on the endothelium for such responses contributed to the Nobel Prize in Medicine being awarded to Furchgott, Murad, and Ignarro [21]. The medial layer of the arterial wall is primarily muscular, comprising VSMCs. These cells help maintain vascular tone and provide an essential structural component. VSMCs are integral to many processes implicated in atherosclerosis [22]. They may contribute to the presence of hypertension or be involved in the reaction to it. The migration of VSMCs from the media to the intima as well as proliferation of VSMCs once present there is a hallmark of the atherosclerotic process. VSMCs also provide the main source of extracellular matrix which forms the fibrous cap. In this role, VSMCs provide an essential function, albeit a reaction to a pathologic state. VSMC dysfunction may contribute to the weakening of the fibrous cap due to less collagen synthesis. Interestingly, such decreases in collagen production are repressed by inflammatory stimuli such as interleukins (IL-1). VSMCs also elaborate matrix metalloproteinases (MMPs), which contribute to the remodeling process [6]. Interestingly, MMP expression appears in part regulated by pro-inflammatory signals such as IL-1 [23, 24]. Two other cell types represent critical players in atherosclerosis, even if they are not cellular constituents of the artery wall under normal conditions. These atheroma-associated cells include monocytes/macrophages and lymphocytes, predominantly T cells [1]. Monocytes are attracted from the circulation to sites of endothelial injury, going through a process of rolling, attachment, and entry into the vessel wall [12]. Extensive data suggest chemokines, or chemoattractant cytokines, as an important signal attracting monocytes and other inflammatory cells to sites of inflammation and injury [25]. Members of this large complex family of proteins are induced by different signals, including inflammatory cytokines acting on various cells, such as EC. Secreted chemokines bind to chemokine receptors present on specific inflammatory cells. Moving down concentration gradients of chemo-

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kines, monocytes adhere and enter the vessel wall, and take up lipid, especially low-density lipoproteins (LDL). T lymphocytes are also critical atheroma-associated cells, providing important signals, for example inflammatory cytokines that contribute to monocyte, VSMC, and EC activation [26]. 23.1.4

Atherosclerosis as an Inflammatory Disorder

As suggested above, an extensive literature has now established atherosclerosis as a chronic inflammatory disease [1]. Although the exact proximal signals remain unclear, with posited contributors ranging from possible infectious etiologies to oxidized LDL, inflammatory cells and inflammatory signals are implicated in essentially every step of atherogenesis and atherosclerosis. Extensive efforts have identified many inflammatory mediators that participate in these responses. Interestingly, these basic science studies now intersect with epidemiologic studies which find that levels of non-specific markers of inflammation like c-reactive protein (c-RP) predict the risk of future cardiovascular events [27]. Interestingly, c-RP levels are predictive above and beyond total cholesterol/HDL ratios, and decreased cardiovascular events through lipid-lowering are paralleled by decreases in c-RP levels [28, 29]. Although a multitude of questions persist about the clinical significance of inflammatory markers, their striking correlation with atherosclerosis underscores the prospect of inflammation as a therapeutic target, a notion that has been raised for PPAR agonists. 23.1.5

Atherosclerosis as a Metabolic Disorder

Coincident with expanding recognition of the dynamic nature of the vasculature and its pathobiology has been the increasing attention to the interaction between various aspects of metabolism and vascular responses [8]. The most obvious connection in this regard has been lipid metabolism and its now well-established role in atherosclerosis. This extends beyond the extensive data establishing LDL as a risk factor, pathogenic mediator, and therapeutic target to other lipoprotein particles [30]. Triglycerides, carried predominantly in VLDL particles (when synthesized by the liver) and chylomicrons (when particles are assembled in the gut) are composed primarily of fatty acids (FAs) [31, 32]. Establishing if triglycerides represent independent risk factors for atheroclerosis has been challenging [33, 34]. Although triglycerides are clearly associated with cardiovascular events, this association tends to weaken or disappear when other parameters are taken into account. This is not surprising given the close interaction between other risk factors like obesity, diabetes, the post-menopausal state and increased triglyceride levels [35, 36]. Triglycerides are also associated, although inversely, with high-density lipoproteins [30]. HDL is thought to participate in reverse cholesterol transport, moving lipoproteins from the periphery back to the liver [37]. The extensive connections

23.2 PPAR in the Vasculature

between PPARs and lipoprotein metabolism are discussed in detail elsewhere in this book, but remain relevant to the connections between PPARs and atherosclerosis since as PPAR agonists may exert their effects on atherosclerosis through changes in lipids. Recent trials suggest that even in patients with coronary artery disease (CAD) with average to low LDL levels, modestly increased triglycerides, and low HDL, fibrate treatment can decrease cardiovascular events [38]. The possibility that these may occur in part through PPARa activation has been raised [9, 39]. Beyond lipoproteins, the metabolic effects on atherosclerosis are also strikingly evident among diabetic patients [40]. Diabetes mellitus is now considered a cardiovascular risk equivalent based on data that patients with diabetes but no known CAD have the same risk of a future cardiovascular event as non-diabetics with a prior MI [41–43]. Increased cardiovascular risk also extends to patients with the insulin resistance syndrome (syndrome X) and even so-called “pre-diabetes”. A significant percentage of these insulin-resistant subjects will go on to manifest frank diabetes [44, 45]. The fact that so many of these patients will have clinically significant atherosclerosis has suggested the hypothesis that the metabolic aspects of these various syndromes – high triglycerides, low HDL, increased post-prandial hyperglycemia, and lipemia – may exert effects on the vasculature long before hyperglycemia diagnostic of diabetes becomes apparent [46]. Supportive of this hypothesis is the observation that perhaps as many as half of the patients by the time they are diagnosed with diabetes have already had an MI [47, 48]. Considerable attention has been paid to the potential effects, either protective or harmful, of anti-diabetic medications on the vasculature [49, 50].

23.2

PPAR in the Vasculature 23.2.1

PPARs in Vascular Biology and Atherosclerosis

From this broad overview, we can now consider the potential role of PPAR pathways in atherosclerosis. PPARs have been considered elsewhere in this book; the focus here is on the potential part these key transcriptional regulators of various aspects of metabolism (Tab. 23.1) may play in vascular responses, especially those relevant to atherosclerosis. To some extent, this issue has been greatly influenced by the ongoing use of PPAR agonists as therapeutic agents in patients with high risk for atherosclerosis – either those with dyslipidemia receiving fibrates or diabetic patients receiving thiazolidinediones. Early experiments examining these direct effects of PPAR agonists in the vasculature sought to first determine if PPARs were present in the various cellular components of the arterial wall and/or atheroma-associated cells, befiore considering if PPAR-regulated targets with relevance to vascular biology existed. Work has now evolved into testing if such effects occur in vivo, including humans. Thus, prior and emerging clinical trial data

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23 PPARs in Atherosclerosis Tab. 23.1 Putative ligands, cellular expression patterns, and pathways in which PPARs are thought to be involved.

PPARa

PPARc

PPARd

Ligands

Fibrates Fatty acids

Thiazolidinediones Fatty acids Hydroxyoctadenoic acids

Carba/prostacyclins Fatty acids

Expression

Liver Muscle Vascular: ECs, SMCs Atheroma-associated: MPs, lymphocytes

Adipose tissue Vascular: ECs, SMCs Atheroma-associated: MPs, lymphocytes

Ubiquitous

Pathways

FA oxidation Lipid metabolism/transfer Energy balance

Adipogenesis Lipid metabolism/transfer Glucose homeostasis

Inflammation? Lipid metabolism/transfer

PPAR expression in vascular and atheroma associated cells is now well-established, raising the prospect of synthetic and endogenous PPAR ligands having direct effects on vascular responses. EC, endothelial cell; SMC, smooth muscle cell; FA, fatty acid; MP, macrophages.

regarding cardiovascular events in patients receiving PPAR agonists can be viewed through this lens. To what extent did changes in cardiovascular events seen in those clinical trials stem from indirect changes in metabolism induced by PPAR agonists versus possible effects of directly activating PPARs in vascular cells. Beyond these pharmacologic issues, a pertinent question also exists regarding the role of endogenous PPAR activation in vascular responses. 23.2.2

Examining Evidence for PPAR in Vascular Responses

To a certain degree, the data regarding PPARs in the vasculature have been limited by several critical issues. One is the inherent complexity of PPAR action, with multiple levels of control that can vary among different cell types or conditions [51]. These issues include differences in respect to ligands, and the effects of those ligands at different concentrations, differences in PPAR action dependent on cell type, for example due to the presence or absence of certain co-activators or the relative levels of PPAR themselves, and variance in promoter response elements, to name a few. Published results may also differ because of different experimental protocols. Thus, perhaps not surprisingly, various and at times divergent effects have been reported in different and sometimes the very same vascular or atheroma-associated cell types. Furthermore, it remains challenging to ever establish that a given effect occurs in response to nuclear receptor activation, and not some other up- or downstream mechanism. These issues must be kept in mind in interpreting many of the results reported in the PPAR literature in general and certainly with regard to PPARs in the vasculature. Thus, elements of confusion, contradiction, and unre-

23.3 PPARc in Vascular Biology and Atherosclerosis

solved issues persist in the nascent world of PPARs and their agonists in the vasculature. These issues, perhaps a product of the dizzying pace of progress, will be noted as they arise in the review of the data that follows.

23.3

PPARc in Vascular Biology and Atherosclerosis 23.3.1

In vitro Evidence

PPARc expression is now recognized in essentially all vascular and atheroma-associated cells seen in the vessel wall [52]. This includes VSMCs, ECs, monocyte/macrophages, and more recently T cells. PPARc is also expressed in human atherosclerotic lesions [53]. With the presence of these nuclear receptors established in these cellular settings, the question arises as to the evidence for regulation of relevant targets in the vascular pathways described above. PPARc expression has been linked to a variety of integral responses in VSMCs. Among these, regulation of matrix metalloproteinase 9 (MMP-9) is perhaps an example of a now canonical PPARc-regulated target gene, with evidence of trans-repression of MMP-9 induction seen across many levels in both VSMCs [54] and monocytes/macrophages [55]. PPARc agonists repress MMP-9 mRNA induction, protein levels, and gelatinolytic activity on zymograms, a commonly used tool to study MMP responses [54]. Similar PPARc effects have been reported for the MMP-9 promoter [55]. A net effect of decreased MMP-9 action seems apparent given the absence of PPARc regulation of tissue inhibitors of MMPs (known as TIMPs) [54]. PPARc agonists also limit VSMC migration [54] and proliferation [56] with effects on cell cycling [57]. These changes in VSMC responses may contribute to thiazolidinedione effects in cardiovascular settings in which VSMC matrix remodeling may be at work, for example in-stent restenosis [58, 59]. They may also play a role in the inhibition seen in intimal : medial hyperplasia reported with thiazolidinediones [60]. PPARc agonists also exert effects on MMP-9 in monocyte/macrophages, where MMPs [61] have been implicated in plaque rupture; it remains to be seen if PPARc activation contributes to plaque stabilization. In addition to similar effects through PPARc activation on MMPs in monocytes and macrophages, PPARc activation also has other effects in these cells as well. Among the earliest reports of PPARc action in inflammatory cells were studies indicating that PPARc agonists could limit cytokine induction in monocytes/macrophages [62, 63]. More recent work establishes PPARc (and PPARa, discussed below) expression in T lymphocytes [64–66], with a variety of effects reported, including apoptosis and inhibition of cytokine production [67]. Provocative results were also reported for PPARc-mediated induction of CD36, a receptor for oxidized LDL uptake [68, 69]. These effects, which appeared to contribute to foam cell formation, raised concerns for possible pro-atherogenic effects through thiazolidinediones. This would not necessarily be the case, given other

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possible off-setting effects through sequestering oxidized LDL by CD36 induction in other tissues, and concurrent changes in cholesterol efflux [70]. Furthermore, the nature of the increased lipid in these monocytes was never established and could well have been triglyceride-rich lipoprotein, and not the lipid particles typically associated with foam cells. In fact, subsequent work has established that PPARc, and apparently PPARa activators also induce the expression of ABC-1, a protein implicated in cholesterol efflux [71–73]. ABC-1 is the gene disrupted in Tangier’s disease, a clinical condition associated with markedly decreased HDL levels [74, 75]. Endothelial cells also express PPARc as shown by RT-PCR [76], Western blotting and Northern analysis [77]. The anti-inflammatory effects suggested above have also been implicated in ECs on a different limb of inflammation – chemokine expression. We have reported that PPARc agonists limit IFNc induction of specific subsets of chemokines implicated in atherosclerosis, at least among those tested. Three different well-established PPARc agonists limited induction of three different CXC chemokines (IP-10, Mig, and ITAC), but not the CC chemokine MCP-1 [78]. Further analysis found consistent PPARc effects on the IP-10 promoter and in functional responses to lymphocytes expressing the CXC chemokine receptor. Although no effect was seen on MCP-1, this does not exclude PPARc effects on the MCP-1 pathway. Two groups have reported responses that either clearly or likely occur by repression of the receptor for MCP-1 – CCR2 [79, 80]. Interestingly, MCP-1 levels were not changed in mouse models of atherosclerosis treated with PPARc agonists, although MCP-1 levels do appear to fall in patients treated with TZDs [81]. PPARc agonists may also induce apoptosis in EC [82], although these effects were observed primarily with 15-deoxy-D12, 14 prostaglandin J2 (15-deoxy-D12, 14PGJ2), which also has PPAR-independent effects [83]. Another relevant PPARc target in ECs highlights some of the important issues noted earlier regarding challenges of establishing effects in vitro and comparing these to clinical responses. A variety of groups have found that PPARc agonists could induce plasminogen activator inhibitor-1 (PAI-1), a pro-coagulant protein implicated in atherosclerosis [77, 84, 85]. Increased PAI-1 expression through PPARc has been reported in EC in angiogenesis studies [84], in response to putative natural but not synthetic ligands [86] and in adipocytes [85]. In contrast, others find a decrease in PAI-1 in EC. Regardless, in human studies, serum PAI-1 levels appear to fall, possibly due to improved insulin sensitivity, or improved levels of glucose and/or triglycerides [87]. Thus different effects may be seen in vitro while clinical responses may vary due to pleiotropic drug effects, including differences between different agonists as well as PPAR-independent effects. 23.3.2

