Sphingolipids and Metabolic Disease

Sphingolipids and Metabolic Disease

ADVANCES IN EXPERIMENTAL MEDICINE AND BIOLOGY Editorial Board: NATHAN BACK, State University of New York at Buffalo IRUN R. COHEN, The Weizmann Institute of Science ABEL LAJTHA, N.S. Kline Institute for Psychiatric Research JOHN D. LAMBRIS, University of Pennsylvania RODOLFO PAOLETTI, University of Milan Recent Volumes in this Series Volume 712 CYSTEINE PROTEASES OF PATHOGENIC ORGANISMS Mark W. Robinson and John P. Dalton Volume 713 CELL FUSION IN HEALTH AND DISEASE, I: CELL FUSION IN HEALTH Thomas Dittmar Volume 714 CELL FUSION IN HEALTH AND DISEASE, II: CELL FUSION IN DISEASE Thomas Dittmar Volume 715 BACTERIAL ADHESION: BIOLOGY, CHEMISTRY AND PHYSICS Dirk Linke and Adrian Goldman Volume 716 MAST CELL BIOLOGY: CONTEMPORARY AND EMERGING TOPICS  $ODVGDLU0*LO¿OODQDQG'HDQ'0HWFDOIH Volume 717 KAINATE RECEPTORS: NOVEL SIGNALING INSIGHTS Antonio Rodriguez-Moreno and Talvinder S. Sihra Volume 718 BRAIN INSPIRED COGNITIVE SYSTEMS 2010 Ricardo Sanz, Jaime Gomez and Carlos Hernandez Volume 719 HOT TOPICS IN INFECTION AND IMMUNITY IN CHILDREN VIII Nigel Curtis Volume 720 HUMAN CELL TRANSFORMATION Johng S. Rhim and Richard Kremer Volume 721 SPHINGOLIPIDS AND METABOLIC DISEASE L. Ashley Cowart

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Sphingolipids and Metabolic Disease Edited by L. Ashley Cowart, PhD Department of Biochemistry and Molecular Biology, Medical University of South Carolina, and Ralph H. Johnson Veteran’s Administration, Charleston, South Carolina, USA

Springer Science+Business Media, LLC Landes Bioscience

Springer Science+Business Media, LLC Landes Bioscience Copyright ©2011 Landes Bioscience and Springer Science+Business Media, LLC All rights reserved. 1RSDUWRIWKLVERRNPD\EHUHSURGXFHGRUWUDQVPLWWHGLQDQ\IRUPRUE\DQ\PHDQVHOHFWURQLFRUPHFKDQLFDO LQFOXGLQJSKRWRFRS\UHFRUGLQJRUDQ\LQIRUPDWLRQVWRUDJHDQGUHWULHYDOV\VWHPZLWKRXWSHUPLVVLRQLQZULWLQJ IURPWKHSXEOLVKHUZLWKWKHH[FHSWLRQRIDQ\PDWHULDOVXSSOLHGVSHFL¿FDOO\IRUWKHSXUSRVHRIEHLQJHQWHUHG DQGH[HFXWHGRQDFRPSXWHUV\VWHPIRUH[FOXVLYHXVHE\WKH3XUFKDVHURIWKHZRUN Printed in the USA. Springer Science+Business Media, LLC, 233 Spring Street, New York, New York 10013, USA http://www.springer.com Please address all inquiries to the publishers: Landes Bioscience, 1806 Rio Grande, Austin, Texas 78701, USA Phone: 512/ 637 6050; FAX: 512/ 637 6079 http://www.landesbioscience.com The chapters in this book are available in the Madame Curie Bioscience Database. http://www.landesbioscience.com/curie Sphingolipids and Metabolic Disease, edited by L. Ashley Cowart. Landes Bioscience / Springer Science+Business Media, LLC dual imprint / Springer series: Advances in Experimental Medicine and Biology. ISBN: 978-1-4614-0649-5 :KLOHWKHDXWKRUVHGLWRUVDQGSXEOLVKHUEHOLHYHWKDWGUXJVHOHFWLRQDQGGRVDJHDQGWKHVSHFL¿FDWLRQVDQGXVDJH RIHTXLSPHQWDQGGHYLFHVDVVHWIRUWKLQWKLVERRNDUHLQDFFRUGZLWKFXUUHQWUHFRPPHQGDWLRQVDQGSUDFWLFH DWWKHWLPHRISXEOLFDWLRQWKH\PDNHQRZDUUDQW\H[SUHVVHGRULPSOLHGZLWKUHVSHFWWRPDWHULDOGHVFULEHG LQWKLVERRN,QYLHZRIWKHRQJRLQJUHVHDUFKHTXLSPHQWGHYHORSPHQWFKDQJHVLQJRYHUQPHQWDOUHJXODWLRQV DQGWKHUDSLGDFFXPXODWLRQRILQIRUPDWLRQUHODWLQJWRWKHELRPHGLFDOVFLHQFHVWKHUHDGHULVXUJHGWRFDUHIXOO\ UHYLHZDQGHYDOXDWHWKHLQIRUPDWLRQSURYLGHGKHUHLQ

Library of Congress Cataloging-in-Publication Data Sphingolipids and metabolic disease / edited by L. Ashley Cowart. p. ; cm. -- (Advances in experimental medicine and biology ; v. 721) ,QFOXGHVELEOLRJUDSKLFDOUHIHUHQFHV ISBN 978-1-4614-0649-5 1. Sphingolipidoses. 2. Sphingolipids--Pathophysiology. I. Cowart, Lauren Ashley. II. Series: Advances in experimental medicine and biology ; v. 721. 0065-2598 [DNLM: 1. Metabolic Diseases--pathology. 2. Sphingolipids--metabolism. W1 AD559 v.721 2011 / WD 200] RC632.S67S64 2011 616.3’9--dc23 2011021157

PREFACE

5HFHQW\HDUVKDYHZLWQHVVHGDQH[SORVLRQLQWKHLQFLGHQFHRIREHVLW\DQGLWVVHTXHODH LQFOXGLQJ7\SH  GLDEHWHV DQG WKH PHWDEROLF V\QGURPH:KLOH RULJLQDOO\ FRQILQHG WR developed countries, increasing prosperity in developing countries has broadened the ZRUOGZLGHLQFLGHQFHRIWKHVHGLVRUGHUV0RUHRYHUZKLOHGLDJQRVHVRI7\SHGLDEHWHV ZHUHRULJLQDOO\DOPRVWH[FOXVLYHO\FRQILQHGWRDGXOWSRSXODWLRQVWKHULVHLQFKLOGKRRG REHVLW\KDVSUHFLSLWDWHGDPDUNHGLQFUHDVHRIWKLVGLVRUGHULQFKLOGUHQ7KXVLWLVFUXFLDO to explore all possible therapeutic and preventive methods to attenuate pathological processes associated with these disorders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sphingolipids in these disease processes. Chapter 1: Sphingolipid Metabolism and Analysis in Metabolic Disease, by Sarah ( %ULFH DQG /$VKOH\ &RZDUW 7KLV FKDSWHU SUHVHQWV DQ RYHUYLHZ RI VSKLQJROLSLG PHWDEROLVP DQG LWV UHJXODWLRQ IROORZHG E\ FDYHDWV DQG WHFKQLFDO FRQVLGHUDWLRQV IRU sphingolipid measurement. Chapter 2: Sphingolipids and Cardiovascular Diseases, by Xian-Cheng Jiang, Ira -*ROGEHUJDQG7DH6LN3DUN7KLVFKDSWHUDGGUHVVHVFXUUHQWNQRZOHGJHRIWKHUROHV RI VSKLQJROLSLGV LQ G\VIXQFWLRQ RI WKH FDUGLRYDVFXODU V\VWHP LQFOXGLQJ OLSRSURWHLQ metabolism, atherosclerosis, and cardiomyopathy. Chapter 3: Heart Sphingolipids in Health and Disease, by Marcin Baranowski and Jan Górski. This chapter continues the cardiovascular theme by addressing novel v

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PREFACE

PHFKDQLVPVRIUHJXODWLQJVSKLQJROLSLGELRV\QWKHVLVLQWKHKHDUWLQGLDEHWHVDVZHOODV WKHSURWHFWLYHUROHRIVSKLQJROLSLGVLQLVFKHPLDUHSHUIXVLRQLQMXU\ Chapter 4: Blood Sphingolipids in Homeostasis and Pathobiology, by Samar M. +DPPDG 7KLV FKDSWHU DGGUHVVHV WKH FOLQLFDO DVVHVVPHQW RI EORRG VSKLQJROLSLGV IRU diagnostic purposes. Chapter 5: Adipose Tissue and Ceramide Biosynthesis in the Pathogenesis of Obesity, by Fahumiya Samad, Leylla Badeanlou, Charmi Shah, and Guang Yang. This chapter GLVFXVVHVFKDQJHVLQVSKLQJROLSLGVWKDWRFFXUDVDUHVXOWRIREHVLW\DQGKRZWKHVHFKDQJHV PHGLDWHLQIODPPDWLRQDQGFDUGLRYDVFXODUULVN Chapter 6: Sphinoglipids and Hepatic Steatosis,E\%HQMDPLQ7%LNPDQDQG6FRWW $ 6XPPHUV 7KLV FKDSWHU GLVFXVVHV KRZ PDQLSXODWLRQ RI VSKLQJROLSLG PHWDEROLVP LQIOXHQFHVWULDF\OJ\OHUROPHWDEROLVPLQWKHFRQWH[WRIIDWW\OLYHU Chapter 7: Glycosphingolipids and Insulin Resistance, by Johannes M. Aerts and FROOHDJXHV7KLVFKDSWHUGLVFXVVHVWKHUROHVRIJO\FRVSKLQRJOLSLGVLQLQVXOLQVLJQDOLQJDQG KRZSKDUPDFRORJLFDOUHGXFWLRQRIJO\FRVSKLQRJOLSLGV\QWKHVLVDPHOLRUDWHVV\PSWRPV RIWKHPHWDEROLFV\QGURPH Chapter 8: Glycosphingolipids and Kidney Disease, by Andrew R. Mather and /HDK - 6LVNLQG7KLV FKDSWHUFRQWLQXHV D IRFXV RQ JO\FRVSKLQJROLSLGV LQ WKH FRQWH[W RINLGQH\SDWKRORJ\$OWKRXJKWKHLPSOLFDWLRQRIJO\FRVSKLQJROLSLGVLQNLGQH\GLVHDVH DVVRFLDWHGZLWKGLDEHWHVLVVWLOOFRQMHFWXUDOWKHUROHWKHVHOLSLGVSOD\LQDVSHFWUXPRI NLGQH\GLVRUGHUVDVGLVFXVVHGLQWKLVFKDSWHUMXVWLILHVIXUWKHULQYHVWLJDWLRQLQWKLVKLJKO\ novel and underexplored area. Chapter 9: Sphingolipid Synthetic Pathways are Major Regulators of Lipid HomeostasisE\7LOOD6:RUJDOO7KLVILQDOFKDSWHUSUHVHQWVFRPSHOOLQJILQGLQJVWKDW VSKLQJROLSLGVPD\UHJXODWHFKROHVWHUROKRPHRVWDVLVWKURXJK65(%3DQGOLSLGHIIOX[7KH cross-talk between sphingolipids and lipoprotein metabolism presents rich opportunities IRUWKHUDSHXWLFVDLPHGDWDPHOLRUDWLQJG\VOLSLGHPLDDVVRFLDWHGZLWKPHWDEROLFV\QGURPH that promotes atherosclerosis. 2XU JRDO LQ WKLV YROXPH ZDV WR FRPSLOH FKDSWHUV SUHVHQWLQJ EURDG RYHUYLHZV RI WLVVXHVSHFLILFHIIHFWVRIVSKLQJROLSLGVZKLOHHPSKDVL]LQJLQWHUUHODWHGQHVVRIFHOOXODU processes and cross-talk between organs. :HKRSH\RXHQMR\WKHYROXPH L. Ashley Cowart, PhD Department of Biochemistry and Molecular Biology, Medical University of South Carolina, and Ralph H. Johnson Veteran’s Administration, Charleston, South Carolina, USA

ABOUT THE EDITOR...

L. ASHLEY COWARTREWDLQHGKHU%6LQ%LRORJ\LQIURP)XUPDQ8QLYHUVLW\ LQ*UHHQYLOOH6RXWK&DUROLQDDQGKHU3K'LQ%LRFKHPLVWU\LQIURP9DQGHUELOW 8QLYHUVLW\ LQ 1DVKYLOOH 7HQQHVHH$IWHU SRVWGRFWRUDO ZRUN LQ WKH ODERUDWRU\ RI 'U <XVXI+DQQXQVKHMRLQHGWKHIDFXOW\DWWKH0HGLFDO8QLYHUVLW\RI6RXWK&DUROLQDLQ &KDUOHVWRQ6RXWK&DUROLQDZKHUHVKHFXUUHQWO\KROGVWKHSRVLWLRQRI$VVLVWDQW3URIHVVRU RI%LRFKHPLVWU\DQG0ROHFXODU%LRORJ\'U&RZDUW¶VPDLQUHVHDUFKIRFXVLVWKHUHJXODWLRQ RI VSKLQJROLSLG PHWDEROLVP LQ GLDEHWHV DQG REHVLW\ DQG WKH UROHV RI VSKLQJROLSLGV LQ pathological outcomes associated with these disorders. In addition to research, she is involved with graduate admissions and education.

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PARTICIPANTS

Johannes M. Aerts Academic Medical Center  'HSDUWPHQWRI0HGLFDO%LRFKHPLVWU\ 8QLYHUVLW\RI$PVWHUGDP Amsterdam The Netherlands

1RUD%LMO Academic Medical Center  'HSDUWPHQWRI0HGLFDO%LRFKHPLVWU\ 8QLYHUVLW\RI$PVWHUGDP Amsterdam The Netherlands

Jan Aten Academic Medical Center  'HSDUWPHQWRI3DWKRORJ\ 8QLYHUVLW\RI$PVWHUGDP Amsterdam The Netherlands

%HQMDPLQ7%LNPDQ Program in Cardiovascular and Metabolic Disorders Duke-NUS Graduate Medical School Singapore

Leylla Badeanlou 7RUUH\3LQHV,QVWLWXWHIRU0ROHFXODU Studies 6DQ'LHJR&DOLIRUQLD USA Marcin Baranowski 'HSDUWPHQWRI3K\VLRORJ\ 0HGLFDO8QLYHUVLW\RI%LDá\VWRN %LDá\VWRN Poland Florence M. Bietrix Academic Medical Center  'HSDUWPHQWRI0HGLFDO%LRFKHPLVWU\ 8QLYHUVLW\RI$PVWHUGDP Amsterdam The Netherlands

5ROI*%RRW Academic Medical Center  'HSDUWPHQWRI0HGLFDO%LRFKHPLVWU\ 8QLYHUVLW\RI$PVWHUGDP Amsterdam The Netherlands Sarah E. Brice 'HSDUWPHQWRI%LRFKHPLVWU\ and Molecular Biology 0HGLFDO8QLYHUVLW\RI6RXWK&DUROLQD Charleston, South Carolina USA L. Ashley Cowart 'HSDUWPHQWRI%LRFKHPLVWU\ and Molecular Biology 0HGLFDO8QLYHUVLW\RI6RXWK&DUROLQD and Ralph H. Johnson Veteran’s Administration Charleston, South Carolina USA ix

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PARTICIPANTS

Nick Dekker Academic Medical Center  'HSDUWPHQWRI0HGLFDO%LRFKHPLVWU\ 8QLYHUVLW\RI$PVWHUGDP Amsterdam The Netherlands

Samar M. Hammad 'HSDUWPHQWRI5HJHQHUDWLYH0HGLFLQH and Cell Biology 0HGLFDO8QLYHUVLW\RI6RXWK&DUROLQD Charleston, South Carolina USA

0DUFRYDQ(LMN Academic Medical Center  'HSDUWPHQWRI0HGLFDO%LRFKHPLVWU\ 8QLYHUVLW\RI$PVWHUGDP Amsterdam The Netherlands

Xian-Cheng Jiang 'HSDUWPHQWRI&HOO%LRORJ\ 6WDWH8QLYHUVLW\RI1HZ
Ira J. Goldberg 'HSDUWPHQWRI0HGLFLQH  'LYLVLRQRI3UHYHQWLYH0HGLFLQH and Nutrition Columbia University New York, New York USA Jan Górski 'HSDUWPHQWRI3K\VLRORJ\ 0HGLFDO8QLYHUVLW\RI%LDá\VWRN %LDá\VWRN Poland

0LUMDP/DQJHYHOG Academic Medical Center  'HSDUWPHQWRI,QWHUQDO0HGLFLQH 8QLYHUVLW\RI$PVWHUGDP Amsterdam The Netherlands Elisa Lombardo Academic Medical Center  'HSDUWPHQWRI0HGLFDO%LRFKHPLVWU\ 8QLYHUVLW\RI$PVWHUGDP Amsterdam The Netherlands

Albert K. Groen Academic Medical Center  'HSDUWPHQWRI0HGLFDO%LRFKHPLVWU\ 8QLYHUVLW\RI$PVWHUGDP Amsterdam The Netherlands

Andrew R. Mather 'HSDUWPHQWRI0HGLFLQH  'LYLVLRQRI*HQHUDO,QWHUQDO Medicine/Geriatrics 0HGLFDO8QLYHUVLW\RI6RXWK&DUROLQD Charleston, South Carolina USA

Johanna Groener Academic Medical Center  'HSDUWPHQWRI0HGLFDO%LRFKHPLVWU\ 8QLYHUVLW\RI$PVWHUGDP Amsterdam The Netherlands

5RHORI2WWHQKRII Academic Medical Center  'HSDUWPHQWRI0HGLFDO%LRFKHPLVWU\ 8QLYHUVLW\RI$PVWHUGDP Amsterdam The Netherlands

PARTICIPANTS

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+HUPHQ62YHUNOHHIW /HLGHQ,QVWLWXWHRI&KHPLVWU\  'HSDUWPHQWRI%LRRUJDQLF6\QWKHVLV Gorlaeus Laboratories Leiden University Leiden The Netherlands

Leah J. Siskind 'HSDUWPHQWRI0HGLFLQH  'LYLVLRQRI*HQHUDO,QWHUQDO Medicine/Geriatrics 0HGLFDO8QLYHUVLW\RI6RXWK&DUROLQD Charleston, South Carolina USA

Tae-Sik Park Lee Gil Ya Cancer and Diabetes Institute *DFKRQ8QLYHUVLW\RI0HGLFLQH and Science Inchon South Korea

Scott A. Summers Program in Cardiovascular and Metabolic Disorders Duke-NUS Graduate Medical School Singapore and 7KH6WHGPDQ&HQWHUIRU1XWULWLRQ and Metabolism Research Duke University Medical Center Durham, North Carolina USA

Cindy van Roomen Academic Medical Center  'HSDUWPHQWRI0HGLFDO%LRFKHPLVWU\ 8QLYHUVLW\RI$PVWHUGDP Amsterdam The Netherlands Fahumiya Samad 7RUUH\3LQHV,QVWLWXWHIRU0ROHFXODU Studies 6DQ'LHJR&DOLIRUQLD USA Mireille Serlie Academic Medical Center  'HSDUWPHQWRI,QWHUQDO0HGLFLQH 8QLYHUVLW\RI$PVWHUGDP Amsterdam The Netherlands Charmi Shah 7RUUH\3LQHV,QVWLWXWHIRU0ROHFXODU Studies 6DQ'LHJR&DOLIRUQLD USA

Tom Wennekes /LHGHQ,QVWLWXWHRI&KHPLVWU\  'HSDUWPHQWRI%LRRUJDQLF6\QWKHVLV Gorlaeus Laboratories Leiden University Leiden The Netherlands Tilla S. Worgall 'HSDUWPHQWRI3DWKRORJ\ Columbia University New York, New York USA Guang Yang 7RUUH\3LQHV,QVWLWXWHIRU0ROHFXODU Studies 6DQ'LHJR&DOLIRUQLD USA

CONTENTS

1. SPHINGOLIPID METABOLISM AND ANALYSIS IN METABOLIC DISEASE ............................................................................1 Sarah E. Brice and L. Ashley Cowart Abstract......................................................................................................................................... 1 Introduction .................................................................................................................................. 1 Sphingolipid Metabolism............................................................................................................. 3 Sphingolipids and Lipotoxicity ................................................................................................... 6 Approaches to the Study of Sphingolipids in Lipotoxicity ....................................................... 9 Conclusion .................................................................................................................................. 11

2. SPHINGOLIPIDS AND CARDIOVASCULAR DISEASES: LIPOPROTEIN METABOLISM, ATHEROSCLEROSIS AND CARDIOMYOPATHY..........................................................................19 Xian-Cheng Jiang, Ira J. Goldberg and Tae-Sik Park Abstract....................................................................................................................................... 19 Introduction ................................................................................................................................ 19 Sphingolipid Biosynthesis.......................................................................................................... 20 Role of Sphingomyelin (SM) in Lipoprotein Metabolism ...................................................... 22 Sphingomyelin and Atherosclerosis .......................................................................................... 24 Ceramide in Lipotoxic Cardiomyopathy ................................................................................. 31 Conclusion .................................................................................................................................. 32

3. HEART SPHINGOLIPIDS IN HEALTH AND DISEASE .................................41 Marcin Baranowski and Jan Górski Abstract....................................................................................................................................... 41 Introduction ................................................................................................................................ 41 Characterization of Myocardial Sphingolipid Metabolism ................................................... 43 PPARs as Regulators of Sphingolipid Metabolism in the Heart ........................................... 44 Exercise Modulates Myocardial Ceramide Metabolism ........................................................ 45 xiii

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CONTENTS

Sphingolipids in Ischemia/Reperfusion Injury of the Heart .................................................. 48 Ceramide as a Mediator of Lipotoxicity in the Heart............................................................. 50 Conclusion and Future Prospects ............................................................................................. 54

4. BLOOD SPHINGOLIPIDS IN HOMEOSTASIS AND PATHOBIOLOGY ................................................................................57 Samar M. Hammad Abstract....................................................................................................................................... 57 Introduction ................................................................................................................................ 57 Blood Sphingolipids in Homeostasis......................................................................................... 58 Blood Sphingolipids in Pathobiology ....................................................................................... 60 Conclusion .................................................................................................................................. 63

5. ADIPOSE TISSUE AND CERAMIDE BIOSYNTHESIS IN THE PATHOGENESIS OF OBESITY....................................................67 Fahumiya Samad, Leylla Badeanlou, Charmi Shah and Guang Yang Abstract....................................................................................................................................... 67 Introduction ................................................................................................................................ 67 Remodeling of Adipose and Plasma Ceramide in Obesity ..................................................... 69 Regulation of Ceramide Metabolism in Obesity ..................................................................... 71 Novel Links Between Sphingolipid Metabolism and Plasminogen Activator Inhibitor 1 (PAI-1) ............................................................................................................. 72 Contribution of Ceramide Biosynthesis to Body Weight Regulation and Energy Metabolism .................................................................................................... 73 6SKLQJROLSLGVDV0HGLDWRUVRI$GLSRVH,QÀDPPDWLRQLQ2EHVLW\ ......................................... 74 Contribution of Ceramide Biosynthesis to Obesity Mediated Insulin Resistance ............... 75 Contribution of Ceramide Biosynthesis to Hepatic Steatosis ................................................ 77 Ceramide and Cardiovascular Disease .................................................................................... 79 Conclusion .................................................................................................................................. 80

6. SPHINGOLIPIDS AND HEPATIC STEATOSIS ................................................87 %HQMDPLQ7%LNPDQDQG6FRWW$6XPPHUV Abstract....................................................................................................................................... 87 Introduction ................................................................................................................................ 87 Sphingolipid Levels in the Steatotic Liver ............................................................................... 89 Modulation of Sphingolipid Synthesis Impacts Hepatic Steatosis ........................................ 89 Factors Associated with NAFLD Induce Sphingolipid Synthesis .......................................... 91 Possible Mechanisms ................................................................................................................. 94 Conclusion .................................................................................................................................. 94

CONTENTS

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7. GLYCOSPHINGOLIPIDS AND INSULIN RESISTANCE ...............................99 -RKDQQHV0$HUWV5ROI*%RRW0DUFRYDQ(LMN-RKDQQD*URHQHU1RUD%LMO Elisa Lombardo, Florence M. Bietrix, Nick Dekker, Albert K. Groen, 5RHORI2WWHQKRII Cindy van Roomen, Jan Aten, Mireille Serlie, 0LUMDP/DQJHYHOG7RP:HQQHNHVDQG+HUPHQ62YHUNOHHIW Abstract....................................................................................................................................... 99 Introduction .............................................................................................................................. 100 Insulin Resistance..................................................................................................................... 100 Glycosphingolipids ................................................................................................................... 103 (Glyco)Sphingolipids and Insulin Resistance ........................................................................ 107 Pharmacological Modulation of Glycosphingolipids and Insulin Resistance .................... 110 Conclusion ................................................................................................................................ 113

8. GLYCOSPHINGOLIPIDS AND KIDNEY DISEASE ......................................121 Andrew R. Mather and Leah J. Siskind Abstract..................................................................................................................................... 121 Introduction .............................................................................................................................. 121 Metabolism of Sphingolipids................................................................................................... 122 Metabolism of Glycosphingolipids ......................................................................................... 123 Glycosphingolipid Subcellular Localization and Transport ................................................ 125 Functions of Glycosphingolipids............................................................................................. 125 Fabry’s Disease ......................................................................................................................... 127 Polycystic Kidney Disease ....................................................................................................... 127 Kidney Cancer.......................................................................................................................... 128 Glomerulonephritis .................................................................................................................. 129 Diabetic Nephropathy and Metabolic Syndrome .................................................................. 130 Conclusion ................................................................................................................................ 131

9. SPHINGOLIPID SYNTHETIC PATHWAYS ARE MAJOR REGULATORS OF LIPID HOMEOSTASIS ............................................139 Tilla S. Worgall Abstract..................................................................................................................................... 139 Introduction .............................................................................................................................. 140 SREBPs are Key Transcription Factors of Protein That Synthesize Cholesterol, Fatty Acids and Phospholipids........................................................................................ 140 Sphingolipids Synthesis is Required to Increase SREBP in Lipid Depletion ..................... 140 Cholesterol, Unsaturated Fatty Acids and Ceramides Inhibit SREBP ............................... 142 $%&$DQG$%&*DUH.H\/LSLG(IÀX[5HFHSWRUV7KDW'HWHUPLQH Anti-Atherogenic High-Density Lipoprotein (HDL) Levels......................................... 143 Sphingolipids Affect ABCA1 and ABCG1 by Different Mechanisms ................................. 143 Sphingomyelin Induces Lipoprotein Patterns That Correlate with Increased Cardiovascular Risk ........................................................................................................ 145 Conclusion ................................................................................................................................ 146

INDEX........................................................................................................................149

ACKNOWLEDGEMENTS

7KH(GLWRUZLVKHVWRDFNQRZOHGJHIXQGLQJIURPWKH'HSDUWPHQWRI9HWHUDQ¶V $IIDLUVDQGWKH1,+&REUHLQ/LSLGRPLFVDQG3DWKRELRORJ\DWWKH0HGLFDO8QLYHUVLW\ RI 6RXWK &DUROLQD 6KH DOVR ZLVKHV WR DFNQRZOHGJH KHU 3K' PHQWRU 'U -RUJH &DSGHYLODDW9DQGHUELOW8QLYHUVLW\IRUKLVSDWLHQFHDQGDGKHUHQFHWRWKHKLJKHVW VWDQGDUGVIRUVFLHQFHLQOLSLGELRFKHPLVWU\,QDGGLWLRQWKLVZRUNZRXOGQRWKDYH EHHQSRVVLEOHZLWKRXWWKHWUDLQLQJPHQWRUVKLSDQGFRQWLQXHGVXSSRUWIURP'U<XVXI A. Hannun and Dr. Lina M. Obeid.

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CHAPTER 1 SPHINGOLIPID METABOLISM AND ANALYSIS IN METABOLIC DISEASE Sarah E. Brice*,1 and L. Ashley Cowart1,2 Department of Biochemistry and Molecular Biology, Medical University of South Carolina, Charleston, USA; Ralph H. Johnson Veteran’s Administration, Charleston, South Carolina, USA *Corresponding Author: Sarah E. Brice—Email: [email protected] 1 2

Abstract:

6SKLQJROLSLGVDUHDQLPSRUWDQWFODVVRIVWUXFWXUDODQGVLJQDOLQJPROHFXOHVZLWKLQWKH cell. As sphingolipids have been implicated in the development and pathogenesis RI LQVXOLQ UHVLVWDQFH DQG WKH PHWDEROLF V\QGURPH LW LV LPSRUWDQW WR XQGHUVWDQG their regulation and metabolism. Although these lipids are initially produced through a common pathway, there is no “generic” sphingolipid. Indeed, the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chromatography-mass spectrometry approaches, allows investigators to undertake DIXQFWLRQDOFKDUDFWHUL]DWLRQRIDOORUSDUWRIWKHVSKLQJROLSLGRPHLQWKHLUV\VWHPV RILQWHUHVW

INTRODUCTION 0DPPDOLDQVSKLQJROLSLGPHWDEROLVPFRQVLVWVRIDFRPSOH[QHWZRUNRILQWHUORFNLQJ SDWKZD\V)LJ 7KHEDVLFFXUUHQF\RIVSKLQJROLSLGPHWDEROLVPLVWKHVSKLQJRLGEDVH GLK\GUR[\DPLQRDONDQHDQGLWVGHULYDWLYHV WKLVEDVHPD\EHVXEMHFWWRWKHDGGLWLRQ RIDIDWW\DFLGKHDGJURXSRUSKRVSKDWHPRLHW\UHYLHZHGLQUHIV1-3). This intricate

Sphingolipids and Metabolic Disease, edited by L. Ashley Cowart. ©2011 Landes Bioscience and Springer Science+Business Media, LLC 1

Figure 1. 0HWDEROLVP RI VLPSOH VSKLQJROLSLGV

2 SPHINGOLIPIDS AND METABOLIC DISEASE

SPHINGOLIPID METABOLISM AND ANALYSIS IN METABOLIC DISEASE

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ZHERILQWHUDFWLRQVSURYLGHVWKHFHOOZLWKQXPHURXVSRLQWVRIUHJXODWLRQDOORZLQJSUHFLVH FRQWURORIWKHVHLPSRUWDQWELRDFWLYHPHWDEROLWHV Insulin resistance and the metabolic syndrome are two increasingly important public health concerns in Western nations. In these pathologies, excessive accumulation RI DGLSRVH WLVVXH FRPELQHG ZLWK G\VUHJXODWLRQ RI SODVPD IUHH IDWW\ DFLG OHYHOV OHDGV to ectopic lipid accumulation in nonadipose tissues (steatosis).4-6 Although cells can ELRFKHPLFDOO\ VHTXHVWHU VRPH RI WKH H[FHVV OLSLG DV WULJO\FHULGHV WKLV PHFKDQLVP LV eventually overwhelmed and lipids spill over into nonoxidative pathways.7-9 This can OHDGWRDEHUUDQWFHOOXODUVLJQDOLQJPLWRFKRQGULDOG\VIXQFWLRQDQGOLSRWR[LFFHOOGHDWK10 Fatty acid oversupply and oxidative stress are two points at which metabolic syndrome and diabetes may touch sphingolipid metabolism and much attention has been paid in UHFHQW\HDUVWRSRWHQWLDOPRGXODWLRQRIVSKLQJROLSLGPHWDEROLVPLQWKHVHGLVHDVHVWDWHV11 6RPHLQYHVWLJDWRUVKDYHIRFXVHGRQWKHSKHQRW\SLFHIIHFWVRIFRUUHFWLQJG\VUHJXODWLRQ RIVSKLQJROLSLGOHYHOVZKLOHRWKHUVKDYHDWWHPSWHGWRUHVROYHWKHPROHFXODUHIIHFWVRI aberrant sphingolipid metabolism in several cell systems relevant to diabetes and metabolic syndrome.12-146\VWHPVRISDUWLFXODULQWHUHVWLQFOXGHWKHSDQFUHDWLF` cells, skeletal muscle DQGWKHKHDUW3RSXODUPRGHOVIRUGLDEHWHVDQGREHVLW\LQFOXGHWKH=XFNHUGLDEHWLFIDWW\ =') PRXVHDQGUDWGLHWLQGXFHGREHVLW\WR[LFDQWLQGXFHGGLDEHWHVDQGYDULRXVFHOO culture systems.8,15-19 Interestingly, while commonalities occur across model systems, there DUHDOVRVRPHWLVVXHVSHFL¿FHIIHFWVDVZLOOEHGHVFULEHGEHORZDQGLQRWKHUFKDSWHUV SPHINGOLIPID METABOLISM (QWU\ LQWR VSKLQJROLSLG PHWDEROLVP EHJLQV ZLWK WKH FRQGHQVDWLRQ RI VHULQH DQG SDOPLWR\OFRHQ]\PH$&R$ E\WKHHQ]\PHVHULQHSDOPLWR\OWUDQVIHUDVH637 WRIRUP 3-ketodihydrosphingosine (Fig. 1).20,21 This enzyme, which localizes to the endoplasmic UHWLFXOXPLVWKRXJKWWRH[LVWDVDN'DPXOWLPHUFRPSRVHGRIIRXUVHWVRIGLPHUV22,23 'LPHUV PD\ IRUP EHWZHHQ WKH 637/& VXEXQLW DQG 637/& DQGRU 637/&24 $GGLWLRQDOO\ELQGLQJRIWKHHLWKHURIWKHSXWDWLYHVPDOOVXEXQLWVVV637DDQGVV637E KDV EHHQ VKRZQ WR VWLPXODWH 637 DFWLYLW\ DQG 250250'/ IDPLO\ SURWHLQV KDYH recently been claimed to associate with the SPT complex and modulate sphingolipid homeostasis.25-27 7KH VXEXQLW FRPSRVLWLRQ RI WKH HQ]\PH LQFOXGLQJ ELQGLQJ RI VPDOO VXEXQLWV KDV EHHQ VKRZQ WR PRGXODWH IDWW\ DF\O&R$ FKDLQ OHQJWK VSHFL¿FLWLHV DV described below. Several compounds inhibit SPT; two that are commonly encountered in the literature are myriocin and L-cycloserine.28 Myriocin (ISP-1/thermozymocidin) LVW\SLFDOO\WKHLQKLELWRURIFKRLFHGXHWRLWVKLJKVSHFL¿FLW\IRU63729,30 L-cycloserine, ZKLFKLVOHVVFRPPRQO\XVHGHI¿FLHQWO\LQKLELWV637EXWDOVRDFWVDVDEURDGVSHFWUXP LQKLELWRURIS\ULGR[DO¶SKRVSKDWHGHSHQGHQWHQ]\PHV31,32 6LJQL¿FDQWO\UHFHQWVWXGLHVKDYHLQFUHDVHGDZDUHQHVVRIWKHV\QWKHVLVRIQRQFDQRQLFDO VSKLQJRLGEDVHVXWLOL]LQJDOWHUQDWLYHDPLQRDFLGVRUIDWW\DF\O&R$V)RUH[DPSOHDOWKRXJK VHULQHLVWKHDPLQRDFLGFDQRQLFDOO\FRQVLGHUHGWRIRUPDEDVLVIRUVSKLQJROLSLGVDUHFHQW study demonstrated that alanine could be incorporated in some cases.33,34 In the hereditary sensory neuropathy Type 1 (HSAN1) mouse model, a mutation in SPTLC1 allows the SPT HQ]\PHWRXVHDODQLQHDQGJO\FLQHIRUPLQJWR[LFVSKLQJROLSLGPHWDEROLWHV34,35 Similarly, it KDVORQJEHHQDSSUHFLDWHGWKDWZKLOHSDOPLWR\O&R$LVWKHSUHIHUUHGIDWW\DF\OFKDLQGRQRU IRU637PLFURVRPDO637VKRZVDFWLYLW\WRZDUGDQXPEHURISK\VLRORJLFDOO\DYDLODEOH IDWW\DFLGV36-40 It has been demonstrated that total SPT in cellular extracts typically has its

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SPHINGOLIPIDS AND METABOLIC DISEASE

Table 1.&KDLQOHQJWKVSHFL¿FLWLHVRIGLK\GURFHUDPLGHV\QWKDVHLVRIRUPV44 CerS1 Carbon Chain Length

