Leptin and Leptin Antagonists

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GERTLER

INTELLIGENCE UNITS

Arieh Gertler

MIU

Leptin and Leptin Antagonists

Leptin and Leptin Antagonists

Medical Intelligence Unit

Leptin and Leptin Antagonists Arieh Gertler, PhD

Institute of Biochemistry, Food Science and Nutrition The Hebrew University of Jerusalem Rehovot, Israel

Landes Bioscience Austin, Texas USA

Leptin and Leptin Antagonists Medical Intelligence Unit Landes Bioscience Copyright ©2009 Landes Bioscience All rights reserved. No part of this book may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopy, recording, or any information storage and retrieval system, without permission in writing from the publisher. Printed in the USA. Please address all inquiries to the publisher: Landes Bioscience, 1002 West Avenue, Austin, Texas 78701, USA Phone: 512/ 637 6050; Fax: 512/ 637 6079 www.landesbioscience.com ISBN: 978-1-58706-320-6 While the authors, editors and publisher believe that drug selection and dosage and the specifications and usage of equipment and devices, as set forth in this book, are in accord with current recommendations and practice at the time of publication, they make no warranty, expressed or implied, with respect to material described in this book. In view of the ongoing research, equipment development, changes in governmental regulations and the rapid accumulation of information relating to the biomedical sciences, the reader is urged to carefully review and evaluate the information provided herein.

Library of Congress Cataloging-in-Publication Data

Leptin and leptin antagonists / [edited by] Arieh Gertler. p. ; cm. -- (Medical intelligence unit) Includes bibliographical references and index. ISBN 978-1-58706-320-6 I. Gertler, Arieh. II. Series: Medical intelligence unit (Unnumbered : 2003) [DNLM: 1. Leptin. 2. Leptin--Antagonists. 3. Leptin--antagonists & inhibitors. 4. Receptors, Leptin. WK 185 L6113 2009] QP572.L48L467 2009 572'.633--dc22 2008051305

Dedication I would like to dedicate this book to my beloved wife Anna who always encouraged me along the way.

About the Editor...

ARIEH GERTLER is a Professor-Emeritus in Agricultural Biochemistry, holder of the Karl Bach Chair in Agricultural Biochemistry. His main interests include the physiological and molecular action of several cytokines such as growth hormone, prolactin and placental lactogen. In the last decade his main research effort was in the field of leptin and culminated in development of leptin mutants devoid of agonistic activity but retaining their full capacity of binding to leptin receptors and just acting as potent competitive leptin antagonists. Upon his retirement in 2003 Arieh Gertler founded a small biotech company (Protein Laboratories Rehovot) which produces recombinant proteins for research purposes. Arieh Gertler received all his academic degrees (BSc, MSc and PhD) in the Hebrew University of Jerusalem, Israel.

CONTENTS Preface......................................................................................................... xv

Part I: Molecular Aspects of Leptin Action 1. Leptin Signal Transduction—A 2009 Update .............................................1 Walter Becker Leptin and the Leptin Receptor.............................................................................1 JAK Kinase .................................................................................................................2 Activation of STAT3 ................................................................................................3 Leptin-Regulated Genes ..........................................................................................4 Activation of Other STAT Factors .......................................................................4 Activation of the ERK Pathway .............................................................................7 Activation of the IRS/PI3K/PDE3B Pathway...................................................8 Activation of ATP-Sensitive K+ Channels ..........................................................8 Regulation of AMPK (AMP-Activated Protein Kinase) and mTOR (Mammalian Target of Rapamycin) ..........................................9 Perspective...................................................................................................................9 2. Insights in the Activated LR Complex and the Rational Design of Antagonists ................................................................................15 Frank Peelman, Lennart Zabeau and Jan Tavernier Leptin as a Disease-Promoting Factor: Rationale for Leptin Antagonists ......................................................................................16 Structure of Leptin and Its Receptor Homology with the IL-6 and G-CSF Receptor Systems..........................................................................16 Evidence for Receptor Oligomerisation and Higher Order Clustering ................................................................................................17 Three Binding Sites in Leptin...............................................................................18 Models of the CRH2-Leptin Complex............................................................. 20 Models for the Ig-Like and CRH1 Domains....................................................21 Homology Model for a Hexameric 2:4 Leptin:LR Complex....................... 22 Homology Models for the Fibronectin Type III Domains........................... 22 Mechanism of LR Activation .............................................................................. 24 Development of Leptin-Based Antagonists ......................................................25 Optimization of Leptin-Based Antagonists..................................................... 26 Concluding Remarks ............................................................................................. 26 3. Study of Leptin: Leptin Receptor Interaction by FRET and BRET .........30 Julie Dam, Cyril Couturier, Patty Chen and Ralf Jockers Activation Mechanism of OB-R Studied with Biochemical Methods .......31 Methodological Introduction to FRET/BRET ...............................................33 Activation Mechanism of OB-R Monitored by FRET and BRET ............ 34 The OB-R BRET Assay, a Screening Tool for the Identification of New OB-R Ligands ......................................................................................39 Conclusion ................................................................................................................39

Part II: Leptin Involvement in Physiological and Pathological Processes 4. Is Leptin a Pro- or Anti-Apoptotic Agent? ................................................43 Srujana Rayalam, Mary Anne Della-Fera, Suresh Ambati and Clifton A. Baile Apoptosis: A Basic Biologic Phenomenon ........................................................ 44 Anti-Apoptotic Effects of Leptin ........................................................................45 Pro-Apoptotic Effects of Leptin ..........................................................................47 Conclusions ............................................................................................................. 49 5. Leptin Actions in the Gastrointestinal Tract .............................................54 Sandra Guilmeau, Thomas Aparicio, Robert Ducroc and André Bado Gastric Leptin Directly Activates Vagal Afferent Neurons...........................55 Leptin and Intestinal Physiology .........................................................................55 Leptin in Gastrointestinal Pathologies ............................................................. 58 Conclusions and Perspectives ...............................................................................59 6. Leptin as a Novel Marker in Breast and Colorectal Cancer .......................63 Eva Surmacz and Mariusz Koda Leptin and Breast Cancer ......................................................................................63 Leptin and Colorectal Cancer............................................................................. 68 Summary and Perspectives ....................................................................................69 7. The Role of Leptin in Cardiac Physiology and Pathophysiology ..............73 Morris Karmazyn, Daniel M. Purdham, Venkatesh Rajapurohitam and Asad Zeidan Leptin Synthesis and Structure ............................................................................74 Leptin Resistance ....................................................................................................74 Is Leptin a Possible Link between Obesity and Increased Cardiovascular Risk? .........................................................................................74 Expression of Leptin Receptors in Cardiovascular Tissues ...........................75 Effect of Leptin on Cardiomyocyte Function ..................................................76 Cardiomyocyte Hypertrophic Effects of Leptin ..............................................76 Leptin as a Cardioprotective Agent ................................................................... 77 Post Receptor Leptin Signaling .......................................................................... 77 Conclusions: Potential of Leptin Modulators as Therapeutic Agents ........78 8. The Role of Leptin in Bone Development and Growth .............................83 Efrat Monsonego Ornan and Michal Ben-Ami The Effect of Leptin on the Skeleton ..................................................................83 Leptin and Growth .................................................................................................85 Central Effect of Leptin.........................................................................................85 Peripheral/Direct Effect of Leptin ..................................................................... 86 Synopsis .................................................................................................................... 88

9. Involvement of Leptin in Arterial Hypertension .......................................91 Jerzy Beltowski Physiological Effects of Leptin Relevant for Blood Pressure Regulation...... 92 Selective and Peripheral Leptin Resistance ...................................................... 95 Prohypertensive Effects of Chronic Hyperleptinemia................................... 98 Conclusions and Future Perspectives .............................................................. 102 10. Involvement of Leptin in the Endometrial Function ...............................108 Ana Cervero and Carlos Simon Overview of the Leptin System ......................................................................... 108 Leptin System in the Endometrium................................................................. 109 Leptin System in the Implantation Process.................................................... 109 Leptin System in the Endometriosis .................................................................111 Summary and Conclusions ................................................................................ 113 11. The Use of Leptin for the Treatment of Lipodystrophy ...........................116 Angeline Y. Chong, Elaine K. Cochran and Phillip Gorden Metabolic Effects of Leptin Therapy.................................................................117 Endocrine Effects of Leptin Therapy ............................................................... 120 Hepatic and Muscular Effects of Leptin Therapy ......................................... 123 Conclusion ............................................................................................................. 124 12. Use of Anti-Leptin or Anti-Leptin Receptor Antibodies as Blockers of Immune Response ................................................................................126 Giuseppe Matarese and Veronica De Rosa Leptin Has Multiple Functions in Immunity ............................................... 127 Leptin Is Involved in the Development of Various Diseases ...................... 127 Immunotherapeutic Applications Targeting Leptin: Current Evidence and Hypotheses .............................................................. 127 Leptin Neutralization: Novel Strategies to Block Autoimmunity and to Improve Leptin Resistance Observed in Obesity........................ 129 Leptin-Receptor Neutralization ....................................................................... 131 Conclusions and Future Perspectives .............................................................. 131 13. Use of Leptin Antagonists as Anti-Inflammatory and Anti-Fibrotic Reagents ......................................................................133 Eran Elinav and Arieh Gertler Results .....................................................................................................................134 Chronic Hepatitis and Fibrosis ......................................................................... 136 Conclusions ........................................................................................................... 138 14. The Role of Leptin during Early Life in Imprinting Later Metabolic Responses ................................................................................141 Mark H. Vickers, Stefan O. Krechowec, Peter D. Gluckman and Bernhard H. Breier Background............................................................................................................ 141 Leptin and Developmental Programming...................................................... 142 Evidence from Animal Models ......................................................................... 143

Epidemiological and Clinical Evidence........................................................... 146 Leptin in Early Life and Catch-Up Growth .................................................. 147 Potential Mechanisms ......................................................................................... 148 Developmental Programming and Gender Differences in Leptin Sensitivity........................................................................................ 150 Leptin in the Perinatal Period—A Therapeutic Window of Intervention?................................................................................................ 150 Extrapolation from Animal Models to the Clinical Setting .......................152 Discussion ...............................................................................................................153 Index .........................................................................................................163

EDITOR Arieh Gertler

Institute of Biochemistry, Food Science and Nutrition The Hebrew University of Jerusalem Rehovot, Israel Email: [email protected] Chapter 13

CONTRIBUTORS Note: Email addresses are provided for the corresponding authors of each chapter. Suresh Ambati Department of Animal and Dairy Science University of Georgia Athens, Georgia, USA Chapter 4

Thomas Aparicio INSERM, U773 Centre de Recherche Biomédicale Bichat Beaujon Paris, France Chapter 5

André Bado INSERM, U773 Centre de Recherche Biomédicale Bichat Beaujon Paris, France Email: [email protected] Chapter 5

Clifton A. Baile Department of Animal and Dairy Science University of Georgia Athens, Georgia, USA Email: [email protected] Chapter 4

Walter Becker Institute of Pharmacology and Toxicology Medical Faculty of the RWTH Aachen University Aachen, Germany Email: [email protected] Chapter 1

Jerzy Beltowski Department of Pathophysiology Medical University Lublin, Poland Email: [email protected] Chapter 9

Michel Ben-Ami Department of Biochemistry and Nutrition The Hebrew University Jerusalem, Israel Chapter 8

Bernhard H. Breier Liggins Institute and The National Research Centre for Growth and Development The University of Auckland Auckland, New Zealand Chapter 14

Ana Cervero Fundación IVI Valencia, Spain Chapter 10

Patty Chen Institut Cochin Department of Cell Biology Université Paris Descartes Paris, France Chapter 3

Angeline Y. Chong Clinical Endocrinology Branch National Institute of Diabetes, Digestive and Kidney Diseases National Institutes of Health Bethesda, Maryland, USA Chapter 11

Elaine K. Cochran Clinical Endocrinology Branch National Institute of Diabetes, Digestive and Kidney Diseases National Institutes of Health Bethesda, Maryland, USA Chapter 11

Cyril Couturier Centre National de la Recherche Scientifique Université Lille Lille, France Chapter 3

Julie Dam Institut Cochin Department of Cell Biology Université Paris Descartes Paris, France Chapter 3

Mary Anne Della-Fera Department of Animal and Dairy Science University of Georgia Athens, Georgia, USA Chapter 4

Veronica De Rosa Laboratorio di Immunologia Istituto di Endocrinologia e Oncologia Sperimentale Consiglio Nazionale delle Ricerche Napoli, Italy Chapter 12

Robert Ducroc INSERM, U773 Centre de Recherche Biomédicale Bichat Beaujon Paris, France Chapter 5

Eran Elinav Gastroenterology and Liver Institute Tel Aviv Sourasky Medical Center (TASMC) Tel Aviv, Israel Email: [email protected] Chapter 13

Peter D. Gluckman Liggins Institute and The National Research Centre for Growth and Development The University of Auckland Auckland, New Zealand Chapter 14

Phillip Gorden Clinical Endocrinology Branch National Institute of Diabetes, Digestive and Kidney Diseases, National Institutes of Health Bethesda, Maryland, USA Email: [email protected] Chapter 11

Sandra Guilmeau INSERM, U773 Centre de Recherche Biomédicale Bichat Beaujon Paris, France Chapter 5

Ralf Jockers Institut Cochin Department of Cell Biology Université Paris Descartes Paris, France Email: [email protected] Chapter 3

Morris Karmazyn Department of Physiology and Pharmacology Schulich School of Medicine and Dentistry University of Western Ontario London, Ontario, Canada Email: [email protected] Chapter 7

Mariusz Koda Department of Pathomorphology Medical University of Bialystok Bialystok, Poland Chapter 6

Stefan O. Krechowec Liggins Institute and The National Research Centre for Growth and Development The University of Auckland Auckland, New Zealand Chapter 14

Giuseppe Matarese Laboratorio di Immunologia Istituto di Endocrinologia e Oncologia Sperimentale Consiglio Nazionale delle Ricerche Napoli, Italy Email: [email protected] Chapter 12

Efrat Monsonego Ornan Department of Biochemistry and Nutrition The Hebrew University Jerusalem, Israel Email: [email protected] Chapter 8

Frank Peelman Department of Medical Protein Research, VIB Department of Biochemistry Ghent University Ghent, Belgium Chapter 2

Daniel M. Purdham Department of Physiology and Pharmacology Schulich School of Medicine and Dentistry University of Western Ontario London, Ontario, Canada Chapter 7

Venkatesh Rajapurohitam Department of Physiology and Pharmacology Schulich School of Medicine and Dentistry University of Western Ontario London, Ontario, Canada Chapter 7

Srujana Rayalam Department of Animal and Dairy Science University of Georgia Athens, Georgia, USA Chapter 4

Carlos Simon Fundacion IVI Valencia, Spain Email: [email protected] Chapter 10

Eva Surmacz Sbarro Institute for Cancer Research and Molecular Medicine Temple University Philadelphia, Pennsylvania, USA Email: [email protected] Chapter 6

Jan Tavernier Department of Medical Protein Research, VIB Department of Biochemistry Ghent University Ghent, Belgium Email: [email protected] Chapter 2

Mark H. Vickers Liggins Institute and The National Research Centre for Growth and Development The University of Auckland Auckland, New Zealand Email: [email protected] Chapter 14

Lennart Zabeau Department of Medical Protein Research, VIB Department of Biochemistry Ghent University Ghent, Belgium Chapter 2

Asad Zeidan Department of Physiology and Pharmacology Schulich School of Medicine and Dentistry University of Western Ontario London, Ontario, Canada Chapter 7

PREFACE The discovery of leptin, the obese (ob) gene product which is not expressed as a functional protein in ob/ob mice, focused the scientific community’s attention on its role as an anorexic hormone involved in the negative regulation of food intake. Almost 14 years after this breakthrough discovery and over 14,000 leptin-related publications later, leptin is now known to participate in a wide range of biological functions that include, in addition to its early envisaged function as an adipostat, glucose metabolism, glucocorticoid synthesis, CD4+ T-lymphocyte proliferation, cytokine secretion, phagocytosis, hypothalamic-pituitary-adrenal axis regulation, reproduction, cardiovascular pathology, bone formation, apoptosis and angiogenesis. In short, it is now well-documented that leptin acts like a cytokine hormone with many pleiotropic effects. Furthermore, in recent years, it has become more and more apparent that many of leptin’s effects are acquired not only through its central action, but also through its systemic action on a peripheral level. This book focuses mainly on the relatively novel aspects of leptin’s actions. In parallel to the discovery and exploration of leptin’s physiological action, extensive research has been aimed at clarifying leptin signal transduction. Although many transduction pathways have been discovered, the structural aspects of the leptin:leptin receptor interaction have remained mostly speculative and based mainly on models, due to the lack of any valid structural information on the leptin receptor. Nevertheless, modeling of this interaction has enabled a better understanding of the leptin:leptin receptor interaction and has led to the rational development of leptin antagonists. This book is divided into two parts: Part I deals with the molecular aspects of leptin’s action, whereas Part II is devoted to various central and peripheral physiological activities, with an emphasis on its potential involvement in different pathologies. In the first chapter of this book Becker sums up the recently acquired knowledge on leptin’s action. He provides updated information on various leptin-activated transduction signaling pathways, including novel and relatively little investigated phenomena such as activation of ATP-sensitive K+ channels and AMPK-mediated effects leading to activation of mTOR (mammalian target of rapamycin). This chapter also gives an updated compilation of leptin-induced genes (or: “target genes”). In Chapter 2, Tavernier and his colleagues contribute deep insight into the possible models of the leptin:leptin receptor interaction. They review the experiments which led to the hypothesis that leptin interaction with its receptor resembles that of interleukin 6, namely that the leptin:leptin receptor complex is a hexamer composed of two leptin and four leptin receptor molecules. This model led to the identification of leptin’s site III interaction site, which interacts with the Ig domain of the receptor, a breakthrough discovery that led to the development of leptin antagonists. In the third chapter of the Part I, Jockers and his colleagues review the resonance energy transfer (RET)

methodologies used to study the leptin:leptin receptor interaction, which led to the conclusion that leptin receptors exist as preformed homodimers. Upon leptin binding, the receptors undergo a conformational change and possibly, further aggregation, leading to their activation. This activation can, however, be prevented by leptin antagonists. These authors also review the use of RET technology for the screening of small molecules affecting the leptin:leptin receptor interaction. The second part of this book is devoted to various aspects of leptin’s action beyond its direct regulatory effect on food intake. In the last 40 years, apoptosis has become a major field of study. Leptin’s involvement as an anti-apoptotic agent was described in as early as 1999 and in Chapter 4, Baile and his colleagues summarize leptin’s dual involvement: via direct anti-apoptotic action, mostly in the periphery, and indirect pro-apoptotic action, which affects mainly the adipose tissue and most likely mediated via increased sympathetic activity. Although adipose tissue is the main source of leptin, in 1998, Bado and his colleagues had identified expression of leptin gene in the stomach, the distribution of its receptors throughout the gastrointestinal tract, and the production of leptin by gastric epithelial cells within the gastric mucosa in rodents and humans. In Chapter 5, Bado and his colleagues review these aspects of leptin action, suggesting that gut leptin may act locally to influence gastrointestinal functions. The association between obesity and cancer is now well established, based mostly on epidemiological data; however, the precise underlying mechanism remains elusive. In Chapter 6, Surmacz and Koda review the putative involvement of leptin in breast and colorectal cancer. Compiling a wide array of studies using several in-vivo and in-vitro models, they conclude that leptin acts as a mitogen and survival factor, and may promote anchorage-independent growth, migration and invasion of breast and colorectal cancer cells. Although the association between circulating leptin levels and cancer is yet unclear, the authors suggest that leptin effects can be attributed to overexpression of leptin receptors in breast and colorectal cancer tissues as well as enhanced intratumoral leptin synthesis. Leptin’s involvement in cardiac physiology and pathology is the subject of a relatively new field of investigation originating from the well-documented relationship between obesity and increased risk of cardiovascular disease. This field is reviewed in Chapter 7 by Karmazyn and his colleagues, who highlight leptin’s hypertropic effects in cardiomyocytes and discuss the complex and sometime controversial role of leptin in cardiac pathology. These authors also outline the novel RhoA/ROCK transduction pathway activated by leptin. The next chapter (Chapter 8) reviews the controversial issue of leptin’s putative role in bone elongation. The authors, Monsonego-Ornan and Ben-Ami discuss its specific involvement in the process of endochondral ossification, and review the still unresolved question of whether these effects are limited to central leptin action or are also mediated by systemic leptin, acting in the periphery and

affecting chondrocyte proliferation, differentiation, mineralization and apoptosis. The issue of leptin’s putative involvement in arterial hypertension is reviewed in Chapter 9 by Beltowski. The author discusses the dual contradictory acute activity of exogenously administered leptin versus chronic hyperleptinemia, sums up the various related molecular pathways affected by leptin and suggests possible therapeutic strategies and a relationship to obesity. Involvement of leptin in endometrial function is then reviewed by Cervero and Simon in Chapter 10. These authors begin by outlining the leptin system in the endometrium, then discuss leptin’s controversial role in implantation by comparing humans and mice, and conclude with an evaluation of leptin’s function in endometrial pathologies. Chapter 11 is unique in addressing the use of leptin as a therapeutic agent in lipodystrophy. The authors, Chong, Cochran and Gorden review leptin’s various effects on glycemic control, lipid metabolism and body composition. They then address the various effects of leptin therapy on gonadal function, growth hormone, thyroid and adrenal axis, and the muscle and liver, and conclude with a discussion on the possible utilization of leptin in various therapies. Chapters 12 and 13 are devoted to reviewing leptin’s effects on the immune system. First, Matarese and Rosa in Chapter 12, provide an extensive review of leptin’s involvement in modulating the immune response, with a special emphasis on its Th1-promoting effects, which have been linked to enhanced susceptibility to experimentally induced autoimmune diseases. They suggest that leptin also exerts a negative signal for the proliferation and expansion of regulatory T cells (Tregs), a specific subset of cells involved in the control of immune and autoimmune responses. They also review the possibility of using either anti-leptin or anti-leptin receptor antibodies as blockers, i.e., the action that is being blocked is the breaking of self-tolerance. Then, in Chapter 13, Elinav and Gertler report on the first use of a recently developed competitive leptin antagonist acting as an anti-inflammatory agent in mice models of acute and chronic T-cell-mediated liver inflammation and chronic liver fibrosis. Their recent results suggest that this beneficial effect may be mediated by both the direct modulation of T-cells and the inhibition of hepatic stellate cells activation and function. Leptin’s involvement in early postnatal imprinting has led to new insight into developmental programming. This highly novel aspect of leptin’s action is reviewed extensively in the final chapter of this book by the Auckland group, Vickers, Krechowec, Gluckman and Breier. In the last five years, it has been shown that at least in rodents, leptin acts as an important neurotrophic factor promoting the early postnatal maturation of neural pathways within the hypothalamus. The authors review experimental evidence, originating largely from their own work, which shows that therapeutic intervention with leptin in the rodents’ early postnatal life can potentially reverse or substantially ameliorate the consequences of developmental malprogramming, and that this effect is highly influenced by both gender and postnatal diet.

In conclusion, my colleagues who contributed to this book and I hope that this extensive review of the recent advances in leptin research will be of help and interest to the scientific community at large, particularly those whose field of study involves this multifaceted hormone. Arieh Gertler, PhD Institute of Biochemistry, Food Science and Nutrition The Hebrew University of Jerusalem Rehovot, Israel

Chapter 1

Leptin Signal Transduction— A 2009 Update Walter Becker*

Abstract

L

eptin is an adipocyte-secreted hormone that informs the brain about the status of the body’s energy stores. Leptin controls energy homeostasis through effects on satiety and energy expenditure but also regulates other processes, including reproduction, glycemic control, immune function and wound healing. The leptin receptor exists in multiple alternatively spliced isoforms, of which only the long form (LEPRb) associates with Janus kinase 2 ( JAK2) to mediate intracellular signaling. Upon leptin binding, LEPRb initiates multiple intracellular signal transduction pathways that result in the activation of STAT family transcription factors, extracellular signal-regulated kinases (ERK), phosphoinositol-3 kinase, AMP-activated kinase and ATP-sensitive potassium channels. This chapter gives a delineation of our current knowledge about leptin signal transduction, with a particular focus on the role of individual signaling pathways in vivo and the changes in gene expression induced by leptin.

Leptin and the Leptin Receptor

Leptin is a 16 kDa polypeptide secreted from adipocytes that is often referred to as an adipokine because of it is structurally related to the long-chain four helix bundle family of cytokines, which includes interleukin 6 (IL-6), oncostatin M and others. The cytokine character of leptin is reflected by the pleiotropic actions of leptin and the widespread expression of the leptin receptor. In addition to its central function as a regulator of food intake and energy expenditure in hypothalamic nuclei, leptin is involved in many additional physiological processes. Adequate leptin levels are required to permit energy consuming processes such as reproduction, angiogenesis, wound healing, hematopoiesis, bone development and activation of the immune systems (see also part 2 of this book).1-3 Leptin also regulates glucose homeostasis and lipid metabolism independently of its central weight regulatory function, partly via direct action on pancreatic -cells and hepatocytes.4-6 Cloning of the leptin receptor identified it as a single membrane-spanning receptor of the class I cytokine family.7 The murine leptin receptor exists in at least six isoforms that are alternative splicing products derived from a single Lepr gene.7 Each of LEPRa—LEPRf are identical in their extracellular domain that binds leptin with an affinity in the nanomolar range.8 The molecular interaction of leptin with the extracellular domain of the leptin receptor is dealt with in detail in the ensuing chapters.9,10 The shortest isoform (LEPRe) lacks the transmembrane region and forms a soluble, secreted form of the receptor. The extracellular domains of the membrane-bound receptors can also shed into the circulation by the action of surface proteases.11 These soluble receptors determine the proportion of free and protein-bound leptin in the circulation and appear to alter leptin clearance without directly affecting leptin action.12,13 *Walter Becker—Institute of Pharmacology and Toxicology, Medical Faculty of the RWTH Aachen University, Wendlingweg 2, 52074 Aachen, Germany. Email: [email protected]

Leptin and Leptin Antagonists, edited by Arieh Gertler. ©2009 Landes Bioscience.

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Leptin and Leptin Antagonists

Four splicing variants of the leptin receptor, i.e., LEPRa, LEPRc, LEPRd and LEPRf, share the membrane-spanning region and 29 intracellular amino acids and diverge thereafter, comprising only 3-11 additional residues at their cytosolic domain. Only LEPRb has an extended intracellular domain of approximately 300 amino acids, which comprise the typical structural elements of cytokine receptors.7 LEPRa is the most abundant isoform of the leptin receptor and exhibits some signalling capacity in overexpression systems.14 However, the specific lack of LEPRb in Lepr db/db mice results in an obesity phenotype very similar to that in the leptin-deficient Lepob/ob mice, indicating that LEPRb is crucial for the function of leptin in vivo. Furthermore, LEPRa does not form functional heterodimers or -oligomers with LEPRb.15,16 Taken together, there is no evidence that any of the short isoforms of the leptin receptor can elicit intracellular effects and this review focuses on leptin signaling via LEPRb. It should however be noted that LEPRa and LEPRc have been proposed to play a role in leptin uptake or efflux from cerebrospinal fluid and in receptor-mediated transport of leptin through the blood brain barrier.17,18 This review summarizes the current knowledge about the intracellular signal transduction pathways initiated by LEPRb (see Fig. 1). Mechanism of negative regulation of leptin signaling are not within the scope of this chapter but are covered by excellent recent reviews.19-21

JAK Kinase

Like other cytokine receptors, LEPRb does not have intrinsic tyrosine kinase activity but signals by activating a noncovalently associated tyrosine kinase of the Janus kinase family, JAK2.22 Association of LEPRb with JAK2 does not depend on ligand binding, but rather the receptor polypeptide and the kinase form a constitutive complex.22,23 In contrast to the receptors of the IL-6 receptor family, LEPRb do not form heteromeric complexes with other receptor chains and does not require nonsignaling receptor subunits such as CNTFR, IL11R or IL-6R for JAK activation.24 JAKs bind to the membrane-proximal region of cytokine receptors, which contains an essential

Figure 1. Intracellular signal transduction pathways regulated by LEPRb. The cartoon illustrates the activation of different pathways by recruitment of SH2-domain containing proteins to the intracellular tyrosine residues of LEPRb (pTyr985, pTyr1077, pTyr1138) or JAK2. See the text for further details.

Leptin Signal Transduction—A 2008 Update

3

and conserved so-called box1 motif and a less well defined box2 motif that also contributes to JAK binding.24 The box1 sequence is located in the short piece of sequence common to the long and the short isoforms of the leptin receptor, but additional residues specific to LEPRb have been identified that are required for JAK activation.23,25 Recently, the activation of JAK2-independent pathways by LEPRb has been described and attributed to kinases of the Src family.129 However, it remains to be determined whether other JAK family kinases may compensate for the lack of JAK2 in this experimental system. Binding of leptin to the extracellular domain of LEPRb leads to trans-autophosphorylation of the associated JAK on at least 13 tyrosines, including Tyr1007 and Tyr1008 in the activation loop that cause the activation of these kinases.26,27 The requirement of at least two JAK molecules within a receptor complex has been experimentally shown using chimeric LEPRb constructs.23,28 Although it is not fully understood how cytokine receptors transmit the signal through the membrane, conformational changes of the receptor somehow propagate through the membrane and orientate the JAK molecules correctly to allow their reciprocal phosphorylation.29 In addition to autophosphorylation, activated JAK2 also phosphorylates three conserved intracellular tyrosine residues on LEPRb (Tyr985, Tyr1077 and Tyr1138 in murine LEPRb).30-34 In some species including human, LEPRb contains additional tyrosines in the cytoplasmic domain that are not evolutionary conserved and thus are unlikely to play a role in signal transduction. The phosphorylated tyrosine residues in LEPRb and in JAK2 then provide docking sites for signal transduction proteins with specialized phosphotyrosines-binding domains called Src homology 2 (SH2) domains. In general, SH2 domains from different proteins specifically recognize phosphotyrosines in different sequence motifs. Thus, each tyrosine phosphorylation site recruits specific downstream signaling proteins depending on the surrounding amino acids.

Activation of STAT3

The best known downstream targets of the JAKs are the members of the STAT (Signal Transducers and Activators of Transcription) family of transcription factors. STATs are transiently recruited to specific phosphotyrosine motifs, are themselves phosphorylated on a single tyrosine residue by the receptor-associated JAK kinases, dimerize, translocate to the nucleus and modulate the transcription of target genes. LEPRb is most closely related with IL-6-type cytokine receptors (gp130, OSMR, LIFR; ref. 19), which signal via activation of STAT3, and the phosphorylation of STAT3 on Tyr705 is indeed the most robust downstream effect of leptin receptor activation. Tyrosine phosphorylation and/or nuclear translocation of STAT3 upon leptin treatment was observed in most if not all leptin-responsive cells, including hypothalamic neurons, hepatocytes, hepatic stellate cells, T-cells, insulin-secreting-cells, macrophages, endothelial cells and many others.35-42 In addition to phosphorylation of Tyr705, phosphorylation of Ser727 in the activation domain appears to be necessary for the full transcriptional activity of STAT3 at least in certain systems and was shown to be biologically important in STAT3S727A knock-in mice.43,44 Leptin-induced phosphorylation of STAT3 on Ser727 has not yet been extensively studied but was observed in the J744.2 macrophage cell line (ref. 41) and in RINm5F insulinoma cells (unpublished results from our lab). Numerous protein kinases have been implicated in the phosphorylation of Ser727 in different systems (ref. 45), but the effect of leptin in the macrophages was dependent on the activation of the ERK (extracellular signal-regulated kinase) pathway.41 LEPRb contains a canonical STAT3 binding motif (box3, Tyr-x-x-Gln) at position 1138-1141 that is essential for the leptin-induced activation of STAT3 in vitro and in vivo.46,47 Homologous replacement of Tyr1138 with serine in transgenic mice (Lepr S1138) completely abolished leptin-induced activation of STAT3 and provided an excellent model for assessing the specific contribution of this pathway to the different biological effects of leptin.47-49 Lepr S1138 homozygous mice were obese and hyperphagic like Lepr db/db, indicating that activation of STAT3 is indispensable for the hypothalamic effects of leptin on appetite control and regulation of energy expenditure. However, Lepr S1138/S1138 mice are less hyperglycemic compared to Lepr db/db mice and display nearly normal reproductive function.47,49 These elegant studies provide clear evidence that important effects of

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Leptin and Leptin Antagonists

leptin such as fertility and glycemic control are mediated via STAT3-independent effector systems. Using a different approach to elucidate the requirement of STAT3 for the effects of leptin, Buettner et al inhibited STAT3 activation by stereotaxic intracerebroventricular application of a peptide inhibitor of STAT3 activation.50 This acute inhibition of leptin-induced STAT3 activation in the hypothalamus prevented the effect of leptin on food intake and hepatic glucose metabolism. Surprisingly, the restoration of the luteinizing hormone (LH) surge in food-deprived female rats by leptin was also abolished by inhibition of STAT3 activation in the hypothalamus. This observation contrasts with the fertility of the Lepr S1138/S1138 mice but can possibly be explained by the fact that the peptide inhibited activation of STAT3 by cytokines other than leptin. Recently, mice with a specific deletion of STAT3 in Lepr expressing cells were shown to exhibit normal fertility.51 Concerning peripheral leptin effects, mice with an adipocyte-specific disruption of STAT3 have increased adiposity and an impaired lipolytic effect of leptin.52 In contrast, leptin stimulates vascular smooth muscle cell proliferation independent of STAT3 activation.53 Taken together, these results clearly establish STAT3 as a key effector of leptin’s physiological functions but show that other pathways are also involved. Furthermore, many effects of leptin have not yet been studied in the Lepr S1138 mice, e.g., the immune-modulatory function and the action on pancreatic -cells.

Leptin-Regulated Genes

The requirement of STAT3 signalling for the weight lowering effect of leptin raises the question which target genes of STAT3 are involved (Table 1). Leptin is well known to increase transcription of the proopiomelanocortin gene (Pomc) in a specialized population of hypothalamic neurons.54 Proopiomelanocortin is the precursor for –melanocyte-stimulating hormone (-MSH), which has anorectic effects by activating melanocortin receptors (MC3R, MC4R). Pomc gene expression is directly induced by leptin via a STAT3 response element in the promoter in vitro and in vivo.55 Interestingly, genetic inactivation of STAT3 in POMC neurons caused only mild obesity and did not completely abolish the appetite-suppressing effect of leptin, indicating that STAT3-dependent effects in other cells are also involved.56 A second well-characterized effect of leptin is the negative regulation of the orexigenic peptides neuroeptide Y (NPY) and agouti-related protein (AgRP). Interestingly, genetic disruption of STAT3 in hypothalamic AgRP/NPY neurons revealed that STAT3 in these neurons contributes also to the regulation of energy homeostasis.128 Different mouse models have yielded conflicting results concerning the role of STAT3 in the leptin-induced downregulation of NPY and AgRP.47,128 Another effect of leptin directly transmitted through STAT3 is the upregulation of thyreotropin-releasing hormone (TRH) that enhances thyroid function and results in an increased energy expenditure.57-59 Activation of the JAK/STAT pathway by leptin is expected to result in extensive changes in gene expression. Apart from the above-mentioned neuropeptides, surprisingly few leptin-induced genes have been linked to the multitude of leptin effects in the different target organs. Although numerous leptin-regulated transcripts have been identified in various tissues, many of these changes are a consequence of metabolic reprogramming (e.g., ref. 60). Among the direct targets of leptin, suppressor of cytokine signaling 3 (SOCS3) is a feedback inhibitor that is rapidly induced after activation of STAT3 and downregulates receptor activity by inhibiting the receptor-associated JAK kinase.61 Most of the known leptin-induced genes are regulated by STAT3, including several genes encoding inflammation-related proteins (-fibrinogen, plasminogen activator, tissue-type (tPA), pancreatitis-associated protein, lipocalin-2, preprotachykinin, superoxide dismutase 2, see Table 1).62 However, in most cases the functional consequences of their upregulation by leptin have not been defined in vivo.

Activation of Other STAT Factors

In addition to STAT3, leptin can induce tyrosine phosphorylation and activation of STAT1, STAT5 and STAT6.33,46,62 The biological role of these STAT factors in leptin signalling is less clear than that of STAT3. STAT1 is recruited to the same docking site as STAT3 (Tyr1138 in mouse LEPRb) and can form heterodimers with STAT3 once activated.46 This promiscuity of the box3

Experimental Evidence Gene Symbol

Gene Product

Tissue or Cell Line

Method

Upregulation of...

Pathway

Ref

Pomc

proopiomelanocortin

Hypothalamus AtT20*, HEK*

LEPRb-mut, dnSTAT3

mRNA, promoter activity

STAT3

47,55

Trh

thyreotropin-releasing hormone

Hypothalamus 293T*

ChIP

STAT3 binding, Promoter activity STAT3

58,59

Socs3

suppressor of cytokine signalling 3 (SOCS3)

Hypothalamus 32D cells* INS1

ChIP, LEPRb-mut, EMSA

mRNA STAT3/5 binding, Promoter activity

STAT3 (STAT5)

58,31,122

Fos

c-fos proto-oncogene#

Hypothalamus 293T*

LEPRb-mut, PD98059

Protein mRNA

ERK

78,31

Egr1

early growth response 1

CHO*

dnSHP2

Promoter activity

ERK

75

Il1b

Interleukin 1β (IL-1β)#

Hypothalamus microglia cells

Lepr db/db Peptide inh.

mRNA, Secreted IL-1β

Non-STAT3§ STAT3

123, 124

Il1rn

IL-1 receptor antagonist#

HepG2*

PD98059, U0126

Promoter activity

ERK

125

ERK, STAT3

38,126

Timp1

Tissue inhibitor of metalloproteinase-1#

LX-2

PD098059, EMSA

mRNA, STAT3 binding

$

Leptin Signal Transduction—A 2008 Update

Table 1. Leptin target genes and signaling pathways involved in their regulation

continued on next page

5

6

Table 1. Continued Experimental Evidence Gene Symbol

Gene Product

Tissue or Cell Line

Method

Upregulation of...

Pathway

Ref

Pap

Pancreatitis-associated protein#

RINm5F*, PC12*

dnSTAT3 LEPRb-mut

mRNA, promoter activity

STAT3

127

Lcn2

Lipocalin-2#

RINm5F*, HIT-T15*

LEPRb-mut

mRNA, promoter activity

STAT3

61

#

Tac1

preprotachykinin

LEPRb-mut

mRNA, promoter activity

STAT3

61

Sod2

superoxide dismutase 2# RINm5F*

RINm5F*, HIT-T15*

LEPRb-mut

mRNA

STAT3

61

Fbgn

fibrinogen β#

RINm5F*

LEPRb-mut

mRNA

STAT3

61

Plat

plasminogen activator, tissue-type (tPA)#

RINm5F*

LEPRb-mut

mRNA

STAT3

61

Leptin and Leptin Antagonists

*cell line ectopically expressing recombinant LEPRb; §the reported upregulation of IL-1β in Lepr db/db mice implies a tyrosine-independent mechanism and is in contrast with the study by Pinteaux (ref. 124); $indirect binding via association with Sp1; #gene products involved in inflammatory processes. Evidence for the contribution of either the JAK/STAT3 or the ERK pathway was obtained using LEPRb mutants lacking specific tyrosine residues (LEPRb-mut), overexpression of dominant negative mutants of STAT3 (dnSTAT3) or SHP2 (dnSHP2), specific inhibitors of ERK activation (PD98059, U0126) or by detecting promoter binding of STAT3 to the promoter of the target gene by chromatin immunoprecipitation (ChIP) or electrophoretic mobility shift assay (EMSA). In one study, STAT3 activation was blocked with help of a specific STAT3 peptide inhibitor (peptide inh). The stimulatory effect of leptin was determined as upregulation of mRNA levels (Northern blot or RT-PCR), promoter activity (reporter gene assay), protein levels (ELISA or immunocytochemistry) or enhanced binding of transcription factors to the promoter.

Leptin Signal Transduction—A 2008 Update

7

motif is also known for the gp130 receptor subunit of the IL-6 type cytokine receptors that also activates both STAT3 and STAT1 via the same phosphotyrosines.24 In a direct side-by-side comparison in the insulinoma cell line RINm5F, leptin induced a much stronger tyrosine phosphorylation of STAT1 than IL-6 under conditions of similar STAT3 phosphorylation.33 Although the activation of STAT1 might suggest that leptin can induce an IFN-like response, leptin did not upregulate the paradigmatic STAT1 target gene, IRF1 (interferon regulatory factor 1) under these conditions.62 Like in IL-6 signaling, the STAT1 response is probably suppressed via a STAT3-dependent mechanism and would only take effect in the absence of STAT3.63 Consistently, leptin strongly activates STAT1 in STAT3-deficient but not in wild-type mouse adipocytes.52 However, leptin induced tyrosine phosphorylation of STAT1 in normal rat adipose tissue.64 STAT5 is activated by leptin in many cell lines, including HIT-T15 and RINm5F insulinoma cells, neuronal GT1-7 cells, enterocyte-like CACO2 cells, H35 hepatoma cells and LX-2 hepatic stellate cells.33,65-68 However, early in vivo-studies failed to detect leptin-induced STAT5 phosphorylation in the hypothalamus of mice and rats.35,69 Recent studies have now succeeded to demonstrate STAT5 phosphorylation in mouse hypothalamus and nuclear translocation of STAT5 in rat hypothalamic nuclei.70,71 Either Tyr1077 or Tyr1138 in LEPRb can mediate the activation of STAT5, with no preferential activation of either STAT5A and STAT5B.33,68 Phosphorylation of Tyr1077 in LEPRb proved difficult to detect with most phosphotyrosine-specific antibodies (refs. 31,72) and was controversial until recently. Gong et al68 have now unambiguously shown that this residue is phosphorylated after receptor activation. Notably, the amino acids around Tyr1077 are phylogenetically conserved in vertebrates, indicating that the role of this tyrosine in signaling is not redundant.33 So far no target gene nor any effect of leptin is known to be regulated via STAT5. In vitro, leptin can activate STAT5-dependent promoters and thus might have gene regulatory effects overlapping with those other STAT5 recruiting hormones such as growth hormone (GH) or prolactin, given that the receptors are expressed on the same cells (e.g., hepatocytes or pancreatic -cells).33 It will be difficult to reveal the physiological effects of leptin-induced STAT5 activation because the targeted mutation of Tyr1077 in transgenic mice will still allow STAT5 activation via Tyr1138. Taken together, even if the poorly characterized activation of STAT6 is neglected, LEPRb activates a broader spectrum of STAT factors than most other cytokine receptors.

Activation of the ERK Pathway

Like many other cytokines, leptin activates the RASRAFMEKERK pathway.14,31,73 Other members of the MAPK family (p38, JNK) have also been reported to be activated by leptin (e.g., refs. 74,75), but the relevant pathways have not been well characterized. Although the complete chain of reactions leading to the activation of ERK1 and ERK2 has not been specifically dissected in leptin signaling, LEPRb most likely exploits the same pathway as the signal transducing subunit of the IL-6-type cytokine receptors, gp130.24 Phosphorylation of the most proximal intracellular tyrosine residue, Tyr985 in LEPRb or Tyr759 in gp130, creates a binding site for the carboxyterminal SH2 domain of the tyrosine phosphatase SHP2.72,76 SHP2 becomes itself phosphorylated on C-terminal tyrosines, which then recruit the adapter protein GRB2 (growth factor receptor-bound protein-2) to the receptor complex.31 In the canonical ERK pathway, GRB2 forms a complex with SOS, the GTP exchange factor for RAS and initiates the RAFMEKERK pathway, wherein each kinase activates the downstream kinase by phosphorylation. Leptin can also induce a lower level of ERK activation independent of Tyr985 and SHP-2, possibly mediated by direct binding of GRB2 to JAK2.31 Interestingly, catalytically inactive SHP2 did not support LEPRb mediated ERK activation.77 Of the many possible downstream effects of ERK, upregulation of the immediate early genes egr-1 and c-fos has been demonstrated in cell culture and in vivo in the hypothalamus (Table 1).77,78 The same genes are also upregulated by IL-6 via Tyr759 in gp130, SHP2 and ERK.79 Analysis of the Lepr S1138/S1138 knock-in mice confirmed that upregulation of c-fos does not depend on STAT3 activation.80 It is not clear how activation of ERKs translates into physiological effects of leptin,

8

Leptin and Leptin Antagonists

but upregulation of c-fos is a marker for neuronal activity.78 Knock-in mice homozygous for a Tyr985Leu point mutation have not yet revealed a biological function of leptin-induced ERK activation.81 These mice exhibit increased leptin sensitivity, consistent with the known role of Tyr985 as a binding site for the site for autoinhibitory SOCS3, and their phenotype demonstrates that Tyr985 is not essential for regulation of growth or reproduction. Another function of ERK1/2 is the phosphorylation and activation of RSK (ribosomal protein S6 kinase). Phosphorylation of S6 by RSK enhances cap-dependent translational initiation and protein synthesis.70 Leptin-dependent phosphorylation of S6 has been demonstrated in vivo in the hypothalamus (ref. 82) and in vitro-studies have shown that Tyr985 and ERK activation are required for this effect of LEPRb.70

Activation of the IRS/PI3K/PDE3B Pathway

PI3K (phosphoinositide 3-kinase) is a key signaling molecule that transmits downstream effects of insulin. Activation of PI3K by receptor tyrosine kinases is mediated via phosphorylation of IRS (insulin receptor substrate) proteins, which then associate with the SH2 domain of the regulatory subunit of PI3K, p85. Leptin reportedly stimulates tyrosine phosphorylation of IRS1 and IRS2 and activation PI3K in different cell types.14,64,88 This pathway does not depend on STAT3 activation but is initiated by direct binding of IRS proteins to phosphorylated JAK2.73 Recently, leptin was found to recruit IRS4, which binds to phosphorylated Tyr1077 and can also associate with p85 to recruit PI3K.83 Although leptin signaling via the IRSPI3K pathway suggests that leptin may have insulin-like effects in cells that express both receptors, the crosstalk between these hormones is complicated by many indirect effects. In liver, the acute lipid-lowering effect of leptin and inhibition of gluconeogenesis depend on PI3K activity.84,85 However, results obtained in myoblasts and even in different hypothalamic neurons are inconsistent and indicate that cell type-specific mechanisms determine the actual interaction between these pathways.86-90 It is also important to note that the magnitude of PI3K stimulation in response to leptin in vivo is much lower than that seen with insulin.64 At least in some cell types, leptin increases the levels of PIP3 (phosphoinositide3,4,5 trisphosphate), the reaction product of PI3K, mainly by inhibition of the lipid phosphatase, PTEN.91 Downstream of PI3K, PIP3 stimulates protein kinases such as PDK1 and PKB/Akt. PKB/Akt has been implicated in insulin-induced phosphorylation and activation of membrane-associated phosphodiesterase 3B (PDE3B).92 PDE3B activation reduces intracellular cAMP levels and thus leptin-induced activation of PDE3B antagonizes the cAMP-mediated effects of glucagon-like peptide-1 (GLP-1) in pancreatic -cells and glucagon in hepatocytes.92,93 Leptin-induced activation of PDE3B has also been shown in the hypothalamus and intracerebroventricular injection of the PDE3 inhibitor, cilostamide, blocked the inhibitory effect of leptin on food intake.94 In vivo studies implicate the LEPRPI3KPDE3B pathway in the suppression of NPY neurons in the arcuate nucleus.95-97 Thus, this pathway may be responsible for STAT3-independent the gene regulatory effects in the hypothalamus. In contrast, activation of PI3K appears less important in POMC neurons, because genetic ablation of IRS2 in POMC neurons did not cause obesity.98 SH2B1 (a.k.a. SH2-B) is an SH2-domain containing adapter protein that increases the leptin-induced tyrosine kinase activity of JAK2 by binding to the autophosphorylated pTyr830.99,100 In addition, SH2B1 enhances the activation of IRS-dependent pathways by recruiting IRS proteins to the receptor complex.100,101 Transgenic mouse models have revealed an important role of neuronal SH2B1 in the control of leptin sensitivity and energy homeostasis.102

Activation of ATP-Sensitive K Channels

Activation of ATP-sensitive K channels by leptin was first observed in certain hypothalamic neurons and pancreatic -cells.103,104 Hyperpolarization due to the enhanced K conductance results in reduced neuronal firing and inhibition of insulin secretion from -cells, respectively. Activation of the ATP-sensitive K channels depends on the IRSPI3KPIP3 pathway and is mediated by direct binding of PIP3 to the ATP binding site of the channel.91,105,107 In the hypothalamus, this pathway

Leptin Signal Transduction—A 2008 Update

9

can explain the leptin-induced hyperpolarization of NPY/AgRP neurons, whereas depolarization of POMC neurons must obviously be accomplished by a different pathway. As far as known, the activation of ATP-sensitive K channels is a unique feature of LEPRb as compared with other cytokine receptors and may reflect the predominantly neuronal action of leptin.

Regulation of AMPK (AMP-Activated Protein Kinase) and mTOR (Mammalian Target of Rapamycin)

AMPK acts as a sensor of cellular energy status and also regulates whole body energy homeostasis by integrating nutrient and hormonal signals in the hypothalamus.108 Leptin regulates AMPK activity in a tissue-specific manner: leptin activates AMPK in muscle and liver, causing suppression of ATP-consuming metabolic pathways (e.g., hepatic glucose production, fatty acid synthesis) and stimulation of ATP-regenerating pathways (e.g., oxidation of intracellular fatty acids).109,110 Thereby leptin improves glucose tolerance and has an antisteatotic effect that protects tissues from the lipotoxicity that is a consequence of leptin deficiency.111 Interestingly, the oral antidiabetic drug, metformin, acts via stimulation of hepatic AMPK activity and thus has leptin-like effects.112 The mechanism of AMPK activation by leptin is unknown but requires JAK kinase activity and does not appear to depend on intracellular tyrosine motifs.110 Surprisingly, no effect of leptin was observed in insulinoma cell lines although AMPK was readily activated by glucose deprivation.33,113 In contrast to its action in muscle and liver cells, leptin reduces AMPK activity in hypothalamic neurons and thus suppresses the stimulatory effect of AMPK on food intake.114,115 However, recent results indicate that the targeted deletion of AMPK activity in POMC and NPY/AgRP neurons did not affect the appetite suppressing effect of leptin but specifically prevented glucose sensing.116 One downstream effect of AMPK is the inhibition of the protein kinase mTOR, which also integrates responses to changes in cellular energy status. Intracerebroventricular administration of leptin was reported to activate hypothalamic mTOR, possibly by preventing its inhibition by AMPK. Inhibition of mTOR by rapamycin caused an increase in food intake, demonstrating the role of this pathway in appetite regulation.80 Further studies will be necessary to fully elucidate the role of mTOR in leptin signaling.

Perspective

In the recent years, the analysis of transgenic mouse models has advanced our understanding of the signaling pathways that are important for the weight regulatory effect of leptin. In contrast, very little is known about the molecular mechanisms by which potentially negative effects are controlled, in particular enhanced immune responses in autoimmune diseases.117 The development of leptin antagonists to block the unwanted effects of leptin emphasizes the need to understand the mechanisms by which LEPRb produces these effect (refs.118,119, see also the other chapters of this book120,121). Although several inflammation-related genes have been found to be upregulated by leptin (Table 1), their role in vivo has yet to be determined. The analysis of transgenic mice with specific mutations of individual tyrosine residues in the intracellular part of LEPRb should offer valuable insight in the molecular mechanisms of leptin’s immune-modulatory and other peripheral effects and provide potential new targets for drug development.

Acknowledgements

I am grateful to Hans-Georg Joost for having introduced me to the study of leptin and its receptor. I wish to thank all past and present members of my lab and collaborating groups for contributing to our effort to understand leptin signaling. This work was supported by the Deutsche Forschungsgemeinschaft (SFB 542 TP-B3). This article is dedicated to Professor Hans-Georg Joost on occasion of his 60th birthday.

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57. Harris M, Aschkenasi C, Elias CF et al. Transcriptional regulation of the thyrotropin-releasing hormone gene by leptin and melanocortin signaling. J Clin Invest 2001; 107:111-120. 58. Guo F, Bakal K, Minokoshi Y et al. Leptin signaling targets the thyrotropin-releasing hormone gene promoter in vivo. Endocrinology 2004; 145:2221-2227. 59. Huo L, Münzberg H, Nillni EA et al. Role of signal transducer and activator of transcription 3 in regulation of hypothalamic trh gene expression by leptin. Endocrinology 2004; 145:2516-2523. 60. Liang CP, Tall AR. Transcriptional profiling reveals global defects in energy metabolism, lipoprotein and bile acid synthesis and transport with reversal by leptin treatment in ob/ob mouse liver. J Biol Chem 2001; 276:49066-49076. 61. Bjørbæk C, Lavery HJ, Bates SH et al. SOCS3 mediates feedback inhibition of the leptin receptor via Tyr985. J Biol Chem 2000; 275:40649-40657. 62. Hekerman P, Zeidler J, Korfmacher S et al. Leptin induces inflammation-related genes in RINm5F insulinoma cells. BMC Mol Biol 2007; 8:41. 63. Costa-Pereira AP, Tininini S, Strobl B et al. Mutational switch of an IL-6 response to an interferon-gamma-like response. Proc Natl Acad Sci USA 2002; 99:8043-8047. 64. Kim YB, Uotani S, Pierroz DD et al. In vivo administration of leptin activates signal transduction directly in insulin-sensitive tissues: overlapping but distinct pathways from insulin. Endocrinology 2000; 141: 2328-2339. 65. Kaszubska W, Falls HD, Schaefer VG et al. Protein tyrosine phosphatase 1B negatively regulates leptin signaling in a hypothalamic cell line. Mol Cell Endocrinol 2002; 195:109-118. 66. Morton NM, Emilsson V, Liu Y-L et al. Leptin action in intestinal cells. J Biol Chem 1998; 273:26194-26201. 67. Wang Y, Kuropatwinski KK, White DW et al. Leptin receptor action in hepatic cells. J Biol Chem 1997; 272:16216-16223. 68. Cao Q, Mak KM, Ren C et al. Leptin stimulates tissue inhibitor of metalloproteinase-1 in human hepatic stellate cells: respective roles of the JAK/STAT and JAK-mediated H2O2-dependant MAPK pathways. J Biol Chem 2004; 279:4292-4304. 69. McCowen KC, Chow JC, Smith RJ Leptin signaling in the hypothalamus of normal rats in vivo. Endocrinology 1998; 139: 4442-4447. 70. Gong Y, Ishida-Takahashi R, Villanueva EC et al. The long form of the leptin receptor regulates STAT5 and ribosomal protein S6 via alternate mechanisms. J Biol Chem 2007; 282:31019-31027. 71. Mütze J, Roth J, Gerstberger R et al. Nuclear translocation of the transcription factor STAT5 in the rat brain after systemic leptin administration. Neurosci Lett 2007; 417:286-291. 72. Li C, Friedman JM. Leptin receptor activation of SH2 domain containing protein tyrosine phosphatase 2 modulates Ob receptor signal transduction. Proc Natl Acad Sci USA 1999; 96:9677-9682. 73. Myers MG. Leptin receptor signaling and the regulation of mammalian physiology. Recent Prog Horm Res 2004; 59:287-304. 74. Shin HJ, Oh J, Kang SM. Leptin induces hypertrophy via p38 mitogen-activated protein kinase in rat vascular smooth muscle cells. Biochem Biophys Res Commun 2005; 329:18-24. 75. Cui H, Cai F, Belsham DD. Leptin signaling in neurotensin neurons involves STAT, MAP kinases ERK1/2 and p38 through c-Fos and ATF1. FASEB J 2006; 20:2654-2656. 76. Carpenter LR, Farruggella TJ, Symes A et al. Enhancing leptin response by preventing SH2-containing phosphatase 2 interaction with Ob receptor. Proc Natl Acad Sci USA 1998; 95:6061-6066. 77. Bjørbaek C, Buchholz RM, Davis SM et al. Divergent roles of SHP-2 in ERK activation by leptin receptors. J Biol Chem 2001; 276:4747-4755. 78. Elias CF, Aschkenasi C, Lee C et al. Leptin differentially regulates NPY and POMC neurons projecting to the lateral hypothalamic area. Neuron 1999; 23:775-786. 79. Kim H, Baumann H. Dual signaling role of the protein tyrosine phosphatase SHP-2 in regulating expression of acute-phase plasma proteins by interleukin-6 cytokine receptors in hepatic cells. Mol Cell Biol 1999; 19:5326-5338. 80. Münzberg H, Jobst EE, Bates SH et al. Appropriate inhibition of orexigenic hypothalamic arcuate nucleus neurons independently of leptin receptor/STAT3 signaling. J Neurosci 2007; 27:69-74. 81. Björnholm M, Münzberg H, Leshan R et al. Mice lacking inhibitory leptin receptor signals are lean with normal endocrine function. J Clin Invest 2007; 117:1354-1360. 82. Cota D, Proulx K, Smith KA et al. Hypothalamic mTOR signaling regulates food intake. Science 2006; 312:927-930. 83. Wauman J, De Smet AS, Catteeuw D et al. Insulin Receptor Substrate 4 Couples the Leptin Receptor to Multiple Signaling Pathways. Mol Endocrinol 2008; 22(4):965-977. 84. Huang W, Dedousis N, Bhatt BA et al. Impaired activation of phosphatidylinositol 3-kinase by leptin is a novel mechanism of hepatic leptin resistance in diet-induced obesity. J Biol Chem 2004; 279:21695-21700.

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85. Anderwald C, Müller G, Koca G et al. Short-term leptin-dependent inhibition of hepatic gluconeogenesis is mediated by insulin receptor substrate-2. Mol Endocrinol 2002; 16:1612-1628. 86. Kitamura T, Kitamura Y, Kuroda S et al. Insulin-induced phosphorylation and activation of cyclic nucleotide phosphodiesterase 3B by the serine-threonine kinase Akt. Mol Cell Biol 1999; 19:6286-6296. 87. Szanto I, Kahn CR. Selective interaction between leptin and insulin signaling pathways in a hepatic cell line. Proc Natl Acad Sci USA. 2000; 97:2355-2360. 88. Kellerer M, Koch M, Metzinger E et al. Leptin activates PI-3 kinase in C2C12 myotubes via janus kinase-2 ( JAK-2) and insulin receptor substrate-2 (IRS-2) dependent pathways. Diabetologia 1997; 40:1358-1362. 89. Xu AW, Kaelin CB, Takeda K et al. PI3K integrates the action of insulin and leptin on hypothalamic neurons. J Clin Invest 2005; 115:951–958. 90. Benomar Y, Roy AF, Aubourg A et al. Cross down-regulation of leptin and insulin receptor expression and signalling in a human neuronal cell line. Biochem J 2005; 388:929-939. 91. Ning K, Miller LC, Laidlaw HA et al. A novel leptin signalling pathway via PTEN inhibition in hypothalamic cell lines and pancreatic beta-cells. EMBO J 2006; 25: 2377-2387. 92. Zhao AZ, Shinohara MM, Huang D et al. Leptin induces insulin-like signaling that antagonizes cAMP elevation by glucagon in hepatocytes. J Biol Chem 2000; 275:11348-11354. 93. Zhao AZ, Bornfeldt KE, Beavo JA. Leptin inhibits insulin secretion by activation of phosphodiesterase 3B. J Clin Invest 1998; 102:869-873. 94. Zhao AZ, Huan JN, Gupta S et al. A phosphatidylinositol 3-kinase phosphodiesterase 3B-cyclic AMP pathway in hypothalamic action of leptin on feeding. Nat Neurosci 2002; 5:727-728. 95. Niswender KD, Morton GJ, Stearns WH et al. Intracellular signalling. Key enzyme in leptin-induced anorexia. Nature 2002; 413:794-795. 96. Morrison CD, Morton GJ, Niswender KD et al. Leptin inhibits hypothalamic Npy and Agrp gene expression via a mechanism that requires phosphatidylinositol 3-OH-kinase signaling. Am J Physiol Endocrinol Metab 2005; 289:E1051-1057. 97. Kohno D, Nakata M, Maekawa F et al. Leptin suppresses ghrelin-induced activation of neuropeptide Y neurons in the arcuate nucleus via phosphatidylinositol 3-kinase- and phosphodiesterase 3-mediated pathway. Endocrinology 2007; 148:2251-2263. 98. Choudhury AI, Heffron H, Smith MA. The role of insulin receptor substrate 2 in hypothalamic and beta cell function. J Clin Invest 2005; 115:940-950. 99. Maures TJ, Kurzer JH, Carter-Su C. SH2B1 (SH2-B) and JAK2: a multifunctional adaptor protein and kinase made for each other. Trends Endocrinol Metab 2007; 18:38-45. 100. Li Z, Zhou Y, Carter-Su C et al. SH2B1 enhances leptin signaling by both Janus kinase 2 Tyr813 phosphorylation-dependent and -independent mechanisms. Mol Endocrinol 2007; 21:2270-2281. 101. Duan C, Li M, Rui L. SH2-B promotes insulin receptor substrate 1 (IRS1)- and IRS2-mediated activation of the phosphatidylinositol 3-kinase pathway in response to leptin. J Biol Chem 2004; 279:43684-43691. 102. Ren D, Zhou Y, Morris D et al. Neuronal SH2B1 is essential for controlling energy and glucose homeostasis. J Clin Invest 2007; 117:397-406. 103. Spanswick D, Smith MA, Groppi VE et al. Leptin inhibits hypothalamic neurons by activation of ATP-sensitive potassium channels. Nature 1997; 390:521-525. 104. Kieffer TJ, Heller RS, Leech CA et al. Leptin suppression of insulin secretion by the activation of ATP-sensitive K channels in pancreatic beta-cells. Diabetes 1997; 46(6):1087-1093. 105. Plum L, Ma X, Hampel B et al. Enhanced PIP3 signaling in POMC neurons causes KATP channel activation and leads to diet-sensitive obesity. J Clin Invest 2006; 116:1886-1901. 106. Harvey J, McKay NG, Walker KS et al. Essential role of phosphoinositide 3-kinase in leptin-induced K(ATP) channel activation in the rat CRI-G1 insulinoma cell line. J Biol Chem 2000; 275:4660-4669. 107. MacGregor GG, Dong K, Vanoye CG et al. Nucleotides and phospholipids compete for binding to the C terminus of KATP channels. Proc Natl Acad Sci USA 2002; 99:2726-2731. 108. Claret M, Smith MA, Batterham RL et al. AMPK is essential for energy homeostasis regulation and glucose sensing by POMC and AgRP neurons. J Clin Invest 2007; 117:2325-2336. 109. Minokoshi Y, Kim YB, Peroni OD et al. Leptin stimulates fatty-acid oxidation by activating AMP-activated protein kinase. Nature 2002; 415:339-343. 110. Uotani S, Abe T, Yamaguchi Y. Leptin activates AMP-activated protein kinase in hepatic cells via a JAK2-dependent pathway. Biochem Biophys Res Commun 2006; 351:171-175. 111. Unger RH. The hyperleptinemia of obesity-regulator of caloric surpluses. Cell 2004; 117:145-146. 112. Misra P. AMP activated protein kinase: a next generation target for total metabolic control. Expert Opin Ther Targets 2008; 12:91-100.