In vivo Evidence

At least four different studies have reported various PPARc agonists in vivo decrease atherosclerosis in mouse models [81, 88–90]. The first of these, by Glass and colleagues, studied the effects of rosiglitazone and an experimental PPARc

23.4 PPARa in Vascular Biology and Atherosclerosis

agonist, GW7845, in LDL receptor-deficient animals [81]. The extent of lesions was decreased with both agents, although interestingly, only in male mice. The explanation for this gender difference is unclear, but the female mice were more insulin resistant. This difference between males and females was not observed in other studies employing other models (apoE-deficient and LDLR-deficient mice on high fat or high fructose) and other PPARc agonists. The ongoing clinical use of PPARc ligands (pioglitazone or Actos, rosiglitazone or Avandia) as insulin-sensitizing agents in humans affords the possibility of asking if these drugs exert similar effects on atherosclerosis. Such questions are particularly germane given the very high risk for ischemic cardiovascular disease that patients with diabetes face. Despite these opportunities, establishing that the effects of any PPAR agonist derives from activation of its purported nuclear receptor target remains difficult. These agents may influence atherosclerosis indirectly, by improving metabolic status, directly but independent of PPAR activation, or directly through activation of its cognate PPAR. Early studies in humans suggest that these agents can in fact improve both serum and vascular surrogate markers of abnormal vascular responses [60, 87, 91, 92]. In terms of lipids, decreased triglycerides and improved HDL levels have been seen, often with changes that rival or exceed those seen in trials demonstrating decreased cardiac events through HDL-raising therapies [93]. Questions persist if there may also be differential effects on various parameters such as lipids among PPARc agonists. Beyond this, improvements in inflammatory markers have also been seen, as recently reported. Such effects include changes in markers such as c-reactive protein (C-RP) [94], currently under intensive study as a novel predictor of cardiovascular risk. Arterial reactivity may be improved after TZD treatment, a potentially significant finding given evidence for endothelial dysfunction as a surrogate marker for early changes in atherosclerosis, although much of this data has been in abstract form. Perhaps most impressive have been the improvements in carotid intimal thickness seen in relatively short time frames in studies with limited numbers of patients. These results have contributed to a variety of ongoing studies focusing on cardiovascular endpoints in patients being treated with these agents.

23.4

PPARa in Vascular Biology and Atherosclerosis 23.4.1

In vitro Evidence

The central role for PPARa in fatty acid metabolism – controlling enzymes involved with b-oxidation as well as various targets involved in lipid metabolism – places PPARa in a position to be relevant to a host of vascular issues [95]. Such a role is furthered by evidence that certain FAs can act as ligands for PPARa [51, 96]. FAs, present in various lipoproteins to a variable degree and with a varying nature (chain length, degree of saturation, cis/trans conformation), have been implicated in many

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aspects of EC, VSMC, monocyte/macrophage and even lymphocyte responses [97– 100]. PPARa has been reported in all such cellular settings, thus targets in these locations may come under regulation by synthetic and natural PPAR agonists, exerting direct effects on vascular responses. Such action could occur independently or in synergism with PPARa’s myriad effects on lipid metabolism, which range from regulation of apolipoproteins involved in HDL metabolism to enzymes of lipid metabolism (e.g., lipoprotein lipase), to even possible effects of other lipid drugs on HDL levels (e.g., statin-induction of HDL). Our focus here is on the potential direct vascular effects. Prior observations from our group revealed that certain fatty acids, including the x-3 FA docosohexanoic acid, could limit adhesion molecule expression, especially VCAM-1, in ECs [101]. We asked if these effects might be occurring through PPARa activation. In fact, PPARa agonists can limit inflammatory cytokine-induced VCAM1 expression, with effects that do not derive from cellular toxicity or changes in mRNA stability. These effects are demonstrable at the level of the VCAM-1 promoter through likely NFjB effects, as well as in functional assays of adhesion [86]. Although we found this effect to be specific to PPARa, others report that PPARc agonists can also limit adhesion molecule expression [102, 103]. More recent evidence indicates that oxidation of some x-3 FA, like eicosopentanoic acid (EPA), can limit leukocyte adhesion through PPARa-dependent mechanisms evident in vivo (Fig. 23.1) [104]. These effects, specific to oxidized EPA and evident in classic PPARa transactivation assays, were absent when such in vivo adhesion studies were repeated in the genetic absence of PPARa (Fig. 23.1) [104]. Other PPARa-regulated targets in ECs include endothelin 1 as well as various enzymes involved in oxidative processes including superoxide dismutase and phox (p47, p22) [92, 105]. The effects of PPAR agonists in general appear to be an antioxidative effect, as also suggested by studies in humans [91, 92]. In contrast to these potentially anti-atherosclerotic effects, at least two reports indicate that certain oxidized lipoproteins may induce adhesion molecule expression through PPARa-dependent mechanisms [106], although perhaps only under certain conditions such as in the presence of specific lipolytic enzymes [107]. Integrating these apparently contradictory effects into a unified model remains currently beyond our grasp but certainly worthy of further investigation, and likely a function of the complexity alluded to earlier. PPARa is also expressed in VSMCs. Evidence from Staels and colleagues indicates that PPARa activation can limit IL-6-induced changes in those cells [108]. PPARs clearly play a role in inflammatory cell differentiation and signaling, thus providing another setting in which they are likely players in atherosclerosis. In monocyte/macrophages PPARa has been reported to induce apoptosis, but only after cytokine stimulation, in contrast to PPARc agonists, which induced apoptosis in the absence of such stimuli [109]. Mentioned earlier was the possible effect of PPARa on ABC-1 regulation [72]. Recent work highlights the effects of PPARs in T lymphocytes, although the results have varied, in part due to different agonists, concentrations, and cell types employed [65, 67, 110, 111]. We found PPARa activation limits inflammatory cytokine production, including TNFa, IL-2, and

23.4 PPARa in Vascular Biology and Atherosclerosis

Fig. 23.1 Effect of oxidized EPA on leukocyte adhesion in mesenteric venules in wild-type and PPARa-deficient mice. Wild-type or PPARa-deficient mice (PPARa–/–) were given an intraperitoneal injection of vehicle (Veh) alone, native EPA, or oxidized EPA (oxEPA) one hour prior to injection of LPS. Five hours later mice were anesthetized and mesenteric venules were observed using intra-vital microscopy. (A) Adherent leukocytes were deter-

mined (n = 5–7 for each group of mice). * P < 0.03 compared with Veh + LPS (wild-type) and oxidized EPA + LPS (PPARa–/–). Similar results were seen for leukocyte rolling. (B) Representative photographs of leukocytes interacting with the vessel wall (arrows) in LPS stimulated wild-type and PPARa–/– mice, after indicated treatments, are shown. The effects of oxidized EPA are abrogated in the genetic absence of PPARa [104].

IFNc [67]. This would argue for a significant proximal effect of PPARa activators on cytokine production by T lymphocytes, which is of potential importance given the critical part of lymphocytes in interacting with and activating macrophages in atherosclerotic plaque. 23.4.2

In vivo Evidence

In vivo mouse data had previously been somewhat lacking. This may have been a result of the toxicity that mice experience in response to fibrate-induced peroxisomal proliferation. PPARa-deficient mice were crossed to the apoE deficiency model

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of atherosclerosis, with the surprising result that these mice had less not more atherosclerosis, despite the published record supporting an anti-inflammatory and antiatherosclerotic roles for PPARa in the vasculature [112]. One might have anticipated that atherosclerosis should have been accentuated, not decreased. Several factors may have contributed to this result. This important experiment did require the crossing of different strains, which could have contributed to the results. Interestingly, this same group has reported in abstract form that bone marrow transplantation of PPARa-deficient bone marrow ameliorates atherosclerosis in the LDL receptor-deficient mouse, potentially more consistent with the brunt of the in vitro data [113]. If so, perhaps involvement of PPARa in apoE pathways may have contributed to different responses in the absence of apoE. Anomalous results in mouse models of atherosclerosis have engendered general questions regarding the suitability of this model for broad extrapolation to human atherosclerosis [114, 115]. For example, hepatic effects are certainly fundamentally different in mice versus humans. Rigorous examination of this interesting data will also have to consider the prospect that PPARa can, in certain settings or in certain cells, promote pro-atherosclerotic and pro-inflammatory responses. Recent work demonstrated that treatment of apoE-deficient mice with the PPARa agonist fenofibrate decreased lesion size, an effect that was even greater when these mice also expressed human aPoAI [128]. In terms of humans, clinical trials with fibrates from PPARa agonists can also be reviewed, asking if the responses seen might have derived from PPARa action, either because of indirect effects on lipid parameters or directly through PPARa activation in vascular or inflammatory cells themselves. PPARa activation accounts at least in part for the effects of fibrates on HDL increases (inducing apoAI transcription) or triglyceride lowering (by inducing lipoprotein lipase, downregulating apoCIII expression, and inducing b-oxidation) [116–118]. Interestingly, some of these trials may suggest the possibility of direct PPARa effects in the vasculature exactly because of minimal lipid changes despite positive results. VA-HIT (Veterans Administration HDL Intervention Trial) is one such case in point [38]. In that trial, veterans with relatively low to average LDL levels (110 mg dL–1; obviating an ethical requirement for statin treatment), low HDL (32 mg dL–1), and only modestly increased triglycerides (160 mg dL–1) were given the putative PPARa ligand gemfibrozil or placebo. At the end of the study, primary cardiovascular endpoints were significantly decreased. Given that HDL levels only rose *3 points to a mean of 35 mg dL–1, and that post-hoc analyses fail to show an impact of triglyceride lowering on event reduction, the possibility of a direct effect of the drug can be entered as a possible hypothesis. Other studies with fibrates, e.g. bezafibrate, have had somewhat less impressive results, although this may derive from bezafibrates lesser activity against PPARa [119]. Positive angiographic results have been seen with fenofibrate in DAIS (Diabetes Atherosclerosis Intervention Study) [120]. The notion that fibrates might bind to and activate PPARa raises the intriguing issue of what substances produced endogenously by the body might be capable of similar responses. We have found that lipolysis of triglyceride-rich lipoproteins generates specific PPAR ligands in a manner determined by the lipase, the lipid substrate acted upon, and the PPAR being targeted. Lipoprotein lipase (LPL), the

23.6 Conclusion

main enzyme implicated in triglyceride metabolism, can act on VLDL to release specific PPARa ligands as well as distal PPARa effects. Of note, these distal responses included decreases in inflammation, suggesting that intact triglyceride metabolism may limit inflammation and/or atherosclerosis through PPARa [129].

23.5

PPARd in Vascular Biology and Atherosclerosis

Less is known about PPARd pathways and effects, but this is rapidly changing [121– 123]. There is considerable interest in the potential role for PPARd given its essentially ubiquitous expression. Like PPARa, certain fatty acids bind to the activated PPARd. Studies using PPARd (and PPARa) ligands have been performed in an interesting rhesus monkey model that spontaneously develops an insulin resistancelike syndrome among some but not all members of the colony [124, 125]. More recent work suggests that PPARd may mediate pro-inflammatory responses, including the promotion of lipid accumulation in macrophages [126], although other reports suggest effects on cholesterol efflux through PPARd activation [127]. No doubt ongoing work will provide further insights into the potential role of PPARd in the vasculature.

23.6

Conclusion

Dramatic progress has been made over the past decade regarding the role of PPAR in metabolism; even more impressive has been the rapidly evolving evidence for PPAR signaling in vascular biology and atherosclerosis. Both sets of data continue to argue that PPAR are likely relevant for vascular responses and the pathogenesis of atherosclerosis, either indirectly by changing metabolic parameters or directly by activating PPAR. Despite these advances, a multitude of issues remains unresolved. Most fundamentally, potential pro-atherosclerotic effects through PPAR activation have been suggested by some reports. Better understanding of PPAR effects, especially in different cellular settings, should address this. No doubt the field will continue to be driven by the ongoing emergence of new synthetic PPAR ligands, including ones with greater potency, dual PPARa/PPARc ligands, and PPARd activators. A central issue persists regarding the identity of true endogenous PPAR ligands, with a sense that perhaps those naturally occurring mediators have not yet been identified. Understanding the nature and generation of such natural PPAR ligands may provide considerable insight into in vivo physiologic pathways that maintain insulin sensitivity, raise HDL, lower triglycerides, and may limit inflammation. Ultimately, all these issues will return to the central question regarding the effects such pathways have on atherosclerosis in humans, and its treatment.