18:0, 20:0

CerS2 20:0, 22:0, 24:0, 24:1, 26:0

CerS3 14:0, 22:0, 24:0

CerS4 18:0, 22:0, 24:0

CerS5 14:0, 16:0, 18:0, 18:1

CerS6 14:0, 16:0

greatest activity toward palmitoyl-CoA (C16 ZLWKIDWW\DFLGVZLWKFKDLQOHQJWKVRIPRUH than the optimal sixteen carbons being utilized more readily than those with shorter chain lengths.36,39 Additionally, cisGRXEOHERQGVIRXQGLQQDWXUDOO\RFFXUULQJXQVDWXUDWHGIDWW\ acids, have been shown to inhibit SPT activity in vitro and in vivo, while activities toward transXQVDWXUDWHGIDWW\DFLGVZHUHPRUHVLPLODUWRPD[LPDODFWLYLW\IRUDJLYHQFKDLQ length.386SHFL¿FDOO\637/&637/&VV637DGHPRQVWUDWHGPD[LPDODFWLYLW\WRZDUG palmitoyl CoA (C16 ZKLOHVXEVWLWXWLRQRIVV637EIRUVV637DVKLIWHGWKLVSUHIHUHQFHWRZDUG stearoyl-CoA (C18).27&RPSOH[HVFRPSRVHGRI637/&637/&VV637DRQWKHRWKHU hand, utilized myristoyl-CoA (C14 VRPHZKDWLQSUHIHUHQFHWRSDOPLWR\O&R$ZKHUHDV LQFOXVLRQRIVV637EUHGXFHGRYHUDOODFWLYLW\FRPSDUHGWRWKH637/&637/&VV637D FRPSOH[EXWEURDGHQHGWKHVXEVWUDWHVSHFL¿FLW\WRLQFOXGHDOOVDWXUDWHGIDWW\DFLGVRI HYHQQXPEHUHGFKDLQOHQJWKVEHWZHHQIRXUWHHQDQGWZHQW\FDUERQV27 Thus, although all FRPELQDWLRQVRIVXEXQLWVUHWDLQDFWLYLW\WRZDUGSDOPLWR\O&R$FKDQJHVLQWKHVXEXQLW SUR¿OHRIFHOOXODU637FRXOGSRWHQWLDOO\DOORZLQFRUSRUDWLRQRIDOWHUQDWLYHIDWW\DFLGV $IWHU LWV IRUPDWLRQ E\ 637 NHWRGLK\GURVSKLQJRVLQH LV UHGXFHG WR IRUP dihydrosphingosine.417KLVFRPSRXQGLVWKHQ1DF\ODWHGE\RQHRIVL[GLK\GURFHUDPLGH V\QWKDVHV&HU6IRUPHUO\NQRZQDV/RQJHYLW\$VVXUDQFH*HQHVRU/$66HVSULRUWRWKHLU IXQFWLRQDOFKDUDFWHUL]DWLRQ ZKLFKDFWRQGLK\GURFHUDPLGHLQDFKDLQOHQJWKGHSHQGHQW manner (Table 1).42-48,QGHHGZKLOHWKHSUHGRPLQDQWDF\OFKDLQOHQJWKVRIPDPPDOLDQ ceramide are C16- and C24VDWXUDWHG IDWW\ DFLGV LQYHVWLJDWRUV KDYH HQFRXQWHUHG IDWW\ DFLGVRIFKDLQOHQJWKVUDQJLQJIURP&14 to C32.1,42 Ceramides are mostly saturated and may contain _ and/or t-hydroxyl groups.1,427KLVSURYLGHVDQDGGLWLRQDOSRLQWRIFRQWURO ZKHUHWKHFHOOPD\H[HUWVSHFL¿FPDQLSXODWLRQVRIWKHFKDLQOHQJWKSUR¿OHRIVSKLQJROLSLG VSHFLHV7KHPDLQWHQDQFHDQGGLIIHUHQWLDOUHJXODWLRQRIQXPHURXVJHQHVHQFRGLQJ&HU6 ZLWKGLIIHUHQWVXEVWUDWHSUHIHUHQFHVVXSSRUWVWKHVSHFXODWLRQWKDWPRGL¿FDWLRQRIVSHFL¿F chain lengths may be an important cellular process.42 $IWHUV\QWKHVLVE\RQHRIWKH&HU6GLK\GURFHUDPLGHLVUHGXFHGE\GLK\GURFHUDPLGH GHVDWXUDVH'(*6'(6 WRIRUPFHUDPLGHWKH¿UVWOLSLGLQWKLVSDWKZD\NQRZQWR KDYHLPSRUWDQWVLJQDOLQJIXQFWLRQV49 Ceramide has been implicated in stress responses, VHQHVFHQFHFHOOF\FOHDUUHVWDSRSWRVLVDQGGLIIHUHQWLDWLRQ47,50-53 It acts through a number RI WDUJHW RU HIIHFWRU SURWHLQV LQFOXGLQJ FDWKHSVLQ ' -1. .65 S 3.&c, PP1, 33$5DF5DIDQG5EUHYLHZHGLQUHI54 ,QGXFWLRQRIDSRSWRVLVDNH\IXQFWLRQ RI FHUDPLGH VLJQDOLQJ RFFXUV WKURXJK FHOO W\SHVSHFL¿F PHFKDQLVPV 0DQ\ RI WKHVH LQYROYHWKHDFWLYDWLRQRISURWHLQNLQDVHVRUSURWHDVHVRUSURPRWLRQRIPLWRFKRQGULDOSRUH IRUPDWLRQ55-60 Notably, ceramide produced through de novo sphingolipid synthesis may HYRNHDVRPHZKDWGLIIHUHQWLQWUDFHOOXODUUHVSRQVHWKDQFHUDPLGHGHULYHGIURPVDOYDJH SDWKZD\VDQGWKHVHSDWKZD\VPD\EHGLIIHUHQWLDOO\UHJXODWHG61-63 &HUDPLGHDFWVDVDEXLOGLQJEORFNIRUWKHRWKHUEUDQFKHVRIVSKLQJROLSLGPHWDEROLVP LQZKLFKLWPD\EHPRGL¿HGZLWKKHDGJURXSVSKRVSKRU\ODWHGRUGHJUDGHGWRVSKLQJRVLQH which is an eighteen carbon amino alcohol with an unsaturated hydrocarbon tail.64 )RUPDWLRQRIVSKLQJRVLQHLVPHGLDWHGE\VHYHUDOFHUDPLGDVHHQ]\PHV65-67 Acid, neutral

SPHINGOLIPID METABOLISM AND ANALYSIS IN METABOLIC DISEASE

5

Table 2.,QWUDFHOOXODUORFDOL]DWLRQH[SUHVVLRQDQGVSHFL¿FLWLHVRIPRXVHFHUDPLGDVHV67,170 Intracellular Localization

Substrate 6SHFL¿FLW\

Protein

Gene

Acid Ceramidase

mAC/ASAH1

Lysosome

C6 -C16-Cer

Neutral Ceramidase Akaline Ceramidase 1 Alkaline Ceramidase 2

mNC/ASAH2

Plasma Membrane Endoplasmic Reticulum Golgi

C14 -Cer and Longer

Endoplasmic Reticulum/ Golgi

C20:1-Cer/DHCer and shorter (unsaturated)

Alkaline Ceramidase 3

ACER1/ASAH3 ACER2/ ASAH3L/ maCER1 ACER3/PHCA/ aPHC

C24:1-Cer and Longer, unsaturated C14 -Cer and longer

Highest Expression Kidney, lung, heart, brain Kidney, liver, heart Skin Placenta

Unknown

and alkaline ceramidases have distinct expression patterns, intracellular topologies and VXEVWUDWHSUHIHUHQFHVIRUDF\O&R$FKDLQOHQJWKVDQGVDWXUDWLRQ7DEOH UHYLHZHGLQ UHI67 5HODWLYHFRQWULEXWLRQVRIWKHK\GURO\WLFDQGGHQRYRSDWKZD\VYDU\ZLGHO\E\ cell type. For example, C2C12 myotubes rely very heavily on de novo sphingolipid V\QWKHVLVZKLOHRWKHUFHOOOLQHVVWURQJO\SUHIHUK\GURO\WLFPHWDEROLVP68 ,PSRUWDQWO\VSKLQJRVLQHPD\EHSKRVSKRU\ODWHGE\VSKLQJRVLQHNLQDVH6. WRIRUP sphingosine-1-phosphate (S1P).69 S1P is an important signaling molecule imputed with both intracellular and extracellular signaling properties.70-727KHH[WUDFHOOXODUHIIHFWVRI 63DUHPHGLDWHGE\LQWHUDFWLRQZLWKDIDPLO\RIDWOHDVW¿YH*SURWHLQFRXSOHGUHFHSWRUV named S1P1-S1P5UHYLHZHGLQUHI73).The S1P receptors signal through heterotrimeric *SURWHLQVZLWKVXEW\SHVSHFL¿F*SURWHLQFRXSOLQJOHDGLQJWRXQLTXHVLJQDOLQJSDWKZD\V IRUHDFKUHFHSWRUVXEW\SH747KHLQWUDFHOOXODUWDUJHWVRI63KDYHQRW\HWEHHQLGHQWL¿HG 7ZRLVRIRUPVRI6.6SK.DQG6SK.DUHUHVSRQVLEOHIRUSURGXFWLRQRI6375,76 These LVRIRUPVDUHGLIIHUHQWLDOO\H[SUHVVHGLQYDULRXVWLVVXHVLQWKHERG\DQGWKH\PD\VHUYH VHSDUDWHVLJQDOLQJIXQFWLRQVDQGEHVXEMHFWWRGLVWLQFWPRGHVRIUHJXODWLRQ76-79 S1P generally SURPRWHVFHOOSUROLIHUDWLYHDQGDQWLDSRSWRWLFSDWKZD\V80-82 Important signaling activities RI63LQFOXGHVWLPXODWLRQRILQRVLWROWULVSKRVSKDWHLQGHSHQGHQWFDOFLXPPRELOL]DWLRQ LQKLELWLRQRIFDVSDVHDFWLYLW\DFWLYDWLRQRIQRQUHFHSWRUW\URVLQHNLQDVHVDQGWKH5DI 0..(5.VLJQDOLQJFDVFDGHVDQGVWLPXODWLRQRI3/'via MAPK and AP-1.54,83 ,QDQRWKHULPSRUWDQWEUDQFKRIWKHSDWKZD\FHUDPLGHUHFHLYHVDSKRVSKRFKROLQH head group via WKH DFWLRQ RI VSKLQJRP\HOLQ V\QWKDVH IRUPLQJ VSKLQJRP\HOLQ DQG D ELRORJLFDOO\DFWLYHSRRORIGLDF\OJO\FHURO84,857KHUHDUHWZRNQRZQIRUPVRIVSKLQJRP\HOLQ synthase (SMS1 and SMS2).86 Sphingomyelin (choline phosphoceramide) comprises RIPHPEUDQHSKRVSKROLSLGVLQPDPPDOLDQFHOOV87 ,QDQRWKHUSRUWLRQRIWKHSDWKZD\FHUDPLGHPD\EHPRGL¿HGZLWKDFDUERK\GUDWHKHDG JURXSIRUPLQJJO\FRVSKLQJROLSLGVVXFKDVJOXFRV\OFHUDPLGHDQGJDODFWRV\OFHUDPLGH88 2YHUIRXUKXQGUHGFRPSOH[VXJDUFRQWDLQLQJVSHFLHVPD\EHJHQHUDWHGLQWKLVPDQQHU 7KHFHUDPLGHSRUWLRQRIWKHVHOLSLGVLVHPEHGGHGLQWKHSODVPDPHPEUDQHZKLOHWKH sugar moieties extend into the extracellular space.887KHVHOLSLGVVHUYHDVSUHFXUVRUVIRU V\QWKHVLVRIJDQJOLRVLGHVDQGFHUHEURVLGHVZKLFKDUHPRUHFRPSOH[JO\FRVSKLQJROLSLGV89-92

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SPHINGOLIPIDS AND METABOLIC DISEASE

Table 3./RFDOL]DWLRQRIPRXVHVSKLQJRP\HOLQDVHVZLWKNQRZQin vivo activity171,172 Protein

Gene

Secretory sphingomyelinase (S-Smase) Lysosomal acid sphingomyelinase (L-SMase) Neutral sphingomyelinase 2 (nSMase2) Neutral sphingomyelinase 3 (nSMase3)

SMPD1

SMPD3 SMPD4

Intracellular Localization/Secreted Secreted Lysosome, translocates to the plasma membrane under stress Plasma membrane Endoplasmic reticulum, Golgi

Ceramide-1-phosphate (C1P) and ceramide kinase (CERK), which are still being FKDUDFWHUL]HG PD\ IRUP DQ LPSRUWDQW OLQN EHWZHHQ VSKLQJROLSLGV DQG HLFRVDQRLG biosynthesis.93,94 CERK has high structural and amino acid homology to the sphingosine kinase (SPHK) enzymes; it is primarily expressed in heart, kidney, brain and hematopoetic cells.952QO\RQHLVRIRUPRI&(5.KDVEHHQLGHQWL¿HGEXWHOLPLQDWLRQRIWKLVNLQDVH GRHVQRWHOLPLQDWH&3VXJJHVWLQJWKHH[LVWHQFHRIDQDOWHUQDWLYHLVRIRUPRUV\QWKHWLF SDWKZD\IRUWKLVOLSLG&3KDVEHHQLPSOLFDWHGLQDOOHUJLFDQGLQÀDPPDWRU\VLJQDOLQJ PHPEUDQHIXVLRQDQGSURVWDJODQGLQV\QWKHVLV93,94,96,97 C1P promotes cPLA2 activation DQGSURGXFWLRQRIDUDFKLGRQLFDFLGDQGLWLQKLELWV33DQG33$LQYLWUR93,94 7KHSURGXFWVRIWKHVHELRV\QWKHWLFSDWKZD\VPD\DOVREHPRGL¿HGDQGGHJUDGHG 63PD\EHGHSKRVSKRU\ODWHGE\HLWKHUVSHFL¿F63SKRVSKDWDVHVRUPHPEHUVRIWKH OLSLGSKRVSKDWHSKRVSKRK\GURODVHIDPLO\98,99 Alternatively, the cell may shunt lipids out RIWKHVSKLQJROLSLGSDWKZD\E\GHJUDGLQJ63via S1P lyase, which cleaves the C2-C3 ERQGRI63WR\LHOGKH[DGHFDQDODQGSKRVSKRHWKDQRODPLQH100 Under stress conditions, sphingomyelin may be broken down to release ceramide and phosphocholine.101,102 ,QWULJXLQJO\FHUDPLGHSURGXFHGE\WKLVSDWKZD\PD\KDYHGLVWLQFWIXQFWLRQDOUROHVIURP WKDWSURGXFHGE\GHQRYRV\QWKHVLVDQGWKHVXEFHOOXODUORFDOL]DWLRQRIWKHVHHQ]\PHVPD\ KDYHIXQFWLRQDOLPSRUWDQFH7DEOH 61-63 Finally, glycosphingolipids may be degraded by exohydrolases such as glucosidase and galactosidase; this degradation results in the VWHSZLVHUHOHDVHRIWHUPLQDOPRQRVDFFKDULGHVIURPWKHROLJRVDFFKDULGHFKDLQ103,104 8SUHJXODWLRQ RI VSKLQJROLSLG PHWDEROLVP RFFXUV LQ PDQ\ VWUHVV UHVSRQVHV 6WLPXOL WKDW FDQ WULJJHU GH QRYR VSKLQJROLSLG V\QWKHVLV DQG FHUDPLGH IRUPDWLRQ include chemotherapeutic agents, oxidized LDL, excess substrate and heat stress.105-110 Salvage-based sphingolipid metabolism is triggered by phorbol esters, which stimulate PKC, oxidative stress and TNF-_.63,101,111-116$GGLWLRQDOO\WKHJHQHUDWLRQRIFHUDPLGHIURP sphingomyelin can be stimulated by Fas ligand.117,WKDVEHHQSURSRVHGWKDWGLIIHUHQWLDO DFWLYDWLRQRIVSHFL¿FFHUDPLGHJHQHUDWLQJSDWKZD\VLVIDFLOLWDWHGE\WKHGLVWLQFWWRSRORJ\ RIWKHLQGLYLGXDOHQ]\PHV SPHINGOLIPIDS AND LIPOTOXICITY %DVHGRQWKHZRUNRIQXPHURXVUHVHDUFKHUVVSKLQJROLSLGVKDYHEHHQVKRZQWRDFW LQWKHGHYHORSPHQWDQGSDWKRORJ\RIPHWDEROLFV\QGURPH(OHYDWHGFHUDPLGHKDVEHHQ PHFKDQLVWLFDOO\DVVRFLDWHGZLWKWKHGHYHORSPHQWRILQVXOLQUHVLVWDQFH118-120 Currently, it LVEHOLHYHGWKDWLQVFHQDULRVRISDOPLWDWHRYHUVXSSO\FHUDPLGHDFWLYDWHV33$121-123 This UHVXOWVLQWKHGHSKRVSKRU\ODWLRQRI$NW3.%VXEVHTXHQWO\UHGXFLQJJOXFRVHWUDQVSRUW

SPHINGOLIPID METABOLISM AND ANALYSIS IN METABOLIC DISEASE

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and activating glycogen synthase kinase.124,125 Alternatively, ceramide may act to stabilize WKHUHSUHVVLYHSURWHLQSURWHLQLQWHUDFWLRQRI3.&c with Akt/PKB, preventing release and DFWLYDWLRQRI$NW3.%XSRQLQVXOLQVWLPXODWLRQ126,QKLELWLRQRIGHQRYRVSKLQJROLSLG V\QWKHVLVUHVWRUHGJOXFRVHXSWDNHDQGSKRVSKRU\ODWLRQRI$NW3.%LQVNHOHWDOPXVFOHRI DQLPDOVLQIXVHGZLWKODUGRLO127$GGLWLRQDOO\WUHDWPHQWZLWKVHYHUDOLQKLELWRUVRIGHQRYR FHUDPLGHV\QWKHVLVRYHUFDPHWKHLQKLELWRU\HIIHFWRISDOPLWDWHRQ$NW3.%DQG*6.` SKRVSKRU\ODWLRQLQ&&P\RWXEXOHVZKLFKDUHDPRGHORIKXPDQVNHOHWDOPXVFOH128,129 Furthermore, heterozygous DES1 knockouts had enhanced insulin sensitivity and they developed less insulin resistance in response to treatment with synthetic glucocorticoids.127 (OHYDWHG VSKLQJROLSLG OHYHOV DOVR VHHP WR SOD\ D UROH LQ WKH GHYHORSPHQW RI characteristic pathologies seen in the heart in metabolic syndrome. For example, mice overexpressing GPI-tagged lipoprotein lipase (LpL-GPI) had elevated cardiac ceramide OHYHOVDVZHOODVFDUGLDFK\SHUWURSK\LQFUHDVHGOHIWYHQWULFXODUV\VWROLFGLDPHWHUDQG GHFUHDVHGIUDFWLRQDOVKRUWHQLQJ130)XUWKHUPRUHH[SUHVVLRQRIWKHFDUGLDFIDLOXUHPDUNHUV $1)DQG%13ZDVHOHYDWHGLQWKHVHPLFH$OORIWKHVHSDWKRORJLHVZHUHFRUUHFWHGE\ P\ULRFLQWUHDWPHQW$GGLWLRQDOO\WKHDEHUUDQWSDWWHUQVRIVXEVWUDWHXWLOL]DWLRQVHHQLQ WKHVHKHDUWVZKLFKPLUURUDVLJQL¿FDQWSDWKRORJ\RIWKHKHDUWLQPHWDEROLFV\QGURPH were corrected with myriocin treatment. Attenuating de novo sphingolipid synthesis DOVRUHGXFHGSKRVSKRU\ODWLRQRI$.7DQGLWVGRZQVWUHDPWDUJHW*6.`, which acts to inhibit cardiac hypertrophy and glycogen synthesis in the heart.12,130-132 Furthermore, HOHYDWHGOHYHOVRIFHUDPLGHKDYHEHHQVKRZQWRSURPRWHP\RFDUGLDODSRSWRVLVLQ=') rats.133,QSDUWLFXODUDFFXPXODWLRQRIFHUDPLGHLVDVVRFLDWHGZLWKLQKLELWLRQRISURVXUYLYDO signaling kinases, including Akt/PKB and PKCc DQG VWLPXODWLRQ RI SURDSRSWRWLF SURWHLQNLQDVHVVXFKDVFMXQ1WHUPLQDOSURWHLQNLQDVH-1. 134-136 These actions UHVXOWLQVXSSUHVVLRQRI%FODFWLYDWLRQRIFDVSDVHVDQGRWKHUSURDSRSWRWLFHYHQWV ,WKDVEHHQVKRZQLQDYDULHW\RIFHOOW\SHVWKDWLQFUHDVLQJFRQFHQWUDWLRQVRISDOPLWDWH LQWKHH[WUDFHOOXODUHQYLURQPHQWLQGXFHVDQLQFUHDVHLQIRUPDWLRQRIFHUDPLGHRURWKHU VSKLQJROLSLGVUHYLHZHGLQUHI54). This is attributable both to increased substrate supply and changes in gene expression. 17,130, 137,138 For example, cardiac ceramide levels were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fa/fa=')UDWVZKLFKKDYHHOHYDWHGSODVPDIUHHIDWW\DFLGOHYHOV17 Heart muscle IURP/S/RYHUH[SUHVVLQJPLFHZKLFKKDGLQFUHDVHGXSWDNHRIIDWW\DFLGVLQWRFHOOVDOVR FRQWDLQHGH[FHVVFHUDPLGHVWKLVZDVFRUUHFWHGDIWHUVL[ZHHNVRIWUHDWPHQWZLWKP\ULRFLQ ZKLFK LQKLELWV VHULQH SDOPLWR\OWUDQVIHUDVH130 This response seems to be widespread DPRQJWLVVXHVLQWKHERG\IRUH[DPSOHWUHDWPHQWRISULPDU\DVWURF\WHVZLWKSDOPLWDWH LQFUHDVHGDFWLYLW\DQGSURWHLQOHYHOVRI637140 However, palmitate supplementation may increase sphingolipids other than pro-apoptotic ceramides, potentially acting to protect the cell. For example, it has been shown that treatment with palmitate results in an increase in S1P in C2C12 myotubes.141 Further supporting this perspective, a drastic reduction in SPTLC1 H[SUHVVLRQLQ/P\RWXEHVZKLFKDUHDPRGHORIVNHOHWDOPXVFOHDFWXDOO\VHQVLWL]HG cells to palmitate-induced cell death.147KLVPD\EHPHGLDWHGE\LQFUHDVHGURXWLQJRI

8

SPHINGOLIPIDS AND METABOLIC DISEASE

IDWW\DFLGVLQWRGLDF\OJO\FHURO'$* ZKLFKKDVSRWHQWVLJQDOLQJSURSHUWLHV5HGXFLQJ H[SUHVVLRQRI637/&LQSDOPLWDWHWUHDWHGFHOOVDOVRLQFUHDVHGOHYHOVRI3.&e, which phosphorylates IRS1 at Ser1,110. +XPDQVWXGLHVKDYHJHQHUDOO\VXSSRUWHGWKH¿QGLQJVLQDQLPDODQGFHOOFXOWXUHV\VWHPV 6NHOHWDOPXVFOHIURPREHVHLQVXOLQUHVLVWDQWKXPDQVFRQWDLQLQFUHDVHGOHYHOVRIFHUDPLGH DQGH[SRVXUHRIKXPDQVNHOHWDOPXVFOHWRH[FHVVSDOPLWDWHUHVXOWVLQWKHGHYHORSPHQWRI insulin resistance and elevated ceramide levels.11,142-145$UHFHQWFDUHIXOO\FRQWUROOHGVWXG\ GHPRQVWUDWHGHOHYDWLRQRIORQJFKDLQFHUDPLGHVLQVNHOHWDOPXVFOHIURPLQVXOLQUHVLVWDQW patients.143,QSDUWLFXODUVLJQL¿FDQWLQFUHDVHVZHUHVHHQLQ&14-, C16- and C18:0-ceramides as well as S1P and total ceramide levels. 4XDQWL¿FDWLRQRIVSKLQJROLSLGOHYHOVLQKXPDQKHDUWVKDVSURGXFHGOHVVFRQFOXVLYH UHVXOWVRQGLIIHUHQFHVEHWZHHQOHDQREHVHDQGGLDEHWLFVXEMHFWV146 However, obesity, alone or in combination with Type-2 diabetes, was associated with increased expression RIWKHVHULQHSDOPLWR\OWUDQVIHUDVHVXEXQLWV637DQG637WKHVSKLQJRVLQHNLQDVH SPHK1, alkaline/neutral ceramidase, acid ceramidase and neutral sphingomyelinase LQULJKWDWULDOWLVVXHIURPSDWLHQWVUHFHLYLQJHOHFWLYHFRURQDU\E\SDVVJUDIWVXUJHU\ $GGLWLRQDOO\H[SUHVVLRQRIWKHVHHQ]\PHVZDVKLJKHULQSDSLOODU\YHQWULFXODU PXVFOH than in atrial muscle, suggesting that ceramide turnover is higher in ventricular tissue than in the atria. Intriguingly, overweight or obesity was associated with increased CPT1 and PDK4 in the human myocardium, while FAT/CD36 and LPL levels remained constant. 7KLVUHVXOWVLQLQFUHDVHGSRWHQWLDOIRUIDWW\DFLGR[LGDWLRQZLWKRXWDVLPLODUXSUHJXODWLRQ RI XSWDNH FDSDFLW\ %DVHG RQ WKHVH GDWD LW KDV EHHQ SURSRVHG WKDW XSUHJXODWLRQ RI sphingolipid metabolic enzymes may represent a stabilizing mechanism that allows VSKLQJROLSLGPHWDEROLVPWRFRPSHWHIRUWKHDYDLODEOHIDWW\DF\O&R$ It has been hypothesized that elevated triglyceride (TG) stores are an important VRXUFHRIIDWW\DFLGVIRUFHUDPLGHRYHUSURGXFWLRQLQPHWDEROLFV\QGURPHDQGDFXWHIDWW\ acid oversupply.1436WRUDJHRIH[FHVVIDWW\DFLGVLQ7*LVSURSRVHGWRLQLWLDOO\VHUYH DVDSURWHFWLYHPHFKDQLVPVHTXHVWHULQJOLSLGVDZD\IURPWKHV\QWKHVLVRIELRDFWLYH metabolites.137,147,148+RZHYHUDVFHOOXODU7*FRQWHQWSHDNVIDWW\DFLGVHVFDSHIURP IXWLOH 7* F\FOLQJ LQWR RWKHU SDWKZD\V VXFK DV '$* RU VSKLQJROLSLG SURGXFWLRQ $OWKRXJKWKLVUHODWLRQVKLSKDVQHYHUEHHQGH¿QLWLYHO\WHVWHGWKHFRUUHODWLYHGDWDDUH KLJKO\ VXJJHVWLYH )RU H[DPSOH 7* FRQWHQW LQ IDWW\ DFLGWUHDWHG SDQFUHDWLF LVOHWV IURPWKH=')UDWLVDERXWWKUHHWLPHVWKDWLQZLOGW\SHLVOHWVWUHDWHGZLWKIDWW\DFLG DQGOHSWLQWKHPDJQLWXGHRIWKLVFKDQJHLVVLPLODUWRWKHWZRWRWKUHHIROGLQFUHDVH LQWKHUDWHRIFHUDPLGHIRUPDWLRQLQWKHVHLVOHWV5,17,1493RZHUIXOO\RYHUH[SUHVVLRQRI SHULOLSLQZKLFKSUHYHQWVEUHDNGRZQRI7*DQGWKXVÀRZRIIDWW\DFLGVLQWRVSKLQJROLSLG metabolism, attenuated palmitate-induced insulin resistance.137 Additionally, elevation RI FHUDPLGHV LQ LQVXOLQUHVLVWDQW VNHOHWDO PXVFOH ZDV DFFRPSDQLHG E\ D VLJQL¿FDQW LQFUHDVH LQ LQWUDP\RFHOOXODU 7* DQG GRZQUHJXODWLRQ RI DGLSRVH WULJO\FHULGH OLSDVH DQGKRUPRQHVHQVLWLYHOLSDVHWKH¿UVWWZRHQ]\PHVRI7*EUHDNGRZQ150 These data VXJJHVWWKDWFHUDPLGHVLQFUHDVHZKHQEXONIDWW\DFLGXSWDNHRYHUZKHOPVWKHDELOLW\RI the cell to store FFA as neutral lipid. ,QWULJXLQJO\WKHFRPSRVLWLRQRIIDWW\DFLGVLQWKHEORRGRUPHGLDFDQSURIRXQGO\ LPSDFWWKHHIIHFWRIIDWW\DFLGRYHUVXSSO\RQFHOOV151 In particular, oleate appears to DWWHQXDWHWKHSDWKRORJLFDOHIIHFWVRIH[FHVVSDOPLWDWH)RUH[DPSOHFHUDPLGHVHHPV WR LQKLELW LQVXOLQ VLJQDOLQJ WKURXJK D 33$GHSHQGHQW PHFKDQLVP LQ VLWXDWLRQV RI palmitate, but not oleate, oversupply.123$GGLWLRQDOO\LQIXVLRQZLWKODUGRLOZKLFKLV KLJKLQVDWXUDWHGIDWW\DFLGVDQGLVFRPSRVHGRISDOPLWDWHDQGROHDWH

SPHINGOLIPID METABOLISM AND ANALYSIS IN METABOLIC DISEASE

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disrupted glucose uptake and Akt/PKB phosphorylation in a manner that could be FRUUHFWHG E\ LQKLELWLRQ RI VHULQH SDOPLWR\OWUDQVIHUDVH ZKLOH LQIXVLRQ ZLWK VR\ RLO ZKLFKLVKLJKLQXQVDWXUDWHGIDWW\DFLGVDQGFRQWDLQVSDOPLWLFDFLGDQGROHLF acid, did not.129,PSRUWDQWO\RYHUVXSSO\RIXQVDWXUDWHGIDWW\DFLGVUHVXOWVLQEXLOGXS RI '$* EXW QRW FHUDPLGHV152 It has also been shown that treatment with palmitate resulted in increased SK1 message levels, which resulted in elevated S1P levels and SK enzyme activity, in C2C12 myotubes, an in vitro skeletal muscle model.141 In contrast, cosupplementation with oleate attenuated the increase in SK1 mRNA.141%HFDXVHRI WKHGLIIHUHQWHIIHFWVRIH[SRVXUHWRVSHFL¿FIDWW\DFLGVUHVXOWVVKRXOGEHLQWHUSUHWHG LQWKHOLJKWRIWKHIDWW\DFLGFRPSRVLWLRQRIWKHVSHFL¿FGLHWLQIXVLRQRUFHOOFXOWXUH conditions used in any particular study. APPROACHES TO THE STUDY OF SPHINGOLIPIDS IN LIPOTOXICITY 6SKLQJROLSLGRPLFVWKHVWXG\RIWKHFRPSOHWHVSKLQJROLSLGSUR¿OHRIDFHOOÀRZV QDWXUDOO\ IURP WKH LQWHUFRQQHFWHGQHVV RI VSKLQJROLSLG SDWKZD\V153 %HFDXVH RI WKH PHWDEROLFWLHVEHWZHHQDOOSRUWLRQVRIVSKLQJROLSLGPHWDEROLVPFKDQJHVLQUHJXODWLRQ RUÀX[LQRQHSDUWRIWKHSDWKZD\FDQSURIRXQGO\FKDQJHWKHVSKLQJROLSLGRPHDVD whole.47,154-1567KLVUHPRGHOLQJPD\DFWWRDPSOLI\RUWRDWWHQXDWHWKHHIIHFWVRIDWUHDWPHQW RU PXWDWLRQ EH\RQG WKH SUR[LPDO SRLQW RI FRQWDFW ZLWK WKH VSKLQJROLSLG SDWKZD\ Furthermore, although compounds may be lumped together in broad categories, e.g., FHUDPLGHVIRUSXUSRVHVRIGLVFXVVLRQGLVWLQFWHQ]\PHVSURGXFHDQGDFWRQVSKLQJROLSLGV FRQWDLQLQJGLIIHUHQWIDWW\DF\OFKDLQOHQJWKV43,44 Emerging evidence suggests that the cell GLVWLQJXLVKHVEHWZHHQFKDLQOHQJWKVIRUVLJQDOLQJSXUSRVHV)RUH[DPSOH&18-ceramide was implicated as a key pro-apoptotic molecule in a head and neck squamous cell carcinoma model, while C16FHUDPLGHZDVIRXQGWRSURWHFWDJDLQVWFHOOGHDWK157,158 ,WKDVEHHQVXJJHVWHGWKDWGLIIHUHQWLDOUHJXODWLRQRIFHUDPLGHFKDLQOHQJWKVFRXOG EH GXH WR WKH LQÀXHQFH RI FKDLQ OHQJWK RQ ELRSK\VLFDO SURSHUWLHV RI WKH PHPEUDQH bilayer or by direct interaction with downstream players in signaling pathways. The SURSHUWLHVRIVSHFL¿FOLSLGVPD\DOVRGLFWDWHWKHLUWUDQVSRUWDQGURXWLQJ159 For example, the sphingoid bases sphingosine and dihydrosphingosine are rapidly exchanged between DGMDFHQWPHPEUDQHVXUIDFHVERUGHULQJWKHF\WRVRODQGDFURVVPHPEUDQHVWKLVLVGXH to the partially charged amino group on the head group and the single anchoring acyl FKDLQ,QFRQWUDVWFHUDPLGHZKLFKSRVVHVVHVWZRDF\OFKDLQVFDQERWKGLIIXVHODWHUDOO\ DORQJDPHPEUDQHDQGPRYHDFURVVLWVIDFHVEXWLWFDQQRWVSRQWDQHRXVO\PRYHEHWZHHQ PHPEUDQHV$GGLWLRQDOO\VSKLQJROLSLGVPD\EHVRUWHGDWWKH*ROJLIRUURXWLQJWRWKH plasma membrane, as opposed to routing to the endoplasmic reticulum, and additional OLSLGVSHFL¿FURXWLQJE\SURWHLQVDQGSK\VLFDOFKHPLFDOVHJUHJDWLRQERWKSOD\LPSRUWDQW UROHVLQFHOOXODUIXQFWLRQPHPEUDQHFRPSRVLWLRQDQGOLSLGURXWLQJUHYLHZHGLQVHH UHI159). Furthermore, ceramide N-acyl chain length has been demonstrated to dictate LWVHIIHFWVRQPHPEUDQHELRSK\VLFDOSURSHUWLHVLQFOXGLQJPLVFLELOLW\ZLWKWKHPDMRU PHPEUDQHFRQVWLWXHQWVSKLQJRP\HOLQDQGFRQVHTXHQWRUJDQL]DWLRQDQGVWDELOL]DWLRQRI membrane domains.160$VUROHVIRUVSKLQJROLSLGVRIVSHFL¿FFKDLQOHQJWKVEHFRPHEHWWHU XQGHUVWRRGUHYHDOLQJDQVZHUVPD\EHJOHDQHGIURPERWKDTXDQWLWDWLYHDVVHVVPHQW RIFKDQJHVLQOHYHOVRIWKHPDMRUVSKLQJROLSLGVSHFLHVDVZHOODVD¿QHUUHVROXWLRQRI changes in chain length distribution within species.