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113. Leclerc I, Woltersdorf WW, da Silva Xavier G et al. Metformin, but not leptin, regulates AMP-activated protein kinase in pancreatic islets: impact on glucose-stimulated insulin secretion. Am J Physiol Endocrinol Metab 2004; 286:E1023-1031. 114. Minokoshi Y, Alquier T, Furukawa N et al. AMP-kinase regulates food intake by responding to hormonal and nutrient signals in the hypothalamus. Nature 2004; 428:569-574. 115. Andersson U, Filipsson K, Abbott CR et al. AMP-activated protein kinase plays a role in the control of food intake. J Biol Chem 2004; 279:12005-12008. 116. Claret M, Smith MA, Batterham RL et al. AMPK is essential for energy homeostasis regulation and glucose sensing by POMC and AgRP neurons. J Clin Invest 2007; 117:2325-2336. 117. La Cava A, Matarese G. The weight of leptin in immunity. Nat Rev Immunol 2004; 4:371-379. 118. Peelman F, Iserentant H, Eyckerman S et al. Leptin, immune responses and autoimmune disease. Perspectives on the use of leptin antagonists. Curr Pharm Des 2005; 11:539-548. 119. Gertler A. Development of leptin antagonists and their potential use in experimental biology and medicine. Trends Endocrinol Metab 2006; 17:372-378. 120. Elinav E, Gertler A. Use of leptin antagonists as anti-inflammatory and anti-fibrotic reagents. In: Gertler A, ed. Leptin and Leptin Antagonists. Austin: Landes Bioscience, 2009; 133-140. 121. Matarese G, DeRosa V. Use of anti-leptin or anti-leptin receptor antibodies as blockers of immune response. In: Gertler A, ed. Leptin and Leptin Antagonists, Austin: Landes Bioscience, 2009; 126-132. 122. Laubner K, Kieffer TJ, Lam NT et al. Inhibition of preproinsulin gene expression by leptin induction of suppressor of cytokine signaling 3 in pancreatic beta-cells. Diabetes. 2005; 54:3410-3417. 123. Hosoi T, Okuma Y, Nomura Y. Leptin regulates interleukin-1beta expression in the brain via the STAT3-independent mechanisms. Brain Res 2002; 949:139-146. 124. Pinteaux E, Inoue W, Schmidt L et al. Leptin induces interleukin-1beta release from rat microglial cells through a caspase 1 independent mechanism. J Neurochem 2007; 102:826-833. 125. Dreyer MG, Juge-Aubry CE, Gabay C et al. Leptin activates the promoter of the interleukin-1 receptor antagonist through p42/44 mitogen-activated protein kinase and a composite nuclear factor kappa B/ PU.1 binding site. Biochem J 2003; 370:591-599. 126. Lin S, Saxena NK, Ding X et al. Leptin increases tissue inhibitor of metalloproteinase I (TIMP-1) gene expression by a specificity protein 1/signal transducer and activator of transcription 3 mechanism. Mol Endocrinol 2006; 20:3376-3388. 127. Broekaert D, Eyckerman S, Lavens D et al. Comparison of leptin- and interleukin-6-regulated expression of the rPAP gene family: evidence for differential co-regulatory signals. Eur Cytokine Netw 2002; 13:78-85. 128. Gong L, Yao F, Hockman K et al. Signal transducer and activator of transcription-3 is required in hypothalamic agoutirelated protein/neuropeptide Y neurons for normal energy homeostasis. Endocrinol 2008; 149:3346-3354. 129. Jiang L, Li Z, Rui L. Leptin stimulates both JAK2-dependent and JAK2-independent signaling pathways. J Biol Chem 2008; 283:28066-28073.

Chapter 2

Insights in the Activated LR Complex and the Rational Design of Antagonists Frank Peelman, Lennart Zabeau and Jan Tavernier*

Introduction

T

he hormone leptin plays an important role in the control of body weight. Leptin is mainly produced and secreted by adipocytes as a 16 kDa nonglycosylated polypeptide and plasma leptin levels positively correlate with body fat energy stores.1,2 To a lesser extent, leptin is also expressed in other tissues such as the epithelium of the stomach, placenta, skeletal muscle and brain.3,4 Spontaneous loss of function mutations in the leptin encoding ob gene (for example in ob/ ob mice) give rise to a complex syndrome that includes morbid obesity, hypothermia, infertility, hyperglycemia, decreased insulin sensitivity and hyperlipidemia.5 Leptin turned out to be a quite pleiotropic cytokine and its effects are not restricted to energy homeostasis, but also include neuroendocrine function,6 angiogenesis,7 bone formation,8 reproduction9 and immune responses.10 Leptin mediates its effects by binding and activation of the leptin receptor (LR), encoded by the db gene.11 Loss of function mutations in the db gene lead to a phenotype that is comparable to that of the ob/ob mouse. The LR is a single-membrane spanning class I cytokine receptor. Like all members of the class I cytokine receptor family, the receptor has no intrinsic kinase activity and uses cytoplasmic-associated Janus kinase 2 ( JAK2) for intracellular signalling. In a generally accepted model, leptin-binding leads to formation of an activated receptor complex, allowing JAK2 cross-phosphorylation. JAK2 then rapidly phosphorylates several tyrosine residues in the cytosolic domain of the receptor (in the case of the mouse LR, tyrosines at positions 985, 1077 and 1138). Phosphorylated tyrosines 1077 and 1138 bind STAT5 (signal transducer and activator of transcription 5), while tyrosine 1138 further recruits STAT1 and STAT3.12,13 Although other STATs can be recruited, STAT3:STAT3 dimers are the most dominant after leptin stimulation. Once recruited, STATs themselves become a substrate for JAKs and homo- or heterodimerize upon phosphorylation, translocate to the nucleus and modulate transcription of target genes. Other signalling pathways activated by the LR include MAPK14 and phosphoinositide 3 kinase pathways.15 Thus far, six LR isoforms have been identified (LRa-f ): one long form (LRb or LRlo) and four short forms (LRa,c,d,f ) are generated by alternative splicing. A sixth, soluble form (LRe) is a result of ectodomain shedding and/or alternative splicing in respectively men and mice. High expression of LRlo, the major signalling isoform, is observed in certain nuclei of the hypothalamus,16 a region of the brain involved in the regulation of body weight. Expression could also be shown in several other cell types including liver, pancreas, lung, kidney, adipose tissues, endothelial cells and cells of the immune system, thereby forming the basis of several peripheral biological functions of leptin. *Corresponding Author: Jan Tavernier—Department of Medical Protein Research, VIB, and Department of Biochemistry, Faculty of Medicine and Health Sciences, Ghent University, A. Baertsoenkaai 3, B-9000 Ghent, Belgium. Email: [email protected]

Leptin and Leptin Antagonists, edited by Arieh Gertler. ©2009 Landes Bioscience.

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Leptin as a Disease-Promoting Factor: Rationale for Leptin Antagonists

It has become clear over the last few years that leptin plays a role in both innate and adaptive immunity (reviewed in ref. 10). In innate immunity, leptin promotes secretion of inflammatory cyto-kines and the activation of macrophages, neutrophils and natural killer cells. Functions in adaptive immunity include thymic homeostasis, naïve CD4+ cell proliferation and promotion of T helper 1 (TH1) responses. Moreover, leptin can act as a negative signal for the expansion of CD4+CD25high regulatory T-cells (TRegs), a T-cell subpopulation known to dampen immune reactions.17 Leptin is involved in the onset and/or progression of several T-cell controlled autoimmune diseases, like Crohn’s disease,18 rheumatoid arthritis,19 multiple sclerosis20 and autoimmune hepatitis.21 Leptin or LR deficiency can protect against onset of experimentally induced diseases in rodents. In leptin deficient animals, leptin administration results in a switch from TH2 to TH1 controlled responses. Furthermore, administration to wild type mice worsens the clinical manifestations in these models for autoimmune diseases. In some of these diseases, in situ production of the cytokine could be shown in active inflammatory lesions, thereby representing a significant local source of leptin. Overweight is a risk factor for postmenopausal breast cancer. The LR is expressed on breast cancer cells and promotes their growth in vitro.22 Cleary et al crossed MMTV-TGF-alpha mice, which develop mammary tumors, with ob/ob or db/db mice.23 In the MMTV-TGF-alpha female mice, tumor incidence increases with increased body weight and vice versa. However, both the obese MMTV-TGF-alpha/Lep(ob)Lep(ob) and MMTV-TGF-alpha/Lep(db)Lep(db) female mice do not develop mammary tumors, strongly supporting the idea that leptin is a necessary factor for mammary tumor development. The involvement of leptin in immune diseases and breast cancer provided a rationale for the development of leptin antagonists. Different strategies can be used to reduce leptin’s activities. Anti-leptin antibodies or soluble LR that scavenge free leptin in circulation24 and blocking antibodies against the LR.25 Another approach is the use of leptin mutants or synthetic peptides derived from leptin that block LR activation.26-28 In this chapter, we discuss recent insights in the mechanism of LR activation and how these led to the development of leptin antagonists. We summarize how these insights can be used to guide the optimization of leptin antagonists.

Structure of Leptin and Its Receptor Homology with the IL-6 and G-CSF Receptor Systems

The structure of leptin revealed by crystallography showed that leptin is a four helix-bundle cytokine: 4 α-helices are arranged in a typical up-up-down-down fold.29 The leptin structure shows the highest similarity with the long chain α-helical cytokines of the interleukin-6 (IL-6) family, including IL-6, leukaemia inhibitory factor (LIF), ciliary neurotrophic factor (CNTF), oncostatin M (OSM) and with granulocyte-colony stimulating factor (G-CSF). To a lesser extent, it also resembles the other long chain α-helical cytokines, such as growth hormone (GH) and prolactin. The LR belongs to the class I cytokine receptor family, which typically contains a so-called cytokine receptor homology (CRH) domain in its extracellular domain. This CRH structure consists of two barrel-like domains, each around 100 amino acids in length, which resemble the fibronectin type III (FN III) fold. Two conserved disulfide bridges are found in the N-terminal domain, while a WSXWS motif is characteristic for the C-terminal part. The LR contains two such CRH domains, CRH1 and CRH2, which are separated by an immunoglobulin-like (Ig) domain and followed by two membrane proximal FN III domains (Fig. 1). Based on sequence similarity and overall architecture of the ectodomains, the LR is most related to the G-CSF receptor and the glycoprotein 130 (gp130) family receptors, including gp130, LIF and OSM receptors. Unique to the LR is the presence of an additional N-terminal CRH module and two, instead of three, FN III domains (Fig. 1). The N-terminal CRH domain seems to be preceded by an additional domain of

Insights in the Activated LR Complex and the Rational Design of Antagonists

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Figure 1. Topology of the LR, compared with the topology of the erythropoietin (Epo) receptor, the gp130 family receptors and the G-CSF receptor.

approximately 100 residues, which shows no clear sequence homology to other domains. The disulfide pattern and secondary structure suggest that it might be a degenerated Ig-like domain.30 The CRH2 domain is the main high-affinity leptin binding site.31-33 The other domains do not show detectable leptin binding when expressed in vitro. All domains are necessary for receptor activation, except for CRH1, deletion of which reduces the full receptor activation capacity by about 50%.31,33,34

Evidence for Receptor Oligomerisation and Higher Order Clustering LR Oligomerisation in the Absence of Ligand

Many cytokine receptors exist as inactive, preformed complexes on the cellular surface. Examples include the receptors for Epo,35-37, GR38 and IL-6.39 There is a growing body of evidence that also the LR appears as ligand-independent oligomers: purified soluble extracellular LR domain from baculovirus-infected insect cells behaves as dimers in SDS-PAGE and gelfiltration experiments.40,41 This clustering could also be demonstrated with membrane-bound receptors.42,43 White and coworkers extended these findings and showed that LR long and short homo-oligomerize in the absence of ligand, while hetero-oligomerisation between both isoforms was only observed in the presence of leptin.42 This may help to explain why the long form is able to signal in the presence of an excess signal-deficient short forms as seen in many tissues. A quantitative bioluminescence resonance energy transfer (BRET) approach illustrated that in living cells 60% of the LR exists as constitutive dimers.44 Using a series of LR deletion and cysteine to serine mutants, we recently demonstrated that this clustering most likely involves disulphide bridges between residues of the CRH2 domain.43

LR Becomes Activated Upon Higher Order Clustering

We examined the requirements for leptin signalling in more detail with a complementation-of-signalling strategy.33 Here, the LR was made signalling deficient in two ways: in the LR-F3 mutant all cytoplasmic tyrosines were mutated to phenylalanines, while in the LR ∆box1 mutant

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two prolines necessary for JAK activation were replaced by alanines. While these mutants are both unable to signal via the JAK/STAT pathway, a clear STAT3-dependent signal is generated upon leptin stimulation when they are co-expressed in cells. Assuming that JAK/STAT signalling requires at least two JAK kinases and one tyrosine residue, the complementation can only be explained by the presence of at least three LR chains in the leptin:LR complex. The complementation of signalling is completely lost when the extracellular domains of the mutants are replaced by that of the strict homodimeric EpoR, suggesting that the higher order clustering is determined by the extracellular domains of the LR.

Three Binding Sites in Leptin

4-Helix bundle cytokines activate their receptors by contacting two or more receptor subunits through multiple binding sites in the cytokine. This orientates the extracellular domains of the receptor chains in the right position for receptor activation. Epo and GH bind to their receptors through two binding sites.45,46 Binding site I is found at the fourth helix (helix D) and contacts with the CRH domain of a first receptor. Binding site II is formed by the surfaces of the anti-parallel first and third helix (helices A and C) and binds the CRH domain of a second receptor. The homodimeric Epo and GH receptors thus use the same CRH binding epitope to bind to two totally different binding sites in their cytokine ligand.45,46 Cytokines of the IL-6 family and G-CSF contain a third receptor binding site at the N-terminus of helix D.47,48 This binding site III binds to an Ig-like domain in the receptor. In the IL-6 receptor complex, IL-6 uses three binding sites: binding site I binds to the CRH domain of the IL-6Rα. Binding site II binds to the CRH domain of a first gp130 chain, leading to a heterotrimeric IL-6:IL-6Rα:gp130 complex. Two trimers subsequently form a hexamer, in which binding site III of IL-6 contacts the immunoglobulin-like domain of a second gp130 chain (Fig. 2). The G-CSF:G-CSF receptor system does not have a binding site I or an α-receptor chain. This receptor complex is 2:2 tetramer. Binding site II of G-CSF binds to the CRH domain of a first G-CSF receptor chain, while binding site III binds to the Ig-like domain of the second G-CSF receptor chain (Fig. 3). Structural superposition of the leptin crystal structure with other four helix bundle cytokines was used to identify the position of possible binding sites I, II or III in leptin. Residues in these areas were mutated and the leptin mutants were tested in LR activation assays and in an assay that determines their binding to CRH2. A predicted binding site II is found in the middle of helices A and C (for a schematic representation of the secondary structures within leptin, see Fig. 4). Mutations in this site show a clearly decreased affinity for the CRH2 domain, suggesting that binding site II interacts with this domain. Surprisingly, the mutants did not show a large decrease

Figure 2. The 2:2:2 IL-6:IL-6Rα:gp130 complex.

Insights in the Activated LR Complex and the Rational Design of Antagonists

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Figure 3. The 2:2 G-CSF:G-CSF receptor complex.

in EC50 value or maximal LR activation. This finding is unexpected and suggests that the loss of affinity for CRH2 might be compensated by other leptin:receptor interactions. The contribution of such additional interactions to high affinity binding might explain why several studies report a slightly higher affinity of leptin for the full-length LR than for CRH2 (summarized in ref. 49). The predicted binding site III is found around the N-terminus of helix D and contains residues in the A-B and C-D loops. Several mutations in binding site III led to a strong decrease in the maximal LR activation. Binding site I is found in the helical face of helix D and the A-B loop. Mutations in binding site I in this study had a less pronounced effect, with moderately decreased maximal receptor activation potential. While mutations in binding sites I and III both decrease maximal receptor activation potential, they do not affect binding to CRH2 or the EC50 value for LR activation. Niv-Spector et al used a different approach to identify binding site III in leptin.27 In the viral IL-6:gp130 complex, the binding site III interaction involves a hydrophobic strand at the N-terminus of the gp130 Ig-like domain that interacts with a hydrophobic strand in the viral IL-6. Using hydrophobic cluster analysis, similar hydrophobic strands were predicted at residues 39 to 42 in leptin and at residues 325-328 in the LR Ig-like domain. Mutations of two or more residues to alanine in these predicted strands in leptin abolished LR activation capacity. These do not affect the secondary structure of the protein, or binding to the LR or the isolated CRH2 domain. Similarly, mutation of residues 325-328 to alanines in the predicted strand at the N-terminus of the Ig-like domain drastically reduces LR activation. Based upon the mutagenesis of leptin and analogy with other receptor systems, we propose the following interaction scheme for leptin/receptor complex: Binding site II in leptin interacts with the CRH2 domain (Fig. 5). Binding site III interacts with the Ig-like domain of a second LR chain. Since no α-receptor chain is necessary for leptin, it might interact with a third or fourth LR chain. These predicted interactions of leptin and its

Figure 4. Schematic representation of the secondary structures within the leptin molecule. Boxes represent helices, lines the connecting loops. Numbers are the positions of the beginning and end of the helices.

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Figure 5. The hexameric 2:4 leptin:LR complex.

receptor were further investigated by homology modelling and mutagenesis of the LR domains and modelling of the leptin-receptor complex.

Models of the CRH2-Leptin Complex

Several homology models of leptin bound to CRH2 have been presented.32,50,51 Hiroike et al presented a model of a 2:2 leptin/CRH2 complex based upon a crystal structure of a 2:2 complex of G-CSF with the G-CSF receptor CRH.50 In this model, each leptin molecule contacts two different CRH2 domains via a major and minor binding site.50 However, the minor site in the G-CSF later turned out to be an artefact of crystallization. 48 Nonetheless, the major interface in the model corresponds with the interface proposed in the models described below. Sandowski et al built a homology model for the complex of human leptin with the CRH2 domain, based on the crystal structures of the gp130 CRH domain and of GH bound to the CRH of its receptor.32 This model was later used to model the CRH2 of the chicken LR.52 We built a homology model of the mouse leptin:CRH2 domain using a superposition of the structures of several cytokine:CRH complexes as a guide for the alignment and the structure of the G-CSF:G-CSF receptor complex as template.51 The leptin CRH2 interface model resembles in many aspects the model of Hiroike et al.50 The recombinant chicken CRH2 domain, expressed in E. coli was extensively mutated by Niv-Spector et al and the leptin binding properties of the mutants were tested in vitro.52 We made mutants of the recombinant mouse CRH2 domain, expressed in COS-1 cells and tested the effect of the mutations on leptin binding.51 The same mutations were introduced in the full-length LR and their effect on LR JAK/STAT signalling was tested. Both mutagenesis studies demonstrate the importance of a region of four consecutive hydrophobic residues in CRH2: 501-IFFL-504 in mouse LR CRH2, 504-VFLL-507 in chicken LR CRH2 (Fig. 6). Mutations at these residues affect leptin binding. In all homology models, the region forms a central part of the interaction surface with leptin. In the mouse leptin:CRH2 homology model, the four hydrophobic residues become buried upon leptin binding (Fig. 6). The residues make contact with L13 and L86 in mouse leptin. L86 mutants have lower affinity for CRH2 and the L86S mutant has a drastically increased EC50 value for LR activation.51 Leptin residues that were predicted to be part of binding site II by structural superposition with other cytokines,28 all interact with the CRH2 domain in the model: D9,

Insights in the Activated LR Complex and the Rational Design of Antagonists

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Figure 6. Model for the leptin:CRH2 complex, with indication of the mutations in leptin (yellow) or CRH2 (blue) that affect the KD of the interaction. A color version of this image is available at www.landesbioscience.com/curie.

T12, L13, K15, T16 in helix A, N78, N82, D85, L86 in helix C. Mutations of these residues lead to lower affinity for CRH2 in in vitro binding assays28,51 (Fig. 6).

Models for the Ig-Like and CRH1 Domains

In the gp130 receptor family and the G-CSF receptor, the Ig-like domain interacts with binding site III in the cytokine ligand. We built homology models for the Ig-like domain and pinpointed possible binding site III interaction residues by alignment/superposition with Ig-like domains of gp130 family receptors and the G-CSF receptor. The residues were mutated in the mouse LR and the effects of the mutations on LR activation were tested. Several of these mutations have a drastic effect on the maximal response to leptin stimulation, without affecting the EC50 value, similar to the effect seen with the leptin binding site III mutants. These mutants form a continuous cluster on the surface of the Ig-like domain, at a position that superposes with the binding site III-interacting region in the Ig-like domain of the IL-6 family and G-CSF receptors (Fig. 7). We therefore propose that this cluster of residues interacts with leptin binding site III. The area of the cluster in the Ig-like domain has a positive electrostatic surface potential, that is probably compatible with the negative electrostatic surface potential found in our predicted binding site III in leptin, which is situated around the N-terminus of helix D (Fig. 7). The Study of Niv-Spector et al predicts binding site III around residue 39-42 in leptin, in contrast with our study, which places binding site III around the N-terminus of helix D. The two predicted binding sites III are actually quite distant from each other in the crystal structure. When the leptin crystal structure is superposed onto the G-CSF molecule in the crystal structure of the G-CSF receptor complex, residues 39-42 do not come near the Ig-like domain. Similar structural

22

Leptin and Leptin Antagonists

superposition of the leptin crystal structure onto the IL-6 molecule in the crystal structure of the extracellular part of the IL-6 receptor complex, brings residues 39-42 at binding site I, in contact with the IL-6Rα. In contrast, the region surrounding the N-terminus of helix D approaches the Ig-like domains in both superpositions. We therefore propose that residues 39-42 are part of binding site I and that mutations at these position affect LR activation by affecting binding site I. Niv-Spector et al used hydrophobic cluster analysis to predict a hydrophobic strand at the N-terminus of the Ig-like domain (residues 325-VFTT-328) that might be involved in binding site III interactions and this in analogy with the virus Il-6/gp130 complex. Mutation of residues 325-328 to four alanines abolishes LR activation. However, homology modelling of the CRH1 domain demonstrates that these residues are probably an integral part of the structure of CRH1 and that most of the hydrophobic residues in the strand are buried inside CRH1. It is therefore unlikely that residues 325-328 are part of binding site III. Moreover, deletion of the entire CRH1 domain, including residues 325-328 only leads to 50% reduction of the maximal LR activation.34 The role of the CRH1 domain remains elusive. A Q269P mutation in the CRH1 domain causes the obesity in the fa/fa rat, with defective and partially constitutive LR signalling.53-55 In our model for CRH1, the Q269P mutation leads to severe steric clashes between the introduced proline residue and the first tryptophan of the CRH1 WSXWS motif. This probably affects the stability or the correct folding of this domain. It cannot be excluded that the CRH1 domain might be more important at lower, physiologically relevant LR expression levels and that some effects of CRH1 deletion are not detected at the high LR expression levels in in vitro overexpression systems.

Homology Model for a Hexameric 2:4 Leptin:LR Complex

Based upon mutagenesis data for leptin and its receptor, we proposed a hexameric model for the leptin:LR complex. 2:2 tetramer and 2:4 hexamer leptin:LR complexes were built using the crystal structure of the IL-6 receptor complex as a guide for modelling.34 In the tetramer model, the leptin binding site II:CRH2 interaction is modelled as described above. Binding site III is situated around the N-terminus of helix D and interacts with the Ig-like domain of a second LR chain. Binding site III and the Ig domain might attract each other by an opposing electrostatic surface potential. The binding site II and III interactions would allow the formation of a 2:2 leptin:LR tetramer complex, as found for the G-CSF receptor complex. However, the tetramer model seems to contradict some previous findings: 1. The LR can oligomerize via disulfide bridges. The tetramer model does not allow disulfide bridge formations between LR chains. Disulfide bridges cannot be introduced by simply moving or rotating the receptor chains or addition of models of the FN III domains. 2. Our JAK/STAT complementation assay suggests that the leptin:LR complex must contain more than two receptor chains.33 3. Mutations at positions 39-42 in leptin can have a very strong effect on LR activation capacity. F41 belongs to our predicted binding site I and other mutations in this putative binding site I also affect LR activation. None of the residues 39-42 has an interaction partner in the tetramer model. These three issues can be resolved by considering a hexamer leptin:LR complex (Fig. 8). In the IL-6 receptor complex, binding site I interacts with the CRH domain of the IL-6Rα. We created a hexameric 2:4 leptin:LR model complex by putting CRH2 of the LR at the positions of the IL-6Rα CRH. F41 and binding site I residues in leptin now all interact with the additional CRH2 domains.

Homology Models for the Fibronectin Type III Domains

FN III domains have no detectable affinity for leptin, but are absolutely essential for signalling.43 When these domains are expressed as soluble proteins, they appear as disulfide linked oligomers on SDS-PAGE. The LR contains two conserved cysteines, on positions 672 and 751. Mutation

Insights in the Activated LR Complex and the Rational Design of Antagonists

23 Figure 7, left. A) Model for the Ig-like domain of the LR, with indication of the mutations that affect LR activation (top). The area around the cluster has a positive electrostatic surface potential (blue) (bottom).34 B) Model for mouse leptin with indication of the binding site III mutations that affect LR activation (top). The area around S120 and T121 (circled) has a negative surface potential (red) (bottom).

Figure 8, above. A 2:4 hexameric model for the leptin:LR complex.34 Side view, with indication of the mutations in binding sites II that affect binding to CRH2 (purple), or mutations in binding site I (orange) or III (red) that affect maximal LR activation.34,52

Figure 9, right. Homology model of the tandem FN III domains of the mouse LR. Two cysteines are exposed at the surface and potentially capable of inter-chain disulfide bridges.

24

Leptin and Leptin Antagonists

of C751 to serine has limited effect on ligand binding and receptor activation, the C672S mutant exhibits a marked reduction in STAT3-dependent signalling. The double mutant is completely devoid of biological activity, although leptin binding remains unaffected. The FN III domains connect the leptin interacting CRH2 and Ig-like domains with the transmembrane domain and thus may act as levers that communicate LR rearrangements induced by leptin binding to the transmembrane and intracellular domains. A receptor variant with an extracellular domain consisting of only the FN III domains shows a marked increase in ligand-independent signalling. This illustrates that these domains can position the intracellular domains in such a way that JAK activation and thus signalling are possible. Homology models for the LR FN III domains were built using the crystal structure of type III repeats 7-10 of fibronectin56 as a template. In Figure 9, the FN III domains are illustrated. The cysteine residues are found on the surface, accessible for possible inter-chain disulphide formation.

Mechanism of LR Activation

The following models for LR activation can be proposed: Leptin first binds to the CRH2 domain of a first LR via its binding site II. After this first high affinity binding step, two models are possible: Model 1: The bound leptin molecule binds to a second LR chain via CRH2:binding site I interactions (Fig. 10). These trimeric complexes subsequently interact with a third LR chain or another trimer complex via binding site III and the Ig-like domain: Model 2: The dimeric leptin:LR complexes form tetramers via interactions of binding site III and the Ig-like domain (Fig. 11). Subsequently, leptin binding site I interacts with the CRH2 domain of additional LR chains: These models are in line with the presence of three binding sites, the oligomeric nature of the LR and the JAK/STAT complementation assay, but remain hypothetical at present. It is also not

Figure 10. Model 1.

Figure 11. Model 2.

Insights in the Activated LR Complex and the Rational Design of Antagonists

25

clear why the LR has developed such a complicated activation process, when other homomeric receptors, such as the Epo, GH and G-CSF receptors work by much simpler mechanisms. The LR exists as preformed oligomers, possibly linked by disulphide bridges. Leptin binding could lead to a spatial reorganisation of receptor chains in the preformed complex, resulting in correct positioning and activation of the cytoplasmic associated JAK kinases. This hypothesis is supported by BRET experiments. In cells expressing short LR forms fused to luciferase and YFP, leptin treatment resulted in a marked enhancement in energy transfer signals, possibly reflecting specific conformational changes.44

Development of Leptin-Based Antagonists

Insight into the binding sites of leptin and its interaction with the LR has led to opportunities for the development for leptin antagonists. Mutations that affect the initial binding step via site II can affect the EC50 value for LR activation, as shown by the L86S leptin mutant.51 Mutations that affect the next interaction steps via binding sites I or III do not affect the EC50 value but affect the maximal LR activation capacity and can even lead to mutants that avidly bind to the receptor without activating it, as is the case for the S120A/T121A mutant and for mutations at position 39-42. Such mutants are potential LR antagonists: the leptin mutant will bind to CRH2 without subsequent LR activation and will block binding of wild type leptin by competitive binding. Several such mutants have been proposed as leptin antagonists (for an overview, see ref. 49). A first antagonistic leptin mutant is the R128Q human leptin mutant, developed by Verploegen et al.57 The mutant binds normally to the LR, but fails to trigger a proliferative response in LR expressing Ba/F-3 cells. R128Q leptin induces weight gain in mice. However, when the R128Q mutation is introduced in leptin of other species, such as sheep or chicken, it does not always result in an antagonist and sometimes even in a weak agonist.58 Surprisingly, injection of the R128Q mutant in rats resulted in a strong dose-dependent decrease in food intake.59 The human leptin mutant R128Q leptin is therefore not a suitable tool for investigating the physiological actions of leptin. R128 is not part of any of the three binding sites, but is largely buried in the leptin structure. The effects of the R128Q mutation are probably indirect, possibly via binding site I or III. The S120A/T121A mutant was tested in our mutagenesis study of leptin binding site III.28 The mutant showed no LR activation in a JAK/STAT signalling-based luciferase assay in Hek293T cells, while its binding to the CRH2 domain was unaffected. The mutant acted as an inhibitor of wild type human and mouse leptin in the JAK/STAT signalling assay. When injected in mice, it showed a clear induction of weight gain, suggesting that the S120A/T121A mutant is an antagonist in vitro and in vivo. Both the human and the mouse S120A/T121A mutant can inhibit mouse or human LR activation. While the R128Q mutant shows LR activation at higher concentrations in an in vitro JAK/STAT signalling assay, this is not the case for comparable concentrations of the S120A/T121A mutant. Niv-spector et al found that the mutations at residues 39-42 in human and ovine leptin led to leptin mutants that were unable to activate the LR, while retaining normal secondary structure and LR binding.27 These mutants potently antagonize leptin-induced proliferation of Ba/F-3 stably expressing the LR. In a similar way, mouse and rat leptin can be transformed into potent antagonists by introduction of the 39-42 mutations.27 While all the 39-41 mutants are devoid of agonistic activity, the S120A/T121A mutant shows some low agonistic effect in the very sensitive Ba/F-3 proliferation assay.27 Our homology model of a hexameric LR complex suggests that residues 39-42 might be part of a binding site I. This would mean that mutations in binding site I, as well as mutations in binding site III (S120A/T121A) both can block receptor activation and lead to antagonistic leptin molecules. In fact, any molecule that avidly binds to CRH2 without activating the receptor will potentially be an antagonist. This is supported by the work of Gonzalez and Leavis.26 These authors showed that the synthetic peptide LPA-2, corresponding to helix C of human leptin (residues 70-95) is sufficient for high affinity (0,6.10–10 M) binding to the LR. The peptide antagonizes LR activation in vitro and in vivo: intrauterine injection of the peptide reduced the number of implantation sites

26

Leptin and Leptin Antagonists

and uterine horns with implanted embryos60 and local injection of LPA-2 in mammary fat pads blocked mammary tumor growth.61

Optimization of Leptin-Based Antagonists

The aforementioned antagonists all work by binding to CRH2 and blocking the binding of leptin. Full antagonism requires that almost all receptors are blocked. The antagonists are therefore used at concentrations that exceed their KD or their IC50 for antagonsism. In the bloodstream, this translates in a requirement for µg/ml concentrations of antagonist. Unfortunately, leptin has a short circulation half-life, with reported values ranging from 5.4 minutes in rats to 25 minutes in humans.62-64 The same most likely holds true for the antagonistic leptin mutants. Several options exist for extending the half-life of leptin (or leptin mutants): an antibody against leptin can be co-injected with the antagonist thereby drastically increasing its half-life in circulation.57 Another option is the use of fusion proteins, where the antagonist is coupled to a molecule that has a long circulation half-life, such as albumin or the constant chains of Ig. In the leptin:LR complexes, the leptin N-and C-termini point away from the complex, allowing the fusion to other proteins. A fusion protein of leptin S120A/T121A to mouse albumin retains its antagonistic properties, while a fusion protein of leptin S120A/T121A to the Fc portion of a mouse IgG1 becomes slightly agonistic, possibly by the bridging effect of the Fc molecule (Peelman et al, unpublished results). The most favourable solution for extending the half-life of leptin antagonists might be the pegylation of the leptin mutants. Covalent modification with high molecular weight polyethylene glycol (PEG) chains is a very efficient method for improving the pharmacokinetics of biomolocules65 and has been shown to increase the half-life of wild-type leptin.66,67 A branched polyethylene glycol (PEG) N- hydroxysuccinimide (NHS), molecular weight 40 kDa, was used for pegylation. This pegylation reagent covalently binds to amines and leads to very efficient PEGylaytion (>30% of leptin PEGylated), with one or two PEG molecules per labelled leptin;68 our unpublished results). Unfortunately, pegylation of leptin antagonist mutants drastically decreases their antagonistic potency by more than six fold;68 (our unpublished results). A reason for this decrease in efficiency might be that modification of certain lysine residues blocks the interaction with the LR. K5 and K15 for example are part of the predicted leptin:CRH2 interaction interface and modification of these residues would almost certainly decrease binding to CRH2. A solution might be the specific deletion of certain lysines in leptin. Homology models of the leptin:receptor complex are useful guidelines for rational choices for such mutagenesis. Another way to increase the potency of the antagonists would be the increase of the affinity for the receptor. The leptin residues that are important for binding to CRH2 have been thoroughly mapped by mutagenesis studies. This information can be used to guide directed evolution, e.g., through degenerated primers as a strategy to improve the affinity for the CRH2 domain.

Concluding Remarks

The disease promoting role of leptin in animal models for autoimmune diseases and breast cancer has raised interest in leptin antagonists. Injection of anti-leptin antibodies or soluble LR antagonizes leptin’s action by reducing the bioavailable leptin. An alternative approach might be the use of neutralising anti-LR antibodies that block activation. With the exception of CRH1, every extracellular domain of the LR is indispensible for LR activation. Blocking antibodies can thus be targeted against the FN III domains, the CRH2 domain or the Ig-like domains. The study of the leptin:LR interaction has led to the development of binding site I or III leptin mutants, that bind but do not activate the LR and thus work as competitive inhibitors. However, the leptin mutants have very short half-lives in circulation, so modifications, such as pegylation, hyperglycosylation or coupling to a partner with high half-life will be needed to increase their efficiency. The new insights into leptin interaction with its receptor can be used to optimize such modifications. If leptin antagonism turns out to have therapeutic potential, the effect of leptin antagonists on body weight control, glucose metabolism, bone formation and other processes that are regulated by leptin are a concern. Many of these functions are regulated centrally in the hypothalamus. It

Insights in the Activated LR Complex and the Rational Design of Antagonists

27

is at present unclear whether it is feasible to make leptin antagonists that do not have access to targets in the hypothalamus. Leptin is transported through the blood brain barrier by an unknown transporter, possibly involving megalin or the short form of the LR. However, leptin responsive neurons that express the LR or show STAT3 activation can be labelled by BBB impermeable fluorescent tracers.69,70 ARC neurons might therefore make direct contact with the blood-circulation by projections through the BBB. If this scenario is true, it may be almost impossible to discriminate between central and peripheral functions and avoid weight gain while treating leptin-involved autoimmune diseases. A lot of unknowns remain to be solved before leptin antagonists can be considered to be of possible therapeutic value. Leptin antagonists form a new tool that will provide new insights, both in the role of leptin in disease and in the mechanism of leptin.

References

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51. Iserentant H, Peelman F, Defeau D et al. Mapping of the interface between leptin and the leptin receptor CRH2 domain. J Cell Sci 2005; 118(Pt 11):2519-2527. 52. Niv-Spector L, Raver N, Friedman-Einat M et al. Mapping leptin-interacting sites in recombinant leptin-binding domain (LBD) subcloned from chicken leptin receptor. Biochem J 2005; 390(Pt 2):475-484. 53. da Silva BA, Bjorbaek C, Uotani S et al. Functional properties of leptin receptor isoforms containing the gln→pro extracellular domain mutation of the fatty rat. Endocrinology 1998; 139(9):3681-3690. 54. Iida M, Murakami T, Ishida K et al. Substitution at codon 269 (glutamine→proline) of the leptin receptor (OB-R) cDNA is the only mutation found in the Zucker fatty (fa/fa) rat. Biochem Biophys Res Comm 1996; 224(2):597-604. 55. White DW, Wang DW, Chua SC et al. Constitutive and impaired signaling of leptin receptors containing the Gln Pro extracellular domain fatty mutation. Proc Natl Acad Sci USA 1997; 94(20):10657-10662. 56. Leahy DJ, Aukhil I, Erickson HP. 2.0 A crystal structure of a four-domain segment of human fibronectin encompassing the RGD loop and synergy region. Cell 1996; 84(1):155-164. 57. Verploegen SA, Plaetinck G, Devos R et al. A human leptin mutant induces weight gain in normal mice. FEBS Letters 1997; 405(2):237-240. 58. Raver N, Vardy E, Livnah O et al. Comparison of R128Q mutations in human, ovine and chicken leptins. Gen Comp Endocrinol 2002; 126(1):52-58. 59. Brunner L, Whitebread S, Leconte I et al. A peptide leptin antagonist reduces food intake in rodents. Int J Obes Relat Metab Disord 1999; 23(5):463-469. 60. Ramos MP, Rueda BR, Leavis PC et al. Leptin serves as an upstream activator of an obligatory signaling cascade in the embryo-implantation process. Endocrinology 2005; 146(2):694-701. 61. Gonzalez RR, Cherfils S, Escobar M et al. Leptin signaling promotes the growth of mammary tumors and increases the expression of vascular endothelial growth factor (VEGF) and its receptor type two (VEGF-R2). J Biol Chem 2006; 281(36):26320-26328. 62. Klein S, Coppack SW, Mohamed-Ali V et al. Adipose tissue leptin production and plasma leptin kinetics in humans. Diabetes 1996; 45(7):984-987. 63. Vila R, Adan C, Rafecas I et al. Plasma leptin turnover rates in lean and obese Zucker rats. Endocrinology 1998; 139(11):4466-4469. 64. Zeng J, Patterson BW, Klein S et al. Whole body leptin kinetics and renal metabolism in vivo. The Am J Physiol 1997; 273(6 Pt 1):E1102-1106. 65. Hamidi M, Azadi A, Rafiei P. Pharmacokinetic consequences of pegylation. Drug Delivery 2006; 13(6):399-409. 66. Hukshorn CJ, Menheere PP, Westerterp-Plantenga MS et al. The effect of pegylated human recombinant leptin (PEG-OB) on neuroendocrine adaptations to semi-starvation in overweight men. EEur J Endocrinol 2003; 148(6):649-655. 67. Hukshorn CJ, Saris WH, Westerterp-Plantenga MS et al. Weekly subcutaneous pegylated recombinant native human leptin (PEG-OB) administration in obese men. J Clin Endocrinol Metab 2000; 85(11):4003-4009. 68. Solomon G, Niv-Spector L, Gonen-Berger D et al. Preparation of leptin antagonists by site-directed mutagenesis of human, ovine, rat and mouse leptin's site III: Implications on blocking undesired leptin action in vivo. Annals NY Acad Sci 2006; 1091:531-539. 69. Cheunsuang O, Morris R. Astrocytes in the arcuate nucleus and median eminence that take up a fluorescent dye from the circulation express leptin receptors and neuropeptide Y Y1 receptors. Glia 2005; 52(3):228-233. 70. Faouzi M, Leshan R, Bjornholm M et al. Differential accessibility of circulating leptin to individual hypothalamic sites. Endocrinology 2007; 148(11):5414-5423.

Chapter 3

Study of Leptin:

Leptin Receptor Interaction by FRET and BRET Julie Dam, Cyril Couturier, Patty Chen and Ralf Jockers*

Abstract

U

nderstanding the molecular mechanism of the leptin:leptin receptor (OB-R) interaction and the OB-R activation process is crucial for the development of drugs that target OB-Rs. Recently developed resonance energy transfer (RET)-based assays participated significantly in the establishment of the current model of OB-R activation. According to this model, OB-Rs exist as preformed homodimers in the basal state. Leptin binding induces a ligand-induced conformational change within these dimers, which triggers receptor activation by facilitating the transphosphorylation of receptor-associated janus kinase 2. The concomitant formation of tetrameric complexes (dimers of dimers) has also been suggested but still remains to be firmly established. RET-based techniques also hold great potential for the screening of small molecular weight compounds targeting the OB-R.

Introduction

Leptin is a member of the cytokine family having a four α-helical bundle structure. It is a well-known anorexigenic hormone secreted mainly into the bloodstream by adipose tissue and controls food intake and energy homeostasis primarily by acting at the hypothalamic arcuate nucleus (ARC). Mutations leading to a functional defect in either leptin or its receptor (OB-R) result in a complex syndrome that includes morbid obesity. Besides the adipostatic function, leptin also plays a direct role in the peripheral system regulating metabolism, hematopoiesis, immunity and reproduction. Among the six different types of OB-R, which result from alternative splicing or proteolysis, two main isoforms were mainly studied. The long functional isoform OB-Rb, mainly expressed in the ARC, displays a full intracellular domain (302 residues) with docking sites for the Janus Tyrosine Kinase ( JAK2) and Signal Transducer and Activator of Transcription 3 (STAT3). The short isoform OB-Ra is ubiquitously expressed and has a truncated intracellular domain (34 residues) lacking the STAT3 binding site but is still able to interact and activate JAK2. Whereas leptin functions have been broadly documented, the molecular mechanism of OB-R activation remains elusive. Nevertheless, the structure of several other cytokines and growth factors were investigated extensively during the last decade, in order to define the manner in which these molecules interact with receptors and to reveal the mechanism of signal transduction across the membrane. The crystal structure of leptin/OB-R is unavailable but the successful use of biochemical, biophysical and molecular modeling techniques brought new insights into the leptin activation mechanism that will be presented in this article.

*Corresponding Author: Ralf Jockers—Institut Cochin, Université Paris Descartes, CNRS (UMR 8104), Department of Cell Biology, Paris, France. Email: [email protected]

Leptin and Leptin Antagonists, edited by Arieh Gertler. ©2009 Landes Bioscience.

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Activation Mechanism of OB-R Studied with Biochemical Methods Lessons from Other Cytokine Receptors

For over a decade, the model of growth hormone (GH)-induced receptor dimerization has served as a dogma for cytokine receptor activation.1 This model was supported by the crystal structure of the 2:1 complex between the purified extracellular domain of the GH receptor (GHR) and the GH2 whereas the complex with GH antagonists revealed a 1:1 stoichiometry favoring the notion that the unbound receptor is a monomer.3,4 Moreover, the model was corroborated by the observation that GH but not the antagonist induces the formation of covalently disulfide-linked dimers.5,6 However, the GH-induced dimerization paradigm was then challenged by several studies demonstrating by co-immunoprecipitation that the GHR exists at the plasma membrane as dimer in the absence of ligand,7 that dimerization itself is insufficient for GHR activation8,9 and that a GH antagonist can bind to receptor dimers at the cell surface.10,11 More recently, the crystal structure of unliganded GHR being a dimer, contributed to establish a model of GHR activation involving a slight rotation of subunits within a dimeric receptor upon ligand binding.12 In the case of the erythropoietin receptor (EpoR), crystallographic data from the extracellular domain is also available confirming its dimeric form in the resting state. EpoR adopts distinct dimeric configurations dependent on being unliganded,13 Epo-bound14 or bound to agonistic15 or antagonistic16 peptides. The open scissors-like configuration of the preformed dimer is envisioned to keep the cytoplasmic domain apart in an inactive state and ligand occupancy would bring the extracellular and cytoplasmic domains into close proximity to allow signaling. These data were further confirmed by fragment complementation assay.17 Concerning more complex Cytokine receptors like Granulocyte Colony-Stimulating factor (G-CSF) or Interleukin-6 (IL-6) type cytokines and their receptors gp130, Leukemia Inhibitory Factor Receptor (LIFR), Ciliary Neurotrophic Factor Receptor (CNTFR) and Oncostatin M Receptor (OSMR), the oligomeric state of the activated complex seems to be of higher order. From studies of the GH/GHR complex, it was generally admitted that cytokines were recognized by their receptors at two sites equivalent to site I and site II of GH. This dogma was not valid for IL-6 type cytokines where three distinct receptor binding sites (I-II-III) have been clearly demonstrated by binding and mutagenesis studies.18-21 The organization of three binding epitopes suggested the formation of higher order complexes.22 The stoichiometry of the signaling complex is different between G-CSFR and IL-6R. G-CSF was shown to form a 2:2 tetrameric complex mediated by binding site II and III.23-25 The IL-6 receptor complex was shown to form a 2:2:2 hexameric assembly composed of two IL-6 ligands in complex with two gp130 chains and two specific IL-6Rα chains at three different binding interfaces.26 On the other hand, without the IL6Rα chain, the viral homolog of IL-6 and gp-130 molecules form a tetrameric 2:2 structure consisting of two sets of 1:1 complexes from vIL-6 and human Ig–CRH domains of gp130.27

The Leptin/OB-R System Studied with Biochemical Methods

OB-R is a member of the class I cytokine receptor family with a larger N-terminal extracellular domain than GHR and EpoR. The extracellular domain consists of more domains than necessary for ligand binding. These extra domains are probably involved in receptor activation and signal transmission into the cell. The extracellular domain is composed of two so-called cytokine receptor homology (CRH) domains, a membrane distal domain CRH1 and a proximal domain CRH2. These domains are separated by an immunoglobulin (Ig) domain and followed by two fibronectin like (FNIII) domains. Binding studies with recombinant OB-R subdomains and molecular modeling of the leptin/OB-R complex indicated the existence of three different binding sites similar to the IL6-system.28-34 Indeed, Leptin and OB-R show the highest structural similarity to G-CSF and to the IL-6 family. Similarly to these cytokines, several observations suggested that OB-R exists as a dimer. Cross-linking of OB-R and leptin revealed western blot bands with apparent molecular weights corresponding to monomers, dimers and higher order oligomers.35 Dimers were detected even with the soluble receptor composed of the whole OB-R extracellular domain but truncated of its transmembrane and intracellular domain.36 Further evidence for ligand-independent

32

Figure 1, legend viewed on following page.

Leptin and Leptin Antagonists

Study of Leptin: Leptin Receptor Interaction by FRET and BRET

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homo-oligomerization of OB-R were provided by co-immunoprecipitation experiments.37,38 Moreover, dimer formation of OB-R would explain why coexpression of wild type OB-R inhibits the partially constitutive activity of the N269P receptor mutant.39 Importantly, complementation assays of two inactive receptors mutated in their functional intracellular domain (either no JAK2 interaction or no STAT3 docking site) also provided strong evidence for higher order clustering of OB-R.40 However, in vitro binding studies were insufficient to demonstrate such a complex as the leptin binding domain of OB-R appeared to be a monomer forming a stable complex with leptin in a 1:1 stoichiometric ratio, as revealed by gel-filtration experiments and SPR analysis.30 Altogether, despite major efforts to determine the activation mechanism of OB-R with biochemical methods, contradictory results were obtained and many questions remained unanswered. Most biochemical assays provided rather indirect information and were limited by the requirement of receptor solubilization and the use of receptor mutants and isolated subdomains. More recently, resonance energy transfer (RET) techniques were used to obtain more direct information of the oligomeric state of OB-R and the dynamics of its activation in living cells.

Methodological Introduction to FRET/BRET

RET techniques such as fluorescence RET (FRET) and bioluminescence RET (BRET) are methods of choice to study oligomerisation and activation of transmembrane receptors in living cells. They rely on a nonradiative energy transfer between an energy donor and an energy acceptor. To fulfill the conditions for energy transfer, the emission spectrum of the donor must overlap with the excitation spectrum of the acceptor (Fig. 1A).41,42 These approaches can be performed using genetically engineered fusion-proteins thus enabling the monitoring of protein-protein interactions and molecular rearrangements (i.e., conformational changes). One protein is fused to the donor and the other to the acceptor. If the two fusion proteins do not interact, only light emitted from the energy donor excitation can be monitored (Fig. 1B). If the two fusion proteins interact and position the energy donor and acceptor within a distance smaller than 10 nm, an additional light signal corresponding to the acceptor reemission due to the resonance energy transfer can be detected42-44 (Fig. 1B). In the FRET method both energy donor and acceptor are different variants of the green fluorescent proteins. The cyan fluorescent protein (CFP) is often used as energy donor and the yellow fluorescent protein (YFP) as energy acceptor. FRET measurements require an external excitatory light source for donor excitation. In the BRET method, the CFP Figure 1, viewed on previous page. A) Emission and excitation spectra of energy donor and acceptor. The resonance energy transfer occurs only if the donor and the acceptor display overlapping emission and excitation spectra respectively. B) Resonance Energy Transfer (RET) principle. The donor molecule emits light when it is excited by an external light source or in the presence of the luciferase substrate. Two possible events can occur depending on the orientation and distance of donor versus acceptor dipoles. If they do not interact, are further than 10 nm from each other, or are unfavorably oriented, there is no RET and the excited donor will emit at the donor emission wavelength. On the contrary, if there is interaction between donor and acceptor dipoles positioned at less than 10 nm from each other, RET will occur from the donor to the acceptor resulting in the excitation of the acceptor which then emits at the acceptor emission wavelength. C) FRET and BRET principle. In RET techniques, the proteins of interest X and Y are fused to donor or acceptor molecules. In the FRET event, the donor can be the Cyan Fluorescent Protein (CFP) which when excited at 433 nm transfers its energy by resonance to the acceptor, the Yellow Fluorescent Protein (YFP) which then emits at 530 nm. In the BRET method, the donor of energy is the enzyme Renilla luciferase (Rluc), which by oxidizing its substrate, coelenterazine, transmits part of the energy to the YFP acceptor, which reemits fluorescent light at 530 nm. D) Donor Saturation Curve. In the donor saturation assay, a donor at a constant concentration is progressively saturated by increasing concentrations of acceptor. When there are specific interactions between the donor and the acceptor, the saturation curve will reach a plateau (BRETmax). The BRET50 value (correlated to relative affinity between donor and acceptor) is defined as the acceptor/donor ratio at half-maximal BRETmax. Conversely, the saturation curve evolves linearly with low BRET signals when donor and acceptor do not interact specifically.

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is replaced by a luciferase that generates light in the presence of its corresponding substrate. In a typical BRET experiment, the luciferase from Renilla reniformis (Rluc) is used as energy donor and YFP as energy acceptor (Fig. 1C). For a given donor/acceptor couple, RET intensity depends on the distance between the donor and acceptor (<10 nm), the relative orientation of the two dipoles and their molar ratio. The dependency of RET on the acceptor/donor ratio is nicely illustrated in donor saturation assays.45 In this assay a fixed amount of energy donor is coexpressed with increasing amounts of energy acceptor. For specific interactions, RET increases rapidly reaching a plateau that represents the saturation of all donor molecules with acceptor molecules (Fig. 1D). RET resulting from nonspecific interactions is also dependent on the acceptor/donor ratio. However, in contrast to RET signals of specific interactions, these signals are typically smaller, increase linearly and do not saturate (Fig. 1D). For specific and saturable RET donor saturation curves, two important parameters can be determined: the maximal BRET (BRETmax) and the half-maximal BRET, called BRET50 value. Comparison of different BRET50 values that have been obtained under identical experimental conditions, may provide information about the relative affinities of these interactions. BRET and FRET techniques are complementary approaches. Whereas FRET methods are extensively used to determine molecular interactions and its dynamics at the subcellular level,46 BRET methods are still not able to reach such a high subcellular resolution due to the weak amount of light emitted by the luciferase.47,48 FRET methods are more sensitive to experimental artifacts as the use of an external light source to excite the energy donor can directly induce acceptor emission, cell autofluorescence and photobleaching of donor and acceptor.46 More sophisticated FRET-derived methods such as fluorescence lifetime imaging (FLIM) and fluorescence recovery after photobleaching (FRAP) have then been developed to circumvent this problem.49 Due to a more favorable signal-to-noise ratio, BRET-based assays generally tend to be approximately 10 times more sensitive50 than FRET-based assays, which might be relevant in some cases where low endogenous receptor expression levels are required.

Activation Mechanism of OB-R Monitored by FRET and BRET Lessons from Other Cytokine Receptors

FRET and more recently BRET methodologies have been used to study hormone or cytokine receptor mechanism of activation in intact cells. FRET measurements demonstrated basal homodimerization of GHR but gave discordant results on GH-induced fluorescence signals, depending on experiments being carried out either on plasma membrane enriched regions12 or in intact cells.51 FRET was also used to study homodimerization or heterodimerization of other cytokine/hormone receptors52-54 or ligand-induced conformational changes within predimerized receptors.55-57 For example, placental lactogen (PL) caused the transient heterodimerization of GHR and prolactin (PRL) receptors (PRLR) (2.5 to 3 min after PL stimulation), whereas GH or PRL had no effect at all.58 BRET assays were more recently developed and only some cytokine receptors have been analyzed by this technique. Indeed, BRET experiments confirmed the preformed homodimeric conformation of GHR.12 Several studies on PRLR showed increased BRET signals upon ligand binding of PRLR short and/or long isoforms suggesting homo- and heterodimerization between distinct PRLR isoforms.59,60 A more recent study pointed out that contrary to an intact PRLR, deletion of the extracellular S2 domain of PRLR generated ligand-independent BRET signals, which were associated to constitutive activity.61

FRET and BRET Studies with OB-R OB-R Exists as Preformed Dimer

In 2003, the BRET assay was first applied to OB-R. The question of the oligomeric state was studied by transfecting OB-Ra and OB-Rb fused to Rluc or YFP in HeLa, COS-7 or HEK-293 cells.45 A significant basal RET was detected between OB-Ra-Rluc and OB-Ra-YFP as well as

Study of Leptin: Leptin Receptor Interaction by FRET and BRET

35

between OB-Rb-Rluc and OB-Rb-YFP in intact cells and crude membranes. The basal BRET indicates the formation of constitutive pre-existing dimers for both OB-R isoforms. Donor saturation experiments where a constant amount of OB-Ra-RLuc is progressively saturated with increasing amounts of OB-Ra-YFP showed unambiguously a specific interaction between OB-Ra protomers even in the absence of ligand (Fig. 2A). The fitting analysis of the saturation curve indicated that a significant fraction of OB-R (∼60%) is engaged in dimers. The existence of a pre-existing homo-multimeric OB-R is consistent with the abovementioned biochemical studies. In 2005, these experiments were complemented by FRET experiments. Leptin-independent oligomerization of OB-R was studied in real-time in HEK-293 cells by measuring FRET using either fluorescence lifetime microscopy (FLIM) or acceptor photobleaching (APB) methods.62 The experiments were performed with transiently transfected OB-Ra and OB-Rb tagged at the C-terminus with CFP or YFP. The interaction of OBRa/OBRa, OBRb/OBRb and OBRa/OBRb were tested. The detection of a significant basal FRET signal confirmed once again the existence of pre-associated OB-R a/ OB-Ra and OB-Rb/OB-Rb homodimers. In contrast, there was no evidence for the formation of OB-Ra/OB-Rb heterocomplexes.62 However, the absence of RET does not necessarily mean that the two proteins do not interact as the distance and/or orientation of the donor/acceptor couple might be unfavorable for RET within the heterodimer. The large differences in the length of the intracellular domains of OB-Ra and OB-Rb isoforms may indeed position the Rluc and YFP at a sub-optimal distance. Results from biochemical experiments regarding the existence of OB-Ra/OB-Rb heterodimers were unfortunately also inconclusive. OB-R a/OB-Rb heterodimers were not detected by co-immunoprecipitation in one study35 and only upon leptin exposure in another.37 The absence of a dominant-negative effect of the signaling-incompetent OB-Ra isoform on the signaling-competent OB-Rb isoform further argues against the existence of OB-Ra/ OB-Rb heterodimers.63 Taken together, whereas the formation of OB-Ra/OB-Rb heterodimers is still questionable, available experimental evidence strongly indicates the existence of preformed OB-Ra and OB-Rb homodimers.

Mechanism of OB-R Activation Monitored by RET

Leptin binding to OB-R is believed to trigger a structural change in the organization or orientation of the receptor’s extracellular domain that is transmitted along the single transmembrane spanning α-helix to the intracellular juxtamembrane domain. These intramolecular rearrangements that may or may not be accompanied by further receptor clustering, allow a more avid and productive association of JAK2 followed by JAK2 autophosphorylation, receptor phosphorylation and STAT3 recruitment. Both, FRET and BRET techniques have been applied to confirm or challenge this molecular model of OB-R activation. We and others observed that the RET signal detected with OB-R is enhanced by leptin exposure with some differences between OB-Ra and OB-Rb.45,62 The leptin-induced increase of RET signal could reflect either recruitment of new protomers into oligomers or conformational changes in the pre-oligomerized receptor. Further RET data analysis attempted to distinguish between both possibilites. To unravel the mechanism of OB-R activation, BRET saturation experiments were performed in the presence and absence of ligand (Fig. 2A).45 Since the majority of OB-R is trapped in intracellular compartments, few receptors (5-20%) reach the cellular surface.64 Hence, the change in BRET signal was investigated both for receptors exposed at the plasma membrane and for total receptors including intracellular OB-R. Saponin was used to gently permeabilize cell membranes and allowed the 16kDa-leptin to enter the cell. Both surface and total receptors displayed similar dose response curves with identical EC50 suggesting that intracellular receptors are competent for ligand stimulation similarly to receptors at the cell surface. Similar results were obtained with crude membrane preparations, indicating that leptin-induced BRET is not the result of receptor redistribution in intracellular compartments. Leptin incubation did increase the BRETmax of the donor saturation curve with OB-Ra but did not modify the shape of the curve nor the BRET50 (correlated to apparent affinity). This excluded de novo formation of new dimers from monomers upon leptin binding but rather reflected a conformational change of OB-Ra that would modify the relative orientation and distance of Rluc and YFP moieties. However, our BRET data cannot

36

Leptin and Leptin Antagonists

Figure 2. A) BRET donor saturation curve of the OB-Ra-Rluc/OB-Ra-YFP pair at the basal level (− leptin) and upon leptin stimulation (+ leptin) (adapted from45). B) Evolution of FRET efficiency with time after addition of a saturating leptin concentration for the OB-Ra-CFP/OB-Ra-YFP and OB-Rb-CFP/OB-Rb-YFP homodimers and for the OB-Ra-CFP/OB-Rb-YFP pair (adapted from62). C) Two models of OB-R activation are currently proposed. In model 1, after leptin binding, a conformational change occurs within the OB-R homodimer leading to a 2:2 complex. In model 2, two OB-R homodimers undergo a conformational change and dimerize into a tetrameric receptor in order to assemble into a hexameric 2:4 complex. D) Dose response curve. OB-R conformational change is monitored by the BRET assay with increasing concentrations of either an agonist (plain line) or an antagonist (dashed line). The competition test is carried out with 20 nM leptin which are displaced by increasing concentrations of an antagonist (dotted line).

exclude the model where leptin induces assemblage of a complex of higher order than a dimer. The analysis of BRET donor saturation assay is difficult if there is higher order oligomerization involving more than 1 RLuc:1YFP, i.e., for example dimerization of a pre-existing dimer into a tetramer or even conformational rearrangement of preformed tetramer. Altogether, these BRET data exclude a dimerization of monomeric OB-R upon leptin stimulation and are in favor of a view

Study of Leptin: Leptin Receptor Interaction by FRET and BRET

37

where pre-existing OB-Ra dimers undergo conformational changes upon leptin binding (Fig. 2C, model 1), but cannot reject any further higher order complexes. The observation of the leptin-dependent increase of FRET efficiency with OB-Ra was similar to BRET data.62 In APB experiments on OB-Ra, leptin application slightly increased FRET efficiency within two minutes and remained sustained with time (Fig. 2B). However, in the FLIM assay leptin exposure to OB-Ra did not change the high basal FRET efficiency. The shorter intracellular fragment of OB-Ra probably constrained the fusion proteins in close proximity even before interacting with the ligand. The authors hypothesized that for this reason, the conformational change resulting from ligand binding might not be easily detectable. For OB-Rb, with both APB and FLIM techniques, the increase of FRET efficiency is significant but transient with a peak at three minutes after leptin addition, followed by a fast decrease to the basal level (Fig. 2B). This was not observed in BRET experiments where leptin stimulation did not have any effect on the BRET signal. BRET suggests, at a first glance, no additional dimerization/conformational change of leptin-stimulated OB-Rb. This hypothesis is rather unlikely. The absence of RET signal is often inconclusive and does not necessarly reflect absence of association or of conformational changes. More likely, (i) the orientation/distance of fusion protein constructs with OB-Rb were not favorable for a detectable change in BRET signal or (ii) the high flexibility of the long intracellular domain of OB-Rb could limit transmission of conformational change to Rluc and YFP fused at the long C-terminus or (iii) BRET signals monitored only after 5 minutes of leptin stimulation could have missed fast transient signals (whereas FRET assays was kinetically studied). An analytical conversion of raw FLIM data into FRET population tried to unravel the activation mechanism of OB-R. The analysis showed that leptin-induced decrease in the population of OB-R displaying no FRET signal suggested de novo oligomerization, whereas an increase in the high FRET population could be favorable to transient conformational changes. Hence, the population analysis seemed to favor both events. The fast decrease of FRET signal just after the transient peak was hypothesized by the authors to result either from dissociation of the receptors, from a switch to an inactive conformation, or from receptor internalization abolishing the signal. The transient FRET signal for OB-Rb which could be correlated to a very short activation state of the receptor was rather surprising. However, it is possible that a transient conformational change and/or transient oligomerization of the receptor would be sufficient for Jak2 activation and transphosphorylation. Collectively, FRET data supported a model where a conformational change of OB-R dimers occurs in addition to de novo oligomerization of receptors upon ligand interaction to the receptor extracellular domain (Fig. 2C, model 2). The FRET results supported and the BRET analysis could not rule out two structural models of leptin/OB-R complex (2:2 or 2:4 stoichiometry) which were built using either the G-CSFR65 or the IL-6R system32 (Fig. 2C). J. Tavernier and his group documented that leptin binding to OB-R resembles the interaction between interleukin 6 (IL6) and its receptor and suggested the existence of a novel, previously unidentified leptin binding site III responsible for the formation of an active 2:4 leptin/OB-R complex. Similar to IL6 and G-CSF, leptin’s site III could be hypothesized to play a pivotal role in OB-R oligomerization and in its subsequent activation. The formation of an active multimeric complex through site III was envisioned to occur through the interaction of leptin with the Ig domain of OB-R (Fig. 2C, model 2).32,40 On the other hand, binding site II was identified to be important for high affinity interaction between leptin and the OB-R CRH2 domain.29 BRET and FRET assays, used to investigate the effect of leptin mutations occurring at binding site III, supported the multimeric model. A. Gertler’s group identified an hydrophobic strand in the leptin A-B loop as a major component of binding site III.31 Mutations in this region did not change the monomeric state of leptin nor modified its binding capacity to the receptor as shown by SPR and binding assay with radiolabeled leptin. Contrary to cell exposure to wild-type leptin, which showed a transient FRET signal with OB-Rb, exposure to leptin mutein (L39A/D40A/

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Leptin and Leptin Antagonists

Figure 3. A) Conformational change of OB-Ra monitored by BRET. Stimulation by an agonist induces a change in BRET signal as a consequence of the relative rearragement between OB-Ra fused to Rluc and OB-Ra fused to YFP, reflecting the ligand-induced conformational change in the OB-R dimer. B) Flow chart for the screening of a compound library by the BRET assay. Cells co-expressing the BRET partners OBR-Rluc/OBR-YFP are loaded in 96 or 384 well plates containing a compound library. After 10 minutes of incubation, either no leptin or leptin will be added to each well for 5 minutes. The BRET signal is then read after the addition of the substrate coelenterazine.

Study of Leptin: Leptin Receptor Interaction by FRET and BRET

39

F41A) did not trigger any FRET increase despite its ability to interact with the receptor. Even simultaneous addition of leptin with 10-fold excess of the mutein inhibited leptin action. BRET assays were also carried out with the same mutein to investigate its capacity to increase BRET signals of the OB-Ra homodimer. Similarly, the mutein was not able to cause an elevation of the BRET signal even at high ligand concentrations (1000 nM) as seen in the dose-response curves (Fig. 2D). The competition curve confirmed that the mutein compete with leptin for binding to the receptor but was not able to trigger any change in BRET signal (Fig. 2D). In the same way, the inability of mutein to induce RET reflecting its incapacity to trigger any conformational change/ oligomerization was correlated to the absence of receptor activation by the mutein as observed in functional studies (reporter gene assays, activation of STAT3 and MAPK pathways and proliferation assays of BAF/3 cells).31 It appears that the leptin mutations in binding site III convert the agonistic activity of leptin into antagonistic activity. These data supported a mutational analysis of J. Tavernier’s team who showed that mutations of S120 and T121 in the N-terminal region of helix D contributed to binding site III and transform leptin into an antagonist.28 As mutations of binding site III abrogate any leptin-induced increase of RET signal as well as any functional activity of OB-R but without modifying ligand binding abilities, it is probable that site III would be at the basis of both conformational changes and/or higher order oligomerization if they do occur. Based on the currently available data, the following model of OB-R activation can be proposed. Two leptin molecules are likely to interact through site I, site II (high affinity) with an OB-R dimer. Additional binding through binding site III would trigger both a conformational change of preformed OB-R dimers as well as dimer recruitment into tetramers to form a hexameric complex (2leptin:4OB-R) through binding site III (Fig. 2C, model 2). However, there is not enough data (solely one study by FRET) that would rule out a simple mechanism where binding site III is involved exclusively in the conformational change of OB-R dimer without any further receptor clustering (Fig. 2C, model 1). Nevertheless, binding site III is believed to be strictly necessary for OB-R activation required for the first steps of signal transduction. Hence, leptin mutations at site III convert leptin agonistic properties into antagonistic functions.