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PPARs: Nuclear Hormone Receptors Involved in the Control of Inflammation Liliane Michalik, Nguan Soon Tan, Walter Wahli, and Béatrice Desvergne

24.1

Introduction

PPARs were first isolated in the early 1990s based on sequence homology with previously known nuclear hormone receptors. Since their discovery in mouse and Xenopus laevis [1, 2], the three isotypes, named PPARa (NR1C1), PPARb (PPARd, FAAR, NUC1, NR1C2), and PPARc (NR1C3), have been identified in the rat and human as well. Due to alternative usage of two different promoters on the same gene, two PPARc isoforms are produced, called PPARc1 and 2. Like the other members of the large nuclear receptor family, PPARs are organized in four functional domains. The N-terminal A/B domain carries phosphorylation sites and a ligand-independent transactivation function. The C domain is the DNA binding domain and contains the classical zinc finger motif of the nuclear receptors. The D domain, also called hinge region, separates the C domain from the C-terminal E/ F region, the latter being responsible for the ligand binding and ligand-dependent transactivation function of the receptor. Upon ligand binding and dimerization with their obligate partner RXR (NR2B), PPARs bind to the PPAR response elements present in the promoter of their target genes, and consequently activate their transcription. Most of these target genes code for enzymes and proteins involved in lipid and intermediary metabolism. PPARa controls genes implicated in lipid catabolism, including the degradation of inflammatory molecules, whereas PPARc activates genes implicated in lipid storage and adipocyte differentiation [3]. So far, only two PPARb target genes are known, which activation leads to cell survival in keratinocytes [72]. In addition, this isotype has recently been shown to be involved in the reverse cholesterol transport [4] and in the control of keratinocyte proliferation, differentiation, and migration [5, 6]. PPARs were initially isolated as orphan nuclear receptors. Since then, several natural and synthetic ligands were identified. The synthetic molecules fibrates and thiazolidinediones (TZDs) have proven their efficiency in the treatment of hyperlipidemia and hyperglycemia, respectively. Among the natural ligands, fatty acids bind PPARs, therefore leading to the concept of transcriptional gene regulation by fatty acids through their nuclear receptor PPARs [3].

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24 PPARs: Nuclear Hormone Receptors Involved in the Control of Inflammation

The aim of this review is to focus on the involvement of PPARs in the regulation of inflammation. First, the expression profile of PPARs and its regulation by inflammatory cytokines will be summarized. We will then describe the ligand binding properties of each isotype and their respective involvement in inflammatory processes. Finally, we will discuss the possible use of PPARs as therapeutic targets in the treatment of inflammatory disorders.

24.2

PPAR Expression Profiles and Modulation by Cytokines

PPARa is mainly expressed in organs with a high rate of fatty acid b-oxidation, like liver, heart, kidney, brown adipose tissue, and to a lesser extent, small intestine, skeletal muscle, thymus, and testes [3, 7]. PPARa is also present in endothelial and aortic smooth muscle cells (SMCs) and in human monocytes, where its expression is upregulated during their differentiation into macrophages. In vivo, several studies have established that PPARa is present in murine and human atherosclerotic lesions, where it seems to co-localize with foam cells, SMCs, and macrophages (for review, see Refs [8, 9]). PPARb is ubiquitously expressed, although with varying levels in different tissues. In rodents, high levels of PPARb are found during embryonic development and its presence seems to be related to a decrease in proliferation and to engagement into the differentiation process [3, 7]. Elevated PPARb expression was also observed in colorectal cancer, head and neck carcinoma, and inflammatory hyperproliferative diseases such as psoriasis. The third PPAR isotype, PPARc, shows a more restricted expression pattern. It is present at high levels in the adipose tissue, and to a lesser extent in colon, retina, and immune system [3, 7]. Like PPARa, PPARc is present in endothelial and smooth muscle cells, and its expression co-localizes with macrophages, foam cells, and SMCs in atherosclerotic lesions. Although absent from isolated human monocytes, PPARc is strongly induced during macrophage differentiation (for review, see Refs [8, 9]). PPAR expression can be modulated by lipid derivatives and cytokines in various cell types. PPARc expression is downregulated by the inflammatory cytokines TNFa, IL-1a, IL-1b, and IL-6 in rat adipocytes [10], whereas it is increased following exposure to oxidized LDL (oxLDL) in human monocytes [11], or upon exposure to the anti-inflammatory cytokine IL-4 in monocytes and macrophages [12]. Recently, PPARb expression was shown to be strongly increased in mouse keratinocytes in wound healing [5] and in primary keratinocytes upon TNFa or INFc treatment [6]. The TNFa-dependent stimulation of PPARb expression requires the activation of the stress-associated kinase cascade, which in turn targets an AP-1 site in the PPARb promoter [6]. Despite being the first PPAR to be isolated, the regulation of PPARa expression by inflammatory cytokines is the least studied. PPARa expression is upregulated by glucocorticoid, an anti-inflammatory steroid [13]. Although to a lesser extent than that of PPARb, PPARa expression also increases in the keratinocytes of the wound edges during the inflammatory phase of

24.3 Fatty Acids and their Metabolites are PPAR Ligands

skin healing [5]. The pathway leading to the stimulation of PPARa expression in this situation is not yet characterized, but as for PPARb, TNFa treatment of primary mouse keratinocytes in vitro slightly increases the expression of PPARa. This effect is not dependent on PPARb-elevated expression as it also occurs in PPARb-null keratinocytes [6].

24.3

Fatty Acids and their Metabolites are PPAR Ligands

The ligand binding domain of the PPARs (E/F domain) is located in the C-terminal part of the protein and contains a ligand-dependent transactivation function [3]. Its overall structure is similar to that of the other members of the nuclear receptor family, and consists of 13 a-helices and a small four-stranded b-sheet. The ligand binding hydrophobic pocket of PPAR appears large compared with that of other nuclear receptors. This difference permits PPARs to interact with a broad variety of structurally distinct natural and synthetic compounds. However, as illustrated below, each PPAR isotype presents a selective activation profile by these ligands. Since the initial description of PPARs as orphan nuclear receptors, several approaches, including transactivation assays, direct binding assays, stimulation of the receptor interaction with a cofactor or heterodimerization partner, allowed the identification of natural and synthetic PPAR ligands [14]. Among the synthetic ligands, the fibrates and the TZDs are PPARa and PPARc selective ligands, respectively (Tab. 24.1). These molecules are of high therapeutic interest, since fibrates are potent hypolipidemic drugs, while TZDs are useful hypoglycemic agents in the treatment of type II diabetes. Additionally, it is interesting to note that some non-steroid anti-inflammatory drugs (NSAIDS) were identified as PPAR ligands, and that activation of PPARs could contribute to their anti-inflammatory effects. In addition to be very attractive therapeutic targets, PPARs also show interesting ligand binding properties with regard to naturally occuring molecules (Tab. 24.1). Fatty acids were the first natural ligands described for PPARs, which established that fatty acids are not only energy storage molecules, but that they can also act as “hormones” in order to regulate their own metabolism, through the regulation of gene transcription via PPARs. Thus, PPARs can be viewed as metabolic sensors able to turn on an appropriate metabolic response to various physiological situations, through the regulation of gene expression [15, 16]. PPARa can be activated by a wide variety of saturated and unsaturated fatty acids, including palmitic, arachidonic, and linoleic acids. On the other hand, short saturated fatty acids are weak PPARa agonists. After this first ligand characterization, several natural PPARa agonists were identified among the fatty acid-derived eicosanoids. PPARa was first proposed as a regulator of inflammation when leukotriene B4 (LTB4), a lipoxygenase arachidonate metabolite, was identified to be a ligand of this receptor [17]. Since then, it has been shown that the 8S-hydroxyeicosatetraenoic acid (8S-HETE), another metabolite of the lipoxygenase pathway, and

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24 PPARs: Nuclear Hormone Receptors Involved in the Control of Inflammation Tab. 24.1 Natural and synthetic PPAR ligands.

Unsaturated fatty acids x-3 PUFAs a-Linolenic c-Linolenic x-6 PUFAs Linoleic Arachidonic x-9 mUFAs Oleic Palmitoleic Eicosanoids LTB4 8S-HETE 9-HODE 13-HODE 15-Deoxy-D12, 14-PGJ2 Synthetic ligands Hypolipidemic agents Clofibric acid GW 2331 (fibrate analog) GW 2433 (fibrate analog) ETYA (arachidonic acid analog) Hypoglycemic agents Rosiglitazone (BRL) Troglitazone Hypoglycemic and hypolipidemic agents L-165041 L-165461 NSAIDs Indomethacin Fenoprofen Ibuprofen

PPARa

PPARb

PPARc

+ +

+/– +

+ +

+ +

+ +

+ +

+ +

+/– +

+/– +

+ +

+/–

+/– + + +

–

– +

+ + + +

+ +

–

– –

– –

+ +

+ +

+/– +/– + + +

Interaction between these compounds and PPARs was analyzed using various approaches. + Efficient binding; +/– weak binding ; – No binding detected. Absence of any sign means that the compound has not been tested with the corresponding PPAR isotype. Note: This table is not exhaustive and only some of the compounds related to the topic of this review are mentioned.

oxidized phospholipids derived from oxLDL were able to bind to PPARa as well (Tab. 24.1) [18–21]. Although synthetic selective ligands are now described for the PPARb isotype [4, 22, 23], natural selective agonists remain to be identified. Like the PPARa isotype, PPARb can be activated by fatty acids, the polyunsaturated fatty acids being the most potent among them (Tab. 24.1) [18, 20]. Gathering studies also suggest that inflammatory bioactive lipids may serve as ligands of this receptor. More par-

24.4 PPARs and the Control of the Inflammatory Response

ticularly, the cyclooxygenase-2 (COX-2)-generated eicosanoid prostacyclin appears to serve as a natural ligand for PPARb [18, 24]. Recently, we demonstrated that total organic lipid extract from inflammation-activated keratinocytes can activate this receptor [6]. PPARc can be activated by polyunsaturated fatty acids, such as linoleic, arachidonic, and eicosapentaenoic acids (Tab. 24.1). Fatty acid metabolites derived from COX activity, such as the 15-deoxy-D12, 14 prostaglandin J2 (15-deoxy-D12, 14PGJ2), or from the lipoxygenase pathways, such as the 15-HETE and 9- or 13-hydroxyoctadenoic acid (HODE), are also natural PPARc ligands (Tab. 14.1). So far the most potent natural ligand of PPARc is an oxidized phospholipid, hexadecylazeaoyl phosphatidylcholine, which binds to the receptor with a Kd value in the nanomolar range [20, 25–27]. It is thus remarkable that all three PPARs present some affinity for molecules that are released in an inflammatory context. Together with the modulation of their expression by cytokines, it supports the hypothesis that PPARs play a role in various aspects of inflammation processes.

24.4

PPARs and the Control of the Inflammatory Response 24.4.1

Anti-inflammatory Properties of PPARa

As mentioned above, the first indication that PPARa could participate in the control of inflammatory responses was the finding that LTB4, a potent chemotactic inflammatory mediator, is a natural PPARa ligand. It is thought that, upon binding to LTB4, activated PPARa controls the catabolism of this pro-inflammatory molecule, therefore limiting the duration of the inflammatory response. In agreement with this hypothesis, in vivo results demonstrated that the duration of an arachidonic acid or LTB4-induced inflammatory response was longer in the PPARa-null mouse compared with the wild-type animal. These data strongly suggest that a feedback loop exists, in which the binding of LTB4 to PPARa induces its own degradation through the x- and b-oxidation pathways [17]. In the PPARa-null mice, this catabolic pathway is not upregulated, therefore leading to a longer inflammatory response. Whether the same mechanism of self-induced degradation exists for other fatty acid-derived inflammatory molecules is still unknown. Since then, several additional data have validated the anti-inflammatory function of PPARa. PPARa is present in human aortic SMCs, and treatment of these cells with PPARa agonists resulted in decreased IL-1-mediated expression of COX-2 and production of IL-6 and prostaglandins, thus culminating in reducing the inflammatory response in vascular SMCs [28]. Consistent with the in vitro data, fibrates lower the plasma levels of acute-phase proteins, like IL-6, TNFa and INFc in human patients with atherosclerosis [28, 29]. Accordingly, aorta from PPARa-null mice exhibit an exacerbated inflammatory response upon inflammatory stimuli, asso-

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24 PPARs: Nuclear Hormone Receptors Involved in the Control of Inflammation

ciated to an increased production of IL-6. In addition, they are unable to respond to an anti-inflammatory fibrate treatment compared with their wild-type counterparts [30]. In parallel, observations were made in splenocytes, where the LPS-induced production of IL-6 and IL-12 is significantly higher in PPARa-mutated compared with wild-type cells. Again, fibrate treament inhibits the production of interleukins in the wild-type but not in the PPARa-null splenocytes [31]. The inhibitory action of fibrate on COX-2 expression also occurred in primary human hepatocytes, suggesting that PPARa is also involved in the control of the liver inflammatory response [32]. Finally, an impaired recruitment of inflammatory cells to the wound bed in the early phase of the skin healing process was observed in PPARa-null mice [5]. Altogether, these anti-inflammatory effects of PPARa in various models and cell types strongly suggest that PPARa is a general modulator of inflammation. Some of the PPARa anti-inflammatory effects occur through the repression of several genes involved in inflammation, via negative interferences between PPARa and the NFjB and AP-1 pathways (Fig. 24.1) [30]. Besides their involvement in the control of cell growth and apoptosis, the NFjB and AP-1 transcription factors play a major role in the regulation of many genes implicated in inflammation. Among the members of the NFjB family, the most frequent heterodimer is composed of the p50 and p65 subunits whereas AP-1 mainly acts as a Jun/Fos heterodimer. In transient transfection assays, overexpression and activation of PPARa strongly inhibits both NFjB and AP-1 induction of the IL-6 promoter [30]. Reciprocally, p65 and c-Jun are able to repress PPARa transactivation of a PPRE-containing promoter. As shown by pull-down assays, PPARa physically interacts with p65 and c-jun, suggesting that this reciprocal inhibition is mediated through a direct interaction between PPARa and the two transcription factors [30]. Further investigation demonstrated that PPARa can also interfere with the NFjB pathway through the induction of the expression of the IjBa inhibitory protein (Fig. 24.1). Indeed, the fibrate-induced expression of IjBa in human primary SMCs and hepatocytes leads to the inactivation of NFjB through sequestration in the cytoplasm compartment [32]. In vivo experiments confirmed that fibrate treatment increases expression of IjBa in the liver of wild-type animals, in a PPARa-dependent manner [32]. Finally, and in agreement with previous observations [31, 33], activation of PPARa in this model induced nuclear inactivation or delayed NFjB nuclear entry. Therefore, besides its direct interaction with p65, PPARa also negatively interferes with the NFjB pathway through induced expression of the IjBa inhibitory protein, which may promote accelerated nuclear inactivation of NFjB [32]. The oxidative stress pathway also triggers an inflammatory response through activation of the NFjB pathway. PPARa activators were shown to regulate the maintenance of the cellular redox balance, and thus the maintenance of low levels of oxidative-stress-induced cytokine production [31]. In the same line of thought, PPARa agonists increase the activity of the antioxidant enzyme catalase in liver and kidney [34]. However, the natural PPARa ligands LTB4 and 8S-HETE induce an increase in nitric oxide synthase (NOS) activity in a murine macrophage cell