10

SPHINGOLIPIDS AND METABOLIC DISEASE

7KH FKRLFH RI DSSURDFK LQ GHWHUPLQLQJ VSKLQJROLSLG OHYHOV FDQ VLJQL¿FDQWO\ impact results. Although there have been numerous exciting technological advances in sphingolipidomic analysis,161,162DOVRUHYLHZHGLQUHIV153 and 163) not all labs KDYHDFFHVVWRIDFLOLWLHVWRSHUIRUPWKHVHDQDO\VHVDWWKHLUKRPHLQVWLWXWLRQV,QVXFK situations, thin-layer chromatography and the DAG kinase assay are two relatively HDV\DQGFRPPRQO\HPSOR\HGRSWLRQV+RZHYHUWKHUHOLDELOLW\RIUHVXOWVLVKLJKO\ FRQWLQJHQW RQ DSSURSULDWH H[SHULPHQWDO GHVLJQ DQG D UHDVRQDEOH XQGHUVWDQGLQJ RI WKHSLWIDOOVDVVRFLDWHGZLWKWKHFKRVHQDSSURDFKHVSHFLDOO\LQWKHFDVHRIWKH'$* kinase assay.164,165([WUDFWLRQPHWKRGVDQGVROYHQWV\VWHPVIRULVRODWLRQRISDUWLFXODU sphingolipid species by TLC have been reviewed in detail elsewhere and will not be GLVFXVVHGIXUWKHU162,166-169+RZHYHUWKH'$*NLQDVHPHWKRGPHULWVIXUWKHUFRPPHQW DVLWLVERWKUHODWLYHO\FRPPRQO\XVHGDQGFDQSURGXFHPLVOHDGLQJUHVXOWVLIFRQGXFWHG improperly.164,165 %ULHÀ\WKH'$*NLQDVHFRQYHUWV'$*WRSKRVSKDWLGLFDFLGDQGLWZDVGHPRQVWUDWHG that this reaction can be used to quantitatively measure DAG levels in crude cellular extract.164,165 Later, it was discovered that DAG kinase will phosphorylate ceramide to &3LQDPDQQHUWKDWLVVLPLODUO\DPHQDEOHWRTXDQWLWDWLRQ:KHQSHUIRUPHGFDUHIXOO\ WKH'$*NLQDVHDVVD\FDQUDSLGO\DQGVLPXOWDQHRXVO\TXDQWLI\PDVVOHYHOVRIFHUDPLGH DQG'$*ZKLFKDUHVHSDUDWHGE\7/&DQGLGHQWL¿HGE\FRPSDULVRQWROLSLGVWDQGDUGV Results produced by this method have compared well with those generated by other PHWKRGV,QFRQWUDVWIDLOXUHWRXVHDQH[FHVVRIHQ]\PHRUWRDOORZWKHUHDFWLRQWRJR WRFRPSOHWLRQZLOOVNHZGDWDREWDLQHGIURPWKLVDVVD\WKLVLVGXHWRHIIHFWVRIUHDFWLRQ NLQHWLFVDQGFRPSHWLWLRQEHWZHHQ'$*DQGFHUDPLGHDVVXEVWUDWHVIRUWKH'$*NLQDVH Furthermore, it should be appreciated that mild alkaline hydrolysis, which is included in some protocols to eliminate diglycerides and glycerolipids, will also hydrolyze 1-O-acyl ceramides, which can reduce detected ceramides by ten to twenty percent. $GGLWLRQDOO\ WKH %OLJK'\HU H[WUDFWLRQ HI¿FLHQWO\ LVRODWHV &3 GLK\GURFHUDPLGH phosphates and phosphatidic acids but not short chain C1Ps, hydroxylated ceramides RUO\VR3$)LQDOO\WKHFKRLFHRIVROYHQWV\VWHPLPSDFWVWKHVHSDUDELOLW\RIGLVWLQFW &3VE\FKDLQOHQJWK7KXVZKLOHWKH'$*NLQDVHPHWKRGFDQEHDQH[WUHPHO\XVHIXO WRROWKHUHVXOWVVKRXOGEHLQWHUSUHWHGFLUFXPVSHFWO\EDVHGRQWKHVSHFL¿FSURWRFROXVHG )LQDOO\WKHDYDLODELOLW\RINQRFNRXWPLFHVL51$DQGLQKLELWRUVKDVRSHQHGWKHGRRU WRVWXGLHVH[DPLQLQJWKHHIIHFWVRIUHGXFLQJOHYHOVRUDFWLYLW\RILQGLYLGXDOVSKLQJROLSLG PHWDEROLFHQ]\PHV+RZHYHUWKHLQWHUFRQQHFWHGQHVVDQGPHWDEROLWHVSHFL¿FHIIHFWV RIVSKLQJROLSLGPHWDEROLVPSUHVHQWDVLJQL¿FDQWFDYHDWIRUVXFKVWXGLHV:KLOHWKHVH PRGHOV FDQ EH H[FHOOHQW WRROV WKH\ VKRXOG EH XVHG MXGLFLRXVO\ DQG WKH GDWD PXVW EH LQWHUSUHWHG ZLWK FDXWLRQ 7KLV SRLQW LV GHPRQVWUDWHG E\ D UHFHQW VWXG\ RI &HU6 knockdown in a cancer cell line.47'RZQUHJXODWLRQRI&HU6SURWHLQOHYHOVDQGDFWLYLW\ only produced a minor decrease in very-long chain ceramides, which are the main SURGXFWVRIWKLVHQ]\PH$GGLWLRQDOO\&HU6NQRFNGRZQLQGXFHGDQLQFUHDVHLQOHYHOV RIORQJFKDLQFHUDPLGHVGXHWRUHYHUVHFHUDPLGDVHDFWLYLW\DVZHOODVXSUHJXODWLRQRI RWKHU GLK\GUR FHUDPLGH V\QWKDVH LVRIRUPV $V VWDWHG DERYH ORQJ FKDLQ FHUDPLGHV KDYHEHHQLPSOLFDWHGLQFHOOGHDWKVLJQDOLQJ7KXVSHUWXUELQJRQHFRPSRQHQWRIWKH sphingolipid biosynthetic pathway activated compensatory mechanisms, resulting in XQIRUHVHHQFKDQJHVLQPHWDEROLWHOHYHOVHQ]\PHDFWLYLWLHVDQGFHOOXODUSURJUDPPHV )RUWKLVUHDVRQLQYHVWLJDWLRQVRIPHWDEROLFGLVWXUEDQFHVLQVSKLQJROLSLGELRV\QWKHVLV VKRXOGWDNHDGYDQWDJHRIVSKLQJROLSLGRPLFDSSURDFKHVWRH[DPLQHWKHHIIHFWVRIWKHVH GLVWXUEDQFHVZLWKD¿QHUHVROXWLRQ

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+RUQHPDQQ7:HL<YRQ(FNDUGVWHLQ$,VWKHPDPPDOLDQVHULQHSDOPLWR\OWUDQVIHUDVHDKLJKPROHFXODUPDVV complex? Biochem J 2007; 405:157-164. +RUQHPDQQ75LFKDUG65XWWL0)HWDO&ORQLQJDQGLQLWLDOFKDUDFWHUL]DWLRQRIDQHZVXEXQLWIRUPDPPDOLDQ VHULQHSDOPLWR\OWUDQVIHUDVH-%LRO&KHP %UHVORZ'.&ROOLQV65%RGHQPLOOHU%HWDO2UPIDPLO\SURWHLQVPHGLDWHVSKLQJROLSLGKRPHRVWDVLV Nature 2010; 463:1048-1053. 26. Han S, Lone MA, Schneiter R et al. Orm1 and Orm2 are conserved endoplasmic reticulum membrane proteins regulating lipid homeostasis and protein quality control. Proc Natl Acad Sci USA 2010; 107:5851-5856. +DQ**XSWD6'*DEOH.HWDO,GHQWL¿FDWLRQRIVPDOOVXEXQLWVRIPDPPDOLDQVHULQHSDOPLWR\OWUDQVIHUDVH WKDWFRQIHUGLVWLQFWDF\O&R$VXEVWUDWHVSHFL¿FLWLHV3URF1DWO$FDG6FL86$ +DQDGD.1LVKLMLPD0)XMLWD7HWDO6SHFL¿FLW\RILQKLELWRUVRIVHULQHSDOPLWR\OWUDQVIHUDVH637 DNH\ HQ]\PHLQVSKLQJROLSLGELRV\QWKHVLVLQLQWDFWFHOOV$QRYHOHYDOXDWLRQV\VWHPXVLQJDQ637GHIHFWLYH mammalian cell mutant. Biochem Pharmacol 2000; 59:1211-1216. 0L\DNH < .R]XWVXPL < 1DNDPXUD 6 HW DO 6HULQH SDOPLWR\OWUDQVIHUDVH LV WKH SULPDU\ WDUJHW RI D sphingosine-like immunosuppressant, ISP-1/myriocin. Biochem Biophys Res Commun 1995; 211:396-403. ,NXVKLUR++D\DVKL+.DJDPL\DPD+5HDFWLRQVRIVHULQHSDOPLWR\OWUDQVIHUDVHZLWKVHULQHDQGPROHFXODU PHFKDQLVPVRIWKHDFWLRQVRIVHULQHGHULYDWLYHVDVLQKLELWRUV%LRFKHPLVWU\ :RQJ'7)XOOHU5:0ROOR\%%,QKLELWLRQRIDPLQRDFLGWUDQVDPLQDVHVE\/F\FORVHULQH$GY(Q]\PH Regul 1973; 11:139-154. :LOOLDPV5'6JRXWDV'6=DDWDUL*6HWDO,QKLELWLRQRIVHULQHSDOPLWR\OWUDQVIHUDVHDFWLYLW\LQUDEELWDRUWD by L-cycloserine. J Lipid Res 1987; 28:1478-1481. *DEOH.*XSWD6'+DQ*HWDO$GLVHDVHFDXVLQJPXWDWLRQLQWKHDFWLYHVLWHRIVHULQHSDOPLWR\OWUDQVIHUDVH causes catalytic promiscuity. J Biol Chem 2010; 285(30):22846-52. (LFKOHU)6+RUQHPDQQ70F&DPSEHOO$HWDO2YHUH[SUHVVLRQRIWKHZLOGW\SH637VXEXQLWORZHUV GHVR[\VSKLQJROLSLGOHYHOVDQGUHVFXHVWKHSKHQRW\SHRI+6$1-1HXURVFL 35. Penno A, Reilly MM, Houlden H et al. Hereditary sensory neuropathy type 1 is caused by the accumulation RIWZRQHXURWR[LFVSKLQJROLSLGV-%LRO&KHP +ROOHUDQ :0 :LOOLDPV 0/ *DR :1 HW DO 6HULQHSDOPLWR\O WUDQVIHUDVH DFWLYLW\ LQ FXOWXUHG KXPDQ keratinocytes. J Lipid Res 1990; 31:1655-61. 0HUULOO$+-U:DQJ(0XOOLQV5(.LQHWLFVRIORQJFKDLQVSKLQJRLG EDVHELRV\QWKHVLVLQLQWDFW/0 FHOOVHIIHFWVRIYDU\LQJWKHH[WUDFHOOXODUFRQFHQWUDWLRQVRIVHULQHDQGIDWW\DFLGSUHFXUVRUVRIWKLVSDWKZD\ Biochemistry 1988; 27:340-345. :LOOLDPV5'6JRXWDV'6=DDWDUL*6(Q]\PRORJ\RIORQJFKDLQEDVHV\QWKHVLVE\DRUWDLQGXFWLRQRI VHULQHSDOPLWR\OWUDQVIHUDVHDFWLYLW\LQUDEELWDRUWDGXULQJDWKHURJHQHVLV-/LSLG5HV 0HUULOO $+ -U 1L[RQ ': :LOOLDPV 5' $FWLYLWLHV RI VHULQH SDOPLWR\OWUDQVIHUDVH NHWRVSKLQJDQLQH V\QWKDVH LQPLFURVRPHVIURPGLIIHUHQWUDWWLVVXHV-/LSLG5HV 0HUULOO $+ -U &KDUDFWHUL]DWLRQ RI VHULQH SDOPLWR\OWUDQVIHUDVH DFWLYLW\ LQ &KLQHVH KDPVWHU RYDU\ FHOOV Biochim Biophys Acta 1983; 754:284-291. 41. Kihara A, Igarashi Y. FVT-1 is a mammalian 3-ketodihydrosphingosine reductase with an active site that IDFHVWKHF\WRVROLFVLGHRIWKHHQGRSODVPLFUHWLFXOXPPHPEUDQH-%LRO&KHP 42. Pewzner-Jung Y, Ben-Dor S, Futerman AH. When do Lasses (longevity assurance genes) become &HU6 FHUDPLGH V\QWKDVHV " ,QVLJKWV LQWR WKH UHJXODWLRQ RI FHUDPLGH V\QWKHVLV - %LRO &KHP  281:25001-25005. /DYLDG(/$OEHH/3DQNRYD.KROP\DQVN\,HWDO&KDUDFWHUL]DWLRQRIFHUDPLGHV\QWKDVHWLVVXHGLVWULEXWLRQ VXEVWUDWHVSHFL¿FLW\DQGLQKLELWLRQE\VSKLQJRVLQHSKRVSKDWH-%LRO&KHP 0L]XWDQL<.LKDUD$,JDUDVKL<0DPPDOLDQ/DVVDQGLWVUHODWHGIDPLO\PHPEHUVUHJXODWHV\QWKHVLVRI VSHFL¿FFHUDPLGHV%LRFKHP- 0L]XWDQL<.LKDUD$,JDUDVKL</$66ORQJHYLW\DVVXUDQFHKRPRORJXH LVDPDLQO\WHVWLVVSHFL¿F GLK\GUR FHUDPLGHV\QWKDVHZLWKUHODWLYHO\EURDGVXEVWUDWHVSHFL¿FLW\%LRFKHP- /DKLUL6)XWHUPDQ$+/$66LVDERQD¿GHGLK\GURFHUDPLGHV\QWKDVHWKDWVHOHFWLYHO\XWLOL]HVSDOPLWR\O&R$ as acyl donor. J Biol Chem 2005; 280:33735-33738. 6SDVVLHYD6'0XOOHQ7'7RZQVHQG'0HWDO'LVUXSWLRQRIFHUDPLGHV\QWKHVLVE\&HU6GRZQUHJXODWLRQ OHDGVWRDXWRSKDJ\DQGWKHXQIROGHGSURWHLQUHVSRQVH%LRFKHP- 6FKLIIPDQQ6=LHEHOO66DQGQHU-HWDO$FWLYDWLRQRIFHUDPLGHV\QWKDVHE\FHOHFR[LEOHDGVWRDVHOHFWLYH LQGXFWLRQRI& FHUDPLGH%LRFKHP3KDUPDFRO 0LFKHO&YDQ(FKWHQ'HFNHUW*5RWKHU-HWDO&KDUDFWHUL]DWLRQRIFHUDPLGHV\QWKHVLV$GLK\GURFHUDPLGH GHVDWXUDVHLQWURGXFHVWKHWUDQVGRXEOHERQGRIVSKLQJRVLQHDWWKHOHYHORIGLK\GURFHUDPLGH-%LRO Chem 1997; 272:22432-22437. 9HQDEOH 0( /HH -< 6P\WK 0- HW DO 5ROH RI FHUDPLGH LQ FHOOXODU VHQHVFHQFH - %LRO &KHP  270:30701-30708.

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-D\DGHY6/LX%%LHODZVND$(HWDO5ROHIRUFHUDPLGHLQFHOOF\FOHDUUHVW-%LRO&KHP 52. Obeid LM, Linardic CM, Karolak LA et al. Programmed cell death induced by ceramide. Science 1993; 259:1769-1771. 5LERQL/3ULQHWWL$%DVVL5HWDO$PHGLDWRUUROHRIFHUDPLGHLQWKHUHJXODWLRQRIQHXUREODVWRPD1HXURD FHOOGLIIHUHQWLDWLRQ-%LRO&KHP &RZDUW/$6SKLQJROLSLGVSOD\HUVLQWKHSDWKRORJ\RIPHWDEROLFGLVHDVH7UHQGV(QGRFULQRO0HWDE 20:34-42. 55. Nica AF, Tsao CC, Watt JC et al. Ceramide promotes apoptosis in chronic myelogenous leukemia-derived K562 cells by a mechanism involving caspase-8 and JNK. Cell Cycle 2008; 7:3362-3370. 3DUN0$=KDQJ*1RUULV-HWDO5HJXODWLRQRIDXWRSKDJ\E\FHUDPLGH&'3(5.VLJQDOLQJ$XWRSKDJ\ 2008; 4:929-931. 57. Ferrari D, Pinton P, Campanella M et al. Functional and structural alterations in the endoplasmic reticulum and mitochondria during apoptosis triggered by C2-ceramide and CD95/APO-1/FAS receptor stimulation. Biochem Biophys Res Commun 2010; 391:575-581. /HL;=KDQJ6%RKUHU$HWDO&DOFLXPLQGHSHQGHQWSKRVSKROLSDVH$L3/$EHWD PHGLDWHGFHUDPLGH generation plays a key role in the cross-talk between the endoplasmic reticulum (ER) and mitochondria during ER stress-induced insulin-secreting cell apoptosis. J Biol Chem 2008; 283:34819-34832. 1RYJRURGRY6$6]XOF=0/XEHUWR&HWDO3RVLWLYHO\FKDUJHGFHUDPLGHLVDSRWHQWLQGXFHURIPLWRFKRQGULDO permeabilization. J Biol Chem 2005; 280:16096-16105. 60. Lin CF, Chen CL, Chang WT et al. Bcl-2 rescues ceramide- and etoposide-induced mitochondrial apoptosis WKURXJKEORFNDJHRIFDVSDVHDFWLYDWLRQ-%LRO&KHP 1LHWR )/ 3HVFLR /* )DYDOH 12 HW DO 6SKLQJROLSLG PHWDEROLVP LV D FUXFLDO GHWHUPLQDQW RI FHOOXODU IDWH LQ QRQVWLPXODWHG SUROLIHUDWLQJ 0DGLQ'DUE\ FDQLQH NLGQH\ 0'&.  FHOOV - %LRO &KHP  283:25682-25691. 62. Kitatani K, Idkowiak-Baldys J, Hannun YA. The sphingolipid salvage pathway in ceramide metabolism and signaling. Cell Signal 2008; 20:1010-1018. %HFNHU.3.LWDWDQL.,GNRZLDN%DOG\V-HWDO6HOHFWLYHLQKLELWLRQRIMX[WDQXFOHDUWUDQVORFDWLRQRISURWHLQ NLQDVH&EHWD,,E\DQHJDWLYHIHHGEDFNPHFKDQLVPLQYROYLQJFHUDPLGHIRUPHGIURPWKHVDOYDJHSDWKZD\ J Biol Chem 2005; 280:2606-2612. :LWWHQEHUJ -% 7KH VHSDUDWLRQ RI VSKLQJRVLQH DQG UHODWHG FRPSRXQGV E\ UHYHUVHG SKDVH SDUWLWLRQ chromatography. J Biol Chem 1955; 216:379-390. %UDG\ 52 )RUPLFD -9 .RYDO *- 7KH HQ]\PDWLF V\QWKHVLV RI VSKLQJRVLQH ,, )XUWKHU VWXGLHV RQ WKH PHFKDQLVPRIWKHUHDFWLRQ-%LRO&KHP %UDG\52.RYDO*-7KHHQ]\PDWLFV\QWKHVLVRIVSKLQJRVLQH-%LRO&KHP 0DR&2EHLG/0&HUDPLGDVHVUHJXODWRUVRIFHOOXODUUHVSRQVHVPHGLDWHGE\FHUDPLGHVSKLQJRVLQHDQG sphingosine-1-phosphate. Biochim Biophys Acta 2008; 1781:424-434. *LOODUG%.&OHPHQW5*0DUFXV'09DULDWLRQVDPRQJFHOOOLQHVLQWKHV\QWKHVLVRIVSKLQJROLSLGVLQGH novo and recycling pathways. Glycobiology 1998; 8:885-890. 6WRIIHO :+ %UXQR +HLPDQQ *HUKDUG +RSSH6H\OHU¶V =HLWVFKULIW IXU SK\VLRORJLVFKH &KHPLH  354:1311-1316. .HQQHG\6.DQH.$3\QH1-HWDO7DUJHWLQJVSKLQJRVLQHSKRVSKDWHVLJQDOOLQJIRUFDUGLRSURWHFWLRQ Curr Opin Pharmacol 2009; 9:194-201. 0DFH\ND00LOVWLHQ66SLHJHO66SKLQJRVLQHSKRVSKDWHWKH6ZLVVDUP\NQLIHRIVSKLQJROLSLGVLJQDOLQJ J Lipid Res 2009; 50 Suppl:S272-S276. +DLW 1& $OOHJRRG - 0DFH\ND 0 HW DO 5HJXODWLRQ RI KLVWRQH DFHW\ODWLRQ LQ WKH QXFOHXV E\ sphingosine-1-phosphate. Science 2009; 325:1254-1257. 73. Rosen H, Gonzalez-Cabrera PJ, Sanna MG et al. Sphingosine 1-phosphate receptor signaling. Annu Rev Biochem 2009; 78:743-768. 74. Means CK, Brown JH. Sphingosine-1-phosphate receptor signalling in the heart. Cardiovasc Res 2009; 82:193-200. .RKDPD72OLYHUD$(GVDOO/HWDO0ROHFXODUFORQLQJDQGIXQFWLRQDOFKDUDFWHUL]DWLRQRIPXULQHVSKLQJRVLQH kinase. J Biol Chem 1998; 273:23722-23728. /LX+6XJLXUD01DYD9(HWDO0ROHFXODUFORQLQJDQGIXQFWLRQDOFKDUDFWHUL]DWLRQRIDQRYHOPDPPDOLDQ VSKLQJRVLQHNLQDVHW\SHLVRIRUP-%LRO&KHP /LX+7RPDQ5(*RSDUDMX6.HWDO6SKLQJRVLQHNLQDVHW\SHLVDSXWDWLYH%+RQO\SURWHLQWKDWLQGXFHV apoptosis. J Biol Chem 2003; 278:40330-40336. 78. Maceyka M, Sankala H, Hait NC et al. SphK1 and SphK2, sphingosine kinase isoenzymes with opposing IXQFWLRQVLQVSKLQJROLSLGPHWDEROLVP-%LRO&KHP +RIPDQQ/35HQ66FKZDOP6HWDO6SKLQJRVLQHNLQDVHDQGUHJXODWHWKHFDSDFLW\RIPHVDQJLDOFHOOV to resist apoptotic stimuli in an opposing manner. Biol Chem 2008; 389:1399-1407.

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6ULYDVWDYD6&KDQ&$SSOLFDWLRQRIPHWDEROLFÀX[DQDO\VLVWRLGHQWLI\WKHPHFKDQLVPVRIIUHHIDWW\DFLG toxicity to human hepatoma cell line. Biotechnol Bioeng 2008; 99:399-410. -DIIUH]RX-3/HYDGH7%HWWDLHE$HWDO'DXQRUXELFLQLQGXFHGDSRSWRVLVWULJJHULQJRIFHUDPLGHJHQHUDWLRQ through sphingomyelin hydrolysis. EMBO J 1996; 15:2417-2424. &DVWLOOR66/HY\07KDLNRRWWDWKLO-9HWDO5HDFWLYHQLWURJHQDQGR[\JHQVSHFLHVDFWLYDWHGLIIHUHQW sphingomyelinases to induce apoptosis in airway epithelial cells. Exp Cell Res 2007; 313:2680-2686. =DJHU5$&RQUDG'6%XUNKDUW.&HUDPLGHDFFXPXODWLRQGXULQJR[LGDQWUHQDOWXEXODULQMXU\PHFKDQLVPV and potential consequences. J Am Soc Nephrol 1998; 9:1670-1680. +XZLOHU$3IHLOVFKLIWHU-YDQGHQ%RVFK+-%LRO&KHP &DFLFHGR-0%HQMDFKDUHRZRQJ6&KRX(HWDO3DOPLWDWHLQGXFHGDSRSWRVLVLQFXOWXUHGERYLQHUHWLQDO SHULF\WHVUROHVRI1$'3 +R[LGDVHR[LGDQWVWUHVVDQGFHUDPLGH'LDEHWHV 2VDZD<8FKLQDPL+%LHODZVNL-HWDO5ROHVIRU&FHUDPLGHDQGVSKLQJRVLQHSKRVSKDWHLQUHJXODWLQJ KHSDWRF\WHDSRSWRVLVLQUHVSRQVHWRWXPRUQHFURVLVIDFWRUDOSKD-%LRO&KHP 117. Pru JK, Hendry IR, Davis JS et al. Soluble Fas ligand activates the sphingomyelin pathway and induces DSRSWRVLV LQ OXWHDO VWHURLGRJHQLF FHOOV LQGHSHQGHQWO\ RI VWUHVVDFWLYDWHG S0$3.  (QGRFULQRORJ\ 2002; 143:4350-4357. /RQJ6'3HNDOD3+/LSLGPHGLDWRUVRILQVXOLQUHVLVWDQFHFHUDPLGHVLJQDOOLQJGRZQUHJXODWHV*/87 gene transcription in 3T3-L1 adipocytes. Biochem J 1996; 319(Pt 1):179-184. 119. Brindley DN, Wang CN, Mei J et al. Lipids 1999; 34 Suppl:S85-S88. 6FKPLW]3HLIIHU&&UDLJ'/%LGHQ7-&HUDPLGHJHQHUDWLRQLVVXI¿FLHQWWRDFFRXQWIRUWKHLQKLELWLRQ RIWKHLQVXOLQVWLPXODWHG3.%SDWKZD\LQ&&VNHOHWDOPXVFOHFHOOVSUHWUHDWHGZLWKSDOPLWDWH-%LRO Chem 1999; 274:24202-24210. 7HUXHO7+HUQDQGH]5/RUHQ]R0&HUDPLGHPHGLDWHVLQVXOLQUHVLVWDQFHE\WXPRUQHFURVLVIDFWRUDOSKDLQ brown adipocytes by maintaining Akt in an inactive dephosphorylated state. Diabetes 2001; 50:2563-2571. 122. Dobrowsky RT, Kamibayashi C, Mumby MC et al. Ceramide activates heterotrimeric protein phosphatase 2A. J Biol Chem 1993; 268:15523-15530. :X<6RQJ3;X-HWDO$FWLYDWLRQRISURWHLQSKRVSKDWDVH$E\SDOPLWDWHLQKLELWV$03DFWLYDWHG protein kinase. J Biol Chem 2007; 282:9777-9788. &D]]ROOL5&DUSHQWHU/%LGHQ7-HWDO$UROHIRUSURWHLQSKRVSKDWDVH$OLNHDFWLYLW\EXWQRWDW\SLFDO SURWHLQ NLQDVH &]HWD LQ WKH LQKLELWLRQ RI SURWHLQ NLQDVH %$NW DQG JO\FRJHQ V\QWKHVLV E\ SDOPLWDWH Diabetes 2001; 50:2210-2218. 6WUDWIRUG6+RHKQ.//LX)HWDO5HJXODWLRQRILQVXOLQDFWLRQE\FHUDPLGHGXDOPHFKDQLVPVOLQNLQJ FHUDPLGHDFFXPXODWLRQWRWKHLQKLELWLRQRI$NWSURWHLQNLQDVH%-%LRO&KHP 126. Fox TE, Houck KL, O’Neill SM et al. Ceramide recruits and activates protein kinase C zeta (PKC zeta) within structured membrane microdomains. J Biol Chem 2007; 282:12450-12457. +ROODQG:/%UR]LQLFN-7:DQJ/3HWDO,QKLELWLRQRIFHUDPLGHV\QWKHVLVDPHOLRUDWHVJOXFRFRUWLFRLG VDWXUDWHGIDWDQGREHVLW\LQGXFHGLQVXOLQUHVLVWDQFH&HOO0HWDE &KDYH]-$ 6XPPHUV 6$ &KDUDFWHUL]LQJ WKH HIIHFWV RI VDWXUDWHGIDWW\DFLGV RQ LQVXOLQ VLJQDOLQJDQG ceramide and diacylglycerol accumulation in 3T3-L1 adipocytes and C2C12 myotubes. Arch Biochem Biophys 2003; 419:101-109. &KDYH]-$.QRWWV7$:DQJ/3HWDO$UROHIRUFHUDPLGHEXWQRWGLDF\OJO\FHUROLQWKHDQWDJRQLVPRI LQVXOLQVLJQDOWUDQVGXFWLRQE\VDWXUDWHGIDWW\DFLGV-%LRO&KHP 130. Park TS, Hu Y, Noh HL et al. Ceramide is a cardiotoxin in lipotoxic cardiomyopathy. J Lipid Res 2008; 49:2101-2112. 131. Soltys CL, Buchholz L, Gandhi M et al. Am J Physiol Heart Circ Physiol 2002; 283:H1056-H1064. /LQ&)&KHQ&/&KLDQJ&:HWDO*6.EHWDDFWVGRZQVWUHDPRI33$DQGWKH3,NLQDVH$NWSDWKZD\ DQGXSVWUHDPRIFDVSDVHLQFHUDPLGHLQGXFHGPLWRFKRQGULDODSRSWRVLV-&HOO6FL =KRX<7*UD\EXUQ3.DULP$HWDO/LSRWR[LFKHDUWGLVHDVHLQREHVHUDWVLPSOLFDWLRQVIRUKXPDQREHVLW\ Proc Natl Acad Sci USA 2000; 97:1784-1789. +HUQDQGH] 20 'LVFKHU '- %LVKRSULF 1+ HW DO 5DSLG DFWLYDWLRQ RI QHXWUDO VSKLQJRP\HOLQDVH E\ K\SR[LDUHR[\JHQDWLRQRIFDUGLDFP\RF\WHV&LUF5HV +UHQLXN'*DUD\0*DDUGH:HWDO,QKLELWLRQRIF-XQ1WHUPLQDONLQDVHEXWQRWF-XQ1WHUPLQDO kinase 2, suppresses apoptosis induced by ischemia/reoxygenation in rat cardiac myocytes. Mol Pharmacol 2001; 59:867-874. 136. Ruvolo PP. Ceramide regulates cellular homeostasis via diverse stress signaling pathways. Leukemia 2001; 15:1153-1160. 3LFNHUVJLOO//LWKHUODQG*-*UHHQEHUJ$6HWDO.H\UROHIRUFHUDPLGHVLQPHGLDWLQJLQVXOLQUHVLVWDQFH in human muscle cells. J Biol Chem 2007; 282:12583-12589. 138. Todd MK, Watt MJ, Le J et al. Thiazolidinediones enhance skeletal muscle triacylglycerol synthesis ZKLOHSURWHFWLQJDJDLQVWIDWW\DFLGLQGXFHGLQÀDPPDWLRQDQGLQVXOLQUHVLVWDQFH$P-3K\VLRO(QGRFULQRO Metab 2007; 292:E485-E493.

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2NHUH,&&KDQGOHU030F(OIUHVK7$HWDO$P-3K\VLRO+HDUW&LUF3K\VLRO++ 140. Blazquez C, Geelen MJ, Velasco G et al. The AMP-activated protein kinase prevents ceramide synthesis de novo and apoptosis in astrocytes. FEBS Lett 2001; 489:149-153. 141. Hu W, Bielawski J, Samad F et al. Palmitate increases sphingosine-1-phosphate in C2C12 myotubes via XSUHJXODWLRQRIVSKLQJRVLQHNLQDVHPHVVDJHDQGDFWLYLW\-/LSLG5HV 7KUXVK$%%ULQGOH\'1&KDERZVNL$HWDO6NHOHWDOPXVFOHOLSRJHQLFSURWHLQH[SUHVVLRQLVQRWGLIIHUHQW EHWZHHQOHDQDQGREHVHLQGLYLGXDOVDSRWHQWLDOIDFWRULQFHUDPLGHDFFXPXODWLRQ-&OLQ(QGRFULQRO0HWDE 2009; 94:5053-5061. 143. Coen PM, Dube JJ, Amati F et al. Insulin resistance is associated with higher intramyocellular triglycerides in type I but not type II myocytes concomitant with higher ceramide content. Diabetes 2010; 59:80-88. 0RUR&*DOJDQL-(/XX/HWDO,QÀXHQFHRIJHQGHUREHVLW\DQGPXVFOHOLSDVHDFWLYLW\RQLQWUDP\RFHOOXODU lipids in sedentary individuals. J Clin Endocrinol Metab 2009; 94:3440-3447. 145. Straczkowski M, Kowalska I, Baranowski M et al. Increased skeletal muscle ceramide level in men at ULVNRIGHYHORSLQJW\SHGLDEHWHV'LDEHWRORJLD %DUDQRZVNL0%ODFKQLR=DELHOVND$+LUQOH7HWDO0\RFDUGLXPRIW\SHGLDEHWLFDQGREHVHSDWLHQWV LVFKDUDFWHUL]HGE\DOWHUDWLRQVLQVSKLQJROLSLGPHWDEROLFHQ]\PHVEXWQRWE\DFFXPXODWLRQRIFHUDPLGH J Lipid Res 2010; 51:74-80. /LX/6KL;%KDUDGZDM.*HWDO'*$7H[SUHVVLRQLQFUHDVHVKHDUWWULJO\FHULGHFRQWHQWEXWDPHOLRUDWHV lipotoxicity. J Biol Chem 2009; 284:36312-36323. 148. Gubern A, Barcelo-Torns M, Casas J et al. Lipid droplet biogenesis induced by stress involves triacylglycerol synthesis that depends on group VIA phospholipase A2. J Biol Chem 2009; 284:5697-5708. 6KLPDEXNXUR 0 :DQJ 0< =KRX <7 HW DO 3URWHFWLRQ DJDLQVW OLSRDSRSWRVLV RI EHWD FHOOV WKURXJK OHSWLQGHSHQGHQWPDLQWHQDQFHRI%FOH[SUHVVLRQ3URF1DWO$FDG6FL86$ :DWW0-YDQ'HQGHUHQ%-&DVWHOOL/$HWDO$GLSRVHWULJO\FHULGHOLSDVHUHJXODWLRQRIVNHOHWDOPXVFOH lipid metabolism and insulin responsiveness. Mol Endocrinol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accharomyces cerevisiae. J Biol Chem 2009; 284:7588-7596. 1HPRWR61DNDPXUD02VDZD<HWDO6SKLQJRVLQHNLQDVHLVRIRUPVUHJXODWHR[DOLSODWLQVHQVLWLYLW\ RI KXPDQ FRORQ FDQFHU FHOOV WKURXJK FHUDPLGH DFFXPXODWLRQ DQG $NW DFWLYDWLRQ - %LRO &KHP  284:10422-10432. /L=+DLOHPDULDP7.=KRX+HWDO,QKLELWLRQRIVSKLQJRP\HOLQV\QWKDVH606 DIIHFWVLQWUDFHOOXODU sphingomyelin accumulation and plasma membrane lipid organization. Biochim Biophys Acta 2007; 1771:1186-1194. 6HQNDO &( 3RQQXVDP\ 6 %LHODZVNL - HW DO $QWLDSRSWRWLF UROHV RI FHUDPLGHV\QWKDVHJHQHUDWHG &FHUDPLGHYLDVHOHFWLYHUHJXODWLRQRIWKH$7)&+23DUPRI(5VWUHVVUHVSRQVHSDWKZD\V)$6(% J 2010; 24:296-308. 6HQNDO&(3RQQXVDP\65RVVL0-HWDO5ROHRIKXPDQORQJHYLW\DVVXUDQFHJHQHDQG&FHUDPLGH in chemotherapy-induced cell death in human head and neck squamous cell carcinomas. Mol Cancer Ther 2007; 6:712-722. YDQ0HHU*+RHW]O66SKLQJROLSLGWRSRORJ\DQGWKHG\QDPLFRUJDQL]DWLRQDQGIXQFWLRQRIPHPEUDQH proteins. FEBS Lett 2010; 584:1800-1805. :HVWHUOXQG%*UDQGHOO30,VDNVVRQ<-HWDO&HUDPLGHDF\OFKDLQOHQJWKPDUNHGO\LQÀXHQFHVPLVFLELOLW\ with palmitoyl sphingomyelin in bilayer membranes. Eur Biophys J 2010; 39:1117-1128. %LHODZVNL-3LHUFH-66QLGHU-HWDO&RPSUHKHQVLYHTXDQWLWDWLYHDQDO\VLVRIELRDFWLYHVSKLQJROLSLGVE\ KLJKSHUIRUPDQFHOLTXLGFKURPDWRJUDSK\WDQGHPPDVVVSHFWURPHWU\0HWKRGV0RO%LRO 162. Levery SB. Glycosphingolipid structural analysis and glycosphingolipidomics. Methods Enzymol 2005; 405:300-369. 0HUULOO$+-U6WRNHV7+0RPLQ$HWDO6SKLQJROLSLGRPLFVDYDOXDEOHWRROIRUXQGHUVWDQGLQJWKHUROHV RIVSKLQJROLSLGVLQELRORJ\DQGGLVHDVH-/LSLG5HV6XSSO66 %LHODZVND$3HUU\'.+DQQXQ<$'HWHUPLQDWLRQRIFHUDPLGHVDQGGLJO\FHULGHVE\WKHGLJO\FHULGH kinase assay. Anal Biochem 2001; 298:141-150. 9DQ9HOGKRYHQ33%LVKRS:5<XULYLFK'$HWDO&HUDPLGHTXDQWLWDWLRQHYDOXDWLRQRIDPL[HGPLFHOODU assay using E. coli diacylglycerol kinase. Biochem Mol Biol Int 1995; 36:21-30.

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CHAPTER 2 SPHINGOLIPIDS AND CARDIOVASCULAR DISEASES: Lipoprotein Metabolism, Atherosclerosis and Cardiomyopathy Xian-Cheng Jiang,*,1 Ira J. Goldberg2 and Tae-Sik Park3 Department of Cell Biology, Downstate Medical Center, State University of New York, Brooklyn, New York, New York, USA; 2Department of Medicine, Division of Preventive Medicine and Nutrition, Columbia University, New York, New York, USA; 3Lee Gil Ya Cancer and Diabetes Institute, Gachon University of Medicine and Science, Inchon, South Korea *Corresponding Author: Xian-Cheng Jiang—Email: [email protected] 1

Abstract

+HDUW GLVHDVH LV ZLGHO\ EHOLHYHG WR GHYHORS IURP WZR SDWKRORJLFDO SURFHVVHV Circulating lipoproteins containing the nondegradable lipid, cholesterol, accumulate within the arterial wall and perhaps are oxidized to more toxic lipids. Both lipid accumulation and vascular reaction to the lipids lead to the gradual thickening RI WKH YDVFXODU ZDOO $ VHFRQG PDMRU SURFHVV WKDW LQ VRPH FLUFXPVWDQFHV LV D SULPDU\HYHQWLVWKHGHYHORSPHQWRIDORFDOLQÀDPPDWRU\UHDFWLRQ7KLVPLJKWEH DUHDFWLRQWRYHVVHOZDOOLQMXU\WKDWDFFRPSDQLHVLQIHFWLRQVLPPXQHGLVHDVHDQG SHUKDSVGLDEHWHVDQGUHQDOIDLOXUH,QWKLVFKDSWHUZHZLOOIRFXVRQWKHUHODWLRQVKLS EHWZHHQGHQRYRV\QWKHVLVRIVSKLQJROLSLGVDQGOLSLGPHWDEROLVPDWKHURVFOHURVLV and cardiomyopathy.