The OB-R BRET Assay, a Screening Tool for the Identification of New OB-R Ligands

The BRET technique is particularly well adapted for high throughput screening purposes of therapeutic molecules as described previously.66,67 The assay can be performed in 96- or 384-well plates, is fast, nonradioactive and homogeneous (Fig. 3). The number of putative therapeutic domains where treatment with OB-R ligands might be beneficial expanded steadily over the last ten years and clearly exceeds its original field of application on the control of energy homeostasis (see also the other chapters of this book). Treatment with OB-R antagonists is likely to be beneficial in the treatment of cancer and in T-cell-dependent autoimmune diseases including multiple sclerosis. Despite this large therapeutic potential, only very few OB-R ligands are known today. Several leptin mutants have been designed and shown to have agonistic or antagonistic properties.28,30-33,68 The in vivo potential of these first-generation OB-R ligands is currently under investigation. The next step will be the development of drugable, small molecular weight compounds. The OB-R BRET assay might be of particular interest for the screening of these second-generation compounds. Due to the high “flexibility” of the OB-R BRET assay, several different compound classes could be identified. Among these compounds will be competitive agonists and antagonists but also noncompetitive allosteric compounds. Furthermore, inhibitors of protein-protein interactions (dimerization inhibitors in this case) may also be detected as these compounds are expected to decrease the basal BRET signal due to the dissociation of the two protomers of the dimer.

Conclusion

RET supported by biochemical and modeling studies could partially unravel the molecular mechanism of OB-R activation. RET methods over other biochemical techniques have the advantages of adding a dynamic dimension in real time and in intact cells to investigate how OB-R is activated

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Leptin and Leptin Antagonists

by leptin. All the studies have led to the compelling view that OB-R exists as a preformed dimer (or oligomer) eliminating the picture where the monomeric form is involved in receptor activation. However, further mechanistic events are more questionable and two models are running in competition. Leptin interacting with OB-R extracellular domains would trigger either conformational changes of OB-R dimers and/or higher order clustering of OB-R dimers in an hexameric complex. While FRET would support both events, BRET would be in favor of a conformational change without possibly ruling out the other model. The unclear results so far leave the question still open. The BRET assay applied to OB-R has the advantage of being able to be adapted to high throughput screening of a large library of compounds in order to identify drugs capable of triggering, interfering or improving the OB-R activation and hence signaling. The behavior of a leptin mutein in contrast to wild-type leptin constitutes a validation of the BRET assay as a possible tool to investigate agonistic/ antagonistic properties of compounds. Indeed, mutations in leptin binding site III, do not give rise to any BRET or FRET signal. This correlates perfectly to the incapacity of the mutein to trigger signaling and function and points out to its antagonistic properties.

Acknowledgements

This work was supported by grants from Sanofi-Aventis, Association pour la recherche contre le cancer (N˚ 3315, 3970), AFERO, La Ligue Contre Le Cancer (Comité de Paris N/Ref : R04/75-102), INSERM, CNRS (Programme National de Recherches sur le Diabète, PNRD) and the University Paris Descartes.

References

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16. Livnah O, Johnson DL, Stura EA et al. An antagonist peptide-EPO receptor complex suggests that receptor dimerization is not sufficient for activation. Nat Struct Biol 1998; 5:993-1004. 17. Remy I, Wilson IA, Michnick SW. Erythropoietin receptor activation by a ligand-induced conformation change. Science 1999; 283:990-3. 18. Barton VA, Hudson KR, Heath JK. Identification of three distinct receptor binding sites of murine interleukin-11. J Biol Chem 1999; 274:5755-61. 19. Simpson RJ, Hammacher A, Smith DK et al. Interleukin-6: structure-function relationships. Protein Sci 1997; 6:929-55. 20. Inoue M, Nakayama C, Kikuchi K et al. D1 cap region involved in the receptor recognition and neural cell survival activity of human ciliary neurotrophic factor. Proc Natl Acad Sci USA 1995; 92:8579-83. 21. Hudson KR, Vernallis AB, Heath JK. Characterization of the receptor binding sites of human leukemia inhibitory factor and creation of antagonists. J Biol Chem 1996; 271:11971-8. 22. Bravo J, Heath JK. Receptor recognition by gp130 cytokines. EMBO J 2000; 19:2399-411. 23. Aritomi M, Kunishima N, Okamoto T et al. Atomic structure of the GCSF-receptor complex showing a new cytokine-receptor recognition scheme. Nature 1999; 401:713-7. 24. Layton JE, Hall NE, Connell F et al. Identification of ligand-binding site III on the immunoglobulin-like domain of the granulocyte colony-stimulating factor receptor. J Biol Chem 2001; 276:36779-87. 25. Tamada T, Honjo E, Maeda Y et al. Homodimeric cross-over structure of the human granulocyte colony-stimulating factor (GCSF) receptor signaling complex. Proc Natl Acad Sci USA 2006; 103:3135-40. 26. Boulanger MJ, Chow DC, Brevnova EE et al. Hexameric structure and assembly of the interleukin-6/ IL-6 alpha-receptor/gp130 complex. Science 2003; 300:2101-4. 27. Chow D, He X, Snow AL et al. Structure of an extracellular gp130 cytokine receptor signaling complex. Science 2001; 291:2150-5. 28. Peelman F, Van Beneden K, Zabeau L et al. Mapping of the leptin binding sites and design of a leptin antagonist. J Biol Chem 2004; 279:41038-46. 29. Iserentant H, Peelman F, Defeau D et al. Mapping of the interface between leptin and the leptin receptor CRH2 domain. J Cell Sci 2005; 118:2519-27. 30. Niv-Spector L, Raver N, Friedman-Einat M et al. Mapping leptin-interacting sites in recombinant leptin-binding domain (LBD) subcloned from chicken leptin receptor. Biochem J 2005; 390:475-84. 31. Niv-Spector L, Gonen-Berger D, Gourdou I et al. Identification of the hydrophobic strand in the A-B loop of leptin as major binding site III: implications for large-scale preparation of potent recombinant human and ovine leptin antagonists. Biochem J 2005; 391:221-30. 32. Peelman F, Iserentant H, De Smet AS et al. Mapping of binding site III in the leptin receptor and modeling of a hexameric leptin receptor complex. J Biol Chem 2006; 281:15496-504. 33. Solomon G, Niv-Spector L, Gonen-Berger D et al. Preparation of leptin antagonists by site-directed mutagenesis of human, ovine, rat and mouse leptin’s site III: implications on blocking undesired leptin action in vivo. Ann N Y Acad Sci 2006; 1091:531-9. 34. Solomon G, Reicher S, Gussakovsky EE et al. Large-scale preparation and in vitro characterization of biologically active human placental (20 and 22K) and pituitary (20K) growth hormones: placental growth hormones have no lactogenic activity in humans. Growth Horm IGF Res 2006; 16:297-307. 35. Devos R, Guisez Y, Van der Heyden J et al. Ligand-independent dimerization of the extracellular domain of the leptin receptor and determination of the stoichiometry of leptin binding. J Biol Chem 1997; 272:18304-10. 36. Liu C, Liu XJ, Barry G et al. Expression and characterization of a putative high affinity human soluble leptin receptor. Endocrinology 1997; 138:3548-54. 37. White DW, Tartaglia LA. Evidence for ligand-independent homo-oligomerization of leptin receptor (OB-R) isoforms: a proposed mechanism permitting productive long-form signaling in the presence of excess short-form expression. J Cell Biochem 1999; 73:278-88. 38. Nakashima K, Narazaki M, Taga T. Leptin receptor (OB-R) oligomerizes with itself but not with its closely related cytokine signal transducer gp130. FEBS Lett 1997; 403:79-82. 39. White DW, Wang DW, Chua SC Jr et al. Constitutive and impaired signaling of leptin receptors containing the gln → pro extracellular domain fatty mutation. Proc Natl Acad Sci USA 1997; 94:10657-62. 40. Zabeau L, Defeau D, Van der Heyden J et al. Functional analysis of leptin receptor activation using a Janus kinase/signal transducer and activator of transcription complementation assay. Mol Endocrinol 2004; 18:150-61. 41. Haugland RP, Yguerabide J, Stryer L. Dependence of the kinetics of singlet-singlet energy transfer on spectral overlap. Proc Natl Acad Sci USA 1969; 63:23-30. 42. Wu P, Brand L. Resonance energy transfer: methods and applications. Anal Biochem 1994; 218:1-13.

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43. Xu Y, Piston DW, Johnson CH. A bioluminescence resonance energy transfer (BRET) system: application to interacting circadian clock proteins. Proc Natl Acad Sci USA 1999; 96:151-6. 44. Pfleger KD, Eidne KA. Illuminating insights into protein-protein interactions using bioluminescence resonance energy transfer (BRET). Nat Methods 2006; 3:165-74. 45. Couturier C, Jockers R. Activation of the leptin receptor by a ligand-induced conformational change of constitutive receptor dimers. J Biol Chem 2003; 278:26604-11. 46. Piston DW, Kremers GJ. Fluorescent protein FRET: the good, the bad and the ugly. Trends Biochem Sci 2007; 32:407-14. 47. Xu X, Soutto M, Xie Q et al. Imaging protein interactions with bioluminescence resonance energy transfer (BRET) in plant and mammalian cells and tissues. Proc Natl Acad Sci USA 2007; 104:10264-9. 48. Coulon V, Audet M, Homburger V et al. Subcellular imaging of dynamic protein interactions by bioluminescence resonance energy transfer. Biophys J 2008; 94:1001-9. 49. Trugnan G, Fontanges P, Delautier D et al. [FRAP, FLIP, FRET, BRET, FLIM, PRIM...new techniques for a colourful life]. Med Sci (Paris) 2004; 20:1027-34. 50. Arai R, Nakagawa H, Tsumoto K et al. Demonstration of a homogeneous noncompetitive immunoassay based on bioluminescence resonance energy transfer. Anal Biochem 2001; 289:77-81. 51. Biener Ramanujan E, Ramanujan VK, Herman B et al. Spatio-temporal kinetics of growth hormone receptor signaling in single cells using FRET microscopy. Growth Horm IGF Res 2006; 16:247-57. 52. Clayton AH, Walker F, Orchard SG et al. Ligand-induced dimer-tetramer transition during the activation of the cell surface epidermal growth factor receptor-A multidimensional microscopy analysis. J Biol Chem 2005; 280:30392-9. 53. Giese B, Roderburg C, Sommerauer M et al. Dimerization of the cytokine receptors gp130 and LIFR analysed in single cells. J Cell Sci 2005; 118:5129-40. 54. Tenhumberg S, Schuster B, Zhu L et al. gp130 dimerization in the absence of ligand: preformed cytokine receptor complexes. Biochem Biophys Res Commun 2006; 346:649-57. 55. Martin Fernandez M, Clarke DT, Tobin MJ et al. Preformed oligomeric epidermal growth factor receptors undergo an ectodomain structure change during signaling. Biophys J 2002; 82:2415-27. 56. Sivaprasad U, Canfield JM, Brooks CL. Mechanism for ordered receptor binding by human prolactin. Biochemistry 2004; 43:13755-65. 57. Krishnaveni MS, Hansen JL, Seeger W et al. Constitutive homo- and hetero-oligomerization of TbetaRII-B, an alternatively spliced variant of the mouse TGF-beta type II receptor. Biochem Biophys Res Commun 2006; 351:651-7. 58. Biener E, Martin C, Daniel N et al. (2003). Ovine placental lactogen-induced heterodimerization of ovine growth hormone and prolactin receptors in living cells is demonstrated by fluorescence resonance energy transfer microscopy and leads to prolonged phosphorylation of signal transducer and activator of transcription (STAT)1 and STAT3. Endocrinology 2003; 144:3532-40. 59. Tan D, Johnson DA, Wu W et al. Unmodified prolactin (PRL) and S179D PRL-initiated bioluminescence resonance energy transfer between homo- and hetero-pairs of long and short human PRL receptors in living human cells. Mol Endocrinol 2005; 19:1291-303. 60. Qazi AM, Tsai Morris CH, Dufau ML. Ligand-independent homo- and heterodimerization of human prolactin receptor variants: inhibitory action of the short forms by heterodimerization. Mol Endocrinol 2006; 20:1912-23. 61. Tan D, Huang KT, Ueda E et al. S2 deletion variants of human PRL receptors demonstrate that extracellular domain conformation can alter conformation of the intracellular signaling domain(dagger). Biochemistry 2006; 47:479-89. 62. Biener E, Charlier M, Ramanujan VK et al. Q uantitative FRET imaging of leptin receptor oligomerization kinetics in single cells. Biol Cell 2005; 97:905-19. 63. White DW, Kuropatwinski KK, Devos R et al. Leptin receptor (OB-R) signaling. Cytoplasmic domain mutational analysis and evidence for receptor homo-oligomerization. J Biol Chem 1997; 272:4065-71. 64. Belouzard S, Delcroix D, Rouille Y. Low levels of expression of leptin receptor at the cell surface result from constitutive endocytosis and intracellular retention in the biosynthetic pathway. J Biol Chem 2004; 279:28499-508. 65. Hiroike T, Higo J, Jingami H et al. Homology modeling of human leptin/leptin receptor complex. Biochem Biophys Res Commun 2000; 275:154-8. 66. Boute N, Jockers R, Issad T. The use of resonance energy transfer in high-throughput screening: BRET versus FRET. Trends Pharmacol Sci 2002; 23:351-4. 67. Bacart JCC, Jockers R, Bach S et al. The BRET technology and its application to screening assays. Biotechnol J, in press 2008. 68. Salomon G, Niv-Spector L, Gussakovsky EE et al. Large-scale preparation of biologically active mouse and rat leptins and their L39A/D40A/F41A muteins which act as potent antagonists. Protein Expr Purif 2006; 47:128-36.

Chapter 4

Is Leptin a Pro- or Anti-Apoptotic Agent? Srujana Rayalam, Mary Anne Della-Fera, Suresh Ambati and Clifton A. Baile*

Abstract

A

poptosis, the regulated destruction of a cell, is characterized by biological and morphological changes and involves a large web of integrating pathways and factors. Apoptosis is necessary to eliminate excess cells and cells that hinder development and hence the importance of apoptotic pathways and apoptotic agents in removing adipocytes for the treatment of obesity has been recently explored. Leptin was widely recognized for its ability to regulate adipose tissue mass by influencing food intake and energy expenditure. Recent findings, however, demonstrated that leptin treatment initiated apoptosis in adipose tissue. Leptin-induced adipocyte apoptosis was a surprising finding, as adipocytes were thought to be extremely stable; however, both pro- and anti-apoptotic effects of leptin have been demonstrated in several cell types. In particular, anti-apoptotic effects have been shown in certain types of cancer cells and are correlated with the presence of leptin receptors. While leptin’s effects on energy balance, including induction of adipocyte apoptosis, are primarily mediated by the central nervous system, it is possible that anti-apoptotic effects of leptin are mediated through autocrine or paracrine effects. In this chapter both anti-apoptotic and pro-apoptotic effects of leptin in several cell types are reviewed.

Background/Introduction

Leptin is a cytokine-like hormone that is expressed primarily in adipose tissue, with low levels also detected in the skeletal muscle, placenta and the brain.1 Leptin is a nonglycosylated protein with a molecular mass of 16 KD.2 Its expression in adipose tissue is up-regulated by a variety of factors, such as glucocorticoids, acute infection and pro-inflammatory cytokines and is down-regulated by cold exposure, adrenergic stimulation and growth hormone.1 Leptin was originally discovered as the missing protein in the genetically obese ob/ob mouse3 and it clearly plays a role in regulation of body fat content, in part through effects on eating behavior, body temperature, physical activity, lipid mobilization and adipose tissue apoptosis.4-6 However, it also is involved in the regulation of bone formation.7,8 In addition, leptin has a concomitant fluctuation with seasonal changes and is probably involved in seasonal control of body fat.9 Thus, leptin is a hormone with multiple functions in most of the body systems.10 Consistent with leptin having structural similarities to cytokines, the leptin receptor (ObR) belongs to the cytokine receptor class I superfamily.11 Several isoforms of ObR (a, b, c, d, e) with different lengths of C-termini have been identified.12 The long form of the receptor, ObRb mediates most leptin functions and the short form, ObRa, facilitates transport of leptin via the blood- brain barrier.13 Leptin receptors are widely expressed throughout the brain in areas such as the cortex, *Corresponding Author: Clifton A. Baile—Department of Animal and Dairy Science and Department of Foods and Nutrition, University of Georgia, Athens, GA 30602, USA. Email: [email protected]

Leptin and Leptin Antagonists, edited by Arieh Gertler. ©2009 Landes Bioscience.

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cerebellum, brainstem, basal ganglia and hippocampus2 and it is generally accepted that the hypothalamus is the critical action site for leptin’s effects on energy balance.14,15 Further, the presence of leptin receptor mRNA in the meninges and the microcirculation implies that leptin receptors at one or all of these sites are responsible for transporting leptin into or out of the CNS.16 The finding of structural similarity between leptin and its receptor and cytokine-receptor systems that control hematopoiesis prompted investigations showing that leptin stimulated blood cell formation and proliferation.17 In contrast, Prins et al suggested that leptin might decrease adipocyte volume and number via its CNS action, thereby influencing adipose tissue mass.18 Thus, contradictory findings on the effects of leptin and leptin receptors on cell proliferation and apoptosis have been reported. Before discussing the anti- and pro-apoptotic effects of leptin, we will briefly discuss the types of apoptotic pathways and molecular events leading to this process.

Apoptosis: A Basic Biologic Phenomenon

Apoptosis is considered the principal mechanism of “programmed cell death” in mammalian tissues. The term “apoptosis” was first devised in 1972 after identifying morphologically similar cell deaths in many pathological conditions as well as in normal tissue.19 Apoptosis, also conceptualized as a self-directed cellular “suicide”, is different from other types of cell death like necrosis or oncosis.20 However, other forms of programmed cell death, namely aponecrosis,21 autophagy22 and paraptosis23 have also been described recently. The morphological features of apoptosis are similar across cell types and species.24 Cell shrinkage, chromatin condensation, cellular budding and rapid phagocytosis by macrophages or adjacent cells are the typical events of apoptosis that occur in fixed sequence. Light and electron microscopy have been used to identify the various morphological changes that occur during apoptosis.25 The rapid increase in the number of publications on apoptosis over the past two decades followed identification of several genes and molecular pathways that are involved in the execution of apoptosis. Apoptosis is a coordinated and energy-dependent process involving a cascade of molecular events. Two apoptotic pathways identified to date are the extrinsic, or death receptor pathway and the intrinsic, or mitochondrial pathway. However, these two pathways are linked and molecules in one pathway influence the other.26

Extrinsic Pathway

This pathway is triggered by the binding of an extracellular ligand to a death receptor which belongs to tumor necrosis factor (TNF) receptor gene superfamily.27 Death factors, Fas ligand (FasL) or tumor necrosis factor (TNF), bind to their death receptors and induce apoptosis, killing the cells within hours. Members of the TNF receptor family carry cysteine-rich extracellular domains and have a cytoplasmic domain of about 80 amino acids called the “death domain”.28 FasL and Fas receptor interaction leads to binding of cytoplasmic adapter protein, FADD (Fas associated death domain), and TNFα/TNF receptor interaction leads to binding of TRADD (TNFR1-associated death domain) with recruitment of FADD.29,30 FADD subsequently binds to the prodomain of caspase-8 and a complex, termed death-inducing signaling complex (DISC), is formed which further activates caspase-8. Caspase-8 then activates a series of downstream caspases that result in cleavage of structural and regulatory intracellular proteins, ultimately leading to apoptosis.31

Intrinsic Pathway

The stimuli activating this pathway are not receptor mediated, but the signals produced act within the cell and lead to mitochondrial initiated events. Oligomerization of two pro-apoptotic proteins, Bax and Bak in the mitochondrial outer membrane activates this pathway resulting in an opening of the mitochondrial permeability transition (MPT) pore, loss of the mitochondrial transmembrane potential and release of cytochrome c, second mitochondria–derived activator of caspase (Smac/DIABLO), Omi stress-regulated endoprotease/high temperature requirement protein A2 (Omi/HtrA2) and the serine protease HtrA2/Omi.32-34 These proteins activate the caspase-dependent mitochondrial pathway. The release of a second group of pro-apoptotic proteins, apoptosis inducing factor (AIF) and endonuclease G from the mitochondria is a late

Is Leptin a Pro- or Anti-Apoptotic Agent?

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event that occurs after the cell has committed to die. AIF translocates to the nucleus and causes DNA fragmentation into approximately 50-300 b pieces and condensation of peripheral nuclear chromatin.35 The activation of the execution caspases, caspase-3, caspase-6 and caspase-7 begins the process of apoptosis. These caspases activate cytoplasmic endonucleases and proteases that degrade and cleave nuclear material and cytoskeletal proteins like cytokeratins, poly(ADP-ribose) polymerase (PARP), the nuclear matrix protein NuMA and others36 resulting in morphological and biochemical changes seen in apoptotic cells.

Anti-Apoptotic Effects of Leptin

Although the discovery of leptin was based on its ability to regulate adipose tissue mass, leptin was later considered a multifunctional factor in the development of hematopoietic, reproductive and immunologic functions.37,38 Mitogenic effects of leptin on breast cancer cells,39 colonic epithelium,40 gastric mucosal cells41 and osteoblastic cells 7 have also been reported recently. Although most cases of obesity are characterized by an increase in leptin levels, leading to resistance to leptin’s effects on body fat regulation42 some cell types may not become resistant to leptin. For example, it has been reported that cancer cells are responsive to leptin, thus the high circulating levels of leptin that occur with obesity may be associated with the increased incidence of cancer in obese individuals.43 Leptin has been shown to activate estrogen receptor α (ERα) through mitogen activated protein kinase pathway (MAPK) in breast cancer cells44 and high levels of interleukin-1alpha (IL-1α) produced by breast cancer cells stimulated the expression of leptin.45 Increased leptin further induced the transcription of aromatase, followed by activation of activator protein 1 (AP-1), signal transducers and activators of transcription 3 (STAT3) and extracellular signal regulated kinase 2 (ERK 2) which promoted estradiol synthesis46 (Fig. 1). Confirming these findings, leptin abrogated anti-cancerous effects of estrogen antagonists in vitro leading to proliferation of breast cancer cells that are positive for ERα.47 Thus it is possible that elevated serum leptin levels might lead to the development of antiestrogen resistance in obese individuals suffering from breast cancer. Recent studies show that leptin affects the risk of clinically relevant prostate cancer through testosterone, a well-established mitogen.48 Leptin also stimulated cell proliferation, migration and angiogenesis in prostate cancer cells by increasing the expression of vascular endothelial growth factor (VEGF), transforming growth factor (TGF-β1) and basic fibroblast growth factor (bFGF). In addition, leptin increased the migration of normal epithelial cells.49 Therefore, high leptin levels associated with obesity may be a risk factor in prostate cancer patients.50 Similarly leptin stimulated proliferation of insulin-secreting tumor cell lines;51 although when pancreatic cells are treated with leptin in vitro, the mitogenic effects are not observed. In fact the growth of pancreatic cells was significantly reduced with leptin treatment.43 Leptin also serves as a survival signal in a variety of cell types, including T-cells, macrophages and neutrophils, by enhancing proliferative and antiapoptotic activities. Some of the mechanisms involved in leptin’s antiapoptotic effects in eosinophils include delayed cleavage of proapoptotic Bax proteins, mitochondrial release of cytochrome c and second mitochondria-derived activator of caspase.52 Leptin is believed to play an important role in regulating the onset of puberty;53 e.g., treatment of female mice with leptin accelerated the maturation of the reproductive tract leading to an early onset of the estrous cycle and reproductive capability.54,55 A surge in plasma leptin concentration observed in prepubertal males (humans) further supports the role of leptin in puberty.56 Leptin replacement therapy has been successful in restoration of spermatogenesis and reproductive function57 in leptin-deficient ob/ob mice. Since the identification of leptin receptors on germ cells and Leydig cells within the testis,58,59 a direct regulatory role for leptin in reproduction had been suggested, as the impaired spermatogenic process in the leptin-deficient mouse was due to elevated germ cell apoptosis.60 Treatment with exogenous leptin had advanced the onset of attenuation of ovarian apoptosis and enhancement of folliculogenesis61 and maturity.62 As a balancing act of homeostasis in developing corpora lutea, leptin acts as a pro- or anti-apoptotic factor, which

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Figure 1. Leptin binds to its receptor OB-R, which becomes phosphorylated by MAPK or ERK. These intracellular kinases probably activate STAT3 by phosphorylation of its tyrosine residue. STAT3 is translocated to the nucleus, where it induces transcription of AP-1 that induces transcription of aromatase. Aromatase changes androgens into estrogens in adipose tissue. Estradiol activates its membrane receptors (ERα) and contributes to proliferation of breast epithelial cells. ERα is sensitized for estrogen stimulation by MAPK and ERK, which can transactivate this receptor independently of estrogens. Apart from induction of aromatase, STAT3 may contribute to cell proliferation by activation of other genes.46 Abbreviations: OB-R: leptin receptor, P: phosphate residue, AP-1: transcription activator protein 1, E2: estradiol, ERα: estrogen receptor α , ERK1/2: extracellular signal-regulated kinase 1/2, MAPK: mitogen-activated protein kinase. Reproduced from Sulkowska M, et al. Pathol Oncol Res 2006; 12(2):69-72.46

reverses the anti-apoptotic action of IGF-1 in order to protect cells from excessive apoptosis in maintaining appropriate cell number.63 Leptin promotes cognitive function and neural survival during adverse conditions. During conditions of energy depletion, AMPK reacts to minimize neural insult and enhances neurogenesis, but in severe conditions, it redirects neuronal fate towards apoptosis.64 Under such conditions of impaired learning and memory, restoration of leptin function inhibits AMPK activation, thereby improving cognitive function,65 inhibiting neural apoptosis and inducing neural survival.64 The differential effects of leptin on cancer cells are thought to be mediated by leptin receptors. A number of cancer cell lines were reported to express leptin receptor isoforms, including breast cancer, choriocarcinoma and colon carcinoma cells.39,66,67 However, the expression and involvement of intracellular mediators like STAT and JAK in the activation of leptin and expression of leptin receptor needs further investigation. The cytokine-inducible inhibitors of STAT signaling,68 like suppressors of cytokine signaling (SOCS) that limit cellular response to leptin,69 might contribute to the differential effects of leptin on different types of cancer cells. Thus, circulating leptin might act as a growth factor for development of several cancers in vivo supporting the link between obesity and risk of cancer proliferation (Table 1).

Is Leptin a Pro- or Anti-Apoptotic Agent?

Pro-Apoptotic Effects of Leptin

47

Although leptin can regulate adiposity indirectly by influencing food intake and energy expenditure,70 interest in leptin stems from its weight reducing effects in rodents.3 Both central and peripheral administration of leptin resulted in a dose-dependent decrease in body weight in both ob/ob (genetically obese mice) and wild-type mice3,71 and this reduction was greater in ob/ ob mice than pair-fed mice.72 The weight reducing effect of leptin in rodents was later shown to be partly a result of induction of adipocyte apoptosis.4 Extreme depletion of adipose depots and prolonged recovery time for repletion of fat stores following long-term leptin treatment suggests that leptin decreases adiposity not only by stimulating lipolysis but also by triggering apoptosis.73,74 In contrast, as discussed in the previous section, leptin acts as either an anti-apoptotic agent or a mitogen in several other cell lines. Apoptosis has been studied extensively in cancer cells, but until recently apoptosis has not been considered a part of normal adipocyte life cycle. Adipocytes were considered an extremely stable cell type and Prins et al18 first reported that fat mass is balanced by cell acquisition and deletion and that apoptosis plays an important role in the regulation of adipose tissue mass in humans. This report triggered several studies to investigate adipocyte apoptosis, including reports that ICV (intracerebroventricular) leptin induced adipocyte—specific apoptosis in rats.4 However, the mechanism through which leptin induces apoptosis in adipocytes is not fully understood. Studies indicate that the effective dose of leptin when administered centrally is much lower than effective peripheral doses.75 In addition, we have shown that leptin does not act directly on adipocytes to cause apoptosis.76 So it is possible that leptin’s effects on adipocyte apoptosis are mediated via increased sympathetic activity. Moreover, the beta-2 adrenergic agonist, clenbuterol, increased adipose tissue apoptosis in vivo,77 which further supports the hypothesis that leptin might be acting through the CNS to induce adipocyte apoptosis via adrenergic stimulation. Adrenergic stimulation activated uncoupling protein 1 (UCP-1), which uncouples mitochondrial respiration in brown adipose tissue (BAT)78 and white adipose tissue (WAT).79 Interestingly, mRNA levels of uncoupling protein 2 (UCP-2), which is present in white adipose tissue and not BAT, increased more than 10-fold upon leptin treatment. This report supports the possibility that UCP-2 might be playing an important role in leptin induced adipocyte apoptosis.80 Uncoupling of mitochondrial respiration in adipose tissue promotes free radical generation, like reactive oxygen species (ROS), which are directly involved with the induction of apoptosis.81 Leptin has also been found to increase ROS in endothelial cells by increasing fatty acid oxidation via protein kinase A activation82 and by stimulating polymorphonuclear neutrophils in response to infection.83 Leptin has also been shown to induce the expression of angiopoietin (Ang-2) in adipose tissue, which coincided with initiation of apoptosis in adipose endothelial cells leading to adipose tissue regression.84 Likewise, leptin administration also decreased Bcl-2/Bax ratio, while an increase in the Bcl-2/Bax ratio in the fat pads during recovery is seen to prevent cell loss as the tissues show a tendency to return to control levels.74 Leptin has also been reported to induce β-cell apoptosis by enhanced release of IL-β and suppressed release of IL-1 receptor antagonist in human pancreatic islets,85 thus contributing to the loss of β-cells in diabetes.86 While we cannot assume leptin-induced adipose apoptosis to be mediated only centrally, when tested in in vitro experiments, leptin did not act directly to induce adipocyte apoptosis,76 thus supporting the hypothesis that leptin acts primarily centrally by initiating neural or humoral signals to induce adipocyte apoptosis (Fig. 2). In addition, peroxisome proliferator-activated receptor γ (PPARγ), which activates adipocyte differentiation through transactivation of adipocyte specific genes, also triggers both apoptotic and anti-apoptotic pathways87 and leptin caused a 70-80% increase in PPARγ expression in the epididymal fat pad.4,88 Apart from regulating feeding behavior, leptin might also control intestinal function through regulation of apoptosis in jejunal and ileal mucosa.89,90

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Table 1. Anti-apoptotic effects of leptin Model

Action of Leptin

Reference

Hepatocarcinoma

Stimulated cell proliferation; apoptosis was suppressed by down regulating Bax proteins

Chen et al 200791

Esophageal adenocarcinoma OE33 cells

Enhanced cell proliferation and inhibited apoptosis by stimulating EGFR and ERK phosphorylation

Beales and ogunwobi, 200792

Stimulates cell proliferation and inhibits apoptosis via ERK, p38 MAPK, PI-3 kinase/Akt and JAK-2 dependent activation of COX-2 and PGE2 production

Ogunwobi et al 200693

Cultured chicken ovarian cells

Sirotkin and Stimulated antiapoptotic proteins Bcl2 and inhibited expression of all markers of cytoplasmic apoptosis (Bax, grossmann, 200794 ASK-1, p53)

Hepatocellular Offers cyto-protection with inhibition of ROS and carcinoma, HEPG2 cells enhanced activity of superoxide dismutase (SOD) Rodent pancreatic beta cells, BRIN-BD11 beta cell-line

Human colonic cancer, T(84), HT29/cl.19A, caco-2 cell-lines

Balasubramanian et al 200795

Protects cells against apoptosis by upregulating Bcl-2 m RNA expression

Brown and dunmore, 200796

Stimulates STAT3 and STAT5b phosphorylation

Briscoe et al 200197

Enhances proliferation via MAPK and PI3-K pathways

Hoda et al 200798

Ogunwobi Stimulates cell proliferation and inhibits apoptosis which involves JAK2, PI3 kinase and JNK and activation and Beales, 2007 99 of STAT3 and AP-1 Hepatic stellate cells (HSCs)

Protects from apoptosis through its interaction with the Qamar et al apoptotic pathway proximal to mitochondrial activation 2006100

Thymus cells

Inhibits apoptosis through a mechanism independent of Mansour activation of JAK-2, but depends on the engagement of et al 2006101 IRS-1/PI-3 kinase pathway

Human eosinophils

Serves as a survival factor for human eosinophils, blocks apoptosis through delayed cleavage of Bax and mitochondrial release of cytochrome c

Conus et al 200552

Neutrophils

Serves as a survival cytokine for neutrophils by protecting from apoptosis through signaling cascade involving PI3-K and MAPK dependent pathways, delayed cleavage of Bid and Bax proteins

Bruno et al 2005102

Neuroblastoma cells

Inhibits apoptosis via potent down-regulation of caspase-10 and TNF-related apoptosis-inducing ligand

Russo et al 2004103

Prostate cancer (DU145, Enhances proliferation and inhibits apoptosis through PI3K and MAPK leptin-receptor-activated pathways PC3 human prostate cancer cells)

Somasundar et al 2004104

Is Leptin a Pro- or Anti-Apoptotic Agent?

49

Figure 2. Proposed mechanisms of action for induction of adipose tissue apoptosis by leptin acting on central nervous system receptors. Leptin acts on its receptors in the hypothalamus to increase sympathetic nervous system output. Activation of β -adrenergic receptors on adipocytes increases lipolysis. As fatty acids undergo β -oxidation in the mitochondria, increased uncoupling protein (UCP1 in brown adipose tissue and possibly UCP2 in white adipose tissue) activity discharges the proton gradient, resulting in increased heat production and also increased production of reactive oxygen species (ROS). Increased levels of ROS can lead to increased mitochondrial membrane permeability, with cytochrome C leaking into the cytoplasm resulting in activation of the caspase cascade that ultimately leads to apoptotic cell death. Increased SNS activation also leads to reduced levels of insulin and reduced phosphorylation of protein kinase B (AKT), which shifts the balance away from pathways involved in cell survival to pathways involved in induction of apoptosis.

Conclusions

Leptin promotes cell proliferation and has anti-apoptotic effects in a variety of cancer and normal cell types. Supraphysiological levels of leptin can cause dramatic reductions in body weight and fat in laboratory rodents and leptin-induced adipose tissue apoptosis could help explain the observation of slower recovery of body weight after leptin treatment is terminated. To summarize, leptin mediates anti-apoptotic effects directly through leptin receptors in many tissues by stimulating the expression of anti-apoptotic proteins leading to enhanced cell proliferation. However, the pro-apoptotic effects of leptin in adipocytes are mediated indirectly through the sympathetic nervous system that leads to both activation of the beta-adrenergic pathway and inhibition of insulin signaling pathways. Activation of the beta-adrenergic pathway results in increased beta-oxidation of fatty acids in the mitochondria and increased UCP activity, leading to increased production

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of ROS which further activate the caspase cascade, ultimately leading to apoptotic cell death. Inhibition of insulin mediated PI3K activation results in reduced phosphorylation of AKT resulting in the stimulation of several apoptotic pathways. Apoptosis, which has not been well studied in adipocytes, now appears to be an important component in the loss of body fat caused by leptin administration. Better understanding of mechanisms involved in leptin’s pro-apoptotic effects on adipose tissue could lead to development of more effective treatments for obesity and possibly also other disorders of metabolism.

References

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58. El-Hefnawy T, Ioffe S, Dym M. Expression of the leptin receptor during germ cell development in the mouse testis. Endocrinology 2000; 141(7):2624-2630. 59. Caprio M, Fabbrini E, Ricci G et al. Ontogenesis of leptin receptor in rat Leydig cells. Biol Reprod 2003; 68(4):1199-1207. 60. Bhat GK, Sea TL, Olatinwo MO et al. Influence of a leptin deficiency on testicular morphology, germ cell apoptosis and expression levels of apoptosis-related genes in the mouse. J Androl 2006; 27(2):302-310. 61. Paczoska-Eliasiewicz HE, Proszkowiec-Weglarz M, Proudman J et al. Exogenous leptin advances puberty in domestic hen. Domest Anim Endocrinol 2006; 31(3):211-226. 62. Almog B, Gold R, Tajima K et al. Leptin attenuates follicular apoptosis and accelerates the onset of puberty in immature rats. Mol Cell Endocrinol 2001; 183(1-2):179-191. 63. Gregoraszczuk EL, Ptak A. In vitro effect of leptin on growth hormone (GH)- and insulin-like growth factor-I (IGF-I)-stimulated progesterone secretion and apoptosis in developing and mature corpora lutea of pig ovaries. J Reprod Dev 2005; 51(6):727-733. 64. Dagon Y, Avraham Y, Magen I et al. Nutritional status, cognition and survival: A new role for leptin and AMP kinase. J Biol Chem 2005; 280(51):42142-42148. 65. Randt CT, Quartermain D, Goldstein M et al. Norepinephrine biosynthesis inhibition: Effects on memory in mice. Science 1971; 172(982):498-499. 66. Attoub S, Noe V, Pirola L et al. Leptin promotes invasiveness of kidney and colonic epithelial cells via phosphoinositide 3-kinase-, rho- and rac-dependent signaling pathways. FASEB J 2000; 14(14):2329-2338. 67. O’Neil JS, Burow ME, Green AE et al. Effects of estrogen on leptin gene promoter activation in MCF-7 breast cancer and JEG-3 choriocarcinoma cells: selective regulation via estrogen receptors alpha and beta. Mol Cell Endocrinol 2001; 176(1-2):67-75. 68. Naka T, Narazaki M, Hirata M et al. Structure and function of a new STAT-induced STAT inhibitor. Nature 1997; 387(6636):924-929. 69. Wang J, Campbell IL. Cytokine signaling in the brain: Putting a SOCS in it? J Neurosci Res 2002; 67(4):423-427. 70. Zhang T, Lin Y, Han C et al [Molecular cloning of rat OB gene and its expression in Escherichia coli]. Wei Sheng Wu Xue Bao 1999; 39(3):215-219. 71. Ahima RS, Flier JS. Leptin. Annu Rev Physiol 2000; 62:413-437. 72. Pelleymounter MA, Cullen MJ, Baker MB et al. Effects of the obese gene product on body weight regulation in ob/ob mice. Science 1995; 269(5223):540-543. 73. Della-Fera MA, Qian H, Baile CA. Adipocyte apoptosis in the regulation of body fat mass by leptin. Diabetes Obes Metab 2001; 3(5):299-310. 74. Gullicksen PS, Hausman DB, Dean RG et al. Adipose tissue cellularity and apoptosis after intracerebroventricular injections of leptin and 21 days of recovery in rats. Int J Obes Relat Metab Disord 2003; 27(3):302-312. 75. Satoh N, Ogawa Y, Katsuura G et al. Sympathetic activation of leptin via the ventromedial hypothalamus: leptin-induced increase in catecholamine secretion. Diabetes 1999; 48(9):1787-1793. 76. Ambati S, Kim HK, Yang JY et al. Effects of leptin on apoptosis and adipogenesis in 3T3-L1 adipocytes. Biochem Pharmacol 2007; 73(3):378-384. 77. Page KA, Hartzell DL, Li C et al. beta-Adrenergic receptor agonists increase apoptosis of adipose tissue in mice. Domest Anim Endocrinol 2004; 26(1):23-31. 78. Nicholls DG, Locke RM. Thermogenic mechanisms in brown fat. Physiol Rev 1984; 64(1):1-64. 79. Yoshitomi H, Yamazaki K, Abe S et al. Differential regulation of mouse uncoupling proteins among brown adipose tissue, white adipose tissue and skeletal muscle in chronic beta 3 adrenergic receptor agonist treatment. Biochem Biophys Res Commun 1998; 253(1):85-91. 80. Fleury C, Neverova M, Collins S et al. Uncoupling protein-2: A novel gene linked to obesity and hyperinsulinemia. Nat Genet 1997; 15(3):269-272. 81. Negre-Salvayre A, Hirtz C, Carrera G et al. A role for uncoupling protein-2 as a regulator of mitochondrial hydrogen peroxide generation. FASEB J 1997; 11(10):809-815. 82. Yamagishi SI, Edelstein D, Du XL et al. Leptin induces mitochondrial superoxide production and monocyte chemoattractant protein-1 expression in aortic endothelial cells by increasing fatty acid oxidation via protein kinase A. J Biol Chem 2001; 276(27):25096-25100. 83. Caldefie-Chezet F, Poulin A, Tridon A et al. Leptin: A potential regulator of polymorphonuclear neutrophil bactericidal action? J Leukoc Biol 2001; 69(3):414-418. 84. Cohen B, Barkan D, Levy Y et al. Leptin induces angiopoietin-2 expression in adipose tissues. J Biol Chem 2001; 276(11):7697-7700. 85. Maedler K, Sergeev P, Ehses JA et al. Leptin modulates beta cell expression of IL-1 receptor antagonist and release of IL-1beta in human islets. Proc Natl Acad Sci USA 2004; 101(21):8138-8143.

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86. Donath MY, Ehses JA, Maedler K et al. Mechanisms of beta-cell death in type 2 diabetes. Diabetes 2005; 54 (Suppl 2):S108-113. 87. Chawla A, Lazar MA. Peroxisome proliferator and retinoid signaling pathways coregulate preadipocyte phenotype and survival. Proc Natl Acad Sci USA 1994; 91(5):1786-1790. 88. Qian H, Hausman GJ, Compton MM et al. Leptin regulation of peroxisome proliferator-activated receptor-gamma, tumor necrosis factor and uncoupling protein-2 expression in adipose tissues. Biochem Biophys Res Commun 1998; 246(3):660-667. 89. Utsumi H, Iwakiri R, Wu B et al. Intracerebroventricular administration of leptin-induced apoptosis in the rat small intestinal mucosa. Exp Biol Med (Maywood) 2003; 228(10):1239-1244. 90. Lin T, Sakata H, Ootani A et al. Apoptosis in rat jejunal mucosa is regulated partly through the central nervous system, which controls feeding behavior. J Gastroenterol Hepatol 2005; 20(8):1285-1291. 91. Chen C, Chang YC, Liu CL et al. Leptin induces proliferation and anti-apoptosis in human hepatocarcinoma cells by up-regulating cyclin D1 and down-regulating Bax via a Janus kinase 2-linked pathway. Endocr Relat Cancer 2007; 14(2):513-529. 92. Beales IL, Ogunwobi OO. Leptin synergistically enhances the anti-apoptotic and growth-promoting effects of acid in OE33 oesophageal adenocarcinoma cells in culture. Mol Cell Endocrinol 2007; 274(1-2):60-68. 93. Ogunwobi O, Mutungi G, Beales IL. Leptin stimulates proliferation and inhibits apoptosis in Barrett’s esophageal adenocarcinoma cells by cyclooxygenase-2-dependent, prostaglandin-E2-mediated transactivation of the epidermal growth factor receptor and c-Jun NH2-terminal kinase activation. Endocrinology 2006; 147(9):4505-4516. 94. Sirotkin AV, Grossmann R. Leptin directly controls proliferation, apoptosis and secretory activity of cultured chicken ovarian cells. Comp Biochem Physiol A Mol Integr Physiol 2007; 148(2):422-429. 95. Balasubramaniyan V, Shukla R, Murugaiyan G et al. Mouse recombinant leptin protects human hepatoma HepG2 against apoptosis, TNF-alpha response and oxidative stress induced by the hepatotoxin-ethanol. Biochim Biophys Acta 2007; 1770(8):1136-1144. 96. Brown JE, Dunmore SJ. Leptin decreases apoptosis and alters BCL-2: Bax ratio in clonal rodent pancreatic beta-cells. Diabetes Metab Res Rev 2007; 23(6):497-502. 97. Briscoe CP, Hanif S, Arch JR et al. Fatty acids inhibit leptin signalling in BRIN-BD11 insulinoma cells. J Mol Endocrinol 2001; 26(2):145-154. 98. Hoda MR, Keely SJ, Bertelsen LS et al. Leptin acts as a mitogenic and antiapoptotic factor for colonic cancer cells. Br J Surg 2007; 94(3):346-354. 99. Ogunwobi OO, Beales IL. Cyclo-oxygenase-independent inhibition of apoptosis and stimulation of proliferation by leptin in human colon cancer cells. Dig Dis Sci 2007; 52(8):1934-1945. 100. Qamar A, Sheikh SZ, Masud A et al. In vitro and in vivo protection of stellate cells from apoptosis by leptin. Dig Dis Sci 2006; 51(10):1697-1705. 101. Mansour E, Pereira FG, Araujo EP et al. Leptin inhibits apoptosis in thymus through a janus kinase-2-Independent, insulin receptor substrate-1/phosphatidylinositol-3 kinase-dependent pathway. Endocrinology 2006; 147(11):5470-5479. 102. Bruno A, Conus S, Schmid I et al. Apoptotic pathways are inhibited by leptin receptor activation in neutrophils. J Immunol 2005; 174(12):8090-8096. 103. Russo VC, Metaxas S, Kobayashi K et al. Antiapoptotic effects of leptin in human neuroblastoma cells. Endocrinology 2004; 145(9):4103-4112. 104. Somasundar P, Frankenberry KA, Skinner H et al. Prostate cancer cell proliferation is influenced by leptin. J Surg Res 2004; 118(1):71-82.

Chapter 5

Leptin Actions in the Gastrointestinal Tract Sandra Guilmeau, Thomas Aparicio, Robert Ducroc and André Bado*

Introduction

T

he primary physiological role of leptin is to communicate to the central nervous system (CNS) the abundance of available energy stores and to restrain food intake and induce energy expenditure.1-5 Thus, it was expected that the absence of leptin leads to increased appetite and food intake that result in morbid obesity. Notably, only rare cases of severe early childhood obesity have been associated with leptin deficiency and the remainder of the obese population typically has elevated leptin levels. The failure of leptin to induce weight loss in these cases is thought to be the result of leptin resistance. Leptin exerts its biological actions through interaction with leptin receptors Ob-Rs, encoded by the db gene.6,7 These receptors are member of the gp130 family of cytokine receptors and occur in several isoforms.8 Actually, leptin can be considered as a multifunctional hormone that regulates not only body weight homeostasis but also neuroendocrine function, reproduction,9 immune function and nutrients absorption. This pleiotropic action of leptin is consistent with the various reported sites of leptin production. Initially thought to be adipocyte-specific, the ob gene has been found in a variety of other tissues.9 Relevant to this chapter, the leptin gene has been identified in the stomach and its receptors were found distributed throughout the gastrointestinal tract. Recent data suggest that gut leptin may act locally to influence gastrointestinal functions. This chapter will focus on the stomach-derived leptin and its implication in the regulation of some gut functions. Gastric epithelial cells within gastric mucosa have been reported to produce leptin in rodents and humans.10-15 The leptin-secreting cells were identified as pepsinogen-secreting chief cells and endocrine P cells.13,16 Ultrastructural studies further showed that leptin protein is present along the rough endoplasmic reticulum-Golgi-granules secretory pathways both in chief and endocrine cells.12,17 During development,18 leptin production in the stomach starts at the onset of suckling in neonatal rats and it markedly increases at the transition from liquid to solid-food intake.14,19 This observation underlies the physiological importance of stomach-secreted leptin and the potential role that it could play in the control of gut-derived regulation of food intake in neonates. Leptin secretion in the stomach is regulated by the nutritional status, acetylcholine-released by the vagus nerve and intestinal hormones i.e., cholecystokinin, secretin.10,12,20 In addition, gastric leptin shows diurnal variations influenced by food intake rhythms and the changes occurring just before the beginning of the feeding period are opposite to those of the appetite-stimulating peptide ghrelin.21 The stomach serves as a reservoir in which ingested food accumulates and undergoes chemical and enzymatic digestion. Among the several types of secretory epithelial cells located within the gastric mucosa are endocrine cells, mucous cells secreting mucus that protects the epithelium, parietal cells secreting gastric acid and chief cells secreting pepsinogen, a proteolytic enzyme. Despite *Corresponding Author: André Bado—INSERM, U773, Centre de Recherche Biomédicale Bichat Beaujon; UFR de Médecine Paris 7—Denis Diderot; IFR02 Claude Bernard, 16 rue Henri Huchard, BP 416, F-75018, Paris, France. Email: [email protected]

Leptin and Leptin Antagonists, edited by Arieh Gertler. ©2009 Landes Bioscience.

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this nature of gastric juice, gastric leptin can be found intact in gastric and intestinal secretions as free leptin and leptin bound to protein.22 The nature of this protein that confers to leptin its resistance in the gastric juice even at pH2, has been identified by Cammisotto and coworkers.23 This leptin-binding protein was shown to be structurally similar to the extracellular domain of the leptin receptor, Ob-Re.23 Such a finding is consistent with previous data reporting that soluble form of leptin receptor Ob-Re, circulates in plasma and is capable to bind to leptin.24 These soluble leptin receptors occurring in the secretory granules in both gastric exocrine and endocrine cells, are produced by ectodomain shedding of membrane-bound forms of leptin Ob-Ra and Ob-Rb receptors.25 This process was suggested to be ensured by proprotein-convertase (PC), probably PC-7 which has various proteolytic sites on the leptin receptor molecule.23Such a generation of Ob-Re has been previously reported in adipose tissue both in vitro and in vivo25. Thus, leptin secreted in the gastrointestinal lumen can be detected as free leptin and leptin associated with Ob-Re as previously described for plasma leptin. This association is likely to protect leptin from degradation. Because no posttranslational modifications of leptin occurs in vivo, soluble OB-Re can be an important factor regulating leptin’s bioavailability and its interactions with membrane spanned leptin receptors.

Gastric Leptin Directly Activates Vagal Afferent Neurons

The first question rose by the discovery of gastric leptin and is secretion in the lumen, was whether it can act locally to generate signals that contribute to meal-induced termination of food intake. One main function of the vagus nerve is to convey primary afferent informations from the gastro-intestinal mucosa to the brain stem. Afferents vagal nerves terminating near to the mucosa are in a position to monitor the composition of the luminal contents. Interestingly, leptin receptors Ob-Rb, Ob-Ra colocalized with STAT3 signalling proteins have been described nodose ganglion in the rat and human.26-28 Moreover, these leptin receptors also occurred in the intestinal vagal mechanoreceptors and neurons of the enteric nervous systems.29,30 The vagal leptin receptors are responsive to gastric lumen as evidenced by the increased in tonic activity of gastric-related neurons in the nucleus solitarius after oral administration of leptin.31,32 These data are in straight line with electrophysiological data identifying two types of leptin-responsive afferent fibers with one type requiring low doses of CCK to be activated by leptin.33,34 Moreover, single cell calcium imaging and patch clamp electrophysiological studies using vagal afferent neurones in culture,35,36 demonstrated that these above effects result from direct interaction of leptin with membrane-bound leptin receptors on the vagus nerve. Indeed, application of leptin evokes cytosolic calcium in about 25% of the neurons and acutely depolarised the cell membranes of a subpopuation vagal afferent neurones.37 Some of these neurons expressing leptin receptors (OB-Rs) colocalize with CCK receptors and, are also responsive to mechanical distension suggesting that they are likely to facilitate leptin mediation of short-term satiety. Taken together, these data indicate that the vagus nerve is the primary target of leptin produced by the stomach and secreted into the gastric juice. This neurocrine action of leptin on vagal afferent neurons may provide basis to explain the earlier reported leptin-CCK potentiating effect in the firing frequency of vagal terminals, in neuronal activity in the NTS, on food intake and, on body weight.

Leptin and Intestinal Physiology

In addition to the presence of leptin in intestinal lumen, leptin receptors were found at the apical side of the enterocytes all along the small and large intestine, which argues for the viewpoint that leptin, acting from the luminal side, affects biological functions of the intestinal epithelium.

Enteroendocrine Cell Secretions Are Regulated by Leptin

Earlier studies have demonstrated that CCK-1 receptor antagonists can prevent the inhibition of food intake and stimulation of pancreatic exocrine secretions induced by peripheral leptin, suggesting that endogenously released CCK is involved.38,39 Thus, in vivo in the rat, direct delivery of leptin in the duodenum, leads to an increase in plasma CCK at levels comparable to those

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induced by feeding.22,40 This action of duodenal leptin was subsequently showed to result from direct activation of leptin receptors on duodenal CCK-producing endocrine I cells and to involve MAPKinase signalling pathways. This ability of leptin to induce release of CCK leptin generates a positive feedback loop because CCK is reported to stimulate the release of gastric leptin.40 CCK is one of the meal-generated molecules that initiate satiety signals which are conveyed by the viscero-sensitive vagal afferent neurons to the nucleus of the NTS and then in the hypothalamus. In this way, the leptin-stimulation of duodenal CCK secretion may suggest that, under physiological conditions, both peptides may potentiate their own actions by cross-stimulating their secretions. This is in accordance with the reported dampening of CCK or leptin inhibitory action on food intake when either peptide is absent or their receptors are functionally inactive.22,41 Leptin was also reported to stimulate the secretion of glucagon-like peptide 1 (GLP-l) in vitro in endocrine cells in culture and in vivo in rodents.42 GLP-1 is a hormone secreted from endocrine L cells, which are localized in the distal ileum and colon, after nutrient ingestion. GLP-1 acts through a specific G-protein-coupled receptor to potently stimulate glucose-dependent insulin secretion. It also inhibits food intake and reduces body weight after long-term administration. Interestingly, several studies have demonstrated that circulating GLP-1 levels are reduced in obese individuals, either with or without concomitant type 2 diabetes and this impairment can be partially reversed by weight loss.43 That leptin stimulates GLP-1 secretion from the endocrine L cells provides evidence for the existence of an adipocyte-enteroendocrine axis in the regulation of nutrient homeostasis.

Leptin and Intestinal Absorption of Nutrients

The small intestinal lining is composed of functionally and morphologically polarized enterocytes that play a central role in the absorption of nutrients after digestion. In the intestine, the chyme undergoes hydrolysis by proteolytic enzymes from pancreatic, bile and intestinal juices, pursuing the primo digestion started in the stomach. Nutrients thus degraded into smaller molecules cross the intestinal brush-border by active transport, passive diffusion, or facilitated processes. The arrival of the meal in the intestine stimulates the release of gastrointestinal hormones that control the absorption of nutrients and are also signals for induction of postprandial satiety. Thus, gut leptin rapidly secreted in the lumen after a meal, may represent a key molecule controlling the intestinal absorption of nutrients.

Leptin Regulates the H+-Coupled Peptide Cotransporter PepT-1

Under physiological conditions, dietary proteins are degraded in a series of steps by hydrolytic enzymes originating from the stomach, pancreas and small intestine. This results in a mixture of free amino acids and small peptides that is efficiently absorbed by enterocytes. These small peptides are cleared from the intestinal lumen by the brush-border transporter PepT-l (SLC15A1) which cotransports di- and tri-peptides peptides with protons.44,45 Luminal, but not basolateral, leptin increases the absorption of dipeptides through PepT-l in the enterocyte1ike Caco2 cells in vitro.46 These results were confirmed in vivo in rats in which direct administration of leptin in the jejunum (mimicking gastric leptin) rapidly increases absorption of di-peptides. The mechanism of this short-term action involves increased recruitment of membrane PepT-l molecules from an intracellular preformed pool to the apical membrane.46 Leptin has also a long-term effect consisting in activation of the transcription of PepT1 gene and/or enhanced of PepT1 mRNA stability to reconstitute cytoplasmic pool of PepT1 transporter. It has been also reported that induction of hyperleptinemia in non-obese animals up-regulates PepT1 transporter activity and expression.47 In addition leptin-deficient ob/ob mice exhibited a dramatic decrease in PepT1 activity and expression and replacement of leptin in these mice restores PepT1 expression and activity.47 These data indicate that leptin is key controller of oligopeptides PepT-1 transporter. From a physiological point of view, the facilitation of protein absorption through PepT-1 activation by gut leptin is consistent with reports of a satiety effect of dietary proteins and is in line with the aminostatic hypothesis.

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Leptin and Intestinal Absorption of Fats

A major function of intestinal cells is absorbing large amount of dietary lipids. After a digestive phase, the free fatty-acid (FA) lipolytic products are absorbed by the enterocytes, in which sequential events result in their packaging as chylomicrons. The formation and secretion of these intestinal lipoproteins are key steps in the transport of dietary fats. The assembly of triglycerides (TG)-rich lipoproteins within the enterocytes involves multiple pathways including (i) the uptake of FFAs by several specific carriers, such as fatty-acid transporter (FAT) and its human homolog CD36; (ii) their translocation from the brush-border membrane to the endoplasmic reticulum by intestinal and liver fatty-acid binding proteins (I- and L-FABPs); and (iii) their esterification in TG and subsequent assembly with apolipoprotein (apoB, apoA-IV) to form lipoprotein particles. Leptin appears to play a role in the regulation of the synthesis of apolipoproteins. In fact, leptin administrated to fat-loaded ob/ob mice induced STAT5 DNA binding and reduced apolipoprotein transcript leve1s in the mice jejunum.48 Leptin was also involved in the regulation of circulating apo-AIV by suppressing apoAIV synthesis in the small intestine. It remains unknown, however, if this function is assumed by leptin trafficking in the lumen of the intestine.49 In the enterocyte-like Caco2 cells, leptin was reported to reduce the output of de novo-synthesized apolipoprotein ApoB-100 and ApoB-48, as well as that of newly formed chylomicrons and of low-density lipoproteins, supporting a role for leptin in the reduction of intestinal TG secretion into the circulation. Moreover, I-FABP expression was decreased by leptin in Caco2 cells.50 These effects of leptin on FA uptake and assembly in the enterocyte are likely to be involved in the regulation of energy homeostasis.

Leptin Regulates Intestinal Absorption of Sugars

Dietary carbohydrates are digested in the intestine through the action of amylase and intestinal brush-border membrane disaccharidases into monosaccharides, D-glucose, D-galactose and D-fructose. Sodium-dependent glucose transporter 1 (SGLT-l) is the specific transporter for D-glucose and D-galactose, whereas D-fructose is transported into the enterocytes by GLUT-5. Once in the enterocytes, the monosaccharides exit the cell across the basolateral membrane transporter GLUT-2. In the jejunum, it was shown that leptin can inhibit the active absorption of galactose mediated by the Na+ glucose cotransporter SGLT-l without affecting the passive component of the absorption. In particular luminal addition of leptin on rat jejunum, isolated in Ussing chambers, rapidly and dramatically decreased active glucose transport.51 This rapid inhibition of glucose entry into the enterocyte by luminal leptin involves a reduced recruitment of SGLT-l from an intracellular preformed pool to the apical membranes. The effects are dependent of leptin-receptor-coupled activation of protein kinase C.51,52 The inhibition of active glucose transport by serosal leptin was slower and probably mediated by endogenously-released CCK. On the other hand, systemic leptin administration to rats after massive small bowel resection was shown to increase the amounts of GLUT-5 protein with no change in the levels of SGLT-153 indicating that leptin may have therapeutic effect in the small bowel syndrome. Because the small intestine is now recognized as an insulin-sensitive and gluconeogenic organ,54,55 a better understanding of the mechanisms by which leptin affects the intestinal absorption of monosaccharides may have physiological relevance in the management of diabetes, in particular non-insulin-dependent diabetes mellitus (NIDDM).

Leptin Controls Colonic Absorption of Short-Chain Fatty Acid

Butyrate, a short-chain fatty acid (SCFA), is one of the products of the microbial digestion of carbohyydrates and dietary fibers in the large bowel, which represents a dominant energy source for the colonocytes. Gut leptin is involved in the regulation of butyrate uptake by the intestinal epithelial cells through the proton-linked monocarboxylate transporter type 1 (MCT-l).56,57 Indeed, in the human intestinal Caco2 cells, luminal leptin increases butyrate uptake by increasing MCT-l mRNA levels and the amounts of MCT-l protein on the apical membrane.58 Such a control of MCT-l activity by leptin, which affects the availability of SCFA in the mucosa, probably modulates the intracellular events regulating normal differentiation

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and proliferation in the colonic mucosa. Along this line, it has been reported that in vitro leptin can protect cancer HT-29 cells from butyrate-induced apoptosis,59 suggesting potential implications for the diseased colon. This mitogenic and anti-apoptotic effect of leptin on intestinal epithelial cells were further showed to involve both the NF-kappaB and ERK-1/2 pathways. More investigations are needed to establish the relevance of these current results in the pathophysiology of colon cancer.

Leptin in Gastrointestinal Pathologies Stomach

Leptin is involved in maintaining gastric epithelial cell integrity and gastroprotection. In rats, systemic leptin was found to be effective in attenuating both ethanol- and aspirin-induced damage to the gastric mucosa. This gastric cytoprotective effect of leptin involved an increase in blood flow, local production of nitric oxide and prostaglandin E2 (PGE2) and vagus nerve-dependent mechanisms.60 In humans, it has been observed that gastric inflammation induced after Helicobacter pylori infection raises gastric leptin expression. Cure of H. pylori infection reduced gastric leptin expression, with a concomitant increase in body mass index.61-64 Since there is a positive relationship between H. pylori and gastric cancer, leptin could contribute to mucosal homeostasis and to abnormal proliferation in gastric cancer. Yet, this still remains to be demonstrated.

Colon Cancers

Epidemiology studies associates elevated leptin levels with colon cancer in men but not women.65 The mechanisms for this association have not been fully demonstrated.66-69 Leptin and Ob-Rb expression have been found in several human colon cancer cell lines and human colon adenomas and adenocarcinomas.65,70-72 Several lines of evidence suggest that leptin may be involved in carcinogenesis. First, leptin promotes the proliferation of some cancer cell lines, notably the human colon cancer cell line HT-29.73,59 Second, leptin is able to induce angiogenesis through interaction with Ob-R expressed on the surface of endothelial cells.74 Third, in vitro, leptin promotes invasiveness of colon cancer cells in collagen gel75 Fourth, leptin increases the secretion of metalloproteinases, key enzymes for tumoral invasion, by cultured cells.76 Although there is consensual data about the in vitro ability of leptin to stimulate growth of human colon cancer cells, the in vivo effect of systemic leptin on normal colon epithelial cell growth remains controversial. Thus, leptin has been shown to enhance the development of adenomatous lesions in genetically predisposed mice. In contrast, leptin did not increase cell proliferation in vivo in mice77 and did not promote the growth of colon cancer xenografts in mice.78 Microarray analysis reveals that leptin induces autocrine/paracrine cascades to promote survival and proliferation of colon epithelial cells in an Apc genotype-dependent fashion.79 Importantly, leptin induced IGF-mediated pathway gene expression changes and their protein products in colon epithelial cells possessing an Apc mutation (IMCE). Thus it was shown that leptin up-regulates IGFBP-6, IGF-1 and Crim1, a putative transmembrane protein with an IGF-binding protein motif and down-regulates IGF binding protein IGFBP-2,-3,-4,-5 and Nov expression.78 These data are strongly suggestive for a link between the elevated levels of growth factors and the increased risk of colon cancer associated with obesity. On the other hand, the finding that leptin levels were lower in colon cancer patients despite similar body mass index to the control subjects suggests that other mechanisms may be involved. Further studies are required to more precisely determine the involvement of leptin in colon cancers.