24.4 PPARs and the Control of the Inflammatory Response

Fig. 24.1 Mechanisms of gene repression by PPARa via the NFjB and AP-1 pathways. Repression of gene expression by PPAR in the context of inflammation regulation is an indirect mechanism due to negative interference

with the NFjB and AP-1 pathways. This might occur through increased expression of IjBa and/or via direct interaction between PPARa and p65 and c-Jun, respectively. Adapted from Ref. [9].

line, therefore activating a pro-inflammatory pathway [35]. Whether these effects are PPARa dependent remains to be investigated. 24.4.2

PPARb and the Keratinocyte Response to Inflammation

Due to its broad pattern of expression, to the lack of identified selective ligands and to the early lethality of the PPARb-null mice, PPARb functions have remained an enigma for long. Recently, PPARb has been implicated in lipid metabolism and adiposity, in mouse embryo implantation and in myelination [36–38]. However, the best characterized function of PPARb so far is its implication in mouse keratinocyte response to inflammation. In the mouse, PPARb is absent from the interfollicular epidermis [5]. Most interestingly, its expression is rapidly and strongly reactivated in the keratinocytes at the wound edges of a skin injury, and the PPARb+/– mice exhibit a delay in skin wound closure [5]. Since these results suggested that PPARb may play an important role in the keratinocytes during stress, the function of PPARb has been further investigated in primary mouse keratinocyte cultures [6]. In such a model, we showed that keratinocyte differentiation triggered by signals from apoptotic cells is PPARb independent. In contrast, PPARb reactivation is clearly associated with signals triggered by necrotic keratinocytes, and is required for keratinocyte differentiation in a skin inflammatory context. More precisely, TNFa and IFNc, two major pro-inflammatory cytokines produced after a skin injury, mediate the reactivation of PPARb expression [6]. TNFa induces the expression of PPARb through the stress-associated kinase cascade invoked in response to inflammation, and this effect is mediated via an AP-1

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24 PPARs: Nuclear Hormone Receptors Involved in the Control of Inflammation

Fig. 24.2 PPARb regulates cell differentiation, survival, and migration in keratinocytes after wounding. After epidermal wounding, high levels of pro-inflammatory cytokines such as TNFa are produced in the wound bed. TNFa activates the neutral sphingomyelinase (NSmase) via FAN (factor associated to neutral sphingomyelinase). The ceramide generated by

N-Smase then activates the stress-associated kinase cascade which subsequently induces the expression of PPARb via an AP-1 site in the promoter of the PPARb gene. PPARb in turn stimulates the expression of target genes which remain to be identified, leading to keratinocyte differentiation and increased resistance to TNFa-induced apoptosis.

site in the promoter of the PPARb gene (Fig. 24.2). Subsequently, expression and activation of PPARb not only leads to keratinocyte differentiation but also confers increased resistance to TNFa-induced apoptosis. Consistent with these data and previous studies [5, 36], ablation of a PPARb allele in mouse results in uncontrolled proliferation and dramatically increased apoptosis in the epidermis [6]. Thus, PPARb in keratinocytes translates inflammatory signals at the cell surface into the regulation of specific gene expression, which leads to an appropriate keratinocyte response in inflammatory conditions and rapid healing of a skin wound [6]. In this context, PPARb is a beneficial transcription factor required for a normal healing process. However, an aberrant PPARb expression may contribute to inflammatory skin disorders like psoriasis, in which its precise role and mechanism of action remain to be elucidated.

24.4 PPARs and the Control of the Inflammatory Response

24.4.3

PPARc is Involved in the Control of Inflammation

The functions of the PPARc isotype have been extensively studied in terms of adipogenesis, lipid storage, and glucose metabolism, and many in vitro and in vivo results have shown that PPARc is a major player in the adipocyte differentiation program. Evidence that PPARc may have an impact in the control of inflammation is recent and still controversial. One of the first clues suggesting that PPARc plays a role in this context was the observation that its expression is strongly increased during macrophage differentiation. However, the absence of PPARc from isolated monocytes, together with recent findings based on the use of PPARc-null stem cells, suggest that this receptor is not necessary for the monocyte/macrophage differentiation process [39, 40]. Many observations support the hypothesis of an anti-inflammatory role of PPARc. The treatment of macrophages with PPARc agonist was reported to inhibit the expression of inducible nitric oxyde synthase (iNOS), leading to an antiinflammatory response [35, 41]. In addition, PPARc ligands are able to reduce the IFNc-induced production of chemokines by endothelial cells, possibly with beneficial effects on the initial development of atherosclerotic lesions due to attenuated recruitment of immune cells [42]. Similarly, PPARc ligand inhibits the phorbol ester-induced production of proinflammatory cytokines such as TNFa, IL-6, and IL1b by human peripheral blood monocytes [43]. In contrast to these data, in a different study, troglitazone was unable to decrease the level of cytokines produced by a murine macrophage cell line or by human peripheral blood monocytes, even after differentiation into macrophages [44]. In vivo, troglitazone treatment of db/db mice had no anti-inflammatory effect upon LPS-induced inflammation, raising doubt about the possible utilization of these compounds in the treatment of acute inflammation [44]. With respect to these data, it is important to note that many inhibitory actions of PPARc agonists on macrophage cytokine production may occur via a PPARc-independent mechanism [40]. The mechanisms underlying the inhibition of gene expression, and thus the anti-inflammatory action of PPARc, have been investigated. The PPARc-induced inhibition of the expression of iNOS, MMP-9, and scavenger receptor A in macrophages is mediated by antagonizing the activities of AP-1, STAT and NFjB [41]. Via similar mechanisms as for PPARa, PPARc was demonstrated to physically interact with the p50 and p65 subunits of NFjB in vitro, therefore suggesting that the inhibition of LPS-induced production of IL-12 in murine macrophages upon oxLDL exposure involves inhibition of the NFjB pathway [45]. More recently, a model of competition for limiting co-factors between PPARc and STAT, AP-1 or NFjB has been proposed for the repression of iNOS expression by PPARc [46]. However, with regards to putative gene trans-repression mediated via PPARc, it should be noted that in many cases the concentrations of PPARc ligands required to get an inhibitory effect on gene expression largely exceed those needed to bind the receptor. In addition, some of the effects of PPARc ligand treatment are clearly PPARc-independent [40]. Therefore, negative gene regulation by PPARc agonists should not be systematically attributed to PPARc activation.

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24 PPARs: Nuclear Hormone Receptors Involved in the Control of Inflammation

24.5

Are PPARs Good Targets for the Treatment of Inflammatory Disorders?

The overall picture of the consequences of PPAR activation on the control of inflammatory responses suggests that they may have interesting anti-inflammatory effects in various models and cell types. However, one major limitation of the studies described above is that PPAR ligands may also have PPAR-independent effects. Some of the consequences of LTB4 in vivo or on some cell types are due to its binding to its membrane and not to its nuclear receptor [47], and PGJ2 was shown to have PPARcindependent anti-inflammatory activities via the IjB kinase [48–50]. Moreover, LTB4 and 8S-HETE, two natural PPARa agonists, were shown to stimulate nitrite accumulation in murine macrophages, indicating an increase in the NOS activity, whereas, in the same study, the synthetic PPARa ligand Wy14,643 and several PPARc activators had the opposite effect [35]. These examples illustrate the complexity of the regulation of inflammatory processes, and the difficulty in interpreting data obtained with regard to the function of PPARs in this context. However, some specific approaches are likely to be beneficial as discussed below. 24.5.1

PPARs in Skin Inflammatory Disorders

Because PPARb was demonstrated to be a major regulator of the keratinocyte response to inflammatory signals [6], it is tempting to hypothesize that PPARs and the anti-inflammatory properties of their ligands might have a beneficial consequence in wound repair and in psoriasis, a chronic inflammatory skin disease. The expression of PPARa and c was shown to be reduced in human psoriatic lesions, indicating that the expression of PPARs is modulated in this pathology [51]. Treatment with the PPARc agonist troglitazone was able to improve the situation for patients with chronic, stable psoriatic plaques, suggesting that PPARs might be of clinical interest in psoriasis [52]. However, since PPARc ligands are known to have PPARc-independent anti-inflammatory properties, further investigation will be necessary to determine whether in this particular case, PPARc is the target of troglitazone. Even though no clinical data are yet available, PPARa agonists may also improve psoriatic lesions, since PPARa ligands modulate keratinocyte differentiation [53, 54]. As mentioned above, the role of PPARb in psoriatic lesions remains to be characterized. 24.5.2

PPARs and the Progression of Atherosclerosis

Despite the complexity mentioned above, several observations suggest that activating PPARs has beneficial effects on the vascular wall, by slowing down the progression of atherosclerosis (Fig. 24.3). Atherosclerosis is a chronic disease that leads to the final obstruction of an artery due to the build-up of an atheromatous plaque on the vessel wall (for review, see Ref. [55]). The initial event in this pro-

24.5 Are PPARs Good Targets for the Treatment of Inflammatory Disorders?

Fig. 24.3 PPARa and PPARc mediated effects in atherogenesis. The consequences of treatment with PPARa and PPARc agonists in various cell types involved in the progression of atherosclerosis are summarized. Even though the general picture suggests anti-atherogenic PPAR function, caution should be exercised due to the limitations mentioned in this review. Clinical observations however suggest a

protective effect of PPARa and PPARc ligands in the progression of atherosclerosis. Blue boxes: PPARa; red boxes: PPARc. iNOS, inducible nitric oxyde synthase; MMP-9, matrix metalloproteinase 9; ET-1, endothelin-1; VCAM-1, vascular cell adhesion molecule-1, MCP-1, monocyte-chemoattractant protein-1; SMC, smooth muscle cells.

cess is the differentiation of circulating monocytes into macrophages in the intima of the vascular wall. These macrophages in turn differentiate into lipid-loaded foam cells, in a process which includes internalization of oxLDL via the scavenger receptor CD36. Consequently, these macrophages/foam cells produce cytokines like the macrophage colony-stimulating factor (M-CSF), IL-1, and TNFa that, in addition to an inflammatory response, promote SMC proliferation. Necrotic macrophages and foam cells liberate their intracellular components, leading to the formation of the fibrous cap of the lesion. Eventually, the rupture of the plaque leads to the obstruction of the artery. Both the PPARa and c isotypes are expressed in human atherosclerotic lesions [56, 57].

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24 PPARs: Nuclear Hormone Receptors Involved in the Control of Inflammation

Paradoxically, PPARc can mediate both pro or anti-atherogenic effects (Fig. 24.3). Its pro-atherogenic effect would be due to the promotion of macrophage to foam cell transition. 9- and 13-HODE components of the oxLDL internalized by the macrophages via the CD36 scavenger receptor can activate PPARc. Since the CD36 receptor is a PPARc target gene, activation of PPARc induces an increase of its expression, therefore promoting internalization of oxLDL, which provides more PPARc ligand, thereby generating a positive feedback loop that further stimulates differentiation of macrophages into foam cells [40, 58]. However, recent observations suggested that the internalization of lipids by the macrophages is counterbalanced by cholesterol reverse transport, leading to a net antiatherogenic effect of PPARc in macrophages [59]. In addition, PPARc activation may inhibit local inflammation in the vascular wall through reduction of the production of the monocyte chemoattractant protein 1 (MCP-1) and chemokines by endothelial cells, and of pro-inflammatory cytokines by human monocytes [42, 43, 60]. PPARc activators, as well as PPARa agonists, promote apoptosis in monocytes, therefore decreasing the source of inflammatory cytokines and matrix-degrading enzymes [61]. Finally, PPARc activators interfere with SMC proliferation via inhibition of the production of the vasoactive peptide endothelin-1 (ET-1) by endothelial cells [62] and with plaque rupture via inhibition of MMP-9 metalloproteinase expression in macrophages [41, 57]. Altogether, these results show that PPARc may inhibit the progression of atherosclerosis at various levels. PPARc agonists have indeed protective effects against atherosclerosis in murine models [63–65]. Troglitazone decreases the thickness of arterial wall in human carotids [66] and prevents restenosis in rat through vascular SMC growth inhibition [67]. PPARa may also modulate many aspects of this pathological process (Fig 24.3). PPARa activators inhibit the cytokine-induced production of vascular cell adhesion molecule-1 (VCAM-1) by endothelial cells, therefore decreasing leukocyte recruitment to the lesion [33]. They also interfere with the production of inflammatory cytokines in various cell types, as described above, and with the expression of tissue factor in human monocytes [68]. Similar to PPARc activators, PPARa agonists can inhibit the activation and proliferation of human SMC via inhibition of the vasoactive peptide ET-1 production by endothelial cells [62]. Consistent with these observations, convincing anti-atherosclerotic properties of the PPARa activators fibrates have been observed in clinical studies, in addition to their hypolipidemic properties that reduce a cardiovascular disease risk factor. Fenofibrate treament decreases the plasma concentrations of acute-phase proteins such as IL-6, fibrinogen, and C-reactive protein in hyperlipidemic patients [28], and of IFNc and TNFa in patients with atherosclerosis and hyperlipoproteinemia [29], therefore having beneficial consequences on the development of atherosclerosis. Indeed, a doubleblind clinical trial performed with the fibrate Gemfibrozil showed a decrease in the number of cardiovascular events in patients with coronary disease and low levels of HDL-cholesterol [69].