INTRODUCTION &RURQDU\DUWHU\GLVHDVHLVZLGHO\EHOLHYHGWRGHYHORSIURPWZRSDWKRORJLFDOSURFHVVHV Circulating lipoproteins containing the nondegradable lipid, cholesterol, accumulate within the arterial wall and perhaps are oxidized to more toxic lipids. Both lipid accumulation DQGYDVFXODUUHDFWLRQWRWKHOLSLGVOHDGWRWKHJUDGXDOWKLFNHQLQJRIWKHYDVFXODUZDOO$ VHFRQGPDMRUSURFHVVWKDWLQVRPHFLUFXPVWDQFHVLVDSULPDU\HYHQWLVWKHGHYHORSPHQW

Sphingolipids and Metabolic Disease, edited by L. Ashley Cowart. ©2011 Landes Bioscience and Springer Science+Business Media, LLC 19

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SPHINGOLIPIDS AND METABOLIC DISEASE

RI D ORFDO LQÀDPPDWRU\ UHDFWLRQ 7KLV PLJKW EH D UHDFWLRQ WR YHVVHO ZDOO LQMXU\ WKDW DFFRPSDQLHVLQIHFWLRQVLPPXQHGLVHDVHDQGSHUKDSVGLDEHWHVDQGUHQDOIDLOXUH /LSLGDFFXPXODWLRQLVDVVRFLDWHGZLWKDQXPEHURIRWKHUGLVHDVHV7KHVHLQFOXGH nonalcoholic liver disease, Type 2 diabetes due to reduced insulin secretion1 and skeletal muscle insulin resistance2DQGVRPHIRUPVRIFDUGLRP\RSDWK\3 Several mechanisms have EHHQSURSRVHGWRH[SODLQKRZOLSLGDFFXPXODWLRQOHDGVWRRUJDQG\VIXQFWLRQ GLUHFW WR[LFHIIHFWVRIQHXWUDOGURSOHWVRUIDWW\DFLGV)$V RQP\R¿EULOODUIXQFWLRQ4 (2) reactive R[\JHQVSHFLHV526 JHQHUDWHGDVDWR[LFE\SURGXFWRIOLSLGR[LGDWLRQ4 (3) FA-induced apoptosis3   GLDF\OJO\FHURO LQGXFHG DFWLYDWLRQ RI VLJQDOLQJ SDWKZD\V VXFK DV WKRVH mediated by protein kinase Cs.5 When the balance between oxidation and FA uptake is DOWHUHGH[FHVV)$PXVWHQWHUSDWKZD\VIRUQRQR[LGDWLYHPHWDEROLVP7KLVDQRPDORXV OLSLGPHWDEROLVPLVWKRXJKWWROHDGWRG\VIXQFWLRQRIQRQDGLSRVHWLVVXHV2QHDOWHUQDWLYH route to utilize the FA surplus is via the sphingolipid biosynthetic pathway. '\VUHJXODWLRQRIWKHVSKLQJROLSLGELRV\QWKHWLFSDWKZD\VDVVRFLDWHGZLWKH[FHVV)$ uptake and accumulation may be a central metabolic derangement in lipotoxicity and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¿FLHQF\RIVSKLQJRP\HOLQ V\QWKDVH606 WKHODVWHQ]\PHIRU60ELRV\QWKHVLVGHFUHDVHVSODVPDPHPEUDQH SM levels and decreases atherosclerosis in a mouse model.9&DUGLDFG\VIXQFWLRQFDXVHG by lipid accumulation in heart10DQGLQVXOLQUHVLVWDQFHDVVRFLDWHGZLWKGLHWDU\RULQIXVHG saturated FAs11LVDPHOLRUDWHGE\LQKLELWLRQRIFHUDPLGHELRV\QWKHVLV7KHVHREVHUYDWLRQV HPSKDVL]HWKHQHHGIRUDEHWWHUXQGHUVWDQGLQJRIVSKLQJROLSLGPHWDEROLVPLQFDUGLRYDVFXODU GLVHDVHV,QWKLVFKDSWHUZHZLOOIRFXVRQWKHUHODWLRQVKLSEHWZHHQGHQRYRV\QWKHVLVRI sphingolipids and lipid metabolism, atherosclerosis and cardiomyopathy. SPHINGOLIPID BIOSYNTHESIS 7KH ELRFKHPLFDO V\QWKHVLV RI 60 RFFXUV WKURXJK D VHULHV RI UHDFWLRQV LQYROYLQJ WKHHQ]\PHVVHULQHSDOPLWR\OWUDQVIHUDVH637 NHWRVSKLQJDQLQHUHGXFWDVHFHUDPLGH synthase, dihydroceramide desaturase and sphingomyelin synthase (Fig. 1). Located in the endoplasmic reticulum (ER) membrane, SPT is the rate-limiting enzyme in the pathway.12 Mammalian SPT contains two subunits, Sptlc1 and Sptlc2, encoding 53- and 63-kDa proteins, respectively.13,14 The subunits are homologous (about 20% identity)13,14 DQG WKLV LV SUREDEO\ UHOHYDQW WR WKHLU IRUPDWLRQ RI D KHWHURGLPHU Both Sptlc1 and Sptlc2 have a single, highly hydrophobic N-terminal transmembrane domain.13,14 Neither appears to be glycosylated.13 Indirect immunocytochemical analysis ZLWK HSLWRSHWDJJHG 6SWOF LQGLFDWHV WKDW WKH 1 DQG &WHUPLQL RI ERWK VXEXQLWV DUH oriented to the lumen and cytosol, respectively.15 A third possible subunit, Sptlc316 has KRPRORJ\ZLWK6SWOFDQGKRPRORJ\ZLWK6SWOF2YHUH[SUHVVLRQRI6SWOF LQ+HNFHOOVOHGWRDIROGLQFUHDVHLQFHOOXODU637DFWLYLW\

SPHINGOLIPIDS AND CARDIOVASCULAR DISEASES

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Figure 1.6SKLQJROLSLGELRV\QWKHVLVLQPDPPDOV637VHULQHSDOPLWR\OWUDQVIHUDVH606VSKLQJRP\HOLQ synthase.

6LOHQFLQJRI6SWOFH[SUHVVLRQLQ+HS*FHOOVUHVXOWHGLQDVLJQL¿FDQWUHGXFWLRQRI cellular SPT activity, but not a complete loss.16 The authors speculated that Sptlc3 was DQLVRIRUPRI6SWOFHDFKELQGLQJ6SWOFLQGHSHQGHQWO\DQGWKDWYDU\LQJWKHDPRXQWV RI6SWOFDQGPLJKWEHDFHOOXODUPHFKDQLVPWRDGMXVW637DFWLYLW\WRPHHWWLVVXH VSHFL¿FUHTXLUHPHQWVIRUVSKLQJROLSLGV ,Q\HDVW637LVFRPSRVHGRIDKHWHURGLPHURIKLJKO\UHODWHGVXEXQLWV/FES and Lcb2p, and a third subunit, Tsc3p, which increases enzyme activity markedly DQG LV UHTXLUHG IRU JURZWK DW HOHYDWHG WHPSHUDWXUHV17 Recently, 2 proteins, ssSPTa and ssSPTb, which despite sharing no homology with Tsc3p, substantially enhances WKHDFWLYLW\RIPDPPDOLDQ637H[SUHVVHGLQHLWKHU\HDVWRUPDPPDOLDQFHOOVZHUH LGHQWL¿HG7KHVHSURWHLQVDUHHYLGHQFHIRUDQHYROXWLRQDULO\FRQVHUYHGIDPLO\RIORZ PROHFXODUZHLJKWSURWHLQVWKDWFRQIHUIXOO637HQ]\PHDFWLYLW\18 The small subunits RIPDPPDOLDQ637FRQIHUGLVWLQFWDF\O&R$VXEVWUDWHVSHFL¿FLWLHV18 SMS is located mainly in the cis-, medial-Golgi19-21 and plasma membranes.22-24 7KHUHPD\DOVREHDIRUPRI606LQWKHtrans-Golgi network25 and the nucleus.26 606DFWLYLW\KDVEHHQIRXQGLQFKURPDWLQDQGFKURPDWLQDVVRFLDWHG606PRGL¿HVWKH SM content.27 Two SMS genes, SMS1 and SMS2, have been cloned and characterized IRUWKHLUFHOOXODUORFDOL]DWLRQ28,29606LVIRXQGLQWKHtrans-Golgi apparatus, while 606LVSUHGRPLQDQWO\IRXQGLQWKHSODVPDPHPEUDQHV28,30 and also Golgi apparatus.28,30 606DQGH[SUHVVLRQSRVLWLYHO\FRUUHODWHVZLWKOHYHOVRIFHOOXODU60DQG60LQ OLSLGUDIWV31-33 7KH VSKLQJROLSLG ELRV\QWKHVLV SDWKZD\ LPSDFWV FHOOXODU SURGXFWLRQ RI DW OHDVW three bioactive lipids: ceramide, diglyceride, and sphingosine-1-phosphate; and two structure-related lipids: sphingomyelin and phosphatidylcholine. In this chapter, we IRFXVRQVSKLQJRP\HOLQDQGFHUDPLGH

22

SPHINGOLIPIDS AND METABOLIC DISEASE

ROLE OF SPHINGOMYELIN (SM) IN LIPOPROTEIN METABOLISM 6SKLQJRP\HOLQ60 LVDPDMRUFRPSRQHQWRIFHOOPHPEUDQHVDQGERWKDWKHURJHQLF and anti-atherogenic lipoproteins. Chylomicrons, very low density lipoprotein (VLDL) and low density lipoprotein (LDL) are atherogenic lipoproteins, while high density lipoproteins (HDL) are antiatherogenic ones. Chylomicrons, VLDL and LDL are enriched with SM.1 SM and Chylomicron/VLDL Metabolism 7KHSULPDU\PHWDEROLFUROHRI9/'/DQGFK\ORPLFURQVLVWRWUDQVSRUWHQGRJHQRXV DQGGLHWDU\WULJO\FHULGHXVHGIRUHQHUJ\VWUXFWXUDOOLSLGVDQGVWRUDJH7ULJO\FHULGHORFDWHG RQWKHVHSDUWLFOHVLVK\GURO\]HGLQWRIUHH)$VE\OLSRSURWHLQOLSDVH/S/ DVVRFLDWHGZLWK WKHOXPLQDOVXUIDFHRIFDSLOODULHV34,35 In vitro studies indicate that SM in these lipoproteins LQKLELWVWKHDFWLRQRI/S/)LJ .XNVLVHWDO36FRPSDUHG/S/K\GURO\VLVRI7*XVLQJ WKUHHHPXOVLRQVDVVXEVWUDWHVIRUWKHHQ]\PHSKRVSKDWLG\OFKROLQH3& 7*3&607* DQG3&SKRVSKDWLG\OHWKDQRORDPLQH3( 7*7KH\IRXQGWKDW60FRQWDLQLQJHPXOVLRQKDG the lowest TG clearance rate in vitro. Saito et al37 and Arimoto et al38IRXQGWKDW60EXWQRW FKROHVWHUROVLJQL¿FDQWO\LQKLELWHG7*OLSRO\VLVE\/S/LQD60FRQFHQWUDWLRQGHSHQGHQW manner. Lobo and Wilton39 and Cantin et al40 showed that cholesterol inclusion in the 3&VXUIDFHFRDWRID7*HPXOVLRQVWLPXODWHGOLSRO\VLVE\/S/EXWWKDWWKLVVWLPXODWLRQ was eliminated by adding SM.

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SPHINGOLIPIDS AND CARDIOVASCULAR DISEASES

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SM and Apolipoprotein E-Containing Lipoprotein Metabolism Apolipoprotein E (ApoE) is a protein which has anti-atherogenic property. Bound to SODVPDOLSRSURWHLQVLWLVDOLJDQGIRUFHOOXSWDNHE\WKH/'/UHFHSWRU/'/5 WKH/'/ UHFHSWRUUHODWHGSURWHLQ/53 DQGKHSDUDQVXOIDWHSURWHRJO\FDQV41,42$SR(DOVRLQÀXHQFHV WKHDVVHPEO\DQGVHFUHWLRQRIOLSRSURWHLQV43,44DQGFKROHVWHUROHIÀX[WR+'/45 Arimoto et al reported that SM in lipoproteins delays remnant clearance by decreasing the binding RIDSR(WRFHOOPHPEUDQHUHFHSWRUV46 Sphingomyelinase causes particle aggregation in DUWL¿FLDOOLSLGHPXOVLRQVRI7*3&60E\GHJUDGDWLRQRI60WRFHUDPLGH$SR(SUHYHQWV this aggregation,47LPSO\LQJDQLQWHUDFWLRQEHWZHHQ60DQGDSR(7RFODULI\WKHUROHRI SM in lipoprotein uptake, Morita et al48 prepared lipid emulsions containing triolein, PC DQG60DVPRGHOSDUWLFOHVRIUHFRQVWLWXWHGOLSRSURWHLQV,QFRUSRUDWLRQRI60LQWRWKH HPXOVLRQVXUIDFHUHGXFHGWKHELQGLQJFDSDFLW\RIDSR(ZLWKRXWFKDQJLQJLWVDI¿QLW\IRU WKHUHFHSWRUVRQWKHVXUIDFHRIKHSDWRF\WHV6XUIDFH60UHGXFHGDSR(PHGLDWHGXSWDNH RIHPXOVLRQVE\+HS*FHOOVEHFDXVHRIWKHGHFUHDVHGDPRXQWRIELQGLQJDSR(7KH VWLPXODWRU\HIIHFWRI/S/RQHPXOVLRQXSWDNHZDVGHFUHDVHGE\UHSODFLQJVXUIDFH3&ZLWK 607KHVHUHVXOWVVXJJHVWWKDW60LQGXFHGFKDQJHVLQWKHELQGLQJSURSHUWLHVRIDSR(DQG /S/FRUUHODWHZLWKGHFUHDVHGKHSDWLFXSWDNHRIOLSLGSDUWLFOHV/XFLFHWDOIRXQGWKDWDSR( H[SUHVVLRQDOVRLQFUHDVHG60VHFUHWLRQIURPPDFURSKDJHVDQGWKLV60ZDVFRORFDOL]HG with apoE in secreted lipoprotein particles.49$OWKRXJKWKHLQYLYRVLJQL¿FDQFHRIWKHVH ¿QGLQJVWRDSR(FRQWDLQLQJOLSRSURWHLQPHWDEROLVPLVQRW\HWHVWDEOLVKHGWKH\PD\EH KLJKO\UHOHYDQWGXULQJWKHLQFUHDVHLQWKH60FRQWHQWRISODVPDOLSRSURWHLQVWKDWRFFXUV IROORZLQJDODUJHOLSLGORDG50RUDSR(GH¿FLHQF\51 SM and HDL Metabolism HDLs are well-known anti-atherogenic lipoproteins. HDL particles are involved in the SURFHVVRIUHYHUVHFKROHVWHUROWUDQVSRUWZKLFKUHPRYHVFKROHVWHUROIURPSHULSKHUDOWLVVXHV and cells. The cholesterol on HDL becomes cholesterol ester (CE) through the activity RIOHFLWKLQFKROHVWHURODF\OWUDQVIHUDVH/&$7 7KH&(RI+'/PD\WKHQEHGHOLYHUHG to liver or steroidogenic tissues by the scavenger receptor B1 (SR-B1)52,53SULPDU\IDWH  RUWUDQVIHUUHGYLDSODVPDFKROHVWHU\OHVWHUWUDQVIHUSURWHLQ&(73 WR/'/DQG7*ULFK OLSRSURWHLQVLQH[FKDQJHIRU7*54 7KHVHSURFHVVHVPD\EHDIIHFWHGE\WKHSUHVHQFHRI60LQ+'/60LQKLELWV/&$7 by decreasing its binding to HDL55 (Fig. 2). A negative correlation between the SM FRQWHQWRI+'/DQG/&$7DFWLYLW\ZDVREVHUYHGLQVWXGLHVZLWKSURWHROLSRVRPHVRU reconstituted HDL.56 Rye, Hime and Barter57 IRXQG WKDW 60 LQÀXHQFHV WKH VWUXFWXUH RI GLVFRLGDO DQG VSKHULFDO+'/DQGFRQ¿UPHGWKDW60LQKLELWVWKH/&$7UHDFWLRQ 0DFURSKDJHFKROHVWHUROHIÀX[SOD\VDQLPSRUWDQWUROHLQUHYHUVHFKROHVWHUROWUDQVSRUW an anti-atherogenic process.58+RZHYHUWKHFDSDFLW\RIPRXVHSODVPDWRPHGLDWHFKROHVWHURO HIÀX[IURPPRXVHPDFURSKDJHVVXSSOHPHQWHGZLWKGLIIHUHQWFRQFHQWUDWLRQVRI60ZDV QRWDOWHUHG7KLVUHVXOWLQGLFDWHGWKDWSODVPD60OHYHOVPLJKWQRWKDYHDGLUHFWHIIHFWRQ FKROHVWHUROHIÀX[/LDQG-LDQJXQSXEOLVKHGREVHUYDWLRQ  65%,LVWKH¿UVWPROHFXODUO\GH¿QHGUHFHSWRUIRU+'/DQGFDQPHGLDWHWKHVHOHFWLYH XSWDNHRI&(LQWRFHOOV6XEEDLDKHWDO59LQYHVWLJDWHGWKHHIIHFWRI60LQOLSRSURWHLQVRQ WKHVHOHFWLYHXSWDNHLQWKUHHGLIIHUHQWFHOOOLQHV65%,WUDQVIHFWHG&+2FHOOVKHSDWRF\WHV

24

SPHINGOLIPIDS AND METABOLIC DISEASE

+HS*  DQG DGUHQRFRUWLFDO FHOOV <%6  7KH\ IRXQG WKDW 60 LQ WKH OLSRSURWHLQV UHJXODWHVWKH65%,PHGLDWHGVHOHFWLYHXSWDNHRI&(SRVVLEO\E\LQWHUDFWLQJZLWKWKH VWHUROULQJRUZLWK65%,LWVHOI $NH\IXQFWLRQRI+'/60PD\EHWRUHJHQHUDWH+'/GXULQJQRUPDOOLSLGPHWDEROLVP VXFKDVDIWHUOLYHU65%H[WUDFWV&(IURPVSKHULFDO+'/DQGUHOHDVHVVRPHOLSLGIUHH DSR$SURWHLQ$GGLWLRQRI60HQULFKHGSKRVSKROLSLGVDQGFKROHVWHUROWRDSR$SURGXFHV D QHZ OLSRSURWHLQ VSHFLHV RSWLPL]HG WR DFFHSW FKROHVWHURO IURP FHOOV WKXV UHVWDUWLQJ HDL growth.60 SPHINGOMYELIN AND ATHEROSCLEROSIS $WKHURVFOHURVLVLVDQLQÀDPPDWRU\GLVHDVHFKDUDFWHUL]HGE\WKHSURGXFWLRQRID ZLGHUDQJHRIFKHPRNLQHVDQGF\WRNLQHV$WKHURJHQHVLVLVLQLWLDWHGE\WKHLQWHUDFWLRQRI cholesterol-rich lipoproteins with the arterial wall.61 Many processes have been implicated in early atherogenesis, including lipoprotein oxidation,62,63 lipoprotein retention and aggregation,64-67 endothelial alteration,61 monocyte recruitment, macrophage chemotaxis DQGIRDPFHOOIRUPDWLRQ61 and smooth muscle cell migration and alteration.61 7ZRVHWVRIHYLGHQFHLQGLFDWHWKDWERWKWKHDPRXQWRI60LQWKHDRUWLFZDOODQG WKH60OHYHOVLQSODVPDDUHFORVHO\UHODWHGWRWKHGHYHORSPHQWRIDWKHURVFOHURVLV  60DFFXPXODWHVLQDWKHURPDVIRUPHGLQKXPDQDQGDQLPDOPRGHOV68-73 LDL extracted IURPKXPDQDWKHURVFOHURWLFOHVLRQVLVPXFKULFKHULQ60WKDQ/'/IURPSODVPD74-77 A VXEVWDQWLDODPRXQWRIWKH60IRXQGLQDUWHULHVDQGDWKHURVFOHURWLFOHVLRQVDSSHDUVWRDULVH IURPV\QWKHVLVLQWKHDUWHULDOWLVVXHV78,7960FRQFHQWUDWLRQLVDOVRVLJQL¿FDQWO\LQFUHDVHG LQPDFURSKDJHVWUHDWHGZLWKDFHW\O/'/SOXVDQDF\O&R$FKROHVWHURODF\OWUDQVIHUDVH (ACAT) inhibitor.80   (YHQ LQ DWKHURVFOHURWLF OHVLRQV WKH UDWH RI 60 IRUPDWLRQ LV UHODWLYHO\ VORZ FRPSDUHG ZLWK WKH UDWH RI WRWDO FKROLQHFRQWDLQLQJ SKRVSKROLSLG synthesis,81VXJJHVWLQJWKDWDGGLWLRQDOIDFWRUVFRQWULEXWHWRLQWLPDO60DFFXPXODWLRQ 7KH UDWLR RI 60 WR 3& LV LQFUHDVHG E\ IROG LQ 9/'/ IURP K\SHUFKROHVWHUROHPLF rabbits.82 ApoE knockout (KO) mice are a well known atherogenic model. Plasma SM OHYHOVLQWKHVHPLFHDUHIROGKLJKHUWKDQLQZLOGW\SHPLFH51 and this may contribute to the increased atherosclerosis.83,84:HDOVRIRXQGWKDWKXPDQSODVPD60OHYHOVDQG 603& UDWLR DUH LQGHSHQGHQW ULVN IDFWRUV IRU FRURQDU\ KHDUW GLVHDVH6,85 Moreover, 60ULFK   GLHW VLJQL¿FDQWO\ LQFUHDVHV SODVPD 60 OHYHOV /'/ DJJUHJDWLRQ DQG atherosclerotic lesions in LDL receptor KO mice.86 All these data suggest that plasma 60SOD\VDFULWLFDOUROHLQWKHGHYHORSPHQWRIDWKHURVFOHURVLV60RQ/'/UHWDLQHG in atherosclerotic lesions is hydrolyzed by an arterial wall sphingomyelinase, which promotes aggregation by converting SM to ceramide51,76 (Fig. 3). 7KHUHDUHWZRZD\VRISUHYHQWLQJWKLVDWKHURJHQLFHYHQWWKH¿UVWEHLQJWRUHGXFH sphingomyelinase levels. Indeed, it has recently been reported that apoE KO mice ODFNLQJVSKLQJRP\HOLQDVHKDYHGHFUHDVHGGHYHORSPHQWRIHDUO\DWKHURVFOHURWLFOHVLRQV DQGPRUHLPSRUWDQWGHFUHDVHGUHWHQWLRQRIDWKHURJHQLFOLSRSURWHLQVFRPSDUHGZLWK DSR( .2 PDWFKHG IRU VLPLODU OLSRSURWHLQ OHYHOV87 7KH VHFRQG ZD\ RI SUHYHQWLQJ atherogenicity is by reducing SM levels in the atherogenic lipoproteins through LQKLELWLRQRIWKH60ELRV\QWKHVLVSDWKZD\LQWKHOLSRSURWHLQSURGXFLQJWLVVXHVVXFK as the liver and small intestine.

SPHINGOLIPIDS AND CARDIOVASCULAR DISEASES

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Figure 3. 5ROH RI VSKLQJRP\HOLQ LQ DJJUHJDWLRQ RI DWKHURJHQLF OLSRSURWHLQ *HQHUDWLRQ RI FHUDPLGH E\ sphingomyelinase aggregates intimal atherogenic lipoproteins and leads to early atherogenesis. Inhibition RI 60 GH QRYR V\QWKHVLV GHFUHDVHV VSKLQJRP\HOLQDVH VXEVWUDWH 60 WKXV GHFUHDVLQJ DJJUHJDWLRQ RI intimal lipoproteins.

SM and Cholesterol Metabolism *LYHQWKDWDOOSDWKZD\VRIFKROHVWHUROHIÀX[UHTXLUHFKROHVWHUROWUDQVSRUWWRWKHSODVPD PHPEUDQHVWKHLGHQWL¿FDWLRQRIPROHFXOHVPHGLDWLQJRUUHJXODWLQJWKLVWUDQVSRUWSURFHVVLV an important goal. Leventhal et al,88 reported that lysosomal sphingomyelinase is involved LQFKROHVWHUROWUDQVSRUWIURPO\VRVRPHVWRWKHSODVPDPHPEUDQHV6SKLQJRP\HOLQDVH hydrolyzes SM in late endosomes and lysosomes.89 Because SM avidly binds cholesterol,90,91 SM hydrolysis by sphingomyelinase enables cholesterol transport through preventing FKROHVWHUROVHTXHVWUDWLRQE\60,QWHUHVWLQJO\KXPDQVZLWKVSKLQJRP\HOLQDVHGH¿FLHQF\ (Types A and B Niemann-Pick disease) have low plasma HDL cholesterol levels,92 which FRXOGUHVXOWIURPGHIHFWLYHFKROHVWHUROHIÀX[93 ,Q PDFURSKDJHV $%&$ H[SRUWV FKROHVWHURO DQG SKRVSKROLSLG WR OLSLGIUHH apolipoproteins, while ABCG1 and SR-BI export cholesterol to phospholipid-containing acceptors.94$%&$GHSHQGHQWFKROHVWHUROH[SRUWLQYROYHVDQLQLWLDOLQWHUDFWLRQRIDSR$, ZLWKOLSLGUDIWPHPEUDQHGRPDLQV95,96 ABCA1 expression causes a change in overall OLSLGSDFNLQJRIWKHSODVPDPHPEUDQHSUREDEO\WKURXJKLWV$73DVHUHODWHGIXQFWLRQV 6XFKUHRUJDQL]DWLRQE\$%&$HIIHFWLYHO\H[SDQGVWKHQRQUDIWPHPEUDQHIUDFWLRQVDQG FRQVHTXHQWO\SUHFRQGLWLRQVFHOOVIRUFKROHVWHUROHIÀX[97 ABCG1 exports cholesterol to

26

SPHINGOLIPIDS AND METABOLIC DISEASE

HDL and other phospholipid-containing acceptors. These include particles generated GXULQJOLSLGDWLRQRIDSR$,E\$%&$VXJJHVWLQJWKDWWKHWZRWUDQVSRUWHUVFRRSHUDWH in cholesterol export.98$%&*LVPDLQO\IRXQGLQWUDFHOOXODUO\LQWKHEDVDOVWDWHZLWK OLWWOH FHOO VXUIDFH SUHVHQWDWLRQ %XW RQ VWLPXODWLRQ IRU H[DPSOH E\ OLYHU ; UHFHSWRU (LXR) agonist treatment, ABCG1 redistributes to the plasma membranes and increases FKROHVWHUROPDVVHIÀX[WR+'/9965%,IDFLOLWDWHVFKROHVWHUROHIÀX[IURPPDFURSKDJHV100 ,QDFWLYDWLRQRIPDFURSKDJH65%,SURPRWHVDWKHURVFOHURWLFOHVLRQGHYHORSPHQWLQDSR( KO mice.101,QWKHOLYHU$%&$PDNHVDPDMRUFRQWULEXWLRQWRWKH+'/LQWKHFLUFXODWLRQ /LYHUVSHFL¿F$%&$.2PLFHGHFUHDVHSODVPD+'/E\DERXWFRPSDUHGZLWK controls.102,10365%,LVDPXOWLIXQFWLRQDOUHFHSWRUFDSDEOHRIELQGLQJDZLGHDUUD\RI QDWLYHDQGPRGL¿HGOLSRSURWHLQV65%,LVDEXQGDQWO\H[SUHVVHGLQOLYHUDQGVWHURLGRJHQLF WLVVXHVZKHUHLWPHGLDWHVWKHVHOHFWLYHXSWDNHRIFKROHVWHU\OHVWHUVIURP+'/104,105 A GH¿QLWLYHUROHIRU65%,LQ+'/PHWDEROLVPDQGUHYHUVHFKROHVWHUROWUDQVSRUWLQYLYR KDVEHHQGHPRQVWUDWHGXVLQJGLIIHUHQWWUDQVJHQLFDQGNQRFNRXWPRXVHPRGHOV$%&* is also expressed in the liver,106EXWLWVIXQFWLRQLQWKHOLYHULVVWLOOQRWZHOOFKDUDFWHUL]HG 7KHDSLFDOPHPEUDQHVRILQWHVWLQDOFHOOVDUHHQULFKHGLQ60DQGFKROHVWHURO107 LQGLFDWLYHRIWKHSUHVHQFHRIOLSLGUDIWV108,109/LSLGUDIWVRQWKHHQWHURF\WHDSLFDOPHPEUDQH SOD\DQLPSRUWDQWUROHLQFKROHVWHURODEVRUSWLRQDQGWUDI¿FNLQJ110,111 $%&$$%&*DQG65%,DUHORFDWHGLQWKHSODVPDPHPEUDQHUDIWV65%, 112,113 RUDVVRFLDWHGZLWKPHPEUDQHOLSLGUDIWV$%&$DQG$%&* 94-97,114,WLVWKHUHIRUH FRQFHLYDEOHWKDWIXQGDPHQWDOFKDQJHVLQ60OHYHOVRIWKHSODVPDPHPEUDQHVLQÀXHQFH WKHIXQFWLRQVRIWKHVHSURWHLQVDQGDOWHUFKROHVWHUROKRPHRVWDVLV,QSHULSKHUDOWLVVXHV 60UHJXODWLRQRIUHYHUVHFKROHVWHUROWUDQVSRUWSURWHLQVFRXOGDIIHFWDWKHURVFOHURVLV (QKDQFHGDSR$,GHSHQGHQWFKROHVWHUROHIÀX[E\$%&$IURP60GH¿FLHQW&KLQHVH hamster ovary (CHO) cells has been reported.115 Serine Palmitoyltransferase (SPT) and Atherosclerosis Plasma SM and other sphingolipid intermediates play important roles in the GHYHORSPHQW RI FRURQDU\ DUWHU\ GLVHDVH ,W LV FRQFHLYDEOH WKDW UHJXODWLRQ RI 60 ELRV\QWKHVLVFDQDOWHU60OHYHOVLQWKHSODVPDDQGRQWKHPHPEUDQHWKXVLQÀXHQFLQJ WKHSURFHVVRIDWKHURVFOHURVLV+RZLV60V\QWKHVLVUHJXODWHGLQYLYRDQGZKLFKVWHSV are critical? SPT Inhibitors 0\ULRFLQ VSKLQJRIXQJLQV DQG OLSR[DP\FLQ DUH SRWHQW DQG KLJKO\ VHOHFWLYH QDWXUDOO\RFFXUULQJLQKLELWRUVRI637LQKLELWLQJIXQJDODQGPDPPDOLDQ637LQFHOOIUHH preparations with IC50 values in the nanomolar range116-118 (Fig. 4). Structurally they UHVHPEOHWKHWUDQVLHQWLQWHUPHGLDWHSRVWXODWHGWRIRUPLQWKHFRQGHQVDWLRQRI/VHULQH DQGSDOPLWR\O&R$&RQVLVWHQWZLWKWKLVWKHLQKLELWRU\DFWLYLW\RIVSKLQJRIXQJLQ% is highly dependent on its stereochemistry.119 Myriocin-linked resins bind the Sptlc1/ Sptlc2 complex tightly.120$OOWKHLQKLELWRUVVLJQL¿FDQWO\LQKLELW60DFFXPXODWLRQLQ both cultured cells and in vivo.116-118

SPHINGOLIPIDS AND CARDIOVASCULAR DISEASES

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Figure 4. 6WUXFWXUHV RI VSKLQJRVLQH DQG LQKLELWRUV RI VHULQH SDOPLWR\OWUDQVIHUDVH

The Effect of SPT Inhibition in Mouse Atherosclerosis Park et al7DQG+RMMDWWLHWDO8 have reported that myriocin treatment (oral administration DQGLQWUDSHULWRQHDOLQMHFWLRQUHVSHFWLYHO\ GHFUHDVHVSODVPD60OHYHOVDQGDWKHURVFOHURVLV in apoE KO mice. Myriocin treatment also can induce lower lipid levels in apoE KO mice.121 However, both administration methods led to reduced atherosclerosis, whereas only oral DGPLQLVWUDWLRQRIP\ULRFLQORZHUHGSODVPDFKROHVWHUROOHYHOV7 Oral administration may reduce cholesterol absorption in small intestine. When wild type and apoE KO animals ZHUHWUHDWHGZLWKP\ULRFLQWKHPLFHDEVRUEHGVLJQL¿FDQWO\OHVVFKROHVWHUROWKDQFRQWUROV with no observable pathological changes in the small intestine. Myriocin treatment also increased insulin sensitivity.11 Thus, myriocin has direct anti-atherosclerosis vascular HIIHFWVDQGDOVRKDVWKHSRWHQWLDOWRIXQFWLRQDVDSODVPDOLSLGORZHULQJDJHQW Homozygous Sptlc1 and Sptlc2 KO mice are embryonic lethal, whereas heterozygous YHUVLRQVRIERWKDQLPDOV6SWOF /< and Sptlc2 /<, are healthy. Compared with wild type mice, Sptlc1

< and Sptlc2 /– mice had: (1) decreased liver Sptlc1 and Sptlc2 mRNA by 44% and 57%, respectively and (2) decreased liver SPT activity by 45% and 60%, respectively. However, these animals had no change in plasma SM, triglyceride, total cholesterol, phospholipids and liver SM levels.123 +HWHUR]\JRXV6SWOFDVXEXQLWRI637 NQRFNRXWPLFHDOVRDEVRUEHGVLJQL¿FDQWO\OHVV FKROHVWHUROWKDQFRQWUROV7RXQGHUVWDQGWKHPHFKDQLVPWKHSURWHLQOHYHOVRI1LHPDQQ3LFN &OLNH13&/ $%&*DQG$%&$WKUHHNH\IDFWRUVLQYROYHGLQLQWHVWLQDOFKROHVWHURO absorption were measured. NPC1L1 and ABCA1 were decreased, whereas ABCG5 was LQFUHDVHGLQWKH637GH¿FLHQWVPDOOLQWHVWLQH60OHYHOVRQWKHDSLFDOPHPEUDQHZHUH DOVRPHDVXUHGDQGWKH\ZHUHVLJQL¿FDQWO\GHFUHDVHGLQ637GH¿FLHQWPLFHFRPSDUHGZLWK controls.1227KHVHUHVXOWVVXJJHVWHGWKDW637GH¿FLHQF\PLJKWUHGXFHLQWHVWLQDOFKROHVWHURO DEVRUSWLRQE\DOWHULQJ13&/DQG$%&*SURWHLQOHYHOVLQWKHDSLFDOPHPEUDQHVRI HQWHURF\WHV WKURXJK ORZHULQJ DSLFDO PHPEUDQH 60 OHYHOV 7KXV PDQLSXODWLRQ RI 637 DFWLYLW\FRXOGSURYLGHDQRYHODOWHUQDWLYHWUHDWPHQWIRUG\VOLSLGHPLD

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Regulation of SPT A. Transcriptional and posttranscriptional regulation: Although the mechanism XQGHUO\LQJWKHUHJXODWLRQRI637LVODUJHO\XQNQRZQVWXGLHVGHVFULEHGEHORZKDYH begun to provide insight into transcriptional and posttranscriptional regulation. Sptlc1 and Sptlc2 mRNA and SPT enzyme activity levels increase in response to VHYHUDOW\SHVRILQÀDPPDWRU\DQGVWUHVVVWLPXOL,QWUDSHULWRQHDODGPLQLVWUDWLRQ RIHQGRWR[LQWR6\ULDQKDPVWHUVVWLPXODWHV637DFWLYLW\WRIROGLQWKHOLYHU VSOHHQDQGNLGQH\ZLWKDFRQFRPLWDQWLQFUHDVHLQWKHOHYHOVRI6SWOFP51$ and SM.124,1256LPLODUFKDQJHVDUHREVHUYHGXSRQDGPLQLVWUDWLRQRILQWHUOHXNLQ` ,/ DQLQÀDPPDWRU\F\WRNLQH124,UUDGLDWLRQRIHSLGHUPDOFHOOVZLWKXOWUDYLROHW OLJKWDOVRDIIHFWV6SWOFDQG6SWOFP51$OHYHOV1267KHH[SUHVVLRQRI6SWOF P51$LVDOVRXSUHJXODWHGLQWKHSDQFUHDWLFLVOHWVRIOHSWLQUHFHSWRUGH¿FLHQW obese fa/fa rats. This is suggested to be a response to an increase in intracellular IDWW\DFLG127 % $FWLYLW\ UHJXODWLRQ &RQVLVWHQW ZLWK WKH XELTXLWRXV SUHVHQFH RI 60 LQ mammalian cells, SPT enzyme activity has been detected in many tissues and cell preparations.1287KHOHYHOVRI637DFWLYLW\YDULHVZLWKGHYHORSPHQW )RUH[DPSOHLQUDWOXQJ637DFWLYLW\LQFUHDVHVSURJUHVVLYHO\IURPWKHIHWDO to neonatal period and reaches a plateau at the adult stage.1297KHOHYHOVRI 637DFWLYLW\LQVHYHUDODQLPDOWLVVXHVDUHDIIHFWHGE\GLHW130,131$PDMRUPRGH RI UHJXODWLRQ RI 637 DFWLYLW\ RFFXUV WKURXJK VXEVWUDWH VXSSO\ ,W KDV EHHQ reported that the greatest activity was obtained with palmitoyl-CoA supply.132 3HQWDGHFDQR\ODQG KHSWDGHFDQR\O&R$V DUH DOVR HIIHFWLYH ,Q PDPPDOLDQ FHOOVSDOPLWR\O&R$LVRQHRIWKHPRVWDEXQGDQWDF\O&R$W\SHV7KHUHIRUH SDOPLWR\O&R$LVWKHSUHGRPLQDQWDF\O&R$VXEVWUDWHRI637LQYLYR133 Sphingomyelin Synthase (SMS) and Atherosclerosis 606LVWKHODVWHQ]\PHIRU60ELRV\QWKHVLV,WVDFWLYLW\GLUHFWO\LQÀXHQFHV603& and ceramide, as well as diacylglycerol (DAG) levels.12 Manipulating SMS activity also can regulate plasma and membrane SM levels. 7KH(IIHFWRI6062YHUH[SUHVVLRQDQG'H¿FLHQF\RQ0RXVH/LSRSURWHLQ0HWDEDOLVP 7RHYDOXDWHWKHLQYLYRUROHRI606LQ60PHWDEROLVP606.2DQG606 OLYHUVSHFL¿FWUDQVJHQLF/7J PLFHZHUHFUHDWHGDQGWKHLUSODVPD60DQGOLSRSURWHLQ metabolism were characterized.134 On a chow diet, SMS2 KO mice had reduced SODVPD60OHYHOVEXWQRVLJQL¿FDQWFKDQJHVLQWRWDOFKROHVWHUROWRWDOSKRVSKROLSLGV RU WULJO\FHULGHFRPSDUHGZLWK ZLOG W\SH OLWWHUPDWHV2Q D KLJKIDWGLHW606 .2 PLFH VKRZHG D VLJQL¿FDQW GHFUHDVH LQ SODVPD 60 OHYHOV ZKHUHDV 606/7J PLFH VKRZHGDVLJQL¿FDQWLQFUHDVHLQWKRVHOHYHOVEXWQRVLJQL¿FDQWFKDQJHVLQRWKHUOLSLGV $WKHURJHQLF OLSRSURWHLQV IURP 606/7J PLFH GLVSOD\HG D VLJQL¿FDQWO\ VWURQJHU WHQGHQF\WRZDUGDJJUHJDWLRQDIWHUPDPPDOLDQVSKLQJRP\HOLQDVHWUHDWPHQWFRPSDUHG ZLWKFRQWUROV0RUHRYHU606GH¿FLHQF\VLJQL¿FDQWO\LQFUHDVHGSODVPDDSR(OHYHOV