Intestinal Inflammation

One of the emerging roles of leptin is its role in the regulation of inflammatory processes. There is, indeed, a large body of evidence that leptin-mediated signal pathways play an active role in innate and adaptative immunity through the alteration of various target genes transcription.80,81 Interestingly, leptin-deficient ob/ob mice are reported to be resistant to experimental induction of colonic inflammation and the replacement of leptin converted their resistance to disease into

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susceptibility.82,83 This resistance to intestinal inflammation was further demonstrated to be associated with reduced cytokines secretion, increased apoptosis of lamina propria lymphocytes (LPL) and to largely involve T-cell mediated intestinal autoimmunity.83,84 On the other hand, human inflamed colonic cells have been shown to exhibit a strong leptin immunoreactivity that is concentrated at subapical part of the colonic cells, whereas normal colonic epithelial cells do not show this.85 This supports the idea that fat- and gut-derived leptin may be a key component in the control of intestinal inflammation processes. Whether these up-regulation of leptin upon tissue injury and particularly, whether the increased colon leptin is causative in inflammatory responses or simply a marker of inflammation, has not been formally proven and remain to be further investigated. These results make leptin, a hormone and a cytokine, a good candidate to link neuroendocrine and immune systems to metabolic status.86

Conclusions and Perspectives

Leptin is an important regulator of food intake and energy expenditure by actions on receptors initially thought to be located in regions of hypothalamus that regulate feeding behaviour. There is now growing evidence that leptin acting peripherally could contribute to control the energy homeostasis through regulation of gastrointestinal hormones that control short-term feeding and intestinal absorption of nutrients. A better understanding of the role of leptin in the physiology of gastrointestinal functions will provide a basis for the determination of its relevance in several diseases states such as obesity, diabetes and its link with inflammation and cancer of the gastrointestinal tract.

Acknowledgements

The authors’ work was supported by the Institut National de la Santé et de la Recherche Médicale (INSERM) and by Fondation pour la Recherche Médicale (FRM).

References

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47. Hindlet P, Bado A, Farinotti R et al. Long-term effect of leptin on H+-coupled peptide cotransporter 1 activity and expression in vivo: evidence in leptin-deficient mice. J Pharmacol Exp Ther 2007; 323:192-201. 48. Morton NM, Emilsson V, Liu YL et al. Leptin action in intestinal cells. J Biol Chem 1998; 273:26194-201. 49. Doi T, Liu M, Seeley RJ et al. Effect of leptin on intestinal apolipoprotein AIV in response to lipid feeding. Am J Physiol Regul Integr Comp Physiol 2001; 281:R753-759. 50. Dube N, Delvin E, Yotov W et al. Modulation of intestinal and liver fatty acid-binding proteins in Caco-2 cells by lipids, hormones and cytokines. J Cell Biochem 2001; 81:613-620. 51. Ducroc R, Guilmeau S, Akasbi K et al. Luminal leptin induces rapid inhibition of active intestinal absorption of glucose mediated by sodium-glucose cotransporter 1. Diabetes 2005; 54:348-354. 52. Lostao MP, Urdaneta E, Martinez-Anso E et al. Presence of leptin receptors in rat small intestine and leptin effect on sugar absorption. FEBS Lett 1998; 423:302-306. 53. Pearson PY, O’Connor DM, Schwartz MZ. Novel effect of leptin on small intestine adaptation. J Surg Res 2001; 97:192-195. 54. Mithieux G. The new functions of the gut in the control of glucose homeostasis. Curr Opin Clin Nutr Metab Care 2005; 8:445-449. 55. Mithieux G, Gautier-Stein A, Rajas F et al. Contribution of intestine and kidney to glucose fluxes in different nutritional states in rat. Comp Biochem Physiol B Biochem Mol Biol 2006; 143:195-200. 56. Halestrap AP, Price NT. The proton-linked monocarboxylate transporter (MCT) family: structure, function and regulation. Biochem J 1999; 343(Pt 2):281-299. 57. Jackson VN, Price NT, Halestrap AP. cDNA cloning of MCT1, a monocarboxylate transporter from rat skeletal muscle. Biochim Biophys Acta 1995; 1238:193-196. 58. Buyse M, Sitaraman SV, Liu X et al. Luminal leptin enhances CD147/MCT-1-mediated uptake of butyrate in the human intestinal cell line Caco2-BBE. J Biol Chem 2002; 277:28182-28190. 59. Rouet-Benzineb P, Aparicio T, Guilmeau S et al. Leptin counteracts sodium butyrate-induced apoptosis in human colon cancer HT-29 cells via NF-kappaB signaling. J Biol Chem 2004; 279:16495-16502. 60. Konturek PC, Brzozowski T, Sulekova Z et al. Enhanced expression of leptin following acute gastric injury in rat. J Physiol Pharmacol 1999; 50:587-595. 61. Nishi Y, Isomoto H, Uotani S et al. Enhanced production of leptin in gastric fundic mucosa with Helicobacter pylori infection. World J Gastroenterol 2005; 11:695-699. 62. Konturek JW, Konturek SJ, Kwiecien N et al. Leptin in the control of gastric secretion and gut hormones in humans infected with Helicobacter pylori. Scand J Gastroenterol 2001; 36:1148-1154. 63. Francois F, Roper J, Goodman AJ et al. The association of gastric leptin with oesophageal inflammation and metaplasia. Gut 2008; 57:16-24. 64. Konturek PC, Czesnikiewicz-Guzik M, Bielanski W et al. Involvement of Helicobacter pylori infection in neuro-hormonal control of food intake. J Physiol Pharmacol 2006; 57(Suppl 5):67-81. 65. Pischon T, Nothlings U, Boeing H. Obesity and cancer. Proc Nutr Soc 2008; 67:128-145. 66. Frezza EE, Wachtel MS, Chiriva-Internati M. Influence of obesity on the risk of developing colon cancer. Gut 2006; 55:285-291. 67. Yakar S, Pennisi P, Kim CH et al. Studies involving the GH-IGF axis: Lessons from IGF-I and IGF-I receptor gene targeting mouse models. J Endocrinol Invest 2005; 28:19-22. 68. McTiernan A. Obesity and cancer: the risks, science and potential management strategies. Oncology (Williston Park) 2005; 19:871-81; discussion 881-882, 885-886. 69. Bray GA. The underlying basis for obesity: relationship to cancer. J Nutr 2002; 132:3451S-3455S. 70. Mix H, Widjaja A, Jandl O et al. Expression of leptin and leptin receptor isoforms in the human stomach. Gut 2000; 47:481-486. 71. Slattery ML, Wolff RK, Herrick J et al. Leptin and leptin receptor genotypes and colon cancer: gene-gene and gene-lifestyle interactions. Int J Cancer 2008; 122:1611-1617. 72. Xiao R, Hennings LJ, Badger TM et al. Fetal programming of colon cancer in adult rats: correlations with altered neonatal growth trajectory, circulating IGF-I and IGF binding proteins and testosterone. J Endocrinol 2007; 195:79-87. 73. Hardwick JC, Van Den Brink GR, Offerhaus GJ et al. Leptin is a growth factor for colonic epithelial cells. Gastroenterology 2001; 121:79-90. 74. Rodrigues S, Van Aken E, Van Bocxlaer S et al. Trefoil peptides as proangiogenic factors in vivo and in vitro: implication of cyclooxygenase-2 and EGF receptor signaling. FASEB J 2003; 17:7-16. 75. Attoub S, Noe V, Pirola L et al. Leptin promotes invasiveness of kidney and colonic epithelial cells via phosphoinositide 3-kinase-, rho- and rac-dependent signaling pathways. FASEB J 2000; 14:2329-2338.

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76. Lin S, Saxena NK, Ding X et al. Leptin increases tissue inhibitor of metalloproteinase I (TIMP-1) gene expression by a specificity protein 1/signal transducer and activator of transcription 3 mechanism. Mol Endocrinol 2006; 20:3376-3388. 77. Aparicio T, Guilmeau S, Goiot H et al. Leptin reduces the development of the initial precancerous lesions induced by azoxymethane in the rat colonic mucosa. Gastroenterology 2004; 126:499-510. 78. Fenton JI, Lavigne JA, Perkins SN et al. Microarray analysis reveals that leptin induces autocrine/ paracrine cascades to promote survival and proliferation of colon epithelial cells in an Apc genotype-dependent fashion. Mol Carcinog 2008; 47:9-21. 79. Fenton JI, Hursting SD, Perkins SN et al. Leptin induces an Apc genotype-associated colon epithelial cell chemokine production pattern associated with macrophage chemotaxis and activation. Carcinogenesis 2007; 28:455-64. 80. Otero M, Lago R, Lago F et al. Leptin, from fat to inflammation: old questions and new insights. FEBS Lett 2005; 579:295-301. 81. Fantuzzi G, Sennello JA, Batra A et al. Defining the role of T-cell-derived leptin in the modulation of hepatic or intestinal inflammation in mice. Clin Exp Immunol 2005; 142:31-8. 82. Siegmund B, Lehr HA, Fantuzzi G. Leptin: a pivotal mediator of intestinal inflammation in mice. Gastroenterology 2002; 122:2011-25. 83. Siegmund B, Sennello JA, Jones-Carson J et al. Leptin receptor expression on T-lymphocytes modulates chronic intestinal inflammation in mice. Gut 2004; 53:965-72. 84. Siegmund B, Sennello JA, Lehr HA et al. Development of intestinal inflammation in double IL-10- and leptin-deficient mice. J Leukoc Biol 2004; 76:782-6. 85. Sitaraman S, Liu X, Charrier L et al. Colonic leptin: source of a novel proinflammatory cytokine involved in IBD. FASEB J 2004; 18:696-8. 86. Karagiannides I, Pothoulakis C. Obesity, innate immunity and gut inflammation. Curr Opin Gastroenterol 2007; 23:661-6.

Chapter 6

Leptin as a Novel Marker in Breast and Colorectal Cancer Eva Surmacz* and Mariusz Koda

Abstract

besity, defined as Body Mass Index (BMI) ≥30, constitutes a known risk factor for the development of different neoplasms, including such common diseases as postmenopausal breast cancer and colorectal cancer.1 According to the National Cancer Institute significant excess of body weight increases the risk of postmenopausal breast cancer by 30-50%, while the risk of colorectal cancer is elevated by 50-100% in man and 20-50% in women. The exact mechanism of the obesity-cancer link is not clear, but ongoing research points to the important role of different biologically active substances produced by the adipose tissue. Among them, mitogenic growth factors, steroid hormones, fatty acids and interleukins stand out as chief culprits.1-10 The impact of mitogens such as insulin-like growth factor (IGF-1), or steroids, such as estrogens, in epithelial oncogenesis has been well documented.1,10-14 However, the function of leptin, the principal cytokine produced by fat cells and directly associated with adiposity and BMI,4,15 is still quite obscure.8 In this chapter, we review new evidence from our and other laboratories describing mechanisms of leptin-induced neoplasia in mammary and colorectal tissues. We also address the possibility that leptin and its receptor (LR) may become new biological markers and attractive pharmaceutical targets in breast and colorectal cancer.

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Leptin and Breast Cancer

In the normal breast, leptin regulates mammary gland development and lactation.16-18 These leptin functions seem to involve increased proliferation of normal breast epithelial cells and be mediated by specific signaling pathways, such as the JAK2/STAT3 and ERK1/2 pathways and activation of AP-1-mediated gene transcription.16 However, as demonstrated by a seminal work of Hu et al,16 leptin exerts much greater proliferative effects in breast cancer cells than in normal mammary epithelial cells. Moreover, leptin can stimulate anchorage-independent growth of cancer, but not normal mammary epithelial cells.16 The effects of leptin in breast cancer cells are mediated by the long form of LR whose expression (together with other shorter LR isoforms) has been demonstrated in several breast cancer cell lines.16,19-23 The activation of breast cancer cell growth in response to medium-high physiological doses of leptin (50-500 ng/mL) has been well documented.4,16,19-23 Leptin-dependent cell proliferation was normally preceded by the activation of downstream LR signaling, i.e., the induction of JAK2/ STAT3, ERK1/2, PI-3K/GSK3, PKCα pathways.16,19,20,22,24-28 In different breast cancer cell lines, leptin-dependent cell growth was associated with upregulation of positive cell cycle regulators, such as c-Myc and cyclin D1 and downregulation of negative growth regulators, such as pRb, *Corresponding Author: Eva Surmacz—Sbarro Institute for Cancer Research and Molecular Medicine, Temple University, Philadelphia, PA 19122, USA. Email: [email protected]

Leptin and Leptin Antagonists, edited by Arieh Gertler. ©2009 Landes Bioscience.

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p53 and p21.WAF1/CIP1 22,26,29,30 Detailed studies revealed that in MCF-7 cells, stimulation of cyclin D1 expression by leptin was mediated by STAT3 binding to specific sequences in the cyclin D1 promoter. This STAT3 activity required the presence of transcriptional coactivator SRC1 (a histone acetyltransferase) and a mediator complex Med1 and was associated with changes in histone acetylation and methylation.30 In addition to these classic growth factor effects, leptin has also been shown to increase the expression of peroxisome proliferator-activated receptors α (PPARα) and ψ (PPARψ) in breast cancer cells.28 In the context of breast cancer therapy and drug resistance, it is important to note that leptin can exert anti-apoptotic effects through activation of various biological systems. In a recent study, leptin treatment of MCF-7 cells induced expression of survivin, a member of the inhibitor of apoptosis protein family, through STAT3-dependent transcription.23 Other reports suggested that in several breast cancer cell lines, leptin can upregulate the classic survival pathways of IGF-1/IGF-1 receptor axis and insulin.31 Leptin has also been shown to transactivate Human Epidermal Growth Factor Receptor 2 (HER2) through JAK2 and HER1-depenedent mechanisms.27 Several reports also point to the fact that leptin induces the expression of Vascular Endothelial Growth Factor (VEGF), a strong promoter of neoangiogenesis and tumor metastasis.31,32 Most interestingly, the leptin system appears to crosstalk with Estrogen Receptor α (ER). First, the levels of the long form of LR appear to be expressed at higher levels in ER-positive cells than in ER-negative cells.22,33 In addition, leptin can upregulate aromatase expression in stromal cells isolated from breast adipose tissue34 as well as in MCF-7 breast cancer cells,24 ultimately leading to increased local estrogen synthesis. As shown by our own analysis, in the presence of pure antiestrogens, leptin prevents ER degradation, enhances ER stability and increases ER transcriptional potential.22 Thus, to conclude this section, overabundance of leptin might interfere with various breast cancer therapeutic strategies, including cytotoxic compounds, antiestrogens as well as HER2 and VEGF target drugs (trastuzumab and bevacizumab) (Fig. 1). Tumorigenic potential of leptin, initially noted in an anchorage-independent growth model in vitro,16 was validated using a xenograft in vivo model. In this case, leptin upregulated the expression of E-cadherin and increased cell-cell adhesion in MCF-7 cells, which was associated with enhanced MCF-7 xenograft growth in nude mice.35 The requirement for the leptin system in mammary tumorigenesis was further confirmed by the fact that genetically obese Lepob/Lepob or Leprdb/Leprdb mice with leptin or LR deficiency exhibited decreased development of mammary tumors.16,36 The source of leptin influencing breast cancer cells is being debated. Our recent data clearly demonstrated that leptin can be produced by breast cancer cells in response to various obesity-related stimuli, for instance hypoxia, insulin, IGF-1 and estrogen37 (Fig. 1). In MCF-7 cells, leptin transcription and synthesis in response to hypoxia and insulin is regulated, at least in part, by Hypoxia Inducible Factor (HIF) binding to Hypoxia Response Elements within the leptin promoter.38 Leptin synthesis and secretion by breast cancer cells implicates that leptin can promote neoplastic processes in the breast via autocrine mechanisms.38 The paracrine leptin, produced by adipocytes in mammary gland could also play a role in cancer progression, as mature adipocytes can enhance growth of human breast cancer cell lines.39 The leptin autocrine/paracrine axes may indeed be operative in the context of breast cancer, as both leptin and LR have been found expressed in breast cancer tissues (primary tumors and lymph node metastases).37,40,41 Importantly, the expression of leptin and LR was significantly elevated in cancer versus noncancer mammary tissue and both markers were highly correlated with each other.37,40 Additionally, in intraductal proliferative lesions bordering on breast cancer, leptin expression was higher relative to proliferative lesions without accompanying breast cancer, which again might imply local autocrine leptin action in cancer development.37 The potential for auto- and/or paracrine leptin involvement in breast cancer progression was confirmed by Miyoshi et al who observed shorter relapse-free survival (RFS) in patients characterized by high LR mRNA levels in breast cancer tissues and by high intratumoral and/or serum leptin levels.42

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Figure 1. Potential mechanisms of leptin action in breast and colorectal cancer. Local leptin can be produced by cancer cells or by tumor adipocytes and affect tumors via autocrine or paracrine mechanisms, respectively. Circulating leptin, derived from distant fat tissue depots, could affect tumors in an endocrine fashion.

The relevance of circulating leptin in breast cancer or breast cancer risk is not clear. The available data from limited epidemiological studies are quite conflicting (Table 1). Several authors described increased serum or plasma leptin levels in women with breast cancer. In studies of Tessitore et al higher plasma leptin levels correlated with disease stage and elevated hormonal status (determined by enhanced plasma concentrations of progesterone and estradiol as well as increased expression of ER and progesterone receptors (PgR) in breast cancer tissue).43 In contrast, Chen et al did not find associations between serum leptin levels in breast cancer and ER, PgR, HER2, lymph node metastasis, tumor stage or tumor grade.44 Ozet et al also described higher serum leptin levels in patients with breast cancer, however, leptin did not correlate with disease stage or BMI.45 In two other reports, higher leptin levels in patients with breast cancer correlated with BMI.44,46 In the study of Liu et al, serum leptin was elevated in patients with breast cancer, but the association was not statistically significant.47 Goodwin et al found relationships between relatively high concentrations of plasma leptin in patients with breast cancer and higher tumor stage, grade and negative steroid hormone receptor status.48 On the other hand, they did not observe associations between plasma leptin and prognosis of patients. Interestingly, Miyoshi et al noted correlations between serum leptin in breast cancer patients and intratumoral leptin mRNA expression.42 Some authors reported no correlations or inverse correlations between leptin and breast cancer. Petridou et al observed significantly decreased leptin levels in the group of premenopausal women with breast, compared with controls, but no differences were noted in postmenopausal women.49 In another study, serum leptin in premenopausal women with breast carcinoma in situ were also decreased in comparison with controls, but the differences were not statistically significant.50 In other reports, no relationship between leptin levels and breast cancer incidence

66

Table 1. Reports on significance of leptin and LR in breast cancer Results, Conclusions

Leptin, LR Measurements

References 44

Serum leptin levels significantly elevated in breast cancer patients (especially in those using tamoxifen) vs controls

Leptin 27.0 vs 17.65 ng/mL

45

Higher serum leptin levels in breast cancer patients vs healthy controls

13.57 ± 0.66 vs 9.46 ± 0.60 µg/L

46

Higher, but not significantly, serum leptin concentration in breast cancer patients vs controls; significant increase in serum leptin levels in high-grade cancers

Leptin 10.43 ± 7.55 vs 8.13 ± 3.16 ng/mL; G2 vs G3: 9.03 ± 5.65 vs 14.99 ± 10.96 ng/mL

47

Higher plasma leptin levels correlated with disease stage and hormonal status of breast cancer

Not known

43

No differences between leptin levels in breast cancer patients and controls; in the group of patients with visceral metastases, leptin level was increased, but not statistically

Leptin 38.1 ± 19.5 ng/mL vs 35.6 ± 13.9 ng/mL; 44.0 ± 16.8 ng/mL in visceral metastases

52

No relationships between serum leptin levels and pre or postmenopausal breast cancer

7.8-27.1 ng/mL

51

No significant differences between mean leptin concentrations in the patient and control groups in pre and postmenopausal women

Leptin 10.98 ± 6.50 vs 7.79 ± 3.83 ng/mL (premenopausal); 18.29 ± 17.93 vs 12.59 ± 8.59 ng/ mL (postmenopausal)

53

No differences between prediagnostic plasma leptin levels in postmenopausal breast cancer patients vs controls

Leptin 16.7 vs 17.1 ng/mL

54

Decreased leptin levels in premenopausal breast cancer patients vs controls; no differences in postmenopausal women

Leptin 14.7 ± 2.0 ng/mL vs 23.9 ± 4.1 ng/mL (premenopausal); 25.6 ± 2.1 ng/mL vs 24.6 ± 2.8 ng/mL (postmenopausal)

49

continued on next page

Leptin and Leptin Antagonists

Serum leptin levels significantly increased in cancer cases vs controls; no correlations between Leptin 13.64 ± 1.18 vs 10.07 ± 0.55 ng/mL serum leptin in cancer cases and clinicopathological parameters

Results, Conclusions

Leptin, LR Measurements

References

Decreased serum leptin levels in premenopausal women with breast carcinoma in situ vs controls

Leptin 13.69 ± 1.3 ng/mL vs 16.03 ± 1.7 ng/mL

50

Association between plasma leptin in patients with breast cancer and higher tumor stage, grade and negative steroid hormone receptor status, but no correlation between plasma leptin and overall survival or distant disease-free survival of patients

Leptin in breast cancer patients 15.2 ± 10.1 ng/ mL

48

Correlation between serum leptin in breast cancer patients and intratumoral leptin mRNA expression; high serum leptin level and increased LR expression in tumor tissues correlated with poor prognosis

Leptin in serum and LR mRNA in breast cancer tissue

42

Overexpression of leptin and LR in breast cancer vs normal mammary gland; relationship between LR overexpression and distant metastases and high risk for tumor recurrence

Leptin and LR protein expression in breast cancer and normal mammary gland

40

Overexpression of leptin and LR in primary and metastatic breast cancers vs noncancerous tissues; elevated leptin and LR expression in poorly differentiated tumors

Leptin and LR protein expression in breast cancer and noncancerous breast tissues

37

Expression of long and short isoforms of LR mRNA in majority of breast cancer cases; leptin and LR long form mRNA had no prognostic value; elevated expression of LR short form mRNA correlated with longer relapse-free survival (RFS); ratio of long to short isoform of LR mRNA was associated with a shorter RFS; overall survival was not associated with leptin and LRmRNA expression

Leptin and LR mRNA found commonly expressed in cancer

41

Leptin as a Novel Marker in Breast and Colorectal Cancer

Table 1. Continued

67

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both in pre and postmenopausal women was detected,51-53 even though in the group of patients with visceral metastases, leptin levels were elevated.52 In a prospective study by Stattin et al, where leptin abundance was assessed in prediagnostic plasma from postmenopausal women who were next diagnosed with breast cancer, no significant relationships between leptin plasma levels and breast cancer risk was observed.54 In essence, many studies noted increased circulating leptin levels in breast cancer patients compared with controls, but the association with tumor markers or prognosis has not been established. It is quite clear that more focused and better-controlled studies are necessary to prove or disapprove the role systemic leptin in breast cancer development.

Leptin and Colorectal Cancer

Leptin produced by the adipose tissue is released into the blood and digestive tract lumine and may regulate physiological processes in normal colon as well as participate in colon carcinogenesis. For instance, leptin can enhance defense mechanisms in the large bowel by stimulation of mucus secretion by goblet cells. In this case, leptin activity was mediated by the PKC and PI-3K pathways.55 Additionally, some data suggested that leptin may promote proliferation, migration and renewal of normal intestinal epithelial cells along the crypt-villus axis and during the reparation of the transiently wounded, inflamed colonic mucosa.56 Apart from leptin role in colorectal tissue physiology, there is accumulating evidence that the hormone can be involved in colorectal cancer. Recent in vitro studies suggested that colorectal cancer cells express LR and respond to leptin treatment with LR phosphorylation, activation downstream intracellular signaling pathways, such as MAPK, JAK2, PI-3K, JNK, mTOR and PKC and subsequent induction of DNA synthesis and cell proliferation.56-58 In addition, leptin exerted antiapoptotic effects in colon cancer cells, which was mediated by the activation of NF-χB pathway and transcription factors AP-1 and STAT3.59,60 In colon cancer xenograft model, leptin treatment enhanced tumor volume and somewhat accelerated tumor growth.58 Leptin also promoted migration and invasiveness of human colorectal cancer cells, which may suggest leptin involvement in colorectal cancer metastasis.56,61 The expression of the leptin system has also been demonstrated in colorectal cancer tissues. Recently, we reported a progressive increase in leptin expression by epithelial cells during colorectal carcinogenesis.62,63 Weak or lack of leptin expression was found in normal colorectal mucosa, while higher leptin production was observed in epithelia adjacent to colorectal cancer. In human colorectal carcinomas leptin and LR were significantly overexpressed compared with noncancer tissues.63 The expression of both leptin and LR was correlated with the presence of HIF-1α.62 All these data obtained in human tissues suggest the possible role of local leptin in colorectal cancer development and progression (Table 2). Unfortunately, the role of circulating leptin in colorectal cancer, similarly to the situation in breast cancer, is not clear (Table 2). Stattin et al measured serum leptin levels in men who were diagnosed with colorectal cancer after blood collection and observed significant increase in colon cancer risk in patients with increasing leptin concentrations.64 The authors concluded that leptin might provide a link between obesity and colon cancer by its direct involvement in tumorigenesis. Some results obtained in animal models support this hypothesis. For instance, in carcinogen-treated rats on high-fat diet, serum leptin levels were increased. It correlated with increased colonic cell proliferation, c-fos protein expression and colon carcinogenesis, assessed by aberrant crypt foci formation.65 On the other hand, several analyses of circulating leptin in cancer patients did not reveal any evidence of leptin association with colorectal cancer.43 Arpaci et al and Bolukbas et al found significantly lower serum leptin concentrations in colorectal cancer patients relative to controls.66,67 However, since the assessment was done retrospectively, the possible upregulation of leptin expression during carcinogenesis was not addressed.

69

Leptin as a Novel Marker in Breast and Colorectal Cancer

Table 2. Reports on significance of leptin and LR in colorectal cancer Results, Conclusions

Leptin, LR Measurements

References

Elevated serum leptin associated with high fat diet Leptin in serum of rats caused aberrant crypt foci formation

65

No differences between plasma leptin in colorectal cancer patients vs controls

Not known

43

Significant increase in colon cancer risk with increasing serum leptin levels, but no differences in serum leptin levels in cancer patients vs controls

Leptin 2.59 vs 2.49 ng/mL

64

Serum leptin levels in colon cancer patients significantly lower vs controls

Leptin 8.79 vs 15.95 ng/mL

66

Serum leptin concentration in cancer patients significantly lower vs controls

Leptin in control vs cancer group: 1.8 vs 5.7 ng/mL (women); 1.8 vs 2.5 ng/mL (men)

67

Leptin treatment caused LR activation and growth LR expression and activation of colon cancer cells in colon cancer cell lines and colon tissue

57

Leptin treatment promoted cancer cell invasion

Leptin and LR in colon cancer cell lines and colon cancer tissue

56

Leptin treatment stimulated DNA synthesis and growth of colon cancer cells

LR in colon cancer cell lines

58

Leptin treatment stimulated proliferation and survival of colon cancer cells

LR activation in colon cancer cell lines

59

Leptin treatment stimulated proliferation and inhibited apoptosis of colon cancer cells

LR activation in colon cancer cell lines

60

Leptin overexpression in human colorectal cancer Leptin protein expression in compared with normal colorectal mucosa human colorectal cancer

63

Association between leptin, LR and markers of tissue hypoxia

62

Summary and Perspectives

Leptin and LR protein expression in human colorectal cancer

The evidence that leptin can be involved in breast and colorectal cancers is supported by aggregate results obtained in cellular and animal models. These data clearly demonstrate that leptin is a mitogen, survival factor and may promote anchorage-independent growth, migration and invasion of breast and colorectal cancer cells (Fig. 1). The analysis of human breast and colorectal cancer tissues confirmed that leptin and LR are expressed in these tumors. Importantly, this expression can be induced by obesity-related simuli as well as by tumor hypoxia. In breast and colorectal cancer, a common phenomenon is that the leptin system appears to be expressed at much higher levels in cancer cells than in noncancer tissue. This suggests that leptin and LR are elements of the neoplastic process and as such can be regarded as novel markers and become attractive targets for novel therapeutics.4 Indeed, preliminary results of Gonzalez et al provided evidence that LR antagonists can inhibit mammary tumors in a mouse syngeneic model.32 Thus, the efficacy

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of leptin targeting drugs in human breast cancer management should be explored, especially in the combination with existing therapeutic options. In the meantime, several pending questions regarding the uncertain role of systemic leptin in breast and colorectal cancer etiology should be clarified by larger, better-controlled, prospective studies.

References

1. Calle EE, Thun MJ. Obesity and cancer. Oncogene 2004; 23(38):6365-78. 2. Ahima RS. Central actions of adipocyte hormones. Trends Endocrinol Metab 2005; 16(7):307-13. 3. McTiernan A. Obesity and cancer: the risks, science and potential management strategies. Oncology (Williston Park) 2005; 19(7):871-81; discussion 81-2, 85-6. 4. Garofalo C, Surmacz E. Leptin and cancer. J Cell Physiol 2006; 207(1):12-22. 5. Hou WK, Xu YX, Yu T et al. Adipocytokines and breast cancer risk. Chin Med J (Engl) 2007; 120(18):1592-6. 6. Ray A, Nkhata KJ, Grande JP et al. Diet-induced obesity and mammary tumor development in relation to estrogen receptor status. Cancer Lett 2007;253(2):291-300. 7. Schaffler A, Scholmerich J, Buechler C. Mechanisms of disease: adipokines and breast cancer—endocrine and paracrine mechanisms that connect adiposity and breast cancer. Nat Clin Pract Endocrinol Metab 2007; 3(4):345-54. 8. Surmacz E. Obesity hormone leptin: a new target in breast cancer? Breast Cancer Res 2007; 9(1):301. 9. Vona-Davis L, Howard-McNatt M, Rose DP. Adiposity, type 2 diabetes and the metabolic syndrome in breast cancer. Obes Rev 2007; 8(5):395-408. 10. Vona-Davis L, Rose DP. Adipokines as endocrine, paracrine and autocrine factors in breast cancer risk and progression. Endocr Relat Cancer 2007; 14(2):189-206. 11. Soler JT, Folsom AR, Kaye SA et al. Associations of abdominal adiposity, fasting insulin, sex hormone binding globulin and estrone with lipids and lipoproteins in postmenopausal women. Atherosclerosis 1989; 79(1):21-7. 12. Sisci D, Surmacz E. Crosstalk between IGF signaling and steroid hormone receptors in breast cancer. Curr Pharm Des 2007; 13(7):705-17. 13. Surmacz E. Function of the IGF-I receptor in breast cancer. J Mammary Gland Biol Neoplasia 2000; 5(1):95-105. 14. Donovan EA, Kummar S. Role of insulin-like growth factor-1R system in colorectal carcinogenesis. Crit Rev Oncol Hematol 2008;66(2):91-8. 15. Zhang F, Chen Y, Heiman M et al. Leptin: structure, function and biology. Vitam Horm 2005; 71:345-72. 16. Hu X, Juneja SC, Maihle NJ et al. Leptin—a growth factor in normal and malignant breast cells and for normal mammary gland development. J Natl Cancer Inst 2002; 94(22):1704-11. 17. Neville MC, McFadden TB, Forsyth I. Hormonal regulation of mammary differentiation and milk secretion. J Mammary Gland Biol Neoplasia 2002; 7(1):49-66. 18. Bonnet M, Delavaud C, Laud K et al. Mammary leptin synthesis, milk leptin and their putative physiological roles. Reprod Nutr Dev 2002; 42(5):399-413. 19. Dieudonne MN, Machinal-Q uelin F, Serazin-Leroy V et al. Leptin mediates a proliferative response in human MCF7 breast cancer cells. Biochem Biophys Res Commun 2002; 293(1):622-8. 20. Laud K, Gourdou I, Pessemesse L et al. Identification of leptin receptors in human breast cancer: functional activity in the T47-D breast cancer cell line. Mol Cell Endocrinol 2002; 188(1-2):219-26. 21. Choi JH, Park SH, Leung PC et al. Expression of Leptin Receptors and Potential Effects of Leptin on the Cell Growth and Activation of Mitogen-activated Protein Kinases in Ovarian Cancer Cells. J Clin Endocrinol Metab 2005;90(1):207-10. 22. Garofalo C, Sisci D, Surmacz E. Leptin interferes with the effects of the antiestrogen ICI 182,780 in MCF-7 breast cancer cells. Clin Cancer Res 2004; 10(19):6466-75. 23. Jiang H, Yu J, Guo H et al. Upregulation of survivin by leptin/STAT3 signaling in MCF-7 cells. Biochem Biophys Res Commun 2008; 368(1):1-5. 24. Catalano S, Marsico S, Giordano C et al. Leptin enhances, via AP-1, expression of aromatase in the MCF-7 cell line. J Biol Chem 2003; 278(31):28668-76. 25. Catalano S, Mauro L, Marsico S et al. Leptin induces, via ERK1/ERK2 signal, functional activation of estrogen receptor alpha in MCF-7 cells. J Biol Chem 2004; 279(19):19908-15. 26. Chen C, Chang YC, Liu CL et al. Leptin-induced growth of human ZR-75-1 breast cancer cells is associated with up-regulation of cyclin D1 and c-Myc and down-regulation of tumor suppressor p53 and p21WAF1/CIP1. Breast Cancer Res Treat 2006; 98(2):121-32.

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27. Soma D, Kitayama J, Yamashita H et al. Leptin Augments Proliferation of Breast Cancer Cells via Transactivation of HER2. J Surg Res 2008; 149(1):9-14. 28. Okumura M, Yamamoto M, Sakuma H et al. Leptin and high glucose stimulate cell proliferation in MCF-7 human breast cancer cells: reciprocal involvement of PKC-alpha and PPAR expression. Biochim Biophys Acta 2002; 1592(2):107-16. 29. Yin N, Wang D, Zhang H et al. Molecular mechanisms involved in the growth stimulation of breast cancer cells by leptin. Cancer Res 2004; 64(16):5870-5. 30. Saxena NK, Vertino PM, Anania FA et al. Leptin-induced Growth Stimulation of Breast Cancer Cells Involves Recruitment of Histone Acetyltransferases and Mediator Complex to CYCLIN D1 Promoter via Activation of Stat3. J Biol Chem 2007; 282(18):13316-25. 31. Ray A, Nkhata KJ, Cleary MP. Effects of leptin on human breast cancer cell lines in relationship to estrogen receptor and HER2 status. Int J Oncol 2007; 30(6):1499-509. 32. Gonzalez RR, Cherfils S, Escobar M et al. Leptin signaling promotes the growth of mammary tumors and increases the expression of vascular endothelial growth factor (VEGF) and its receptor type two (VEGF-R2). J Biol Chem 2006; 281(36):26320-8. 33. Morelli C, Garofalo C, Sisci D et al. Nuclear insulin receptor substrate 1 interacts with estrogen receptor alpha at ERE promoters. Oncogene 2004; 23(45):7517-26. 34. Magoffin DA, Weitsman SR, Aagarwal SK et al. Leptin regulation of aromatase activity in adipose stromal cells from regularly cycling women. Ginekol Pol 1999; 70(1):1-7. 35. Mauro L, Catalano S, Bossi G et al. Evidences that leptin up-regulates E-cadherin expression in breast cancer: effects on tumor growth and progression. Cancer Res 2007; 67(7):3412-21. 36. Cleary MP, Phillips FC, Getzin SC et al. Genetically obese MMTV-TGF-alpha/Lep(ob)Lep(ob) female mice do not develop mammary tumors. Breast Cancer Res Treat 2003; 77(3):205-15. 37. Garofalo C, Koda M, Cascio S et al. Increased expression of leptin and the leptin receptor as a marker of breast cancer progression: possible role of obesity-related stimuli. Clin Cancer Res 2006; 12(5):1447-53. 38. Cascio S, Bartella V, Auriemma A et al. Mechanism of leptin expression in breast cancer cells: role of hypoxia-inducible factor-1alpha. Oncogene 2008;27(4):540-7. 39. Manabe Y, Toda S, Miyazaki K et al. Mature adipocytes, but not preadipocytes, promote the growth of breast carcinoma cells in collagen gel matrix culture through cancer-stromal cell interactions. J Pathol 2003; 201(2):221-8. 40. Ishikawa M, Kitayama J, Nagawa H. Enhanced expression of leptin and leptin receptor (OB-R) in human breast cancer. Clin Cancer Res 2004; 10(13):4325-31. 41. Revillion F, Charlier M, Lhotellier V et al. Messenger RNA Expression of Leptin and Leptin Receptors and their Prognostic Value in 322 Human Primary Breast Cancers. Clin Cancer Res 2006; 12(7):2088-94. 42. Miyoshi Y, Funahashi T, Tanaka S et al. High expression of leptin receptor mRNA in breast cancer tissue predicts poor prognosis for patients with high, but not low, serum leptin levels. Int J Cancer 2006; 118(6):1414-9. 43. Tessitore L, Vizio B, Jenkins O et al. Leptin expression in colorectal and breast cancer patients. Int J Mol Med 2000; 5(4):421-6. 44. Chen DC, Chung YF, Yeh YT et al. Serum adiponectin and leptin levels in Taiwanese breast cancer patients. Cancer Lett 2006; 237(1):109-14. 45. Ozet A, Arpaci F, Yilmaz MI et al. Effects of tamoxifen on the serum leptin level in patients with breast cancer. Jpn J Clin Oncol 2001; 31(9):424-7. 46. Han C, Zhang HT, Du L et al. Serum Levels of Leptin, Insulin and Lipids in Relation to Breast Cancer in China. Endocrine 2005; 26(1):19-24. 47. Liu CL, Chang YC, Cheng SP et al. The roles of serum leptin concentration and polymorphism in leptin receptor gene at codon 109 in breast cancer. Oncology 2007; 72(1-2):75-81. 48. Goodwin PJ, Ennis M, Fantus IG et al. Is leptin a mediator of adverse prognostic effects of obesity in breast cancer? J Clin Oncol 2005; 23(25):6037-42. 49. Petridou E, Papadiamantis Y, Markopoulos C et al. Leptin and insulin growth factor I in relation to breast cancer (Greece). Cancer Causes Control 2000; 11(5):383-8. 50. Mantzoros CS, Bolhke K, Moschos S et al. Leptin in relation to carcinoma in situ of the breast: a study of premenopausal cases and controls. Int J Cancer 1999; 80(4):523-6. 51. Sauter ER, Garofalo C, Hewett J et al. Leptin expression in breast nipple aspirate fluid (NAF) and serum is influenced by body mass index (BMI) but not by the presence of breast cancer. Horm Metab Res 2004; 36(5):336-40. 52. Coskun U, Gunel N, Toruner FB et al. Serum leptin, prolactin and vascular endothelial growth factor (VEGF) levels in patients with breast cancer. Neoplasma 2003; 50(1):41-6.

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53. Woo HY, Park H, Ki CS et al. Relationships among serum leptin, leptin receptor gene polymorphisms and breast cancer in Korea. Cancer Lett 2006; 237(1):137-42. 54. Stattin P, Soderberg S, Biessy C et al. Plasma leptin and breast cancer risk: a prospective study in northern Sweden. Breast Cancer Res Treat 2004; 86(3):191-6. 55. Plaisancie P, Ducroc R, El Homsi M et al. Luminal leptin activates mucin-secreting goblet cells in the large bowel. Am J Physiol Gastrointest Liver Physiol 2006; 290(4):G805-12. 56. Attoub S, Noe V, Pirola L et al. Leptin promotes invasiveness of kidney and colonic epithelial cells via phosphoinositide 3-kinase-, rho- and rac-dependent signaling pathways. FASEB J 2000; 14(14):2329-38. 57. Hardwick JC, Van Den Brink GR, Offerhaus GJ et al. Leptin is a growth factor for colonic epithelial cells. Gastroenterology 2001; 121(1):79-90. 58. Aparicio T, Kotelevets L, Tsocas A et al. Leptin stimulates the proliferation of human colon cancer cells in vitro but does not promote the growth of colon cancer xenografts in nude mice or intestinal tumorigenesis in Apc(Min/+) mice. Gut 2005; 54(8):1136-45. 59. Rouet-Benzineb P, Aparicio T, Guilmeau S et al. Leptin counteracts sodium butyrate-induced apoptosis in human colon cancer HT-29 cells via NF-kappaB signaling. J Biol Chem 2004; 279(16):16495-502. 60. Ogunwobi OO, Beales IL. The anti-apoptotic and growth stimulatory actions of leptin in human colon cancer cells involves activation of JNK mitogen activated protein kinase, JAK2 and PI3 kinase/Akt. Int J Colorectal Dis 2007; 22(4):401-9. 61. Sierra-Honigmann MR, Nath AK, Murakami C et al. Biological action of leptin as an angiogenic factor. Science 1998; 281(5383):1683-6. 62. Koda M, Sulkowska M, Kanczuga-Koda L et al. Expression of the obesity hormone leptin and its receptor correlates with hypoxia-inducible factor-1alpha in human colorectal cancer. Ann Oncol 2007; 18 (Suppl 6):vi116-9. 63. Koda M, Sulkowska M, Kanczuga-Koda L et al. Overexpression of the obesity hormone leptin in human colorectal cancer. J Clin Pathol 2007; 60(8):902-6. 64. Stattin P, Lukanova A, Biessy C et al. Obesity and colon cancer: does leptin provide a link? Int J Cancer 2004; 109(1):149-52. 65. Liu Z, Uesaka T, Watanabe H et al. High fat diet enhances colonic cell proliferation and carcinogenesis in rats by elevating serum leptin. Int J Oncol 2001; 19(5):1009-14. 66. Arpaci F, Yilmaz MI, Ozet A et al. Low serum leptin level in colon cancer patients without significant weight loss. Tumori 2002; 88(2):147-9. 67. Bolukbas FF, Kilic H, Bolukbas C et al. Serum leptin concentration and advanced gastrointestinal cancers: A case controlled study. BMC Cancer 2004; 4:29.

Chapter 7

The Role of Leptin in Cardiac Physiology and Pathophysiology

Morris Karmazyn,* Daniel M. Purdham, Venkatesh Rajapurohitam and Asad Zeidan

Abstract

L

eptin, initially identified as the product of the obesity gene in 1994, has received extensive attention especially in terms of its potential role in appetite suppression and regulation of energy expenditure. Leptin is primarily produced by white adiposity tissue and the polypeptide exerts its principal effects on the hypothalamus by acting on its receptors, termed LR or OBR. Plasma leptin levels are greatly elevated in obese individuals which has been closely related to the degree of adiposity. Although once considered to be solely derived from adipose tissue it is now apparent that leptin can be produced by various tissues including those comprising the cardiovascular system. Moreover, identification of LR expression has been demonstrated in numerous cardiovascular tissue as well as blood borne factors such as platelets suggesting that leptin exerts biological effects beyond those initially identified and related to appetite suppression. In terms of the cardiovascular system LR have been identified in both vascular and cardiac tissues. The increased cardiovascular risk associated with obesity is well known and many of the effects of leptin appear to be compatible with its potential role as a contributing factor to increased cardiovascular morbidity associated with obesity. In both myocardial and vascular tissues leptin exerts its effects via multifaceted cell signalling mechanisms. In terms of leptin’s ability to produce vascular or cardiomyocyte hypertrophy or hyperplasia the effects appear to MAPK-dependent. Recent evidence suggests that p38 activation is of particular importance although how this occurs is uncertain. Leptin also activates the RhoA/ROCK pathway resulting in altered actin dynamics which in turn may be important to p38 activation resulting in hypertrophy. Understanding the cell signalling mechanisms underlying the effects of leptin is of major importance in terms of developing therapeutic intervention targeting the leptin system as a novel approach for treating cardiovascular disorders, particularly those associated with hyperleptinemia.

Introduction

The discovery and cloning of the obesity gene (ob) in 19941 has led to a plethora of studies aimed at unravelling the molecular and cellular basis of obesity and its accompanying disorders. Although originally thought to represent a disease reflecting an imbalance between food intake and energy expenditure, the identification of ob and the demonstration of its overexpression in obesity lent credence to the notion of a biochemical and molecular basis for obesity. The finding that ob encodes a circulating anti-satiety polypeptide, subsequently named leptin for the Greek word leptos, meaning thin, was particularly exciting in view of the polypeptide’s potential for treating obesity. *Corresponding Author: Morris Karmazyn—Department of Physiology and Pharmacology Schulich School of Medicine and Dentistry, Medical Sciences Building, University of Western Ontario, London, Ontario N6A 5C1, Canada. Email: [email protected]

Leptin and Leptin Antagonists, edited by Arieh Gertler. ©2009 Landes Bioscience.

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Leptin exerts its effects via membrane-bound LR which include various short forms possessing short intracellular domains (LRa, LRc, LRd, LRf ) and a long form (LRb) which is highly homologous to the type I cytokine receptor family. LRb has a long intracellular domain and its activation is coupled to the activation of the Janus kinase ( JAK)-signal transducers and activators of transcription (STAT) pathway. As will be discussed later, cardiovascular tissues possess a multiplicity of LR linked to various components of cell signaling processes.

Leptin Synthesis and Structure

Leptin is located on chromosome 7q31.1, 4 and 6 in humans, rat and mouse, respectively. The gene spans ∼ 20 kb consisting of three exons and two introns. The promoter region is ∼ 3 kb having TATA box, multiple C/EBP (CCAAT/enhancer binding protein) sites, glucocorticoid response element (GRE) and many cAMP response element-binding protein (CREB) sites. Leptin is a 16 kD, 167 amino acid polypeptide with 21 amino acids signal sequence at the amino-terminus which is cleaved following the translocation of leptin into microsomes and then secreted into the blood stream. Thus, circulating leptin in humans is actually a polypeptide of 146 amino acids having a molecular mass of 16 kD. Mouse leptin is 84% and 83% homologous to human and rat respectively. The polypeptide contains a single intramolecular disulfide bond that is conserved in mouse, rat and human, resulting in a loop at the C-terminal region and a N-terminal region. It has been proposed that the N-terminal region and not the C-terminal region or disulfide bond is essential for leptin’s biological activity and receptor binding activity2 although it has also been suggested that the disulfide bond is of importance for the secretion, stability and solubility of the polypeptide.3

Leptin Resistance

The concept of leptin resistance is important for understanding the biological effects of leptin and also in terms of explaining the failure of leptin as a potential treatment for obesity. The latter reflects leptin resistance at the central level in obese individuals with concomitant chronic hyperleptinemia although it should also be noted that resistance to leptin may also occur at peripheral tissues. Humans develop resistance to leptin-induced effects resulting in decreased central nervous system signalling and no reduction in body weight.4 The precise mechanisms for resistance are not known with certainty although it has been suggested that leptin resistance may be due to defective transport of leptin through the blood brain barrier, a concept supported by the observation that obese individuals have disproportionately low cerebral spinal fluid concentrations of leptin compared with plasma levels.4-6 It has recently been postulated that chronic elevations in leptin results in activation of STAT-3 resulting in elevations of SOCS-3, a natural inhibitor of leptin signalling. This would result in attenuated or abolished leptin induced LR activation and downstream signaling.7 A recent study also suggested that excess NO production in the hypothalamic regions contributes to central leptin resistance.8 There are various examples of development of peripheral resistance to leptin. For instance, leptin loses its restrictive effects on insulin secretion from pancreatic β-cells in obese individuals.9 As will be alluded to below, cardiac myocytes from spontaneously hypertensive and hyperleptinemic rats do not respond to leptin while normotensive controls show a decrease in contraction.10 The mechanism of peripheral resistance is unknown although this does not appear to involve downregulation of LRb.10 Recently, it has been proposed that leptin binding to C reactive protein (CRP) contributes to peripheral leptin resistance.11 However, this concept has been questioned by a number of investigators who failed to demonstrate interaction between leptin and CRP or the ability of CRP to mitigate the function of leptin.12-14

Is Leptin a Possible Link between Obesity and Increased Cardiovascular Risk?

The relationship between obesity and increased risk for the development of cardiovascular disease is well known15-17 and obesity produces distinct changes in myocardial biochemistry,

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structure and function.18,19 Although a clear mechanistic basis for increased cardiovascular risk in obese individuals is uncertain,20 leptin has received some attention as a potential causative, or at least a contributing factor to this phenomenon particularly as it pertains to hypertension21,22 but also to a host of other cardiovascular conditions. One of the manifestations of obesity is increased sympathetic tone which results in catecholamine related cardiovascular dysfunction and as such the ability of leptin to stimulate sympathetic activity could suggest its involvement in this phenomenon.23-25 Leptin has also been shown to directly stimulate catecholamine synthesis in cultured bovine adrenal medullary cells through a mechanism involving tyrosine hydroxylase and MAPK activation.26 This would suggest that leptin-induced catecholamine elevation may occur via two mechanisms, activation of the sympathetic nervous system and direct stimulation of catecholamine synthesis in adrenal medulla. A significant correlation between plasma leptin concentrations and systolic blood pressure has been reported by various investigators although a cause and effect relationship has not been established.27-31 Such as a relationship was also observed in obese children who also demonstrated a strong relationship between plasma leptin and insulin levels suggesting that leptin could be a marker or contributor to insulin resistance in obese subjects.32 Plasma leptin has also been associated with a potential for increased thromboembolic risk in obesity via two potential mechanisms, first through impaired fibrinolysis33 and, secondly, through increased levels of fibrinogen.34 Dietary-mediated reduction in body weight reduces blood pressure as well as leptin concentrations in obese hypertensive individuals.35 The link between leptin and blood pressure appears to be rather convincing although it should be noted that the effects of leptin on vascular reactivity is multifaceted since the polypeptide likely directly produces vasorelaxation via a nitric oxide (NO)-dependent process.36 In this regard, NO production following leptin administration has been shown to be markedly depressed in obese animals.37 Interestingly, dietary-induced obesity in hypertensive rats results in slower recovery from stress induced elevations in blood pressure and heart rate which was associated with myocardial hypertrophy and hyperleptinemia.38

Expression of Leptin Receptors in Cardiovascular Tissues

The first demonstration of the presence of LR gene expression in cardiac tissue was reported in 1996 upon the discovery of the gene encoding the db/db mutation.39 Further characterization of LR isoforms indicated that cardiac tissue expressed LRa, LRb and LRe.10,40 Recent work from the authors’ laboratory suggest that LR gene expression in the heart differs in terms of regional distribution and is also affected by gender.41 Semi-quantitative real-time polymerase chain reaction revealed that in both males and females all three isoforms investigated were expressed in both atria, left and right ventricular walls as the interventricular septum although the greatest gene abundance was found in the atria. In terms of gender differences, LR expression was generally higher in tissues from female rats especially in the right atria.41 The functions of each of the LR isoforms in the heart, the relevance of regional distribution expression patterns or the influence of gender are currently unclear although some potential functions for leptin signaling in the heart will be discussed later in this review. The identification of LR in cardiac tissue was of particular interest since this soluble receptor represents the primary binding protein for leptin in plasma and may thus dictate leptin availability to tissues. It is possible that the presence of LRe in cardiac tissues is a consequence of proteolytic cleavage of the extracellular domains of one of the other isoforms.42,43 Although the function of LRe in the heart is currently unknown, it is interesting to speculate that its local tissue production serves to “fine tune” leptin concentrations in that specific tissue which would be in keeping with its role as a clearance receptor, although evidence for this hypothesis needs to be obtained with further studies. In addition to cardiac tissue, leptin receptors have also been identified in both cerebral and coronary vessels.44,45 With respect to the latter it was proposed that LR-mediated leptin-induced vasodilatation occurs through a nitric-oxide dependent process and which was abolished by hyperleptinemia. This finding emphasizes the potential dual role of leptin on vascular tissue, a direct

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NO-dependent vasodilatation and vasoconstriction occurring secondarily to central stimulation of the sympathetic nervous system. These effects will be discussed below in greater detail.

Effect of Leptin on Cardiomyocyte Function

Under in vivo conditions, the cardiovascular actions of leptin can be predicted based on the central sympathetic stimulatory effect of the polypeptide resulting in sympathetic nervous system-dependent effects such as elevations in blood pressure and positive inotropic and chronotropic effects. However, leptin can exert direct effects on both the heart and blood vessels through LR-dependent cell signalling mechanisms. In isolated ventricular myocytes leptin produces a negative inotropic effect via a NO dependent pathway as the effect was abrogated by NO synthase inhibition with L-NAME and associated with increased NO synthase activity.46 The negative inotropism is also associated with both JAK-STAT as well as MAP kinase p38 activation.10,47 The negative inotropic effect of leptin can also be significantly augmented by ceramide. Leptin has also been shown to stimulate fatty acid oxidation in working perfused rat hearts in the absence of any effect on glucose oxidation while lowering cardiac triglyceride content.48

Cardiomyocyte Hypertrophic Effects of Leptin

Evidence for leptin as a hypertrophic and pro-growth factor stems primarily from studies examining the direct effect of the polypeptide on myocyte preparations. For example, our laboratory reported that leptin produces marked hypertrophy in cultured neonatal rat ventricular myocytes as manifested by increased cells size, elevated protein synthesis and upregulation of a number genetic hypertrophic markers.49 Leptin-induced hypertrophy was associated with MAPK activation including both the p44/42 and p38 pathways whereas the hypertrophy was prevented only by p38 inhibition.49 Although the latter suggests a p38-dependent pathway of leptin-induced hypertrophy the mechanism may be multifaceted and involve other contributing factors. Thus, Xu and coworkers demonstrated that leptin-induced endothelin-1 release from neonatal rat ventricular myocytes results in activation of the endothelin-1 ETA receptor which then stimulate production of reactive oxygen species, the latter inducing cardiomyocyte hypertrophy.50 This study suggests that leptin does not induce hypertrophy directly per se but rather as a consequence of upregulation of other pro-hypertrophic factors. Accordingly, both ETA receptor blockade as well as catalase were effective in abrogating the hypertrophic response.50 In view of the fact that endothelin-1 and other hypertrophic factors such as angiotensin II are upregulated in obesity (reviewed in ref. 51), this study describes an important potential synergistic relationship between various neurohumoral factors in the overall hypertrophic process. This relationship between leptin is further highlighted by recent evidence from our laboratory that leptin mediates the hypertrophic effects of endothelin-1 and angiotensin II in cultured myocytes.52 In this study, the hypertrophic effects of either endothelin-1 or angiotensin II were associated with increased LR expression and release of leptin into the culture medium. Moreover, anti LR antibodies completely abrogated the hypertrophic responses to both endothelin-1 and angiotensin II.52 These results need to be confirmed in other models but if validated they suggest that leptin plays a critical paracrine or autocrine obligatory role in mediating the hypertrophic to both endothelin-1 and angiotensin II and possibly other pro-hypertrophic factors. Leptin has been shown to increase hyperplasia of the murine atrial HL-1 cell line as well as pediatric cardiomyocytes.53 Activation of ERK and phosphatidylinositol 3-kinase was demonstrated and implicated in the increase in cell number. It should be noted that leptin-induced hypertrophy has also been shown in human pediatric ventricular myocytes which was associated with increased ERK, p38 and JAK phosphorylation.54 As will be discussed later in this chapter, activation of the RhoA/ROCK pathway likely plays a unique and critical role in mediating the cardiomyocyte hypertrophic effect of leptin.

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Leptin as a Cardioprotective Agent

Although the hypertrophic/proremodelling effect of leptin is suggestive of an important role in cardiac pathology, salutary effects of the polypeptide have also been demonstrated particularly in terms of its effect in protecting cardiac tissue against hypoxia or ischemia. Indeed, leptin has been shown to exert protection against hypoxia/reoxygenation injury in cultured myocytes55 or in isolated perfused mouse hearts subjected to ischemia and reperfusion in terms of infarct size reduction possibly acting via the PI3-Akt and ERK pathways.56 The rapid upregulation of expression of leptin and LR following initiation of myocardial ischemia41,57 suggests that stimulation of the leptin system represents a potential endogenous cardioprotective mechanism. It has even been suggested that mild obesity and its attendant elevation in leptin levels, may offer cardiac protection following ischemia58 which may explain reported improved outcomes observed in some clinical studies in obese patients following coronary events.59

Post Receptor Leptin Signaling

In general, the complexity and diversity of leptin’s effects is exemplified by its ability to activate several signal transduction pathways. It is beyond the scope of this review to comprehensively discuss leptin-mediated signaling in all tissues or organ systems. Instead, the discussion below focuses on signaling pathways which have been elucidated in cardiovascular tissue or which appears to be particularly relevant for understanding leptin-mediated cardiac signaling and its possible relationship to pathology particularly in view of harnessing these pathways for cardiac therapeutics. For a more general treatise of this subject interested readers can consult various chapters appearing elsewhere in this volume or previous publications.60-63

JAK-STAT Pathway Activation

It is generally accepted that LRb is the fully competent signal transduction isoform of the receptor and that the short-form LRs (a,c,d,f ) while capable of signal transduction, do so to a lesser extent.64,65 The major signalling pathway activated by leptin binding to LRb is the Janus kinase ( JAK)—signal transducer and activator of transcription (STAT) pathway.64 Upon binding of leptin to its receptor JAK1 and JAK2 are both capable of associating with the cytoplasmic domain of LRb, however recently it has been demonstrated that JAK2 activation likely represents the physiologically relevant activated JAK during LR signaling.66 Activation of JAKs results in transphosphorylation of other JAK as well as phosphorylation of tyrosine residues of LRb.67 Recently, protein tyrosine phosphatase 1B (PTP1B) has been shown to be a negative regulator of JAK-STAT signaling.68,69 PTP1B dephosphorylates the consensus recognition motif on JAK2 resulting in inactivation of downstream STAT proteins.70 PTP1B knockout mice exhibit increased leptin sensitivity, STAT3 activation and decreased leptin to body weight ratios.70 Phosphorylation of the cytoplasmic domain of the receptor results in a docking site for STAT protein binding. STAT1, STAT3, STAT5 and STAT 6 have all been associated with leptin signalling in vitro.71-73 Upon binding the receptor complex, STAT is phosphorylated by JAK where it dissociates from the receptor, forms a homo or heterodimer and then translocates to the nucleus to act as a transcription factor.71,72,74 STAT3 can be inhibited by PIAS3, an endogenous protein inhibitor of this transcriptional factor.75 Evidence for JAK-STAT-dependent signalling in cardiac tissue in terms of physiological effects is at present limited but recent evidence suggests that it may be involved in the negative inotropic effect of leptin in cardiomyocytes based on the ability of the JAK2 inhibitor AG-490 to abrogate these effects.10 Interestingly however, the effect of AG-490 was mimicked by the MAPkinase inhibitor SB203580 suggesting that leptin exerts its effects via multiple and likely independent cell signalling pathways.10

Mitogen Activated Protein Kinase Stimulation

Mitogen-activated protein kinase (MAPK) represents an additional target for leptin-mediated effects. In fact, LRa has signal transduction capabilities through MAPK pathways both dependently and independently of JAK phosphorylation.44,64,67 JAK2 phosphorylation of LR tyrosine

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residue-985 (Tyr985) results in docking of a SH2-domain containing protein tyrosine phosphatase (SHP-2), which associates with an adapter molecule, Grb-2, to activate extracellular regulated kinase (ERK) signaling.67 Although ERK activation is possible in the absence of Tyr985, it still requires SHP-2 phosphatase activity.67 SHP-2 activation by leptin-OBR interaction leads to ERK activation, possibly through MEK1, but this has yet not been confirmed.61 Activation of ERK results in alterations in gene expression patterns for several genes including c-fos.44 Another MAPK, p38, has not been studied as extensively as ERK, but has been shown to be activated by leptin in mononuclear cells.76 In contrast, leptin was shown to reduce insulin-induced p38 activation, while having no effect on p38 activation on its own.62 The role of leptin signalling through c-jun NH2-terminal protein kinase ( JNK), has not been well characterized. However, there are 2 reports of leptin activating JNK in endothelial cells77 and in prostate cancer cells.78 In the cardiovascular system, leptin has been demonstrated to activate components of the MAPK pathways. In cultured neonatal myocytes, ERK1/2 and p38, but not JNK were activated by leptin; inhibiting ERK had no effect, while inhibition of p38 completely inhibited leptin-induced cardiomyocyte hypertrophy.49 Leptin has also been shown to induce hyperplasia in the immortalized atrial HL-1 cell line via an ERK dependent pathway.53 The results from studies using the HL-1 cell line are difficult to compare to primary culture of ventricular myocytes since the two models would likely respond to stimuli differently in view of the fact that the primary response of HL-1 cells is hyperplasia, not hypertrophy.

Pivotal Role for the RhoA/ROCK System in Mediating the Hypertrophic Effects of Leptin

Over the past number of years it has become apparent that the Rho/ROCK pathway, a downstream target protein of small GTP-binding protein Rho important for regulation of cell morphology is likely also an important contributor to hypertrophy although the mechanism leading to activation of Rho GTPases and subsequently to cardiac hypertrophy has not been well characterized (reviewed in ref. 79,80). RhoA activates several protein kinases, including Rho kinases (ROCK). This leads to the activation of LIM kinase-2 (LIMK2) resulting in phosphorylation (inactivation) of the actin binding protein cofilin, an important factor in the regulation of actin dynamics which in turn leads to depletion of globular actin (G-actin) pool and enhanced actin polymerization (F-actin). Work from our laboratory has recently shown that leptin is a potent activator of the RhoA/ROCK pathway leading to a decrease in the G/F actin ratio.81 The precise mechanism of how activation of this pathway leads to cardiac hypertrophy is not known with certainty. Interestingly however, activation of RhoA/ROCK by leptin results in the selective translocation of p38, but not other MAPK isoforms, to the nucleus82 a finding in agreement with our initial observation that leptin-induced hypertrophy can be blocked by p38, but not by ERK inhibition.49 Intact caveolae are also critical for both the activation of the RhoA pathway and the resultant p38 translocation and hypertrophy.82 The role of caveolae in mediating the hypertrophic effects of leptin was supported by various lines of evidence.82 First, leptin significantly increased the number of caveolae as well as caveolin-3 protein expression in myocytes. Secondly, OBR were found to be colocalized with caveolae. Lastly, disruption of caveolae with the cholesterol depleting agent methyl-beta-cyclodextrin was found to prevent leptin induced hypertrophy which was reversed by exogenous cholesterol repletion. A summary of the potential role of the RhoA/ROCK system in mediating the hypertrophic effect of leptin is shown in Figure 1.

Conclusions: Potential of Leptin Modulators as Therapeutic Agents

Since the initial identification of leptin as a key satiety regulator there has been a large body of research into this intriguing polypeptide which has been extensively shown to exert a myriad of effects on a large number of tissues. The identification of leptin receptors in different tissues coupled with findings that diverse organs and tissues can produce leptin leads to the conclusion that leptin exerts effects which are substantially more extensive than initially thought. This clearly applies to the cardiovascular system where leptin and its receptors have been identified in many cell types.

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Figure 1. Schematic showing a pivotal role of RhoA/ROCK activation as a mediator of leptin-induced hypertrophy and its interaction with p38 acting via its receptor (LR). Caveolae-dependent activation of RhoA/ROCK results in increased cofilin phosphorylation and altered actin dynamics as demonstrated by a decreased G/F actin ratio. Stimulation of this pathway results in an exclusive and selective translocation of p38 MAPK into the nucleus which results in an increased protein synthesis through an as yet to be identified mechanism.

Indeed, leptin has diverse cardiovascular effects which are mediated by complex cell signalling mechanisms. Identification of leptin-induced cell signalling is important to fully appreciate the basis for the polypeptide’s cardiovascular effects as well as for development of therapeutic targets involving the leptin system. Therefore, a potentially important outcome of such research will be the development of novel therapeutic strategies for the treatment of cardiovascular disease associated with hyperleptinemic conditions such as heart failure. One such approach would be the use of LR blockers to attenuate the potential deleterious effect of leptin on the heart particularly with respect to pathological hypertrophy. Indeed, a recent study in our laboratory has shown that the use of an antibody against LR significantly mitigated hypertrophy, heart failure and extracellular remodelling in rat hearts subjected to sustained coronary artery ligation.83 This finding is encouraging and provides further impetus for the development of selective cardiac therapeutic strategies aimed at mitigating the deleterious effects of leptin on the heart.

Acknowledgements

The work described from the authors’ laboratory is supported by the Canadian Institutes of Health Research. M Karmazyn holds a Canada Research Chair in Experimental Cardiology. DM Purdham was a recipient of a Scholarship from the Heart and Stroke Foundation of Canada. A Zeidan was supported by the Heart and Stroke Foundation of Ontario Program in Heart Failure.