24.6 Conclusion

24.5.3

PPARc Regulates Intestinal Inflammation

In vivo data suggest that PPARc ligands might be useful anti-inflammatory drugs to treat colitis [70, 71]. Epithelial colonic cells express PPARc and are thought to produce pro-inflammatory cytokines implicated in inflammatory bowel disease (IBD). However, the function of PPARc in these cells is not fully characterized. PPARc ligands were shown to attenuate the production of cytokines in colonic cancer cell lines via interference with the NFjB pathway [70]. Consistent with these data, the TZDs were able to reduce colonic inflammation in an in vivo mouse model of intestinal inflammatory disease [70]. The protective effect of TDZs on colitis was further investigated in a different experimental setting, 2,4,6trinitrobenzene sulfonic acid (TNBS)-induced intestinal inflammation, which is a well-validated mouse model for human IBD [71]. TNBS-induced colitis is characterized by severe and deep necrosis of the colon, large areas of ulceration and neutrophil infiltrates. In the conditions used in these experiments, 68% mortality was observed 5 days after TNBS administration. Given in a preventive mode, the PPARc agonists efficiently reduced mortality and decreased the grade of the inflammatory lesions [71], which presented smaller infiltrates and limited edema and necrosis. Similar results were obtained with rosiglitazone administration in a therapeutic mode. Since RXR is the PPAR obligate dimerization partner, the effect of an RXR agonist treatment was also adressed in the same study, and was shown to mimick the effects of rosiglitazone. Most interestingly, a combination of RXR and PPARc agonists had synergistic effects at low doses compared with separate treatments, resulting in a quasi-absence of TNBS-induced inflammatory lesions with modest neutrophil infiltrates. Consistent with a protective role of PPARc and RXR agonists in intestinal inflammatory diseases, both the PPARc+/– and RXRa+/– mice were more susceptible to TNBS-induced colitis [71]. Although not characterized in detail, the underlying mechanism involves limitation of TNFa and IL-1b expression. These data strongly suggest that PPARc and RXR play an important role in the regulation of intestinal inflammation, and that they are therefore interesting targets for the treatment of colitis. Moreover, the benefit of combined treatment with low doses of PPARc and RXR agonists in mouse certainly deserves further investigation.

24.6

Conclusion

Overall, laboratory and clinical research suggests that PPARs may be interesting targets for pharmacological molecules aimed at the treatment of inflammatory disorders. From the basic research point of view, it is interesting to note that the anti-inflammatory effects of PPARs described so far are mainly due to the repression of proinflammatory gene expression, likely through interference with the NFjB or AP-1

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24 PPARs: Nuclear Hormone Receptors Involved in the Control of Inflammation

pathway. Because repression of transcriptional activity is an indirect, and not yet fully characterized mechanism for PPAR activity, it is tempting to think that PPARs might also act through positive regulation of anti-inflammatory gene transcription. For instance, the antioxidant enzyme catalase, which protects cells from oxidative stress, and the NFjB inhibitory protein IjBa are both upregulated upon fibrate treament. Whether they are direct PPARa target genes, or whether some other genes coding for proteins with anti-inflammatory properties are under PPAR control would be worth investigating. The roles of PPARb in hyperproliferative diseases clearly identify this PPAR isotype as an important target for treatments. The high expression level of PPARb in hyperproliferative tissues such as cancers, psoriasis, and wound repair indicated a fundamental role of this receptor in cell proliferation, migration, and/or differentiation. New insights into its regulation and mechanism will have profound implications for tumor biology, as well as for normal cell renewal and tissue repair. From the medical point of view, fibrates and TZDs are drugs of high medical interest. In addition to their hypolipidemic and hypoglycemic properties, respectively, they also have interesting anti-inflammatory effects as seen in this review. The beneficial roles of PPARa and PPARc agonists in atherosclerosis probably result from both their systemic effects, in lowering hyperlipidemia and hyperglycemia, and from their local anti-inflammatory action on the arterial wall. The beneficial effects of TZDs on psoriasis are quite promising, even though they might be PPARc-dependent or not. This raises the question as to how specific a developing pharmacological molecule should be, and how much do we know of the molecular mechanism underlying the effects of a given molecule. The recent finding that a combination of PPARc and RXR ligands has synergistic anti-inflammatory consequences in an animal model of bowel disease is promising too. Lower doses of each compound could be used in a therapeutic formulation, therefore decreasing the side effects due to PPARc ligands such as fluid retention in patients treated with TZDs. Limitation in this case might arise from the use of rexinoids which are able to induce many different pathways. Tremendous progress has been made during the past years, and PPARs are now well recognized as important therapeutic targets. Fibrates and TZDs certainly have protective effects on the development of chronic inflammatory disorders, but whether PPARs are direct targets in this context deserves further investigation. In the specific field of inflammatory diseases however, progress still needs to be made, both at the bench and in the clinic, before using PPARs as specific anti-inflammatory targets.

24.7

Acknowledgements

The authors wish to thank Laurent Gelman for critical reading of the manuscript. The work done in the authors’ laboratory was supported by the Swiss National Science Foundation (grants to Walter Wahli and to Béatrice Desvergne), by the Etat de Vaud, by the Human Frontier Science Program Organization and by the Herbette Foundation.

24.8 References

24.8

References 1

2 3 4

5

6

7

8 9 10

11

12

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14

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PPARs and Cancer J. H. Gill and Ruth A. Roberts

25.1

Introduction

The peroxisome proliferator activated receptors (PPARs) are ligand-activated transcription factors belonging to the superfamily of nuclear hormone receptors. The prototype PPAR, PPARa was cloned from mouse liver by homology to known superfamily members [1] and was subsequently found to mediate the cellular response to the fibrate hypolipidemic drugs [2]. Since then, three subtypes of PPAR have been identified termed a, b/d, and c [1, 3–5] and an increasing diversity of natural and artificial PPAR ligands have been described, including fatty acids, eicosinoids, and insulin sensitizers (reviewed in Ref. [6]). This biological profile has stimulated much interest in PPARs as therapeutic targets in the search for new drugs for obesity, hypolipidemia, and type 2 diabetes (reviewed in Refs [7–9]). Recently, there has been great interest in the suggestion that the activation of PPARs may have anticancer effects. Here, we review the evidence for PPARs in cancer and the potential opportunities for therapeutic intervention.

25.2

The PPAR Family

The PPARs characterized to date have a common organization consisting of six coding exons (Fig. 25.1), encoding the N-terminal A/B domain, the DNA binding domain, the hinge region, and the ligand binding domain. PPARs exhibit 80% and 65% homology in their DNA binding and ligand binding domains, respectively, and are highly conserved in mammalian species. Once activated, PPARs directly regulate gene transcription by forming heterodimers with the retinoid X receptor (RXR) [10]. This complex binds to a specific sequence in the 5' region of target genes, known as a PPAR response element (PPRE) that consists of a direct repeat (DR) of two TGACCT sequences separated by one nucleotide (so called DR1 element). The first natural PPRE was identified upstream of the rat peroxisomal acyl-CoA oxidase (ACO) gene [11] then subsequently in other peroxisomal genes such as bifunctional enzyme (BFE) [12], rat cy-

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Fig. 25.1 Diagrammatic representation of PPAR activation and protein domains of PPAR. PPAR structure has an A/B domain, a DNA binding domain (DBD), a hinge region, and a ligand binding domain (LBD). The DBD and LBD domains are encoded by two exons each, one for each zinc finger or ligand, respectively. ACO, acyl-CoA oxidase; ACS, acyl-

CoA synthetase; ACODH, acyl-CoA dehydrogenase; BFE, bifunctional enzyme; CYP4A1, cytochrome P450 4A1; FATP, fatty acid transport protein; L-FABP, liver fatty acid binding protein; LPL, lipoprotein lipase; LST-1, liverspecific type 1 sugar transporter; PEPCK, phosphoenolpyruvate carboxykinase.

tochrome P4504A1 [13] and in an increasing number of genes implicated in lipid homeostasis (reviewed in Ref. [9]). All these PPREs conform to the DR1 criteria but a detailed analysis of the family of PPAR responsive genes indicates that additional parameters dictate the patterns of activation such as the regions flanking the PPRE [14] and the presence of additional transcriptional repressors and coactivators [15–18]. Although the PPAR isoforms are highly homologous and function by the same mechanism, their ligand specificity [19] and tissue distribution differs and their target genes are different, suggesting unique functions for the different receptor isoforms [5].

25.3

PPARa 25.3.1

Expression and Activation

PPARa is expressed in a variety of tissues, especially the liver and kidneys [3, 4, 20]. Although many chemicals have been demonstrated to activate PPARa, including plasticizers, fibrate hypolipidemic drugs, and natural fatty acids [1, 6, 21–26] a

25.3 PPARa

definitive endogenous ligand has yet to be identified. Two potential candidates are the inflammatory mediator leukotriene B4 (LTB4), and fatty acids or their metabolites, both of which have been demonstrated to bind directly to PPARa [27, 28]. The outcome of PPARa activation has primarily been studied in the liver. Genes upregulated by PPARa include those involved with the regulation of fatty acid degradation, such as acyl-CoA oxidase and cytochrome P4504A [11, 13, 28]. Transcriptional regulation of such genes strongly suggests a role for PPARa in liver lipid homeostasis [28–30]. 25.3.2

PPARa and Cancer

In addition to their many roles in normal tissue physiology, PPARs have been implicated in regulating cancer development, surprisingly both as promoters and inhibitors of the process [31–38]. Activation of PPARa by the peroxisome proliferator (PP) class of chemicals is known to cause rodent hepatocellular carcinoma (reviewed in Ref. [39]). Studies performed using PPARa-null mice support the dependence of this process on PPARa [18, 40–43], although the actual mechanism by which PPARa activation leads to the formation of liver tumors in rodents is at present unclear. One such hypothesis involves the perturbation of hepatocyte growth control, such that activation of PPARa leads to an increase in proliferation and a suppression of cell death [42, 44, 45]. The induction of proliferation by PPARa ligands has been shown to involve regulation of cell cycle control genes, such as CDK4 [46, 47], c-myc, c-fos, and c-jun [47, 48]. Growth regulators such as the interleukins [49, 50] and tumor necrosis factor a (TNFa) [51–55] have been implicated in this response. However, there is as yet no evidence for direct transcription of these growth regulatory genes by PPARa. The suppression of apoptosis by activation of PPARa may result from the release of cytokines, is not dependent on the type of apoptotic pathway, and occurs in a specific population of hepatocytes [46, 49, 50, 55–59]. 25.3.3

Species Differences

Although PPARa ligands clearly cause rodent liver tumors, this appears to be a rodent-specific phenomenon (reviewed in Ref. [9]). Clinical side effects of fibrate PPs are rare and analyses of causes of death during treatment show no evidence of an adverse effect and no evidence of an increase in malignant disease compared with the normal population [60]. When IARC reviewed clofibrate they concluded that the mechanism of liver carcinogenesis in clofibrate treated rats would not be operative in humans [61] based on lack of response in human hepatocytes and on the results of extensive epidemiological studies, particularly the WHO trial on clofibrate comprising 208 000 man-years of observation [62, 63]. Furthermore, a meta-analysis [64] of the results from six clinical trials on clofibrate also found no excess cancer mortality [61].

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Extensive studies have been performed to dissect this species difference in response to PPARa activation. Such marked differences may be explained by quantity of PPARa and quality of PPARa-mediated responses [39, 42, 65–67]. Humans were found to express functional PPARa, but at much lower expression levels than that of the responsive rodent species [42, 66]. The lower expression of PPARa could be attributed to the presence of a truncated, inactive form of PPARa, which appears to be present in most individuals examined to date [39, 66]. In addition, there is a species-specific response with regard to the responsiveness of PPARa target genes, suggesting different patterns of gene regulation compared with the rodent situation [68–70]. Therefore, levels of human PPARa are sufficient to facilitate the action of hypolipidemic drugs, but may be too low to activate the genes responsible for the hepatocarcinogenic effects of PPARa activation [42, 67]. 25.3.4

PPARa as a Therapeutic Target?

Several studies have suggested an indirect role for PPARa in tumorigenesis based on correlation of expression with cancer development. Expression levels of PPARa have been suggested to be associated with the development of prostatic adenocarcinoma [71]. Similarly, activation of PPARa has been shown to cause an upregulation of the immediate early gene cyclooxygenase-2 (COX-2) in murine liver epithelia [72], rabbit corneal epithelia [73] and human colorectal epithelial cell lines [74, 75]. Since approximately 80% of human colorectal carcinomas have increased levels of COX-2, an enzyme involved in prostaglandin breakdown, the PPARa-induced expression of COX-2 is of potential significance for colon carcinogenesis [74–76]. Interestingly, fatty acids are known to be activators of PPARa and dietary fat is a known risk factor for colon carcinogenesis [77]. The regulation of COX-2 by PPARa observed in colorectal cells may also be mirrored in other human epithelia, given that fatty acids have already been shown to upregulate COX-2 in rat mammary gland [78]. These data suggest that activation of PPARa may play a role in colon carcinogenesis, raising the possibility that inhibition of PPARa could have therapeutic potential. However, these data should be viewed with caution in the presence of extensive epidemiological evidence showing that prolonged exposure to PPARa ligands has no effects on tumor incidence [62, 63]. Nonetheless, if further research identifies inhibition of PPARa as a reasonable therapeutic target, a dominant negative isoform of PPARa, hPPARa6/29 has been described that can inhibit PPAR-mediated gene expression [59]. Aside from the possible effects of PPARa ligands on COX-2 expression, activation of PPARa by Wy 14,643 has been shown to inhibit progression of DMBA-induced rodent mammary tumors [79]. Also, PPARa ligands have been shown to result in apoptosis of human monocyte-derived macrophages [80] and to alter chemotaxis in macrophages from peritoneal fluid [81]. These data raise the possibility of targeting tumor macrophages as a means of modulating cancer cell survival signals provided by macrophage-derived cytokines [82].