SPHINGOLIPIDS AND CARDIOVASCULAR DISEASES

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IROG ZKHUHDVOLYHUVSHFL¿F606RYHUH[SUHVVLRQVLJQL¿FDQWO\GHFUHDVHGWKRVH OHYHOVIROG )LQDOO\606.2PRXVHSODVPDSURPRWHGFKROHVWHUROHIÀX[IURP macrophages, whereas SMS2LTg mouse plasma prevented it.134 These results indicated WKDW606LVRQHRIWKHGHWHUPLQDQWVIRUSODVPDDQGOLYHU60OHYHOVLQPLFH SMS and Apoptosis It is believed that disordered apoptosis may occur in atherogenesis, leading to GHDWK RI OLSLGULFK IRDP FHOOV SURPRWLQJ OLSLG FRUH IRUPDWLRQ135 In two previous papers, investigators reported controversial results regarding the relationship between SMS activity and cell apoptosis. Van der Luit et al33 reported that SMS1 gene NQRFNGRZQDOWHUVWKHSODVPDPHPEUDQHUDIWVWUXFWXUHDQGUHGXFHVWKHLQWHUQDOL]DWLRQRI alkyl-lysophospholipid, thereby reducing cell apoptosis rates. Separovic et al136 reported that SMS1 overexpression suppresses cell apoptosis mediated by photo damage; however, WKH\GLGQRWVKRZWKHHIIHFWRI606RYHUH[SUHVVLRQRQPHPEUDQHOLSLGUDIWV%DVHG on previous results,137 it is believed that SMS1 and SMS2 overexpression increases SM OHYHOVLQWKHOLSLGUDIWVRQSODVPDPHPEUDQHVDQGSURPRWHVDPRUHH[WHUQDODSSHDUDQFH IRU71)_UHFHSWRUDZHOONQRZQUHFHSWRULQOLSLGUDIWV 138IROORZLQJ71)_stimulation, thereby promoting CHO cell apoptosis. SMS1 and SMS2 knockdown by siRNA reduces 60OHYHOVLQOLSLGUDIWVRQWKHSODVPDPHPEUDQHDQGUHGXFHVWKHQXPEHURI7/5ZKLFK DUHDOVRZHOONQRZQIRUWKHLUSUHVHQFHLQOLSLGUDIWV 139 that are presented on the plasma PHPEUDQHDIWHU/36VWLPXODWLRQWKHUHE\UHGXFLQJPDFURSKDJHDSRSWRVLV,QGHHGDIWHU /36VWLPXODWLRQ606606NQRFNGRZQPDFURSKDJHVFRQWDLQHGVLJQL¿FDQWO\OHVV 7/5RQWKHFHOOVXUIDFHWKDQFRQWUROPDFURSKDJHV137 0DQLSXODWLRQ RI 606 DFWLYLW\ DOWHUV FHOOXODU '$* OHYHOV DQG WKXV PD\ DOVR FRQWULEXWHWRDSRSWRVLV&HUERQHWDOGHPRQVWUDWHGWKDWSKDUPDFRORJLFDOLQKLELWLRQRI SMS reduces cellular DAG levels and PKC activity.140 SMS1 or SMS2 overexpression VLJQL¿FDQWO\LQFUHDVHV'$*OHYHOVLQ&+2FHOOVZKLOH606RU606JHQHNQRFNGRZQ VLJQL¿FDQWO\UHGXFHV'$*OHYHOVLQ7+3GHULYHGPDFURSKDJHV137 DAG can regulate both conventional and novel PKCs,141DIDPLO\RIVHULQHWKUHRQLQHNLQDVHVWKDWUHJXODWH DGLYHUVHVHWRIFHOOXODUSURFHVVHVLQFOXGLQJSURDSRSWRWLFDQGSURVXUYLYDOSURFHVVHV PKCb is generally considered a growth inhibitory or proapoptotic PKC,142,143 while PKC¡LVFRQVLGHUHGDSURVXUYLYDOIDFWRU142,144 It is possible that in both CHO cells DQG7+3±GHULYHGPDFURSKDJHVUHJXODWLRQRI606RU606DFWLYLW\E\HLWKHUWKH RYHUH[SUHVVLRQRIWKHLUJHQHVRUJHQHNQRFNGRZQFRXOGPRGXODWH'$*PHGLDWHG3.& DFWLYLW\WKHUHE\LQÀXHQFLQJFHOODSRSWRVLV137 0DQLSXODWLRQRI606DFWLYLW\DOVRDOWHUVFHOOXODUFHUDPLGHOHYHOVDQGWKLVPD\ DOVRFRQWULEXWHWRDSRSWRVLV2YHUH[SUHVVLRQRIERWK606DQG606LVDFFRPSDQLHG E\LQFUHDVHGOHYHOVRIFHUDPLGHDVZHOODV60137 Separovic et al reported the same phenomenon when they overexpressed SMS1 in Jurkat cells.136 This may be due the FRPSOH[LW\RIWKLVHQ]\PHZKLFKFDQFDWDO\]HELGLUHFWLRQDOUHDFWLRQ28 The ceramide:SM ratio increases in cells that overexpress SMS compared with controls.137 This may UHSUHVHQWDQRWKHUPHFKDQLVPIRUWKHLQFUHDVHGDSRSWRWLFSRWHQWLDORIWKHVHFHOOVJLYHQ WKDWFHUDPLGHLVDELRDFWLYHOLSLGWKDWLVZHOONQRZQIRUSURPRWLQJFHOODSRSWRVLV145,146 +RZHYHUFHUDPLGHOHYHOVGLGQRWFKDQJHLQ7+3GHULYHGPDFURSKDJHVDIWHUWKH\ZHUH exposed to SMS siRNA.137 This indicates that the ceramide level might not be important in SMS-knockdown–induced macrophage apoptosis.

30

SPHINGOLIPIDS AND METABOLIC DISEASE

SMS and NFgB and MAP Kinase Activation 7KHDFFXPXODWLRQRIPDFURSKDJHGHULYHGIRDPFHOOVLQWKHYHVVHOZDOOLVDOZD\V DFFRPSDQLHGE\WKHSURGXFWLRQRIDZLGHUDQJHRIFKHPRNLQHVF\WRNLQHVDQGJURZWK IDFWRUV147 7KHVH IDFWRUV UHJXODWH WKH WXUQRYHU DQG GLIIHUHQWLDWLRQ RI LPPLJUDWLQJ DQG UHVLGHQWFHOOVHYHQWXDOO\LQÀXHQFLQJSODTXHGHYHORSPHQW2QHRIWKHNH\UHJXODWRUVRI LQÀDPPDWLRQLV1)gB,148ZKLFKKDVORQJEHHQUHJDUGHGDVDQDWKHURJHQLFIDFWRUPDLQO\ EHFDXVHRILWVUHJXODWLRQRIPDQ\RIWKHLQÀDPPDWRU\JHQHVOLQNHGWRDWKHURVFOHURVLV149,150 'HSOHWLRQ RI FKROHVWHURO IURP UDIWV FDXVHV D UHGLVWULEXWLRQ RI 71)_ receptor 1 WRQRQUDIWSODVPDPHPEUDQHSUHYHQWLQJ1)gB activation151 or ligand-induced RhoA activation152DQGVXFKWUHDWPHQWDOVRLQKLELWVLQÀDPPDWRU\VLJQDOVPHGLDWHGE\7/5V139 Studies also suggest that NFgB activation is triggered by SM-derived ceramide.153 On the contrary, it has been also shown that ceramide is not necessary or even inhibits NFgB activation.154 Luberto et al155IRXQGWKDW'DQRQVSHFL¿F606LQKLELWRUEORFNV TNF_-and phorbol ester-mediated NFgB activation that was concomitant with decreased OHYHOVRI60DQG'$*7KLVGLGQRWDIIHFWWKHJHQHUDWLRQRIFHUDPLGHVXJJHVWLQJ60 DQG'$*GHULYHGIURP60V\QWKHVLVDUHLQYROYHGLQ1)gB activation. Hailemariam et al IRXQGWKDW1)gB activation and its target gene expression are attenuated in macrophages IURP606.2PLFHLQUHVSRQVHWR/36VWLPXODWLRQDQGLQ606VL51$WUHDWHG+(. FHOOVDIWHU71)_ simulation.156 In line with attenuated NFgB activation, it was IRXQGWKDW606GH¿FLHQF\VXEVWDQWLDOO\GLPLQLVKHGWKHDEXQGDQFHRIWROOOLNHUHFHSWRU 7/5 0'FRPSOH[OHYHOVRQWKHVXUIDFHRIPDFURSKDJHVDIWHU/36VWLPXODWLRQ and SMS2 siRNA treatment reduced TNF_VWLPXODWHGOLSLGUDIWUHFUXLWPHQWRI71)_ receptor-1 in HEK293 cells.1567KXV606LVDPRGXODWRURI1)gB activation and could play an important role in NFgB-mediated atherogenic process (Fig. 5).

Figure 5. 0HFKDQLVP RI UHGXFHG DWKHURVFOHURVLV E\ GH¿FLHQF\ RI VSKLQJRP\HOLQ V\QWKDVH 

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GHWHUPLQLQJVXEVWUDWHSUHIHUHQFHIRUFDUGLDFHQHUJHWLFV2QHKLJKO\LPSRUWDQWDVSHFWRI the reported work is the discovery that inhibiting sphingolipid biosynthesis may have great WKHUDSHXWLFYDOXHIRUWKHWUHDWPHQWRIDWKHURVFOHURVLVDQGFDUGLDFIDLOXUH7RFRQ¿UPWKLV DSURVSHFWLYHFOLQLFDOVWXG\LQKXPDQSRSXODWLRQVVKRXOGEHIROORZHGWRHYDOXDWHZKHWKHU SODVPD60LVDUHDOULVNIDFWRUIRUDWKHURVFOHURVLV,QDGGLWLRQVSKLQJROLSLGELRV\QWKHVLV LVWLJKWO\OLQNHGWRGHYHORSPHQWRILQVXOLQUHVLVWDQFHREHVLW\DQGDWKHURVFOHURVLV7KH PHFKDQLVPV RI VSKLQJROLSLG ELRV\QWKHVLV LQYROYHG LQ WKHVH FKURQLF GLVHDVHV QHHG WR EH IXUWKHU HOXFLGDWHG LQ GHWDLO 7KXV VSKLQJROLSLG PRGXODWLRQ LQ DWKHURVFOHURVLV DQG DUGLRP\RSDWK\ZLOOSURYLGHDWKHUDSHXWLFUDWLRQDOHWRFXUHWKHSDWLHQWVLQÀLFWLQJZLWK these chronic diseases. REFERENCES 6KLPDEXNXUR0=KRX<7/HYL0HWDO)DWW\DFLGLQGXFHGEHWDFHOODSRSWRVLVDOLQNEHWZHHQREHVLW\DQG diabetes. Proc Natl Acad Sci USA 1998; 95:2498-2502. 2. Koyama K, Chen G, Lee Y et al. Tissue triglycerides, insulin resistance and insulin production: implications IRUK\SHULQVXOLQHPLDRIREHVLW\$P-3K\VLRO( =KRX<7*UD\EXUQ3.DULP$HWDO/LSRWR[LFKHDUWGLVHDVHLQREHVHUDWVLPSOLFDWLRQVIRUKXPDQREHVLW\ Proc Natl Acad Sci USA 2000; 97:1784-1789. '\QWDU'(SSHQEHUJHU(EHUKDUGW00DHGOHU.HWDO*OXFRVHDQGSDOPLWLFDFLGLQGXFHGHJHQHUDWLRQRI P\R¿EULOVDQGPRGXODWHDSRSWRVLVLQUDWDGXOWFDUGLRP\RF\WHV'LDEHWHV 3HWHUVHQ.)6KXOPDQ*,(WLRORJ\RILQVXOLQUHVLVWDQFH$P-0HG6 -LDQJ;&3DXOWUH)3HDUVRQ7$HWDO3ODVPDVSKLQJRP\HOLQOHYHODVDULVNIDFWRUIRUFRURQDU\DUWHU\GLVHDVH Arterioscler Thromb Vasc Biol 2000; 20:2614-2618. 3DUN 76 3DQHN 5/ 0XHOOHU 6% HW DO ,QKLELWLRQ RI VSKLQJRP\HOLQ V\QWKHVLV UHGXFHV DWKHURJHQHVLV LQ apolipoprotein E-knockout mice. Circulation 2004; 110:3465-3471. +RMMDWL05/L==KRX+HWDO(IIHFWRIP\ULRFLQRQSODVPDVSKLQJROLSLGPHWDEROLVPDQGDWKHURVFOHURVLV LQDSR(GH¿FLHQWPLFH-%LRO&KHP /LX-+XDQ&&KDNUDERUW\0HWDO0DFURSKDJHVSKLQJRP\HOLQV\QWKDVH606 GH¿FLHQF\GHFUHDVHV atherosclerosis in mice. Circulation Research. 2009. 10. Park TS, Hu Y, Noh HL et al. Ceramide is a cardiotoxin in lipotoxic cardiomyopathy. J Lipid Res 2008; 49:2101-2112. +ROODQG:/%UR]LQLFN-7:DQJ/3HWDO,QKLELWLRQRIFHUDPLGHV\QWKHVLVDPHOLRUDWHVJOXFRFRUWLFRLG VDWXUDWHGIDWDQGREHVLW\LQGXFHGLQVXOLQUHVLVWDQFH&HOO0HWDE 0HUULOO$+-U-RQHV''$QXSGDWHRIWKHHQ]\PRORJ\DQGUHJXODWLRQRIVSKLQJRP\HOLQPHWDEROLVP Biochim Biophys Acta 1990; 1044:1-12. :HLVV % 6WRIIHO : +XPDQ DQG PXULQH VHULQHSDOPLWR\O&R$ WUDQVIHUDVH²FORQLQJ H[SUHVVLRQ DQG FKDUDFWHUL]DWLRQRIWKHNH\HQ]\PHLQVSKLQJROLSLGV\QWKHVLV(XU-%LRFKHP +DQDGD.+DUD71LVKLMLPD0HWDO$PDPPDOLDQKRPRORJRIWKH\HDVW/&%HQFRGHVDFRPSRQHQWRI VHULQHSDOPLWR\OWUDQVIHUDVHWKHHQ]\PHFDWDO\]LQJWKH¿UVWVWHSLQVSKLQJROLSLGV\QWKHVLV-%LRO&KHP 1997; 272:32108-32114. 
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0DUJJUDI:'=HUWDQL5$QGHUHU)$HWDO7KHUROHRIHQGRJHQRXVSKRVSKDWLG\OFKROLQHDQGFHUDPLGH LQWKHELRV\QWKHVLVRIVSKLQJRP\HOLQLQPRXVH¿EUREODVWV%LRFKLP%LRSK\V$FWD 23. Malgat M, Maurice A, Baraud J. Sphingomyelin and ceramidephosphoethanolamine synthesis by microsomes DQGSODVPDPHPEUDQHVIURPUDWOLYHUDQGEUDLQ-/LSLG5HV YDQ+HOYRRUW$YDQ¶W+RI:5LWVHPD7HWDO&RQYHUVLRQRIGLDF\OJO\FHUROWRSKRVSKDWLG\OFKROLQHRQWKH EDVRODWHUDOVXUIDFHRIHSLWKHOLDO0DGLQ'DUE\FDQLQHNLGQH\ FHOOV(YLGHQFHIRUWKHUHYHUVHDFWLRQRID sphingomyelin synthase. J Biol Chem 1994; 269:1763-1769. $OODQ'2EUDGRUV0-(Q]\PHGLVWULEXWLRQVLQVXEFHOOXODUIUDFWLRQVRI%+.FHOOVLQIHFWHGZLWK6HPOLNL IRUHVWYLUXVHYLGHQFHIRUDPDMRUIUDFWLRQRIVSKLQJRP\HOLQV\QWKDVHLQWKHWUDQVJROJLQHWZRUN%LRFKLP Biophys Acta 1999; 1450:277-287. 26. Albi E, Magni MV. Sphingomyelin synthase in rat liver nuclear membrane and chromatin. FEBS Lett 1999; 460:369-372. $OEL(00&KURPDWLQDVVRFLDWHGVSKLQJRP\HOLQPHWDEROLVPLQUHODWLRQWRFHOOIXQFWLRQ&HOO%LRFKHP Funct 2003; 21:211-215. +XLWHPD.YDQGHQ'LNNHQEHUJ-%URXZHUV-)HWDO,GHQWL¿FDWLRQRIDIDPLO\RIDQLPDOVSKLQJRP\HOLQ synthases. Embo J 2004; 23:33-44. 
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/XFLF'+XDQJ=+*XGH6HWDO5HJXODWLRQRIPDFURSKDJHDSR(VHFUHWLRQDQGVWHUROHIÀX[E\WKH/'/ receptor. J Lipid Res 2007; 48:366-372. 6FKOLWW$+RMMDWL05YRQ*L]\FNL+HWDO6HUXPVSKLQJRP\HOLQOHYHOVDUHUHODWHGWRWKHFOHDUDQFHRI postprandial remnant-like particles. J Lipid Res 2005; 46:196-200. -HRQJ76FKLVVHO6/7DEDV,HWDO,QFUHDVHGVSKLQJRP\HOLQFRQWHQWRISODVPDOLSRSURWHLQVLQDSROLSRSURWHLQ ( NQRFNRXW PLFH UHÀHFWV FRPELQHG SURGXFWLRQ DQG FDWDEROLF GHIHFWV DQG HQKDQFHV UHDFWLYLW\ ZLWK mammalian sphingomyelinase. J Clin Invest 1998; 101:905-912. .ULHJHU0&KDUWLQJWKHIDWHRIWKH³JRRGFKROHVWHURO´LGHQWL¿FDWLRQDQGFKDUDFWHUL]DWLRQRIWKHKLJKGHQVLW\ lipoprotein receptor SR-BI. Annu Rev Biochem 1999; 68:523-558. 53. Van Eck M, Pennings M, Hoekstra M et al. Scavenger receptor BI and ATP-binding cassette transporter A1 in reverse cholesterol transport and atherosclerosis. Curr Opin Lipidol 2005; 16:307-315. %DUWHU3-%UHZHU+%-U&KDSPDQ0-HWDO&KROHVWHU\OHVWHUWUDQVIHUSURWHLQDQRYHOWDUJHWIRUUDLVLQJ HDL and inhibiting atherosclerosis. Arterioscler Thromb Vasc Biol 2003; 23:160-167. 6XEEDLDK39/LX05ROHRIVSKLQJRP\HOLQLQWKHUHJXODWLRQRIFKROHVWHUROHVWHUL¿FDWLRQLQWKHSODVPD OLSRSURWHLQV,QKLELWLRQRIOHFLWKLQFKROHVWHURODF\OWUDQVIHUDVHUHDFWLRQ-%LRO&KHP %ROLQ'--RQDV$6SKLQJRP\HOLQLQKLELWVWKHOHFLWKLQFKROHVWHURODF\OWUDQVIHUDVHUHDFWLRQZLWKUHFRQVWLWXWHG high density lipoproteins by decreasing enzyme binding. J Biol Chem 1996; 271:19152-19158. 5\H.$+LPH1-%DUWHU3-7KHLQÀXHQFHRIVSKLQJRP\HOLQRQWKHVWUXFWXUHDQGIXQFWLRQRIUHFRQVWLWXWHG high density lipoproteins. J Biol Chem 1996; 271:4243-4250. 7DOO$5&KROHVWHUROHIÀX[SDWKZD\VDQGRWKHUSRWHQWLDOPHFKDQLVPVLQYROYHGLQWKHDWKHURSURWHFWLYH HIIHFWRIKLJKGHQVLW\OLSRSURWHLQV-,QWHUQ0HG 6XEEDLDK39*HVTXLHUH/5:DQJ.5HJXODWLRQRIWKHVHOHFWLYHXSWDNHRIFKROHVWHU\OHVWHUVIURPKLJK density lipoproteins by sphingomyelin. J Lipid Res 2005; 46:2699-2705. 0DUTXHV9LGDO3-DXKLDLQHQ00HWVR-HWDO7UDQVIRUPDWLRQRIKLJKGHQVLW\OLSRSURWHLQSDUWLFOHVE\ KHSDWLFOLSDVHDQGSKRVSKROLSLGWUDQVIHUSURWHLQ$WKHURVFOHURVLV 5RVV57KHSDWKRJHQHVLVRIDWKHURVFOHURVLVDSHUVSHFWLYHIRUWKHV1DWXUH :LW]WXP-/6WHLQEHUJ'5ROHRIR[LGL]HGORZGHQVLW\OLSRSURWHLQLQDWKHURJHQHVLV-&OLQ,QYHVW 88:1785-1792. 
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(LVHQEHUJ66WHLQ<6WHLQ23KRVSKROLSDVHVLQDUWHULDOWLVVXH,97KHUROHRISKRVSKDWLGHDF\OK\GURODVH O\VRSKRVSKDWLGH DF\O K\GURODVH DQG VSKLQJRP\HOLQ FKROLQH SKRVSKRK\GURODVH LQ WKH UHJXODWLRQ RI phospholipid composition in the normal human aorta with age. J Clin Invest 1969; 48:2320-2329. 2NZX$.;X;;6KLUDWRUL<HWDO5HJXODWLRQRIWKHWKUHVKROGIRUOLSRSURWHLQLQGXFHGDF\O&R$FKROHVWHURO 2DF\OWUDQVIHUDVH VWLPXODWLRQ LQ PDFURSKDJHV E\ FHOOXODU VSKLQJRP\HOLQ FRQWHQW - /LSLG 5HV  35:644-655. 1RHO&0DUFHO
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&KULVWLDQVHQ . &DUOVHQ - 0LFURYLOOXV PHPEUDQH YHVLFOHV IURP SLJ VPDOO LQWHVWLQH 3XULW\ DQG OLSLG composition. Biochim Biophys Acta 1981; 647:188-195. 'DQLHOVHQ(0+DQVHQ*+/LSLGUDIWRUJDQL]DWLRQDQGIXQFWLRQLQWKHVPDOOLQWHVWLQDOEUXVKERUGHU J Physiol Biochem 2008; 64:377-382. %URZQ'$5RVH-.6RUWLQJRI*3,DQFKRUHGSURWHLQVWRJO\FROLSLGHQULFKHGPHPEUDQHVXEGRPDLQV GXULQJWUDQVSRUWWRWKHDSLFDOFHOOVXUIDFH&HOO )LHOG)-%RUQ(0XUWK\6HWDO&DYHROLQLVSUHVHQWLQLQWHVWLQDOFHOOVUROHLQFKROHVWHUROWUDI¿FNLQJ" J Lipid Res 1998; 39:1938-1950. *DUP\17DLHE1
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*HQJ <- /LEE\ 3 3URJUHVVLRQ RI DWKHURPD D VWUXJJOH EHWZHHQ GHDWK DQG SURFUHDWLRQ $UWHULRVFOHU Thromb Vasc Biol 2002; 22:1370-1380. 136. Separovic D, Hanada K, Maitah MY et al. Sphingomyelin synthase 1 suppresses ceramide production and apoptosis postphotodamage. Biochem Biophys Res Commun 2007; 358:196-202. 'LQJ7/L=+DLOHPDULDP7HWDO606RYHUH[SUHVVLRQDQGNQRFNGRZQLPSDFWRQFHOOXODUVSKLQJRP\HOLQ and diacylglycerol metabolism and cell apoptosis. J Lipid Res 2008; 49:376-385. 138. Ko YG, Lee JS, Kang YS et al. TNF-alphamediated apoptosis is initiated in caveolae-like domains. J Immunol 1999; 162:7217-7223. 7ULDQWD¿ORX 0 0L\DNH . *ROHQERFN '7 HW DO 0HGLDWRUV RI LQQDWH LPPXQH UHFRJQLWLRQ RI EDFWHULD FRQFHQWUDWH LQ OLSLG UDIWV DQG IDFLOLWDWH OLSRSRO\VDFFKDULGHLQGXFHG FHOO DFWLYDWLRQ - &HOO 6FL  115:2603-2611. 140. Cerbon J, del Carmen Lopez-Sanchez R. Diacylglycerol generated during sphingomyelin synthesis LVLQYROYHGLQSURWHLQNLQDVH&DFWLYDWLRQDQGFHOOSUROLIHUDWLRQLQ0DGLQ'DUE\FDQLQHNLGQH\FHOOV Biochem J 2003; 373:917-924. :DNHODP 0- 'LDF\OJO\FHURO²ZKHQ LV LW DQ LQWUDFHOOXODU PHVVHQJHU" %LRFKLP %LRSK\V $FWD  1436:117-126. *ULQHU(0.D]DQLHW]0*3URWHLQNLQDVH&DQGRWKHUGLDF\OJO\FHUROHIIHFWRUVLQFDQFHU1DW5HY&DQFHU 2007; 7:281-294. 0D\RU60D[¿HOG)5,QVROXELOLW\DQGUHGLVWULEXWLRQRI*3,DQFKRUHGSURWHLQVDWWKHFHOOVXUIDFHDIWHU detergent treatment. Mol Biol Cell 1995; 6:929-944. 0XUULHO&/0RFKO\5RVHQ'2SSRVLQJUROHVRIGHOWDDQGHSVLORQ3.&LQFDUGLDFLVFKHPLDDQGUHSHUIXVLRQ targeting the apoptotic machinery. Arch Biochem Biophys 2003; 420:246-254. 145. Hannun YA, Luberto C. Ceramide in the eukaryotic stress response. Trends Cell Biol 2000; 10:73-80. .ROHVQLFN57KHWKHUDSHXWLFSRWHQWLDORIPRGXODWLQJWKHFHUDPLGHVSKLQJRP\HOLQSDWKZD\-&OLQ,QYHVW 2002; 110:3-8. /LEE\3,QÀDPPDWLRQLQDWKHURVFOHURVLV1DWXUH 0D\R0:%DOGZLQ$67KHWUDQVFULSWLRQIDFWRU1)NDSSD%FRQWURORIRQFRJHQHVLVDQGFDQFHUWKHUDS\ resistance. Biochim Biophys Acta 2000; 1470:M55-62. %UDQHQ/+RYJDDUG/1LWXOHVFX0HWDO,QKLELWLRQRIWXPRUQHFURVLVIDFWRUDOSKDUHGXFHVDWKHURVFOHURVLV in apolipoprotein E knockout mice. Arterioscler Thromb Vasc Biol 2004; 24:2137-2142. :KLWPDQ 6& 5DYLVDQNDU 3 'DXJKHUW\ $ ,)1JDPPD GH¿FLHQF\ H[HUWV JHQGHUVSHFL¿F HIIHFWV RQ DWKHURJHQHVLVLQDSROLSRSURWHLQ(PLFH-,QWHUIHURQ&\WRNLQH5HV /HJOHU')0LFKHDX2'RXFH\0$HWDO5HFUXLWPHQWRI71)UHFHSWRUWROLSLGUDIWVLVHVVHQWLDOIRU TNFalphamediated NF-kappaB activation. Immunity 2003; 18:655-664. +XQWHU,1L[RQ*)6SDWLDOFRPSDUWPHQWDOL]DWLRQRIWXPRUQHFURVLVIDFWRU71) UHFHSWRUGHSHQGHQW VLJQDOLQJSDWKZD\VLQKXPDQDLUZD\VPRRWKPXVFOHFHOOV/LSLGUDIWVDUHHVVHQWLDOIRU71)DOSKDPHGLDWHG DFWLYDWLRQRI5KR$EXWGLVSHQVDEOHIRUWKHDFWLYDWLRQRIWKH1)NDSSD%DQG0$3.SDWKZD\V-%LRO Chem 2006; 281:34705-34715. +LJXFKL06LQJK6-DIIUH]RX-3HWDO$FLGLFVSKLQJRP\HOLQDVHJHQHUDWHGFHUDPLGHLVQHHGHGEXWQRW VXI¿FLHQWIRU71)LQGXFHGDSRSWRVLVDQGQXFOHDUIDFWRUNDSSD%DFWLYDWLRQ-,PPXQRO *DPDUG&-'EDLER*6/LX%HWDO6HOHFWLYHLQYROYHPHQWRIFHUDPLGHLQF\WRNLQHLQGXFHGDSRSWRVLV &HUDPLGHLQKLELWVSKRUEROHVWHUDFWLYDWLRQRIQXFOHDUIDFWRUNDSSD%-%LRO&KHP /XEHUWR&
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CHAPTER 3 HEART SPHINGOLIPIDS IN HEALTH AND DISEASE Marcin Baranowski* and Jan Górski 'HSDUWPHQWRI3K\VLRORJ\0HGLFDO8QLYHUVLW\RI%LDá\VWRN%LDá\VWRN3RODQG *Corresponding Author: Marcin Baranowski—Email: [email protected]

Abstract:

,QUHFHQW\HDUVWKHUROHRIVSKLQJROLSLGVLQSK\VLRORJ\DQGSDWKRSK\VLRORJ\RI WKH KHDUW DWWUDFWHG PXFK DWWHQWLRQ &HUDPLGH ZDV IRXQG WR EH LQYROYHG LQ WKH SDWKRJHQHVLVRIFDUGLDFG\VIXQFWLRQLQDQLPDOPRGHOVRILVFKHPLDUHSHUIXVLRQ LQMXU\7\SHGLDEHWHVDQGOLSRWR[LFFDUGLRP\RSDWK\2QWKHRWKHUKDQGDQRWKHU PHPEHURIWKLVOLSLGIDPLO\QDPHO\VSKLQJRVLQHSKRVSKDWHKDVEHHQVKRZQ to possess potent cardioprotective properties. This chapter provides a review RI WKH UROH RI FHUDPLGH DQG RWKHU ELRDFWLYH VSKLQJROLSLGV LQ SK\VLRORJ\ DQG SDWKRSK\VLRORJ\ RI WKH KHDUW :H GHVFULEH WKH UROH RI 33$5V DQG H[HUFLVH LQ UHJXODWLRQRIP\RFDUGLDOVSKLQJROLSLGPHWDEROLVP:HDOVRVXPPDUL]HWKHSUHVHQW VWDWHRINQRZOHGJHRQWKHLQYROYHPHQWRIFHUDPLGHDQGVSKLQJRVLQHSKRVSKDWH LQWKHGHYHORSPHQWDQGSUHYHQWLRQRILVFKHPLDUHSHUIXVLRQLQMXU\RIWKHKHDUW,Q WKHODVWVHFWLRQRIWKLVFKDSWHUZHGLVFXVVWKHHYLGHQFHIRUDUROHRIFHUDPLGHLQ myocardial lipotoxicity.

INTRODUCTION 6SKLQJROLSLGVZHUHGLVFRYHUHGRYHU\HDUVDJRDQGIRUPDQ\GHFDGHVZHUH FRQVLGHUHGWRVHUYHRQO\DVVWUXFWXUDOFRPSRQHQWVRIELRORJLFDOPHPEUDQHV1RZDGD\V PDQ\RIWKHPDUHNQRZQWREHKLJKO\ELRDFWLYHFRPSRXQGVWKDWSOD\DVLJQL¿FDQWUROHLQ VLJQDOWUDQVGXFWLRQDQGUHJXODWLRQRIDKRVWRIFHOOXODUSURFHVVHV&HUDPLGHWKHFHQWUDO PROHFXOHLQVSKLQJROLSLGVWUXFWXUHDQGPHWDEROLVPLVWKHEHVWVWXGLHGPHPEHURIWKLV IDPLO\,WZDV¿UVWUHFRJQL]HGDVDVHFRQGPHVVHQJHULQE\2ND]DNLHWDO1 who VKRZHGWKDWFHUDPLGHPHGLDWHVWKHHIIHFWRI_,25-dihydroxyvitamin D3 on HL-60 cell GLIIHUHQWLDWLRQ&HUDPLGHLVDOVRDSUHFXUVRUIRURWKHUELRDFWLYHVSKLQJROLSLGVLQFOXGLQJ

Sphingolipids and Metabolic Disease, edited by L. Ashley Cowart. ©2011 Landes Bioscience and Springer Science+Business Media, LLC 41

42

SPHINOGLIPIDS AND METABOLIC DISEASE

ceramide-1-phosphate, sphingosine and sphingosine-1-phosphate (S1P) (Fig. 1). Intensive studies conducted over the next two decades revealed that sphingolipids UHJXODWH QXPHURXV FHOOXODU SURFHVVHV VXFK DV FHOO SUROLIHUDWLRQ GLIIHUHQWLDWLRQ DQG apoptosis as well as responses to cytokines and stress.2 Ceramide has also emerged as DSXWDWLYHPHGLDWRURIPXVFOHLQVXOLQUHVLVWDQFHDQGOLSRWR[LFLW\LQFHUWDLQFHOOW\SHV including cardiomyocytes.3,QDGGLWLRQWKHUROHRIVSKLQJROLSLGVLQWKHSDWKRJHQHVLV RIFDUGLDFG\VIXQFWLRQLQLVFKHPLDUHSHUIXVLRQLQMXU\7\SHGLDEHWHVDQGREHVLW\KDV recently attracted much attention.

Figure 1. 6FKHPDWLF UHSUHVHQWDWLRQ RI FHUDPLGH PHWDEROLVP 637VHULQH SDOPLWR\OWUDQVIHUDVH SM-sphingomyelin, SMase-sphingomyelinase, CDase-ceramidase, SPHK-sphingosine kinase, S1P-sphingosine-1-phosphate.