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60. Ahima RS, Osei SY. Leptin signaling. Physiol Behav 2004; 81:223-241. 61. Hegyi K, Fulop K, Kovacs K et al. Leptin-induced signal transduction pathways. Cell Biol Int 2004; 28:159-169. 62. Sweeney G, Keen J, Somwar R et al. High leptin levels acutely inhibit insulin-stimulated glucose uptake without affecting glucose transporter 4 translocation in l6 rat skeletal muscle cells. Endocrinology 2001; 142:4806-4812. 63. Zabeau L, Lavens D, Peelman F et al. The ins and outs of leptin receptor activation. FEBS Lett 2003; 546:45-50. 64. Bjorbaek C, Uotani S, da Silva B et al. Divergent signaling capacities of the long and short isoforms of the leptin receptor. J Biol Chem 1997; 272:32686-32695. 65. Uotani S, Bjorbaek C, Tornoe J et al. Functional properties of leptin receptor isoforms: internalization and degradation of leptin and ligand-induced receptor downregulation. Diabetes 1999; 48:279-286. 66. Kloek C, Haq AK, Dunn SL et al. Regulation of Jak kinases by intracellular leptin receptor sequences. J Biol Chem 2002; 277:41547-41555. 67. Banks AS, Davis SM, Bates SH et al. Activation of downstream signals by the long form of the leptin receptor. J Biol Chem 2000; 275:14563-14572. 68. Cheng A, Uetani N, Simoncic PD et al. Attenuation of leptin action and regulation of obesity by protein tyrosine phosphatase 1B. Dev Cell 2002; 2:497-503. 69. Kaszubska W, Falls HD, Schaefer VG et al. Protein tyrosine phosphatase 1B negatively regulates leptin signaling in a hypothalamic cell line. Mol Cell Endocrinol 2002; 195:109-118. 70. Zabolotny JM, Bence-Hanulec KK, Stricker-Krongrad A et al. PTP1B regulates leptin signal transduction in vivo. Dev Cell 2002; 2:489-495. 71. Baumann H, Morella KK, White DW et al. The full-length leptin receptor has signaling capabilities of interleukin 6-type cytokine receptors. Proc Natl Acad Sci USA 1996; 93:8374-8378. 72. Bendinelli P, Maroni P, Pecori GF et al. Leptin activates Stat3, Stat1 and AP-1 in mouse adipose tissue. Mol Cell Endocrinol 2000; 168:11-20. 73. Briscoe CP, Hanif S, Arch JR et al. Fatty acids inhibit leptin signalling in BRIN-BD11 insulinoma cells. J Mol Endocrinol 2001; 26:145-154. 74. Heim MH. The Jak-STAT pathway: Specific signal transduction from the cell membrane to the nucleus. Eur J Clin Invest 1996; 26:1-12. 75. Chung CD, Liao J, Liu B et al. Specific inhibition of Stat3 signal transduction by PIAS3. Science 1997; 278:1803-1805. 76. van den Brink GR, O’Toole T, Hardwick JC et al. Leptin signaling in human peripheral blood mononuclear cells, activation of p38 and p42/44 mitogen-activated protein (MAP) kinase and p70 S6 kinase. Mol Cell Biol Res Commun 2000; 4:144-150. 77. Bouloumie A, Drexler HC, Lafontan M et al. Leptin, the product of Ob gene, promotes angiogenesis. Circ Res 1998; 83:1059-1066. 78. Onuma M, Bub JD, Rummel TL et al. Prostate cancer cell-adipocyte interaction: leptin mediates androgen-independent prostate cancer cell proliferation through c-Jun NH2-terminal kinase. J Biol Chem 2003; 278:42660-42667. 79. Loirand G, Guerin P, Pacaud P. Rho kinases in cardiovascular physiology and pathophysiology. Circ Res 2006; 98:322-334. 80. Noma K, Oyama N, Liao JK. Physiological role of ROCKs in the cardiovascular system. Am J Physiol Cell Physiol 2006; 290:C661-C668. 81. Zeidan A, Javadov S, Karmazyn M. Essential role of Rho/ROCK-dependent processes and actin dynamics in mediating leptin-induced hypertrophy in rat neonatal ventricular myocytes. Cardiovasc Res 2006; 72:101-111. 82. Zeidan A, Javadov S, Chakrabarti S et al. Leptin-induced cardiomyocyte hypertrophy involves selective caveolae and RhoA/ROCK-dependent p38 MAPK translocation to nuclei. Cardiovasc Res 2008; 77:64-72. 83. Purdham DM, Rajapurohitam V, Zeidan A et al. A neutralizing leptin receptor antibody mitigates hypertrophy and hemodynamic dysfunction in the postinfarcted rat heart. Am J Physiol Heart Circ Physiol 2008; 295:H441-446.

Chapter 8

The Role of Leptin in Bone Development and Growth Efrat Monsonego Ornan* and Michal Ben-Ami

Abstract

T

he link between obesity and osteoporosis—two major public-health problems, has become central to bone research in recent years, ever since leptin was identified as a regulator of both appetite and bone density. In contrast, the effect of leptin on skeletal development and bone elongation, another anticipated link between metabolic status and skeletal growth rate, has been less recognized. Here we summarize the existing data on leptin’s effects on bone elongation—its central effect through the GHRH-GH-IGF-1 axis on pubertal growth and its peripheral, direct roles on growth-plate chondrocytes, including the expression of leptin and its receptor and its effect on each part of the endochondral ossification process. We conclude that the data are still incomplete, as they give only a partial view of leptin’s role in bone development and growth.

The Effect of Leptin on the Skeleton

Leptin was originally identified as a hormone that suppresses appetite via the hypothalamic center. The effect of intra-cerebro-ventricular leptin administration on bone formation was also investigated. Obese mouse models that are deficient in leptin (ob/ob) or leptin receptor (db/db) have increased bone mass because of an increased rate of bone formation, despite hypogonadism and hypercorticism, both of which normally reduce bone mass.1 Intra-cerebro-ventricular injection of leptin to ob/ob mice decreased the rate of bone formation and returned the bone mass to that seen in wild-type mice. This finding was developed further by showing that leptin’s antiosteogenic actions are mediated by the sympathetic nervous system.2 Further evidence that the hypothalamic antiosteogenic network generates a signal of neuronal, not humoral, but nature, was obtained by cross-circulation experiments between ob/ob mice with and without intra-cerebral infusion of leptin.3 Karsenty4 concluded that leptin does not act directly on osteoblasts, which lack leptin receptor (LR). Nevertheless, abundant evidence also exists for direct effects of this hormone on the skeleton. For instance: leptin receptors are expressed on bone cells5 and leptin has direct effects on osteoblast proliferation6 and maturation,7 osteoclast development8 and chondrocyte activity.6 These findings indicate that leptin directly favors net bone formation. Taken together, it can only be said that the effect of leptin on bone formation remains controversial. To evaluate the effect of leptin on growth in children, the influence of leptin on growth-plate chondrocytes needs to be considered separately from its action on osteoblasts and osteoclasts, since the epiphyseal cartilage is the “motor” driving linear growth in children, through the process of endochondral ossification. Endochondral ossification is the major process controlling skeletogenesis: it begins with the formation of cartilage which is later replaced by bone. In the fetus, endochondral ossification occurs in the vertebral column, pelvis and extremities; later in life, it occurs in the growth plate, *Corresponding Author: Efrat Monsonego Ornan—Department of Biochemistry and Nutrition, Faculty of Agricultural, Food and Environmental Quality Sciences, The Hebrew University, P.O. Box 12, Rehovot 76100, Israel. Email: [email protected]

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which is the final target organ for longitudinal growth resulting from chondrocyte proliferation and differentiation.9 The growth plate can be divided into horizontal zones of chondrocytes at different stages of differentiation, creating three layers of cells (Fig. 1): at the epiphyseal end of the growth plate, the resting zone, also called stem-cell zone, contains the resting chondrocytes. Some unknown trigger prompts the stem cells to enter into the proliferating zone. In this matrix-rich zone, the flattened chondrocytes undergo cell divisions in a longitudinal direction and organize themselves in a typical columnar orientation. At some given point, either due to a finite number of cell divisions or to changes in exposure to a local growth factor, proliferating chondrocytes lose their capacity to divide and start to differentiate into hypertrophic chondrocytes, coinciding with an increase in size. This stage is characterized by an increase in intracellular calcium concentration. This is essential for the production of matrix vesicles, which are small membrane-enclosed particles that are released from chondrocytes. The vesicles secrete calcium phosphates, hydroxyapatite and matrix metalloproteinases, resulting in mineralization of the vesicles and their surrounding matrix. The mineralization process, in combination with low oxygen tension, attracts blood vessels from the

Figure 1. Endochondral ossification. H and E-stained section of the epiphyseal growth plate from the proximal tibia of a 2-week-old mouse.12

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metaphysis. Subsequently, the mineralized chondrocytes undergo apoptosis, leaving a scaffold for new bone formation. Cartilage is resorbed by chondroclasts or osteoclasts in the chondro-osseous junction. At the same time, osteoblasts enter the area to lay down new metaphyseal trabecular bone. The combination of chondrocyte proliferation, enlargement of maturing chondrocytes in the hypertrophic zone and production of extracellular matrix is the major contributor to longitudinal bone growth.10,11 In humans, there are at least three distinct endocrine phases of linear growth thoughout life. A high growth rate is observed from fetal life up to about 2 years of age. The second phase is characterized by a period of slower, decelerating growth velocity, up to puberty. The last phase, puberty, is characterized by an increased rate of longitudinal growth, followed by a rapid decrease in growth velocity.13,14 In some mammals, including humans, the growth-plate cartilage is completely replaced by bone at the time of sexual maturation. This event, termed epiphyseal fusion, appears to be triggered when the proliferative capacity of the growth-plate chondrocytes is finally exhausted.15 Optimal growth occurs only in healthy, well-nourished individuals, suggesting endocrine regulation of this process. Disturbances in longitudinal bone growth occur quite frequently with a highly diverse etiology.

Leptin and Growth

The level of leptin, a protein secreted primarily by white adipose tissue that regulates food intake and body weight, is increased in obesity.16,17 Obese children usually show an increase in height velocity and slightly more advanced bone age despite low physiological levels of growth hormone (GH). The height of obese children has been reported to increase with obesity index.18,19 There are some reports of normal height velocity in children without GH in association with marked obesity.20 Taken together, these findings suggest that humoral factors derived from adipose tissue, including leptin, may affect growth in children. Consistent with this hypothesis, it has been shown that femoral and humeral length in ob/ ob mice is shorter than in wild-type mice and that treatment with recombinant leptin or gene therapy rescues this reduction.21-23 Leptin has also been shown to increase the length of the tibia in a wild-type mouse model, even in the presence of low caloric intake.24 Kishida22 showed that the growth plates in ob/ob mice are more fragile and have morphological abnormalities: chondrocytes do not form organized columnar structures and apoptosis and mineralization are increased. There are only a few reports of leptin deficiency in humans. Ozata25 described a leptin-deficient patient showing normal growth. Clement26 reported a mild but significant growth delay during early childhood in humans with mutations in the leptin receptor, despite normal parental height. There are some case reports of children with congenital leptin deficiency who developed leg deformities requiring corrective surgery.27 Growth-plate fragility could contribute to these findings, rather than obesity. Another study conducted on children during recovery from malnutrition showed that leptin concentration increases only when the fat mass reaches a critical point and it is only at that point that the children show catch-up growth.28 These findings suggest that leptin has a significant effect on the dynamic process of growth; this review describes the cellular mechanisms underlying these findings, by evaluating both the central effect and the direct effect of leptin on the endochondral ossification process, considering each part of this process separately.

Central Effect of Leptin Leptin’s Effect on the GHRH-GH-IGF-1 Axis

Proper regulation of the GH axis is crucial for normal growth and development. Hypothalamic GH-releasing hormone (GHRH) stimulates GH release from the pituitary gland and somatostatin inhibits GH release. Circulating GH then targets many tissues, including the liver, leading to the release of insulin-like growth factor 1 (IGF-1). IGF-1 is an essential mediator of growth and also has an inhibitory effect on GH synthesis and secretion by affecting both the pituitary gland and the hypothalamus.

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The neuro-regulation of GH secretion is closely related to an individual’s nutritional status. Obesity is associated with impairments in both spontaneous and GHRH-induced GH secretion, while low body weight enhances GH secretion. Leptin is secreted by adipocytes and serves as a sensor of energy stores which regulates metabolism at the central level; its concentration in the blood decreases during fasting and increases during obesity.29 The belief that leptin can regulate hypothalamo-pituitary functions is well established. Treatment of ob/ob mice with leptin restored fertility through activation of the hypothalamo-pituitary-gonadal axis.30,31 In rats, daily leptin administration fully prevented the fall in serum GH levels occurring after 3 days of fasting and significantly increased the levels of GH and GHRH mRNA. However, leptin treatment did not prevent the fall in IGF-1 levels.32 Central infusion of leptin in a rat model resulted in an increase in both spontaneous pulsatile GH secretion and GH response to GHRH, an increase in pituitary GH mRNA and hypothalamic GHRH mRNA and a reduction in somatostatin mRNA levels.33 Together, these findings suggest that at least some of the dramatic effects of leptin on GH and GH mRNA are mediated at the hypothalamic level. It has been shown that leptin receptors are present in arcuate GHRH-containing neurons34 and it is therefore possible that GHRH secretion is a target for leptin regulation. On the other hand, an in vitro study has shown that leptin inhibits somatostatin synthesis and secretion in cultured fetal rat neurons.35 Thus, it is possible that leptin activity is mediated, at least in part, via inhibition of hypothalamic somatostatin release. It is also possible that the reduction of somatostatin tone within the hypothalamus inhibits GHRH release and GH secretion. The direct effect of leptin on the pituitary has been evaluated in primary pituitary cells and both positive and negative effects have been reported.33

Leptin’s Effect on Pubertal Growth

Puberty begins slightly earlier in obese children36 and their total height gain is lower than in their non-obese counterparts.37 Garcia-Mayor38 reported that leptin appears to increase in boys and girls before the appearance of other reproductive hormones related to puberty. Palmert39 showed that girls with centrally mediated precocious puberty have a moderate elevation of serum leptin levels compared with healthy children. In another study, leptin rose by approximately 50% just before the onset of puberty and decreased to baseline after its initiation.40 These findings suggest a stimulatory effect of leptin on gonadotropin-releasing hormone (GnRH)-secreting neurons and/or secretion of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) from the pituitary. These findings could explain the growth pattern in obese children. Prepubertal obese children are taller and have older bone age because of higher concentrations of leptin and its influence on growth-plate chondrocytes. Onset of puberty is earlier in obese children due to leptin’s stimulatory effect on the secretion of sex hormones. These children have a lower total height gain probably due to earlier growth plate senescence, a process by which the cartilage is completely replaced by bone at the time of sexual maturation partly by the effect of sex hormones on the epiphyseal chondrocytes. As a result, obese children exhibit earlier maturation and slightly lower final height than non-obese children.

Peripheral/Direct Effect of Leptin

One open question is whether leptin regulates bone elongation only through its central effects or by direct binding and signal transduction in growth-plate chondrocytes. Such a direct role might consist of the expression of leptin and its receptor in these cells and of leptin having an effect on each part of the endochondral ossification process.

Leptin’s Production by Cells in the Growth Plate

Leptin is secreted primarily by white adipose tissue. However, recent evidence shows that leptin is also produced and secreted from the growth plate. Kume41 identified high expression of leptin during endochondral ossification. Leptin was expressed in the secondary ossification center of femora of 1-week-old mice, as well as in the primary ossification center of 15-day-old embryos. Leptin was found in hypertrophic chondrocytes adjacent to blood vessels, but not in the resting or proliferating cartilage. In vitro, leptin gene and protein were detected in mouse

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osteoblasts (MC3T3-E1 cell line) and chondrocytes (MCC-5 cell line): immunohistochemistry of MC3T3-E1 and MCC-5 cells revealed leptin in the cytoplasm. Kishida and coworkers also showed leptin expression in the growth plate of 4-week-old mice: in that study, leptin mRNA and protein were observed in resting and prehypertrophic chondrocytes, but not in the proliferation or hypertrophic zone.5,22 Although it is not clear if these amounts are significant compare to the circulating levels of leptin, its local production in the growth plate is crucial as cartilage is one of the least vasculated tissues in the body.

Leptin Receptor’s Expression by the Cells in the Growth Plate

Leptin is known to have a central effect on body weight and bone remodeling by binding to a specific receptor located in the hypothalamus. This effect is mediated through the central nervous system. However, recent studies have shown that in addition, leptin has a direct effect through high-affinity LR in peripheral tissues.5 Some reports have described the presence of LR in cartilaginous skeletal growth centers, articular cartilage and osteoblasts. In the growth plate, LR mRNA and protein were observed in terminal hypertrophic chondrocytes. Leptin was also shown to upregulate its own receptor.42,43 Together, the production of the ligand and its receptor in the growth plate facilitate leptin’s paracrine effect.

The Effect of Leptin on Chondrocyte Proliferation

Both in vivo and in vitro studies have shown that leptin stimulates the proliferation of growth-plate chondrocytes. Bromouridine incorporation into mandibular condyle DNA was increased by leptin. Moreover, leptin dose-dependently increased the width of the proliferating zone of the growth plate in mice.42 Gat-Yablonski24 showed that leptin stimulates proliferation activity in the prehypertrophic chondrocytes of mouse tibial growth plate. Nakajima43 reported

Figure 2. Leptin and endocrine growth, Modified from: Paulev PE, Textbook in Medical Physiology and Pathophsiology, Copenhagen Medical Publishers 1999-2000, Figure 30-1.

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that chondrocytes proliferate, in response to leptin, at a semi-confluent stage in culture and that this proliferation is inhibited by genistein, an inhibitor of tyrosine kinase.

The Effect of Leptin on Chondrocyte Differentiation

Normal skeletal elongation depends on the coupling of proliferation and differentiation within the growth plates. The stimulatory effect of leptin on differentiation has been established both in vivo and in vitro. Maor42 demonstrated increased expression of chondroitin sulfate within the cartilaginous matrix after leptin administration. Gat-Yablonski and coworkers showed high expression of both type II and type X collagen after leptin administration; this effect was suggested to be mediated through JAK/STAT and MAPK pathways and the PTHrP/Ihh feedback loop.44,45 Kishida22 demonstrated low expression of type X collagen in the growth plates of ob/ob mice compared to the wild type and a high level of type X collagen mRNA in ATDC5 cells cultured in the presence of leptin. Nakajima43 showed, in cultured growth-plate chondrocytes, that leptin increases alkaline-phosphatase activity and proteoglycan production at the semi-confluent stage.

The Effect of Leptin on Mineralization

Kishida22 showed premature mineralization of the cartilaginous matrix of ob/ob mice, an effect that was restored by treatment with recombinant leptin. Moreover, in primary cultures of chondrocytes from ob/ob mice, matrix mineralization increased as compared to cultures from wild-type mice. The addition of exogenous leptin to those cultures completely abolished mineralization. In vitro experiments using ATDC5 cells showed that physiological concentrations of leptin inhibit matrix mineralization.

The Effect of Leptin on Chondrocyte Apoptosis

Kishida22 showed that the number of apoptotic chondrocytes is significantly greater in ob/ob mice. These findings are consistent with an in vitro experiment in which ATDC5 cells incubated with physiological levels of leptin showed a decreased rate of apoptosis. Although the data regarding the direct effect of leptin on the growth plate is still limited, it seemes that leptin promote chondrogenesis by inducing the proliferation and differentiation of chondrocytes and inhibiting apoptosis and growth plate mineralization, leading to elevated bone elongation. In contrast to these direct effects, Iwaniec21 suggested that leptin has a central effect on bone growth. They found that in the absence of circulating and hypothalamic leptin, bone length and mass are decreased and that hypothalamic leptin is sufficient to normalize bone growth, even in the absence of measurable serum leptin. Thus it appears that similar to its role in bone formation, leptin has both central and peripheral effects on bone growth. Further studies, using conditional deletions of LR or leptin with specific promoters directed to bone and cartilage tissues, or to different developmental stages, such as mesenchymal condensation during limb buds development, will shed light on the debate regarding central versus direct effect of leptin on growth-plate chondrocytes.

Synopsis

In the last few years, the connection between an organism’s metabolic status and skeletal homeostasis has taken a central place in the field of bone research, starting with Karsenty’s pioneering work revealing the effect of leptin on bone density and followed by a debate regarding its mode of action: central or peripheral. Recent innovative results, from the same group, suggest an endocrine loop between metabolism-regulating organs and the skeleton. Signals from the adipose tissue (leptin) affect bone tissue and the bone, by means of osteocalcin, affects insulin secretion and sensitivity in the pancreas, muscle and adipose tissues.46 Despite these advances, the effect of leptin on skeletal development and bone elongation, another anticipated link between the metabolic status of an organism and skeletal growth rate, has been less documented. In this review, we summarize the existing data regarding leptin’s effect

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on bone elongation and find that these data are insufficient, as they only paint a partial picture. Many questions remain unanswered: 1. What are the dominant pathways via which leptin exerts its effect on chondrocytes? Is it the classical one ( JAK-STAT) in the chondrocytes themselves? Or, is it through the GHRH-GH-IGF-1 axis? 2. What other factors are involved in the precise and complicated process of cross talk between metabolic status and bone development/growth? These questions are of great importance from both basic and clinical points of view. From the practical viewpoint, these concerns are related to the dramatic rise in childhood obesity that is now reaching epidemic proportions.

References

1. Ducy P, Amling M, Takeda S et al. Leptin inhibits bone formation through a hypothalamic relay: a central control of bone mass. Cell 2000; 100(2):197-207. 2. Takeda S. Central control of bone remodeling. Biochem Biophys Res Commun 2005; 328(3):697-699. 3. Elefteriou F, Ahn JD, Takeda S et al. Leptin regulation of bone resorption by the sympathetic nervous system and CART. Nature 2005; 434(7032):514-520. 4. Karsenty G. Convergence between bone and energy homeostases: leptin regulation of bone mass. Cell Metab 2006; 4(5):341-348. 5. Reseland JE, Syversen U, Bakke I et al. Leptin is expressed in and secreted from primary cultures of human osteoblasts and promotes bone mineralization. J Bone Miner Res 2001; 16(8):1426-1433. 6. Cornish J, Callon KE, Bava U et al. Leptin directly regulates bone cell function in vitro and reduces bone fragility in vivo. J Endocrinol 2002; 175(2):405-415. 7. Thomas T, Gori F, Khosla S et al. Leptin acts on human marrow stromal cells to enhance differentiation to osteoblasts and to inhibit differentiation to adipocytes. Endocrinology 1999; 140(4):1630-1638. 8. Holloway WR, Collier FM, Aitken CJ et al. Leptin inhibits osteoclast generation. J Bone Miner Res 2002; 17(2):200-209. 9. Olsen BR, Reginato AM, Wang W. Bone development. Annu Rev Cell Dev Biol 2000; 16:191-220. 10. Karsenty G, Wagner EF. Reaching a genetic and molecular understanding of skeletal development. Dev Cell 2002; 2(4):389-406. 11. Kobayashi T, Kronenberg H. Minireview: transcriptional regulation in development of bone. Endocrinology 2005; 146(3):1012-1017. 12. Naski MC, Ornitz DM. FGF signaling in skeletal development. Front Biosci 1998; 3:d781-794. 13. Kember NF. Cell kinetics and the control of bone growth. Acta Paediatr Suppl 1993; 82(Suppl) 391:61-65. 14. Kember NF, Sissons HA. Quantitative histology of the human growth plate. J Bone Joint Surg Br 1976; 58-B(4):426-435. 15. Gafni RI, Weise M, Robrecht DT et al. Catch-up growth is associated with delayed senescence of the growth plate in rabbits. Pediatr Res 2001; 50(5):618-623. 16. Friedman JM, Halaas JL. Leptin and the regulation of body weight in mammals. Nature 1998; 395(6704):763-770. 17. Zhang Y, Proenca R, Maffei M et al. Positional cloning of the mouse obese gene and its human homologue. Nature 1994; 372(6505):425-432. 18. Falorni A, Bini V, Molinari D et al. Leptin serum levels in normal weight and obese children and adolescents: relationship with age, sex, pubertal development, body mass index and insulin. Int J Obes Relat Metab Disord 1997; 21(10):881-890. 19. Hassink SG, Sheslow DV, de Lancey E et al. Serum leptin in children with obesity: relationship to gender and development. Pediatrics 1996; 98(2 Pt 1):201-203. 20. Tiulpakov AN, Mazerkina NA, Brook CG et al. Growth in children with craniopharyngioma following surgery. Clin Endocrinol (Oxf ) 1998; 49(6):733-738. 21. Iwaniec UT, Boghossian S, Lapke PD et al. Central leptin gene therapy corrects skeletal abnormalities in leptin-deficient ob/ob mice. Peptides 2007; 28(5):1012-1019. 22. Kishida Y, Hirao M, Tamai N et al. Leptin regulates chondrocyte differentiation and matrix maturation during endochondral ossification. Bone 2005; 37(5):607-621. 23. Steppan CM, Crawford DT, Chidsey-Frink KL et al. Leptin is a potent stimulator of bone growth in ob/ob mice. Regul Pept 2000; 92(1-3):73-78. 24. Gat-Yablonski G, Ben-Ari T, Shtaif B et al. Leptin reverses the inhibitory effect of caloric restriction on longitudinal growth. Endocrinology 2004; 145(1):343-350.

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25. Ozata M, Ozdemir IC, Licinio J. Human leptin deficiency caused by a missense mutation: multiple endocrine defects, decreased sympathetic tone and immune system dysfunction indicate new targets for leptin action, greater central than peripheral resistance to the effects of leptin and spontaneous correction of leptin-mediated defects. J Clin Endocrinol Metab 1999; 84(10):3686-3695. 26. Clement K, Vaisse C, Lahlou N et al. A mutation in the human leptin receptor gene causes obesity and pituitary dysfunction. Nature 1998; 392(6674):398-401. 27. Montague CT, Farooqi IS, Whitehead JP et al. Congenital leptin deficiency is associated with severe early-onset obesity in humans. Nature 1997; 387(6636):903-908. 28. Buyukgebiz B, Ozturk Y, Yilmaz S et al. Serum leptin concentrations in children with mild protein-energy malnutrition and catch-up growth. Pediatr Int 2004; 46(5):534-538. 29. Caro JF, Kolaczynski JW, Nyce MR et al. Decreased cerebrospinal-fluid/serum leptin ratio in obesity: a possible mechanism for leptin resistance. Lancet 1996; 348(9021):159-161. 30. Barash IA, Cheung CC, Weigle DS et al. Leptin is a metabolic signal to the reproductive system. Endocrinology 1996; 137(7):3144-3147. 31. Chehab FF, Lim ME, Lu R. Correction of the sterility defect in homozygous obese female mice by treatment with the human recombinant leptin. Nat Genet 1996; 12(3):318-320. 32. LaPaglia N, Steiner J, Kirsteins L et al. Leptin alters the response of the growth hormone releasing factorgrowth hormone—insulin-like growth factor-I axis to fasting. J Endocrinol 1998; 159(1):79-83. 33. Cocchi D, De Gennaro Colonna V, Bagnasco M et al. Leptin regulates GH secretion in the rat by acting on GHRH and somatostatinergic functions. J Endocrinol 1999; 162(1):95-99. 34. Hakansson ML, Brown H, Ghilardi N et al. Leptin receptor immunoreactivity in chemically defined target neurons of the hypothalamus. J Neurosci 1998; 18(1):559-572. 35. Quintela M, Senaris R, Heiman ML et al. Leptin inhibits in vitro hypothalamic somatostatin secretion and somatostatin mRNA levels. Endocrinology 1997; 138(12):5641-5644. 36. Frisch RE, Revelle R. Height and weight at menarche and a hypothesis of menarche. Arch Dis Child 1971; 46(249):695-701. 37. Vignolo M, Naselli A, Di Battista E et al. Growth and development in simple obesity. Eur J Pediatr 1988; 147(3):242-244. 38. Garcia-Mayor RV, Andrade MA, Rios M et al. Serum leptin levels in normal children: relationship to age, gender, body mass index, pituitary-gonadal hormones and pubertal stage. J Clin Endocrinol Metab 1997; 82(9):2849-2855. 39. Palmert MR, Radovick S, Boepple PA. Leptin levels in children with central precocious puberty. J Clin Endocrinol Metab 1998; 83(7):2260-2265. 40. Mantzoros CS, Flier JS, Rogol AD. A longitudinal assessment of hormonal and physical alterations during normal puberty in boys. V. Rising leptin levels may signal the onset of puberty. J Clin Endocrinol Metab 1997; 82(4):1066-1070. 41. Kume K, Satomura K, Nishisho S et al. Potential role of leptin in endochondral ossification. J Histochem Cytochem 2002; 50(2):159-169. 42. Maor G, Rochwerger M, Segev Y et al. Leptin acts as a growth factor on the chondrocytes of skeletal growth centers. J Bone Miner Res 2002; 17(6):1034-1043. 43. Nakajima R, Inada H, Koike T et al. Effects of leptin to cultured growth plate chondrocytes. Horm Res 2003; 60(2):91-98. 44. Ben-Eliezer M, Phillip M, Gat-Yablonski G. Leptin regulates chondrogenic differentiation in ATDC5 cell-line through JAK/STAT and MAPK pathways. Endocrine 2007; 32(2):235-244. 45. Gat-Yablonski G, Shtaif B, Phillip M. Leptin stimulates parathyroid hormone related peptide expression in the endochondral growth plate. J Pediatr Endocrinol Metab 2007; 20(11):1215-1222. 46. Lee NK, Sowa H, Hinoi E et al. Endocrine regulation of energy metabolism by the skeleton. Cell 2007; 130(3):456-469.

Chapter 9

Involvement of Leptin in Arterial Hypertension Jerzy Beltowski*

Abstract

L

eptin is secreted by white adipose tissue and its concentration in plasma is higher in obese than in lean subjects. Recent studies suggest that leptin is involved in cardiovascular complications of obesity including arterial hypertension. Acutely administered leptin has no effect on blood pressure, probably because it concomitantly stimulates sympathetic nervous system and counteracting depressor mechanisms such as natriuresis and nitric oxide-dependent and -independent vasorelaxation. In contrast, chronic hyperleptinemia increases blood pressure because these acute depressor effects are impaired and/or additional nonsympathetic pressor mechanisms appear such as increased production of endothelin-1, overexpression of endothelin and angiotensin II receptors in vascular smooth muscle cells, oxidative stress, nitric oxide deficiency and enhanced renal Na+ reabsorption. Clinical studies have demonstrated that plasma leptin concentration is higher in patients with essential hypertension than in normotensive controls independently of body weight and the significant positive correlation between leptin and blood pressure was observed in both hypertensive and normotensive subjects. Modulating leptin sensitivity and/or leptin level may be a novel approach in the treatment of hypertension.

Introduction

Arterial hypertension is a major health problem worldwide. Although hypertension itself may be completely asymptomatic, it is associated with markedly increased risk of severe and potentially life-threatening complications such as atherosclerosis, ischemic heart disease, cerebral stroke, left ventricular hypertrophy and failure, aortic aneurysm and chronic nephropathy leading ultimately to end-stage renal disease. Although these complications develop only in a subset of patients, high prevalence of hypertension makes it a major cause of many of them. More than 95% of hypertensive patients are classified as having “idiopathic” or essential hypertension, i.e., hypertension of unknown etiology. Nevertheless, it is estimated that in developed countries about 70% of hypertension can be directly attributed to excess body weight, making overweight and obesity the principal cause of hypertension.1 Hypertension is a component of the metabolic syndrome—a cluster of abnormalities commonly observed in patients with obesity, especially of abdominal type.2 In addition, obesity may contribute to the development of many complications also associated with hypertension such as left ventricular hypertrophy, heart failure and nephropathy, independently of high blood pressure.3,4 The pathogenesis of obesity-associated hypertension is multifactorial, however, as in other forms of hypertension excessive vasoconstriction and abnormal renal sodium handling play an essential role.5 These abnormalities result from disturbed balance between neurohormonal systems causing vasoconstriction and Na+-retention such as sympathetic nervous system (SNS) or *Jerzy Beltowski—Department of Pathophysiology, Medical University, ul. Jaczewskiego 8 20-090 Lublin, Poland. Email: [email protected]

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renin-angiotensin-aldosterone (RAA) and counteractive vasodilating and natriuretic mechanisms including atrial natriuretic peptide, nitric oxide, etc. Studies performed during the last decade suggest an important role of adipose tissue hormones or “adipokines” in obesity-related disorders including arterial hypertension. Imbalance between various adipokines such as adiponectin, resistin, visfatin, etc. has been implicated in the pathogenesis of hypertension,6,7 however, most studies focused on leptin—the first and best characterized adipose tissue hormone.8,9 Although a major role of leptin is to regulate food intake and energy expenditure by acting on hypothalamic centers, leptin receptors are abundantly expressed in peripheral tissues including those relevant for the regulation of blood pressure such as vascular wall and the kidney. Moreover, leptin exerts many effects associated with the regulation of blood pressure (BP) including effects on vascular tone and renal sodium handling. Four types of evidence support the role of leptin in the pathogenesis of arterial hypertension: (1) chronic leptin administration10 or transgenic overexpression11 increase BP in experimental animals, (2) plasma leptin concentration is proportional to the amount of adipose tissue and is markedly higher in obese than in lean subjects, (3) some studies indicate that plasma leptin correlates with BP and is higher in hypertensive than in normotensive humans independently of body weight,12 (4) blood pressure is not elevated in leptin-deficient ob/ ob mice despite severe obesity and leptin supplementation increases BP in these animals despite reducing body weight.13 The purpose of this chapter is to provide an overview of physiological effects of leptin in the cardiovascular system and the kidney important for the regulation of blood pressure and to characterize role of leptin in the pathogenesis of hypertension. I will focus mainly on results of experimental studies which addressed this issue. Due to space limitations, other aspects such as extensive discussion of clinical studies, effect of antihypertensive therapy on plasma leptin, role of leptin in secondary forms of hypertension and in end-organ damage associated with hypertension will not be discussed. These issues have been extensively reviewed recently8,9 and some of them are also covered by professor Moris Karmazyn elsewhere in this book.

Physiological Effects of Leptin Relevant for Blood Pressure Regulation Effect of Leptin on Sympathetic Nervous System

Soon after discovery of leptin it became evident that this hormone not only reduces food intake but also increases energy expenditure; the effect mediated by increased sympathetic outflow to brown adipose tissue (BAT) and enhanced thermogenesis.14 Surprisingly, leptin stimulates sympathetic outflow also to other tissues which are not associated with energy expenditure such as the kidney, adrenals and hindlimbs. Several studies have suggested that leptin induces reflex stimulation of the SNS by activating afferent nerve endings in adipose tissue. For example, leptin injected into perirenal adipose tissue increases renal sympathetic nervous activity (SNA) in a dose-dependent manner.15 Thus, leptin released within the adipose tissue could act locally on afferent fibers to stimulate SNS in a paracrine manner. However, the majority of studies strongly suggest that sympathoexcitatory effect of leptin is mediated via the central nervous system. Intracerebroventricularly injected leptin stimulates SNS at doses which do not elevate systemic hormone level and damage of hypothalamic arcuate nucleus abolishes the sympathoexcitatory effect.16 It is generally appreciated that leptin stimulates SNS by the melanocortin pathway, also involved in the regulation of appetite. Thus, leptin increases proopiomelanocortin production by hypothalamic neurons, which then is processed to α-melanocyte-stimulating hormone (α-MSH). Alpha-MSH then activates melanocortin MC3 and MC4 receptors to increase sympathetic drive.17,18 Recently, it has been demonstrated that leptin injected to the hypothalamic arcuate nucleus increases sympathetic outflow to BAT and the kidneys at much lower doses than required to activate SNS when injected intracerebroventricularly, indicating that arcuate nucleus mediates the sympathoexcitatory effect.19 Some clinical studies indicate that plasma leptin concentration correlates with SNS activity in humans.20 Moreover, leptin supplementation prevents adaptive suppression of the SNS which otherwise occurs following calorie restriction.21 Thus, hyperleptinemia may be responsible, at least in part, for increased sympathetic activity in obese subjects.22

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Many experimental studies have demonstrated that although either centrally or peripherally administered leptin stimulates the SNS, blood pressure increases only when hormone is administered centrally.16,23,24 These data suggest that leptin stimulates also counteracting depressor mechanisms outside the central nervous system and indeed, a couple of studies supports this possibility.

Vasodilatory Effect of Leptin

It was first demonstrated almost 10 years ago25 that bolus intravenous leptin injection increases plasma concentration of nitric oxide metabolites, nitrites+nitrates (NOx), in the rat. In addition, although leptin alone had no effect on blood pressure, it increased blood pressure in rats pretreated with NO synthase inhibitor, L-NAME. Conversely, leptin decreased blood pressure in animals in which SNS was pharmacologically inhibited. These data suggest that under normal conditions leptin induces balanced activation of the SNS and vascular NO production resulting in no net changes of arterial pressure. Subsequently, we have demonstrated that intraperitoneally26 or intravenously27 administered leptin increases plasma concentration and urinary excretion of NO metabolites as well as of NO second messenger, cyclic GMP, in a time- and dose-dependent manner. These results are consistent with in vitro studies, in which leptin induced endothelium- and NO-dependent vasorelaxation.28-30 In addition, leptin increased NO production in bovine pulmonary artery endothelial cells, human aortic endothelial cells and isolated rat aortic rings.31,32 Leptin-induced NO production is not mediated by Ca2+/calmodulin-dependent stimulation of endothelial nitric oxide synthase (eNOS) which is a common mechanism of eNOS activation by endothelium-dependent vasodilators such as acetylcholine. Rather, leptin activates eNOS by inducing its phosphorylation by serine-threonine protein kinase B (PKB)/Akt. Indeed, leptin increases eNOS phosphorylation at ser-1117 as well as PKB/Akt phosphorylation at thre-308 and ser-473 suggesting that, similarly to insulin, leptin activates eNOS by PKB/Akt-dependent mechanism.31 However, in contrast to insulin, NO-mimetic effect of leptin is not abolished by inhibitors of phosphatidylinositol 3-kinase (PI3K) either in vitro31 or in vivo,33 indicating that intracellular mechanisms upstream to PKB/ Akt differ for both hormones. Nevertheless, subthreshold concentrations of insulin augment the effect of leptin on PKB/Akt phosphorylation, eNOS phosphorylation and NO release.34 However, not all studies support the role of NO in hemodynamic effect of leptin. For example, leptin had no effect on mesenteric, hindlimb or renal vascular conductance in conscious rats even after administration of α1-adrenoceptor antagonist prazosin or NO synthase inhibitor L-NAME, indicating that lack of effect of leptin does not result from the balanced activation of SNS and NO.35 Similarly, leptin had no effect on the decrease in renovascular conductance induced by the stimulation of splanchnic sympathetic trunk suggesting that, even if leptin stimulates NO production, its hemodynamic role is negligible.36 In addition, acutely administered leptin did not change vascular conductance in several vascular beds in conscious animals.37 Thus, the physiological significance of vascular NO-mimetic effect of leptin is uncertain. It is possible that leptin stimulates NO production mainly in large conduit arteries which do not contribute to total vascular resistance. In addition, leptin may stimulate NO synthesis in nonvascular tissues such as adipocytes.38 It should be noted that leptin-induced NO production was observed only at pharmacological hormone concentrations. Interestingly, Knudson et al39 observed that although leptin at high concentrations elicited NO-dependent relaxation of isolated rat or canine coronary arterioles, it had no effect on coronary blood flow in anaesthetized dogs in vivo if applied at doses raising its level to the “obese” range. Some studies suggest that leptin may regulate vascular tone also in NO-independent manner. Lembo et al28 observed that leptin decreased blood pressure in rats in which both SNS and NO were pharmacologically inhibited. In our in vivo study26 leptin prevented blood pressure elevation induced by L-NAME suggesting that it might stimulate other depressor mechanisms when NOS is inhibited. In humans, leptin infused into the coronary40 or brachial artery41 induced vasorelaxation insensitive to NOS inhibitors. Apart from NO, vascular endothelium releases two other vasodilating substances i.e., prostacyclin (PGI2) and endothelium-derived hyperpolarizing factor (EDHF). EDHF activity may be accounted for by different mechanisms but cytochrome P450-dependent

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arachidonate metabolites such as various isomers of epoxyeicosatetraenoic acid (EET) seem to be the most important.42,43 The results of one study28 suggest that EDHF is involved in vasodilatory effect of leptin in isolated rat mesenteric arteries, whereas other authors29 did not confirm it. Recently, we have demonstrated that either sulfaphenazole (an inhibitor of EET synthesis) or a mixture of apamin and charybdotoxin (inhibitors of small- and intermediate-conductance potassium channels, respectively, commonly used to block EDHF activity), unmask the pressor effect of leptin in rats pretreated with L-NAME.44 These results suggest that leptin may induce EET/ EDHF-dependent vasorelaxation in vivo when NO availability is reduced. Sahin and Bariskaner45 have demonstrated that vasodilatory effect of leptin on isolated rabbit aorta is totally abolished by endothelial denudation but only partially attenuated by either NOS inhibitor, L-NNA, or by hydrogen peroxide (H2O2) scavenger, catalase, suggesting that H2O2 identified as EDHF in some vascular beds46 may also contribute to vasodilatory effect of leptin. Leptin may regulate vascular tone also independently of the endothelium. For example, although leptin itself had no effect on endothelium-denuded rat aortic rings, it significantly attenuated vasoconstrictor effect of angiotensin II by inhibiting angiotensin II-stimulated Ca2+ release from the intracellular stores.47 This effect of leptin is mediated by PI3K and PKBAkt-dependent stimulation of inducible NO synthase (iNOS) in vascular smooth muscle cells and NO produced by this enzyme inhibits angiotensin II-mediated Ca2+ release from the endoplasmic reticulum.48 Leptin also relaxed isolated human internal mammary artery and saphenous vein in endothelium-independent manner.49 Mechanisms of vasodilatory effect of leptin identified so far are summarized on Figure 1.

Acute Natriuretic Effect of Leptin

Serradeil-Le Gal and coworkers50 first identified specific leptin binding sites in the rat renal medulla and demonstrated significant increase in diuresis following intraperitoneal leptin injection in mice. Subsequently, other studies demonstrated that leptin administered intraperitoneally,51 intravenously,27,52 or locally to the renal artery53 increased natriuresis without affecting renal blood flow, glomerular filtration rate and potassium excretion, suggesting that natriuretic effect of leptin results from the inhibition of tubular Na+ reabsorption. Tubular sodium transport is driven by Na+, K+-ATPase contained in the basolateral membranes of tubular cells.54 We have demonstrated that either systemically51 or locally55 administered leptin induces a time- and dose-dependent decrease in Na+, K+-ATPase activity in the renal medulla but not in the renal cortex. The effect confined to the renal medulla is consistent with exclusive localization of leptin receptors in this region, presumably in medullary collecting duct.56 Moreover, potassium-sparing properties of leptin suggest that it inhibits Na+ reabsorption in medullary collecting duct—a terminal part of the nephron not involved in K+ transport. However, the effect of leptin on tubular Na+ transport has not been directly studied so far, either in isolated nephron segments or in cultured tubular cells. Nitric oxide is continuously produced by renal tubular cells and inhibits Na+ transport, in part by reducing Na+, K+-ATPase activity in a cGMP and protein kinase G (PKG)-dependent manner.57,58 Taking into account that leptin stimulates NO production by endothelial cells, it could be hypothesized that NO is also involved in the natriuretic effect of this hormone. Indeed, Villarreal et al59 have demonstrated that administration of L-NAME for 4 days attenuates the natriuretic effect of acutely injected leptin. In contrast, we observed that inhibitors of NO-cGMP-PKG pathway had no effect on the inhibition of renal medullary Na+, K+-ATPase by leptin.55 The reason for these discrepancies is unclear and thus the involvement of NO in acute natriuretic effect of leptin remains controversial. Sweeney et al60 have demonstrated that the inhibitory effect of leptin on Na+, K+-ATPase in cultured 3T3-L1 fibroblasts is abolished by specific inhibitors of PI3K. PI3K is involved in the downregulation of Na+, K+-ATPase in renal tubular cells by mediating its endocytosis from the plasma membrane to inactive intracellular pool.61 These data led us to hypothesize that leptin might inhibit sodium pump in PI3K-dependent manner. Indeed, we observed that two specific PI3K inhibitors, wortmannin and LY294002, prevented the inhibition of renal Na+, K+-ATPase by leptin infused into the renal artery55 as well as the increase in natriuresis after bolus intravenous leptin

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Figure 1. Mechanisms of acute vasodilatory effect of leptin. Leptin stimulates protein kinase B (PKB) in endothelial cells and PKB then phosphorylates and activates endothelial nitric oxide synthase (eNOS) which generates nitric oxide (NO) from L-arginine (L-Arg). NO diffuses to smooth muscle cells and stimulates soluble guanylyl cyclase (sGC) which synthesizes cyclic GMP from GTP; cGMP than induces vasorelaxation. Insulin, by acting on the insulin receptor (IR), also activates PKB-eNOS pathway but, unlike leptin, uses phosphoinositide 3-kinase (PI3K) as an intermediary signaling step. Apart from NO, leptin may stimulate endothelial production of epoxyeicosatetraenoic acids (EETs) and hydrogen peroxide (H2O2) both being endothelium-derived hyperpolarizing factors in various vascular beds. In addition, leptin, by acting directly on smooth muscle cells, inhibits angiotensin II (AngII)-stimulated vasoconstriction. Herein leptin activates inducible NO synthase (iNOS) to generate NO, which inhibits AngII-stimulated calcium release from the endoplasmic reticulum. Abbreviations: Ob-R: leptin receptor. AT1R: angiotensin AT1 receptor, PIP2: phosphatidylinositol bisphosphate, DAG: diacylglycerol, IP3: inositol triphosphate.

injection.27 Interestingly, PKB/Akt, a common downstream pathway of PI3K, is not involved in natriuretic effect of leptin,33 which is consistent with the findings that PI3K downregulates sodium pump by directly interacting with it rather than by activating PKB/Akt.62,63 Blockade of endogenous leptin by a specific antibody reduced natriuresis induced in the rat by mild volume expansion.64 In addition, leptin expression in adipose tissue increases in response to high-salt diet.65 These data suggest that renal effect of leptin may be relevant for maintaining Na+ balance, especially after ingesting high-salt meal.

Selective and Peripheral Leptin Resistance Concept of Selective Leptin Resistance

In contrast to leptin-deficient ob/ob mice, plasma leptin concentration is significantly elevated in animals with dietary-induced obesity as well as in obese humans reflecting the state of hypothalamic leptin resistance. Correia et al66 proposed that obesity is accompanied by selective leptin

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resistance, i.e., impairment of appetite-suppressing effect and preserved sympathoexcitatory activity. This might lead, due to hyperleptinemia, to overactivation of the SNS and BP elevation. This concept has been confirmed by the experiments performed in agouti obese mice. In these animals agouti protein—an endogenous melanocortin receptor antagonist normally expressed only in the skin—is ectopically expressed and blocks the anorectic effect of leptin-melanocortin pathway mediated by hypothalamic MC3 and MC4 receptors. Consequently, agouti mice are obese, hyperinsulinemic and leptin-resistant. In these animals either peripherally66 or intracerebroventricularly67 administered leptin exerts less marked effects on food intake and body weight but stimulates renal SNA similarly as in lean wild-type controls. The similar selective leptin resistance was observed in mice made obese by high-fat diet.68 In addition, in the latter study chronic leptin administration increased BP to the similar extent in lean and obese groups, suggesting that renal SNA is crucial for BP elevation and that acute hypertensive effect of leptin is not affected by leptin resistance. Interestingly, centrally infused synthetic MC3R/MC4R agonist exerts a comparable effects on food intake in rats fed normal-fat or high-fat diets, indicating that stimulation of melanocortin synthesis by leptin rather than signaling pathways downstream from melanocortins is impaired in obesity.70 The mechanism of selective resistance to leptin is incompletely elucidated. It was hypothesized that leptin’s effect is specifically impaired in the arcuate nucleus involved in the regulation of BAT thermogenesis, but not in ventromedial hypothalamus which mediates cardiovascular responses.70

Peripheral Leptin Resistance

Although initial studies suggested that resistance of hypothalamic centers to anorectic effect of circulating leptin is mainly associated with impaired hormone transport across the blood—brain barrier, later studies demonstrated that leptin resistance is also observed following central administration of this hormone due to receptor or postreceptor signaling defects. These data, together with increasing number of peripheral effects of leptin being described, led us to ask if peripheral effects of leptin on vascular NO or natriuresis are preserved or impaired in obesity. To address this issue, we tested the effect of leptin in rats made obese by feeding highly palatable diet for 4 weeks.51,71 This model of obesity is characterized by moderate increase in body weight and plasma leptin concentration but plasma glucose, lipid profile, insulin concentration and basal blood pressure are still normal. Thus, the model represents early uncomplicated phase of obesity and thus the results are not confounded by abnormalities of carbohydrate and lipid metabolism.51,71 We observed that the effects of leptin on NO production, natriuresis and renal Na+, K+-ATPase were impaired in obese animals. Thus, leptin resistance is not confined to the CNS but involves also some peripheral actions of this hormone. Subsequently, other examples of peripheral leptin resistance have been described including some effects on the cardiovascular system (Table 1).72-79

Mechanisms of Peripheral Leptin Resistance

The mechanism of “peripheral” leptin resistance is not clear at present. Prolonged hyperleptinemia associated with obesity may result in the downregulation of leptin receptors or postreceptor signaling mechanisms. For example, reduced number of leptin receptors was observed in the kidney of obese rats80 and dogs.81 Downregulation of Ob-Ra but not Ob-Rb upon prolonged exposure to leptin was also observed in vitro in human aortic smooth muscle cells.82 In contrast, expression of both Ob-Ra and Ob-Rb is enhanced in aortic smooth muscle cells of spontaneously hypertensive rats (SHR) which are hyperleptinemic and leptin resistant suggesting that downregulation of leptin receptors is not universally observed in hyperleptinemic states.74 Resistance to natriuretic effect of leptin was also observed in SHR52 and renal denervation restored sensitivity to leptin in these animals,72 suggesting that increased renal sympathetic activity counteracts natriuretic effect of leptin. Because insulin augments the stimulatory effect of leptin on vascular NO production,34 insulin resistance may contribute to leptin resistance at the vascular level. Among postreceptor signaling pathways, two mechanisms attracted special attention as potential contributors to central leptin resistance: suppressor of cytokine signaling-3 (SOCS-3) and protein tyrosine phosphatase-1B (PTP-1B).83 SOCS-3 is induced upon activation of the leptin receptor and

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Table 1. Examples of peripheral leptin resistance Effect of Leptin Which is Impaired

Cause of Resistance

Mechanism of Resistance

Implications of Resistance Ref.

↑ Systemic NO production

Diet-induced obesity

?

Unbeneficial 71

↑ Natriuresis

Diet-induced obesity

?

Unbeneficial 51

↑ Natriuresis

SHR vs WKY

Increased SNS Unbeneficial 52,72 activity

NO-dependent relaxation of rat mesenteric arteries

Rats fed high-salt vs normal-salt diet

?

Unbeneficial 73

↓ AII-induced contraction of aortic rings

SHR vs WKY

Postreceptor

Unbeneficial 74

↑ NO production by HAECs in vitro

Prolonged exposure to leptin ↑ Expression of SOCS-3

Unbeneficial 75

↓ Acetylcholine-induced relaxation of the coronary arterioles

High-fat diet vs normal diet

?

Beneficial

76

↑ NHE-1 in erythrocytes

Obese vs lean subjects

?

?

77

↓ Myocardial contractility

SHR vs WKY

Postreceptor

Beneficial

78

↑ Platelet aggregation

Obese vs lean humans

?

Beneficial

79

NO: nitric oxide; SHR: spontaneously hypertensive rats; WKY: normotensive Wistar-Kyoto rats; SNS: sympathetic nervous system; AII: angiotensin II; HAECs: human aortic endothelial cells; SOCS-3: suppressor of cytokine signaling-3; NHE-1: sodium/proton exchanger-1.

inhibits leptin signaling by binding to the receptor itself as well as to the key downstream kinase, JAK2. SOCS-3 is overexpressed in the hypothalamus in various models of leptin resistance such as dietary-induced obesity. Recently, it has been demonstrated that stimulatory effect of leptin on NO production in human aortic endothelial cells is impaired upon prolonged exposure to this hormone, which is associated with enhanced SOCS-3 expression.75 PTP-1B dephosphorylates proteins phosphorylated after activation of the leptin receptor such as JAK2 and STAT-3. Both leptin and dietary-induced obesity induce overexpression of PTP-1B in the liver.84,85 However, it is unknown if enhanced activity of this phosphatase contributes to peripheral leptin resistance at the vascular and/or renal level.

EDHF—A Backup Vasodilatory Mechanism in Obesity

If stimulatory effect of leptin on SNS is intact while its influence on NO and natriuresis is impaired, one could speculate that leptin should increase blood pressure in obese animals. Surprisingly, we observed that a single intraperitoneal leptin injection had no effect on BP also in rats made obese by feeding high-calorie diet for 1-month.71 However, leptin increased BP in obese rats pretreated with either apamin+charybdotoxin or sulfaphenazole, suggesting that EDHF/ EET compensate for impaired NO in obese animals.86 Moreover, if feeding rats a high-calorie diet was prolonged to 3 months, leptin elevated BP even without pretreatment with EDHF inhibitors and these inhibitors had no additional effect on BP.44 The stimulatory effect of leptin on NO production was similarly impaired in 1-month and 3-month obese groups. These data indicate that EDHF/EET-dependent mechanism compensates for NO deficiency in 1-month obese group, but this mechanism becomes also impaired in the 3-months obese group (Fig. 2).

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Figure 2. Time-dependent effect of dietary-induced obesity on vasoconstrictor (sympathetic nervous system, SNS) and vasodilatory (nitric oxide, NO and endothelium-derived hyperpolarizing factor, EDHF) mechanisms. Normally, leptin activates SNS and vascular NO resulting in no net changes of blood pressure. In obese animals leptin concentration is increased leading to overstimulation of the SNS. However, effect of leptin on vascular NO is impaired. In rats made obese by 1-month high calorie diet lack of NO is compensated by EDHF and therefore leptin still does not change BP. In contrast, in 3-month obesity effects of leptin on both NO and EDHF are impaired resulting in unbalanced SNS stimulation and BP elevation (reproduced with permission from Bełtowski J, Life Sci 2006; 79:63-71,44 with modification).

These results are consistent with the observations that NO inhibits EDHF under baseline conditions and that EDHF becomes an important vasodilatory mechanism when NO generation is impaired.86 It is unclear why EDHF/EET-dependent vasorelaxation is impaired in the 3-month but not in the 1-month obese group, but several differences between these groups were observed. Dyslipidemia (reduced HDL-cholesterol and increased triglycerides), oxidative stress (increased plasma isoprostanes) and hyperinsulinemia (suggestive of insulin resistance) were observed only in the 3-month obese group; thus one or more of these abnormalities could be responsible for impaired effect of leptin on EDHF in this group.44

Prohypertensive Effects of Chronic Hyperleptinemia

The initial hypothesis about role of leptin in arterial hypertension was that obesity-associated hyperleptinemia results in BP elevation due to excessive activation of the SNS and impaired depressor (vasodilatory and natriuretic) mechanisms. According to this concept, leptin elevates BP exclusively by activating SNS. Indeed, hypertensive effect of intravenously infused leptin was abolished by combined blockade of α- and β-adrenergic receptors.87 Adrenergic antagonists normalize BP also in rats overexpressing leptin.11 Moreover, BP is not elevated in obese and hyperleptinemic MC4R−/− mice88 and exogenous leptin fails to elevate BP in these animals as well as in MC4R−/− mice which are pair-fed and thus nonobese and normally sensitive to leptin.89 Similarly, hypertensive effect of leptin is abolished by synthetic NC3R/MC4R receptor antagonist.17 Given a key role of central melanocortin system in sympathoexcitatory effect of leptin, these data strongly suggest that leptin-induced BP elevation is mediated solely via the SNS. Nevertheless, some studies suggest that SNS activation cannot fully explain the hypertensive effect of leptin90 and thus other peripheral mechanisms should also be considered. Recently, Tümer et al91 have studied the role of leptin in obesity associated hypertension using rat leptin mutein (L39A/D40A/F41A) which acts as leptin antagonist. They examined the effect of leptin antagonist administered intracerebroventricularly on BP in two experimental models: (1) local hypothalamic overexpression of leptin by recombinant adenoviral vector in lean rats with normal plasma leptin concentration, (2) high fat diet-induced obesity. BP was increased in both models but leptin antagonist reduced it only in animals overexpressing leptin in the hypothalamus but not in obese rats. Although authors interpret these findings

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as the evidence that leptin does not contribute to obesity-associated hypertension, an alternative explanation is that peripheral effects of leptin (not inhibited by centrally administered leptin antagonist) may be more important than centrally-mediated sympathoexcitation in obese animals.

Nonsympathetic Hypertensive Effects of Leptin

Several potentially prohypertensive SNS-independent peripheral effects of leptin have been described. For example, leptin stimulates vasoconstrictor endothelin-1 (ET-1) production by cultured endothelial92 and vascular smooth muscle cells.93 Although this effect is not observed in some endothelial cell lines,94 infusion of leptin for 28 days increased serum ET-1 level in the rat.95 Moreover, leptin increases the expression of endothelin ETA receptors in cultured endothelial cells96 and the expression of angiotensinogen and angiotensin AT1 receptors in vascular smooth muscle cells.97 Other authors observed the stimulatory effects of leptin on vascular smooth muscle cell hypertrophy98-99 and proliferation,100,101 indicating that this hormone may increase vascular tone by inducing remodeling of the arteriolar wall. Finally, hyperleptinemia may lead to the development of nephropathy independently of BP elevation.95

Hyperleptinemia, Oxidative Stress, NO Production and Endothelial Function

Recent studies point to the role of oxidative stress in arterial hypertension.102 Oxidative stress, i.e., imbalance between the amount of reactive oxygen species (ROS) formed and antioxidant defense mechanisms, promotes hypertension through scavenging endothelial NO by superoxide anion radical (O2−), which binds NO to form peroxynitrite (ONOO−) and thus removes its tonic vasodilatory effect. Leptin stimulates ROS formation by endothelial cells.103 Moreover, it has been demonstrated that ROS are involved in the pathogenesis of obesity-associated hypertension.104 These data led us to study the effect of chronically elevated leptin on oxidative stress and NO availability. For this purpose, we induced experimental hyperleptinemia in normal healthy rats by administering exogenous hormone at a dose of 0.5 mg/kg/day for 7 days; during this treatment plasma leptin level is elevated to the “obese” range. Although this model does not reproduce all abnormalities associated with obesity, it allows studying the effect of leptin itself without confounding obesity-associated disturbances. Consistently with other studies, we observed increase in blood pressure in leptin-treated rats. Leptin stimulated systemic oxidative stress as evidenced by increase in plasma concentration and urinary excretion of isoprostanes. In addition, whole-body NO production assessed as urinary NOx and cGMP excretion was impaired,105 whereas the level of nitrotyrosine, the marker of peroxynitrite formation, was increased in hyperleptinemic rats. These effects were prevented by coadministration of O2− scavenger, tempol, or NADPH oxidase inhibitor, apocynin106 suggesting that NADPH oxidase-derived superoxide curtails NO availability in leptin-treated animals. However, biochemical parameters such as markers of NO formation are only indirect measures of endothelial function. Effect of leptin on endothelial function measured directly as vascular relaxation in response to endothelium-dependent vasodilators such as acetylcholine was examined in several experimental studies. Although the results of these studies are controversial, they suggest that leptin may indirectly improve endothelial function by beneficially modulating the metabolic profile (e.g., by reducing blood lipids, increasing insulin sensitivity etc.) in aged obese rats, rats with streptozotocin-induced diabetes or leptin-deficient ob/ob mice, but impairs endothelial function in healthy normolipidemic animals with normal insulin sensitivity.32,39,107-109 Several lines of evidence support unfavorable effect of leptin on oxidative stress and NO generation in humans. For example, inverse correlation between plasma leptin and NO production was observed in patients with ischemic heart disease who developed restenosis after coronary angioplasty.110 In one study111 plasma leptin was higher whereas NOx were lower in obese hypertensive than in obese normotensive subjects, suggesting the possible link between hyperleptinemia, NO deficiency and elevated blood pressure. Plasma leptin was also positively correlated with markers of oxidative stress such as the level of oxidized LDL112 or urinary excretion of isoprostanes.113 Finally, an inverse relationship between circulating leptin and endothelium-dependent vasorelaxation was reported in humans.114

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Hyperleptinemia, Oxidative Stress, Intrarenal NO Production and Renal Sodium Handling

NO deficiency promotes blood pressure elevation not only by causing vasoconstriction but also due to the loss of the inhibitory effect of NO on renal tubular Na+ reabsorption.115 Like in the vasculature, one of the mechanisms leading to intrarenal NO deficiency is oxidative stress. Even under physiological conditions superoxide is continuously produced in the kidney, mainly by NADPH oxidase and limits the inhibitory effect of NO on Na+ transport and renal Na+, K+-ATPase.116,117 We observed that total and fractional Na+ excretion was reduced in rats receiving leptin for 7 days despite unchanged glomerular filtration, which indicates enhanced tubular Na+ reabsorption. Consistently with this, Na+, K+-ATPase activity was higher in leptin-treated than in control group.118 Moreover, leptin increased intrarenal oxidative stress and reduced renal NO formation. All these effects were prevented by either tempol or apocynin.119 Taken together, these data indicate that chronic leptin administration induces ROS-mediated NO deficiency not only in blood vessels but also in the kidney, leading to the enhancement of Na+, K+-ATPase-dependent Na+ reabsorption (Fig. 3). Other studies about effect of hyperleptinemia on renal Na+ handling gave conflicting results. For example, Shek et al10 and Kuo et al120 observed that a 5-day systemic leptin infusion had no effect on Na+ excretion. However, in these studies Na+ intake was clamped by saline administration and was

Figure 3. Effect of chronic hyperleptinemia on renal tubular Na + -transporting pumps. Leptin stimulates NADPH oxidase (NOX) which generates superoxide anion radical (O2−). Superoxide reacts with nitric oxide (NO) to form peroxynitrite (ONOO−) and thus curtails the inhibitory effect of NO on active sodium reabsorption. NO stimulates soluble guanylyl cyclase (sGC) to generate cGMP and cGMP inhibits both Na+, K+ -ATPase and ouabain-resistant Na + -ATPase. However, Na +, K+ -ATPase is inhibited in protein kinase G (PKG)-dependent manner whereas Na + -ATPase is inhibited because cGMP stimulates (in PKG-independent manner) phosphodiesterase 2 (PDE2) and decreases cAMP concentration. Apart from NO, cGMP synthesis is stimulated by atrial natriuretic peptide (ANP) binding to membrane receptors (particulate guanylyl cyclase, pGC). Hyperleptinemia induces resistance to ANP by stimulating degradation of cGMP by phosphodiesterase 5 (PDE5). The scheme presents mechanisms operating in different nephron segments; Na +, K+ -ATPase is inhibited by ANP in the medullary collecting duct whereas Na+ -ATPase is expressed only in the proximal tubule. Localization of ANP and leptin receptors demonstrated above does not necessarily reproduce their real localization in polarized tubular cells but was chosen arbitrarily for clarity.

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not reduced despite leptin-induced decrease in food intake. In contrast, in our studies Na+ intake was reduced in leptin-treated animals which could unmask antinatriuretic mechanisms, although leptin-induced changes were not reproduced by pair-feeding itself. Nevertheless, the observation that leptin had no effect on Na+ excretion despite BP elevation10,121 suggests that pressure-natriuresis relationship was impaired in leptin-treated animals. Gunduz et al97 observed that leptin infused at a dose of 20 µg/kg/day for 28 days increased Na+ excretion despite no changes in blood pressure, suggesting chronic natriuretic effect of this hormone. In that study95 leptin was administered for a much longer time than in other studies10,118-120 and induced marked renal damage evidenced by histological lesions and proteinuria. Na+ excretion was measured only once at the end of the experiment in the Gunduz’s study95 and it is unlikely that natriuresis was increased throughout a 4-week leptin administration since in that case a marked Na+ depletion would occur. Thus, it is likely that natriuresis was a late event associated with renal damage.95 At present it is unknown if chronic hyperleptinemia affects renal function in humans. In one study121 plasma leptin inversely correlated with lithium clearance which suggests that high leptin was associated with increased Na+ reabsorption in the proximal tubule. These findings suggest that chronically elevated leptin has antinatriuretic effect in humans.