25.4 PPARc

25.4

PPARc 25.4.1

Expression and Activation

PPARc has a restricted expression profile, being expressed predominantly in adipose tissue and large intestine [34, 38, 83]. The observation that the anti-diabetic thiazolidinediones (TZDs) activated PPARc led to a wave of research and hypotheses regarding a role for PPARc in the regulation of diabetes and obesity [36, 84]. In addition to TZDs, PPARc is activated by endogenous ligands (Fig. 25.2), including fatty acids and prostaglandin metabolites, such as 15-deoxy-D12,14-prostaglandin J2 [83, 84]. Major roles for PPARc have been identified in adipocyte differentiation, monocyte/macrophage development and glucose homeostasis [36, 38, 83, 85] mediated via control of the expression of a large number of genes involved in lipid transport and insulin sensitization (reviewed in Ref. [86]). In humans, three PPARc mRNA have been identified, all of which are derived as splice variants of the same gene. To date, no physiological differences have been discovered between the three different PPARc isoforms [87].

Fig. 25.2 Natural and synthetic ligands for PPARs and possible role in cancer therapy. LA, linoleic acid; AA, arachadonic acid; Trog, troglitazone; PGJ2, 15-deoxy-D12,14-prostaglandin J2.

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25.4.2

PPARc and Cancer

In addition to a role in obesity and diabetes, PPARc may regulate proliferation and differentiation (reviewed in Ref. [88]), suggesting a potential role in tumorigenesis. PPARc expression was shown to be tumor-specific in non-small cell lung cancer, with expression observed in > 50% of primary lung tumors and several immortalized cell lines [32]. Similarly, PPARc expression was observed in > 90% pancreatic adenocarcinomas investigated, in contrast to normal pancreatic duct epithelia which were negative for PPARc at the protein level [89]. In a study of normal versus malignant brain, breast, and prostate cells, PPARc expression was consistently higher in the malignant cells but was not regulated by ligand [90]. As well as evidence from expression studies, activation of PPARc resulted in an inhibition of estrogen production in breast adipose tissue [91] and played a role in tumor regression [92]. Additionally, activation of PPARc inhibited growth of human hepatocellular [93] and esophageal carcinoma cells though cell cycle arrest and induction of apoptosis [94]. The tumor suppressor functions of PPARc ligands are reported to be mediated by modulation of expression of the tumor suppressor PTEN [95], a protein known to have an involvement in many cellular functions including proliferation and apoptosis (reviewed in Ref. [96]). 25.4.3

PPARc as a Therapeutic Target?

Studies on estrogen production suggest a role for PPARc ligands in the treatment of breast cancer [91]. In addition, the observation that activation of PPARc is antiproliferative and correlates with maturational stage in neuroblastoma, vascular smooth muscle cells, and DMBA-induced mammary tumors suggests a potential use for PPARc in the management of advanced stage vascularised tumors [38, 79, 97]. However, the most compelling evidence for PPARc as a therapeutic target for cancer is derived from studies of colorectal tumors. Treatment of colorectal cancer cell lines with PPARc ligands resulted in growth inhibition, promotion of differentiation related markers, and expression of Drg-1 (differentiation-related gene-1), a putative suppressor of metastasis in human colorectal cancer [34, 98]. Activation of PPARc was also found to inhibit expression of COX-2 in colorectal cancer cell lines, which is in opposition to results observed with PPARa [75, 99]. Furthermore, inactivating mutations in PPARc were identified in a subset of colorectal tumors, supporting a role for PPARc as a tumor suppressor of colorectal carcinogenesis [100]. Although the majority of studies have demonstrated a role for PPARc ligands as preventative agents for colorectal cancer, two studies in APCmin/+ mice suggested the converse. These studies showed an increase in tumors or polyps in the colon of these mice after they were fed a diet containing a PPARc agonist for 8 or 5 weeks, respectively [101, 102]. However, this is in contrast to in vitro and murine xenograft studies using human cell lines in which PPARc agonists resulted in

25.5 PPARb

growth inhibition and induction of differentiation [100, 103]. As already described for PPARa, species differences in response between the human cell lines compared with the APCmin/+ mice offer the most likely explanation for this discrepancy. Additionally, one recent paper suggests that the antitumor effects of TZDs are independent of PPARc and are mediated instead by inhibition of transcription [104].

25.5

PPARb 25.5.1

Expression and Activation

Expression of PPARb has been demonstrated in a vast array of tissue and cell types [4, 105, 106]. Unlike PPARa and PPARc, little is known about the physiological role of PPARb and several independent studies have linked this receptor to diverse functions. An involvement for PPARb has been suggested in brain lipid metabolism [107], squamous cell differentiation [105], regulation of other hormone receptors [108], and as a mediator of prostacyclin function in blastocyst implantation in mice [76]. Although several compounds have been demonstrated to activate PPARb non-specifically, including a number of PPARa and c agonists, a selective ligand for PPARb has yet to be elucidated. 25.5.2

PPARb and Cancer

Unlike PPARa and PPARc, little is known about the role of PPARb and its involvement, if any, in the development of cancer. As described earlier, activation of this receptor has been associated with processes independent of those identified for either PPARa or PPARc. The observations that PPARb can repress human PPARa activity [108] and induce PPARc expression [109] would suggest that PPARb can act as a negative regulator of tumorigenesis in certain cell systems. In contrast to this hypothesized antitumorigenic role, PPARb has been suggested to play a role in the development of colorectal cancer [37, 76, 110]. Expression levels of PPARb were shown to be higher in colon carcinoma compared to the adjacent surrounding tissue [37, 76]. Furthermore, PPARb was concentrated in the most differentiated cells at the luminal surface of the mucosal glands in normal mucosa, but was expressed in epithelial cells located throughout the dysplastic glands of the neoplastic tissue [76]. This suggests that PPARb expression alone is not pro-neoplastic [76]. Using serial analysis of gene expression (SAGE), PPARb was identified as a downstream target of the adenomatous polyposis coli (APC) tumor suppressor pathway [111]. Under normal conditions, APC binds to b-catenin and inhibits its ability to form a transcription complex with TCF-4. Conversely, in the majority of colorectal tumors examined, APC is inactivated by truncating muta-

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tions giving rise to elevated transcriptional activity of the b-catenin/TCF-4 complex. Similarly, in those tumors with intact APC genes, mutations of b-catenin that render it resistant to the inhibitory effects of APC occur, thereby demonstrating that inhibition of b-catenin/TCF-4-mediated transcription is critical to the tumor suppressive role of APC [37]. Downregulation of PPARb by APC was demonstrated to be as the result of a direct molecular interaction, since the PPARb promoter contains TCF-4 binding sites, and PPARb promoters were repressed by APC as well as stimulated by mutant b-catenin [37]. 25.5.3

PPARb as a Therapeutic Target?

Evidence for a potential use of PPARb ligands in cancer therapy is derived from studies of colorectal tumor development where non-steroidal anti-inflammatory drugs (NSAIDs) can bind and potentially inhibit PPARb [37], offering an explanation of NSAID-mediated chemoprevention [110]. This theory was supported using a PPARb-null human colorectal cancer cell line [37]. These cells showed no obvious phenotype in vitro, and no increased sensitivity to NSAIDs. However, they were defective in establishing tumors in vivo [37]. Although the absence of PPARb affected tumorigenicity of this colorectal cell line, the authors stressed that this effect may be specific to this cell line, and that the role of PPARb in the chemopreventative effects of NSAIDs may involve more than one mechanism [37]. However, because the APC pathway is mutated with high frequency in colon cancer and overexpression of PPARb has been seen in many colorectal cancers, it seems likely that increased expression of PPARb may contribute to the neoplastic process and thus its modulation may present therapeutic opportunities.

25.6

Future Directions

A review of the last decade of literature reveals an involvement of PPARs in many diverse biological processes from adipocyte differentiation to rat liver cancer. The evidence for a role of PPARs in fat metabolism and energy balance is conclusive whereas the evidence for a role in tumorigenesis is confusing. Nonetheless, it is clear that modulation of PPARc activity has some effect on the regulation of tumors of the colon, opening many avenues for exploratory research. Perhaps most compelling is the tentative connection between high fat diet, cellular energy balance, and cancer of the colon and maybe of the breast. Although the way forward depends on further elucidation of the basic biology of cancer, much is already known about PPAR ligand chemistry and toxicology and the binding specificity of different PPARs [19]. Thus, PPARs provide excellent therapeutic targets since they are receptors with intrinsic thresholds that act directly to modulate gene expression.

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449

Subject Index a AAACT 175 ABC ABCA1 41 ABCC7 41 ABCC8 41 ABCC9 41 ABCE1 41 domain 40 ABC transporter ABCA1 39 ABCA4 39 ABCB4 39 ABCB11 39 ABCC2 39 ABCG1 39 ABC transporters 213 ABC-1 408 ABCA ABCA1 45 ABCA2 45 ABCA3 45 ABCA4 45 ABCA5 45 ABCA6 45 ABCA7 45 ABCA8 45 ABCA9 45 ABCA10 45 ABCA12 45 ABCA13 45 ABCB1 46 family 45 ABCA1 212, 213 HDL-deficiency syndromes such as classical Tangier disease 52 ABCA4 mutations 54 retinitis pigmentosa 54

ABCA4 (ABCR) gene located on chromosome 1p21 54 retinal degeneration 54 ABCC ABCC1 48 ABCC2 48 ABCC3 48 ABCC6 48 ABCC7 48 ABCC8 48 ABCC9 48 ABCC10 48 subfamily 48 ABCF (GCN20) subfamilies 51 ABCG1 212 intracellular mobilization of lipid stores 65 ABCG5 213 ABCG8 213 ACBP 163, 338, 339, 376 expressed at high levels in the larval midgut of fruit fly 160 expression during adipocyte differentiation 159 expression in liver 159 expression in the liver is downregulated by fasting 160 Manduca sexta 160 mRNA 372 plant Arabidopsis thaliana bind saturated and unsaturated C14–C22 acyl-CoA esters 156 regulatory functions 164 silkworm 160 tobacco horn worm 160 yeast 156 ACBP function 168 ACBP ratio 162

450

Subject Index ACS1 32 activation of PPARa 144 acyl-CoA 162, 163, 337 regulatory functions 164 acyl-CoA binding protein (ACBP) 372 *10-kDa cytosolic protein 151 bovine 156 intracellular acyl-CoA transporter 151 acyl-CoA carboxylase 214 acyl-CoA ester 165 acyl-CoA hydrolase 162 peroxisomal 162 acyl-CoA oxidase (ACO) 316, 437 acyl-CoA synthetase activity 346 acyl-CoA synthetases 365 ADD-1 313 ADD-1 (SREBP-1c) 315 adenomatous polyposis coli (APC) tumor suppressor pathway 443 adhesion molecule expression 403 ADIFAB assay 111 adipocyte determination and differentiation factor 1 (ADD1) 159, 313 adipocytes, 3T3-L1 361 adipogenesis 314 adipose tissue 311, 319, 399 adrenoleukodystrophy (ALD) 58 mutations in ABCD1 58 AF-2 domain 181 AF-2 helix 199 A-FABP 263, 274, 275, 278, 279 albumin induced NFjB activation 88 albumin receptors 81 albumin toxicity 79 albumin-bound long-chain fatty acids 80 all-trans retinal (atRAL) 191 all-trans retinol (atROL) 191 anthroyloxy-FA (AOFA) 371 antiparallel b-strands 368 AOX and SCP-2 required for efficient peroxisomal oxidation of certain fatty acids 145 AOX and SCP-x required for efficient peroxisomal oxidation of certain fatty acids 145 AOX deficiency 144 AOX-null mice 144 AP-1 182, 424, 427 APC pathway 444 APC–/+ mice 442 apoA1 232 apoB 232

apoCIII 232 expression 412 apoE 213 apoE–/– E-FABP–/– 130 E-FABP–/– mice 130 fluorescent FA transfer from I-FABP 130 hydrophobic ligands 130 K- or E-FABP 130 tertiary structures 130 apoptosis 407 apoptotic pathway 440 ARA 242 metabolizing enzymes 292 transport 299 arachidonic acid 383 substrate for either cyclooxygenase or lipoxygenase 285 transport of 291 arachidonic acid (Ara) 285 arachidonic acid, 20: 4n-6 (ARA) 242 atherosclerosis 401, 405 atherosclerotic lesions 429 ATP binding cassette (ABC) transporter superfamily 39 A-ZIP/F1 319

b

b-barrel structure 95 BBM 362, 367 BBM vesicles 362 B-FABP 254, 261, 262, 263, 278 expression 261 immunoreactivity 261 ligand 262 localization 261 mRNA 261 mRNA expression 257 bilirubin 383 blood albumin concentrations 79 BMI A/G polymorphism 399 brain lipid binding protein (BLBP) breast cancer 442

253

c C/EBPb 313, 315 C/EBP 313 Caco-2 362, 370 Caco-2 cell 328 Caco-2 intestinal cell [Thr54]I-FABP-transfected cells 125