HEART SPHINGOLIPIDS IN HEALTH AND DISEASE

43

CHARACTERIZATION OF MYOCARDIAL SPHINGOLIPID METABOLISM %RWK FDUGLDF DQG VNHOHWDO PXVFOH H[SUHVVHV GLIIHUHQW HQ]\PHV DQG UHFHSWRUV LQYROYHG LQ VSKLQJROLSLG PHWDEROLVP DQG FDQ WKHUHIRUH V\QWKHVL]H DQG UHVSRQG WR GLIIHUHQWELRDFWLYHVSKLQJROLSLGV7KHUHDUHKRZHYHUFRQVLGHUDEOHGLIIHUHQFHVEHWZHHQ myocardial and skeletal muscle sphingolipid metabolism. As shown on Table 1, the rat KHDUWLVFKDUDFWHUL]HGE\DSSUR[LPDWHO\IROGKLJKHUFRQWHQWRIVSKLQJRVLQH63DQG VSKLQJRP\HOLQDVFRPSDUHGWRWKHVROHXVPXVFOHZKHUHDVWKHOHYHORIFHUDPLGHLVYLUWXDOO\ WKHVDPH7KHDFWLYLW\RIPRVWRIWKHHQ]\PHVLQYROYHGLQVSKLQJROLSLGPHWDEROLVPLV DOVRPXFKKLJKHULQWKHP\RFDUGLXP:HREVHUYHGVLPLODUGLIIHUHQFHVLQWKHFRQWHQWRI sphingoid bases also between human cardiac and skeletal muscle (see Table 1). ,QDGGLWLRQFRQVLGHUDEOHVSHFLHVGLIIHUHQFHVDUHIRXQGLQP\RFDUGLDOVSKLQJROLSLG metabolism. As compared to rat myocardium, human heart is characterized by similar level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

Table 1.6SKLQJROLSLGFRQWHQWDQGDFWLYLW\RIHQ]\PHVUHODWHGWRFHUDPLGHPHWDEROLVP in heart vs skeletal muscle Rat

Human

Soleus

Heart

Vastus Lateralis

Heart

&RQWHQWRIVSKLQJROLSLGV (pmol/mg) Sphingosine Sphinganine Sphingosine-1P Ceramide Sphingomyelin

1.0 0.38 0.25 25 316

2.2 0.55 0.49 23 645

0.33 0.06 0.06 16

1.3 0.14 0.14 17

(Q]\PHDFWLYLW\QPRORI SURGXFWKPJRISURWHLQ SPT al-CDase n-CDase a-CDase n-SMase a-SMase

0.59 1.5 1.2 not detected 16.5 23

0.94 6.0 8.0 2.7 6.6 125

637VHULQH SDOPLWR\OWUDQVIHUDVH DO&'DVHDONDOLQH FHUDPLGDVH Q&'DVHQHXWUDO FHUDPLGDVH a-CDase-acid ceramidase, n-SMase-neutral sphingomyelinase, a-SMase-acid sphingomyelinase. &UHDWHGIURPGDWDLQ0%DUDQRZVNLHWDO-3K\VLRO3KDUPDFRO0%DUDQRZVNLHWDO-/LSLG 5HV%áDFKQLR=DELHOVNDHWDO-&HOO%LRFKHPDQGIURPXQSXEOLVKHGUHVXOWVRI0%DUDQRZVNL

44

SPHINOGLIPIDS AND METABOLIC DISEASE

human but not murine heart.6,7:HIRXQGVLPLODUVSHFLHVGLIIHUHQFHVDOVRIRUH[SUHVVLRQ RIVSKLQJRVLQHNLQDVHLVRIRUPV5 Our data indicate that sphingosine kinase (SPHK) 1 is the dominant subtype in the human myocardium, whereas the rodent heart expresses predominantly SPHK2.8 0RUHRYHUZHKDYHUHFHQWO\VKRZQVLJQL¿FDQWUHJLRQDOGLIIHUHQFHVLQVSKLQJROLSLG metabolism in the human heart.5,QSDWLHQWVXQGHUJRLQJFRURQDU\E\SDVVJUDIWVXUJHU\ SDSLOODU\PXVFOHRIWKHPLWUDODSSDUDWXVZDVFKDUDFWHUL]HGE\ORZHUOHYHORIFHUDPLGH sphinganine and S1P as compared to the right atrial appendage. On the other hand, expression RIHQ]\PHVLQYROYHGLQERWKFHUDPLGHV\QWKHVLVVHULQHSDOPLWR\OWUDQVIHUDVH²637 DQG degradation (ceramidases, sphingosine kinases) was markedly higher in the papillary muscle which suggests higher ceramide turnover in the ventricular tissue. However, SK\VLRORJLFDO DQG SDWKRSK\VLRORJLFDO FRQVHTXHQFHV RI VSHFLHV DV ZHOO DV UHJLRQDO GLIIHUHQFHVLQP\RFDUGLDOVSKLQJROLSLGPHWDEROLVPUHPDLQREVFXUH PPARs AS REGULATORS OF SPHINGOLIPID METABOLISM IN THE HEART $OWKRXJKWKHUROHRIVSKLQJROLSLGVLQWKHKHDUWKDVUHFHQWO\DWWUDFWHGPXFKDWWHQWLRQ UHJXODWLRQ RI P\RFDUGLDO VSKLQJROLSLG PHWDEROLVP LV RQO\ SRRUO\ LQYHVWLJDWHG 7KHUH LVKRZHYHUVRPHHYLGHQFHLQGLFDWLQJWKDWSHUR[LVRPHSUROLIHUDWRUDFWLYDWHGUHFHSWRUV 33$5V SOD\DVLJQL¿FDQWUROHLQWKLVSURFHVV33$5VDUHOLJDQGDFWLYDWHGWUDQVFULSWLRQ IDFWRUVRIWKHQXFOHDUKRUPRQHUHFHSWRUVXSHUIDPLO\7KUHHGLVWLQFW33$5LVRIRUPVWHUPHG _, b and aKDYHEHHQGHVFULEHG7KHIRUPHUWZRDUHKLJKO\H[SUHVVHGLQWKHKHDUWDQGDUH FRQVLGHUHGWREHNH\WUDQVFULSWLRQDOUHJXODWRUVRIIDWW\DFLGPHWDEROLVPLQFDUGLRP\RF\WHV9 Finck et al10GHPRQVWUDWHGWKDWKLJKIDWIHHGLQJRIPLFHZLWKFDUGLDFVSHFL¿FRYHUH[SUHVVLRQ RI33$5_OHDGVWRDFFXPXODWLRQRIFHUDPLGHLQWKHP\RFDUGLXP6XFKHIIHFWZDVQRW observed in wild type animals which suggests that PPAR_ may be involved in regulation RIP\RFDUGLDOFHUDPLGHPHWDEROLVP7RLQYHVWLJDWHWKHPHFKDQLVPRIWKLVSKHQRPHQRQZH H[DPLQHGHIIHFWVRI:<DVHOHFWLYH33$5_ agonist) on sphingolipid metabolism in the rat heart.11$FWLYDWLRQRI33$5_LQKLJKIDWIHGUDWVUHVXOWHGLQDFFXPXODWLRQRI P\RFDUGLDOFHUDPLGHDQGVSKLQJRP\HOLQ7KLVHIIHFWZDVUHODWHGWRVWLPXODWLRQRIWKH GHQRYRVSKLQJROLSLGV\QWKHVLVDVHYLGHQFHGE\HOHYDWHGDFWLYLW\RI637DQGLQFUHDVHG DYDLODELOLW\ RI LQWUDP\RFDUGLDO SDOPLWDWH (Q]\PHV LQYROYHG LQ RWKHU SDWKZD\V LH VSKLQJRP\HOLQDVHVDQGFHUDPLGDVHVZHUHQRWDIIHFWHG7KHPHFKDQLVPRI:< action on SPT activity is unclear. PPAR_ stimulation was shown to upregulate expression RI637LQUHFRQVWUXFWHGKXPDQHSLGHUPLV12ZKLFKVXJJHVWVLQYROYHPHQWRIDWUDQVFULSWLRQDO regulation. However, it remains obscure whether PPAR_DIIHFWV637H[SUHVVLRQGLUHFWO\ or via changes in cellular lipid metabolism. The latter hypothesis is supported by the IDFWWKDWLQVWDQGDUGFKRZIHGUDWVZKLFKGLGQRWVKRZDFFXPXODWLRQRIP\RFDUGLDOIUHH palmitate upon PPAR_ activation, WY-14643 did not increase either ceramide content or SPT activity.11 We also observed a modest increase in ceramide content in the heart RIUDWVWUHDWHGZLWKDVHOHFWLYH33$5b agonist (GW0742) (M. Baranowski, unpublished REVHUYDWLRQ 7KLVLQFUHDVHZDVDFFRPSDQLHGE\HOHYDWLRQLQWKHOHYHORIVSKLQJDQLQH DQGORQJFKDLQIDWW\DF\O&R$ZKLFKVXJJHVWVWKDWWKHUDWHRIGHQRYRFHUDPLGHV\QWKHVLV pathway was augmented upon PPARb activation. ([SUHVVLRQRI33$5a in cardiomyocytes is very low and it is the least investigated RIDOOP\RFDUGLDO33$5LVRIRUPV9 Several studies demonstrated that thiazolidinediones,

HEART SPHINGOLIPIDS IN HEALTH AND DISEASE

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which are selective PPARaDFWLYDWRUVLQGXFHH[SUHVVLRQRIJOXFRVHWUDQVSRUWHUDQG and increase basal and insulin-stimulated glucose uptake in cultured rat cardiomyocytes DVZHOODVLQWKHKHDUWRIGLDEHWLFDQGLQVXOLQUHVLVWDQWURGHQWV13-17=KRXHWDO18IRXQGWKDW DGPLQLVWUDWLRQRIWURJOLWD]RQHWR=XFNHUGLDEHWLFIDWW\=') UDWVUHGXFHGDFFXPXODWLRQ RIP\RFDUGLDOFHUDPLGH7KLVSURPSWHGXVWRH[DPLQHHIIHFWRI33$5a activation on myocardial sphingolipid metabolism. To our surprise, pioglitazone administration LQFUHDVHGFHUDPLGHOHYHOLQWKHKHDUWRIUDWVIHGHLWKHUVWDQGDUGFKRZRUDKLJKIDWGLHW +RZHYHULWVKRXOGEHQRWHGWKDWWKLVHIIHFWZDVPRUHSURQRXQFHGLQWKHODWWHUJURXS$ SODXVLEOHH[SODQDWLRQRIWKHGLVFUHSDQF\EHWZHHQRXUUHVXOWVDQGWKRVHRI=KRXHWDOLVWKDW thiazolidinediones lower cardiac ceramide level only in chronically obese and diabetic DQLPDOVZKLFKVKRZH[FHVVLYHOLSLGGHSRVLWLRQLQWKHKHDUW$FFXPXODWLRQRIP\RFDUGLDO FHUDPLGHREVHUYHGLQRXUVWXG\ZDVOLNHO\DUHVXOWRILWVDXJPHQWHGV\QWKHVLVGHQRYR 7KLVLVVXSSRUWHGE\WKHIDFWWKDWDGPLQLVWUDWLRQRI33$5a agonist did not produce evident FKDQJHVLQVSKLQJRP\DOLQDVHRUFHUDPLGDVHDFWLYLW\ZKHUHDVWKHDFWLYLW\RI637DQGWKH DYDLODELOLW\RILQWUDFHOOXODUSDOPLWDWHZDVPDUNHGO\LQFUHDVHGLQUDWVIHGRQHLWKHUGLHW 3LRJOLWD]RQHDOVRLQGXFHGDPRGHVWHOHYDWLRQLQWKHFRQWHQWRI637SURWHLQ+RZHYHULW GLGQRWPDWFKWKHLQFUHDVHLQHQ]\PHDFWLYLW\WKXVVXJJHVWLQJWKDWWKHHIIHFWRI33$5a DJRQLVWZDVSUHGRPLQDQWO\DUHVXOWRISRVWUDQVODWLRQDOPRGL¿FDWLRQRI637SURWHLQ,W ZDVVKRZQWKDWDFWLYLW\RIWKLVHQ]\PHFDQEHPRGXODWHGLQGHSHQGHQWO\RIFKDQJHVLQ WKHOHYHORILWVP51$RUSURWHLQ19 However, our data do not exclude a possibility that SLRJOLWD]RQHDIIHFWVWKHH[SUHVVLRQRIWKLVHQ]\PHDWWKHWUDQVFULSWLRQDOOHYHO,WLVZLGHO\ DFFHSWHGWKDWLQFUHDVHGDYDLODELOLW\RISDOPLWDWHLQGXFHVDFFXPXODWLRQRIFHUDPLGHLQ cells due to its augmented synthesis de novo.3 However, in vitro studies on rat astrocytes and pancreatic islets showed that incubation with palmitate also increases activity and H[SUHVVLRQRI63720,217KHVHGDWDLQGLFDWHWKDWSDOPLWDWHLQGXFHGDFFXPXODWLRQRIFHUDPLGH LVQRWVROHO\DUHVXOWRILQFUHDVHGDYDLODELOLW\RIVXEVWUDWHIRULWVV\QWKHVLVGHQRYREXWLV DOVRDFRQVHTXHQFHRIDFWLYDWLRQRIWKHUDWHOLPLWLQJHQ]\PHLQWKLVSDWKZD\7KHUHIRUH LWLVOLNHO\WKDWWKHVWLPXODWRU\HIIHFWRISLRJOLWD]RQHRQ637REVHUYHGLQRXUVWXG\ZDV UHODWHGWRDFFXPXODWLRQRIIUHHSDOPLWDWHHYRNHGE\33$5a agonist. Collectively, the results RIRXUH[SHULPHQWVLQGLFDWHWKDW33$5VUHJXODWHP\RFDUGLDOVSKLQJROLSLGPHWDEROLVP SUHGRPLQDQWO\DWWKHOHYHORIGHQRYRV\QWKHVLVVHH7DEOH ,QWHUHVWLQJO\DOOWKUHH 33$5LVRIRUPVVHHPWRDFWLYDWHWKLVSDWKZD\LQWKHKHDUWDWOHDVWLQRXUDQLPDOPRGHOV EXERCISE MODULATES MYOCARDIAL CERAMIDE METABOLISM :H IRXQG WKDW H[HUFLVH H[HUWV SURIRXQG HIIHFWV RQ WKH P\RFDUGLDO FRQWHQW RI VSKLQJROLSLGVDQGDIIHFWVPDQ\SDWKZD\VRIWKHLUPHWDEROLVP4,QWHUHVWLQJO\WKLVHIIHFW WR D ODUJH H[WHQW GHSHQGHG RQ H[HUFLVH GXUDWLRQ :H IRXQG WKDWPLQXWH EXUVW RI H[HUFLVHGHFUHDVHGP\RFDUGLDOFRQWHQWRIFHUDPLGH+RZHYHULWVOHYHOUHWXUQHGWRWKH EDVHOLQHDIWHUPLQXWHVRIUXQQLQJDQGLQFUHDVHGIXUWKHUDWWKHSRLQWRIH[KDXVWLRQ VLJQL¿FDQWO\H[FHHGLQJWKHFRQWHQWIRXQGLQWKHFRQWURODQLPDOV)LJ ,QWHUHVWLQJO\ LW ZDV UHFHQWO\ UHSRUWHG WKDW HQGXUDQFH WUDLQLQJ FRQVLVWLQJ RI PLQXWH H[HUFLVH bouts reduced ceramide level in the murine heart to a similar extent as observed in our study.22$QDO\VHVRIWKHDFWLYLWLHVRIHQ]\PHVLQYROYHGLQFHUDPLGHPHWDEROLVP revealed that the initial reduction in its content observed in our experiment was likely D UHVXOW RI DXJPHQWHG FHUDPLGH GHJUDGDWLRQ VLQFH D FRQFRPLWDQW HOHYDWLRQ LQ WKH DFWLYLW\RIDFLGFHUDPLGDVHDQGWKHOHYHORIVSKLQJRVLQHZDVREVHUYHG7KHVXEVHTXHQW

46

SPHINOGLIPIDS AND METABOLIC DISEASE

Table 2.6XPPDU\RIHIIHFWVLQGXFHGE\DFWLYDWLRQRIGLVWLQFW33$5LVRIRUPVRQ myocardial sphingolipid metabolism PPAR_

PPARb

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High-Fat Diet

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PPARa Standard High-Fat Chow Diet

&RQWHQWRI sphingolipids Sphingosine Sphinganine Sphingosine-1P Ceramide Sphingomyelin

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Enzyme activity SPT al-CDase n-CDase a-CDase n-SMase a-SMase

33$5SHUR[LVRPHSUROLIHUDWRUDFWLYDWHGUHFHSWRU637VHULQHSDOPLWR\OWUDQVIHUDVHDO&'DVHDONDOLQH ceramidase; n-CDase, neutral ceramidase; a-CDase, acid ceramidase; n-SMase, neutral sphingomyelinase; a-SMase, acid sphingomyelinase; CQRVLJQL¿FDQWFKDQJHB, increase; ?, decrease. Created IURPGDWDLQ0%DUDQRZVNLHWDO-3K\VLRO3KDUPDFRO0%DUDQRZVNLHWDO3URVWDJODQGLQV 2WKHU/LSLG0HGLDWDQGIURPXQSXEOLVKHGUHVXOWVRI0%DUDQRZVNL

WUDQVLWLRQIURPLQLWLDOGHFUHDVHLQFHUDPLGHFRQWHQWWRLWVDFFXPXODWLRQDWWKHSRLQWRI H[KDXVWLRQZDVDFRQVHTXHQFHRIJUDGXDOUHGXFWLRQLQWKHDFWLYLW\RIDFLGFHUDPLGDVH DQGVLPXOWDQHRXVLQFUHDVHLQWKHUDWHRIGHQRYRFHUDPLGHV\QWKHVLVDVHYLGHQFHGE\ SURJUHVVLYHDFWLYDWLRQRI637DQGDFFXPXODWLRQRIVSKLQJDQLQH)LJ 7KLVHIIHFWFRXOG EHDUHVXOWRILQFUHDVHGSODVPDQRQHVWHUL¿HGIDWW\DFLG1()$ FRQFHQWUDWLRQZKLFK occurred during prolonged exercise. As already mentioned in the previous section, in YLWURVWXGLHVVKRZHGWKDWLQFXEDWLRQRIWKHFHOOVZLWKSDOPLWDWHLQFUHDVHVDFWLYLW\DQG H[SUHVVLRQRI637,QWHUHVWLQJO\LQRXUVWXG\WKHFKDQJHVLQWKHP\RFDUGLDODFWLYLW\ RI637UHÀHFWHGWKRVHRISODVPD1()$FRQFHQWUDWLRQ It should be mentioned that exercise until exhaustion increased not only SPT activity EXWDOVRFRQWHQWRI637SURWHLQ)LJ +RZHYHUWKHPDJQLWXGHRILQFUHDVHLQWKH HQ]\PHDFWLYLW\ZDVRYHUIROGKLJKHUWKDQWKDWRIHQ]\PHSURWHLQFRQWHQW0RUHRYHU 637P51$OHYHOZDVQRWDIIHFWHGE\WUHDGPLOOUXQQLQJ7DNLQJWRJHWKHULWLQGLFDWHVWKDW H[HUFLVHLQGXFHGDFWLYDWLRQRI637ZDVQRWDUHVXOWRILWVLQFUHDVHGH[SUHVVLRQEXWUDWKHU DFRQVHTXHQFHRISRVWWUDQVODWLRQDOPRGL¿FDWLRQRIVHULQHSDOPLWR\OWUDQVIHUDVHSURWHLQ $FFXPXODWLRQRIVSKLQJRVLQHLVWKRXJKWWREHDQLPSRUWDQWIDFWRUFRQWULEXWLQJWRWKH GHYHORSPHQWRIPXVFOHIDWLJXH23 We showed previously that exercise until exhaustion LQGXFHGDFFXPXODWLRQRIWKLVVSKLQJRLGEDVHLQUDWVNHOHWDOPXVFOHV24 We observed a VLPLODUDOWKRXJKPXFKOHVVSURQRXQFHGHIIHFWDOVRLQWKHKHDUW4 There are many reports showing that sphingosine and to a lesser extent also sphinganine, reduce contractility

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RIFDUGLRP\RF\WHVE\EORFNLQJFDOFLXPUHOHDVHWKURXJKWKHVDUFRSODVPDWLFUHWLFXOXP ryanodine receptor.25,26,WZDVIRXQGWKDWSURORQJHGH[HUFLVHPD\LQGXFHDUHGXFWLRQLQ OHIWYHQWULFXODUV\VWROLFIXQFWLRQDSKHQRPHQRQRIWHQFDOOHGH[HUFLVHLQGXFHGFDUGLDF IDWLJXH27 ,Q YLHZ RI WKH DERYH GDWD LW LV WHPSWLQJ WR VSHFXODWH WKDW DFFXPXODWLRQ RI VSKLQJRVLQHDQGVSKLQJDQLQHFDQFRQWULEXWHWRWKHGHYHORSPHQWRIIDWLJXHQRWRQO\LQ VNHOHWDOPXVFOHEXWDOVRLQWKHKHDUW+RZHYHUIXUWKHUVWXGLHVDUHUHTXLUHGWRGHWHUPLQH WKHSK\VLRORJLFDOUHOHYDQFHRIWKLVHIIHFW SPHINGOLIPIDS IN ISCHEMIA/REPERFUSION INJURY OF THE HEART Krown et al28ZHUHWKH¿UVWWRGHPRQVWUDWHWKDWFHOOSHUPHDEOH&2-ceramide induces DSRSWRWLFFHOOGHDWKLQFDUGLRP\RF\WHV6KRUWO\DIWHUWKLVGLVFRYHU\%LHODZVNDHWDO29 XVLQJWKHUDWFRURQDU\DUWHU\RFFOXVLRQPRGHOIRXQGWKDWLVFKHPLFP\RFDUGLXPZDV FKDUDFWHUL]HGE\LQFUHDVHGFHUDPLGHOHYHOWKDWZDVIXUWKHUHOHYDWHGXSRQVXEVHTXHQW UHSHUIXVLRQ 7KLV VWXG\ SURYLGHG WKH ¿UVW HYLGHQFH WKDW FHUDPLGH PD\ EH LQYROYHG LQ LQGXFWLRQ RI FDUGLRP\RF\WH DSRSWRVLV E\ LVFKHPLDUHSHUIXVLRQ LQMXU\ 7KLV REVHUYDWLRQZDVODWHUFRQ¿UPHGE\RXUDQGRWKHUJURXSVLQSHUIXVHGUDWKHDUWDQGLQ the rabbit myocardium in vivo.30-33 However, the mechanism underlying ischemia/ UHSHUIXVLRQLQGXFHG FHUDPLGH DFFXPXODWLRQ UHPDLQV XQFOHDU 7KHUH LV VRPH GDWD LQGLFDWLQJ WKDW LQFUHDVHG FHUDPLGH SURGXFWLRQ IURP VSKLQJRP\HOLQ LV UHVSRQVLEOH IRU WKLV HIIHFW $UJDXG HW DO32 IRXQG WKDW SUHWUHDWPHQW ZLWK VSKLQJRP\HOLQDVH LQKLELWRU²'FRPSOHWHO\SUHYHQWHGLVFKHPLDLQGXFHGDFFXPXODWLRQRIP\RFDUGLDO FHUDPLGH DQG UHGXFHG FDUGLRP\RF\WH DSRSWRVLV LQ UDEELWV 0RUHRYHU GHSOHWLRQ RI VSKLQJRP\HOLQSRROZDVUHSRUWHGLQLVFKHPLFUHSHUIXVHGUDWKHDUW31 In line with the DERYHREVHUYDWLRQVK\SR[LDUHR[\JHQDWLRQZDVIRXQGWRUDSLGO\DFWLYDWHQHXWUDOEXW not acid) sphingomyelinase in neonatal rat cardiomyocytes.34 Interestingly, pretreatment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n our experiment, IPC resulted LQPDUNHGUHGXFWLRQLQWKHLVFKHPLDUHSHUIXVLRQLQGXFHGDFFXPXODWLRQRIFHUDPLGHLQ WKHSHUIXVHGUDWKHDUW7KHVHUHVXOWVZHUHFRQIRUPHGE\$UJDXGHWDO32 who showed that IPC prevented elevation in ceramide content in ischemic rabbit myocardium. 63KDVUHFHQWO\DWWUDFWHGPXFKDWWHQWLRQDVDQLPSRUWDQWIDFWRUSURWHFWLQJWKHKHDUW DJDLQVWLVFKHPLDUHSHUIXVLRQLQMXU\63LVQRWRQO\DQLQWUDFHOOXODUPHVVHQJHULQIDFWPRVW RILWVHIIHFWVDUHH[HUWHGE\ELQGLQJWRDIDPLO\RISODVPDPHPEUDQH*SURWHLQFRXSOHG UHFHSWRUV635V 63LVQRUPDOO\IRXQGLQKLJKQDQRPRODUFRQFHQWUDWLRQVLQKXPDQ and rodent plasma.35 Interestingly, this mediator acts also in an autocrine and/or paracrine IDVKLRQDVHQGRJHQRXV63FDQEHWUDQVSRUWHGWRWKHH[WUDFHOOXODUVSDFHPRVWOLNHO\ZLWK WKHKHOSRIFDVVHWWHWUDQVSRUWHUV36)LYHVXEW\SHVRIWKH63UHFHSWRUKDYHEHHQLGHQWL¿HG (S1PR1-5). Cardiomyocytes express S1PR1, S1PR2 and S1PR3KRZHYHUWKH¿UVWVXEW\SH ZDVIRXQGWREHSUHGRPLQDQWLQWKHVHFHOOV37

HEART SPHINGOLIPIDS IN HEALTH AND DISEASE

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Karliner et al38SURYLGHGWKH¿UVWHYLGHQFHIRUFDUGLRSURWHFWLYHHIIHFWRI637KH\ VKRZHGWKDWSUHLQFXEDWLRQRIUDWQHRQDWDOFDUGLRP\RF\WHVZLWK63RUWKHJDQJOLRVLGH *0ZKLFKVWLPXODWHVHQGRJHQRXV63SURGXFWLRQYLDDFWLYDWLRQRI63+.SUHYHQWV K\SR[LDLQGXFHGFHOOGHDWK&DUGLRSURWHFWLYHDFWLRQRIH[WUDFHOOXODU63DQG*0ZDV VXEVHTXHQWO\FRQ¿UPHGLQURGHQWPRGHOVRILVFKHPLDUHSHUIXVLRQLQMXU\39,40 The early study by Jin et al40VXJJHVWHGWKDWH[RJHQRXVDQGHQGRJHQRXV63H[HUWVLWVHIIHFWV WKURXJKGLVWLQFWSDWKZD\V7KH\IRXQGWKDWFDUGLRSURWHFWLRQDIIRUGHGE\LQWUDFHOOXODU S1P (generated in response to GM-1) required PKC¡ZKHUHDVWKHDFWLRQRIH[RJHQRXV S1P did not. However, it was recently reported that pertussis toxin (preventing G proteins IURPLQWHUDFWLQJZLWK*SURWHLQFRXSOHGUHFHSWRUV DVZHOODVWKH6351/3 antagonist abolished GM-1 mediated cardioprotection.41 These data indicate that intracellulary SURGXFHG63LVH[SRUWHGIURPFDUGLRP\RF\WHVDQGH[HUWVLWVSURWHFWLYHDFWLRQYLDFHOO VXUIDFH635V7KHUHIRUH3.&¡ seems to mediate GM-1 induced SPHK activation UDWKHUWKDQDFWLRQRILQWUDFHOOXODU63,QWHUHVWLQJO\LQWKHSHUIXVHGUDWKHDUWLVFKHPLD PDUNHGO\LQKLELWHG63+.DFWLYLW\DQGUHGXFHGP\RFDUGLDO63FRQWHQWDQGWKLVHIIHFW ZDV PDLQWDLQHG RYHU WKH SHULRG RI VXEVHTXHQW UHSHUIXVLRQ42 Consistently, deletion RI WKH 63+. JHQH LQFUHDVHG VXVFHSWLELOLW\ RI WKH KHDUW WR LVFKHPLDUHSHUIXVLRQ LQMXU\43 ZKHUHDV DGHQRYLUXVPHGLDWHG 63+. JHQH WUDQVIHU ZDV IRXQG WR LQGXFH SURWHFWLYHHIIHFWDQGDWWHQXDWHSRVWLVFKHPLFKHDUWIDLOXUH44 It was also suggested that FDUGLRSURWHFWLYHSURSHUWLHVRIKLJKGHQVLW\OLSRSURWHLQ+'/ LQYROYH63DVPRVWRI plasma S1P is contained within HDL.45,QWHUHVWLQJO\VSKLQJRVLQHLQIXVHGDWSK\VLRORJLFDO FRQFHQWUDWLRQVKLJKFRQFHQWUDWLRQVDUHFDUGLRWR[LF DOVRH[HUWVSURWHFWLYHHIIHFWLQWKH SHUIXVHGUDWKHDUW+RZHYHUWKHPHFKDQLVPRILWVDFWLRQLVLQGHSHQGHQWRI635VDQG involves cyclic nucleotide-dependent pathways.46 There is a controversy as to which S1PR subtype mediates the cardioprotective action RI63([SHULPHQWVPDGHE\.DUOLQHU¶VJURXSVXJJHVWHGSUHGRPLQDQWUROHRI6351. They IRXQGWKDWDQWLERG\ZKLFKIXQFWLRQVDVD6351VSHFL¿FDJRQLVWDVZHOODVDV\QWKHWLF S1PR1DFWLYDWRUSURWHFWHGDGXOWPRXVHFDUGLRP\RF\WHVIURPK\SR[LDWRWKHVDPHH[WHQW as exogenous S1P.47 In addition, the S1PR1 antagonist VPC23019 blocked protection DIIRUGHGE\63RULWVV\QWKHWLFDQDORJ)7<7KHHIIHFWRI63ZDVPHGLDWHGE\D SKRVSKDWLG\OLQRVLWRONLQDVHDQGOLNHO\LQYROYHGDFWLYDWLRQRI$NW3.%DQGLQKLELWLRQRI glycogen synthase kinase-3`. However, reports by other groups indicate that S1PR2 and/or S1PR3 rather than S1PR1PHGLDWHWKHFDUGLRSURWHFWLYHDFWLRQRI630HDQVHWDO48IRXQG that S1PR2/3 double knockout mice are characterized by markedly increased myocardial LQIDUFWVL]HIROORZLQJLVFKHPLDUHSHUIXVLRQWKDWLVDFFRPSDQLHGE\LPSDLUHGDFWLYDWLRQ RI$NW3.%,QWHUHVWLQJO\QHLWKHU6352 nor S1PR3 deletion alone augmented ischemia/ UHSHUIXVLRQLQMXU\GHPRQVWUDWLQJVRPHUHGXQGDQF\LQ63UHFHSWRUIXQFWLRQ,QDGGLWLRQ Theilmeier et al49 reported that S1P-mediated cardioprotection was completely absent in S1PR3GH¿FLHQWPLFH,WZDVDOVRDEROLVKHGE\SKDUPDFRORJLFDOQLWULFR[LGHV\QWKDVH LQKLELWLRQLPSOLFDWLQJDFUXFLDOUROHRIQLWULFR[LGHLQWKLVSDWKZD\5HFHQWO\/HYNDX¶V JURXS GHPRQVWUDWHG WKDW PLFH ZLWK FDUGLDFVSHFL¿F 6351 GH¿FLHQF\ DUH VXVFHSWLEOH WRLVFKHPLDUHSHUIXVLRQLQMXU\WRWKHVDPHH[WHQWDVZLOGW\SHDQLPDOVZKLFKVWURQJO\ DUJXHVDJDLQVWWKHLQYROYHPHQWRIWKLVUHFHSWRULQ63LQGXFHGFDUGLRSURWHFWLRQ37 The discrepancy between the results by Karliner’s group and those by other investigators PD\UHVXOWIURPGLIIHUHQFHVLQH[SHULPHQWDOPRGHOVVLQFHSURWHFWLYHUROHRI6351 was demonstrated on isolated cardiomyocytes, whereas S1PR2/3-mediated cardioprotection ZDVUHSRUWHGLQWKHLQWDFWKHDUW7KLVQRWLRQLVVXSSRUWHGE\DUHFHQWVWXG\E\+RIPDQQ

50

SPHINOGLIPIDS AND METABOLIC DISEASE

et al50 who demonstrated that selective S1PR1DJRQLVWH[HUWVSURWHFWLYHHIIHFWLQQHRQDWDO UDWFDUGLRP\RF\WHVEXWQRWLQSHUIXVHGUDWKHDUW,QIDFWRQHUHSRUWLQGLFDWHGWKDW6351 VWLPXODWLRQH[DJJHUDWHVP\RFDUGLDOLVFKHPLDUHSHUIXVLRQLQMXU\51 63ZDVDOVRIRXQGWRPHGLDWH,3&LQWKHKHDUW63+.LVDFWLYDWHGDQGWUDQVORFDWHG to the plasma membrane in response to IPC52 which to a large extent prevents decrease in WKHHQ]\PHDFWLYLW\DQGP\RFDUGLDO63OHYHOXSRQLVFKHPLDUHSHUIXVLRQ42 Consistently, SKDUPDFRORJLFDORUJHQHWLFLQKLELWLRQRI63+.DEROLVKHG,3&LQGXFHGFDUGLRSURWHFWLRQ in the murine heart.43,52 A recent report by Vessey et al53 suggests that the mechanism RI ,3& LQYROYHV H[SRUW RI SURGXFHG 63 IURP FDUGLRP\RF\WHV WR WKH H[WUDFHOOXODU VSDFHDQGVWLPXODWLRQRI6351 and/or S1PR3,QWHUHVWLQJO\LWZDVIRXQGWKDWLVFKHPLF postconditioning, similarly to IPC, protected isolated mouse hearts against ischemia/ UHSHUIXVLRQLQMXU\YLD63+.DFWLYDWLRQ54 :HUHFHQWO\IRXQGWKDWSODVPDFRQFHQWUDWLRQRI63LQSDWLHQWVZLWKDFXWHP\RFDUGLDO LQIDUFWLRQXSRQDGPLVVLRQWRLQWHQVLYHKHDUWFDUHXQLWZDVPDUNHGO\ORZHUDVFRPSDUHG to healthy controls.550RUHRYHUIXUWKHUUHGXFWLRQLQ63OHYHOZDVREVHUYHGLQWKHVH SDWLHQWV ZLWKLQ WKH QH[W ¿YH GD\V 7KHUHIRUH RXU GDWD VXJJHVW D VXVWDLQHG UHGXFWLRQ RI WKH SURWHFWLYH HIIHFW RI SODVPD 63 DIWHU WKH LQIDUFWLRQ +RZHYHU WKH PHFKDQLVP XQGHUO\LQJWKLVGHFUHDVHUHPDLQVXQFOHDU7KHPDLQVRXUFHVRISODVPD63DUHSODWHOHWV and erythrocytes.35,WLVWKHUHIRUHOLNHO\WKDW63UHOHDVHIURPWKHVHFHOOVLVUHGXFHGXSRQ P\RFDUGLDOLQIDUFWLRQ+RZHYHUIDFWRUVDIIHFWLQJ63UHOHDVHIURPEORRGFHOOVDUHRQO\ SRRUO\UHFRJQL]HG(DFKSDWLHQWZDVVXEMHFWHGWRVWDQGDUGDQWLSODWHOHWWUHDWPHQWLWLVWKHQ WHPSWLQJWRVSHFXODWHWKDWWKLVWUHDWPHQWUHGXFHGOLEHUDWLRQRI63IURPWKURPERF\WHV,W ZRXOGEHVRIDUXQNQRZQDQGXQGHVLUHGHIIHFWRIWKHVHGUXJVHVSHFLDOO\LQYLHZRIWKH recent report showing that exogenous S1P is cardioprotective also in human myocardial tissue.50

CERAMIDE AS A MEDIATOR OF LIPOTOXICITY IN THE HEART /LSRWR[LFLW\LVWKHSURFHVVWKURXJKZKLFKOLSLGRYHUORDGOHDGVWRFHOOXODUG\VIXQFWLRQ FHOOGHDWKDQGHYHQWXDOO\LPSDLUHGRUJDQIXQFWLRQ56$FFXPXODWLRQRIQHXWUDOOLSLGVZLWKLQ FDUGLRP\RF\WHVLVDKDOOPDUNRIWKHP\RFDUGLXPRIKXPDQVDQGURGHQWVZLWKQRQLVFKHPLF KHDUWIDLOXUH56,57 In recent years a hypothesis suggesting that lipotoxicity may contribute WRWKHGHYHORSPHQWRIFDUGLDFG\VIXQFWLRQKDVHPHUJHG58,59 Direct evidence supporting this QRWLRQFRPHVIURPH[SHULPHQWVRQPLFHZLWKFDUGLDFUHVWULFWHGRYHUH[SUHVVLRQRIORQJFKDLQ acyl coenzyme A synthetase 1 (MHC-ACS1),60IDWW\DFLGWUDQVSRUWSURWHLQ61 and the cell PHPEUDQHDQFKRUHGIRUPRIOLSRSURWHLQOLSDVH/S/*3, 62 All these transgenic animal PRGHOVDUHFKDUDFWHUL]HGE\LQFUHDVHGP\RFDUGLDOIDWW\DFLGXSWDNHOLSLGGHSRVLWLRQLQ WKHKHDUWDQGFDUGLRP\RSDWK\WKDWGHYHORSVLQWKHDEVHQFHRIGLVWXUEDQFHVLQV\VWHPLF PHWDEROLVPRUFDUGLDFIDWW\DFLGR[LGDWLRQ It is still unclear how lipid overload leads to cardiomyopathy, however, there is LQFUHDVLQJHYLGHQFHLQGLFDWLQJWKDWDFFXPXODWLRQRIFHUDPLGHSOD\VDNH\UROHLQWKLV SKHQRPHQRQ :KHQ WKH EDODQFH EHWZHHQ IDWW\ DFLG XSWDNH DQG R[LGDWLRQ LV DOWHUHG H[FHVVIDWW\DFLGVLVGLUHFWHGWRZDUGVV\QWKHVLVRIFRPSOH[OLSLGVVRPHRIZKLFKOLNH FHUDPLGHDUHWR[LF,WZDVIRXQGWKDWLQFUHDVHLQP\RFDUGLDOFHUDPLGHFRQWHQWDFFRPSDQLHV FDUGLDFG\VIXQFWLRQLQVHYHUDOJHQHWLFPRGHOVRIOLSRWR[LFFDUGLRP\RSDWK\VHH7DEOH  7KHVHPRGHOVLQFOXGHPLFHZLWKFDUGLDFVSHFL¿FRYHUH[SUHVVLRQRI33$5a, ACS1 and

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Table 3. Relationship between myocardial ceramide, cardiomyocyte apoptosis DQGKHDUWIXQFWLRQLQYDULRXVDQLPDOPRGHOVRIREHVLW\GLDEHWHVDQGOLSRWR[LF cardiomyopathy

Experimental Model

(IIHFWRQ Ceramide Level

(IIHFWRQ Apoptosis

(IIHFWRQ Heart Function

Q38)$ULFKKLJKIDW diet, rats

C

+LJKVDWXUDWHGIDWGLHW UDWLQIDUFWPRGHORIKHDUW IDLOXUH +LJKVDWXUDWHGIDWGLHW UDWLQIDUFWPRGHORIKHDUW IDLOXUH +LJKVDWXUDWHGIDWGLHW rats

75%

C

C

C

C

C

C

Q38)$ULFKKLJKIDW diet, rats +LJKVDWXUDWHGIDWGLHW wild type mice +LJKVDWXUDWHGIDWGLHW MHC-PPAR_ mice +LJKVDWXUDWHGIDWGLHW rats ob/ob mice Streptozotocin-diabetes, rats

– 39%

C

C

Streptozotocin-diabetes, rats Akita Ins2(WT/C96Y) PLFHJHQHWLFPRGHORI Type 1 diabetes) =XFNHUGLDEHWLFIDWW\UDWV

C

?