Hyperleptinemia and Renal Ouabain-Resistant Na +-ATPase

Apart from Na+, K+-ATPase, renal tubules contain the second sodium pump, ouabain-resistant Na+-ATPase, also referred to as “the second sodium pump.” Unlike Na+, K+-ATPase, Na+-ATPase transports sodium but not potassium, in not inhibited by Na+, K+-ATPase inhibitor, ouabain, but is sensitive to loop diuretics such as furosemide and is expressed only in the proximal tubule but not in other nephron segments. Although this enzyme transport only 10-15% of sodium reabsorbed in the proximal tubule, changes in its activity may cause marked alterations of natriuresis. The mechanisms regulating Na+-ATPase are poorly characterized, however, its activity is affected by many mediators controlling Na+ balance such as angiotensin II, nitric oxide and natriuretic peptides.122 We have demonstrated that acutely administered leptin does not change renal Na+-ATPase activity suggesting that this pump is not involved in acute natriuretic effect of leptin.123 In contrast, Na+-ATPase activity was increased in the renal cortex of rats treated with leptin for 7 days.123 Using pharmacological inhibitors of various signaling pathways, we have demonstrated that, similarly to Na+, K+-ATPase, Na+-ATPase stimulation in hyperleptinemic rats results from O2− -dependent NO deficiency. However, in contrast to Na+, K+-ATPase which is inhibited by NO in the cGMP-PKG dependent manner, Na+-ATPase is inhibited by NO-cGMP pathway through the PKG-independent mechanism. Rather, cGMP stimulates phosphodiesterase 2 which degrades cAMP and removes its tonic stimulatory effect on ouabain-resistant Na+ pump.124 Leptin stimulates ROS formation leading to NO degradation and thus downregulates this inhibitory mechanism. Consequently, the stimulatory effect of cAMP on ouabain-resistant Na+-ATPase is enhanced (Fig. 3).

Role of Leptin in Renal Resistance to Atrial Natriuretic Peptide

Apart from NO, atrial natriuretic peptide (ANP) and a related intrarenally produced peptide, urodilatin, inhibit Na+ reabsorption through the cGMP-PKG dependent mechanism.125 Interestingly, in our model of hyperleptinemia plasma ANP and urinary urodilatin concentrations were increased.105,118 Reduced natriuresis and cGMP generation despite increased ANP suggest that the kidneys become resistant to natriuretic peptides following leptin treatment. Indeed, increase in cGMP and Na+ excretion in response to exogenous ANP was less marked in hyperleptinemic than in control rats.126 This effect was corrected by inhibitor of cGMP-specific phosphodiesterase (PDE5), zaprinast, suggesting that enhanced cGMP degradation by PDE5 is responsible for this phenomenon. In addition, resistance to ANP and its correction by zaprinast were observed in rats made obese by high-calorie diet thus indicating that hyperleptinemia could contribute to renal unresponsiveness to natriuretic peptides observed in obese subjects.127 Although increased PDE5-dependent cGMP degradation is a common mechanism of renal unresponsiveness to ANP in various disease states, the mechanism through which leptin enhances PDE5 is unclear.

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Figure 4. Mechanisms through which leptin oversecreted by adipose tissue in obese subjects contributes to blood pressure elevation by increasing vascular tone (left) and impairing renal Na + excretion (right). Leptin stimulates sympathetic nervous system (SNS) at the level of hypothalamus and norepinephrine (NE) released by sympathetic fibers induces vasoconstriction and enhances tubular Na + reabsorption. In addition, leptin has direct effects on the vascular wall and the kidney. ET-1: endothelin-1; ETAR: endothelin ETA receptors; ANGG: angiotensinogen; AT1R: angiotensin type 1 receptor; SMC: smooth muscle cells; ROS: reactive oxygen species; NO: nitric oxide; ANP: atrial natriuretic peptide.

Interestingly, natriuretic effect of synthetic NO donor was intact in either leptin-treated or obese rats, indicating that various intracellular pools of cGMP mediate the effects of ANP and NO on renal Na+ transport.126 Vascular and renal mechanisms through which chronically elevated leptin may increase blood pressure are summarized on Figure 4.

Conclusions and Future Perspectives

Identification of leptin in 1994 opened a new era in obesity research and also markedly contributed to our understanding of cardiovascular complications of obesity. Many experimental and clinical studies strongly suggest the involvement of leptin in the regulation of blood pressure and in the pathogenesis of arterial hypertension but the precise mechanisms are incompletely elucidated. There is little doubt that leptin stimulates the SNS and may be a major cause of its overactivity in obese subjects. In general, there are two principal mechanisms through which leptin may contribute to BP elevation: (1) unbalanced stimulation of the SNS and resistance to acute depressor (natriuretic and vasodilatory) effects of this hormone, (2) direct prohypertensive effects

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of chronic hyperleptinemia. It is difficult to differentiate between these two possibilities, especially in humans. Although hyperleptinemia has been observed in hypertensive subjects, it is unclear what is more important: excess of leptin per se or leptin resistance reflected by hyperleptinemia. Although much work is still to be done in this field, this effort may result in developing novel therapeutic strategies for obesity-associated hypertension such as ameliorating selective leptin resistance or administration of leptin antagonists.

References

1. Wofford MR, Hall JE. Pathophysiology and treatment of obesity hypertension. Curr Pharm Des 2004; 10:3621-37. 2. Eckel RH, Grundy SM, Zimmet PZ. The metabolic syndrome. Lancet 2005; 365:1415-28. 3. Wong C, Marwick TH. Obesity cardiomyopathy: Pathogenesis and pathophysiology. Nat Clin Pract Cardiovasc Med 2007; 4:436-43. 4. Cignarelli M, Lamacchia O. Obesity and kidney disease. Nutr Metab Cardiovasc Dis 2007; 17:757-62. 5. Hall JE. The kidney, hypertension and obesity. Hypertension 2003; 41:625-33. 6. Frühbeck G. The adipose tissue as a source of vasoactive factors. Curr Med Chem Cardiovasc Hematol Agents 2004; 2:197-208. 7. Matsuzawa Y. The metabolic syndrome and adipocytokines. FEBS Lett 2006; 580:2917-21. 8. Correia ML, Rahmouni K. Role of leptin in the cardiovascular and endocrine complications of metabolic syndrome. Diabetes Obes Metab 2006; 8:603-10. 9. Bełtowski J. Role of leptin in blood pressure regulation and arterial hypertension. J Hypertens 2006; 24:789-801. 10. Shek EW, Brands MW, Hall JE. Chronic leptin infusion increases arterial pressure. Hypertension 1998; 31:409-414. 11. Aizawa-Abe M, Ogawa Y, Masuzaki H et al. Pathophysiological role of leptin in obesity-related hypertension. J Clin Invest 2000; 105:1243-52. 12. Agata J, Masuda A, Takada M et al. High plasma immunoreactive leptin level in essential hypertension. Am J Hypertens 1997; 10:1171-4. 13. Mark AL, Shaffer RA, Correia ML et al. Contrasting blood pressure effects of obesity in leptin-deficient ob/ob mice and agouti yellow obese mice. J Hypertens 1999; 17:1949-53. 14. Haynes WG, Morgan DA, Walsh SA et al. Receptor-mediated regional sympathetic nerve activation by leptin. J Clin Invest 1997; 100:270-8. 15. Tanida M, Iwashita S, Ootsuka Y et al. Leptin injection into white adipose tissue elevates renal sympathetic nerve activity dose-dependently through the afferent nerves pathway in rats. Neurosci Lett 2000; 293:107-10. 16. Dunbar JC, Hu Y, Lu H. Intracerebroventricular leptin increases lumbar and renal sympathetic nerve activity and blood pressure in normal rats. Diabetes 1997; 46:2040-43. 17. da Silva AA, Kuo JJ, Hall JE. Role of hypothalamic melanocortin 3/4-receptors in mediating chronic cardiovascular, renal and metabolic actions of leptin. Hypertension 2004; 43:1312-17. 18. Song CK, Jackson RM, Harris RBS et al. Melanocortin-4 receptor mRNA is expressed in sympathetic nervous system outflow neurons to white adipose tissue. Am J Physiol Regulatory Integrative Comp Physiol 2005; 289:R1467-76. 19. Rahmouni K, Morgan DA. Hypothalamic arcuate nucleus mediates the sympathetic and arterial pressure responses to leptin. Hypertension 2007; 49:647-52. 20. Esler M, Straznicky N, Eikelis N et al. Mechanisms of sympathetic activation in obesity-related hypertension. Hypertension 2006; 48:787-96. 21. Rosenbaum M, Goldsmith R, Bloomfield D et al. Low-dose leptin reverses skeletal muscle, autonomic and neuroendocrine adaptations to maintenance of reduced weight. J Clin Invest 2005; 115:3579-86. 22. Mancia G, Bousquet P, Elghozi JL et al. The sympathetic nervous system and the metabolic syndrome. J Hypertens 2007; 25:909-20. 23. Casto RM, VanNess JM, Overton JM. Effects of central leptin administration on blood pressure in normotensive rats. Neurosci Lett 1998; 246:29-32. 24. Gardiner SM, Kemp PA, March JE et al. Regional haemodynamic effects of recombinant murine or human leptin in conscious rats. Br J Pharmacol 2000; 130:805-10. 25. Frühbeck G. Pivotal role of nitric oxide in the control of blood pressure after leptin administration. Diabetes 1999; 48:903-8. 26. Bełtowski J, Wójcicka G, Borkowska E. Human leptin stimulates systemic nitric oxide production in the rat. Obes Res 2002; 10:939-46.

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27. Bełtowski J, Jochem J, Wójcicka G et al. Influence of intravenously administered leptin on nitric oxide production, renal hemodynamics and renal function in the rat. Regul Pept 2004; 120:59-67. 28. Lembo G, Vecchione C, Fratta L et al. Leptin induces direct vasodilation through distinct endothelial mechanisms. Diabetes 2000; 49:293-7. 29. Kimura K, Tsuda K, Baba A et al. Involvement of nitric oxide in endothelium-dependent arterial relaxation by leptin. Biochem Biophys Res Commun 2000; 273:745-9. 30. Mohammed MM, Myers DS, Sofola OA et al. Vasodilator effects of leptin on canine isolated mesenteric arteries and veins. Clin Exp Pharmacol Physiol 2007; 34:771-4. 31. Vecchione C, Maffei A, Colella S et al. Leptin effect on endothelial nitric oxide is mediated through Akt-endothelial nitric oxide synthase phosphorylation pathway. Diabetes 2002; 51:168-73. 32. Winters B, Mo Z, Brooks-Asplund E et al. Reduction of obesity, as induced by leptin, reverses endothelial dysfunction in obese (Lepob) mice. J Appl Physiol 2000; 89:2382-90. 33. Bełtowski J, Wójcicka G, Jamroz-Wiśniewska A et al. Role of PI3K and PKB/Akt in acute natriuretic and NO-mimetic effects of leptin. Regul Pept 2007; 140:168-77. 34. Vecchione C, Aretini A, Maffei A et al. Cooperation between insulin and leptin in the modulation of vascular tone. Hypertension 2003; 42:166-70. 35. Mitchell JL, Morgan DA, Correia ML et al. Does leptin stimulate nitric oxide to oppose the effects of sympathetic activation? Hypertension 2001; 38:1081-86. 36. Jalali A, Morgan DA, Sivitz WI et al. Does leptin cause functional peripheral sympatholysis? Am J Hypertens 2001; 14:615-18. 37. Rahmouni K, Jalali A, Morgan DA et al. Lack of dilator effect of leptin in the hindlimb vascular bed of conscious rats. Eur J Pharmacol 2005; 518:175-81. 38. Frühbeck G, Gomez-Ambrosi J. Modulation of the leptin-induced white adipose tissue lipolysis by nitric oxide. Cell Signal 2001; 13:827-33. 39. Knudson JD, Dincer UD, Zhang C et al. Leptin receptors are expressed in coronary arteries and hyperleptinemia causes significant coronary endothelial dysfunction. Am J Physiol Heart Circ Physiol 2005; 289:H48-H56. 40. Matsuda K, Teragawa H, Fukuda Y et al. Leptin causes nitric-oxide independent coronary artery vasodilation in humans. Hypertens Res 2003; 26:147-52. 41. Nakagawa K, Higashi Y, Sasaki S et al. Leptin causes vasodilation in humans. Hypertens Res 2002; 25:161-5. 42. Zhou MS, Raij L. Cross-talk between nitric oxide and endothelium-derived hyperpolarizing factor: synergistic interaction? J Hypertens 2003; 21:1449-51. 43. Campbell WB, Falck JR. Arachidonic acid metabolites as endothelium-derived hyperpolarizing factors. Hypertension 2007; 49:590-6. 44. Bełtowski J, Wójcicka G, Jamroz-Wiśniewska A. Role of nitric oxide and endothelium-derived hyperpolarizing factor (EDHF) in the regulation of blood pressure by leptin in lean and obese rats. Life Sci 2006; 79:63-71. 45. Sahin AS, Bariskaner H. The mechanisms of vasorelaxant effect of leptin on isolated rabbit aorta. Fundam Clin Pharmacol 2007; 21:595-600. 46. Ellis A, Triggle CR . Endothelium-derived reactive oxygen species: their relationship to endothelium-dependent hyperpolarization and vascular tone. Can J Physiol Pharmacol 2003; 81:1013-28. 47. Fortuno A, Rodriguez A, Gomez-Ambrosi J et al. Leptin inhibits angiotensin II-induced intracellular calcium increase and vasoconstriction in the rat aorta. Endocrinology 2002; 143:3555-60. 48. Rodríguez A, Fortuno A, Gómez-Ambrosi J et al. The inhibitory effect of leptin on angiotensin II-induced vasoconstriction in vascular smooth muscle cells is mediated via a nitric oxide-dependent mechanism. Endocrinology 2007; 148:324-31. 49. Momin AU, Melikian N, Shah AM et al. Leptin is an endothelial-independent vasodilator in humans with coronary artery disease: Evidence for tissue specificity of leptin resistance. Eur Heart J 2006; 27:2294-9. 50. Serradeil-Le Gal C, Raufaste D, Brossard G et al. Characterization and localization of leptin receptors in the rat kidney. FEBS Lett 1997; 404:185-91. 51. Bełtowski J, Wójcicka G, Górny D et al. Human leptin administered intraperitoneally stimulates natriuresis and decreases renal medullary Na+, K+-ATPase activity in the rat—Impaired effect in dietary-induced obesity. Med Sci Monit 2002; 8:BR221-29. 52. Villarreal D, Reams G, Freeman RH et al. Renal effects of leptin in normotensive, hypertensive and obese rats. Am J Physiol 1998; 275:R2056-60. 53. Jackson EK, Li P. Human leptin has natriuretic activity in the rat. Am J Physiol 1997; 272:F333-38. 54. Feraille E, Doucet A. Sodium-potassium-adenosinetriphosphatase-dependent sodium transport in the kidney: hormonal control. Physiol Rev 2001; 81:345-418.

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55. Bełtowski J, Marciniak A, Wójcicka G. Leptin decreases renal medullary Na+, K+-ATPase activity through phosphatidylinositol 3-kinase dependent mechanism. J Physiol Pharmacol 2004; 55:391-407. 56. Martinez-Anso E, Lostao MP, Martinez JA. Immunohistochemical localization of leptin in rat kidney. Kidney Int 1999; 55:1129-30. 57. Herrera M, Ortiz PA, Garvin JL. Regulation of thick ascending limb transport: role of nitric oxide. Am J Physiol Renal Physiol 2006; 290:F1279-84. 58. Bełtowski J, Marciniak A, Wójcicka G et al. Nitric oxide decreases renal medullary Na+, K+-ATPase activity through cyclic GMP-protein kinase G dependent mechanism. J Physiol Pharmacol 2003; 54:191-210. 59. Villarreal D, Reams G, Samar H et al. Effects of chronic nitric oxide inhibition on the renal excretory response to leptin. Obes Res 2004; 12:1006-10. 60. Sweeney G, Niu W, Kanani R et al. Regulation of the Na, K-pump by leptin in 3T3-L1 fibroblasts. Endocrinology 2000; 141:1277-80. 61. Chibalin AV, Zierath JR, Katz AI et al. Phosphatidylinositol 3-kinase-mediated endocytosis of renal Na+, K+-ATPase α subunit in response to dopamine. Mol Biol Cell 1998; 9:1209-20. 62. Yudowski GA, Efendiev R, Pedemonte CH et al. Phosphoinositide-3 kinase binds to a proline-rich motif in the Na+, K+-ATPase α subunit and regulates its trafficking. Proc Natl Acad Sci USA 2000; 97:6556-61. 63. Efendiev R, Chen Z, Komar RT et al. The 14-3-3 protein translates the Na+, K+-ATPase α1-subunit phosphorylation signal into binding and activation of phosphoinositide 3-kinase during endocytosis. J Biol Chem 2005; 280:16272-77. 64. Villarreal D, Reams G, Freeman R et al. Leptin blockade attenuates sodium excretion in saline-loaded normotensive rats. Mol Cell Biochem 2006; 283:153-7. 65. Dobrian AD, Schriver SD, Lynch T et al. Effect of salt on hypertension and oxidative stress in a rat model of diet-induced obesity. Am J Physiol Renal Physiol 2003; 285:F619-28. 66. Correia ML, Haynes WG, Rahmouni K et al. The concept of selective leptin resistance: evidence from agouti yellow obese mice. Diabetes 2002; 51:439-42. 67. Rahmouni K, Haynes WG, Morgan DA et al. Selective resistance to central neural administration of leptin in agouti obese mice. Hypertension 2002; 39:486-90. 68. Rahmouni K, Morgan DA, Morgan GM et al. Role of selective leptin resistance in diet-induced obesity hypertension. Diabetes 2005; 54:2012-18. 69. Silva AA, Kuo JJ, Tallam LS et al. Does obesity induce resistance to the long-term cardiovascular and metabolic actions of melanocortin 3/4 receptor activation? Hypertension 2006; 47:259-64. 70. Münzberg H, Flier JS, Bjorbaek C. Region-specific leptin resistance within the hypothalamus of diet-induced obese mice. Endocrinology 2004; 145:4880-9. 71. Bełtowski J, Wójcicka G, Jamroz A. Stimulatory effect of leptin on nitric oxide production is impaired in dietary-induced obesity. Obes Res 2003; 11:1571-80. 72. Villarreal D, Reams G, Freeman RH. Effects of renal denervation on the sodium excretory actions of leptin in hypertensive rats. Kidney Int 2000; 58:989-94. 73. Jaffar MM, Myers DS, Hainsworth LJ et al. Effects of dietary salt loading on the responses of isolated rat mesenteric arteries to leptin. Am J Hypertens 2005; 18:500-3. 74. Rodríguez A, Frühbeck G, Gómez-Ambrosi J et al. The inhibitory effect of leptin on angiotensin II-induced vasoconstriction is blunted in spontaneously hypertensive rats. J Hypertens 2006; 24:1589-97. 75. Blanquicett C, Graves A, Kleinhenz DJ et al. Attenuation of signaling and nitric oxide production following prolonged leptin exposure in human aortic endothelial cells. J Investig Med 2007; 55:368-77. 76. Knudson JD, Dincer UD, Dick GM et al. Leptin resistance extends to the coronary vasculature in prediabetic dogs and provides a protective adaptation against endothelial dysfunction. Am J Physiol Heart Circ Physiol 2005; 289:H1038-46. 77. Konstantinou-Tegou A, Kaloyianni M, Bourikas D et al. The effect of leptin on Na+-H+ antiport (NHE-1) activity of obese and normal subjects erythrocytes. Mol Cell Endocrinol 2001; 183:11-8. 78. Wold LE, Relling DP, Duan J et al. Abrogated leptin-induced cardiac contractile response in ventricular myocytes under spontaneous hypertension: Role of Jak/STAT pathway. Hypertension 2002; 39:69-74. 79. Corica F, Corsonello A, Lucchetti M et al. Relationship between metabolic syndrome and platelet responsiveness to leptin in overweight and obese patients. Int J Obes 2007; 31:842-9. 80. Coatmellec-Taglioni G, Dausse JP, Giudicelli Y et al. Sexual dimorphism in cafeteria diet-induced hypertension is associated with gender-related difference in renal leptin receptor down-regulation. J Pharmacol Exp Ther 2003; 305:362-7. 81. Gu JW, Wang J, Stockton A et al. Cytokine gene expression profiles in kidney medulla and cortex of obese hypertensive dogs. Kidney Int 2004; 66:713-21. 82. Bohlen F, Kratzsch J, Mueller M et al. Leptin inhibits cell growth of human vascular smooth muscle cells. Vascul Pharmacol 2007; 46:67-71.

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83. Enriori PJ, Evans AE, Sinnayah P et al. Leptin resistance and obesity. Obesity (Silver Spring) 2006; 14(Suppl 5):254S-258S. 84. Lam NT, Lewis JT, Cheung AT et al. Leptin increases hepatic insulin sensitivity and protein tyrosine phosphatase 1B expression. Mol Endocrinol 2004; 18:1333-45. 85. Lam NT, Covey SD, Lewis JT et al. Leptin resistance following over-expression of protein tyrosine phosphatase 1B in liver. J Mol Endocrinol 2006; 36:163-74. 86. Félétou M, Vanhoutte PM. Endothelium-derived hyperpolarizing factor: Where are we now? Arterioscler Thromb Vasc Biol 2006; 26:1215-25. 87. Carlyle M, Jones OB, Kuo JJ et al. Chronic cardiovascular and renal actions of leptin: Role of adrenergic activity. Hypertension 2002; 39:496-501. 88. Tallam LS, Stec DE, Willis MA et al. Melanocortin-4 receptor-deficient mice are not hypertensive or salt-sensitive despite obesity, hyperinsulinemia and hyperleptinemia. Hypertension 2005; 46:326-32. 89. Tallam LS, da Silva AA, Hall JE. Melanocortin-4 receptor mediates chronic cardiovascular and metabolic actions of leptin. Hypertension 2006; 48:58-64. 90. Bernal-Mizrachi C, Weng S, Li B et al. Respiratory uncoupling lowers blood pressure through a leptin-dependent mechanism in genetically obese mice. Arterioscler Thromb Vasc Biol 2002; 22:961-8. 91. Tümer N, Erdös B, Matheny M et al. Leptin antagonist reverses hypertension caused by leptin overexpression, but fails to normalize obesity-related hypertension. J Hypertens 2007; 25:2471-8. 92. Quehenberger P, Exner M, Sunder-Plassmann R et al. Leptin induces endothelin-1 in endothelial cells in vitro. Circ Res 2002; 90:711-18. 93. Chao HH, Hong HJ, Liu JC et al. Leptin stimulates endothelin-1 expression via extracellular signal-regulated kinase by epidermal growth factor receptor transactivation in rat aortic smooth muscle cells. Eur J Pharmacol 2007; 573:49-54. 94. Takahashi K, Totsune K, Kikuchi K et al. Expression of endothelin-1 and adrenomedullin was not altered by leptin or resistin in bovine brain microvascular endothelial cells. Hypertens Res 2006; 29:443-8. 95. Gunduz Z, Dursun N, Akgun H et al. Renal effects of long-term leptin infusion and preventive role of losartan treatment in rats. Regul Pept 2005; 132:59-66. 96. Juan CC, Chuan TY, Lien CC et al. Leptin increases endothelin type A receptor levels in vascular smooth muscle cells. Am J Physiol Endocrinol Metab 2007, in press. 97. Zeidan A, Purdham DM, Rajapurohitam V et al. Leptin induces vascular smooth muscle cell hypertrophy through angiotensin II- and endothelin-1-dependent mechanisms and mediates stretch-induced hypertrophy. J Pharmacol Exp Ther 2005; 315:1075-84. 98. Shin HJ, Oh J, Kang SM et al. Leptin induces hypertrophy via p38 mitogen-activated protein kinase in rat vascular smooth muscle cells. Biochem Biophys Res Commun 2005; 329:18-24. 99. Zeidan A, Paylor B, Steinhoff KJ et al. Actin cytoskeleton dynamics promotes leptin-induced vascular smooth muscle hypertrophy via RhoA/ROCK- and phosphatidylinositol 3-kinase/protein kinase B-dependent pathways. J Pharmacol Exp Ther 2007; 322:1110-6. 100. Oda A, Taniguchi T, Yokoyama M. Leptin stimulates rat aortic smooth muscle cell proliferation and migration. Kobe J Med Sci 2001; 47:141-50. 101. Li L, Mamputu JC, Wiernsperger N et al. Signaling pathways involved in human vascular smooth muscle cell proliferation and matrix metalloproteinase-2 expression induced by leptin: Inhibitory effect of metformin. Diabetes 2005; 54:2227-34. 102. Vaziri ND, Rodríguez-Iturbe B. Mechanisms of disease: Oxidative stress and inflammation in the pathogenesis of hypertension. Nat Clin Pract Nephrol 2006; 2:582-93. 103. Bouloumie A, Marumo T, Lafontan M et al. Leptin induces oxidative stress in human endothelial cells. FASEB J 1999; 13:1231-8. 104. Dobrian AD, Davies MJ, Schriver SD et al. Oxidative stress in a rat model of obesity-induced hypertension. Hypertension 2001; 37:554-60. 105. Bełtowski J, Wójcicka G, Marciniak A et al. Oxidative stress, nitric oxide production and renal sodium handling in leptin-induced hypertension. Life Sci 2004; 74:2987-3000. 106. Bełtowski J, Wójcicka G, Jamroz-Wiœniewska A et al. Antioxidant treatment normalizes nitric oxide production, renal sodium handling and blood pressure in experimental hyperleptinemia. Life Sci 2005; 77:1855-68. 107. Zanetti M, Barazzoni R, Vadori M et al. Lack of direct effect of moderate hyperleptinemia to improve endothelial function in lean rat aorta: Role of calorie restriction. Atherosclerosis 2004; 175:253-9. 108. Bełtowski J. Effect of hyperleptinemia on endothelial nitric oxide production. Atherosclerosis 2005; 178:403-4. 109. Ozer C, Gülen S, Dileköz E et al. The effect of systemic leptin administration on aorta smooth muscle responses in diabetic rats. Mol Cell Biochem 2006; 282:187-91.

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110. Piatti P, Di Mario C, Monti LD et al. Association of insulin resistance, hyperleptinemia and impaired nitric oxide release with in-stent restenosis in patients undergoing coronary stenting. Circulation 2003; 108:2074-81. 111. Golan E, Tal B, Dror Y et al. Reduction in resting metabolic rate and ratio of plasma leptin to urinary nitric oxide: influence on obesity-related hypertension. Isr Med Assoc J 2002; 4:426-30. 112. Porreca E, Di Febbo C, Moretta V et al. Circulating leptin is associated with oxidized LDL in postmenopausal women. Atherosclerosis 2004; 175:139-43. 113. Nakanishi S, Yamane K, Kamei N et al. A protective effect of adiponectin against oxidative stress in Japanese Americans: the association between adiponectin or leptin and urinary isoprostane. Metabolism 2005; 54:194-9. 114. Sundell J, Huupponen R, Raitakari OT et al. High serum leptin is associated with attenuated coronary vasoreactivity. Obes Res 2003; 11:776-82. 115. Wilcox CS. Oxidative stress and nitric oxide deficiency in the kidney: A critical link to hypertension? Am J Physiol Regul Integr Comp Physiol 2005; 289:R913-35. 116. Silva GB, Ortiz PA, Hong NJ et al. Superoxide stimulates NaCl absorption in the thick ascending limb via activation of protein kinase C. Hypertension 2006; 48:467-72. 117. Bełtowski J, Marciniak A, Jamroz-Wiœniewska A et al. Nitric oxide—Superoxide cooperation in the regulation of renal Na+, K+-ATPase. Acta Biochim Pol 2004; 51:933-42. 118. Bełtowski J, Jamroz-Wiśniewska A, Borkowska E et al. Up-regulation of renal Na+, K+-ATPase: the possible novel mechanism of leptin-induced hypertension. Pol J Pharmacol 2004; 56:213-22. 119. Bełtowski J, Jamroz-Wiśniewska A, Borkowska E et al. Antioxidant treatment normalizes renal Na+, K+-ATPase activity in leptin-treated rats. Pharmacol Rep 2005; 57:219-28. 120. Kuo JJ, Jones OB, Hall JE. Inhibition of NO synthesis enhances chronic cardiovascular and renal actions of leptin. Hypertension 2001; 37:670-6. 121. El-Gharbawy AH, Kotchen JM, Grim CE et al. Gender-specific correlates of leptin with hypertension-related phenotypes in African Americans. Am J Hypertens 2002; 15:989-93. 122. Caruso-Neves C, Rangel LB, Lara LS et al. Regulation of the renal proximal tubule second sodium pump by angiotensins. Braz J Med Biol Res 2001; 34:1079-84. 123. Bełtowski J, Jamroz-Wiśniewska A, Nazar J et al. Spectrophotometric assay of renal ouabain-resistant Na +-ATPase and its regulation by leptin and dietary-induced obesity. Acta Biochim Pol 2004; 51:1003-14. 124. Bełtowski J, Borkowska E, Wójcicka G et al. Regulation of renal ouabain-resistant Na+-ATPase by leptin, nitric oxide, reactive oxygen species and cyclic nucleotides: Implications for obesity-associated hypertension. Clin Exp Hypertens 2007; 29:189-207. 125. Bełtowski J, Wójcicka G. Regulation of renal tubular sodium transport by cardiac natriuretic peptides: two decades of research. Med Sci Monit 2002; 8:RA39-52. 126. Bełtowski J, Jamroz-Wisniewska A, Borkowska E et al. Phosphodiesterase 5 inhibitor ameliorates renal resistance to atrial natriuretic peptide associated with obesity and hyperleptinemia. Arch Med Res 2006; 37:307-15. 127. Dessi-Fulgheri P, Sarzani R, Serenelli M et al. Low calorie diet enhances renal, hemodynamic and humoral effects of exogenous atrial natriuretic peptide in obese hypertensives. Hypertension 1999; 33:658-62.

Chapter 10

Involvement of Leptin in the Endometrial Function Ana Cervero and Carlos Simon*

Abstract

L

eptin was discovered in 1994 as the product of the OB gene and was originally thought to be produced by only adipocytes governing energy homeostasis. Nevertheless, it has since been described as a pleiotrophic hormone secreted by many tissues affecting different processes. Numerous data have been published about the potential role of the leptin system in the control of the reproductive function in mammals, which acts directly or indirectly on the embryo or the endometrium. Moreover, several disorders in the leptin system have been related to some reproductive pathologies such as endometriosis, preeclampsia and polycystic ovary syndrome. Nowadays there is controversy in several aspects of the leptin action in reproduction that requires a more in-depth research of this system. In this chapter we will review the main findings in the leptin system related to its expression in the endometrium and its potential function during the implantation process. We will also mention the presence and possible implication of this system in endometriosis.

Introduction

Leptin, the product of the OB gene, was discovered in 1994 by Zhang et al1 as an adipocyte hormone thats secretion was linked to food consumption and energy balance. An early indication about the reproductive role of leptin was that ob/ob female mice (which lack functional leptin) and db/db mice (which lack a functional leptin receptor) were obese and infertile.1,2 Fertility in these ob/ob animals can be restored by an exogenous administration of leptin but not by food restriction,3 indicating that this hormone is per se required for the normal reproductive function.1-3 Later publications have reported that the leptin system is expressed by different tissues in the body, including reproductive tissues and is implicated in different processes between them in the regulation of the reproductive function and acts at endocrine and paracrine levels.4 Nevertheless, certain controversy exists in several aspects of the leptin action in reproduction that requires a more in-depth research of this system. It is likely that leptin and the leptin receptor will be subject of future research work in the reproductive field.

Overview of the Leptin System

Leptin is a 16 KDa nonglycosylated polypeptide of 146 amino acids discovered in 1994 by Zhang et al.1 Leptin is synthesized as a precursor with 167 amino acids which is activated by cleavage in the 21 amino acid residue.1,5 Leptin is a four-helix bundle cytokine and the helix length and disulfide bonds suggest that it is a member of the short-helix cytokine family.6 Leptin presents a C-terminal disulfide bond which is not necessary for its biological function, but it could be important for its secretion, stability and solubility.7 *Corresponding Author: Carlos Simon—Fundación IVI. C. Guadassuar 1 bajo 46015, Valencia, Spain. Email:[email protected]

Leptin and Leptin Antagonists, edited by Arieh Gertler. ©2009 Landes Bioscience.

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The leptin receptor is the product of LEPR or the OB-R gene and belongs to the class I cytokine superfamily of receptors.8 The full-length receptor has a helical structure and a signalling capability similar to that of the IL-6 type receptor.9,10 The cloned leptin receptor contains two homologous segments that are potential ligand binding sites.11 In vitro experiments have demonstrated that only the second domain is functional.11 The most important aspect about the leptin receptor is that its mRNA undergoes alternative splicing in the last exon, leading to several isoforms that differ in the length of their intracitoplasmatic domains.12 The short forms (OB-Rs) have a truncated intracellular domain13 and are considered to lack the signalling capability.14 The function of these short isoforms is still unclear, but it is assumed that they are implicated in different processes such as the clearance of leptin from cells, or they act as a circulating leptin-binding protein.15 The long form (OB-RL) presents a complete intracellular domain, it predominates in the hypothalamus and anterior pituitary and is also expressed in peripheral tissues.16

Leptin System in the Endometrium

The expression of the leptin receptor and its long form (OB-RL) in the human endometrium was described by several groups at more or less the same time.17-19 However, some differences exist in the expression pattern of this receptor throughout the menstrual cycle in these articles. Kitawaki et al17 found that the leptin receptor and its functional long form (OB-RL) were expressed in the endometrium with a peak in the early secretory phase. In contrast, Alfer et al18 reported that the expression of this receptor was low during the early secretory phase and high during the proliferative and late secretory phases. Some years later in 2004, comparable results were reported by Cervero et al.20 In this study it was shown that the total leptin receptor and its long form, OB-RL, underwent a cyclical variation with an increased expression during the late secretory phase (Fig. 1). These data, obtained using real-time PCR, were confirmed by an in situ hybridization analysis. This technique revealed the localization of the OB-RT mRNA mainly at the epithelial and glandular epithelium. With regard to the soluble isoforms of the leptin receptor (HuB219.1 and HuB219.3), Cervero et al20 found that they follow the same expression pattern as the total leptin receptor and its long form, with a peak in the late secretory phase and a minimal expression during the early secretory phase (Fig. 1). All these findings demonstrate that the endometrial leptin receptor is available in the human endometrial epithelium to be activated by its ligand at the time of implantation, which suggests a putative role for this system in this process. In vitro experiments have demonstrated that while the estradiol has no effect on the OB-R mRNA expression, progesterone plus estradiol diminished this expression in both epithelial and stromal endometrial cells.21 Given that no progesterone binding site has been identified in the OB-R gene, this effect has to be mediated by other indirect factors. Certain controversy exists in relation to the expression of leptin mRNA in the normal human endometrium;4 whereas some authors have not detected leptin mRNA in the human endometrium,17,18 others have shown its presence at mRNA and protein levels.19,20 In one of the latest works concerning this topic, leptin mRNA was found in the endometrium as well as in cultured endometrial epithelial cells using nested-PCR.20 In a similar way, the presence of leptin in the murine uterus remains controversial; whereas a study showed that leptin mRNA appears in the luminal and glandular epithelium of the endometrium as well as in the oviduct of pregnant mice,22 another was not able to find it.23 An explanation for such differences is that the leptin mRNA expression is very low and is only detectable by means of nested-PCR or overloading cDNA. This new endometrial leptin could influence the endometrium in an autocrine manner or the embryo in a paracrine manner.

Leptin System in the Implantation Process

Over time, a great deal of evidence has accumulated about the importance of leptin in murine implantation. Some years ago, a study reported that the absence of leptin prevented murine implantation.24 This study consisted in mating ob/ob mice, that had been previously treated with

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Figure 1. Quantitative mRNA analysis of different isoforms of the leptin receptor in the human endometrium throughout the menstrual cycle. A) OB-RT. B) OB-RL. C) HuB219.1 and D) HuB219.3 mRNA expression. Endometrial biopsies were divided into five groups: group I; early-mid proliferative (days 1-8), group II; late proliferative (days 9-14), group III; early secretory (days 15-18), group IV; mid secretory (days 19-22), group V; late secretory (days 23-28). Data were normalized with the GAPDH gene and are represented as a fold-increase of the mRNA expression compared with the group of basal expressions for each receptor form. Copyright 2004, The Endocrine Society, Cervero A, et al. J Clin Endocrinol Metab 2004; 89:2442-2451.

recombinant leptin and withdrawing the treatment at various stages of pregnancy. When leptin treatment was stopped before implantation, the pregnancy rate dramatically decreased.24 These results indicate that leptin is not required for maintaining pregnancy once implantation has been achieved. Some years before, however, another study had been published showing that a lack of leptin did not prevent the implantation and development of the embryo.25 This study made use of a similar design, but employed a higher dose of leptin (50 mg/kg versus 5 mg/kg). This considerable high dose was proposed by Malik et al24 as the explanation of such contradictory results. It is possible that a reserve of leptin remained in the mothers, which would have been sufficient to enable implantation. A further explanation could be the different strains of mice used. Both the aforementioned papers concluded that leptin was not required for pregnancy to proceed once implantation had taken place. The temporal and spatial OB-R expression could be an important mechanism to establish a crucial molecular crosstalk between the endometrium and the blastocyst at the time of implantation. In this sense, OB-R and OB-RL have been found to be differentially regulated in murine implantation sites and interimplantation sites, with a lower expression in the former.23 In 2005, a study described that the disruption of leptin signalling in the endometrium, using leptin peptide antagonists or OB-R antibody, impairs mouse embryo implantation and decreases LIF-R, VEGF-R2, IL-1R tI and the β3 integrin levels.26 However, we have to bear in mind that the contribution of leptin via the embryo cannot be discarded. Therefore, these effects could also be due to the blockade of leptin signalling in the embryo, preventing the blastocyst from acquiring implantation capability and/or secreting essential factors for the implantation process.

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By using an in vitro culture model for studying embryo implantation, leptin has been found to promote mouse blastocyst adhesion and blastocyst outgrowth on a fibronectin matrix27 as well as to stimulate mouse trophoblast cell invasion.28 This trophoblast invasion could be prevented by an inhibitor of metalloproteinases (MMP), which indicates that leptin may play an important role during early pregnancy and that this function is dependent on MMP activity. In view of the parallelism that exists between mice and humans, we could think that this ligand-receptor system is also essential for human embryonic implantation. To date however, no functional experiments have been performed to confirm this hypothesis. A study has been recently published in which the functional implication of the leptin system during the adhesion phase of implantation has been investigated using a heterologous in vitro model.29 RNA interference (RNAi) was performed to induce a consistent and stable silencing of OB-R mRNA and protein in the endometrial cell line HEC-1-A and adhesion assays were carried out with mouse blastocysts. The knockdown of the leptin receptor does not affect the blastocyst adhesion rate. Nevertheless, it should be noted that RNAi only decreases the expression of the targeted protein and it does not produce a complete knockout, so it is possible that a few remaining molecules in the cells are sufficient to maintain the normal function. Moreover, possible actions of this system cannot be excluded in other implantation phases, such as the invasion phase. The leptin produced and secreted by endometrial epithelial cells could act in a paracrine or autocrine manner by triggering a ligand-receptor-mediated effect through the endometrial leptin receptor and by directly or indirectly facilitating the implantation process. It has been reported that the presence of a human blastocyst, which expresses leptin mRNA, does not increase the mRNA expression of OB-RL and the short isoform HuB219.3 in cultured endometrial epithelial cells.20 Nevertheless, we cannot rule out the possibility that other effects, such as the regulation of different genes related to embryonic adhesion, take place through the leptin receptor activation in the endometrium. In this sense it has been found that leptin increases IL-6,30 IL-1β, IL-R tI, IL-1Ra,31 as well as LIF and LIF-R in cultured endometrial cells.32 It is well-known that LIF and LIF-R are mandatory for implantation in mouse.33 Moreover, IL-1 and leptin induce the β3-integrin expression, an adhesion molecule considered to be an endometrial receptivity marker which probably affects the implantation process.34-36 Finally, IL-1 is able to induce the expression of other implantation molecules such as CSF-137and VEGF.38 Following this matter, it should also be noted that leptin has been implicated in the synergistic stimulation of angiogenesis and vascular permeability together with FGF-2 and VEGF.39 Likewise, in vitro studies have shown that leptin increases the expression of the metalloproteinase MMP-2 and the extracellular matrix molecule fFN, as well as the activity of MMP-9 in cytotrophoblast cells.40 Both the angiogenesis process and expression of metalloproteinases are essential for implantation to be successful. Using an in vitro model, Schultz et al28 demonstrated that leptin promotes the invasiveness of trophoblast cells and that this invasion is blocked in the presence of an inhibitor of MMP activity.

Leptin System in the Endometriosis

One of the first papers reporting that the leptin system could be affected in some pathological situation was published in the year 2000.18 In this study it was found that subfertile patients presented an abnormal expression of the functional form of the leptin receptor.18 Nevertheless, data about this issue are contradictory. In that same year, another study was published where endometria from women with and without endometriosis were analyzed and compared.17 In this report no difference was found in the leptin receptor of the RNA expression when both types of endometria where compared.17 Similar results were shown some years later by Lima-Couy et al.41 In this paper, the leptin receptor mRNA expression was analyzed at LH+2 (prereceptive phase) and LH+9 (receptive phase) and this expression was evaluated in endometria from infertile patients with moderate/severe endometriosis and compared with those that were present in endometria from normal fertile patients.41 A higher expression was found at LH+9 in groups with and without endometriosis (Fig. 2).41 This result was obtained using real-time PCR, in situ hybridization and immunohistochemistry, indicating that there is no alteration in the leptin system in the eutopic

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Figure 2. Quantitative mRNA analysis of different isoforms of the leptin receptor in the human endometrium at LH+2 and LH+9 in patients with and without endometriosis. A) OB-RT. B) OB-RL. C) HuB219.3 mRNA expression. Data were normalized with the GAPDH gene and are represented as the relative average value. All isoforms showed the same expression pattern with a significant increase (*P < 0.05) at LH+9 compared to LH+2 in both groups, with and without endometriosis. No differences exist in the expression of any isoform at LH+9 between the endometriosis and control groups. A lower expression is observed at LH+2 in the endometriosis group comparing with the control group (**P < 0.05). Reproduced from Lima-Couy I, Cervero A, Bonilla et al. Mol Hum Reprod 2004; 10:777-782.

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endometrium at either the RNA or the protein level. The expression of leptin mRNA was also evaluated using nested-PCR and a low expression was found in both groups.41 Soon after, another study revealed that the leptin receptor underwent a lower expression in the eutopic endometrium of women with endometriosis compared with the endometrium of fertile women.42 These divergent results between both studies could be due to the different endometriotic stages of the samples analyzed. While the endometria from women with moderate endometriosis were employed in this last paper,42 Lima-Couy’s group41 carried out experiments using moderate/ severe endometriotic tissues. In this sense, it had been previously described that leptin levels in peritoneal fluid were determined by the stage of the disease.43 Differences in the leptin receptor expression are reported when eutopic and ectopic endometria are compared. A lower expression is found in the latter and this reduction is greater as the stage of the disease is more advanced.44 With regard to the leptin expression, the ectopic endometrium presents a higher expression than the eutopic endometrium.44 Moreover, leptin stimulates the leptin receptor expression in ectopic endometrial stromal cells, but not in those derived from the eutopic endometrium.44 Such differences could reflect the different biochemical features of endometriotic cells. In this way it has been verified that ectopic endometriotic tissues are able to develop different mechanisms in order to guarantee their self maintenance and the ability to product estrogens45,46 and progesterone47 between them. In addition, the difference in the gene expression between eutopic and ectopic endometria has been revealed by microarrays works.48,49 As mentioned before, the role of leptin in endometriotic tissues could be mediated indirectly through angiogenic factors, such as VEGF,39 whose expression is increased in the presence of leptin. In this way, the angiogenesis and vascularization of the new tissue is achieved and the ectopic endometrium is able to progress.

Summary and Conclusions

In just over a decade, the study of leptin has demonstrated that this hormone is implicated in different processes including the reproduction process. Several groups have shown data supporting the notion that leptin plays an important role in embryonic development and implantation. Leptin is present in the endometrium as well as in the blastocyst and its receptor is also found in the pre-implantation embryo and in the endometrium. Therefore, the leptin system could be an important mechanism in the crucial cross-talk established between the mother and the embryo during preimplantation development and at the time of implantation. Moreover, the leptin system is apparently mandatory for implantation in mice. Nevertheless, there are no functional proof of this concept in humans. The only data existing on this matter point out that the leptin system is not necessary for the adhesion phase, but other possible actions of this system cannot be excluded in other implantation phases, such as the invasion phase. Future research work is expected to elucidate and understand this complex system, which could also provide new alternatives for certain endometrial pathologies where this molecule may play an important role. Moreover, it is necessary to investigate the potential use of leptin in assisted reproductive technology and embryo culture.

References

1. Zhang Y, Proenca R, Maffei M et al. Positional cloning of the mouse obese gene and its human homologue. Nature 1994; 372:425-432. 2. Chen H, Charlat O, Tartaglia LA et al. Evidence that the diabetes gene encodes the leptin receptor: identification of a mutation in the leptin receptor gene in db/db mice. Cell 1996; 84:491-495. 3. Chehab FF, Lim ME, Lu R. Correction of the sterility defect in homozygous obese female mice by treatment with the human recombinant leptin. Nat Genet 1996; 12:318-320. 4. Cervero A, Horcajadas JA, Dominguez F et al. Leptin system in embryo development and implantation: a protein in search of a function. Reprod Biomed Online 2005; 10:217-223. 5. Ogawa Y, Masuzaki H, Isse N. Molecular cloning of rat obese cDNA and augmented gene expression in genetically obese zucker fatty (fa/fa) rats. J Clin Invest 1995; 96:1647-1652. 6. Kline AD, Becker GW, Churgay LM et al. Leptin is a four-helix bundle: secondary structure by NMR. FEBS Letters 1997; 407:239-242.

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7. Imagawa K, Numata Y, Katsuura G et al. Structure-function studies of human leptin. J Biol Chem 1998; 273:35245-35249. 8. Tartaglia LA, Dembski M, Weng X et al. Identification and expression cloning of a leptin receptor. Cell 1995; 83:1263-1271. 9. Baumann H, Morella KK, White DW et al. The full-length leptin receptor has signalling capabilities of interleukin 6-type cytokine receptors. Proc Natl Acad Sci USA 1996; 93:8374-8378. 10. Tartaglia LA. The leptin receptor. J Biol Chem 1997; 272:6093-6096. 11. Fong TM, Huang RR, Tota MR. Localization of leptin binding domain in the leptin receptor. Mol Pharmacol 1998; 53:234-240. 12. Cioffi JA, Shafer AW, Zupancic TJ et al. Novel B219/OB receptor isoforms: possible role of leptin in hematopoiesis and reproduction. Nat Med 1996; 2:585-589. 13. Campfield LA, Smith FJ, Burn P. The OB protein (leptin) pathway-a link between adipose tissue mass and central neural networks. Horm Metab Res 1996; 28:619-632. 14. Wang Y, Kuropatwinski KK, White DW. Leptin receptor action in hepatic cells. J Biol Chem 1997; 272:16216-16223. 15. Mantzoros CS. The role of leptin in human obesity and disease: a review of current evidence. Ann Intern Med 1999; 130:671-680. 16. Finn PD, Cunningham MJ, Pau KY et al. The stimulatory effect of leptin on the neuroendocrine reproductive axis in the monkey. Endocrinology 1998; 139:4652-4662. 17. Kitawaki J, Koshiba H, Ishihara H. Expression of leptin receptor in human endometrium and fl uctuation during the menstrual cycle. J Clin Endocrinol Metab 2000; 85:1946-1950. 18. Alfer J, Müller-Schöttle F, Classen Linke I. The endometrium as a novel target for leptin: differences in fertility and subfertility. Mol Hum Reprod 2000; 6:595-601. 19. González RR, Caballero-Campo P, Jasper M et al. Leptin and leptin receptor are expressed in the human endometrium and endometrial leptin secretion is regulated by the human blastocyst. J Clin Endocrinol Metab 2000; 85:4883-4888. 20. Cervero A, Horcajadas JA, Martin J et al. The leptin system during human endometrial receptivity and preimplantation development. J Clin Endocrinol Metab 2004; 89:2442-2451. 21. Koshiba H, Kitawaki J, Ishihara H et al. Progesterone inhibition of functional leptin receptor mRNA expression in human endometrium. Mol Hum Reprod 2001; 7:567-72. 22. Kawamura K, Sato N, Fukuda J et al. Leptin promotes the development of mouse preimplantation embryos in vitro. Endocrinology 2002; 143:1922-1931. 23. Yoon SJ, Cha KY, Lee KA. Leptin receptors are down-regulated in uterine implantation sites compared to interimplantation sites. Mol Cell Endocrinol 2005; 232:27-35. 24. Malik NM, Carter ND, Murray JF et al. Leptin requirement for conception, implantation and gestation in the mouse. Endocrinology 2001; 142:5198-5202. 25. Mounzih K, Qiu J, Ewart Toland A et al. Leptin is not necessary for gestation and parturition but regulates maternal nutrition via a leptin resistance state. Endocrinology 1998; 139:5259-5262. 26. Ramos MP, Rueda BR, Leavis PC et al. Leptin serves as an upstream activator of an obligatory signaling cascade in the embryo-implantation process. Endocrinology 2005; 146:694-701. 27. Yang YJ, Cao YJ, Bo SM et al. Leptin-directed embryo implantation: Leptin regulates adhesion and outgrowth of mouse blastocysts and receptivity of endometrial epithelial cells. Anim Reprod 2006; 92:155-67. 28. Schulz LC, Widmaier EP. The effect of leptin on mouse trophoblast cell invasion. Biol Reprod 2004; 71:1963-1967. 29. Cervero A, Domínguez F, Horcajadas JA et al. Embryonic adhesion is not affected by endometrial leptin receptor gene silencing. Fertil Steril 2007; 88:1086-1092. 30. Fukuda J, Nasu K, Sun B et al. Effects of leptin on the production of cytokines by cultured human endometrial stromal and epithelial cells. Fertil Steril 2003; 80:783-7. 31. Gonzalez RR, Leary K, Petrozza JC et al. Leptin regulation of the interleukin-1 system in human endometrial cells. Mol Hum Reprod 2003; 9:151-158. 32. Gonzalez RR, Rueda BR, Ramos MP et al. Leptin-induced increase in leukemia inhibitory factor and its receptor by human endometrium is partially mediated by interleukin 1 receptor signaling. Endocrinology 2004; 145:3850-3857. 33. Stewart CL, Kaspar P, Brunet LJ et al. Blastocyst implantation depends on maternal expression of leukaemia inhibitory factor. Nature 1992; 359:76-9. 34. Lessey BA, Damjanovich L, Coutifaris C et al. Integrin adhesion molecules in the human endometrium. Correlation with the normal and abnormal menstrual cycle. J Clin Invest 1992; 90:188-95. 35. Simón C, Gimeno MJ, Mercader A et al. Embryonic regulation of integrins β3, α4 and α1 in human endometrial epithelial cells in vitro. J Clin Endocrinol Metab 1997; 82:2607-2616.

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36. González RR, Palomino A, Boric A et al. A quantitative evaluation of alpha1, alpha4, alphaV and beta3 endometrial integrins of fertile and unexplained infertile women during the menstrual cycle. A flow cytometric appraisal. Hum Reprod 1999; 14:2485-92. 37. Harty JR, Kauma SW. Interleukin-1 beta stimulates colony-stimulating factor-1 production in placental villous core mesenchymal cells. J Clin Endocrinol Metab 1992; 75:947-50. 38. Lebovic DI, Shifren JL, Ryan IP et al. Ovarian steroid and cytokine modulation of human endometrial angiogenesis. Hum Reprod 2000; 15:67-77. 39. Cao R, Brakenhielm E, Wahlestedt C et al. Leptin induces vascular permeability and synergistically stimulates angiogenesis with FGF-2 and VEGF. Proc Natl Acad Sci USA 2001; 98:6390-6395. 40. Castelluci M, De Matteis R, Meisser A et al. Leptin modulates extracellular matrix molecules and metalloproteinases: possible implications for trophoblast invasion. Mol Hum Reprod 2000; 6:951-958. 41. Lima-Couy I, Cervero A, Bonilla Musoles F et al. Endometrial leptin and leptin receptor expression in women with severe/moderate endometriosis. Mol Hum Reprod 2004; 10:777-782. 42. Kao LC, Germeyer A, Tulac S et al. Expression profiling of endometrium from women with endometriosis reveals candidate genes for disease-based implantation failure and infertility. Endocrinology 2003; 144:2870-81. 43. Mahutte NG, Matalliotakis IM, Goumenou AG et al. Inverse correlation between peritoneal fluid leptin concentrations and the extent of endometriosis. Hum Reprod 2003; 18:1205-9. 44. Wu MH, Chuang PC, Chen HM et al. Increased leptin expression in endometriosis cells is associated with endometrial stromal cell proliferation and leptin gene up-regulation. Mol Hum Reprod 2002; 8:456-64. 45. Noble LS, Simpson ER, Johns A et al. Aromatase expression in endometriosis. J Clin Endocrinol Metab 1996; 81:174-9. 46. Bulun SE, Zeitoun KM, Takayama K et al. Estrogen biosynthesis in endometriosis: molecular basis and clinical relevance. J Mol Endocrinol 2000; 25:35-42. 47. Tsai SJ, Wu MH, Lin CC et al. Regulation of steroidogenic acute regulatory protein expression and progesterone production in endometriotic stromal cells. J Clin Endocrinol Metab 2001; 86:5765-73. 48. Matsuzaki S, Canis M, Vaurs-Barriere C et al. DNA microarray analysis of gene expression profiles in deep endometriosis using laser capture microdissection. Mol Hum Reprod 2004; 10:719-28. 49. Wu Y, Kajdacsy-Balla A, Strawn E et al. Transcriptional characterizations of differences between eutopic and ectopic endometrium. Endocrinology 2006; 147:232-46.

Chapter 11

The Use of Leptin for the Treatment of Lipodystrophy Angeline Y. Chong, Elaine K. Cochran and Phillip Gorden*

Abstract

L

eptin was the first of the adipocyte-secreted hormones, later termed adipokines, to be identified. It is used clinically to treat hypoleptinemic states, such as congenital leptin deficiency and lipodystrophy. In this chapter, we discuss the effects of leptin administration in patients with lipodystrophy. Leptin markedly improves hyperglycemia, hypertriglyceridemia and insulin resistance. It normalizes menses in women and increases testosterone secretion in men. It also ameliorates hepatic and muscular steatosis. Studying the effects of leptin replacement in lipodystrophy may lead to the discovery of new and broader clinical applications of leptin therapy.

Introduction Discovery of Leptin

Leptin was discovered when the gene mutation in the ob/ob mouse, an animal model of obesity, was identified.1 Leptin is a polypeptide of 146 amino acids that is encoded by the obese (ob) gene.2-6 Ob/ob mice make a defective leptin product and are extremely obese and hyperphagic.1 Since leptin administration causes a dramatic reduction in weight and food intake in these mice,7-9 it was hoped that leptin could be used to treat obesity in humans. As leptin is produced and secreted by fat cells, leptin levels are proportional to fat tissue mass and are high in obese humans. Exogenous leptin has limited effect in inducing weight loss, suggesting that obese humans have leptin resistance.10 Nevertheless, the discovery of leptin was pivotal for a number of reasons. It identified adipose tissue as an endocrine organ and was the first of the adiopocyte-derived hormones, later termed adipokines, to be recognized. It led to the discovery of a monogenic form of obesity in rodents and humans in which mutations in the leptin gene cause congenital leptin deficiency. Finally, it presented a form of treatment for various disorders: congenital leptin deficiency, lipodystrophy and insulin resistance secondary to insulin receptor abnormalities. Individuals who lack circulating leptin because of mutations in the ob gene are severely obese.11-13 Leptin administration in these patients induces satiety and significant weight loss.13-16 Patients with Rabson-Mendenhall syndrome have extreme insulin resistance caused by mutations in the insulin receptor gene. When they are given exogenous leptin, they experience improvements in hyperglycemia, hyperinsulinemia, glucose and insulin tolerance.17 Lipodystrophy is characterized by loss of adipose tissue and low leptin levels. The etiology may be congenital or acquired and the involvement may be generalized or partial. Patients with lipodystrophy have extreme insulin resistance, diabetes, dyslipidemia and hepatic steatosis. Exogenous recombinant leptin has been used successfully to treat the metabolic, endocrine and hepatic abnormalities of lipodystrophy. *Corresponding Author: Phillip Gorden—10 Center Dr., CRC Room 6-5940, Bethesda, Maryland 20892, USA. Email: [email protected]

Leptin and Leptin Antagonists, edited by Arieh Gertler. ©2009 Landes Bioscience.

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Leptin Signaling

Leptin has structural similarities to the IL-6 cytokine family.2-6 The leptin receptor (OBR) in turn is a member of the class I cytokine receptor family. OBR is a transmembrane protein and is found in almost all tissues. In the central nervous system, OBRs are highly expressed in the hypothalamus. It is unclear whether most of the effects of leptin in humans are mediated centrally or peripherally. The primary signal transduction pathway of OBR is through the Janus kinases/signal transducers and activators of transcription ( JAK/STAT) cascade. Binding of leptin to OBR causes the phosphorlyation of JAKs and OBR. This phoshorylation allows the association of STATs, which then become substrates for JAKs. After being phosphoylated, STATs translocate to the nucleus and regulate gene expression. Other intracellular signaling cascades that are activated by leptin are the mitogen-activated protein kinase pathway, the phosphoinositide 3-kinase/phosphodiesterase 3B/cyclic AMP pathway and the 5′-AMP-activated protein kinase (AMPK) pathway. Leptin signaling regulates lipid and glucose oxidation at a number of levels.2-4,18,19 In skeletal muscle, leptin activates AMPK, which then inactivates acetyl-CoA carboxylase (ACC). The inactivation of ACC results in the disinhibition of carnitine palmitoyl-transferase 1 (CPT-1), an enzyme needed for the translocation of fatty acids into mitochondria. By ultimately increasing CPT-1 activity, leptin increases fatty acid oxidation. Through its effects on the hypothalamus, leptin also stimulates fatty acid oxidation by activating the alpha-adrenergic system. In contrast to its actions in skeletal muscle, leptin suppresses AMPK activity in the hypothalamus.20 Another way that leptin affects lipid metabolism is by regulating gene transcription. In rats, leptin has been demonstrated to stimulate expression of enzymes of fatty acid oxidation and decrease expression of enzymes of fatty esterification.21-23

Metabolic Effects of Leptin Therapy Effect on Glycemic Control

Leptin significantly improves hyperglycemia and insulin sensitivity in patients with lipodystrophy. In our first paper on leptin therapy, we studied the effect of leptin on hyperglycemia in patients with various forms of lipodystrophy (eight generalized and one partial). Eight female patients who had lipodystrophy and diabetes mellitus had a mean glycosylated hemoglobin value of 9.1 ± 0.5% at baseline. These eight patients experienced a 1.9% mean decrease in glycosylated hemoglobin levels after four months of leptin replacement (P = 0.001).31 In all nine patients, plasma glucose levels during an insulin tolerance test significantly decreased after four months of leptin therapy (P < 0.02). The K constant rate of glucose disposal rose from 0.007 to 0.017 (P = 0.04), demonstrating increased insulin sensitivity. In addition, plasma glucose levels during an oral glucose tolerance test were significantly lower at four months compared with baseline (P < 0.01). In a study of 15 patients with generalized lipodystrophy (GL) who all had diabetes (DM), leptin led to significant reductions in fasting glucose (from 205 ± 19 to 126 ± 11 mg/dL, P < 0.001) and glycosylated hemoglobin (from 9.0 ± 0.4 to 7.1 ± 0.5%, P < 0.001) after 12 months of treatment.24,25 Insulin sensitivity increased with leptin such that the K constant determined from insulin tolerance tests more than doubled (from 0.0074 to 0.015, P < 0.001). Improvement in insulin sensitivity was reflected in oral glucose tolerance test results. While oral glucose tolerance test results were markedly abnormal at baseline, both fasting and two-hour glucose levels were significantly reduced after four months of leptin treatment. These improvements were sustained over 12 months of therapy. In a subset of six patients with GL and DM who were not treated with exogenous insulin, a similar drop in fasting and two-hour glucose levels was seen when compared with the entire cohort. Furthermore, in this subset of non-insulin-treated patients, endogenous insulin levels peaked earlier and the total amount of insulin secretion increased after 12 months of leptin treatment, consistent with enhanced insulin responsiveness and sensitivity. Diabetic management also changed dramatically with leptin replacement. Before the initiation of leptin, 9 of 15 patients were on a mean insulin dose of 916 units a day. During therapy, six patients became euglycemic and were able to discontinue insulin. The three patients who still required insulin were able to decrease their mean dose by 65%. Eight

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patients were on oral antidiabetic agents at baseline and six of them were able to discontinue these medications within 12 months of leptin replacement. Another research group reported similar improvements in glycemic control when leptin was administered to patients with GL. Ebihara et al studied seven Japanese patients with GL, two with acquired generalized lipodystrophy (AGL) and five with congenital generalized lipodystrophy (CGL).26 These patients experienced marked reductions in fasting glucose within seven days of leptin therapy (from 172 ± 20 to 120 ± 12 mg/dL, P < 0.05). Improvements in plasma glucose levels on oral glucose tolerance tests were achieved by one month and were sustained at two and four months. Antidiabetic medication could be reduced or discontinued completely by four months. By two months, leptin replacement significantly increased insulin sensitivity as measured by glucose infusion rates during hyperinsulinemic-euglycemic clamp studies. Interestingly, insulin secretion during oral glucose tolerance testing increased by one month in AGL patients, but did not change in CGL patients. The authors proposed that the different responses of insulin secretion to leptin replacement could be attributed to the longer duration of diabetes and more depressed β-cell function in CGL patients. We found that leptin therapy affected glycemic control to a somewhat different degree in a study of six patients with Dunnigan-type familial partial lipodystrophy (FPLD).27 In patients with FPLD, glycosylated hemoglobin levels did not change significantly after 12 months of treatment (from 8.4 ± 0.6 to 8.0 ± 0.4%, P = 0.069). While there was a significant drop in fasting glucose with leptin (from 190 ± 26 to 151 ± 15 mg/dL, P = 0.006), there was no significant improvement in oral glucose tolerance test results. A sustained increase in insulin sensitivity, however, was observed on insulin tolerance tests (P < 0.01). In comparison to the GL patients we reported in Javor et al.,24 the six study subjects with FPLD were older, had higher leptin levels, had been diagnosed with diabetes for a longer time and had lower insulin secretion for a similar degree of hyperglycemia. We have suggested that the lack of significant improvement in glycosylated hemoglobin levels and glucose tolerance seen in FPLD is secondary to waning endogenous insulin secretion. An effect of leptin on glycemic control has been observed in the pediatric population as well. In a French study of 7 children ages 2.4 to 13.6 years with CGL, fasting glucose was normal at baseline in all subjects and did not significantly change after four months of leptin replacement.28 However, there was a trend towards lower fasting insulin (from 135.2 ± 84 pmol/L to 90.8 ± 78 pmol/L, P = 0.2) and a significant increase in the fasting glucose to insulin ratio (from 0.24 ± 0.08 to 1.04 ± 1.52, P = 0.04). There was an increase in insulin sensitivity after four months of leptin as measured by peripheral glucose uptake on hyperinsulinemic-euglycemic clamp studies or quantitative insulin sensitivity check index (Q UICKI). Interestingly, leptin appears to have some effect in lipodystrophy induced by highly active antiretroviral therapy (HAART). Lee et al administered leptin for two months to seven men with HIV infection and HAART-induced lipodystrophy.29 Leptin led to statistically significant decreases in fasting insulin levels and HOMA-IR (homeostasis model assessment of insulin resistance) values.