Subject Index A-FABP and hormonesensitive lipase (HSL) 126 CRBP-I 126 electroporation of A-FABP into 3T3-L1 126 HSL A-FABP complex 126 calcium 286 binding 287 calcium-binding protein 285 CAP (c-Cbl-associating protein) 319 CAP gene 319 carnitine palmitoyltransferase I (CPT I) 163, 384 Cav-1 365 expression 366 Cav-3 365 caveolin-1 363 caveolins (Cav) 365 Cay-1-null mice 366 CBP 181, 198 CCAAT/enhancer binding protein (C/EBP) 313 CD36 5, 15, 127, 213, 243, 298, 299, 344, 346, 347, 348, 351, 355, 366, 374, 376, 429 adipocyte differentiation 9 as a receptor of S100A8-Ara 303 as a receptor of S100A9-Ara 303 as the dominant scavenger receptor on macrophages 12 binding and uptake of oxidatively modified low-density lipoproteins 12 binds long-chain FA 3 by a translocation 352 distribution 355 downregulating CD36 expression on endothelial cells 14 genetical linkage to insulin resistance in the SHR 23 highly expressed in platelets monocytes/ macrophages and endothelial cells 366 highly glycosylated, integral membrane protein 346 homology to human muscle FA binding protein (M-FABP) 6 implicated in the development and early progression of atherosclerotic lesions 12 in animal models of genetic obesity and diabetes 17 increased expression 17 in mice fed a high-fat diet 17

induced in cardiomyocytes by synthetic PPARa agonists 9 integral membrane glycoprotein 3 lacks the caveolin scaffold recognition sequences 7 long-chain 9 mRNA 354 mRNA is strongly induced by glitazones 9 reversibly bound long-chain FA with high affinity and specificity 16 scavenger receptor 3 translocation 352 CD36 deficiency 10 diabetes type II 11 insulin resistance 11 CD36 gene 8 PPAR activation 9 CD36-deficient human 23 CD36-null mice 19, 22, 25 CD36-overexpressing mice models 24 cellspecific expression activation 271 L-FABP 271 PPAR 271 cellular retinal binding protein I (CRBP-I) a-helical domain 122 A-FABP 122, 123 CRBP-II 123 FABP a-helical domain 122 FABP-membrane complexes 122 FABP-membrane interaction 123 H-FABP 122 I-FABP 122, 123 L-FABP 123 cellular retinoic acid binding proteins I (CRABP-I) 96, 193 cellular retinoic acid binding proteins II (CRABP-II) 96, 193 cellular retinol binding proteins CRBP-I through CRBP-IV 96 cellular retinol binding proteins I 193 c-fos 439 ChoGpl 328, 329, 334 ChoGpl mass 329 cholesterol 135 cholesterol 7a-hydroxylase (CYP7A1) 211 cholesterol efflux 213 cholesterol elimination 212 cholesterol homeostasis 209 cholesterol trafficking 135 cholesteryl ester 334 choline plasmalogen (PlsCho) 332

451

452

Subject Index chylomicron 400 cis-acting elements 217 cis-parinarate (cPnA) 386 cis-parinaric acid (CPA) became fluorescent when bound to PPARc 176 c-jun 439 c-myc 439 CNS 191 co-activators 180 colon carcinogenesis 441 colorectal cancer 443 colorectal carcinogenesis 442 compound F 180 coronary artery disease (CAD) 405 COX-2 182, 292, 423 expression 424, 441 CRABP-I 193, 276 CRABP-II 193, 276, 277, 279, 280 CRBP-I 193 CRBP-II 193 c-reactive protein (c-RP) 404 cross-talk 384 c-terminal ligand binding domain (LBD) 210 cubilin 3600 amino acid protein 86 cyclooxygenase 165 cyclooxygenase (COX) 294 cyclooxygenase-2 (COX-2) 423, 440 CYP26 192 CYP7A1 146 CYP7A1 expression 146 Cystic fibrosis caused by mutations in ABCC7 (CFTR) cytokines 403, 420 cytoplasmic carrier protein 348 cytoplasmic FABP (FABPc) 345 cytosolic phospholipase A2 292

dietary fatty acids 230 dietary lipids 377 DNA binding domain (DBD) 309 docosahexaenoic acid (DHA) or C22:6 cis 4, 7, 10, 13, 16, 19 RXR ligand 201 docosahexaenoic acid, 22: 6n-3 (DHA) 242 DR1 motifs 175 dyslipoproteinemia 232

e

56

d DAUDA (undecanoate (11:0) derivative) L-FABP 124 de novo lipogenesis 215 degranulation of intact PMN 285 15-deoxy D12,14PGJ2 176, 408, 423 comparison of the action 178 15-deoxy-D12,14 prostaglandin J2 310, 423, 441 15-deoxy-D12,14 prostaglandin 408 DHA 242 process of memory formation, vision and reproduction 202

EC 408 EFA 243 E-FABP 261, 263, 274, 275, 278, 279 expression 259 immunoreactivity 259 localization 258 mRNA expression 257 promoter 260 eicosanoid 423 eicosapentaenoic acid 182, 243 eicosopentenoic acid 410 embryo 192 embryogenesis 191 endothelial cells (Ecs) 127, 402 A-FABP–/–animals 129 between I-FABP and sex hormones 129 CD36 128 CD36 nulls 128 FAT-null mouse 128 H-FABP–/– 128 H-FABP–/– mice 128 I-FABP–/– 128 keratinocyte FABP (K-FABP) 129 endothelin 1 410 enterocyte 360, 367 24(S)-25-epoxycholesterol 210 essential fatty acids (EFAs) long-chain polyunsaturated fatty acid (LCPU-FA) derivatives 241 estrogen production 442 ethanolamine glycerophospholipid (EtnGpl) 328 ethanolamine plasmalogen (PlsEtn) 332 EtnGpl 331 expression CD36 352 FAT 352 expression of cyclin D 183

f F-2,6-P2 388 x-3 FA 410

Subject Index FABP 119, 298, 338, 368, 383, 389 binding of FA 388, 392 intestinal 327 intestinal (I-) 327 liver (L-) 327 M-FABP 392 PFK-1 392 FABP and CRBP-II apo CRBP-II 103 CRBP-II 103 iLBP subfamilies 103 solution state structures 102 X-ray data of I-FABP 103 FABP expression alters fatty acid targeting 335 FABP family 277 FABP2 369 FABPc 348 FABP-FA 386 FABPpm 24, 348, 364 FABPpm 40 kDa 243 FAS expression 215 promoter 215 FAT 127, 298, 299, 344, 346, 347, 348, 351, 355, 366, 374, 376 as a receptor of S100A8-Ara 303 as a receptor of S100A9-Ara 303 by a translocation 352 distribution 355 genetical linkage to insulin resistance in the SHR 23 highly glycosylated, integral membrane protein 346 mRNA 354 translocation 352 FAT 88 kDa 243 fat absorption 359 fat cell 317 fat supply 359 FATP 355 mmFATP1 33 mmFATP1–5 33 mmFATP2 33 mmFATP5 33 FATP 63 kDa 243 FATP1 24, 32, 33, 365, 376, 397 5' regulatory region 399 A/G polymorphism 398 A/G substitution 398 AA genotype 399 AG 399 AG genotype 398

chromosome 19p13.1 398 gene 398 GG 399 GG genotype 398 IYTSGTTGXPK 33 mRNA 399 polymorphisms 398 protein 399 transporter 398 FATP2 FATP1 mRNA 35 isoforms 34 mmFATP1 34 mmFATP2 34 mmFATP4 34 mmFATP5 34 orthologs 34 peroxisome matrix 33 FATP2 gene Ala54Thr polymorphism 399 FATP4 365 fatty acid binding protein (aP2) 313 fatty acid binding protein (FABP) 95, 119, 268, 327, 383 brain (B-) type 253 cutaneous type (C)-type 253 epidermal (E-) type 253 heart (H-) type 253 psoriasis-associated (PA)-type 253 fatty acid binding proteins (L-FABP) 317 fatty acid synthase 214 fatty acid targeting 328 fatty acid translocase (FAT) 346 cDNAs 32 mAspAT or CD36 32 fatty acid transport protein (FATP) 313, 365 fatty acid transport protein 4 (FATP4) 363 fatty acid transport proteins (FATP1–5) 346 fatty acid transporter (CD36) 363 fatty acid transporter (FAT) 363 fatty acid uptake 327, 328 fatty acids 119, 253 a metabolic energy source 241 as cellular signaling molecules 241 signaling pathway 267 fatty acyl-CoA 230 binding affinity 227 Kd value 227 ligands 232 PUFAs 228 thioesters 228

453

454

Subject Index fatty streak 402 fenestrated endothelia 82 feto-placental growth and development 250 fibrate hypolipidemic drugs 438 fibroblasts, 3T3 314 x-3 fish oils 182 FLAP 293 fluorescent fatty acid analog cis-parinaric acid (cPnA) 125 cPnA binding to I-FABP 125 L-FABP content 125 L-FABP mRNA expression 125 fluorescent probe ADIFAB 360 fluorescently tagged glucose 389 FRAP 124 free fatty acids 397 fructose-1,6-bisphosphate 386 fructose-6-phosphate 386

g G1P 388 G6P 386 GATA 313 giant sarcolemmal vesicles 345, 348 a2-globulin adipocyte- (A-) 96 basic liver- (Lb-) 96 brain- (B-) 96 epidermal- (E-) 96 FABP 96 heart- (H-) 96 intestinal- (I-) 96 liver- (L-) 96 myelin- (M-) 96 testis-type (T-) 96 glucocorticoid receptor 182 glucokinase-glucose 6-phosphatase 384 glucose 386 glucose transporter (GLUT4) 313, 319 GLUT4 expression 317 glycerol-3-phosphate acyltransferase (GPAT) 337 glycolytic flux 384 glycolytic intermediates 393 gp60 82 gp60-albumin interactions 82 GPAT activity 339 G-proteins 267 GRIP 175 GST-pulldown assay 275 guanylate cyclases 267 GW7845 409

h

H+ antiport 362 hCG promoter 248 hCGb transcript 248 HDL 404 levels 409 HDL-cholesterol 232 heart 345 heart tissue 387 hemophilia 233 HepG2 cells L-FABP+I-FABP 124 15-HETE 423 8S-HETE 425 heterodimeric partner 174 hexokinase I 384 hexokinase-glucose 6-phosphatase 384 H-FABP 254, 261, 262, 277, 278, 279 mRNA 254 mRNA expression 254, 257 HK 389 HMG-CoA reductase 146 HNFa 249 HNF-4a 225 DNA binding domain (DBD) 225 fatty Acyl-CoA ligands 226 in rodents 233 knockout mice 233 ligand binding domain (LBD) 225 long-chain 228 nuclear receptors 225 response elements 225 spliced variants 225 transcriptional activity 228 xenobiotic ligands 230 9-HODE 430 13-HODE 430 13S-HODE 310 HSI 127 HSL 127 human albumin 79 human intestine 366 3-hydroxybutyrate 387 FA binding 391 22(R)-hydroxy-cholesterol 210 24(S)-hydroxycholesterol 210 8(S)-hydroxyeicosatetraenoic acid (8SHETE) 421 hydroxyeicosatetraenoic acids (HETEs) 310 9-hydroxyoctadecadienoic acid (9-HODE) AB domains 184

Subject Index 9-hydroxyoctadienoic acid 423 13-hydroxyoctadienoic acid 423 hydroxyoctadienoic acids (HODEs) 310 hyperproliferative diseases 432 hypertriglyceridemia 232 hypolipidemia 437

i I-BABP I-BABP 98 I-L-FABP 98 I-FABP 368 differential effects 328 I-FABP expression 328 IFNc 411 IL-1 403 IL-1a 420, 427 IL-12 427 IL-2 411 IL-6 182, 319, 420, 423, 427 iLBP 278, 279, 280 as modulators of fatty acid activated nuclear receptor activity 270 expression pattern 269 import into the nucleus 278 subfamilies 269 I-LBP 119 b-barrel structure 121 A-, H-, I-, E-, B- and M-FABPs 122 ADIFAB (acrylodated intestinal fatty acid binding protein) 120 A-FABP 121 anthroyloxy-labeled fatty acid (AOFA) 122 apo I-FABP 121 H-FABP 121 I-FABP 121 I-LBP 120 iLBP family 277 ileal lipid binding protein (I-LBP) liver fatty acid binding protein (L-FABP) 119 INFc 423, 430 inflammatory bowel disease (IBD) 431 inflammatory cytokines 430 inflammatory processes 182 iNOS 182, 427 insulin 352 insulin action 250 insulin resistance 322 insulin resistance syndrome (syndrome X) 405 insulin sensitizers 437 insulin signaling 319

insulin-inducible LCFA uptake 352 insulin-sensitizing drugs (TZDs) 318 intercellular lipid binding proteins 269 interleukin 1 407 intestinal bile acid binding protein (I-BABP) A-FABP 96 ALBP 96 aP2 96 B-FABP 96 BLBP 96 E-FABP 96 I-BABP 96 KLBP 96 Lb-FABP 96 Mal-1 96 P2 (for M-FABP) 96 T-FABP 96 TLBP 96 Z protein 96 intracellular lipid binding proteins (iLBP) iLBP 95 ionic interactions 371 c isotypes 429

k Kennedy pathway 331 ketone body 387 known as PPARb 402

l

b-lactoglobulin 96 LBD 197 LBP 372 upregulated by fatty acids (FAs) 359 L-cells I-FABP 327 L-FABP 327 LCFA 31, 361, 368, 398 diffusional event 298 induced modulation of gene expression 353 influx 362 protein-mediated transport 298 transport 32, 364 transport across the adipocyte membrane 36 transport across the membrane 352 uptake 298, 365 utilization rates 350 LCFA transport non-protein-mediated 31 protein-mediated permeation 31 transport 31