69%

?

164%

B

?

MHC-PPARa mice

40%

B

?

MHC-ACS1 mice

230%

B

?

LpL(GPI) mice

+ 45%

ļ

Ļ

C

C

31%

?

65%

C

166%

14%

C

5HIHUHQFH Baranowski et al. J Physiol Pharmacol 2007 Rennison et al. Am J Physiol Heart Circ Physiol 2007 Morgan et al. Am J Physiol Heart Circ Physiol 2005 Okere et al. Am J Physiol Heart Circ Physiol 2006 Finck et al. Proc Natl Acad Sci USA 2003 Torre-Villalvazo et al. J Nutr 2009 M. Baranowski, unpublished observation +D\DVKLHWDO/LIH Sci 2001 Basu et al. Am J Physiol Heart Circ Physiol 2009 =KRXHWDO3URF1DWO Acad Sci USA 2000 Son et al. J Clin Invest 2007 Chiu et al. J Clin Invest 2001 Park et al. J Lipid Res 2008

38)$ SRO\XQVDWXUDWHG IDWW\ DFLGV 0+&33$5_ FDUGLDFVSHFL¿F RYHUH[SUHVVLRQ RI SHUR[LVRPH SUROLIHUDWRUDFWLYDWHG UHFHSWRU _; MHC-PPARa FDUGLDFVSHFL¿F RYHUH[SUHVVLRQ RI SHUR[LVRPH SUROLIHUDWRUDFWLYDWHG UHFHSWRU a 0+&$&6 FDUGLDFVSHFL¿F RYHUH[SUHVVLRQ RI ORQJFKDLQ DF\O &R$V\QWKHWDVH/S/*3, FDUGLDFVSHFL¿FRYHUH[SUHVVLRQRIWKHFHOOPHPEUDQHDQFKRUHGIRUP RIOLSRSURWHLQOLSDVH CQRVLJQL¿FDQWFKDQJH BDFWLYDWLRQRIFDUGLDFDSRSWRVLV ?LPSDLUPHQWRI KHDUWIXQFWLRQ

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LpL(GPI).60,63,64,QDGGLWLRQ=KRXHWDO18UHSRUWHGWKDWKHDUWRI=')UDWVLVFKDUDFWHUL]HG E\SURJUHVVLYHDFFXPXODWLRQRIFHUDPLGHWKDWSUHFHGHVGHYHORSPHQWRIFDUGLRP\RSDWK\ ,QFUHDVHG P\RFDUGLDO FHUDPLGH FRQWHQW DVVRFLDWHG ZLWK GLDVWROLF G\VIXQFWLRQ ZDV UHFHQWO\ REVHUYHG DOVR LQ $NLWD ,QV:7&<  PLFH D JHQHWLF PRGHO RI QRQREHVH Type 1 diabetes).65,QWHUHVWLQJO\LPSURYHPHQWLQFDUGLDFIXQFWLRQREVHUYHGLQ=')UDWV $NLWD,QV:7&< PLFHDQGLQ0+&$&6PLFHDIWHUSKDUPDFRORJLFDORUJHQHWLF LQWHUYHQWLRQDGPLQLVWUDWLRQRIWURJOLWD]RQHLQVXOLQDQGRYHUH[SUHVVLRQRIGLDF\OJO\FHURO DF\O WUDQVIHUDVH  LQ =') UDWV $NLWD ,QV:7&<  PLFH DQG 0+&$&6 PLFH respectively) was associated with decreased myocardial ceramide level.18,22,65 However, GH¿QLWLYHHYLGHQFHIRUWKHNH\UROHRIFHUDPLGHLQOLSRWR[LFFDUGLRP\RSDWK\ZDVSURYLGHG by Park et al647KH\IRXQGWKDWLQKLELWLRQRI637ZLWKP\ULRFLQSUHYHQWHGP\RFDUGLDO DFFXPXODWLRQRIFHUDPLGHEXWQRWRIRWKHUOLSLGV UHGXFHGPRUWDOLW\UDWHLPSURYHGKHDUW IXQFWLRQDQGUHGXFHGH[SUHVVLRQRIFDUGLDFIDLOXUHPDUNHUVLQ/S/*3, PLFH6LPLODU HIIHFWZDVLQGXFHGE\KHWHUR]\JRXVGHOHWLRQRI/&%JHQHHQFRGLQJRQHRI637VXEXQLWV  ZKLFKH[FOXGHVLQYROYHPHQWRIQRQVSHFL¿FSKDUPDFRORJLFDOHIIHFWVRIP\ULRFLQ &DUGLRP\RF\WHDSRSWRVLVLVRQHRIWKHPHFKDQLVPVXQGHUO\LQJGHYHORSPHQWRI GLDEHWLFFDUGLRP\RSDWK\DQGKHDUWIDLOXUH66,677KHUHIRUHFRQVLGHULQJWKHSURDSRSWRWLF DFWLRQRIFHUDPLGHLWVFDUGLRWR[LFHIIHFWVDUHFRPPRQO\FRQVLGHUHGWREHPHGLDWHG SULPDULO\ E\ DFWLYDWLRQ RI SURJUDPPHG FHOO GHDWK $FFXPXODWLRQ RI FHUDPLGH ZDV IRXQGWREHDVVRFLDWHGZLWKP\RFDUGLDODSRSWRVLVLQ=')UDWVDVZHOODVLQPLFHZLWK FDUGLDFVSHFL¿FRYHUH[SUHVVLRQRI33$5a or ACS118,60,63 (see Table 3). In addition, interventions leading to a decrease in heart ceramide simultaneously reduced FDUGLRP\RF\WH DSRSWRVLV LQ =') UDWV DQG LQ 0+&$&6 PLFH18,22 Moreover, in YLWUR VWXGLHV RQ LVRODWHG UDW FDUGLRP\RF\WHV UHYHDOHG WKDW LQFXEDWLRQ RI WKH FHOOV ZLWKSDOPLWDWHLQGXFHGFHUDPLGHDFFXPXODWLRQDQGDFWLYDWLRQRIDSRSWRVLVWKDWZDV DWWHQXDWHGE\LQKLELWLRQRIFHUDPLGHV\QWKHVLV68,69 It should be noted, however, that a recent report by Park et al64 indicates that FDUGLRWR[LFLW\RIFHUDPLGHPD\UHVXOWIURPLWVHIIHFWRQP\RFDUGLDOJOXFRVHDQGIDWW\DFLG PHWDEROLVPUDWKHUWKDQLQGXFWLRQRIDSRSWRWLFORVVRIFDUGLRP\RF\WHV7KH\XVHG/S/*3,  PLFHZKLFKZHUHFKDUDFWHUL]HGE\LPSDLUHGV\VWROLFIXQFWLRQLQFUHDVHGUDWHRIIDWW\DFLG R[LGDWLRQDQGUHGXFHGJOXFRVHR[LGDWLRQUDWHLQWKHKHDUW$FFXPXODWLRQRIP\RFDUGLDO ceramide was observed as well, however, it was not accompanied by cardiomyocyte apoptosis. Interestingly, pharmacological (with myriocin) or genetic (heterozygous GHOHWLRQRI/&%JHQH LQKLELWLRQRIGHQRYRFHUDPLGHV\QWKHVLVQRUPDOL]HGIDWW\DFLG DQGJOXFRVHR[LGDWLRQUDWHLQWKHKHDUWDVZHOODVV\VWROLFIXQFWLRQ7KHVHLQWHUYHQWLRQV also corrected the mismatch between myocardial glucose uptake and oxidation, which OLNHO\FRQWULEXWHGWRWKHGHYHORSPHQWRIFDUGLRP\RSDWK\LQWKLVPRGHO3DUNHWDODOVR UHSRUWHGWKDWLQFXEDWLRQRIKXPDQFDUGLRP\RF\WH$&FHOOVZLWK&FHUDPLGHLQGXFHG GRZQUHJXODWLRQRIJOXFRVHWUDQVSRUWHUDQGXSUHJXODWLRQRIS\UXYDWHGHK\GURJHQDVH kinase 4, atrial natriuretic peptide and brain natriuretic peptide gene expression. These changes were consistent with those observed in LpL(GPI) mice that show elevated myocardial ceramide level. The above data strongly suggest that ceramide is able to modulate cardiomyocyte energetic substrate metabolism via transcriptional regulation RIWKHUHOHYDQWSURWHLQV ,Q DGGLWLRQ LW ZDV UHFHQWO\ UHSRUWHG WKDW P\RFDUGLXP RI RERE PLFH DQG UDWV FKURQLFDOO\IHGKLJKVDWXUDWHGIDWGLHWGLGQRWVKRZHYLGHQFHRIFDUGLRP\RF\WHDSRSWRVLV

HEART SPHINGOLIPIDS IN HEALTH AND DISEASE

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Figure 3. (IIHFW RI REHVLW\ RSHQ EDUV  DQG REHVLW\ FRPELQHG ZLWK 7\SH  GLDEHWHV KDWFKHG EDUV  RQ P51$ OHYHO RI HQ]\PHV UHODWHG WR FHUDPLGH PHWDEROLVP LQ WKH KXPDQ KHDUW VWDWLVWLFDOO\ VLJQLILFDQW FKDQJH YV OHDQ VXEMHFWV ‚VWDWLVWLFDOO\ VLJQLILFDQW FKDQJH YV REHVH QRQGLDEHWLF SDWLHQWV 637VHULQH SDOPLWR\OWUDQVIHUDVH 63+.VSKLQJRVLQH NLQDVH DOQ&'DVHDONDOLQHQHXWUDO FHUDPLGDVH a-CDase-acid ceramidase, n-SMase-neutral sphingomyelinase, a-SMase-acid sphingomyelinase. 'DWD UHGUDZQ IURP 0 %DUDQRZVNL HW DO 0\RFDUGLXP RI 7\SH  GLDEHWLF DQG REHVH SDWLHQWV LV FKDUDFWHUL]HGE\DOWHUDWLRQVLQVSKLQJROLSLGPHWDEROLFHQ]\PHVEXWQRWE\DFFXPXODWLRQRIFHUDPLGH J Lipid Res 2010; 51:74-80. 5

GHVSLWHDFFXPXODWLRQRIFHUDPLGH707KHVHGDWDIXUWKHULQGLFDWHWKDWLQFUHDVHGP\RFDUGLDO FHUDPLGHOHYHOVGRQRWQHFHVVDULO\OHDGWRDFWLYDWLRQRIDSRSWRVLV8QIRUWXQDWHO\KHDUW IXQFWLRQZDVQRWDVVHVVHGLQWKHVHH[SHULPHQWV $OWKRXJK GDWD IURP DQLPDO H[SHULPHQWV VWURQJO\ VXJJHVW WKDW FHUDPLGH PD\ EH LQYROYHGLQSDWKRJHQHVLVRIKHDUWG\VIXQFWLRQDVVRFLDWHGZLWKREHVLW\DQGGLDEHWHVLW remains unknown whether similar relationship is present also in the human heart. We DWWHPSWHGWRDQVZHUWKLVTXHVWLRQLQRXUUHFHQWVWXG\XVLQJVDPSOHVRIWKHULJKWDWULDO DSSHQGDJHREWDLQHGIURPSDWLHQWVXQGHUJRLQJFRURQDU\E\SDVVJUDIWVXUJHU\5:HIRXQG WKDWFRPSDUHGZLWKOHDQVXEMHFWVP\RFDUGLXPRIRYHUZHLJKWSDWLHQWVZDVFKDUDFWHUL]HG E\PDUNHGXSUHJXODWLRQRIVSKLQJROLSLGPHWDEROLFHQ]\PHVH[SUHVVLRQ)LJ 7KHVH enzymes included neutral sphingomyelinase, SPT subunits, ceramidases and SPHK1. ,QWHUHVWLQJO\ P51$ OHYHO RI VRPH JHQHV XSUHJXODWHG E\ RYHUZHLJKW ZDV UHGXFHG LI concomitant diabetes was present, however, their expression was still higher than in OHDQVXEMHFWV)LJ ,QDGGLWLRQZHREVHUYHGHOHYDWHG'1$IUDJPHQWDWLRQOHYHOD PDUNHURIDSRSWRVLV LQWKHKHDUWRIRYHUZHLJKWQRQGLDEHWLFSDWLHQWVWKDWZDVLQFUHDVHG IXUWKHULQRYHUZHLJKW7\SHGLDEHWLFVXEMHFWV+RZHYHUWRRXUVXUSULVHWKHUHZDVQR DFFXPXODWLRQRIP\RFDUGLDOFHUDPLGHLQHLWKHUJURXS7KLVZDVOLNHO\GXHWRWKHIDFW WKDWFDUGLDFH[SUHVVLRQRIHQ]\PHVLQYROYHGLQV\QWKHVLVDQGGHJUDGDWLRQRIFHUDPLGH was regulated in concert. Our results suggest, that in contrast to rodents, obesity and Type 2 diabetes do not induce ceramide accumulation in the human heart, or at least in WKHDWULXP,QDGGLWLRQFHUDPLGHGRHVQRWVHHPWREHDPDMRUIDFWRULQFDUGLRP\RF\WH DSRSWRVLVREVHUYHGLQSDWLHQWVVXIIHULQJIURPWKHVHGLVHDVHV

54

SPHINOGLIPIDS AND METABOLIC DISEASE

CONCLUSION AND FUTURE PROSPECTS $V UHYLHZHG KHUH HYLGHQFH IURP DQLPDO PRGHOV VWURQJO\ VXJJHVW WKDW FHUDPLGH DFFXPXODWLRQSOD\VDFDVXDOUROHLQWKHSDWKRJHQHVLVRIKHDUWG\VIXQFWLRQ+RZHYHUWR date, it remains an open question whether a similar relationship is present also in the human P\RFDUGLXP0RUHRYHUIXUWKHUUHVHDUFKLVUHTXLUHGWRIXOO\HOXFLGDWHWKHPHFKDQLVPV XQGHUO\LQJFDUGLRWR[LFHIIHFWVRIFHUDPLGH6XFKLQIRUPDWLRQPLJKWDOORZGHYHORSPHQW RIQHZDSSURDFKHVWRSUHYHQWLRQDQGWUHDWPHQWRIP\RFDUGLDOGLVHDVHLQSDWLHQWVZLWK diabetes and obesity. In contrast to ceramide, S1P plays a critical role in maintaining FDUGLDFFHOOVXUYLYDODQGIXQFWLRQ63LWVHOIDVZHOODVDJRQLVWVRILWVFRJQDWHUHFHSWRUV ZHUH IRXQG WR EH SRWHQW FDUGLRSURWHFWLYH DJHQWV DJDLQVW LVFKHPLDUHSHUIXVLRQ LQMXU\ RIWKHKHDUWLQH[SHULPHQWDODQLPDOV7KHIDFWWKDWWKHVHFRPSRXQGVDUHHIIHFWLYHDOVR ZKHQDSSOLHGGXULQJUHSHUIXVLRQPDNHVWKHPSRWHQWLDOFDQGLGDWHVIRUSKDUPDFRORJLFDO SRVWFRQGLWLRQLQJWKHUDS\DIWHUP\RFDUGLDOLVFKHPLD8QGRXEWHGO\PDQ\QHZH[FLWLQJ GLVFRYHULHVLQWKH¿HOGRIP\RFDUGLDOVSKLQJROLSLGVLJQDOLQJDUHWREHH[SHFWHGLQWKH QHDUIXWXUH ACKNOWLEDGEMENTS 7KLVZRUNZDVVXSSRUWHGE\WKH3ROLVK0LQLVWU\RI6FLHQFHDQG+LJKHU(GXFDWLRQ (grant no. N N402 470937). REFERENCES 2ND]DNL7%LHODZVND$%HOO50HWDO5ROHRIFHUDPLGHDVDOLSLGPHGLDWRURIDOSKDGLK\GUR[\YLWDPLQ 'LQGXFHG+/FHOOGLIIHUHQWLDWLRQ-%LRO&KHP 2. Yang J, Yu Y, Sun S et al. Ceramide and other sphingolipids in cellular responses. Cell Biochem Biophys 2004; 40:323-50. 3. Summers SA. Ceramides in insulin resistance and lipotoxicity. Prog Lipid Res 2005; 45:42-72. %DUDQRZVNL0=DELHOVNL3%ODFKQLR$HWDO(IIHFWRIH[HUFLVHGXUDWLRQRQFHUDPLGHPHWDEROLVPLQWKHUDW KHDUW$FWD3K\VLRO2[I  %DUDQRZVNL0%ODFKQLR=DELHOVND$+LUQOH7HWDO0\RFDUGLXPRIW\SHGLDEHWLFDQGREHVHSDWLHQWVLV FKDUDFWHUL]HGE\DOWHUDWLRQVLQVSKLQJROLSLGPHWDEROLFHQ]\PHVEXWQRWE\DFFXPXODWLRQRIFHUDPLGH J Lipid Res 2010; 51:74-80. /L&0+RQJ6%.RSDO*HWDO&ORQLQJDQGFKDUDFWHUL]DWLRQRIWKHIXOOOHQJWKF'1$DQGJHQRPLFVHTXHQFHV encoding murine acid ceramidase. Genomics 1998; 50:267-74. 7. Li CM, Park JH, He X et al. The human acid ceramidase gene (ASAH): structure, chromosomal location, mutation analysis and expression. Genomics 1999; 62:223-31. /LX+6XJLXUD01DYD9(HWDO0ROHFXODUFORQLQJDQGIXQFWLRQDOFKDUDFWHUL]DWLRQRIDQRYHOPDPPDOLDQ VSKLQJRVLQHNLQDVHW\SHLVRIRUP-%LRO&KHP 
HEART SPHINGOLIPIDS IN HEALTH AND DISEASE

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15. Oakes ND, Kennedy CJ, Jenkins AB et al. A new antidiabetic agent, BRL 49653, reduces lipid availability and improves insulin action and glucoregulation in the rat. Diabetes 1994; 43:1203-10. &DUOH\ $1 6HPHQLXN /0 6KLPRQL < HW DO 7UHDWPHQW RI W\SH  GLDEHWLF GEGE PLFH ZLWK D QRYHO 33$5JDPPDDJRQLVWLPSURYHVFDUGLDFPHWDEROLVPEXWQRWFRQWUDFWLOHIXQFWLRQ$P-3K\VLRO(QGRFULQRO Metab 2004; 286:E449-55. 17. Liu LS, Tanaka H, Ishii S et al. The new antidiabetic drug MCC-555 acutely sensitizes insulin signaling in isolated cardiomyocytes. Endocrinology 1998; 139:4531-9. =KRX<7*UD\EXUQ3.DULP$HWDO/LSRWR[LFKHDUWGLVHDVHLQREHVHUDWVLPSOLFDWLRQVIRUKXPDQREHVLW\ Proc Natl Acad Sci USA 2000; 97:1784-9. +DQDGD.6HULQHSDOPLWR\OWUDQVIHUDVHDNH\HQ]\PHRIVSKLQJROLSLGPHWDEROLVP%LRFKLP%LRSK\V$FWD 2003; 1632:16-30. 6KLPDEXNXUR0+LJD0=KRX<7HWDO/LSRDSRSWRVLVLQEHWDFHOOVRIREHVHSUHGLDEHWLFIDIDUDWV5ROH RIVHULQHSDOPLWR\OWUDQVIHUDVHRYHUH[SUHVVLRQ-%LRO&KHP 21. Blazquez C, Geelen MJ, Velasco G et al. The AMP-activated protein kinase prevents ceramide synthesis de novo and apoptosis in astrocytes. FEBS Lett 2001; 489:149-53. /LX/6KL;%KDUDGZDM.*HWDO'*$7H[SUHVVLRQLQFUHDVHVKHDUWWULJO\FHULGHFRQWHQWEXWDPHOLRUDWHV lipotoxicity. J Biol Chem 2009; 284:36312-23. 6DEEDGLQL5$'DQLHOL%HWWR'%HWWR57KHUROHRIVSKLQJROLSLGVLQWKHFRQWURORIVNHOHWDOPXVFOHIXQFWLRQ a review. Ital J Neurol Sci 1999; 20:423-30. 'REU]\Q$*RUVNL-(IIHFWRIDFXWHH[HUFLVHRQWKHFRQWHQWRIIUHHVSKLQJDQLQHDQGVSKLQJRVLQHLQGLIIHUHQW VNHOHWDOPXVFOHW\SHVRIWKHUDW+RUP0HWDE5HV 0F'RQRXJK30
56

SPHINOGLIPIDS AND METABOLIC DISEASE

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CHAPTER 4 BLOOD SPHINGOLIPIDS IN HOMEOSTASIS AND PATHOBIOLOGY Samar M. Hammad Department of Regenerative Medicine and Cell Biology, Medical University of South Carolina, Charleston, South Carolina, USA Email: [email protected]

Abstract:

6SKLQJROLSLGVKDYHHPHUJHGDVNH\VLJQDOLQJPROHFXOHVLQYROYHGLQWKHUHJXODWLRQRI DYDULHW\RIFHOOXODUIXQFWLRQVLQFOXGLQJFHOOJURZWKDQGGLIIHUHQWLDWLRQSUROLIHUDWLRQ DQGDSRSWRWLFFHOOGHDWK6SKLQJROLSLGVLQEORRGFRQVWLWXWHSDUWRIWKHFLUFXODWLQJ lipoprotein particles (HDL, LDL and VLDL), carried by serum albumin and also SUHVHQW LQ EORRG FHOOV DQG SODWHOHWV 5HFHQW OLSLGRPLF DQG SURWHRPLF VWXGLHV RI plasma lipoproteins have provided intriguing data concerning the protein and OLSLG FRPSRVLWLRQ RI OLSRSURWHLQV LQ WKH FRQWH[W RI GLVHDVH 6SKLQJROLSLGV KDYH been implicated in several diseases such as cancer, obesity, atherosclerosis and VSKLQJROLSLGRVHV KRZHYHU HIIRUWV DGGUHVVLQJ EORRG VSKLQJROLSLGRPLFV DUH VWLOO OLPLWHG7KHGHYHORSPHQWRIPHWKRGVWRGHWHUPLQHOHYHOVRIFLUFXODWLQJELRDFWLYH VSKLQJROLSLGVLQKXPDQVDQGYDOLGDWLRQRIWKHVHPHWKRGVWREHDURXWLQHFOLQLFDO laboratory test could be a pioneering approach to diagnose disease in the population. This approach would probably evolve to be analogous in implication to determining “good” and “bad” cholesterol and triglyceride levels in lipoprotein classes.

INTRODUCTION 6SKLQJROLSLGVRQFHGHHPHGPDLQO\VWUXFWXUDOFRPSRQHQWVRIFHOOPHPEUDQHVKDYH HPHUJHGDVNH\VLJQDOLQJPROHFXOHVLQYROYHGLQWKHUHJXODWLRQRIDUDQJHRIFHOOXODU IXQFWLRQVLQFOXGLQJFHOOJURZWKDQGGLIIHUHQWLDWLRQSUROLIHUDWLRQDQGDSRSWRWLFFHOOGHDWK1-4 Ceramide (Cer), the central molecule in sphingolipid metabolism, is generated by either GH QRYR V\QWKHVLV RU WKURXJK WKH DFWLRQ RI VSKLQJRP\HOLQDVHV 60DVHV  D IDPLO\ RI phospholipases.57KH&HUIRUPHGIURPVSKLQJRP\HOLQ60 WXUQRYHUPLJKWEHK\GURO\]HG by ceramidases to liberate sphingosine (Sph). The latter can be re-acylated to Cer or phosphorylated to sphingosine 1-phosphate (S1P) by sphingosine kinase (SK).1,4,6 Several Sphingolipids and Metabolic Disease, edited by L. Ashley Cowart. ©2011 Landes Bioscience and Springer Science+Business Media, LLC 57

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SPHINGOLIPIDS AND METABOLIC DISEASE

sphingolipid metabolites, particularly Cer, Sph, S1P and ceramide 1-phosphate (Cer1P), have been recognized as bioactive signaling molecules that regulate cell growth and death.1-4,7-12,QJHQHUDOFHOOXODUDFFXPXODWLRQVRI&HUDQG6SKZKLFKRFFXULQUHVSRQVH WRVWUHVVVXFKDVH[SRVXUHWRWXPRUQHFURVLVIDFWRUDOSKD71)_) or oxidative stress, are associated with apoptotic responses.13,QFRQWUDVWDFFXPXODWLRQRI63LVXVXDOO\ DPRGXODWRURIFHOOJURZWK14DQGFDQSURWHFWFHOOVIURPDSRSWRVLV1 Studies addressing the VSKLQJROLSLGRPHRIEORRGDUHOLPLWHG7KLVFKDSWHUUHYLHZVFXUUHQWOLWHUDWXUHRQEORRG VSKLQJROLSLGVXQGHUQRUPDODQGDEQRUPDOFRQGLWLRQVDQGKLJKOLJKWVHIIRUWVDGGUHVVLQJ WKHLPSRUWDQFHRIGHWHUPLQLQJEORRGVSKLQJROLSLGOHYHOVDVELRPDUNHUVRIGLVHDVH BLOOD SPHINGOLIPIDS IN HOMEOSTASIS Interest in blood sphingolipids has been broadened by the development and clinical DSSOLFDWLRQRIWKHLPPXQRVXSSUHVVLYHGUXJ)7<ZKLFKWDUJHWV63UHFHSWRUVUHVXOWLQJ in lymphocyte sequestration.15 It has since become increasingly essential to explore the PHWDERORPLFSUR¿OHRIEORRGVSKLQJROLSLGVXQGHUQRUPDODQGDEQRUPDOFRQGLWLRQVDQG to determine the mechanisms by which sphingolipid biosynthesis and turnover regulate FHOOIXQFWLRQDQGSDWKRELRORJ\ 6SKLQJROLSLGVDUHWKHPRVWVWUXFWXUDOO\GLYHUVHDVZHOODVFRPSOH[FDWHJRU\RIOLSLGV ,Q DGGLWLRQ WR QXPHURXV YDULDWLRQV LQ WKH VSKLQJRLG EDVHV 1DF\O OLQNHG IDWW\ DFLGV and head groups, sphingolipids contain long-chain hydrocarbon groups.7 Furthermore, sphingolipids are insoluble in water and have both hydrophobic and hydrophilic properties.7 'HWHFWLRQRIVSKLQJROLSLGVKDVEHHQKDPSHUHGE\WKHLUODFNRIFKURPRSKRUHVQHFHVVDU\ IRUWUDGLWLRQDO89DQGÀXRUHVFHQFHGHWHFWLRQRIKLJKSHUIRUPDQFHOLTXLGFKURPDWRJUDSK\ +3/& WHFKQLTXHV7KHDSSOLFDWLRQRI+3/&FRXSOHGZLWKWDQGHPPDVVVSHFWURPHWU\ +3/&0606 KDVUHFHQWO\SURYLGHGDQHIIHFWLYHDQDO\WLFDOWRROIRUWKHGHWHUPLQDWLRQ RIVSKLQJROLSLGRPLFSUR¿OHVRIYDULHGELRORJLFDOPDWHULDOV16 however, studies addressing WKHVSKLQJROLSLGRPHRIEORRGDUHVWLOOVFDUFH In a recent study using HPLC-MS/MS we have analyzed a comprehensive sphingolipid SUR¿OHLQ³QRUPDO´KXPDQVHUXPDQGSODVPDLQDQHIIRUWWRHVWDEOLVKDUHIHUHQFHUDQJHIRU FLUFXODWLQJVSKLQJROLSLGVSHFLHVLQEORRGRIKHDOWK\KXPDQV17 We simultaneously analyzed the sphingoid bases (C18:1, C18:0) Sph and dihydrosphingosine (dhSph); sphingoid base SKRVSKDWHV63DQGGLK\GURVSKLQJRVLQHSKRVSKDWHGK63 PROHFXODUVSHFLHVRI&HU dihydroceramides (dhCer), Cer1P, SM, hexosylceramide (HexCer) and lactosylceramide /DF&HU FRYHULQJDEDVLFPHWDERORPLFSUR¿OH17:HIRXQGWKDWWKHVSKLQJROLSLGV60 /DF&HU+H[&HU&HUDQG&HU3FRQVWLWXWHDQGRIWRWDO sphingolipids, respectively. The abundant circulating SM, LacCer, HexCer and Cer are C16-SM, C16-LacCer, C24-HexCer and C24&HUUHVSHFWLYHO\,QWHUHVWLQJO\XQGHUIDVWLQJ FRQGLWLRQVOHYHOVRI&16-SM and the very long-chain LacCers (C24:1, C26:1) increased, whereas OHYHOVRIWKHORQJFKDLQ&HUV&16-C20) and C26&HU3GHFUHDVHGFRPSDUHGWRIHGVWDWH 7KHVWXG\DOVRUHYHDOHGJHQGHUGLIIHUHQFHVDQGVKRZHGWKDWOHYHOVRI&18- and C18:1-SM, C18&HU3DQGWRWDOGK&HUVDUHKLJKHULQIHPDOHVWKDQPDOHVXQGHUIDVWLQJFRQGLWLRQV17 7KLVJHQGHUGLIIHUHQFHFRXOGSUREDEO\EHGXHWRGLIIHUHQFHVLQWKHOLSRSURWHLQSUR¿OH VXFKDVWKHKLJKHUQXPEHURI+'/SDUWLFOHVLQIHPDOHV18 Future studies should be able WRSURYLGHPRUHLQIRUPDWLRQDERXWWKHHIIHFWVRIVWDUYDWLRQDQGRYHUIHHGLQJRQOHYHOVRI ELRDFWLYHVSKLQJROLSLGVDVZHOODVH[SDQGRXUNQRZOHGJHRIVSKLQJROLSLGSUR¿OHVXQGHU disease conditions.

BLOOD SPHINGOLIPIDS IN HOMEOSTASIS AND PATHOBIOLOGY

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6SKLQJROLSLGV LQ WKH EORRG DUH IRXQG LQ FLUFXODWLQJ OLSRSURWHLQ SDUWLFOHV 9/'/ LDL and HDL) and in blood cells and platelets and also bound to serum albumin.19 7KHLQIRUPDWLRQDERXWWKHORFDWLRQDQGGLVWULEXWLRQRIVSKLQJROLSLGFODVVHVDQGVSHFLHV in lipoprotein particles are still obscure. In our recent study, HPLC-MS/MS was also XVHGWRDQDO\]HWKHOHYHORIVSKLQJRLGEDVHVDQGWKHLUSKRVSKDWHVDVZHOODVOHYHOV RIVSKLQJROLSLGVSHFLHVRI&HUDQG60LQ9/'//'/DQGWKHVXEFODVVHV+'/+'/ and HDL3.177KHPDMRUFDUULHURI&HUDQGGK6SKLQWKHFLUFXODWLRQLV/'/ZLWK DQGRIWRWDOOLSRSURWHLQDVVRFLDWHG&HUDQGGK6SKUHVSHFWLYHO\17 In an analysis XVLQJIDVWSHUIRUPDQFHOLTXLGFKURPDWRJUDSK\)3/& :HLVQHUHWDOVKRZHGWKDWWKH PDMRU60LQOLSRSURWHLQFODVVHVLV60IROORZHGE\6020 which corresponds to C16- and C24:1-SMs containing C18:1 sphingoid backbone, respectively. In VLDL, LDL, +'/DQG+'/SDUWLFOHVZKHWKHULVRODWHGE\)3/&RUSUHSDUDWLYHXOWUDFHQWULIXJDWLRQ C1660ZDVIRXQGWREHWKHPDMRU60VSHFLHVLQWKHEORRGIROORZHGE\&24:1-SM.17,20 ,WKDVEHHQDOVRIRXQGWKDW&24-Cer is the most abundant Cer species in all lipoprotein FODVVHVLQFOXGLQJVXEFODVVHVRI+'/17,207KHFRQFHQWUDWLRQRI60DQG&HUVSHFLHVSHU OLSRSURWHLQSDUWLFOHUHÀHFWVWKHVL]HRIWKHSDUWLFOHZLWKWKHODUJHUVL]HSDUWLFOHFRQWDLQLQJ KLJKHUFRQWHQWRI60DQG&HUVSHFLHV ,WKDVEHHQHVWDEOLVKHGWKDWH[WUDFHOOXODU63ELQGVPHPEHUVRIWKH63UHFHSWRU IDPLO\63 RQWDUJHWFHOOVLQGXFLQJGLIIHUHQWLDWLRQPLJUDWLRQDQGPLWRJHQHVLV21,22 1XPHURXV VWXGLHV VKRZHG WKDW 63 UHJXODWHV YDULRXV IXQFWLRQV RI FHOOV LQYROYHG LQ vascular remodeling, including endothelial cells, smooth-muscle cells, lymphocytes, monocytes and platelets.12,23-27,WZDVGHWHUPLQHGWKDWPRUHWKDQRIWKH63LQEORRG is associated with LDL, VLDL and HDL particles.195HVXOWVIURPRXUUHFHQW+3/&06 06DQDO\VHVRIOLSRSURWHLQVVKRZHGWKDWRIOLSRSURWHLQDVVRFLDWHG63LVFDUULHG RQ+'/SDUWLFOHVZKLFKDUHDOVRWKHPDMRUFDUULHUVRIGK63DQG6SKZLWKDQG RIWRWDOOLSRSURWHLQDVVRFLDWHGGK63DQG6SKUHVSHFWLYHO\17 Others have also shown that the smallest lipoprotein particles, HDL3, are enriched in S1P but poor in SM compared to the larger HDL2 particles.28 $QDUUD\RIVXSHUODWLYHVWXGLHVVXJJHVWHGWKDW+'/DVVRFLDWHG63PHGLDWHVPDQ\RI WKHEHQH¿FLDOHIIHFWVRI+'/RQWKHFDUGLRYDVFXODUV\VWHPLQFOXGLQJWKHV\QWKHVLVRISRWHQW anti-atherogenic and anti-thrombotic molecules (e.g., nitric oxide and prostacyclin).23,29,30 7KHUHLVHPHUJLQJOLWHUDWXUHKRZHYHUWRVXJJHVWWKDW63PD\DOVREHSURLQÀDPPDWRU\30,31 and pro-atherogenic32 DQG ZDV HYHQ FRQVLGHUHG D ELRPDUNHU RI REVWUXFWLYH FRURQDU\ artery disease.33 In concurrence, we have recently demonstrated that HDL3, which FRQWDLQVKLJKHUDPRXQWVRI63WKDQ+'/VLJQL¿FDQWO\LQFUHDVHVSODVPLQRJHQDFWLYDWRU LQKLELWRU VHFUHWLRQ IURP DGLSRF\WHV DQG WKXV PD\ QHJDWLYHO\ PRGXODWH ¿EULQRO\VLV in vivo.34%HFDXVHRIWKHUROHRI+'/LQWKHUHYHUVHFKROHVWHUROWUDQVSRUWIURPSHULSKHUDO tissues to the liver, the larger diameter HDL2 particles viewed as more atheroprotective compared to the smaller sized HDL3 particles.350RUHRYHUWKHPRGL¿FDWLRQRIWKHFRUH OLSLGFRQWHQWRI+'/SDUWLFOHVZDVVKRZQWRDOWHUWKHFRQIRUPDWLRQRIDSROLSRSURWHLQ$, GRPDLQVWKDWDUHFULWLFDOIRU+'/WRDFWDVOLSLGDFFHSWRUV36,37 More recently, it has been VKRZQWKDWWKHVWDELOLW\RIWKH1WHUPLQDOKHOL[EXQGOHGRPDLQRIDSROLSRSURWHLQ$,DQG WKHK\GURSKRELFLW\RILWV&WHUPLQDOGRPDLQDUHLPSRUWDQWGHWHUPLQDQWVRIERWKQDVFHQW +'/SDUWLFOHVL]HDQGUDWHRILWVIRUPDWLRQ38 It is intriguing then to hypothesize that the ORFDWLRQRI63LVDVFULWLFDODVLWVDPRXQWLQGHWHUPLQLQJLWVEHQH¿FLDOFKDUDFWHULVWLFV 5HFHQWVWXGLHVVXJJHVWWKDWPRUHHODERUDWHDQDO\VHVRIOLSRSURWHLQVXEFODVVHVPD\OHDG WR IXUWKHU LPSURYHPHQWV LQ FDUGLRYDVFXODU GLVHDVH ULVN HYDOXDWLRQ DQG LPSRUWDQWO\ LQ LGHQWL¿FDWLRQRIDSSURSULDWHWDUJHWVIRUWKHUDSHXWLFLQWHUYHQWLRQUHYLHZHGLQUHI 