Effect on Lipid Metabolism

The severe dyslipidemia seen in lipodystrophy patients tends to improve on leptin therapy. In our study of nine female patients with various forms of lipodystrophy, all nine had hypertriglyceridemia.31 We observed an average 60% drop in fasting triglyceride levels after four months of leptin treatment (P < 0.001). Fasting free fatty acid levels fell from a mean of 1540 ± 407 to 790 ± 164 µmol/L over the same time (P=0.05). The 15 patients in our study of long-term leptin use in generalized lipodystrophy had severe hypertriglyceridemia at baseline with a mean triglyceride level of 1380 ± 500 mg/dL.24,25 Leptin replacement led to a 63% drop in serum triglycerides to 516 ± 236 mg/dL (P < 0.001). The largest reductions in triglyceride levels were observed after four months and were more gradual thereafter. Over 12 months of therapy, there were significant reductions in low density lipoprotein (LDL) levels (from 139 ± 16 to 85 ± 7 mg/dL, P = 0.01) and total cholesterol levels (from 284 ± 40 to

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167 ± 21 mg/dL, P < 0.001). High density lipoprotein (HDL) levels were low at baseline and did not significantly change over the 12 months of treatment (from 31 ± 3 to 29 ± 2 mg/dL, P = 0.9). The Japanese study of leptin replacement in GL recapitulated our findings and demonstrated a rapid and significant drop in fasting triglyceride levels within 7 days of leptin treatment (from 700 ± 272 to 260 ± 98 mg/dL, P < 0.05).26 In our report of six patients with Dunnigan-type FPLD on leptin, all subjects had marked hypertriglyceridemia prior to therapy (mean serum triglycerides 749 ± 331 mg/dL).27 At 4 months, there was a 65% decrease in mean triglycerides to 260 ± 58 mg/dL. One patient had high triglycerides at baseline and lower levels at four months; however, she had variable triglyceride levels thereafter because of noncompliance with leptin and lipid-lowering drugs. When this patient was excluded from the analysis, there was a significant decrease in triglycerides over the 12-month course of leptin (P = 0.026). We observed a significant drop in total cholesterol levels (280 ± 49 to 231 ± 41 mg/dL, P = 0.012), but a decrease of borderline significance in LDL levels (135 ± 4 to 118 ± 8 mg/dL, P = 0.052). Similar to the GL patients in our long-term study,24,25 HDL levels in the FPLD subjects were low prior to leptin and did not change over 12 months of treatment (40 to 36 mg/dL, P = 0.468). The hypertriglyceridemia of children with CGL appears to improve with leptin therapy as well. In their study of seven children with CGL, Beltrand et al reported significant reductions in serum triglycerides with a mean 40% drop in z score (from 6.84 ± 3.18 to 2.49 ± 0.94, P = 0.017) after 4 months of leptin treatment.28 Cholesterol and free fatty acid levels were within reference ranges at baseline and remained so during leptin replacement. Patients with HAART-induced lipodystrophy seem to have a more modest response to leptin. In these patients, Lee et al found an increase in HDL that was associated with a change in visceral fat mass, but no difference in triglycerides or LDL after 2 months of leptin administration.29

Effect on Weight and Body Composition

Leptin replacement in lipodystrophic patients is associated with changes in weight and body composition. Our long-term study of 15 GL patients demonstrated a significant decrease in total body weight with leptin replacement (from 61.8 to 57.4 kg, P =0.02).24,25 Two years later we specifically evaluated body composition in a study of 14 patients with lipodystrophy, 12 with GL and 2 with PL.30 Leptin replacement was associated with a reduction in weight from 60.8 kg at baseline to 53.2 kg at 12 months (P < 0.001). Our study showed that patients lost both fat mass and lean body mass on leptin replacement. Mean fat mass decreased from 5.4 kg at baseline to 4.0 kg at 12 months (P < 0.001). Lean body mass dropped from 51.2 kg at baseline to 48.3 kg at 4 months (P < 0.003). The reduction in lean body mass from baseline to 12 months did not reach statistical significance, most likely because there were fewer patients studied at 12 compared with 4 months (8 patients compared with 12). In our report of six FPLD patients on leptin, there was a moderate reduction in weight (from 62.1 to 60.2 kg, P = 0.032) and body mass index (23.3 to 22.7 kg/m2, P = 0.024).27 However, the change in percent body fat did not reach statistical significance (from 21.6 to 20.2%, P = 0.069). Ebihara et al reported a trend towards decreased body weight after four months of leptin in their seven Japanese patients with GL, but the change was not statistically significant (from 40.9 ± 3.5 to 38.1 ± 3.1 kg, P = 0.55).26 The study of leptin treatment in children with CGL did not reveal a significant change in weight (from 37.7 ± 15.8 to 37.4 ± 16.6 kg, P = 0.61) or fat mass (from 2.04 ± 0.8 to 2.41 ± 2.1 kg, P = 0.21) with therapy.28 In adult men with HAART-induced lipodystrophy, leptin was associated with modest decreases in body weight, body mass index, percent body fat and total fat mass, largely because of reductions in truncal fat.29 Leptin therapy appears to reduce resting energy expenditure (REE) in patients with lipodystrophy. Our initial report of eight GL patients and one PL patient on leptin showed a significant decrease in REE from 1920 to 1580 kcal/day (P = 0.003).31 When we studied 15 patients with exclusively GL, we again observed a significant reduction in REE from 1929 to 1611 kcal per day (P < 0.001).24 Our body composition study of 12 patients with GL and two with PL demonstrated a significant decrease in REE with leptin replacement (from 1818 to 1551 kcal/day, P = 0.037).30

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Table 1. Metabolic effects of leptin therapy in lipodystrophy Plasma glucose Insulin sensitivity Serum triglycerides LDL cholesterol HDL cholesterol Body weight Fat mass Lean body mass Resting energy expenditure Food intake and appetite

↓↓↓ ↑↑↑ ↓↓↓↓ ↓↓ ↔ ↓ ↓ ↓ ↓↓ ↓↓↓

However, when we analyzed six FPLD patients on leptin therapy, we did not find a significant change in REE.27 Leptin also seems to decrease food intake and appetite in lipodystrophic patients. When we followed 9 female patients with various forms of lipodystrophy, there was a significant reduction in self-reported daily caloric intake from 2680 to 1600 after 4 months of leptin treatment (P = 0.005).31 Likewise, our study on the effect of leptin on body composition in 12 GL patients and 2 PL patients revealed a significant decrease in caloric intake by 4 months (from 3170 to 1739 kcal/ day, P = 0.019).30 At baseline, these patients had voracious appetites and demonstrated food-seeking behavior between meals. On leptin, their appetites abated and they felt full after meals. In a study of eight females with hypoleptinemia and lipodystrophy, exogenous leptin decreased satiation time (time to eating cessation during a meal) and increased satiety time (time to eating again after consuming a standardized amount of food).32 In the study of seven Japanese patients with GL, all subjects described improved satiety within one to two days of leptin therapy.26 The metabolic effects of leptin therapy in patients with lipodystrophy (non-HAART-induced) are summarized in Table 1.

Endocrine Effects of Leptin Therapy Effect on Gonadal Function

Leptin plays a role in pituitary hormone regulation, especially in the hypothalamic-pituitary-gonadal axis. In vitro, leptin stimulates the frequency of pulsatile gonadotropin-releasing hormone (GnRH) secretion from rat hypothalamic explants and GnRH-secreting neurons.33 In a nine year-old girl with congenital leptin deficiency, the nocturnal pattern of gonadotropin secretion became pulsatile with leptin therapy, consistent with early puberty.14 Leptin was reported to increase luteinizing hormone (LH) and testosterone levels and induce puberty in a 27 year-old man with congenital leptin deficiency.16 More evidence of the role of leptin in the hypothalamic-pituitary-gonadal axis comes from a study of healthy men who were fasted. Fasting decreased LH pulsatility and serum testosterone levels, but leptin administration normalized LH pulsatility and restored testosterone levels to baseline.34 In lipodystrophic women, leptin appears to increase serum estradiol, decrease testosterone, improve LH responses to luteinizing hormone-releasing hormone (LHRH) stimulation and normalize menses. In a report of seven women of reproductive age with lipodystrophy, serum estradiol concentrations increased nearly five-fold (P = 0.002) after four months of recombinant leptin therapy.35 Serum testosterone concentrations decreased by 63% (P = 0.055). There was no change in sex hormone binding globulin (SHBG) or progesterone levels after four months of leptin. The LH response to an LHRH test also normalized. Prior to leptin therapy, patients had attenuated LH secretion after LHRH administration. After four months of leptin treatment, the LH response to LHRH was significantly more robust (P < 0.001). Unlike the LH response, the follicle stimulating hormone (FSH) response did not change with leptin therapy. These hormonal changes appeared

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Table 2. Effects of leptin therapy on gonadal function in lipodystrophic females Serum estradiol Serum testosterone LH response to LHRH stimulation Sex hormone binding globulin Regularity of menses Ovarian volume

↑↑ ↓↓↓ ↑↑ ↑ ↑↑↑ ↔

to be clinically relevant. Only one of five patients who had intact reproductive systems had regular menstrual cycles prior to leptin replacement. By the fourth month of therapy, all five had normal menses. Six women had intact ovaries and ultrasound imaging revealed polycystic ovaries at baseline; however, ovarian volume did not change after four months of leptin replacement. Our group published a larger follow-up study of pituitary function on 10 women and 4 men with GL who were treated with leptin for 12 and 8 months, respectively.36 In the female patients, serum free testosterone levels fell from a mean of 39.6 ± 11 to 18.9 ± 4.5 ng/dL (P < 0.01). The mean total testosterone level also decreased significantly from 92 ± 30 to 54.8 ± 8.8 ng/dL (P < 0.01). Unlike the previous study, there was a significant increase in SHBG over 12 months. There was a trend towards higher serum estradiol levels with leptin therapy (from 44.5 ± 10 to 73.9 ± 25 pg/dL), but it was not significant. The inability to achieve statistical significance may have been due to the fact that estradiol was not checked at a specific point in the menstrual cycle. When the LH response to LHRH stimulation was analyzed in the entire female group, there was no significant change compared with baseline. However, among the 3 youngest women, ages 12 to 17, the LH response to LHRH was diminished at baseline and significantly increased after 4 months of leptin. In this study, the female subjects had baseline ultrasound imaging consistent with polycystic ovarian syndrome. Again, leptin treatment did not change ovarian volume. Eight out of the 10 women studied had irregular menses or primary amenorrhea at baseline. By the fourth month of leptin treatment, all eight had regular menses. Ebihara et al reported a similar experience in their group of Japanese patients with GL. Four of five female patients who were of reproductive age had hypogonadotropic amenorrhea prior to leptin therapy.26 Regular menses resumed with leptin replacement. An 11-year-old girl in their study had menarche after 12 months of therapy. In the male group of our follow-up study on pituitary function, serum testosterone tended to rise (from 433 ± 110 to 725 ± 184 ng/dL, P = 0.1) and SHBG significantly increased from 18.25 ± 2.6 to 27 ± 1.7 nmol/L (P < 0.04) over 4 months of leptin replacement.36 No changes were seen in the LH response to LHRH administration. Leptin therapy did not seem to affect pubertal development in the male group. A group of hypoleptinemic male patients with lipodystrophy who were not on exogenous leptin were compared with leptin-treated males. All male subjects underwent normal puberty and had an age-appropriate serum testosterone level regardless of whether they had leptin replacement. For example, one leptin-treated patient had a testosterone level of 177 ng/mL at the start of puberty (age 10) and had progressively increasing testosterone levels through puberty into the normal adult range. Thus, in lipodystrophy, leptin replacement does not appear to induce LH secretion before appropriate pubertal timing. The study by Beltrand et al of six boys and one girl with CGL did not demonstrate an adverse effect of leptin on pubertal development either.28 Over the four months of leptin replacement, the authors found no induction or acceleration of puberty. The effects of leptin replacement on gonadal function in lipodystrophic women and men are summarized in Tables 2 and 3.

Effect on the Growth Hormone and Insulin-Like Growth Factor Axes

In both males and females, leptin therapy is associated with an increase in insulin-like growth factor 1 (IGF-1) levels.36 In the 10 females with GL in our follow-up study of leptin and pituitary function, the mean baseline IGF-1 level was 125 ± 24 ng/dL and increased by about 73% over 12 months of leptin treatment (P < 0.02). When we analyzed the 10 females and 4 males in our

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Table 3. Effects of leptin therapy on gonadal function in lipodystrophic males ↑↑ ↑ ↔

Serum testosterone* Sex hormone binding globulin Pubertal development

*Leptin therapy does not affect male pubertal development, however.

follow-up study as a group, we observed an approximately 53% increase in IGF-1 levels after 8 months of leptin replacement (P < 0.03). However, there was no significant change in growth hormone (GH) levels. We believe that the increase in IGF-1 concentration with leptin therapy is due to improved insulin action or enhanced insulin sensitivity.

Effect on the Thyroid Axis

When we first reported the effect of leptin replacement on pituitary hormone function, we saw no change in total triiodothyronine(T3)(from 1.5 ± 0.3 to 1.7 ± 0.6 nmol/L) and free thyroxine (T4)levels after four months of treatment.35 Serum thyrotropin (TSH) and total T4 levels stayed in the normal range, but decreased significantly (TSH from 2.2 ± 1.1 to 1.2 ± 0.7 µU/mL, P < 0.001; total T4 from 126 ± 27 to 92 ± 19 nmol/L, P < 0.001). The percent increase in TSH after thyrotropin-releasing hormone (TRH) stimulation was similar before (560%) and after 4 months (580%) of leptin therapy. Our results suggested that leptin might alter the pituitary set-point for TSH secretion. Our follow-up study of pituitary function did not demonstrate a significant effect of leptin on the thyroid axis over a period of 12 months for females and 8 months for males.36 No changes were found in the mean TSH (from 1.37 ± 0.1 to 1.31 ± 0.5 µU/mL), T3 (from 124 ± 10 to 126.1 ± 11.5 nmol/L) and free T4 levels (from 1.05 ± 0.04 to 1.04 ± 0.04 pmol/L).

Effect on the Adrenal Axis

We have not observed a significant effect of leptin replacement on serum adrenocorticotropic hormone (ACTH) or cortisol levels. After 4 months of leptin , we did not see a change in ACTH (from 6.0 ± 3.4 pmol/L to 4.2 ± 1.2 pmol/L, P = 0.11) or cortisol (from 680 ± 280 to 453 ± 142 nmol/L, P = 0.13) in seven female patients with lipodystrophy.35 The ACTH and cortisol response to corticotropin-releasing hormone (CRH)stimulation was normal before and after four months of leptin replacement, suggesting that leptin therapy does not have a significant effect on the hypothalamic-pituitary-adrenal axis in hypoleptinemic lipodystrophic patients. We had consistent findings in our follow-up study. There was no significant change in serum cortisol and ACTH concentrations over 12 months of leptin replacement in females and over 8 months in males.36 Table 4 is a summary of the effects of leptin therapy on the growth hormone, thyroid and adrenal axes in lipodystrophic patients.

Table 4. Other endocrine effects of leptin therapy in lipodystrophy Serum IGF-1 Serum growth hormone TSH Free T4 Total T3 ACTH Cortisol

↑↑ ↔ ↔ or ↓ ↔ ↔ ↔ ↔

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In lipodystrophy, ectopic fat accumulates in the liver, resulting in hepatic steatosis. Steatosis may eventually progress to nonalcoholic stepatohepatitis (NASH). Leptin therapy is associated with a reduction in hepatic triglyceride content. Using nuclear magnetic resonance spectroscopy, Peterson et al. studied the intrahepatic lipid content of three patients with generalized lipodystrophy.37 Prior to leptin replacement, all three subjects had high liver triglyceride content, ranging from 4.6 to 48% compared with less than 1% in healthy controls. After three months of leptin treatment in two patients and eight months of treatment in one patient, an 86 ± 8% reduction in hepatic triglyceride content was observed (P = 0.008). There was an increase in hepatic insulin responsiveness with a rise in insulin suppression of glucose production during a hyperinsulinemic-euglycemic clamp study (from 40 ± 6% to 82 ± 5%, P < 0.05). Simha et al. showed similar changes in intrahepatic lipid content with leptin therapy.38 They evaluated the effects of leptin in three patients with GL. At baseline, two patients had intrahepatic lipid content that was two to three times that of normal. One patient had high-normal levels. After eight months of leptin replacement, intrahepatic triglyceride content fell from 14.9% to 1.2% in one patient, 5.7 to 2.2% in another patient and 11.1 to 1.5% in the third patient. In our long-term study of 15 patients with GL, leptin therapy significantly reduced liver volumes after 12 months (from 3663 ± 326 to 2190 ± 159 cm3, P < 0.001), representing loss of steatosis.24,25 We later demonstrated that leptin therapy reverses nonalcoholic steatohepatitis in patients with lipodystrophy. In this study, we examined eight patients with GL and two patients with Dunnigan’s FPLD who were treated with leptin for a mean duration of 6.6 months.39 With leptin, there were significant decreases in liver volume (from 3209 ± 348 to 2391 ± 254 cm3, P = 0.007), liver fat by magnetic resonance imaging (from 31 ± 7 to 11 ± 6 %, P = 0.006), alanine aminotransferase (ALT) levels (from 54 ± 13 to 24 ± 4 U/L, P = 0.02) and aspartate aminotransferase (AST) levels (from 47 ± 11 to 22 ± 2 U/L, P = 0.046). Among the eight patients in this study who met histological criteria for nonalcoholic steatohepatitis, there were significant reductions in NASH scores, particularly steatosis (P = 0.006), ballooning injury (P = 0.005) and NASH activity (P = 0.002). There was no change in fibrosis. Nine patients had steatosis at baseline, but only three had steatosis on follow-up biopsy. Of the eight patients with ballooning injury prior to leptin, only three had ballooning on follow-up. Six of the eight patients with NASH at baseline did not meet criteria for NASH on follow-up biopsy. The reductions in NASH scores were strongly correlated with decreases in liver volume, liver fat as determined by MRI, serum transaminases, serum triglycerides, fasting serum insulin and fasting serum glucose. Ebihara et al. noted an improvement in fatty liver with leptin in their Japanese patients with GL.28 They used liver to spleen (L/S) ratios of computed tomography (CT) attenuation value as an indicator of hepatic fat content. The L/S ratio of CT attenuation value in five out of seven patients improved from 0.74 ± 0.10 to 1.09 ± 0.06 by two months. In these five patients with fatty liver, serum ALT decreased from 80.5 ± 24.2 to 32.3 ± 4.6 U/L and AST fell from 42.3 ± 11.1 to 21.5 ± 4.3 U/L after two months. Liver volume of these five patients also decreased with leptin treatment from 1.88 ± 0.12 at baseline to 1.50 ± 0.101L at two months. In the study of leptin replacement in children with CGL, there was a significant drop in mean AST (from 47 ± 41 to 25 ± 7 U/L, P = 0.04) and ALT (from 105 ± 99 to 35 ± 17 U/L, P = 0.02) after four months of leptin.28 Mean liver volume also decreased from 4.53 ± 1.4 at baseline to 3.22 ± 0.9 L (P = 0.002) after four months. There was a concomitant reduction in waist circumference from 65.4 ± 7.4 to 61.7 ± 6.7 cm (P = 0.02). Leptin replacement also decreases muscle triglyceride content in patients with GL. Using nuclear magnetic resonance spectroscopy, Petersen et al observed a 33 ± 3% decrease in muscle triglyceride content in three GL patients after three to five months of leptin therapy (P = 0.006).37 In one patient, the drop in muscle triglyceride content was associated with an approximately 30% decrease in muscle total fatty acyl CoA concentrations. Simha et al noted a decrease in muscle triglyceride content as well in their study of three GL patients.38 Intramyocellular lipid content decreased by about 42% after eight to ten months of leptin replacement.

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Table 5. Hepatic and muscular effects of leptin therapy in lipodystrophy Hepatic triglyceride content Hepatic insulin responsiveness Liver volume NASH score Serum transaminases Muscle triglyceride content

↓↓↓ ↑↑ ↓↓ ↓↓ ↓↓ ↓

The changes seen in liver and muscle with leptin administration are reviewed in Table 5.

Conclusion

Leptin has evolved from the first identified adipokine to a therapeutic agent for a variety of disorders. It is used in humans to treat rare diseases such as congenital leptin deficiency, Rabson-Mendenhall syndrome and lipodystrophy. Leptin has also gained attention as a therapy for women with hypothalamic amenorrhea from weight loss or strenuous exercise.40 More recently, leptin appears to improve insulin resistance and decrease truncal fat mass in HIV patients with HAART-induced lipodystrophy. The effects of leptin in HAART-induced lipodystrophy are less striking compared with non-HIV-related lipodystrophy. It has been proposed that the reason for this finding may be the milder degree of leptin deficiency and metabolic derangements associated with HAART.29 In patients with non-HAART-induced lipodystrophy, leptin has profound metabolic, endocrine and hepatic effects, whether the lipodystrophy is acquired versus congenital, or generalized versus partial. It causes a dramatic reduction in serum glucose and triglyceride levels and markedly improves insulin sensitivity. Leptin therapy normalizes menses in lipodystrophic women and augments testosterone secretion in lipodystrophic men. Finally, leptin administration reduces hepatic and muscular lipid content. As we learn more about the actions of leptin, we may be able to expand its therapeutic uses.

References

1. Zhang Y, Proenca R, Maffei M et al. Positional cloning of the mouse obese gene and its human homologue. Nature 1994; 372(6505):425-432. 2. Bjorbaek C, Kahn BB. Leptin signaling in the central nervous system and the periphery. Recent Prog Horm Res 2004; 59:305-331. 3. Fruhbeck G. Intracellular signalling pathways activated by leptin. Biochem J 2006; 393(Pt 1):7-20. 4. Hegyi K, Fulop K, Kovacs K et al. Leptin-induced signal transduction pathways. Cell Biol Internat 2004; 28(3):159-169. 5. Leshan RL, Bjornholm M, Munzberg H et al. Leptin receptor signaling and action in the central nervous system. Obesity 2006; 14(Suppl 5):208S-212S. 6. Sweeney G. Leptin signalling. Cellular signalling 2002; 14(8):655-663. 7. Campfield LA, Smith FJ, Guisez Y et al. Recombinant mouse OB protein: evidence for a peripheral signal linking adiposity and central neural networks. Science 1995; 269(5223):546-549. 8. Halaas JL, Gajiwala KS, Maffei M et al. Weight-reducing effects of the plasma protein encoded by the obese gene. Science 1995; 269(5223):543-546. 9. Pelleymounter MA, Cullen MJ, Baker MB et al. Effects of the obese gene product on body weight regulation in ob/ob mice. Science 1995; 269(5223):540-543. 10. Heymsfield SB, Greenberg AS, Fujioka K et al. Recombinant leptin for weight loss in obese and lean adults: a randomized, controlled, dose-escalation trial. JAMA 1999; 282(16):1568-1575. 11. Montague CT, Farooqi IS, Whitehead JP et al. Congenital leptin deficiency is associated with severe early-onset obesity in humans. Nature 1997; 387(6636):903-908. 12. Ozata M, Ozdemir IC, Licinio J. Human leptin deficiency caused by a missense mutation: multiple endocrine defects, decreased sympathetic tone and immune system dysfunction indicate new targets for leptin action, greater central than peripheral resistance to the effects of leptin and spontaneous correction of leptin-mediated defects. J Clin Endocrinol Metab 1999; 84(10):3686-3695. 13. Gibson WT, Farooqi IS, Moreau M et al. Congenital leptin deficiency due to homozygosity for the Delta133G mutation: report of another case and evaluation of response to four years of leptin therapy. J Clin Endocrinol Metab 2004; 89(10):4821-4826.

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14. Farooqi IS, Jebb SA, Langmack G et al. Effects of recombinant leptin therapy in a child with congenital leptin deficiency. N Engl J Med 1999; 341(12):879-884. 15. Farooqi IS, Matarese G, Lord GM et al. Beneficial effects of leptin on obesity, T-cell hyporesponsiveness and neuroendocrine/metabolic dysfunction of human congenital leptin deficiency. J Clin Invest 2002; 110(8):1093-1103. 16. Licinio J, Caglayan S, Ozata M et al. Phenotypic effects of leptin replacement on morbid obesity, diabetes mellitus, hypogonadism and behavior in leptin-deficient adults. Proc Natl Acad Sci USA 2004; 101(13):4531-4536. 17. Cochran E, Young JR, Sebring N et al. Efficacy of recombinant methionyl human leptin therapy for the extreme insulin resistance of the Rabson-Mendenhall syndrome. J Clin Endocrinol Metab 2004; 89(4):1548-1554. 18. Ahima RS, Qi Y, Singhal NS et al. Brain adipocytokine action and metabolic regulation. Diabetes 2006; 55(Suppl 2):S145-154. 19. Ceddia RB. Direct metabolic regulation in skeletal muscle and fat tissue by leptin: implications for glucose and fatty acids homeostasis. Int J Obes 2005; 29(10):1175-1183. 20. Minokoshi Y, Alquier T, Furukawa N et al. AMP-kinase regulates food intake by responding to hormonal and nutrient signals in the hypothalamus. Nature 2004; 428(6982):569-574. 21. Zhou YT, Shimabukuro M, Koyama K et al. Induction by leptin of uncoupling protein-2 and enzymes of fatty acid oxidation. Proc Natl Acad Sci USA 1997; 94(12):6386-6390. 22. Unger RH, Zhou YT, Orci L. Regulation of fatty acid homeostasis in cells: novel role of leptin. Proc Natl Acad Sci USA 1999; 96(5):2327-2332. 23. Orci L, Cook WS, Ravazzola M et al. Rapid transformation of white adipocytes into fat-oxidizing machines. Proc Natl Acad Sci USA 2004; 101(7):2058-2063. 24. Javor ED, Cochran EK, Musso C et al. Long-term efficacy of leptin replacement in patients with generalized lipodystrophy. Diabetes 2005; 54(7):1994-2002. 25. Gorden P, Park JY. The clinical efficacy of the adipocyte-derived hormone leptin in metabolic dysfunction. Arch Physiol Biochem 2006; 112(2):114-118. 26. Ebihara K, Kusakabe T, Hirata M et al. Efficacy and safety of leptin-replacement therapy and possible mechanisms of leptin actions in patients with generalized lipodystrophy. J Clin Endocrinol Metab 2007; 92(2):532-541. 27. Park JY, Javor ED, Cochran EK et al. Long-term effi cacy of leptin replacement in patients with Dunnigan-type familial partial lipodystrophy. Metabolism 2007; 56(4):508-516. 28. Beltrand J, Beregszaszi M, Chevenne D et al. Metabolic correction induced by leptin replacement treatment in young children with Berardinelli-Seip congenital lipoatrophy. Pediatrics 2007; 120(2):e291-296. 29. Lee JH, Chan JL, Sourlas E et al. Recombinant methionyl human leptin therapy in replacement doses improves insulin resistance and metabolic profile in patients with lipoatrophy and metabolic syndrome induced by the highly active antiretroviral therapy. J Clin Endocrinol Metab 2006; 91(7):2605-2611. 30. Moran SA, Patten N, Young JR et al. Changes in body composition in patients with severe lipodystrophy after leptin replacement therapy. Metabolism 2004; 53(4):513-519. 31. Oral EA, Simha V, Ruiz E et al. Leptin-replacement therapy for lipodystrophy. N Engl J Med 2002; 346(8):570-578. 32. McDuffie JR, Riggs PA, Calis KA et al. Effects of exogenous leptin on satiety and satiation in patients with lipodystrophy and leptin insufficiency. J Clin Endocrinol Metab 2004; 89(9):4258-4263. 33. Lebrethon MC, Vandersmissen E, Gerard A et al. In vitro stimulation of the prepubertal rat gonadotropin-releasing hormone pulse generator by leptin and neuropeptide Y through distinct mechanisms. Endocrinology 2000; 141(4):1464-1469. 34. Chan JL, Heist K, DePaoli AM et al. The role of falling leptin levels in the neuroendocrine and metabolic adaptation to short-term starvation in healthy men. J Clin Invest 2003; 111(9):1409-1421. 35. Oral EA, Ruiz E, Andewelt A et al. Effect of leptin replacement on pituitary hormone regulation in patients with severe lipodystrophy. J Clin Endocrinol Metab 2002; 87(7):3110-3117. 36. Musso C, Cochran E, Javor E et al. The long-term effect of recombinant methionyl human leptin therapy on hyperandrogenism and menstrual function in female and pituitary function in male and female hypoleptinemic lipodystrophic patients. Metabolism 2005; 54(2):255-263. 37. Petersen KF, Oral EA, Dufour S et al. Leptin reverses insulin resistance and hepatic steatosis in patients with severe lipodystrophy. J Clin Invest 2002; 109(10):1345-1350. 38. Simha V, Szczepaniak LS, Wagner AJ et al. Effect of leptin replacement on intrahepatic and intramyocellular lipid content in patients with generalized lipodystrophy. Diabetes care 2003; 26(1):30-35. 39. Javor ED, Ghany MG, Cochran EK et al. Leptin reverses nonalcoholic steatohepatitis in patients with severe lipodystrophy. Hepatology 2005; 41(4):753-760. 40. Welt CK, Chan JL, Bullen J et al. Recombinant human leptin in women with hypothalamic amenorrhea. N Engl J Med 2004; 351(10):987-997.

Chapter 12

Use of Anti-Leptin or Anti-Leptin Receptor Antibodies as Blockers of Immune Response Giuseppe Matarese* and Veronica De Rosa

Abstract

L

eptin, a hormone produced primarily by adipose cells, is known to be critically involved in regulating nutrient intake and metabolism. Increasing evidence has indicated that leptin also plays crucial role in modulating immune response. Several studies including our recent findings have demonstrated that leptin can enhance the proliferation and production of proinflammatory cytokines in T-cells, macrophages and dendritic cells. More recent studies have suggested that leptin is also a negative signal for the proliferation and expansion of regulatory T-cells (Tregs), a specific subset involved in the control of immune and autoimmune responses. Strategies aimed at interfering with the leptin axis such as leptin- or leptin-receptor neutralizing antibodies have been suggested as feasible molecular tools to dampen chronic inflammation and autoimmunity. We will analyze the most recent advances in the field and the possibility to utilize this approach as novel therapeutic strategy.

Introduction

Over the last century, improved hygienic and nutritional conditions have reduced significantly the incidence of infectious diseases, at least in the most-developed countries.1 In parallel with the improvement in nutritional status, an increase in susceptibility to autoimmune disorders has emerged.1 Recently, it has been proposed that the lifestyle in developed countries, with reduced exposure to environmental pathogens, could be relevant to the increase in the prevalence of autoimmune disorders.1 Conversely, in less-affluent societies, exposure to micro-organisms, pathogens and other environmental influences might promote the development of T-regulatory responses that protect against autoimmune responses.1 Leptin, an adipocyte-derived hormone of the long-chain helical cytokine family, has been proposed recently to act as a link between nutritional status and immune function.2 Leptin has multiple biological effects on nutritional status, metabolism and the neuro-immunoendocrine axis. The circulating concentration of leptin is proportional to fat mass and reduced body fat or nutritional deprivation—associated typically with hypoleptinaemia—is a direct cause of secondary immunodeficiency and increased susceptibility to infections.2 The reason for this association was not apparent until recently. Now, it can be hypothesized that a low concentration of serum leptin increases susceptibility to infectious diseases by reducing T-helper (Th)-cell priming and direct effects on thymic function.3 Furthermore, congenital deficiency of leptin has been found to be associated with increased frequency of infection and related mortality. By contrast, the Th1-promoting effects of leptin have been linked recently to enhanced *Corresponding Author: Giuseppe Matarese—Laboratorio di Immunologia, Istituto di Endocrinologia e Oncologia Sperimentale, Consiglio Nazionale delle Ricerche, Via S. Pansini 5, 80131 Napoli, Italy. Email: [email protected]

Leptin and Leptin Antagonists, edited by Arieh Gertler. ©2009 Landes Bioscience.

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susceptibility to experimentally induced autoimmune diseases, such as experimental autoimmune encephalomyelitis (EAE), type 1 diabetes (T1D), antigen-induced arthritis (AIA), etc.3 These latter observations suggested a novel role for leptin in determining the gender bias of susceptibility to autoimmunity, because female mice and humans, which are relatively hyperleptinaemic, have an increased frequency of autoimmune diseases compared with males, which are relatively hypoleptinaemic.3 In view of these findings, we suggest leptin as novel candidate able to explain at least in part the increased frequency of autoimmune disorders in the more effluent countries and in females. Therefore, recent observation from our and other laboratories have suggested the possibility to utilize either anti-leptin or anti-leptin receptor blocking antibodies to neutralize leptin’s action as possible promoter in breaking self-tolerance.3

Leptin Has Multiple Functions in Immunity

Recent studies have suggested the involvement of leptin in regulating lymphopoiesis and immune function.4 For example, leptin can increase the proliferation and production of a variety of cytokines in T-cells and monocytes/macrophages.4 Moreover, leptin has been shown to modulate immune responses towards the Th1 phenotype and suppress the Th2 effect by stimulating dendritic cell (DC) differentiation and function.3-4 In relation to the important function of leptin in immune response, the leptin receptor expression has been found not only in the central nervous system but also in a variety of immune cells. Encoded by the diabetes (db) gene, the leptin receptor (Ob-R) is a member of class I cytokine receptors and has signalling capability of IL-6 type cytokine receptors.4 Ob-R mRNA gives rise to six different forms of the receptor by alternative splicing but only the long isoform (Ob-Rb) has been recognized to be of prime importance in leptin-mediated signaling. Leptin promotes the survival of T-lymphocytes via the up-regulation of anti-apoptotic proteins such as Bcl-xL.5 Recently, leptin has been shown to suppress the expansion of Foxp3+CD4+CD25+ regulatory T (Treg) cells whereas in vitro neutralization with anti-leptin antibody results in Treg-cell proliferation6-7 (Fig. 1).

Leptin Is Involved in the Development of Various Diseases

In addition to its critical role in protective immune responses, leptin has been suggested to be involved in the development of various diseases including autoimmune diseases and cancer.4,8 The immuno-modulatory effects of leptin in promoting Th1 cytokine production have been linked to enhanced susceptibility to other experimentally-induced autoimmune diseases, such as EAE, T1D mellitus and other experimentally-induced autoimmune disorders.4 Although leptin blockade by neutralization in vivo significantly improves clinical score and delays disease progression by inhibiting pathogenic T-cell autoreactivity in EAE,9 it is unclear whether leptin exerts a direct effect on autoreactive/effector T-cell differentiation, autoantibody production and/or antigen presentation. Recent evidence also indicates a role for leptin in the control of Tregs proliferation in vitro and in vivo; indeed, leptin neutralization with mAbs induced expansion of Tregs upon anti-CD3/CD28 stimulation. This phenomenon is also a mechanism to be taken into account for the observed improvement of clinical course in animal models autoimmunity6 (Fig. 1).

Immunotherapeutic Applications Targeting Leptin: Current Evidence and Hypotheses

Leptin-based therapy is currently administered to a few cases of genetically leptin-deficient individuals and to morbidly obese nonleptin-deficient patients to reduce their food intake.10 This treatment is effective in genetically leptin-deficient individuals in restoring some of the impaired neuroendocrine functions and in controlling food intake and reproductive function.10 Conversely, in nonleptin-deficient obese patients the effect of leptin administration is modest on food intake and weight loss, probably because of the leptin receptor desensitization due to the already high circulating leptin.11 Although the above are the only therapeutic uses of leptin on humans, additional clinical applications could be hypothesized on the basis of the immunoregulatory properties of leptin on CD4+ T-cells. In immunodeficiency associated with reduced food intake such as anorexia

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nervosa or HIV-1 infection, leptin levels do not increase and are often reduced, as well as CD4+ T-cell numbers and function.12 Leptin administration might be suggested to provide help for immunoreconstitution via increased thymic T-cell output and cell-mediated Th1 immune responses. Increased Th1 responses may also be envisaged for resistant tuberculosis in immunocompromised hosts and in the context of vaccination protocols to boost immune responses.12 In animals with reduced leptin levels, which have reduced delayed-type hypersensitivity responses and increased Th2 responses, the administration of leptin completely restores delayed-type hypersensitivity reactivity as well as the Th1 phenotype.3 A possible side effect of leptin therapy in these immunocompromised hosts is the inhibition in the food intake due to the leptin action on the hypothalamus. This side effect can be avoided by using leptin receptor antagonists not able to cross the blood brain barrier such as anti-leptin neutralizing antibodies. With this approach it would be possible to have the effects of leptin on the peripheral tissues including the immune system and not on food intake. Moreover, modulation of circulating leptin levels may be considered as a newer possible strategy to intervene on some inflammatory and autoimmune conditions. This approach could be easily applicable as it would be possible to reduce circulating leptin by caloric deprivation, thus overcoming some disadvantages of other cytokine-based therapies. In addition to starvation, diets rich in polyunsaturated fatty acids (n-3, fish oil) and low in saturated fatty acids and/or are zinc-free could also be considered to diminish circulating leptin with little effects on body fat composition.13 Clinical trials involving starvation to modulate pro-inflammatory responses in human autoimmune diseases have already been reported as successful.14 A better understanding of the role of leptin in the modulation of inflammation and autoimmunity is a promising yet little explored possibility

Figure 1. The figure shows that either leptin- or leptin-receptor desensitization upon T-cell receptor stimulation is able on one side to inhibit effector T-cell expansion and on the other to induce Tregs proliferation. These combined approaches could be utilized to dampen autoimmunity and inflammation though direct neutralization of the leptin axis or via expansion of immunoregulatory Tregs.

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that may lead to the addition of novel interesting possibilities to the armamentarium of the current immunotherapies for inflammation and autoimmune conditions (Fig. 1).

Leptin Neutralization: Novel Strategies to Block Autoimmunity and to Improve Leptin Resistance Observed in Obesity Effect on Autoimmunity: the EAE Model

Previous studies by our group and others have shown the relevance of leptin in the pathogenesis of EAE.15 In particular, it was previously reported that leptin decient ob/ob mice are resistant to induction of the disease, whereas in wild-type, EAE-susceptible controls, a surge of serum leptin precedes acute EAE.15 In addition, in vivo neutralization of leptin is effective at blocking initiation, progression and clinical relapses of EAE, an animal model of Multiple Sclerosis.9 We and others have previously reported that in the central nervous system (CNS) of EAE mice, both infiltrating T-cells and neurons express leptin during the acute phase of the disease and the degree of leptin expression within the lesions correlates with CNS inflammatory score and disease severity.16 Because of the possibility of an autocrine loop sustaining autoreactive Th1 lymphocytes in EAE, we investigated the Delayed Type Hypersensitivity (DTH) response as well as T-cell proliferation and cytokine secretion in response to proteolipid protein peptide (PLP)139-151 in leptin-neutralized mice versus controls.9 Anti-leptin—treated animals showed reduced DTH and T-cell proliferative responses to PLP139-151 peptide associated with a Th2/regulatory-type cytokine shift. This evidence was also supported by increased expression levels of the regulatory T-cell master gene Foxp3 in CD4+ T-cells from mice with EAE.9 Leptin blockade also affected expression of ICAM-1, OX-40 and VLA-4 on CD4+ T-cells. In particular, reduced expression of ICAM-1 was consistent with our previous findings showing that leptin treatment increases surface expression of this adhesion molecule on T-cells.9 This finding suggested the possibility that neutralization of leptin directly affects the cognate interaction leading to reactive and/or autoreactive T-cell activation. Moreover, marked reduction of OX-40 was also observed after leptin blockade. Since OX-40 is an important costimulatory molecule with prosurvival activity for CD4+ T-cells and signalling through this molecule breaks peripheral T-cell tolerance, our data suggest that leptin may affect expression of key molecules on T-lymphocytes involved in the mechanisms of immune tolerance. Surprisingly, we also observed that leptin neutralization induced increased expression of VLA-4, the α4β1 integrin shown to play an integral part in the homing and migration of cells that induce EAE.9 However, experimental evidence has shown that administration of anti–VLA-4 ameliorated EAE only if it was initiated before disease onset, whereas treatment during acute disease exacerbated EAE and enhanced the accumulation of T-cells in the CNS.17 Therefore, we are tempted to hypothesize that the induction of VLA-4 on CD4+ T-cells after leptin neutralization could be associated in part with an increased cell capability to migrate into the CNS and produce regulatory cytokines able to downmodulate EAE. Of note, these data are in agreement with other findings showing that adhesion molecules are increased on regulatory T-cells in experiments of protection from EAE. To further address, at the biochemical level, whether in vivo leptin neutralization interferes with the signalling capacity of autoreactive T-cells, we analyzed several molecular pathways associated with T-cell anergy/activation and cytokine switch.9,18 We found that CD4+ T-cells from mice treated with leptin antagonists showed hyporesponsiveness to PLP139–151 peptide, which was indicated by accumulation of p27Kip-1. This negative cell cycle regulator plays a central role in blocking clonal expansion of T-cells and is therefore critical for anergy induced by blockade of costimulatory pathways.18 We also found that the hyporesponsive state induced by leptin antagonism was associated with marked increase of ERK1/2 phosphorylation, confirming involvement of ERK1/2 in the improvement of EAE. It is interesting to observe that our findings with leptin antagonism seem to involve pathways affected by statins, cholesterol-lowering drugs that have recently been shown to reduce production of leptin by adipocytes,19 promote Th2 responses and improve EAE by disabling downregulation of p27Kip-1 and upregulating phosphorylation of ERK1/2. Finally, we also observed at the biochemical level the induction of phosphorylation of

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the STAT6 transcription factor, well known to be able to induce the transcription of IL-4 and associated with a classical Th2/regulatory-type cytokine response during EAE. Taken together, our results provide a framework for leptin-based intervention in EAE and identify molecules with possible therapeutic potential for the diseases.

Effect on Tregs: Role of Leptin Neutralization

Freshly isolated Tregs produce leptin and express high amounts of ObR. In vitro neutralization with anti-leptin mAb, during anti-CD3 and anti-CD28 stimulation, result in Tregs proliferation that is IL-2-dependent.6 Tregs that proliferated in the presence of anti-leptin mAb had increased expression of Foxp3 and remained suppressive over time. The phenomena appeared secondary to leptin signalling via ObR and, importantly, leptin neutralization reversed the anergic state of the Tregs, as indicated by downmodulation of the p27kip1 and the phosphorylation of the ERK1/2.6 Taken together these findings suggest a potential role for leptin neutralization as novel protocol to expand Tregs in vitro and to utilize them for adoptive immunotherapy in autoimmunity6 (Fig. 1).

Effect on Leptin Resistance: the Obesity Model

Though the discovery of leptin energized the study of energy balance, much of the initial enthusiasm has waned with the realization that obesity is not a condition of leptin insufficiency but instead of leptin resistance.11 Though the existence of leptin resistance is well accepted, it remains ill-defined. Leptin resistance is often described as a state in which circulating levels are elevated coincident with ongoing hyperphagia and obesity. By this standard, most obese individuals are leptin resistant. Leptin resistance is also defined as a failure of exogenously administered leptin to suppress food intake. Leptin resistance can also be defined from a molecular standpoint, as a failure of leptin to activate key signalling molecules within target neurons. Yet leptin activates multiple intracellular signalling molecules of which resistance is often only demonstrated for a few and there is also evidence for variations in leptin activated STAT-3 across the brain, such that leptin sensitivity may vary even within the same individual.11 What is known about leptin resistance is that it involves at least two separate mechanisms, the first being reduced transport across the blood brain barrier and the second a reduced capacity for intracellular signalling within target neurons. A reduction in leptin transport across the blood brain barrier (BBB) has been demonstrated directly in obese animals and additional work documents reduced sensitivity to peripheral leptin signalling prior to loss of central leptin sensitivity, yet it has not been demonstrated that alterations in leptin transport directly influence body weight or food intake in lean or obese animals and thus the role of reduced transport in the aetiology of obesity is not fully clear. For instance, would enhancing transport in obese individuals reduce body weight? These issues are complicated by the fact that the cellular mechanisms of leptin transport and its disruption in obesity are not fully resolved. Obesity is also associated with reduced capacity for leptin signalling within target neuron. This resistance is manifest by an attenuated response to direct brain leptin injections (which bypass the BBB), both in terms of food intake and activation of intracellular signalling. While leptin activates multiple intracellular signalling cascades, leptin resistance is almost exclusively characterized as a reduced activation of the transcription factor STAT-3.11 Progress has been made in identifying potential cellular mediators of biochemical leptin resistance, leading to the identification of two molecules; suppressor of cytokine signalling 3 (SOCS3) and protein tyrosine phosphatase 1B (PTP1B). SOCS3 is a member of a family of proteins produced in response to cytokine signalling which act as intracellular negative feedback signals. SOCS3 expression is induced by STAT-3 signalling and it in turns binds to the leptin receptor and blocks the activation of STAT-3. Based on these observations, it was predicted that SOCS3 might contribute to leptin resistance. Genetic approaches have confirmed this hypothesis, as mice bearing genetic modifications which delete SOCS3 or inhibit its ability to bind the leptin receptor exhibit reduced food intake and body weight and a resistance to diet-induced obesity. PTP1B is likewise implicated in leptin resistance. As a phosphatase, PTP1B binds to and dephosphorylates Janus Kinase 2 ( JAK2), the initial tyrosine kinase mediating leptin receptor signaling. Overexpression of PTP1B in vitro dampens signalling

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from the leptin receptor and mice genetically deficient for PTP1B are lean and hypersensitive to leptin. Deletion of PTP1B exclusively within neurons recapitulates the leptin hypersensitivity and resistance to diet-induced obesity and pharmacological inhibition of PTP1B enhances the effects of central leptin injection. These data collectively indicate that PTP1B acts within the brain to tonically inhibit signalling from the leptin receptor. Starting from the above considerations, it should be considered the possibility to reverse the level of “leptin resistance” in obesity, thanks to the use of anti-leptin neutralizing antibodies followed by administration of recombinant leptin. This novel strategy bases on the possibility that leptin neutralization with antibodies is able to desensitize the ObRb and alters the levels of SOCS3 andPTP1B observed in diet-induced obesity (DIO) mice (our unpublished data); subsequent recombinant leptin administration is then able to inhibit food intake very efficiently as compared with leptin administration only. These preliminary data suggest that this approach can be considered for future strategies in the treatment of human obesity, in which leptin administration alone has not been efficient.

Leptin-Receptor Neutralization

Leptin receptor neutralization could represent an alternative approach to blockade of leptin (Fig. 1). Recently, a series of monoclonal antibodies have been generated against leptin receptor.20 The possibility to utilize leptin receptor blockers has also been considered as alternative approach to leptin blockade. Preliminary data from our laboratory suggest also the possibility to utilize this molecular tool in the treatment of autoimmunity and inflammation. Indeed, ObR blockade has shown an even higher capacity of to induce Tregs expansion in vitro. This marked effect has been considered in the possibility to expand exvivo Tregs from patients with autoimmunity, in which a specific defect in Tregs number has been observed. Another approach could be related to the possibility to utilize directly ObR neutralization in vivo to treat inflammation and autoimmunity (Fig. 1). This approach in any case generates a series of concerns due to the fact that, being ObR short forms expressed ubiquitously, neutralizing monoclonals against the extracellular domain could bind on many tissues and determine potential tissue damage by activating complement. Therefore, the generation of Fab fragments is under consideration to eventually reduce these possible adverse effects.

Conclusions and Future Perspectives

There are still many questions concerning the role of several molecules at the interface between metabolism and immunity in the regulation of the two systems. Significant leaps of knowledge have been done in recent years in the expanding field of study of such molecules. While new information is unveiling the complexity connecting metabolism and immunity, further research is still needed. However, the adipose tissue can no longer be regarded merely as a store of body fat but rather as an active participant in the regulation of essential body processes with prominent roles particularly in the balance of inflammation and immune homeostasis. In this context antagonism of the leptin axis can be considered as novel strategy to control inflammation and also peripheral leptin resistance observed in obesity and related conditions. At the moment significant amount of data has been generated suggesting that this approach could be relevant in novel therapeutic strategies.

Acknowledgements

This work was supported by grants from the Juvenile Diabetes Research Foundation ( JDRF)-Telethon-Italy (n. GJT04008) and from Fondazione Italiana Sclerosi Multipla (FISM) (n. 2002/R/55). The author wish to thank Claudio Procaccini for helpful discussion and revising the manuscript.

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1. Christen U, von Herrath MG. Infections and autoimmunity—good or bad? J Immunol 2005; 174(12):7481-6. 2. Matarese G, La Cava A, Sanna V et al. Balancing susceptibility to infection and autoimmunity: a role for leptin? Trends Immunol 2002; 23(4):182-7. 3. La Cava A, Matarese G. The weight of leptin in immunity. Nat Rev Immunol 2004; 4(5):371-9. 4. Fantuzzi G, Faggioni R. Leptin in the regulation of immunity, inflammation and hematopoiesis. J Leukoc Biol 2000; 68(4):437-46. 5. Sánchez-Margalet V, Martín-Romero C, Santos-Alvarez J et al. Role of leptin as an immunomodulator of blood mononuclear cells: mechanisms of action. Clin Exp Immunol 2003; 133(1):11-9. 6. De Rosa V, Procaccini C, Calì G et al. A key role of leptin in the control of regulatory T-cell proliferation. Immunity 2007; 26(2):241-55. 7. Taleb S, Herbin O, Ait-Oufella H et al. Defective leptin/leptin receptor signaling improves regulatory T-cell immune response and protects mice from atherosclerosis. Arterioscler Thromb Vasc Biol 2007; 27(12):2691-8. 8. Surmacz E. Obesity hormone leptin: a new target in breast cancer? Breast Cancer Res 2007; 9(1):301. 9. De Rosa V, Procaccini C, La Cava A et al. G Leptin neutralization interferes with pathogenic T-cell autoreactivity in autoimmune encephalomyelitis. J Clin Invest 2006; 116(2):447-55. 10. Farooqi IS, Matarese G, Lord GM et al. Beneficial effects of leptin on obesity, T-cell hyporesponsiveness and neuroendocrine/metabolic dysfunction of human congenital leptin deficiency. J Clin Invest 2002; 110(8):1093-103. 11. Coll AP, Farooqi IS, O’Rahilly S. The hormonal control of food intake. Cell 2007; 129(2):251-62. 12. Schaible UE, Kaufmann SH. Malnutrition and infection: complex mechanisms and global impacts. PLoS Med 2007; 4(5):e115. 13. Shay NF, Mangian HF. Neurobiolog y of zinc-influenced eating behavior. J Nutr 2000; 130(5S Suppl):1493S-9S. 14. La Cava A, Matarese G, Ebling FM et al. Leptin-based immune intervention: current status and future directions. Curr Opin Investig Drugs 2003; 4(11):1327-32. 15. Matarese G, Di Giacomo A, Sanna V et al. Requirement for leptin in the induction and progression of autoimmune encephalomyelitis. J Immunol 2001; 166(10):5909-16. 16. Sanna V, Di Giacomo A, La Cava A et al. Leptin surge precedes onset of autoimmune encephalomyelitis and correlates with development of pathogenic T-cell responses. J Clin Invest 2003; 111(2):241-50. 17. Theien BE et al. Discordant effects of anti–VLA-4 treatment before and after onset of relapsing experimental autoimmune encephalomyelitis. J Clin Invest 2001; 107:995-1006. 18. Schwartz RH. T-cell anergy. Annu Rev Immunol 2003; 21:305-334. 19. Youssef S et al. The HMG-CoA reductase inhibitor, atorvastatin, promotes a Th2 bias and reverses paralysis in central nervous system autoimmune disease. Nature 2002; 420:78-84. 20. Fazeli M, Zarkesh-Esfahani H, Wu Z et al. Identification of a monoclonal antibody against the leptin receptor that acts as an antagonist and blocks human monocyte and T-cell activation. J Immunol Methods 2006; 312(1-2):190-200.

Chapter 13

Use of Leptin Antagonists as Anti-Inflammatory and Anti-Fibrotic Reagents Eran Elinav* and Arieh Gertler

Abstract

L

eptin has been implicated as a pro-inflammatory cytokine, involved in the activation of effector T-cells as well as various other components of the innate and adaptive immune response. Leptin-deficient ob/ob mice exhibit resistance to several T-cell-mediated autoimmune disorders including experimental arthritis, hepatitis and experimental allergic encephalomyelitis. Inhibition of the leptin-induced pro-immune response may be useful as a therapeutic tool in T-cell-mediated autoimmune disorder. Herein, we report on the use of our recently developed competitive leptin antagonist as an anti-inflammatory agent in models of acute and chronic T-cell-mediated liver inflammation and chronic liver fibrosis. This beneficial effect may be mediated by both direct T-cell modulatory effects and inhibition of hepatic stellate cell activation and function, leading to alleviation of liver fibrosis. Our results suggest that leptin inhibition may be developed into a rational immunomodulatory therapeutic modality.

Introduction

Leptin possesses potent immunomodulatory properties. Structurally, leptin is similar to the interleukins IL2, IL6 and IL15, making it a member of the cytokine superfamily1 and leptin receptors (LRs) are structurally similar to hematopoietic cytokine receptors.2 LRs are found on CD4 and CD8 lymphocytes, monocytes,3 natural-killer (NK) lymphocytes4 and hepatic stellate cells (HSCs).5 Leptin enhances T-cell proliferation and pro-inflammatory cytokine secretion via activation of JAK/STAT signaling.6 Leptin-deficient ob/ob mice are resistant to several Th1-mediated immune disorders, including allergic experimental encephalomyelitis,7 concanavalin A (ConA) hepatitis, experimental arthritis and autoimmune nephritis, but they are extremely vulnerable to lipopolysaccharide-induced hepatic damage.8 Leptin replenishment reverses these disorders.5,9,10 Leptin increases both inflammatory and pro-fibrogenic responses in the liver caused by hepatotoxic chemicals. Effects of leptin on T-cell immunity have also been documented in humans. Correlation studies in patients with rheumatoid arthritis, systemic lupus erythematosis and multiple sclerosis suggest leptin’s role in autoimmune diseases.11 Moreover, there is increasing evidence that leptin is involved in the pathogenesis of various autoimmune diseases. Of particular relevance to the present study is the role of leptin in inflamed colonic epithelial cells which express and release leptin into the intestinal lumen. Leptin has been reported to induce epithelial wall damage and neutrophilic infiltration, a characteristic histological finding in acute intestinal inflammation and inflammatory bowel disease (IBD).12 Moreover, a key role for intestinal leptin in an experimental immune model of chronic intestinal inflammation in mice has been demonstrated. In this model, leptin effects *Corresponding Author: Eran Elinav—Gastroenterology and Liver Institute, Tel Aviv Sourasky Medical Center (TASMC), Tel Aviv, 64239, Israel. Email: [email protected]

Leptin and Leptin Antagonists, edited by Arieh Gertler. ©2009 Landes Bioscience.

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appear to be mediated through LR transduction pathways on Th1 T-lymphocytes.13 Leptin has also been recently documented as a negative regulator of naturally occurring Foxp3+CD4+CD25+ regulatory T (Treg) cells in humans, which express high amounts of LR. Further, this effect could be blocked by specific anti-leptin mAb, suggesting leptin blocking as a potential intervention in immune and autoimmune diseases.14 Thus, recent data have established a regulatory function for leptin in immunity similar to that of a pro-inflammatory cytokine, while gene-targeting studies have also demonstrated an essential role for leptin in regulating hematopoiesis and lymphopoiesis.15 Inhibition of leptin’s pronounced pro-inflammatory activity in acute or chronic hepatitis may be achieved by producing leptin antagonists capable of binding, but not activating, LRs. In their pioneering work, Tavernier and his group documented the resemblance between leptin binding to its receptor and the interaction between IL6 and gp13016-18 and suggested the existence of a novel, previously unidentified leptin site III composed of several amino acids,18 including Ser 120 and Thr 121 (see also Zabeau et al in the present book). Mutagenesis of those residues in human and mouse leptin to Ala resulted in the creation of potent leptin antagonists.19 To determine whether, in addition to the N-terminal portion of helix D, other parts of the leptin molecule also contribute to leptin site III, careful analysis of the structures of IL6-receptor complexes vIL6/gp13020 and IL6/IL6Rα/gp130,21 in which site III was first identified, was performed and leptin antagonists (mutants of human/ovine/rat/mouse leptins) which bind to but do not activate LR were developed by our group.22-24 This was achieved using a sensitive bidimensional hydrophobic cluster analysis which identified the LDFI sequence (amino acids 39-42), located in the loop connecting helices A and B, as an additional sequence contributing to leptin’s ability to activate LR, most likely by affecting site III. Mutations of some or all those amino acids to Ala in human/ovine/rat/mouse leptins did not change their binding properties, but abolished their biological activity and converted these muteins into potent antagonists.22,23 So far, over 15 muteins have been prepared and all have exhibited potent antagonistic activity in various in-vitro and in-vivo bioassays. In this paper, we report on the effect of mouse leptin antagonist L39A/D40A/F41A (MLA) on ConA- or thioacetamide (TAA)-induced acute hepatitis and TAA-induced chronic hepatitis with subsequent fibrosis. In ConA-induced hepatitis, massive recruitment of CD4+ and NKT-lymphocytes occurs within hours of ConA administration, resulting in fulminant T-cell immune-mediated hepatitis. In the TAA model, acute administration results in toxic hepatitis, while chronic administration results in the activation of both T-cells and HSCs, resulting in chronic hepatitis which eventually leads to the development of hepatic fibrosis. We believe that these models cover the spectrum of T-cell-mediated hepatic inflammation and as such serve as appropriate models to elucidate the possible anti-inflammatory role of peripheral leptin inhibition. In addition, leptin signaling has been previously shown to be a prerequisite factor in HSC activation and function. Thus, leptin inhibition may also lead to the amelioration or prevention of fibrosis in a direct HSC-mediated (and non-immune mediated) fashion.

Results Acute Hepatitis

Treatment with either ConA (200 µg/g body weight) or TAA (350 mg/kg body weight) given as a single intraperitoneal (i.p.) injection resulted in a dramatic elevation in alanine aminotransferase (ALT) activity after 24 h. In both cases, administration of leptin or MLA alone, in the absence of induction of liver injury, had no effect. However, whereas in the ConA treatment this effect was enhanced by leptin and remarkably attenuated by MLA (Fig. 1a), neither leptin nor MLA had any effect on the dramatic acute-TAA-induced elevation in ALT (Fig. 1b). ConA and especially ConA + leptin treatments drastically increased sickness and mortality and this effect was almost totally reversed by the simultaneous application of MLA (Table 1). Histological analysis reflected both the increase in ALT activity and health observations (Fig. 2). Livers from ConA-treated mice featured the classic combination of T-cell-mediated infiltrates and areas of hepatic necrosis. Livers from leptin + TAA-treated mice featured exacerbated inflammation, manifested as confluent areas

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135

Figure 1. Effect of acute treatment of mice with concanavalin A (ConA) (a) or thioacetamide (TAA) (b) on alanine aminotransferase (ALT) activity in sera after 24 h. Two acute inflammatory experimental models, Con A- (200 mg/kg body weight) and TAA- (350 mg/kg body weight)-induced hepatitis in female 8- to 10-week-old C57bl mice, were employed. In all experiments, mice were i.p. injected with mouse leptin (1.0 µg/g.day) (L) and/or mouse leptin antagonist L39A/D40A/F41A (50 µg/g/day) (A), control (NaCl) (C), control or leptin + leptin antagonists (L + A). Serum ALT activity was used as a marker of liver injury. Serum was collected in glass tubes, centrifuged and ALT was analyzed on the day of sampling using a Kone Progress Selective Chemistry Analyzer.

of widespread necrosis and large amounts of hepatic lymphocytic exudates. In contrast, only mild inflammatory exudates and rare small areas of necrosis were observed in MLA-treated mice. We believe that the difference in MLA’s effect on acute TAA hepatitis vs ConA hepatitis stems from differences in the mechanism of liver injury in these two models. While acute injury in acute TAA hepatitis results mainly from direct hepatotoxic injury, liver injury in ConA hepatitis is immune-related, resulting from the hepatotoxic effects of CD4+ and NK T-lymphocytes. Thus, our results suggest that antagonizing peripheral leptin activity results in immune-modulation of

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Table 1. Effect of acute treatment with ConA (200 mg/kg body weight) or TAA (350 mg/kg body weight) in absence or presence of mouse leptin (1 mg/kg body weight) or mouse leptin antagonist (MLA) D39A/L40A/F41A (50 mg/kg body weight) on health and mortality of mice 24 hours after injection Experimental Treatment1

Healthy (%)

Sick2 (%)

Dead (%)

n

Control

100

0

0

15

Leptin

100

0

0

12

MLA

100

0

0

12

ConA experiment (four experiments)

ConA

0

93

7

15

ConA + Leptin

0

73

27

15

ConA + MLA

93

7

0

15

ConA + Leptin + MLA

86

14

0

15

TAA experiment (one experiment) Control

100

0

0

8

Leptin

100

0

0

8

Antagonist

100

0

0

8

TAA

0

86

14

7

TAA + Leptin

0

86

14

7

TAA + MLA

72

14

14

7

TAA + Leptin + MLA

72

28

0

7

1

For further details, see legend to Figure 1. 2 The sick appearance was defined as the animal being apathetic, relatively immobile and having stiff fur.

the T-cell effect, leading to protection from the immune-mediated ConA injury but no protection from direct hepatotoxic damage.

Chronic Hepatitis and Fibrosis

I.p. injections of 200 mg/kg TAA three times a week resulted in the development of chronic hepatitis and subsequently liver fibrosis, with a mortality rate of approximately 50% after 8 weeks of disease induction (Fig. 3). Co-administration of leptin was associated with significantly enhanced hepatitis and fibrosis, resulting in high mortality rates (approximating 100%) after only 5 weeks of induction. The effect of leptin can probably be attributed to a combination of enhanced inflammation and liver fibrosis. This aggravation in disease severity was almost completely attenuated by co-administration of MLA. Interestingly, while MLA significantly abolished the leptin effect, it only partially prevented the TAA-induced mortality, despite the histological observation that TAA-induced hepatic damage is largely prevented by MLA (Fig. 4). TAA-induced chronic hepatitis and fibrosis were manifested as scattered areas of lymphocytic infiltrates and formation of regenerative nodules after 8 weeks of disease induction. Co-administration of leptin resulted in significant worsening of both inflammation and fibrosis, featuring large infiltrates throughout the liver parenchyma, as well as thick fibrotic bands. Administration of MLA resulted in mild improvement of the hepatic inflammation and attenuation of fibrosis, while co-administration of leptin and leptin antagonist resulted in

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137

Figure 2. Histological evaluation of liver in concanavalin A (ConA)-treated mice. For each mouse, a single liver segment was fixed in 10% buffered formaldehyde and embedded in paraffin for histological analysis. Sections (5 µm) were stained with hematoxylin/eosin. Inflammation severity was assessed by two blinded expert pathologists. Data were derived from blinded analysis of five sections from each of 10 animals per group. MLA, mouse leptin antagonist.

Figure 4. Histological evaluation of liver in mice treated chronically with low doses of thioacetamide (TAA) after 8 weeks. For each mouse, a single liver segment was fixed in 10% buffered formaldehyde and embedded in paraffin for histological analysis. Sections (5 µm) were stained with hematoxylin/eosin.

Figure 5. Sirius red staining of liver sections from mice treated chronically with low doses of thioacetamide (TAA). (A) TAA + leptin or (B) TAA + leptin + mouse leptin antagonist (MLA). For each mouse, a single liver segment was fixed in 10% buffered formaldehyde and embedded in paraffin for histological analysis. Sections (5 µm) were stained with Sirius red and viewed under a light microscope. Red staining represents collagen I deposition. Data were derived from blinded analysis of five sections from each of 10 animals per group.

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Figure 3. Mortality of mice treated chronically with low doses of thioacetamide (TAA) after 4 and 8 weeks of treatment. Female 8- to 10-week-old C57bl mice were used. Induction of chronic liver fibrosis was achieved by i.p. injection of 200 mg/kg TAA (Sigma Co., Rehovot, Israel), three times a week for 8 weeks. Experimental groups (n = 8) were administered mouse leptin i.p. (Lep, 1 µg/g.day in two injections, or Lep (1/2), 0.5 µg/g.day in two injections or 50 µg/g mouse leptin antagonists (MLA), or a combination of the two. All animals were grown under 12-h light-dark cycles, in accordance with regulations of the institutional authority for animal care.

significant attenuation (but not complete resolution) of leptin-induced hepatitis and fibrosis. To further characterize the fibrotic effect of TAA in the liver we performed Sirius red staining, which demonstrated MLA inhibition of the leptin-enhanced TAA fibrotic effect, as indicated by excessive appearance of collagen patches (Fig. 5, see previous page). HSCs are believed to be central players in the pathogenesis of hepatic fibrosis and have also been suggested to be activated by leptin. Thus, we also tested whether in-vitro administration of MLA could block leptin’s pro-fibrogenic effects. As shown in Table 2, MLA indeed abolished leptin-induced HSC activation, as reflected by Proliferating Cell Nuclear Antigen (PCNA) expression, collagen α1 promoter activity (measured as LUC/βGAL ratio) and α-smooth muscle actin (SMA) protein expression. These results hint at a potentially direct MLA-mediated effect on HSC function, in addition to its anti-inflammatory activity.