455

456

Subject Index LCFA uptake 344 LCFAs 360 LCPUFA 243 LDL-R family 88 LDLR family members 89 LEF families 313 leukotriene B4 (LTB4) 421, 439 L-FABP 272, 273, 275, 276, 277, 280, 334, 338, 368 cytosolic 278 differential effects 328 dissociation constants for ligand binding 274 FA binding 386 localize to the nucleus 376 modifications 279 L-FABP-expressing cells 328 L-FABP-expressing L-cells 329, 331 ligand-receptor interactions 267 LIMPII 5 linoleic acid, 18:2n-6 242 c-linolenic acid 243 lipid binding protein (LBP) 309, 359 lipidex assay 110 lipocalins 96 lipogenic malic enzyme genes 317 lipoprotein lipase 242, 412 lipoxygenase 165 5-lipoxygenase (5-LO) 292 5-lipoxygenase activating protein (FLAP) 292 lipoxygenase pathways 423 liver 399 liver X receptor (LXR) 209, 249 long-chain acyl-CoA synthetase 1 (ACS1) 32 long-chain fatty acid (LCFA) hydrolysis 31 LCFA 230, 298, 343, 359, 397 mediators of gene expression 119 precursors for signaling molecules 119 long-chain fatty acyl-CoA (LCACoA) 151 LTB4 423 H-FABP mRNA 247 L-FABP and H-FABP in human placenta syncytiotrophoblasts 247 LXR 64, 214 ABCA1, expression and activity control 61 fatty acid responsive 218 signaling pathways 215 LXR activation response to cellular cholesterol stress 61

LXR agonists 61 LXR heterodimer 210 LXR target genes in lipid homeostasis 209 LXRa 209, 249 by fatty acids 218 LXRa/b–/– 214 LXRa–/– mice 212, 214 LXRa-deficient mice 212 LXRa-knockout mice 212 LXRs require heterodimerization with the retinoid X receptor (RXR) 210 LXXLL co-activators 199 LXXLL helix 198 LXXLL motif 177, 181 lysophosphatidic acid acyltransferase (LAT) 337

m macrophage colony-stimulating factor (M-CSF) 429 macrophages 182 malaria 14 malonyl-CoA 384 mammalian cells 32 MAP kinase 217 MAP kinase pathway 184 mAspAT expression 364 maternal plasma 242 matrix metalloproteinase (MMP) 402 matrix metalloproteinase 9 407 maturity-onset diabetes of the young (MODY)-1 232 MDGI 262, 277 megalin 84 belongs 85 low-density lipoprotein receptor (LDL-R) family 85 megalin and cubulin as cooperative albumin receptor 87 membrane ATP-binding cassette (ABC) and high-density lipoproteins (HDLs) 212 membrane-associated acyl-coenzyme A (CoA) synthetase (fadD) 31 membrane-bound fatty acid transport protein fadL 31 microsomal desaturation 333 microsomal fatty acid x-hydroxylase (CYP4A) 173 microsomal GPAT activity 338 microvillous membranes (MVM) 242

Subject Index mitochondrial 397 mitochondrial aspartate aminotransferase (mAspAT) 32, 364 MMP 402 response 407 MMP-9 407, 427 MODY-1 233 monocyte chemoattractant protein 1 (MCP-1) 430 monocytes 285 motif AGGT-CA 309, 310 MRG 262 multidrug resistance ABCB1 57 ABCC1 57 ABCG2 57 MDR1 57 MRP1 57 multifunctional enzyme 2 (MFE-2) 138 muscle 399 muscle LCFA uptake 345 myocardial infarction 401 myocytes 344

n n-3 LCPUFAs 242 n-6 LCPUFAs 242 Na+ antiport 362 NADPH oxidase 295 NADPH oxidase complex 297 Naka-negative phenotype 11 natural fatty acids 438 NBDS 124 N-COR 181 N-CoR (nuclear receptor co-repressor) 199 neutral lipid 328 neutrophils 285 NFjB 178, 182, 249, 424, 427 NFjB family 424 NFjB pathway 424 NMR spectra 99 non-lipid metabolites 384 non-steroidal anti-inflammatory drugs (NSAIDs) 444 NSAIDs 176, 444 N-terminal transcriptional activation domain (AF-1) 210 nuclear hormone receptor (NHR) 193, 209, 268, 309

o obese ob/ob mouse A-FABP–/– 129

–/–

–/–

A-FABP /apoE 129, 130 ap2–/– 130 ApoE–/– 129 obesity 354, 437 orphan receptor 174, 226 b-oxidation pathway 316 b-oxidation 25, 142, 327 oxidized LDL 408 oxLDL 213, 429, 430

p p/CAF 198 p300 181, 198 p65 424 PAI-1 408 palmitoylcarnitine translocase 384 palmitoyl-CoA 338 peroxisomal b-oxidation 397 peroxisomal b-oxidation of VLCFA-CoA 138 peroxisome proliferation 173 peroxisome proliferator activated receptor c (PPARc) 213 peroxisome proliferator activated receptor (PPARa, c and d) 8, 127, 268, 309, 333, 375, 401, 437 peroxisome proliferator responsive elements (PPREs) 268 peroxisome proliferators (PPs) 173, 230, 234 peroxisomes 316 PfEMP-1 15 PGE2 247 PGJ2 428 phosphatidic acid (PtdOH) 337 phosphatidylinositol 331 phosphatidylinositol 3-kinase (P13-kinase) pathways 217 phosphatidylserine 328 phosphoenolpyruvate kinase (PEPCK) 313 phosphofructokinase-fructose 1,6-phosphatase 384 phosphofructokinase-I (PFK-I) 384 phospholipid 328 phospholipid levels 334, 337 phospholipid mass 329 phospholipid synthesis 331 PKC 184 placenta 243, 311 placental FABPpm (p-FABPpm) 244 placental microvillous and basal membrane preparations 246 H-FABP 247 L-FABP 247 plasma membrane 346

457

458

Subject Index plasma membrane fatty acid binding protein (FABPpm) 298, 363, 364 plasma membrane proteins 32 plasma retinol binding protein (RBP) 96 plasmalogen biosynthesis 333, 334 plasmodium falciparum 14 PlsCho 332 polymorphonuclear neutrophils (PMN) 285 PP hypolipidemic 231 PPAR 248, 273, 278, 310, 420 antagonists at 376 dissociation constants for ligand binding 274 independent effects 428 isoform 275, 277 ligand binding cavity 177 ligands 421 modulated by the action of kinases 183 phosphoproteins 183 PPARc2 184 PPAR and RXR ligands 196 PPAR family 248 PPAR heterodimer 182, 248 PPAR response element (PPRE) 174, 319, 437 PPAR responsive elements (PPRE) 376 PPARb expression 443 PPARc 159, 165, 174, 176, 177, 180, 213, 233, 248, 269, 276, 278, 309, 310, 311, 312, 316, 317, 318, 375, 383, 402, 408, 410, 412, 413, 419, 420, 421, 423, 424, 427, 428, 429, 430, 431, 432, 437, 438, 441, 442, 443, 444 a isoforms 268 activation by arachidonic acid 184 activators 424 agonist 409, 423, 427 associated downregulation of the protein phosphatase PP2A 314 binding sites 176 CD36 383 expression 407, 425 gene 426 human liver 234 ligands 184, 440, 441, 442 ligands LTB4 425 promoter 420 regulation by phosphorylation 185 rodents 234 selective target genes 176 target genes 310 transactivation 272

PPARd activators 413 PPARd expression 317 PPARc ligand 321, 427, 428 PPARd pathways 413 PPARc–/– 315 PPARb/d 375 PPARc–/– embryonic stem (ES) 312 PPARa 353 PPARb+/– 425 PPARc1 314 PPARc2 314 regulator of adipogenesis 311 PPARc-deficient 315 PPARc-null embryos 311 PPARb-null human colorectal cancer cell line 444 PPARb-null keratinocytes 421 PPARc-null mice 311, 423, 425, 439 PPARa-null mouse 317 PPARc-null stem 427 PPARs 202, 248, 375, 401, 406 cholesterol sensor 218 metabolism 413 natural ligands 218 role of carboxylic acid in the activation 177 PPRE 269 pre-adipocytes 312 prostacyclin 423 prostaglandin E1 383 prostaglandins 310 PtdOH acid biosynthesis 339 PtdOH biosynthesis 337, 338 PtdSer 329 PUFAs 232

r RA 192 RA response elements (RAREs) 191, 194 RAL dehydrogenase (RALDH) 191 RALDH2 192 RAR 194, 203, 269, 276 a isoforms 268 RAR mutants 194 RARb 193, 277 RAR-RXR heterodimers 196 receptor for peroxisome proliferators 173 renal FABP 298 repression of cyclin PPARc ligands 183 respiratory burst 285 retinoic acid (RA) 191

Subject Index retinoic acid receptor responsive element (RARE) 268 retinoic acid receptors (RARs) 193, 268 retinoic acid X receptors (RXRs) 268 retinoid X receptor (RXR) 175, 193, 310, 437 agonists 35 FATP1 mRNA 35 retinoids 191 rosglitazone ligand binding domain 176 rosiglitazone 184 RXR 64, 159, 194, 202, 248, 269, 310, 406 a isoforms 268 ABCA1, expression and activity control 61 heterodimer partner 196 ligands 201, 203 RXR heterodimer 182, 210, 248 RXR ligands 432 RXR mutants 194 RXRa 203, 248, 249, 278

s S100 family 285 S100 protein expressed in circulating neutrophils and monocytes 287 S100A8 285, 286 binds fatty acids in a Ca2+-dependent manner 289 expressed in circulating neutrophils and monocytes 287 protein complexes 294 S100A8-Ara complexes specifically secreted from activated human neutrophils 302 S100A8-arachidonic acid complex 127 S100A9 285, 286 binds fatty acids in a Ca2+-dependent manner 289 cell type-specific expression 287 expressed in circulating neutrophils and monocytes 287 gene expression 288 protein complexes 294 S100A9-Ara complexes specifically secreted from activated human neutrophils 302 S100A9-arachidonic acid complex 127 sarcolemmal 351 SCD-1 215 SCD-1–/– mice 215

Schiff bases 388 SCP-2 141, 144, 146 binds most fatty acids and fatty acylCoAs 136 crystal structure of the 139 SCP-2 deficiency attributed to sustained PPAR a activation 143 SCP-2 gene family 138 D-PBE 136 hSLP-1 136 MFE-2 136 SCP-2 136 SCP-x 136, 137 UNC-24 136 SCP-2-null mice 144, 146 SCP-x 141 SCP-x deficiency attributed to sustained PPAR a activation 143 SCP-x thiolase medium straight-chain acyl-CoA substrates 137 2-methylbranched-chain fatty acylCoAs 137 ketocholestanoyl-CoA 137 trihydroxy 137 SCP-x-null mice 144, 146 Ser112 183 serine kinases 267 signal transduction receptors 267 silencing mediator of retinoid and thyroid hormone receptor (SMRT) 181, 199 sitosterolemia b-ABCB subfamily 69 b-ABCB11 69 b-hepatobiliary secretion 69 b-sitosterolemia 65 ABCA1–/– 66 ABCG8 66 skeletal muscle 322 small intestine 359 sphingomyelin 328 SRC-1 181 SREBP cleavage-activating protein (SCAP) 214 SREBP-1c 214, 249 mRNA 215 upregulated by insulin 216 STAT 427 STAT1 314 STAT5 314

459

460

Subject Index STAT5a 314 STAT5b 314 stearoyl-CoA desaturase-1 (SCD-1) 214 sterol absorption 213 sterol carrier protein 2 (SCP-2) non-specific lipid transfer protein 136 sterol homeostasis 64 sterol regulatory element binding proteins (SREBPs) 313 SREBP-1 159, 249 SREBP-1a 214, 249 SREBP-1c 214, 249 SREBP-2 214, 249 subfamily II A- 106 Arg106 in the iLBP of subfamily IV 106 E- 106 E-FABP 106 Gln108 in the CRBPs 106 His108 in the CRBPs 106 I-FABP 106 Lb-FABP 105 L-FABP 105 M-FABP 106 subfamily IV 106 T-FABP 106 subfamily ABCD (ALD) 50 subfamily ABCE 51 subfamily ABCG (White) ABCG1 51 ABCG2 51 ABCG4 51 ABCG5 51 ABCG8 51 sulfo-N-succinimidyl-oleate (SSO) 345 sulfonylurea receptor (ABCC8) 59 sulfonylurea receptor (SUR) 59

t 3T3-L1 365 T lymphocytes 407 Tangier disease 213 target gene of LXR 211 TCF families 313 thiazolidinedione 318 threonine kinases 267 TNFa 182, 185, 319, 407, 411, 420, 421, 423, 426, 427, 429, 430 total phospholipid 328

transcription factor 226, 312 transmembrane 348 triacylglycerol 328 triacylglycerol hydrolase activity 242 triglyceride 334, 360 movement of LCFA 31 utilization 31 triglyceride metabolism 397 trophoblast cells 243, 246 tumor necrosis factor a (TNFa) 440 A-FABP–/– mice 129 aP2–/– mice 129 FABP–/– mouse 129 tumor suppressor 314, 442 tumorigenesis 440 tumors 183 TxB2 247 type 2 diabetes 354, 437 tyrosine kinases 267 tyrosine phosphatases 267 TZD 177, 178, 421, 432, 441

u upregulate COX-2 441

v various ROL dehydrogenases (ROLDH) 191 vascular cell adhesion molecule-1 430 vascular smooth muscle cells (VSMCs) 403 very long-chain acyl-CoA synthetase (VLACS) FATP4 33 vitamin A 191 deficiency 191 levels 191 VLDL-triglyceride particles 400 VLDLs 232 VSMC 410 migration 407

w Wy14,463 176, 321, 428, 441

x xenopus laevis oocytes 364

z zinc-finger motifs promoters 225 zucker rats 355