Conclusions

The presented data demonstrate that inhibition of leptin activity by a competitive leptin antagonist results in attenuation of acute and chronic hepatic inflammation, as well as improvement in leptin-induced exacerbation of liver fibrosis. Interestingly, no significant metabolic effects were observed with the MLA treatment of the chronic disease, including weight gain, hypercholesterolemia, hypertriglyceridemia, or non-alcoholic fatty liver disease (data not shown). This absence of metabolic effects may stem from reduced penetration of the antagonist through the blood-brain barrier, preferential peripheral rather than CNS leptin inhibition, or compensatory

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Table 2. In-vitro effect of MLA on several leptin-induced effects in primary hepatic stellate cells1 (mean ± SEM, n = 3). Leptin concentration was 1 ng/ml and MLA concentration was 50 ng/ml Treatment

α-SMA/GAPH2 Expression4

PCNA/GAPH2 Expression

LUC/βGAL3 Activity Units × 10 –4

Control Leptin MLA Leptin + MLA

0.35 ± 0.21a 1.48 ± 0.20b 0.63 ± 0.07a 0.74 ± 0.10a

1.34 ± 0.19a 2.28 ± 0.14b 0.95 ± 0.09a 1.07 ± 0.10a

1.68 ± 0.07a 2.09 ± 0.11b 1.45 ± 0.10a 1.44 ± 0.09a

1 Hepatic stellate cells were isolated from female Wistar rats by sequential pronase/collagenase digestion followed by density gradient centrifugation. After anesthesia and abdominal exploration, hepatic washout was performed using liver perfusion via the portal vein with 50 ml GBSS (GIBCO BRL, Rockville, MD). Perfusion was followed by 200 ml of GBSS containing 140 mg pronase (Roche Diagnostics, Bazel Switzerland) and 100 mg collagenase (Worthington Biochemical Corporation, Lakewood, NJ). The digested liver was mashed ex vivo and incubated at 37˚C for 25 min in 100 ml of GBSS solution containing 0.025% (w/v) pronase, 0.025% (w/v) collagenase and 20 mg/ml DNase I (Roche Diagnostics). The resultant suspension was filtered through a 150-mm steel mesh and centrifuged on an 8.2% Nycodenz cushion ((Nycomed Pharma AS, Oslo, Norway) at 1400g for 20 min at 4˚C, which produced a stellate cell-enriched fraction in the upper whitish layer. Cells were washed by centrifugation (400g, 4˚C, 10 min) and cultured in DMEM supplemented with 10% (v/v?) fetal calf serum, 100 µg/ml penicillin and 100 µg/ml streptomycin. 2 Total cellular RNA was extracted from liver tissue with EZ-RNA total RNA isolation kit (Biological Industries, Beit Haemek, Israel) and transcribed into cDNA, using the Reverse Transcription System (Sigma Aldrich, Rehovot, Israel). mRNA for α -smooth muscle actin (SMA), Proliferating Cell Nuclear Antigen (PCNA) and GADPH were obtained after 35 cycles of amplification. Following SDS-PAGE and western blot, the respective blots were quantitatively scanned (n = 3). 3 The plasmid ColCAT3.6 contains 3520 bp of rat pro-a1(I) collagen promoter followed by 115 bp of rat α1(I) first exon cloned upstream of the chloramphenicol acetyltransferase gene within the pUC 12 vector (Dr. D. Rowe, Univ. of Connecticut). The promoter region was excised and introduced into GL3-luciferase. The plasmid containing the -378 α 2 (I) collagen promoter upstream of the luciferase gene was from Dr. F. Ramirez (Mount Sinai Medical Center, NY). Primary hepatic stellate cells were transiently transfected using Lipofectamine Plus (Invitrogen) with the various constructs described above. In all transfection experiments, cells were transfected with a vector containing the β -galactosidase gene, to normalize for transfection efficiency. Luciferase (LUC) activity was determined using a standard luminescence reader. 4 Treatments followed by the same letter do not differ significantly (p < 0.05).

counter-regulatory mechanisms that attenuate the metabolic effects of leptin inhibition, while only slightly affecting the immunoregulatory effects. We suggest that this protective effect is immune-mediated in liver inflammation. The mechanisms by which leptin antagonist exerts its immune-mediated protective effect are still under investigation and may involve suppression of CD4 and NKT-lymphocytes, the main T-cell subpopulations believed to mediate acute ConA-induced liver injury. In the TAA model of chronic hepatic fibrosis, we suggest that an additional and direct inhibitory effect on HSC function contributes to attenuation of liver fibrosis. Further research is expected to elucidate some of the cellular mechanisms involved in this immunomodulatory and anti-fibrogenic effect. In the future, peripheral leptin inhibition could potentially be developed into a rational anti-inflammatory and anti-fibrogenic therapeutic modality.

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Acknowledgment

This work was supported by the Israeli Science Foundation, grant no. 521/07 to AG and EE.

References

1. Madej T, Boguski MS, Bryant SH. Threading analysis suggests that the obese gene product may be a helical cytokine. FEBS Lett 1995; 373(1):13-18. 2. Gimble JM, Robinson CE, Wu X et al. The function of adipocytes in the bone marrow stroma: an update. Bone 1996; 19(5):421-428. 3. Sanchez Margalet V, Martin Romero C, Gonzalez Yanes C et al. Leptin receptor (Ob-R) expression is induced in peripheral blood mononuclear cells by in vitro activation and in vivo in HIV-infected patients. Clin Exp Immunol 2002; 129(1):119-124. 4. Motivala SJ, Dang J, Obradovic T et al. Leptin and cellular and innate immunity in abstinent alcoholics and controls. Alcohol Clin Exp Res 2003; 27(11):1819-1824. 5. Saxena NK, Ikeda K, Rockey DC et al. Leptin in hepatic fibrosis: evidence for increased collagen production in stellate cells and lean littermates of ob/ob mice. Hepatology 2002; 35(4):762-771. 6. Cao Q, Mak KM, Ren C et al. Leptin stimulates tissue inhibitor of metalloproteinase-1 in human hepatic stellate cells: respective roles of the JAK/STAT and JAK-mediated H2O2-dependant MAPK pathways. J Biol Chem 2004; 279(6):4292-4304. 7. Matarese G, Di Giacomo A, Sanna V et al. Requirement for leptin in the induction and progression of autoimmune encephalomyelitis. J Immunol 2001; 166(10):5909-5916. 8. Yang S, Lin H, Diehl AM. Fatty liver vulnerability to endotoxin-induced damage despite NF-kappaB induction and inhibited caspase 3 activation. Am J Physiol Gastrointest Liver Physiol 2001; 281(2):G382-392. 9. Ahima RS, Osei SY. Leptin signaling. Physiol Behav 2004; 81(2):223-241. 10. Ikejima K, Honda H, Yoshikawa M et al. Leptin augments inflammatory and profibrogenic responses in the murine liver induced by hepatotoxic chemicals. Hepatology 2001; 34(2):288-297. 11. La Cava A, Matarese G. The weight of leptin in immunity. Nat Rev Immunol 2004; 4(5):371-379. 12. Sitaraman S, Liu X, Charrier L et al. Colonic leptin: source of a novel proinflammatory cytokine involved in IBD. FASEB J 2004; 18(6):696-698. 13. Siegmund B, Sennello JA, Jones-Carson J et al. Leptin receptor expression on T-lymphocytes modulates chronic intestinal inflammation in mice. Gut 2004; 53(7):965-972. 14. De Rosa V, Procaccini C, Cali G et al. A key role of leptin in the control of regulatory T-cell proliferation. Immunity. 2007; 26(2):241-255. 15. Lam QL, Lu L. Role of leptin in immunity. Cell Mol Immunol 2007; 4(1):1-13. 16. Zabeau L, Lavens D, Peelman F et al. The ins and outs of leptin receptor activation. FEBS Lett 2003; 546(1):45-50. 17. Zabeau L, Defeau D, Van der Heyden J et al. Functional analysis of leptin receptor activation using a janus kinase/signal transducer and activator of transcription complementation assay. Mol Endocrinol 2004; 18(1):150-161. 18. Peelman F, Iserentant H, De Smet AS et al. Mapping of binding site III in the leptin receptor and modeling of a hexameric leptin.leptin receptor complex. J Biol Chem 2006; 281(22):15496-15504. 19. Peelman F, Van Beneden K, Zabeau L et al. Mapping of the leptin binding sites and design of a leptin antagonist. J Biol Chem 2004; 279(39):41038-41046. 20. Chow D, He X, Snow AL et al. Structure of an extracellular gp130 cytokine receptor signaling complex. Science 2001; 291(5511):2150-2155. 21. Boulanger MJ, Chow DC, Brevnova EE et al. Hexameric structure and assembly of the interleukin-6/ IL-6 alpha-receptor/gp130 complex. Science 2003; 300(5628):2101-2104. 22. Niv Spector L, Gonen Berger D, Gourdou I et al. Identification of the hydrophobic strand in the A-B loop of leptin as major binding site III: implications for large-scale preparation of potent recombinant human and ovine leptin antagonists. Biochem J 2005; 391(Pt 2):221-230. 23. Salomon G, Niv Spector L, Gussakovsky EE et al. Large-scale preparation of biologically active mouse and rat leptins and their L39A/D40A/F41A muteins which act as potent antagonists. Protein Expr Purif 2006; 47(1):128-136. 24. Gertler A. Development of leptin antagonists and their potential use in experimental biology and medicine. Trends Endocrinol Metab 2006; 17(9):372-378.

Chapter 14

The Role of Leptin during Early Life in Imprinting Later Metabolic Responses Mark H. Vickers,* Stefan O. Krechowec, Peter D. Gluckman and Bernhard H. Breier

Abstract

A

robust regulatory physiologic system has evolved to maintain relative constancy of weight; an equilibrium broken by modern lifestyles leading to the development of obesity, type 2 diabetes and other metabolic disorders. Epidemiological and experimental studies have highlighted a relationship between the periconceptual, fetal and early infant phases of life and the subsequent development of adult obesity and type 2 diabetes, a process referred to as developmental programming. Thus, maternal exposure to unfavorable environmental factors during pregnancy and/or lactation can result in progeny with an increased risk of later obesity and metabolic disease. Indices of good infant nutritional state such as leptin concentrations during critical developmental windows predict the development of a broader and healthier metabolic adaptive capacity. Conversely signals of poor early nutrition induce responses which are maladaptive in later life and lead to the developmental programming of metabolic disease. Thus maintaining a critical leptin level during development may allow the normal maturation of tissues and pathways involved in metabolic homeostasis and a period of relative hypo- or hyperleptinemia may induce some of the metabolic adaptations which underlie developmental programming. Furthermore, nutritional or therapeutic intervention in postnatal life can ameliorate the consequences of developmental malprogramming and, at least in the rodent, developmental programming is potentially reversible by intervention with leptin late in the phase of developmental plasticity. Taken together, recent work highlight the importance of leptin in disorders manifest as a consequence of developmental programming and offer exciting new strategies for therapeutic intervention, whether it be maternal or neonatal intervention or targeted nutritional manipulation in postnatal life.

Background

Over the last two decades the global prevalence of obesity and related metabolic disease including type 2 diabetes has increased markedly. Currently more than half of all adults in both the United Kingdom United States are either overweight or obese. Obesity is strongly associated with the morbidities of type 2 diabetes, hypertension and ischaemic heart disease and represents an enormous burden to the health care system. The last 20 years has also seen a rise of over 40% in the prevalence of childhood obesity with a concomitant increase in early-onset type 2 diabetes. Metabolic disease results from a complex interaction of many factors, including genetic, metabolic, behavioral and environmental influences. However, the recent rate at which these diseases have increased suggests that environmental and behavioral influences, rather than genetic causes, are fueling the present epidemic. In this context, it is of particular relevance that epidemiological and *Corresponding Author: Mark Vickers—Liggins Institute and the National Research Centre for Growth and Development, University of Auckland, Private Bag 92019, Auckland, New Zealand. Email: [email protected]

Leptin and Leptin Antagonists, edited by Arieh Gertler. ©2009 Landes Bioscience.

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experimental studies have highlighted a relationship between the periconceptual, fetal and early infant phases of life and the subsequent development of adult obesity and type 2 diabetes.1-5 Thus, maternal exposure to unfavorable environmental factors during pregnancy and/or lactation can result in progeny with an increased risk of later obesity and related metabolic sequelae.3,6-14 This relationship, referred to as the “developmental origins of health and disease” (DOHaD) model, speculates that the fetus makes predictive adaptations in response to adverse environmental cues in utero resulting in permanent readjustments in homeostatic systems to aid immediate survival and improve success in an adverse postnatal environment. However, when there is a mismatch between the prenatal predictions and postnatal environment, these adaptations, known as predictive adaptive responses (PARS), may ultimately be disadvantageous in postnatal life, leading to an increased risk of chronic noncommunicable disease in adulthood and/or the inheritance of risk factors and a cycle of disease transmission across generations.10,15-18 Recent work has highlighted a prenatal role for the hormone leptin in the programming of adult metabolic disorders.19,20 Maintaining a critical leptin level during early development may facilitate the maturation of tissues and pathways involved in metabolic homeostasis. Conversely a period of relative hypo- or hyperleptinemia may induce maladaptive metabolic changes which contribute to the developmental programming of adult disease.21,22 Although the precise mechanisms are unclear, these metabolic changes are generally considered to be part of an irreversible change in developmental trajectory. However, recent work in the rodent suggests that therapeutic intervention with leptin in early postnatal life can potentially reverse or substantially ameliorate the consequences of developmental malprogramming.20,23 Taken together, these data highlight the importance of leptin in the developmental induction of metabolic disease and offer exciting new strategies for therapeutic intervention, whether it be maternal or neonatal intervention or targeted nutritional manipulation in postnatal life.

Leptin and Developmental Programming

The field of metabolic physiology has been profoundly altered by the discovery of the adipokines. Leptin, the first adipokine to be discovered, was originally identified as an anti-obesity hormone in the leptin deficient ob/ob mouse. In both wildtype and mutant mice, leptin treatment was found to dramatically reduce body weight by inhibiting food intake and stimulating the depletion of body fat.24-27 However, hopes of finding a silver bullet cure for obesity within the human population were quickly dashed as it became readily apparent that leptin deficiency was an extremely rare condition in the general population. Principally produced by the white adipose tissue, systemic leptin levels generally reflect current fat mass and as a consequence hyperleptinemia is typically seen in most cases of obesity. In this context, leptin resistance has been identified as a central feature of the pathogenesis of obesity.28 Although the presence of hyperleptinemia with obesity is now well recognized, a central causal role for leptin resistance in this pathological state remains unclear.29 Rather than acting as the bodies principal anti-obesity hormone leptin contributes to the regulation of energy homeostasis by functioning as a central signal for current and long term energy levels. Specifically, leptin seems to exert a permissive effect on various biological systems, facilitating the activation of energy dependent biological processes when adipose energy stores and leptin levels are high.30 In addition to its effects on food intake and body weight, roles for leptin have been identified in the regulation of reproduction, glucose homeostasis, bone formation, wound healing and the immune system.19,31,32 The hypothalamic region of the brain has been identified as the essential control centre of energy homeostasis. Consistent with leptin’s role in regulating energy metabolism various neural nuclei within the hypothalamus have been identified as the primary targets of leptin activity. In the hypothalamus, leptin regulates energy homeostasis and food intake by activating its long isoform receptor (ObRb), concentrated within the arcuate (ARC), paraventricular (PVN), dorsomedial (DMN) and ventromedial (VMN) nuclei.33 Activation of ObRb initiates a JAK2/STAT3 signalling cascade which regulates the expression of several neuropeptides involved in the control of food intake and energy metabolism such as proopiomelanocortin (POMC) (anorexigenic peptide)

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and neuropeptide Y (NPY) (orexigenic peptide).34,35 In this way leptin activates the expression of POMC in anorexigenic neural circuits and inhibits that of NPY in orexigenic neural circuits.36,37 Recent data on the ontogeny of these pathways indicate that, in rodents, these leptin responsive hypothalamic control circuits are laid out during neonatal life but remain structurally and functionally plastic until 3 weeks of life.38 In a landmark study by Bouret et al it has been shown that leptin acts as a key neurotrophic factor promoting the mature patterning of key neural pathways within the hypothalamus.39,40 Further work also suggests that leptin is involved in the formation of normal brain structure and in the regenerative potency of neural cells.41 In the mouse, a lack of leptin during early life compromises the neuronal organization of hypothalamic nuclei,39 consequently causing a loss of appetite control and a reduction in leptin sensitivity in adulthood. This may explain, at least partially, the development of obesity in adult rodents born to dams made hypoleptinemic by maternal undernutrition during pregnancy.42 Leptin activity in the CNS is dependent on the transport of leptin across the BBB, which is mediated by a saturable receptor-mediated transport system located in the brain microvasculature and choroid plexus.43 The impairment of the BBB transport of leptin has also been proposed as a possible causal mechanism for the development of leptin resistance and obesity.43,44 The concentration of OB-Rb in the hypothalamus initially led investigators to conclude that leptin’s main effects were almost exclusively mediated by its effects in the CNS. However, most tissues, including the adipose tissue, skeletal muscle, liver and pancreas have also been found to express OB-Rb, albeit at levels much lower than the hypothalamus.45-49 Various studies have now found that leptin has direct metabolic effects on peripheral tissues that are independent of leptin’s effects in the CNS.50-54 It is now clear that peripheral leptin activity has a significant effect on the regulation of lipid metabolism, up-regulating lipid oxidation and suppressing lipogenesis within a majority of metabolically active peripheral tissues. Serum levels of leptin vary dramatically during intrauterine and early postnatal life, with a 5 to 10-fold increase in leptin occurring between postnatal days 4 and 10 in female mice.55 Breast milk also contains significant amounts of leptin56 which may also contribute to circulating levels in the neonate. Although cord blood leptin levels tend to reflect neonatal fat mass, low cord blood leptin levels correlate with rapid postnatal weight gain in small for gestational age (SGA) infants.57 The temporal co-expression of OB-Rb and its ligand in mesenchymal tissues during fetal development58 raises the possibility that leptin may act as a paracrine or autocrine factor during fetal development. Since circulating leptin levels in neonates vary according to maternal diet, leptin can be therefore viewed as a critical link between environmental and maternal factors and the developing physiology of the infant.59 Together the central and peripheral effects of leptin appear to act as a negative feedback signal limiting energy intake and stimulating energy expenditure when energy stores are abundant and leptin levels are high.30 The combined effects of leptin act to reduce body weight by depleting body fat through the stimulation of peripheral lipid oxidation and by the induction of a negative energy balance.60,61 Conversely, decreased leptin activity contributes to a positive energy balance that facilitates weight gain and the accumulation of adipose tissue. In the long term, the leptin endocrine axis regulates energy homeostasis and body weight by limiting large fluctuations in adipose tissue energy stores. Defects in the leptin endocrine axis, such as leptin resistance, which may result from disturbances in development in early life, are currently implicated in fostering the development of excess adiposity and obesity in adulthood.

Evidence from Animal Models

Although epidemiological data suggest that developmental programming occurs within the normal range of birth size13 most prospective experimental work has tended to focus on significant restriction of fetal growth based on the assumption that the stimuli that impair fetal growth are likely to be those that trigger developmental programming. In an attempt to elucidate the relationship of early growth restriction with adult onset disease, several approaches have been developed in animals. Gestational and neonatal programming of

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obesity risk and altered leptin sensitivity has been examined in several animal models, primarily in rodents but also in larger animal models such as the sheep6,7,9,62,63 and, to a lesser extent, pigs, guinea pigs and nonhuman primates.64-67 In the rat, obesity and related metabolic sequelae have been induced in offspring by exposure to maternal undernutrition,6,68-70 a maternal low protein (MLP) diet;71-73 maternal uterine artery ligation,74,75 maternal dexamethasone (DEX) treatment,76,77 maternal iron deficiency78,79 or prenatal exposure to the cytokines interleukin (IL)-6 and tumour necrosis factor (TNF)-α.80,81 Alterations in maternal nutrition are the most commonly used experimental models utilised to induce intrauterine growth retardation as it is an experimentally practical and reproducible way to induce nutrient limitation to the fetus and thus change its developmental trajectory. However, small for gestational age does not necessarily imply fetal undernutrition and lower birth weight may represent a different etiology and pathogenesis. In this context growth retardation is not essential to developmental programming, but is merely a surrogate for evidence that fetal development has been impaired. Within the laboratory, fetal undernutrition can most commonly be achieved through maternal dietary restriction during pregnancy. Manipulation of maternal nutrition during pregnancy has been known to alter fetal growth and development for some time.82 At present, rodent models investigating the mechanistic links between maternal undernutrition and adult disease primarily utilise one of two dietary protocols; global undernutrition or isocaloric low protein diets. The MLP diet during pregnancy and lactation is one of the most extensively utilised models of nutritional programming.83-88 This model involves ad-libitum feeding to pregnant rats a low protein diet containing 5-8% (w/w) protein (casein), generally a little under half the protein content but equivalent in energy of a control diet containing 18-20% (w/w) protein.83,89 Offspring from protein restricted mothers are around 15-20% lighter at birth86 and maintenance of a MLP diet during lactation enhances this weight difference and permanently limits later growth. If MLP offspring are cross-fostered to mothers fed a control diet, they exhibit rapid catch-up growth.86 This catch-up growth appears to have a detrimental effect on life span, resulting in premature death which is associated with accelerated loss of kidney telomeric DNA.90 Experimental observations in the MLP diet model of developmental programming highlight many potential mechanisms that may be involved in the pathogenesis of obesity and Type 2 diabetes. These mechanisms include both physical and functional changes to various organ and endocrine systems. For example, recent work has examined adipose tissue gene expression profiling of offspring from MLP rats.91 Analysis of visceral adipose tissue (VAT) revealed a global up-regulation of genes involved in carbohydrate, lipid and protein metabolism in offspring of MLP animals. Thus VAT in the MLP model is marked by dynamic changes in the transcriptional profile of key metabolic genes. The mechanisms by which fetal nutrition affects the development of adult obesity and altered leptin sensitivity are not well understood and the exact nature of maternal factors that change prenatal development and determine postnatal pathophysiology remain uncertain. In particular, the extent to which fetal adipose tissue can be programmed as a consequence of altered maternal nutrition has not been elucidated.92 It has been argued that maternal nutrition is not a limiting factor for human fetal growth except under extreme conditions.93 However, epidemiological studies have found relationships between nutritional status during pregnancy in nonfamine conditions, adverse outcomes at birth and later disease risk.94-96 These observations have raised the possibility that subtle changes in the materno-placental supply of nutrients may alter fetal metabolism and endocrine status with postnatal health consequences compatible with the DOHaD hypotheses.97,98 In addition, despite the detection of leptin in the rat placenta during late gestation,99 it is thought that maternal leptin is the significant source of leptin for the fetus, due to its 10-fold increase in transplacental passage as well as the increased expression of one or more of the short truncated isoforms of the leptin receptor in the placental labyrinth zone, the site of maternal-fetal exchange.100-102 In models of maternal global undernutrition and low protein diets, maternal plasma leptin levels are significantly reduced during gestation compared to normally nourished dams103 (Fig. 1A). The dramatic fall in leptin levels and the absence of plasma leptin patterning during

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late gestation of UN dams may effectively reduce the availability and transportation of maternal leptin necessary for the appropriate growth and development of the fetus.39,104-106,107-109 In the sheep fetus, perirenal adipose tissue appears to be a primary source of leptin in the circulation and leptin gene expression is regulated by both glucocorticoids and thyroid hormones. Developmental changes in circulating and perirenal adipose tissue-derived leptin thus is suggested to mediate the maturational effects of cortisol in utero and have long-term consequences for appetite regulation and the development of obesity.110 Adult rats born to undernourished mothers have elevated postnatal plasma leptin concentrations compared to control offspring concomitant with increased adiposity and reduced locomotor activity.6,111 It has also been demonstrated that nutrient restricted offspring demonstrate increased plasma leptin following sympathetic stimulation, not observed in controls, indicated resetting of adipocyte sensitivity to stress.63 Importantly, there is an interaction whereby the development of diet-induced obesity (DIO) and hyperleptinemia in high fat fed animals is amplified in offspring that were born following maternal undernutrition (Fig. 1B,C).6 Work by Yura et al, using the maternal low protein (MLP) model in the rat and a postnatal high calorie diet, showed increased propensity for weight gain and obesity in male offspring of calorie restricted mothers compared to controls.112 However, direct assessment of altered peripheral and/or central leptin sensitivity in animal models of developmental programming has only recently become a focus of investigation. In a study by Krechowec et al, leptin treatment in adult female rat offspring born following maternal undernutrition, failed to reduce food intake and weight loss was diminished compared to leptin treated offspring of dams fed ad-libitum throughout pregnancy.113 This peripheral leptin resistance observed in offspring of undernourished mothers was independent of diet-induced obesity and was associated with fasting hyperinsulinemia and hypertriglyceridemia. These data suggest that prenatal nutrition can shape future susceptibility to obesity through alterations in leptin sensitivity and changes in energy metabolism during adult life. Work by Desai et al has shown that intrauterine growth restricted (IUGR) rat offspring demonstrate resistance to anorexigenic agents, leptin (6 weeks and 6 months) and sibutramine (8 months), as evidenced by less reduction in food intake and less body weight loss than controls.114 IUGR offspring demonstrated suppressed leptin-induced STAT3 phosphorylation and impaired anorexigenic response to 2 key factors in the central satiety pathway.

Figure 1. A) Maternal plasma leptin levels at day 20 of gestation in normal (AD) and undernourished rat dams (UN). P < 0.05 for effect of maternal undernutrition; B) Demonstration of the interaction between the prenatal and postnatal environment in the development of obesity and leptin resistance: Retroperitoneal fat mass and fasting plasma leptin concentrations in offspring from control C) and undernourished mothers (UN) on either chow or high fat (HF) nutrition. P < 0.001 for effect of maternal nutrition and diet. Maternal nutrition x postnatal diet interaction p < 0.0001. A) Taken from Ikensia et al, unpublished data. B,C) Adapted with permission from: Vickers MH et al. Am J Physical Endocrinal Metab 2000; 279(1): E83-87.6

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Delahaye et al has recently shown that maternal undernutrition drastically reduces the postnatal surge of plasma leptin disturbing hypothalamic wiring as well as gene expression within the anorexigenic POMC neurons in male rat pups.115 These alterations might contribute to the adult metabolic disorders resulting from perinatal growth retardation. It has also been reported that leptin can modulate the hormonal response to stress in young rats either by a direct effect on the HPA axis or indirectly through changing some aspects of maternal behaviour.116,117 Toste et al have shown that leptin injection in the beginning of lactation results in hypothyroidism in the offspring as soon as 30 days of age and this alteration may be the imprinted factor for the programming of a higher thyroid function in adulthood.168 Another area of focus is that of the programming of obesity and Type 2 diabetes in those offspring born to diabetic mothers. Maternal diabetes, particularly with poor glycemic control, causes fetal hyperglycemia which, in turn induces fetal hyperinsulinemia.118-122 It has been proposed that fetal hyperinsulinemia, during critical windows of development, induces insulin and leptin resistance by down-regulation of the leptin and insulin receptors. Developmentally programmed central insulin and leptin resistance can lead to appetite dysregulation, while pancreatic leptin and insulin resistance can lead to hyperinsulinemia and hypertrophy of adipocytes, both of which are causally related to increased obesity in postnatal life.119,123,124 Inducing a supraphysiological leptin surge by administering leptin to offspring of normal pregnancies may have relevance to models of neonatal overnutrition. One of the interesting observations from human epidemiological studies is that the relationship between birth weight and adult metabolic abnormalities is “U” shaped.125 Maternal high-fat diet programs a hypothalamic leptin resistance in offspring, which, however, fails to increase the body weight gain until adulthood.42 Animal models of neonatal overnutrition have also demonstrated a link between excessive weight gain early in life and later metabolic complications.126 Neonatal leptin treatment to AD male offspring subsequently placed on a high fat diet exacerbates the degree of leptin resistance in these animals.127 It is possible that the mechanism underlying the long-term effects of neonatal overnutrition on energy homeostasis may depend upon a period of hyperleptinemia during a critical stage of development.

Epidemiological and Clinical Evidence

The early work by Barker and colleagues focused on the relationship between birth weight and adult disease in geographically localized populations.1,13 Barker and colleagues demonstrated a relationship between low birth weight and an increased propensity for hypertension, obesity, insulin resistance and dyslipidemia in later life. From these initial observations, the importance of early development and, in particular, the effect of poor nutrition on birth weight and the development of adult disease was addressed in studies of famine exposure. The most widely reported of these being the Dutch Hunger Winter of 1944-1945 where reduced maternal caloric intake in late gestation was associated with increased adult adiposity in adult life.93,128 The importance of leptin in developmental programming related metabolic disorders is now well recognised with evidence that both central and peripheral mechanisms are involved. A number of studies have shown that obesity resultant from developmental programming may involve a U-shaped relationship between disease prevalence and birth weight, with a higher prevalence of adult obesity occurring in individuals who are of either low (e.g., those exposed to famine during the Dutch Hunger Winter of 1944-45) or high birth weights (e.g., offspring of mothers with gestational diabetes).129-131 Low birthweight and rapid postnatal weight gain, or catch-up growth, are independent risk factors for the development of obesity and diabetes during adult life.131,132 Fetal plasma leptin concentration is correlated with weight, length and head circumference at birth, but maternal leptin concentration shows a negative correlation with fetal growth in humans.133,134 SGA children have been shown to have low cord blood and plasma leptin levels135 which are associated with rapid postnatal weight gain and a predisposition to develop the metabolic syndrome in adult life.136 Conversely, maternal obesity and/or gestational diabetes results in elevated cord leptin levels and children also born at increased risk of developing the metabolic syndrome.137

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The timing and magnitude of gestational food restriction are critical in determining the obese phenotype. For example, from the Dutch Famine study, the rate of obesity was higher in men exposed in the first half of gestation and lower in men exposed in the last trimester of gestation as compared to non-exposed men.128 Thus while fetal exposure to a substrate limited environment at most stages of development appears to lead to adult dysregulation of metabolism, the precise mechanisms responsible may vary with the timing of exposure. In addition, there is increasing evidence of intergenerational effects, with babies born at both ends of the birthweight spectrum being prone to excess weight gain, which then, in girls, predisposes them to diabetes in pregnancy, which, in turn, promotes an accelerating cycle of early diabetes in subsequent generations.138 Prenatal events are compounded by postnatal over-nutrition or lifestyle choices which are in conflict with the programming of the fetus. However, the relative importance of the interactions between the pre and postnatal environment is not easily discernable from human studies due to the lack of controls for dietary content and quantity or the severity of obesity or undernutrition in the mothers and offspring.139 Thus, animal studies have become the mainstay for investigating the interactions between pre and postnatal influences on leptin sensitivity.

Leptin in Early Life and Catch-Up Growth

Work by Plagemann et al argues that it may not be fetal undernutrition and low birth weight per se that predisposes to adult onset obesity; rather it is the overfeeding of underweight newborns that may substantially contribute to their long term risk.140 This concurs with recent papers by Ross and Desai relating to population survival effects in models of drought and famine during pregnancy. When low birth-weight offspring are permitted rapid catch-up growth by nutrient availability, these offspring will demonstrate evidence of increase body weight and body fat and leptin resistance as adults. Conversely, if catch-up growth is delayed by nutrient restriction, these offspring exhibit normal body weight, body fat and plasma leptin levels as adults.9,141 This fits with the rodent data described above demonstrating that prevention of early catch-up growth can reverse glucose tolerance and obesity in models of low birth-weight associated diabetes.132 Thus the degree of newborn nutrient enhancement and timing of catch-up growth in newborns may determine the programming of orexigenic hormones and obesity in offspring. These findings suggest a perinatally acquired malprogramming and disorganisation of the hypothalamic systems governing appetite and energy homeostasis which manifests as appetite disorders, leptin insensitivity and obesity in adult life. This appears to be reflected in recent studies comparing breast fed and bottle fed infants that suggest that the lactation period is a critical window in the early development of obesity risk in humans.131,142 Breast fed babies appear to be at a reduced risk of obesity compared to those who were formula fed. It is proposed that this protective effect of breastfeeding may be related to differences in substrate intakes with breast milk and standard infant formulae.142 Whereas the protein intake of breast-fed infants decreases with age and closely matches the requirements for protein during the early months of life, the protein intake of formula-fed infants exceeds requirements after the first 1-2 months of life.143 Thus, as bottle fed infants have a higher total and protein caloric intake than breast fed infants, the level of nutrition during lactation may have long term consequences for appetite regulation. This may relate to circulating leptin levels which are higher in breast fed infants during the first 4 months of life and may have a role in subsequent obesity risk.131 Lopez et al have reported that perinatal overfeeding (using small litters, SL) does not induce alterations in either the anorectic response to central leptin administration or expression of leptin receptors and neuropeptides in adult rats.144 These data suggest that leptin resistance in adult SL rats may be related to impaired leptin transport across the blood-brain barrier (BBB). Impairment of this transport mechanism may be triglyceride-mediated as reported by Banks et al.145 High levels of plasma triglycerides can inhibit the transport of leptin across the BBB and so could be key in the onset of the peripheral leptin resistance, which is a hallmark of obesity. Th is work postulated that triglyceride-induced resistance to leptin transport across the BBB initially evolved to limit the signal of an anorectic to the brain during starvation.

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Potential Mechanisms

The plasticity of the systems which control energy homeostasis, including the leptin axis appears to greatest during early developmental stages, hence the prenatal and early perinatal period may be considered the most important time in which extrinsic factors may permanently alter future adult metabolic processes.139 In this context, it has been suggested that neonatal leptin exposure can modulate the hormonal response to stress in young rats either by a direct effect on the HPA axis or indirectly through changing some aspects of maternal behaviour.116 In addition to these effects, leptin plays an important role in brain development. Brain volume, weight and DNA content are reduced in adult Lepob/Lepob mice compared to wild-type controls107,146 and these impairments in Lepob/Lepob mice can be rescued by the neonatal administration of leptin in juveniles. The work by Bouret et al indicated that the neuronal circuit related to energy regulation in the hypothalamus remains immature until the neonatal period and that leptin is necessary for the maturation of these neural circuits.39,147 Recent studies have suggested that leptin may also act on the fetal cerebral cortex, including the cingulate cortex, which is involved in motor and cognitive processes and that leptin may affect maintenance and differentiation of neural stem cells, glial-restricted progenitor cells and/or neuronal lineage cells.41 These studies showed that leptin not only has homeostatic functions in adults, but also regulates brain development in the prenatal and neonatal periods. These findings suggest that leptin is related to formation of the normal brain structure and regenerative potency of neural cells as well as the predisposition to homeostatic dysfunction, reduced locomotor activity or impairment of cognitive function. Although in the study by Vickers et al, neonatal leptin treatment had an effect on body weight during the period of treatment, data confirming that this weight loss was a consequence of reduced milk intake were not available.20 Daily intraperitoneal injections of 1mg/kg leptin from day 7 to day 10 in C57BL/6J mice has been previously reported to have no effect on milk intake or body weight.148 When 1 µg of leptin was administered intracerebroventricularly to 17 day old pups it markedly increased oxygen consumption and by 28 days intracerebroventricular leptin inhibited food intake.148 In addition, the observed differences in response to neonatal leptin treatment on weight gain may have been a direct result of differential effects of leptin on energy expenditure in AD and UN pups. Work by Stehling et al has shown that treatment with murine leptin from neonatal days 7 to 16 can reduce juvenile fat storage solely by increasing energy expenditure and independent of its effects on food intake.149 It has also been shown that, despite adult-like effects of leptin treatment on appetite-related neuropeptides proopiomelanocortin (POMC) and neuropeptide Y (NPY) expression in neonates, leptin does not regulate food intake during early development.150 On the contrary, there is evidence suggesting that leptin promotes swallowing activity and hyperphagia, therefore contributing to the rapid growth and weight gain of the newborn, but this evidence is still inconclusive.151-153

Epigenetics

Epigenetics is the study of heritable alterations in gene expression rather than gene sequence. Epigenetic alterations are involved in the modulation of tissue-specific gene expression and genomic imprinting. Mechanisms include posttranslational modifications of core histones and DNA methylation. The patterning of the epigenome is established during gametogenesis and early embryogenesis and is particularly sensitive to disruptive environmental influences during this time.154 In this context, a disruption of prenatal epigenetic patterning provides a key mechanism through which adverse environmental stimuli can generate stable developmental changes which will be maintained into adulthood. However, the relative contribution of altered epigenetic patterning to the link between an adverse early life environment and an increased risk of obesity, leptin resistance and type 2 diabetes in adulthood has yet to be determined. Altered DNA methylation patterns have been proposed to serve as potential biomarkers for exposure to adverse prenatal environmental stimuli and could help in the development of adult risk management strategies.155 Recent work from mouse models, human monozygotic twin studies and large-scale profiling suggests that epigenetically determined phenotypes and epigenetic inheritance are more common

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than previously appreciated.156 Owing to their inherent malleability, epigenetic mechanisms, such as cytosine methylation, are susceptible to nutritional cues, particularly during early development (see reviews by Junien et al157,158 and Waterland and Jirtle159). It is therefore possible that individuals with adult obesity and type 2 diabetes exhibit aberrant methylation patterns for example, due to maternal undernutrition and subsequent metabolic disturbances during fetal/postnatal development. The extent to which the environment or genotype is involved in programmed changes in leptin sensitivity and subsequent postnatal metabolic disorders still remains unclear.160 Molecular mechanisms involved might include alterations of the fetal epigenome in response to maternal nutrition. Such epigenetic alterations in gene expression may be environmentally led, heritable, but potentially reversible alterations in gene expression that do not involve changes in primary DNA sequence.155 Such potential mechanisms underlying epigenetic modification of tissue function resulting in a predisposition to altered programming of leptin and insulin signalling are discussed by Holness et al.155 For example, activation of the leptin receptor also induces expression of suppressor of cytokine signaling-3 (SOCS-3). This protein inhibits further leptin signal transduction and also potently inhibits signalling by the insulin receptor. Altered methylation of the SOCS-3 may therefore have lasting effects on the leptin-insulin feedback loop (the adipoinsular axis) and adversely impact on developmental programming. The application of epigenomic approaches and the determination of targets (e.g., imprinted or non-imprinted genes) and methylation sites for early nutritional effects on epigenetic gene regulation are an exciting and important new area of investigation.161 Recent work by Lillycrop et al has shown that unbalanced prenatal nutrition using the maternal low protein diet model induces persistent, gene-specific epigenetic changes that alter mRNA expression in adult offspring.71 These changes can be reversed by folic acid supplementation during pregnancy suggesting that that changes in DNA methylation may reflect an impaired supply of methyl donors from the mother. This work also raises the possibility of therapeutic strategies to increase availability of methyl donors and thus prevent or ameliorate the effects of environmental insults in early life. Maternal nutrition and the availability of dietary methyl donors (methionine, choline) and cofactors (folic acid, vitamin B12 and pyridoxal phosphate) during critical periods, including the preimplantation period, are perceived to influence DNA methylation and subsequent gene expression patterns.159,162 There is significant interest in the impact of maternal diets, particularly those that could alter homocysteine or folate bioavailability around the time of conception and implantation. The longer-term consequences of maternal diets lacking these critical methyl-donors are hypothesized to include not only a predisposition towards obesity but may also contribute to premature ‘epigenetic ageing’.159 There is already good evidence that DNA methylation patterns are altered in cancer and there is growing interest on their potential involvement in the acquisition of risk of noncommunicable adult-onset metabolic diseases, particularly insulin resistance and type 2 diabetes mellitus. Gluckman et al have shown that the effects of neonatal leptin on hepatic gene expression and epigenetic status in adulthood are directionally dependent on the animal’s nutritional status in utero. These results demonstrate that, during mammalian development, the direction of the response to one cue can be determined by previous exposure to another, suggesting the potential for a discontinuous distribution of environmentally induced phenotypes, analogous to the phenomenon of polyphenism.163 Leptin only exerts these important effects during a narrow window in postnatal development: the neonatal period before leptin becomes involved in the acute regulation of food intake in adults.39 It is thus highly likely that levels of leptin and of particular nutritional stimuli during this precise developmental period have long-term consequences because of the inappropriate epigenetic remodeling of chromatin.164 Experimentally, rodent models have already demonstrated the persistence of programming effects through several generations, transmitted by either maternal or paternal lines, indicative of the potential importance of epigenetic factors in the intergenerational inheritance of the pro-

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grammed phenotype and provides a basis for the inherited association between low birth weight and obesity.165

Developmental Programming and Gender Differences in Leptin Sensitivity

A number of studies have highlighted the complex relationship between gender and energy homeostasis. Serum leptin levels in humans are higher in females than males and this is partly attributed to the greater subcutaneous fat depots in females. Moreover, sex steroids may not only modulate adipocyte leptin secretion but also the sensitivity of the hypothalamus to leptin. Testosterone has an inhibitory effect on leptin secretion while estrogen has a stimulatory effect. Recent studies suggest that male rats are more sensitive to the anorectic effects of insulin whereas females are more responsive to alterations in serum leptin. Part of the increased leptin sensitivity in females appears to be due estrogen dependant effects in the arcuate nucleus of the hypothalamus.166 Male mice are also more susceptible to dietary induced weight gain than female mice.167 Taken together these findings suggest that although serum leptin levels are higher in female rats this does not necessarily indicate leptin resistance but rather an altered set point in relation to leptin’s effect on energy homeostasis. It is perhaps not surprising then that neonatal leptin treatment of male and female pups results in different long-term effects on energy homeostasis. The combined data suggest a complex interplay between programming, postnatal diet and neonatal leptin treatment in male and female rats. UN females were more susceptible than UN males to dietary high-fat induced weight gain. Neonatal leptin treatment amplified the effects of a high fat diet in AD males but not AD females, however this effect was independent of food intake and may reflect a lasting effect of neonatal leptin exposure on energy utilisation.

Leptin in the Perinatal Period—A Therapeutic Window of Intervention?

Recent work suggests that, at least in the rat, developmental metabolic programming is potentially reversible by an intervention late in the phase of developmental plasticity (Fig. 2).20 Circulating leptin levels vary dramatically during intrauterine and early postnatal life, with a 5-10-fold increase in leptin occurring between postnatal day 4 and 10 in female mice; the so-called leptin surge.55 Leptin treatment to neonatal female rats following maternal undernutrition prevented the development of the programmed phenotype in adulthood. The complete normalisation of the “programmed” phenotype by neonatal leptin treatment implies that leptin has effects that can reverse the prenatal adaptations resulting from relative fetal undernutrition.20 In contrast, the study by Yura et al showed that treatment of male control offspring with leptin in the neonatal period resulted in a modest increase in the risk for obesity as compared to saline treated controls.112 This concurs with a previous study of neonatal leptin to normal rats which showed programmed hyperleptinemia and hyperinsulinemia in adulthood, which lead to leptin resistance by reducing the expression of the hypothalamic leptin receptor.168 However, in these studies, only offspring of normal control dams were treated with leptin thus developmental-programming-related mediated effects of leptin were not investigated and may simply reflect the effects of an abnormal leptin surge to normal rat offspring or gender related effects. Nonetheless, the rodent data highlights the importance of this critical leptin window in reprogramming the postnatal phenotype. The mechanisms underlying the observation of reversibility following neonatal leptin intervention are not yet understood. As described earlier, the perinatal period is the most important time in which extrinsic factors may be able to permanently alter metabolic set-points.139 Recent work has highlighted the plasticity of arcuate nucleus projections in the neonatal period and showed the importance of leptin as a signal for the development of hypothalamic circuits.39,147 Hypothalamic arcuate nucleus projections that regulate body weight mature during the first two weeks after birth in rodents.39,109,147 In leptin-deficient (ob/ob) mice these projections remain immature. Exogenous leptin has a neurotrophic effect on these hypothalamic projections but only

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Figure 2. A) Diet induced weight gain (∆ high fat—chow fed) at postnatal day 170 in female AD and UN animals treated with either saline or leptin in the neonatal period. Neonatal leptin in UN animals normalised diet-induced weight gain to match that of AD animals. Neonatal leptin had no effect on diet-induced weight gain in AD animals. UN Saline (•), UN Leptin (O), AD Saline (■), AD Leptin (). AD saline versus AD leptin N.S., UN leptin vs AD saline N.S., UN leptin versus AD leptin N.S.; UN saline p < 0.001 versus all other groups. N.S. = not significant. b) Representative DEXA scans from adult UN offspring at postnatal day 170 treated as neonates with either saline (top) or leptin (bottom). Reproduced with permission from: Vickers MH et al. Endocrinology 2005; 146(10): 4211-4216;20 copyright 2005, The Endocrine Society.

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during the neonatal period.39 Although animals administered leptin during this crucial neonatal period had no detectable leptin in their circulation as adults, their feeding behaviour was closer to that seen of wild-type than in untreated ob/ob littermates. This lead to the conclusion that leptin may actually be dispensable for acute metabolic regulation in the adult.169 This work also indicated that if there was an absence of leptin during a crucial postnatal period, the brain would be hardwired for obesity. Pico et al have shown that the intake of physiological doses of leptin during lactation in rats prevents obesity in later life. The animals that received leptin during lactation become more protected against fat accumulation in adult life and seem to be more sensitive to the short- and long-term regulation of food intake by leptin. Thus, leptin plays an important role in the earlier stages of neonatal life, as a component of breast milk, in the prevention of later obesity.170 The same group has also recently shown that oral supplementation with physiological doses of leptin during lactation in rats improves insulin sensitivity and affects food preferences later in life.171 Work by Stocker et al, utilizing the MLP model, showed that administration of leptin during pregnancy and lactation to protein-restricted dams produced male offspring with an increased metabolic rate and a reduced susceptibility to insulin resistance and obesity when fed a high-fat diet postnatally.172,173 A possible mechanism proposed by the authors related to activity of the placental enzyme 11β-HSD2; the physiological "barrier" protecting the fetus from exposure to maternal glucocorticoids. 11β-HSD2 was reduced by the low-protein diet; this reduction was prevented by treating the dams with leptin. In this study, control offspring were not treated with leptin so direct programming effects were not able to be differentiated although maternal leptin treatment to offspring of normal dams during lactation has been shown to result in adult offspring that are more susceptible to overweight with resistance to the anorectic effect of leptin.174 In addition to the paradigm of leptin treatment, dietary manipulations have recently been shown to attenuate programming-induced metabolic sequelae. Work by Jimenez-Chillaron et al has highlighted the consequences of catch-up growth following prenatal undenutrition. Using a mouse model of undernutrition, they showed that males that were small at birth and exhibited early postnatal catch-up growth developed glucose intolerance and obesity by 6 months of age. In contrast, low birthweight mice without catch-up growth remained smaller than controls and glucose intolerance and obesity was prevented.132 Work by Wyrwoll et al utilised the dexamethasone (Dex) model in the rat (Dex treatment from day 13 to term) with offspring then cross-fostered to mothers on either a standard diet or a diet high in omega-3 fatty acids and remained on these diets postweaning. Maternal Dex reduced birthweight and delayed the onset of puberty in offspring. Hyperleptinemia (associated with elevated leptin mRNA expression in adipose tissue) was evident in offspring by 6 months of age in Dex-exposed animals consuming a standard diet, but these effects were completely blocked by the high omega-3 diet.175 These results demonstrate that manipulation of postnatal diet can limit adverse outcomes of developmental programming, with programmed hyperleptinemia prevented by a postnatal diet enriched with omega-3 fatty acids. This raises the possibility that dietary supplementation with omega-3 fatty acids may provide a viable therapeutic option for preventing and/or reducing adverse programming outcomes in humans. Of note, the recent reports on leptin intervention in normal animals during the neonatal period highlight that the dose, timing and gender can either potentiate or ameliorate the postnatal pathophysiology associated with alterations in leptin sensitivity and obesity. Physiologic doses appear to be protective against postnatal obesity170,171 but supraphysiologic leptin doses are reported to induce an obese and leptin resistant phenotype in adult life.168,176,177

Extrapolation from Animal Models to the Clinical Setting

Although recent studies in the rodent have highlighted a critical postnatal period whereby leptin treatment can modify the development of neural circuitry, it is important to recognise that developmental events that occur postnatally in the rodent hypothalamus occur in utero in primates, including humans.169,178 Thus, it is difficult to directly extrapolate the rodent results to the clinical setting. However, recent work has investigated the impact of increased maternal nutrition on

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the appetite regulatory network in species in which this network develops before birth, as in the human. In species such as the human and sheep, there is evidence that the synthesis and secretion of adipocyte-derived hormones, such as leptin, are regulated in fetal life.62 Exposure to increased nutrition before birth alters the responses of the central appetite regulatory system to signals of increased adiposity after birth.179 Bispham et al have shown that, irrespective of maternal nutrition in late gestation, term fetuses sampled from ewes with nutrient restriction in early gestation possess more adipose tissue, whereas when ewes were fed to appetite throughout gestation, fetal adipose tissue deposition and leptin mRNA abundance were both reduced. These changes suggested that offspring of nutrient restricted mothers were at increased risk of developing obesity in later life.92 These observations suggest that the increased incidence of obesity in adults born to mothers exposed to the Dutch famine during early pregnancy may be a direct consequence of adaptations in the endocrine sensitivity of fetal adipose tissue. Factors that control fetal fat accretion are not known. The close association between increased fetal leptin and enlarged fat depot makes leptin a good marker of prenatal obesity. The temporal co-expression of the OB-Rb and its ligand in mesenchymal tissues during fetal development thus raises the possibility that leptin may act as a paracrine or autocrine factor during fetal life. Fetal hyperleptinemia in conjunction with the alterations of placental lipid storage and transport may provide a link between maternal diabetes and fetal obesity.135 However, before considering leptin as a prenatal growth factor, it must be noted that neonates born with total congenital leptin deficiency have a normal birth weight.135 This suggests that the lack of both placental and fetal leptin synthesis can be overcome successfully in utero. Experimental support is provided with the findings that leptin withdrawal from 0.5 to 19.5 days of rat pregnancy with mothers and fetuses homozygous for the ob/ob genotype does not affect pregnancy outcome. Leptin has known to play a permissive role in the initiation of puberty although the link between developmental programming, leptin and pubertal onset is not well defined. In the MRC 1946 chort in the United Kingdom, earlier puberty was related to smaller size at birth and rapid growth between 0 and 2 years.180 Obese children have higher leptin levels, a proven permissive factor in initiating LH pulsatility. Obesity could also affect the rate of progression through puberty as nutrition and SHBG may act respectively as an accelerator and brake on peripheral sex steroid action. Early weight gain and early pubertal development might also be associated with loss of the pubertal growth spurt perhaps through obesity-related suppression of GH secretion. Trans-generational recurrence of low birth weight, early catch-up weight gain, earlier menarche and shorter adult stature have been observed in women and could contribute to the strong heritability in age at menarche.180 Both maternal undernutrition and high fat nutrition during pregnancy have been shown to significantly advance the age of puberty in male and female rat offspring, the effects of which are exacerbated by a postnatal high fat diet.181,182 However, DEX treatment to dams during pregnancy has been reported to delay the onset of puberty in rat offspring despite the presence of hyperleptinemia.175

Discussion

The risk of developing some chronic diseases in adulthood is influenced not only by genetic and adult lifestyle factors, but also by environmental factors acting in early life. These factors act through the processes of developmental plasticity and possibly epigenetic modification and can be distinguished from pathological developmental disruption.16 The fetus responds to challenges such as nutrient restriction in ways that help to ensure its survival, but this “developmental plasticity” may have long-term sequelae that may not be beneficial in adult life. Whereas long-standing or permanent alterations in appetite control for example, are induced by experience during critical periods of development, the same experience occurring outside of the critical period has little or no lasting effect, indicating that the plasticity depends on windows of opportunity during early life when development can be altered in response to the external environment. This plasticity is clearly shown in the development of the hypothalamic feeding circuitry. Brain development continues well into the first years of life and thus both the

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intrauterine and postnatal environments have a major impact on the physiologic, metabolic and neural development of pathways regulating energy homeostasis.183 Epidemiological studies in humans and animal studies have demonstrated that maternal undernutrition, obesity and diabetes during gestation and lactation can all produce obesity and altered leptin sensitivity in offspring, the effects of which can be amplified with rapid catch-up growth and exposure to an obesogenic postnatal dietary environment. It is notable that the variety of different insults in fetal life (caloric, protein, iron, fat-fed) produce comparable detrimental consequences that occur in adult life, suggestive of a common mechanism that may underlie the developmental programming of adult disease. These manipulations appear to promote obesity in offspring by reprogramming the development of central neural pathways involved in the regulation of food intake, energy expenditure and storage.139 Given its strong neurotrophic properties, it is likely that leptin is a major effector of these developmental changes during the perinatal period. Experimental data also suggest that this activity is restricted to a critical neonatal period that precedes leptin’s acute regulation of food intake in adults.39,147 The current data fit with the PAR hypothesis proposed by Gluckman and Hanson.18 Following the PAR hypothesis, in response to a given in utero or early postnatal nutritional plane (either high or low), cellular processes are induced to cope with a predicted nutritional environment (either high or low). This hypothesis suggests that disease only manifests when the actual nutritional environment diverges from that which was predicted. Since the development of critical pathways involved in energy homeostasis in rodents continue well into the postnatal period, it can be modified by both pre and early postnatal environmental manipulation (e.g., prevention of catch-up growth) and thus obesity can be potentiated, reversed or attenuated postnatally.139 It is possible that similar principles hold true for humans although the timing of pathway development would occur earlier than in rodents. Although the mechanisms underlying developmental programming of obesity are yet to be fully elucidated, the process has been considered irreversible. Recent findings indicate that, at least in the rat, there is an early postnatal window during which the process can be reversed. It is possible that maintaining a critical leptin level during development allows the normal maturation of pathways and tissues involved in metabolic regulation and that a period of relative hypo- or hyperleptinemia may result in the adverse metabolic sequelae that underlie developmental programming. To fully understand the mechanism by which leptin modifies the desired developmental target, it will necessitate an understanding of the mechanisms underlying the timing and amplitude of the leptin surge, optimising the developmental response to exogenous leptin and a detailed understanding of the role of leptin in the development of the neural circuits controlling appetite regulation and energy homeostasis.

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Index A Absorption 54, 56, 57, 59 Actin 73, 78, 79, 138, 139 Adipocyte 1, 4, 43, 44, 47, 54, 56, 108, 116, 126, 145, 150, 153 Adipokine 1, 92, 116, 124, 142 Adipose tissue 7, 15, 30, 43-47, 49, 50, 55, 63, 68, 73, 85, 86, 88, 91, 92, 95, 102, 116, 131, 142-145, 152, 153 Adiposity 4, 47, 63, 73, 143, 145, 146, 153 Agouti-related protein 4 AgRP 4, 9 AKT 49, 50 AMP-activated protein kinase 9, 46, 117 Angiotensin II 76, 91, 94, 95, 97, 101 Animal model 26, 68, 69, 116, 127, 129, 143-146, 152 Anti-apoptotic 43, 45-49, 58, 64, 127 Appetite 3, 4, 9, 54, 73, 83, 92, 96, 120, 143, 145-148, 153, 154 Arterial hypertension 91, 92, 98, 99, 102 ATP-sensitive potassium channel 1 Atrial natriuretic peptide 92, 100-102 Autocrine axis Autoimmunity 59, 126-129, 131

B BAT 47, 92, 96 Behaviour 59, 146, 148, 152 Beta (β)-adrenergic pathway 49 Blastocyst 110, 111, 113 Blood-brain-barrier (BBB) 27, 130, 143, 147 Blood pressure 75, 76, 91-93, 96-102 Bone elongation 83, 86, 88, 89 Breast cancer 16, 26, 45, 46, 63-68, 70 Bioluminescence resonance energy transfer (BRET) 17, 25, 30, 33-40

C cAMP 8, 74, 100, 101 Cancer 16, 26, 39, 40, 43, 45-49, 58, 59, 63-70, 78, 127, 149 Cardiomyocyte hypertrophy 73, 76, 78 Cardiovascular risk 73-75

Cartilage 83, 85-88 Caveolae/caveolin-3 78, 79 Cell proliferation 4, 16, 44-46, 48, 49, 58, 63, 68, 129, 133 Ciliary neurotrophic factor (CNTF) 16, 31 Clenbuterol 47 Colorectal 63, 65, 68-70 Cyclic GMP 93, 95 Cytokine receptor 2, 3, 7, 9, 15-17, 31, 34, 43, 54, 74, 117, 127, 133 Cytokine receptor homology (CRH) 16, 18, 20, 22, 31, 122

D Developmental plasticity 141, 150, 153 Developmental programming 141-146, 149, 150, 152-154 Diabetes 47, 56, 57, 59, 99, 116-118, 127, 131, 141, 142, 144, 146-149, 153, 154 Diet-induced obesity 97, 98, 130, 131, 145 Drug resistance 64 Dyslipidemia 98, 116, 118, 146

E Embryo 86, 108-111, 113 Endochondral ossification 83-86 Endometrium 108-113 Endothelial cell 3, 15, 47, 58, 93-95, 97, 99 Endothelial dysfunction Endothelin-1 76, 91, 99, 102 Endothelium-derived hyperpolarizing factor (EDHF) 93, 94, 97, 98 Energy expenditure 1, 3, 4, 43, 47, 54, 59, 73, 92, 119, 120, 143, 148, 154 Energy homeostasis 1, 4, 8, 9, 15, 30, 39, 57, 59, 108, 142, 143, 146-148, 150, 154 Environmental influence 126, 141, 148 Epigenetics 148 Epo 17, 18, 25, 31 Erythropoietin 17, 31 Estrogen 45, 46, 63, 64, 150 Extracellular signal-regulated kinase (ERK) 1, 3, 5-8, 45, 46, 48, 58, 76-78 Extrinsic pathway 44

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F

J

Fibronectin type III 16, 22-24, 26 Fluorescence resonance energy transfer (FRET) 30, 33-37, 39, 40

Janus kinase (JAK) 2-4, 6, 9, 15, 18, 20, 22, 24, 25, 30, 46, 48, 74, 76-88, 89, 117, 130, 133 Janus kinase 2 (JAK2) 1-3, 7, 8, 15, 30, 33, 35, 48, 63, 64, 68, 77, 97, 130, 142

G Gene expression 1, 4, 58, 75, 78, 113, 117, 144-146, 148, 149 Glycoprotein 130 (Gp130) 16 Granulocyte-colony stimulating factor (G-CSF) 16-22, 25, 31, 37 Growth 5, 7, 8, 16, 26, 30, 31, 43, 45, 46, 58, 63, 64, 68, 69, 76, 83-89, 121, 122, 143-148, 152-154 Growth hormone (GH) 7, 16, 18, 20, 25, 31, 34, 43, 83, 85, 86, 89, 121, 122, 153 Growth plate 83-88

H Hepatic fibrosis 134, 138, 139 Hepatic steatosis 116, 123 Hepatic stellate cells 3, 7, 48, 133, 139 Heterodimer 2, 4, 35, 77 High throughput screening 39, 40 Homodimer 30, 35, 36, 39 Hypertrophic chondrocyte 84, 86, 87

I Immunoglobulin (Ig) 16-19, 21, 22, 24, 26, 31, 37 Implantation 25, 108-111, 113, 149 Inflammation 4, 9, 58, 59, 126, 128, 129, 131, 133, 134, 136-139 Insulin-like growth factor-1 (IGF-1) 46, 58, 63, 64, 83, 85, 86, 89, 121, 122 Insulin receptor substrate (IRS) 8, 48 Insulin resistance 75, 96, 98, 116, 118, 124, 146, 149, 152 Interleukin (IL) 1-3, 5-7, 16-19, 21, 22, 31, 37, 45, 47, 63, 109-111, 117, 127, 130, 133, 144 Intestine 55-57 Intrinsic pathway 44

L LEPR 1-9, 109 Leptin 1-9, 15, 16-27, 30, 31, 33-40, 43-50, 54-59, 63-70, 73-79, 83-89, 91-103, 108-113, 116-124, 126-131, 133-139, 141-154 Leptin antagonist 9, 16, 25-27, 98, 99, 103, 129, 133-139 Leptin receptor (LR) 1-3, 15, 27, 30, 43-46, 49, 54-56, 63, 64, 66-69, 73-79, 83, 85-87, 88, 92, 94-97, 100, 108-113, 117, 126-128, 130, 131, 133, 134, 144, 147, 149, 150 Leptin resistance 54, 74, 95-97, 103, 116, 129-131, 142, 143, 145-148, 150 Leptin structure 16, 25 Leukemia inhibitory factor (LIF) 16, 31, 110, 111 Lipodystrophy 116

M Mammalian target of rapamycin (mTOR) 9, 68 Mammary epithelium 63 Mass index 58, 63, 119 Maternal nutrition 144, 145, 149, 152, 153 Melanocyte-stimulating hormone (MSH) 4, 92 Menses 116, 120, 121, 124 Metabolic syndrome 91, 146 Metabolism 1, 4, 26, 30, 50, 86, 88, 96, 117, 118, 126, 131, 142-145, 147 Mineralization 84, 85, 88 Mitogen-activated protein kinase(MAPK) 7, 39, 45, 46, 48, 68, 73, 75-79, 88, 117 Myocardial ischemia 77

165

Index

N

R

NADPH oxidase 99, 100 Na+,K+-ATPase 94, 96, 100, 101 Natriuresis 91, 94-97, 101 Negative inotropic effect 76, 77 Neoplasm 63 Neuropeptide Y (NPY) 4, 8, 9, 143, 148 Nitric oxide 58, 75, 91-95, 97, 98, 100-102 Nutrition 141, 144-147, 149, 152, 153

Reactive oxygen specie (ROS) 47-50, 76, 99-102 Receptor 1-5, 7-9, 15-22, 24-26, 30, 31, 33-37, 39, 40, 43-49, 54-57, 59, 63-65, 67, 73-79, 83, 85-87, 91, 92, 94-100, 102, 108-113, 116, 117, 126-128, 130, 131, 133, 134, 142, 143, 144, 146, 147, 149, 150 Receptor activation 3, 7, 17-19, 24, 25, 30, 31, 39, 40, 111 Receptor antagonist 5, 47, 55, 96, 98, 128 Regulatory T cell (Treg) 16, 126-129, 134 Renal sodium handling 91, 92, 100 RhoA/ROCK activation 79 ROS 47-50, 99-102

O Obesity 2, 4, 8, 15, 22, 30, 43, 45, 46, 50, 54, 58, 59, 63, 64, 68, 69, 73-77, 83, 85, 86, 89, 91, 92, 95-99, 102, 103, 116, 129-131, 141-150, 152-154 Ob/Ob 2, 15, 16, 43, 45, 47, 56-58, 83, 85, 86, 88, 92, 95, 99, 108, 109, 116, 129, 133, 142, 150, 152, 153 OB-R 30, 31, 33-40, 46, 55, 109-111 Oncostatin M (OSM) 1, 16, 31 Ouabain-resistant Na+-ATPase 100, 101 Oxidative stress 91, 98-100

P Paracrine axis 64 Phosphodiesterase 8, 100, 101, 117 Phosphodiesterase 3B 8, 117 Phosphoinositol-3 kinase 1 PI3-Akt 77 PI3K 8, 48, 50, 93-95 POMC 4, 8, 9, 142, 143, 146, 148 PPAR γ 47 Predictive adaptive response 142 Pregnancy 110, 111, 141-147, 149, 152, 153 Proliferative chondrocyte 84, 85, 87, 88 Proopiomelanocortin 4, 5, 92, 142, 148 Protein kinase B 49, 93, 95 Protein-tyrosine phosphatase (SHP2) 5-7, 78 PTHrP/Ihh loop 88

S SH2B adaptor protein 1 8 Signal transducer and activator of transcription (STAT) 1, 3, 4, 7, 15, 18, 20, 22, 24, 25, 30, 46, 74, 76, 77, 88, 89, 97, 117, 130, 133 Stomach 15, 54-56, 58 Sympathetic nervous system (SNS) 49, 75, 76, 83, 91-93, 96-99, 102

T T cell 3, 16, 39, 45, 59, 126-129, 133, 134, 136, 139 Testosterone 45, 116, 120-122, 124, 150 Th1 16, 126-129, 133, 134 Thioacetamide 134-138 Thyreotropin-releasing hormone (TRH) 4, 5, 122 Treg 126-128, 130, 131, 134 Type 2 diabetes 56, 141, 142, 144, 146, 148, 149

U Uncoupling Protein (UCP) 47, 49

V Vagal afferent 55, 56 Vascular smooth muscle cell 4, 91, 94, 99

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Leptin and Leptin Antagonists

Leptin and Leptin Antagonists