60

SPHINGOLIPIDS AND METABOLIC DISEASE

BLOOD SPHINGOLIPIDS IN PATHOBIOLOGY In a recent well-designed clinical study, Sattler et al demonstrated that the S1P content RI+'/LVQHJDWLYHO\DVVRFLDWHGZLWKSODVPDOHYHOVRI63ZKLFKLVQRWERXQGWR+'/LQ KHDOWK\FRQWUROVEXWQRWLQSDWLHQWVZLWKP\RFDUGLDOLQIDUFWLRQ0, RUVWDEOHFRURQDU\ artery disease (CAD).407KHDXWKRUVSURYLGHGHYLGHQFHWKDWSODVPDOHYHOVRI+'/ERXQG 63DUHORZHUDQGWKRVHRIQRQ+'/ERXQG63DUHKLJKHULQSDWLHQWVZLWK0,DQGVWDEOH &$'FRPSDUHGWRKHDOWK\FRQWUROV7KH\VXJJHVWHGWKHUHIRUHWKDWQRQ+'/ERXQG63 PD\VHUYHDVDQRYHOELRPDUNHUIRU&$',QWULJXLQJO\WKH\DOVRVKRZHGWKDW0,SDWLHQWV ZLWKV\PSWRPGXUDWLRQRIOHVVWKDQKKDGWKHKLJKHVWOHYHOVRISODVPD63DVZHOO DV WKH KLJKHVW OHYHOV RI 63 LQ LVRODWHG +'/ 7KH\ FRQFOXGHG WKDW &$'DVVRFLDWHG GHIHFWVLQ63³XSWDNH´E\+'/FRXOGSRWHQWLDOO\DOORZGHOHWHULRXVHIIHFWVRIIUHH6340 $OWHUQDWLYHO\&$'UHODWHGVKLIWVLQWKHGLVWULEXWLRQRI+'/VXEIUDFWLRQVPLJKWFDXVH WKHDWWHQXDWHGELQGLQJRI63WR+'/LQSDWLHQWVZLWK&$' Red blood cells,41,42 vascular endothelial cells43 and platelets44,45 all contribute to EORRG633ODWHOHWVODFNWKHHQ]\PH63O\DVHZKLFKLVUHVSRQVLEOHIRUGHJUDGDWLRQRI S1P; but maintains a highly active Sph kinase,45ZKLFKFRQYHUWV6SKWR637KHUHIRUH platelets are able to store and release S1P upon stimulation.44,457KXVKLJKHUOHYHOVRI 63DUHFRQVWDQWO\IRXQGLQVHUXPWKDQSODVPDGXHWRSODWHOHWDFWLYDWLRQDQGUHOHDVH RI63GXULQJFORWWLQJ1763FDQEHJHQHUDWHGIURPPHPEUDQHVSKLQJROLSLGVDQGWKHLU metabolites, Cer, Sph and SM.46 2XU UHFHQW ¿QGLQJ RI 6. UHOHDVH IURP DFWLYDWHG PDFURSKDJHVLQUHVSRQVHWRPRGL¿HGOLSRSURWHLQLPPXQHFRPSOH[HVFRXOGVLJQL¿FDQWO\ LQÀXHQFHVWXGLHVDGGUHVVLQJPHFKDQLVPVPHGLDWLQJ63IRUPDWLRQH[WUDFHOOXODUO\47 This QRYHOPHFKDQLVPFRXOGKDYHDVLJQL¿FDQWLPSDFWRQVWXGLHVUHODWHGWRLQÀDPPDWLRQDQG LQÀDPPDWLRQUHODWHGGLVHDVHV /LSRSURWHLQ SDUWLFOHV FRQVLVW RI K\GURSKRELF OLSLGV ORFDWHG ZLWKLQ WKH FRUH DQG DPSKLSDWKLF PROHFXOHV LQ WKH VXUIDFH &RPSOH[ VSKLQJROLSLGV VXFK DV 60 H[LVW SUHGRPLQDQWO\LQWKHRXWHUOHDÀHWRIWKHELOD\HURIFHOOPHPEUDQHDVZHOODVWKHK\GURSKRELF RXWHUOD\HURIWKHOLSRSURWHLQSDUWLFOHZLWKIUHHFKROHVWHURODQGSKRVSKROLSLGV487KHVXUIDFH RIWKH/'/SDUWLFOHIRULQVWDQFHFRQWDLQVDSSUR[LPDWHO\PROHFXOHVRI6049 The use RIWHFKQLTXHVFDSDEOHRIUHYHDOLQJGHWDLOHGOLSLGLQWHUDFWLRQVLQFOXGLQJPROHFXODUSDUWLFOH dynamics, is hampered by the large molecular dimensions and complex thermodynamic SURSHUWLHV RI OLSLGV LQ DGGLWLRQ WR WKH VWUXFWXUDO FRPSOH[LW\ RI OLSRSURWHLQ SDUWLFOHV50 Kumpula et al used a structural model to optimize the lipid distributions within lipoprotein SDUWLFOHVEDVHGRQWKHWRWDOPROHFXODUYROXPHVRIWKHFRUHDQGVXUIDFH48 This model ZDVDSSOLHGIRUFRPSRVLWLRQDOGDWDRQHOHYHQOLSRSURWHLQVXEFODVVHVIRURSWLPL]LQJWKH GLVWULEXWLRQRIWKHK\GURSKRELFOLSLGVWULJO\FHULGHDQGFKROHVWHUROHVWHUPROHFXOHV7KH PRGHOUHYHDOHGWKDWSDUWLFOHVL]HGHSHQGHQWSURSRUWLRQRIWKHFRUHOLSLGVPD\ORFDWHLQ WKHVXUIDFHRIOLSRSURWHLQSDUWLFOHV$GGLWLRQDOO\WKH\VKRZHGWKDWWKHFRPSRVLWLRQRIWKH SDUWLFOHVLQÀXHQFHVWKHPROHFXODUFRQWHQWRIWKHVXUIDFH48 Such structural models seem WRSURYLGHDORJLFDOVWUXFWXUDOUDWLRQDOHIRUPHWDEROLFFDVFDGHVLQOLSRSURWHLQPHWDEROLVP ZLWKWKHDFWLYLW\RIFDWDO\WLFHQ]\PHDQGWKHPROHFXODUELQGLQJRIWUDQVSRUWSURWHLQVDW WKHVXUIDFHRIWKHSDUWLFOHV 7ROLQNOLSLGRPLFSUR¿OHVPHDVXUHGLQVHUXPWRWKRVHLGHQWL¿HGLQPDMRUOLSRSURWHLQ FODVVHVDKLHUDUFKLFDO%D\HVLDQUHJUHVVLRQPRGHOZDVGHYHORSHGE\DJURXSIURPWKH 9777HFKQLFDO5HVHDUFK&HQWHURI)LQODQG517KH\IRXQGWKDWWKHDPRXQWRIDOLSLGLQ VHUXPFDQEHDGHTXDWHO\GHVFULEHGE\WKHDPRXQWVRIOLSLGVLQWKHOLSRSURWHLQFODVVHV51

BLOOD SPHINGOLIPIDS IN HOMEOSTASIS AND PATHOBIOLOGY

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7KHDSSOLHGDSSURDFKLIXVHGZLGHO\FRXOGHYHQWXDOO\IDFLOLWDWHG\QDPLFPRGHOLQJRI lipid metabolism at the individual molecular species level. ,WLVHVWDEOLVKHGWKDWVSKLQJROLSLGVLQWHUDFWZLWKFKROHVWHUROWRIRUPPHPEUDQHOLSLG microdomains that mediate signal transduction. Complex membrane sphingolipids such as 60DQGJO\FRVSKLQJROLSLGVPRGXODWHWKHIXQFWLRQRIJURZWKIDFWRUUHFHSWRUVDQGH[WUDFHOOXODU PDWUL[SURWHLQVDQGVHUYHDVELQGLQJVLWHVIRUPLFURRUJDQLVPVDQGWR[LQV7 Accordingly, PDLQWHQDQFHRIWKHPHPEUDQHVWUXFWXUHLVFUXFLDOIRUPHFKDQLFDOVWDELOL]DWLRQDQGDQ\VKLIW LQOLSLGVDV\PPHWU\FDQLQGXFHDYDULHW\RIXQIDYRUDEOHFHOOXODUUHVSRQVHV$OWHUDWLRQV in sphingolipids have been implicated in several diseases.10,11,52-57 For example, elevated SODVPD60OHYHOVZHUHVKRZQWREHFORVHO\UHODWHGWRWKHGHYHORSPHQWRIDWKHURVFOHURVLV9 DQGSODVPD&HUVZHUHDOVRSURSRVHGWRVHUYHDVELRPDUNHUVIRUDWKHURVFOHURVLV8 ,W KDV EHHQ VKRZQ WKDW 60 DQG SKRVSKDWLG\OFKROLQH PRGXODWH WKH IXQFWLRQ RI OLSRSURWHLQV DQG VHUYH DV SUHFXUVRUV IRU D YDULHW\ RI UHJXODWRU\ PROHFXOHV LQFOXGLQJ lysophosphatidylcholine58,59 and Cer.607\SLFDOO\WKHEUHDNGRZQRIFRPSOH[VSKLQJROLSLGV UHVXOWVLQWKHIRUPDWLRQRI&HUWKURXJKWKHDFWLRQRIVSKLQJRP\HOLQDVHVRUJO\FRVLGDVHV DQG &HUV LQ WXUQ FDQ VHUYH DV SUHFXUVRUV IRU PDMRU VSKLQJROLSLGV VXFK DV 60 DQG glucosylceramide.61&HUDPLGHZDVWKHUHIRUHSURSRVHGDVD³FRRUGLQDWRU´RIVWUHVVUHVSRQVHV in eukaryotes.2,62,63 In accordance with this concept, it has been shown that plasma levels RI WKH PRVW DEXQGDQW &HU &24-Cer, increased in coronary artery disease and stroke SDWLHQWVFRPSDUHGWROHYHOVLQFRQWUROVXEMHFWVZLWKRXWFRQVLGHUDEOHFKDQJHVRIRWKHU Cer species.64)DUEHUDQG1LHPDQQ3LFNGLVHDVHVZKLFKDUHWULJJHUHGE\G\VIXQFWLRQRI DFLGFHUDPLGDVHDQGDFLGVSKLQJRP\HOLQDVHDUHDVVRFLDWHGZLWKHOHYDWHGOHYHOVRI&HU and SM.10)XUWKHUPRUHDEQRUPDOLWLHVRIJO\FROLSLGPHWDEROLVPLQ*DXFKHUGLVHDVH)DEU\ disease and metachromatic leukodystrophy have led to an interest in the composition, PHWDEROLVPDQGIXQFWLRQRIWKHJO\FRV\OFHUDPLGHVLQKXPDQEORRG11 Gaucher disease is a glycolipid storage disorder characterized by the accumulation RIJOXFRV\OFHUDPLGH8VLQJGHOD\HGH[WUDFWLRQPDWUL[DVVLVWHGODVHUGHVRUSWLRQLRQL]DWLRQ WLPHRIÀLJKWPDVVVSHFWURPHWU\'(0$/',72)06 DVWXG\DQDO\]HGVSKLQJROLSLGV LQSHULFDUGLDOÀXLGSHULWRQHDOÀXLGDQGVHUXPIURPWZRSDWLHQWVZLWK*DXFKHUGLVHDVH65 7KHUHVXOWVVKRZHGWKDWLQSHULFDUGLDOÀXLGSHULWRQHDOÀXLGDQGVHUXPWKHFHUDPLGH monohexoside/SM ratio was increased in the Gaucher disease patients compared to controls. 7KHVDPHJURXSDQDO\]HGWKHVSKLQJROLSLGVLQWKHFDUGLDFYDOYHVIURPD\HDUROGPDOH SDWLHQWZLWK)DEU\GLVHDVHZKRVXIIHUHGIURPFRQJHVWLYHFDUGLDFIDLOXUH66 Fabry disease is DJO\FROLSLGVWRUDJHGLVRUGHUFDXVHGE\DGHIHFWRIDOSKDJDODFWRVLGDVH$DQGFKDUDFWHUL]HG E\WKHV\VWHPLFGHSRVLWLRQRIJO\FRVSKLQJROLSLGVZLWKWHUPLQDODOSKDJDODFWRV\OPRLHWLHV PDLQO\JORERWULDRV\OFHUDPLGHLQWLVVXHV8VLQJWKLVVHPLTXDQWLWDWLYHDQDO\VLVRI'( MALDI-TOF-MS, it was revealed that ceramide trihexoside species clearly accumulated LQWKHFDUGLDFYDOYHVIURPWKHSDWLHQW66 ,WZDVGRFXPHQWHGWKDWDOOEORRGFHOOVFDQUHPRYHSODVPD6SKZKLFKLVKDUPIXO RU VXSSUHVVLYH WR FHOOXODU IXQFWLRQV DQG FRQYHUW LW LQWR SODVPD 6344 Plasma S1P may play diverse important roles in blood vessels. We have recently shown that QRUPDOSK\VLRORJLFDOOHYHOVRIFLUFXODWLQJ63SOD\DNH\UROHLQYDVFXODUVWDELOLW\DQG permeability.23 Recent work in our laboratory also showed that S1P can be generated in response to monocyte/macrophage activation47 and S1P can also stimulate the secretion RINH\LQÀDPPDWRU\PDUNHUVLQFOXGLQJ&2;71)_ and PGE2.12 In a study designed WRWHVWWKHDELOLW\RIVHUXPVSKLQJROLSLGVWRSUHGLFWREVWUXFWLYHFRURQDU\DUWHU\GLVHDVH VHUXP 63 ZDV SURSRVHG WR EH D UHPDUNDEO\ VWURQJ DQG UREXVW SUHGLFWRU RI ERWK WKH RFFXUUHQFHDQGVHYHULW\RIFRURQDU\VWHQRVLV33

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+DPPDG60&UHOOLQ+*:X%;HWDO'XDODQGGLVWLQFWUROHVIRUVSKLQJRVLQHNLQDVHDQGVSKLQJRVLQH SKRVSKDWHLQWKHUHVSRQVHWRLQÀDPPDWRU\VWLPXOLLQ5$:PDFURSKDJHV3URVWDJODQGLQV2WKHU/LSLG Mediat 2008; 85(3-4):107-114. 9HUKHLM0%RVH5/LQ;+HWDO5HTXLUHPHQWIRUFHUDPLGHLQLWLDWHG6$3.-1.VLJQDOOLQJLQVWUHVVLQGXFHG apoptosis. Nature 1996; 380(6569):75-79. $XJp11LNRORYD.DUDNDVKLDQ0&DUSHQWLHU6HWDO5ROHRIVSKLQJRVLQHSKRVSKDWHLQWKHPLWRJHQHVLV LQGXFHGE\R[LGL]HGORZGHQVLW\OLSRSURWHLQLQVPRRWKPXVFOHFHOOVYLDDFWLYDWLRQRIVSKLQJRP\HOLQDVH ceramidase and sphingosine kinase. J Biol Chem 1999; 274(31):21533-21538. %ULQNPDQQ9/\QFK.5)7<WDUJHWLQJ*SURWHLQFRXSOHGUHFHSWRUVIRUVSKLQJRVLQHSKRVSKDWHLQ transplantation and autoimmunity. Curr Opin Immunol 2002; 14(5):569-75. %LHODZVNL-3LHUFH-66QLGHU-HWDO&RPSUHKHQVLYHTXDQWLWDWLYHDQDO\VLVRIELRDFWLYHVSKLQJROLSLGVE\ KLJKSHUIRUPDQFHOLTXLGFKURPDWRJUDSK\WDQGHPPDVVVSHFWURPHWU\0HWKRGV0RO%LRO +DPPDG603LHUFH-66RRGDYDU)HWDO%ORRGVSKLQJROLSLGRPLFVLQKHDOWK\KXPDQV,PSDFWRIVDPSOH collection methodology. J Lipid Res 2010. -H\DUDMDK (- &URPZHOO :& 2WYRV -' /LSRSURWHLQ SDUWLFOH DQDO\VLV E\ QXFOHDU PDJQHWLF UHVRQDQFH spectroscopy. Clin Lab Med 2006; 26(4):847-870. 0XUDWD16DWR..RQ-HWDO,QWHUDFWLRQRIVSKLQJRVLQHSKRVSKDWHZLWKSODVPDFRPSRQHQWVLQFOXGLQJ lipoproteins, regulates the lipid receptor-mediated actions. Biochem J 2000; 352 Pt 3:809-815. :LHVQHU3/HLGO.%RHWWFKHU$HWDO/LSLGSUR¿OLQJRI)3/&VHSDUDWHGOLSRSURWHLQIUDFWLRQVE\HOHFWURVSUD\ ionization tandem mass spectrometry. J Lipid Res 2009; 50(3):574-585. 21. Pyne S, Pyne NJ. Sphingosine 1-phosphate signalling in mammalian cells. Biochem J 2000; 349(Pt 2):385-402. 22. Hla T. Sphingosine 1-phosphate receptors. Prostaglandins 2001; 64(1-4):135-142. 23. Argraves KM, Gazzolo PJ, Groh EM et al. High density lipoprotein-associated sphingosine 1-phosphate SURPRWHVHQGRWKHOLDOEDUULHUIXQFWLRQ-%LRO&KHP  %RUQIHOGW.(*UDYHV/05DLQHV(:HWDO6SKLQJRVLQHSKRVSKDWHLQKLELWV3'*)LQGXFHGFKHPRWD[LV RIKXPDQDUWHULDOVPRRWKPXVFOHFHOOVVSDWLDODQGWHPSRUDOPRGXODWLRQRI3'*)FKHPRWDFWLFVLJQDO transduction. J Cell Biol 1995; 130(1):193-206. .LPXUD7:DWDQDEH76DWR.HWDO6SKLQJRVLQHSKRVSKDWHVWLPXODWHVSUROLIHUDWLRQDQGPLJUDWLRQRIKXPDQ endothelial cells possibly through the lipid receptors, Edg-1 and Edg-3. Biochem J 2000; 348 Pt 1:71-76. /HH0-7KDQJDGD6&ODIIH\.3HWDO9DVFXODUHQGRWKHOLDOFHOODGKHUHQVMXQFWLRQDVVHPEO\DQGPRUSKRJHQHVLV induced by sphingosine-1-phosphate. Cell 1999; 99(3):301-312. 27. Yatomi Y, Yamamura S, Ruan F et al. Sphingosine 1-phosphate induces platelet activation through an H[WUDFHOOXODUDFWLRQDQGVKDUHVDSODWHOHWVXUIDFHUHFHSWRUZLWKO\VRSKRVSKDWLGLFDFLG-%LRO&KHP 272(8):5291-5297. .RQWXVK$7KHURQG3=HUUDG$HWDO3UHIHUHQWLDOVSKLQJRVLQHSKRVSKDWHHQULFKPHQWDQGVSKLQJRP\HOLQ GHSOHWLRQDUHNH\IHDWXUHVRIVPDOOGHQVH+'/SDUWLFOHVUHOHYDQFHWRDQWLDSRSWRWLFDQGDQWLR[LGDWLYH activities. Arterioscler Thromb Vasc Biol 2007; 27(8):1843-1849. 5RGUtJXH]&*RQ]iOH]'tH]0%DGLPRQ/HWDO6SKLQJRVLQHSKRVSKDWH$ELRDFWLYHOLSLGWKDWFRQIHUV high-density lipoprotein with vasculoprotection mediated by nitric oxide and prostacyclin. Thromb Haemost 2009; 101(4):665-673. /XFNH 6 /HYNDX % (QGRWKHOLDO IXQFWLRQV RI VSKLQJRVLQHSKRVSKDWH &HOO 3K\VLRO %LRFKHP  26(1):87-96. 1L[RQ *) 6SKLQJROLSLGV LQ LQÀDPPDWLRQ SDWKRORJLFDO LPSOLFDWLRQV DQG SRWHQWLDO WKHUDSHXWLF WDUJHWV Br J Pharmacol 2009; 158(4):982-993. 2NDMLPD ) 3ODVPD OLSRSURWHLQV EHKDYH DV FDUULHUV RI H[WUDFHOOXODU VSKLQJRVLQH SKRVSKDWH LV WKLV DQ atherogenic mediator or an anti-atherogenic mediator? Biochim Biophys Acta 2002; 1582(1-3):132-137. 33. Deutschman DH, Carstens JS, Klepper RL et al. Predicting obstructive coronary artery disease with serum sphingosine-1-phosphate. Am Heart J 2003; 146(1):62-68. 34. Lee MH, Hammad SM, Semler AJ et al. HDL3, but not HDL2, stimulates plasminogen activator inhibitor-1 UHOHDVHIURPDGLSRF\WHV²WKHUROHRIVSKLQJRVLQHSKRVSKDWH-/LSLG5HV 5DGHU '- 5HJXODWLRQ RI UHYHUVH FKROHVWHURO WUDQVSRUW DQG FOLQLFDO LPSOLFDWLRQV $P - &DUGLRO  92(4A):42J-49J. 6SDUNV'/'DYLGVRQ:6/XQG.DW]6HWDO(IIHFWVRIWKHQHXWUDOOLSLGFRQWHQWRIKLJKGHQVLW\OLSRSURWHLQ on apolipoprotein A-I structure and particle stability. J Biol Chem 1995; 270(45):26910-26917. &XUWLVV/.%RQQHW'-5\H.$7KHFRQIRUPDWLRQRIDSROLSRSURWHLQ$,LQKLJKGHQVLW\OLSRSURWHLQVLV LQÀXHQFHGE\FRUHOLSLGFRPSRVLWLRQDQGSDUWLFOHVL]HDVXUIDFHSODVPRQUHVRQDQFHVWXG\%LRFKHPLVWU\ 2000; 39(19):5712-5721. 9HGKDFKDODP&&KHWW\361LFNHO0HWDO,QÀXHQFHRIDSROLSRSURWHLQ$32 $,VWUXFWXUHRQQDVFHQW high density lipoprotein (HDL) particle size distribution. J Biol Chem 2010. .UDXVV 50 /LSRSURWHLQ VXEIUDFWLRQV DQG FDUGLRYDVFXODU GLVHDVH ULVN &XUU 2SLQ /LSLGRO    305-311.

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40. Sattler KJ, Elbasan S, Keul P et al. Sphingosine 1-phosphate levels in plasma and HDL are altered in coronary artery disease. Basic Res Cardiol 2010. 41. Camerer E, Regard JB, Cornelissen I et al. Sphingosine-1-phosphate in the plasma compartment regulates EDVDODQGLQÀDPPDWLRQLQGXFHGYDVFXODUOHDNLQPLFH-&OLQ,QYHVW  3DSSX56FKZDE65&RUQHOLVVHQ,HWDO3URPRWLRQRIO\PSKRF\WHHJUHVVLQWREORRGDQGO\PSKE\GLVWLQFW VRXUFHVRIVSKLQJRVLQHSKRVSKDWH6FLHQFH  9HQNDWDUDPDQ./HH<00LFKDXG-HWDO9DVFXODUHQGRWKHOLXPDVDFRQWULEXWRURISODVPDVSKLQJRVLQH 1-phosphate. Circ Res 2008; 102(6):669-676. 
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CHAPTER 5 ADIPOSE TISSUE AND CERAMIDE BIOSYNTHESIS IN THE PATHOGENESIS OF OBESITY Fahumiya Samad,* Leylla Badeanlou, Charmi Shah and Guang Yang Torrey Pines Institute for Molecular Studies, San Diego, California, USA *Corresponding Author: Fahumiya Samad—Email: [email protected]

Abstract:

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INTRODUCTION 2EHVLW\ KDV UHDFKHG HSLGHPLF SURSRUWLRQV LQ ZHVWHUQ VRFLHWLHV ZLWK  RI WKH adult US population being either overweight or obese.12EHVLW\FRQWULEXWHVVLJQL¿FDQWO\ WRPRUELGLW\DQGPRUWDOLW\DVLWLVDPDMRUULVNIDFWRULQWKHHWLRORJ\RIKHDUWGLVHDVH GLDEHWHVDQGFDQFHU:LWKLQWKHODVWGHFDGHWKHDGLSRVHWLVVXHKDVEHFRPHDFHQWUDOIRFXV LQWKHSDWKRJHQHVLVRIREHVLW\DVVRFLDWHGPHWDEROLFDQGFDUGLRYDVFXODUFRPSOLFDWLRQV Sphingolipids and Metabolic Disease, edited by L. Ashley Cowart. ©2011 Landes Bioscience and Springer Science+Business Media, LLC 67

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Another mechanism by which obesity leads to insulin resistance in the liver and muscle is via increased intracellular lipid accumulation in these tissues.21-255HOHDVHRI IUHHIDWW\DFLGV))$V IURPDGLSRVHWLVVXHVWRUHVYLDOLSRO\VLVFRXSOHGZLWKWKHLQDELOLW\ RIWKHDGLSRVHWLVVXHWRVWRUHH[FHVVHQHUJ\UHVXOWVLQHFWRSLFOLSLGDFFXPXODWLRQLQWLVVXHV XQVXLWHGIRUWULJO\FHULGHVWRUDJHVXFKDVWKHOLYHUPXVFOHDQGSDQFUHDV,QÀDPPDWRU\ cytokines and FFAs activate stress signaling pathways including c-Jun N terminal kinase DQG1)Ʉ%WKDWLQWHUIHUHZLWKLQVXOLQVLJQDOLQJDQGFRQWULEXWHWRLQVXOLQUHVLVWDQFHLQ adipose, muscle and liver (Fig. 1). Studies demonstrate that sphingolipid metabolism is altered in adipose tissues in obesity and bioactive sphingolipids such as ceramide and/or its metabolites, sphingosine and sphingosine 1 phosphate (S1P) may provide a common pathway that link both elevated ))$VUHVXOWLQJIURPH[FHVVQXWULHQWVDQGDGLSRVHLQÀDPPDWLRQWRLQFUHDVHGPHWDEROLF and cardiovascular risk. REMODELING OF ADIPOSE AND PLASMA CERAMIDE IN OBESITY 6SKLQJROLSLGPHWDEROLVPLVFRQWUROOHGE\DFRPSOH[QHWZRUNRIKLJKO\UHJXODWHG LQWHUFRQQHFWHGSDWKZD\VOHDGLQJWRWKHSURGXFWLRQRIELRDFWLYHPROHFXOHVLQFOXGLQJ ceramide, sphingosine, S1P and ceramide 1 phosphate (C1P). Ceramide is the central PROHFXOHLQVSKLQJROLSLGPHWDEROLVPDQGWKHFRPPRQSUHFXUVRULQWKHJHQHUDWLRQRI FRPSOH[VSKLQJROLSLGV7KHSURGXFWLRQRIFHUDPLGHLVPHGLDWHGE\GHQRYRV\QWKHVLV YLDVHULQHSDOPLWR\OWUDQVIHUDVH637 DQGFHUDPLGHV\QWKDVH&HU6 RUWKHK\GURO\VLV RI PHPEUDQH VSKLQJRP\HOLQ E\ DFLG RU QHXWUDO VSKLQJRP\HOLQDVH $60DVH RU NSMase).26-28$QXPEHURILVRIRUPVRI&HU6KDYHEHHQLGHQWL¿HGDQGWKHVHHQ]\PHV UHJXODWHWKHIDWW\DFLGFRPSRVLWLRQRIFHUDPLGHOHDGLQJWRWKHJHQHUDWLRQRIPXOWLSOH ceramide species.29 Ceramide is subsequently deacylated to produce sphingosine through WKHDFWLRQRIFHUDPLGDVHVDONDOLQHRUDFLGFHUDPLGDVH DQGVSKLQJRVLQHFDQEHWKHQ phosphorylated to S1P via sphingosine kinase.27,28 Ceramide can be phosphorylated by ceramide kinase to produce C1P and converted to the complex sphingolipid JOXFRV\OFHUDPLGHE\WKHDGGLWLRQRIJOXFRVHPROHFXOHVLQDUHDFWLRQFDWDO\]HGE\ glucosylceramide synthase.30,31 Accumulating evidence suggest that these bioactive sphingolipids (e.g., ceramide, sphingosine, S1P, C1P) serve as signaling molecules LQYROYHG LQ PXOWLSOH VLJQDOLQJ SDWKZD\V UHJXODWLQJ D YDULHW\ RI SK\VLRORJLFDO DQG SDWKRORJLFDO ELRORJLFDO HYHQWV LQFOXGLQJ FHOO JURZWK DQG VXUYLYDO GLIIHUHQWLDWLRQ DSRSWRVLVDQGLQÀDPPDWLRQ27,28 It is now well established that sphingolipid metabolism can be activated by a YDULHW\RIFRQGLWLRQVVXFKDVSURLQÀDPPDWRU\F\WRNLQHVHJ71)_ JURZWKIDFWRUV R[LGDWLYHVWUHVVDQGLQFUHDVHGDYDLODELOLW\RI))$V$OORIWKHVHFRQGLWLRQVFKDUDFWHUL]H WKHORFDOPLOLHXRIWKHREHVHDGLSRVHWLVVXHVXJJHVWLQJWKDWVSKLQJROLSLGPHWDEROLVPPD\ be altered in adipose tissues in obesity. Detailed changes in sphingolipid metabolites SURGXFHGLQWKHDGLSRVHWLVVXHDQGSODVPDRIJHQHWLFDOO\REHVHRERE ZDVUHSRUWHG using a lipidomics approach.32,QWKHOHSWLQGH¿FLHQWREREPLFHWRWDOVSKLQJRP\HOLQ and ceramide were reduced, whereas sphingosine was increased when compared to their lean counterparts. In contrast with sphingolipid levels in adipose tissue, total VSKLQJRP\HOLQFHUDPLGHVSKLQJRVLQHDQG63OHYHOVZHUHDOOHOHYDWHGLQWKHSODVPDRI REREPLFH6LJQL¿FDQWGHFUHDVHVZHUHREVHUYHGLQDGLSRVHWLVVXHVIRU&&DQG &FHUDPLGHZKHUHDVDJHQHUDOLQFUHDVHZDVREVHUYHGLQSODVPDIRUDOOGHWHFWDEOH

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VSHFLHVRIFHUDPLGH+RZHYHUWKHODUJHVWLQFUHDVHRIDOPRVWZDVREVHUYHGIRU C18 ceramide. The observed changes in adipose ceramide levels in the ob/ob mice SDUDOOHOHGLQFUHDVHVLQJHQHH[SUHVVLRQRIHQ]\PHVLQYROYHGLQFHUDPLGHJHQHUDWLRQ (SPT, NSMase and ASMase) and ceramide hydrolysis (Ceramidase). The decrease in ceramide observed in the adipose tissue and the corresponding increase in plasma may UHÀHFWVHFUHWLRQIURPDGLSRVHWLVVXHLQWRWKHFLUFXODWLRQ,QWKLVUHVSHFWLWZDVVKRZQ that in Sprague-Dawley rats, dexamathasone treatment induced a dramatic increase in ceramide within the portal vein, suggesting that elevated circulating ceramide may RULJLQDWHIURPWKHJHQHUDWLRQDQGVHFUHWLRQRIFHUDPLGHIURPDGLSRVHWLVVXHVWRUHV33 Ceramide secretion was also observed in cultured adipocytes (Samad et al, unpublished observations). Since the ob/ob mice lack the satiety hormone leptin and human obesity is FKDUDFWHUL]HG E\ OHSWLQ UHVLVWDQFH DQG LQFUHDVHG OHYHOV RI FLUFXODWLQJ OHSWLQ WKH UHOHYDQFHRIWKHREREPRGHOWRKXPDQREHVLW\PD\EHTXHVWLRQDEOH7KHVHVWXGLHV ZHUHWKHUHIRUHDOVRH[WHQGHGWRDKLJKIDWGLHW+)' LQGXFHGREHVHPRXVHPRGHO which better mimics human obesity. Here, C57BL/6J mice were placed on a high (60% NFDOVIURPIDW RUORZIDWNFDOVIURPIDW GLHWIRUZHHNV,QWKLVPRGHOWKH +)'OHDGWRDVLJQL¿FDQWLQFUHDVHLQFHUDPLGHOHYHOVLQERWKWKHSODVPDDQGDGLSRVH WLVVXHYLDDPHFKDQLVPWKDWLQYROYHVWKHLQGXFWLRQRIHQ]\PHVWKDWLQFUHDVHFHUDPLGH synthesis (SPT, acid-SMase and neutral-SMase).34 Lipidomics analysis revealed that WKH SUHGRPLQDQW FHUDPLGH VSHFLHV LQ DGLSRVH WLVVXHV RI QRUPDO OHDQ PLFH ZDV & (Fig. 2A), whereas C24 ceramide was the dominant species in the plasma (Fig. 2C). HFD-induced obesity resulted in more than 300% increase in C18 ceramide in both

Figure 2. Ceramide expression in lean and diet induced obese (DIO) mice. Panels A and C: Ceramide OHYHOV LQ DGLSRVH WLVVXHV DQG SODVPD RI OHDQ PLFH UHVSHFWLYHO\ 3DQHOV % DQG ' &HUDPLGH H[SUHVVLRQ LQ DGLSRVH WLVVXHV DQG SODVPD RI ',2 PLFH UHVSHFWLYHO\ 2EHVH PLFH ZHUH JHQHUDWHG E\ SODFLQJ PDOH &%/- PLFH RQ D KLJK IDW GLHW  RI WRWDO FDORULHV GHULYHG IURP IDW  IRU  ZHHNV ZKHUHDV OHDQ PLFH ZHUH IHG D ORZ IDW GLHW  WRWDO FDORULHV IURP IDW  $GDSWHG IURP UHIHUHQFH 

ADIPOSE TISSUE AND CERAMIDE BIOSYNTHESIS IN OBESITY

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adipose and plasma, whereas C16 ceramide was induced by approximately 75% (Fig. 2B,D). Interestingly, C18 ceramide which was maximally stimulated in response WRWKH+)'FRQVWLWXWHGDUHODWLYHO\PLQRUVSHFLHVLQWKHSODVPDDQGDGLSRVHWLVVXHRI normal lean mice. The dramatic increase in adipose C18 ceramide correlated with elevated OHYHOV RI FHUDPLGH V\QWKDVH  &HU6  ZKLFK SUHIHUHQWLDOO\ OHDGV WR WKH SURGXFWLRQ RI & FHUDPLGH 7KHVH VWXGLHV VXJJHVW WKDW LQFUHDVHV LQ VSHFL¿F FHUDPLGH VSHFLHV VXFKDV&RU&UDWKHUWKDWWRWDOFHUDPLGHPD\EHVLJQL¿FDQWLQWKHSDWKRJHQHVLV RIREHVLW\DQGWKHPHWDEROLFV\QGURPH$UHFHQWVWXG\VKRZHGWKDW7\SHGLDEHWLF VXEMHFWVKDGKLJKHUFRQFHQWUDWLRQVRISODVPDWRWDODQG&&DQG&FHUDPLGH ZKLFKFRUUHODWHGZLWKVHYHULW\RILQVXOLQUHVLVWDQFH35,QFUHDVHGOHYHOVRIWKHFHUDPLGH metabolites, sphingosine and S1P have also been reported in Type 2 diabetic patients LQGLFDWLQJWKHDFWLYDWLRQRIVSKLQJROLSLGPHWDEROLVPOHDGLQJWRFHUDPLGHJHQHUDWLRQ and degradation in this population.36$GLSRVHFHUDPLGHZDVDOVRHOHYDWHGLQVXEMHFWV ZLWKLQFUHDVHGOLYHUIDWWKDWZDVLQGHSHQGHQWRIREHVLW\37$JLQJLQFUHDVHVWKHULVNIRU LQVXOLQUHVLVWDQFHDQG7\SHGLDEHWHVDQGDGLSRF\WHVIURPROGPLFHH[SUHVVHGKLJKHU OHYHOVRIFHUDPLGHFRPSDUHGWRWKRVHIURP\RXQJPLFH38 Thus, aberrant adipose tissue ceramide accumulation appears to be associated with obesity and may play a role in WKHSDWKRJHQHVLVRIWKHPHWDEROLFV\QGURPH REGULATION OF CERAMIDE METABOLISM IN OBESITY TNF-_ expression is elevated in adipose tissues in obesity39 and this cytokine KDVEHHQVKRZQWRDFWLYDWHJHQHVLQYROYHGLQFHUDPLGHJHQHUDWLRQYLDK\GURO\VLVRI VSKLQJRP\HOLQ$60DVHDQG160DVH DQGWKHGHQRYRSDWKZD\RIFHUDPLGH637  generation in other cell systems.28,QWUDSHULWRQHDOLQMHFWLRQRI71)_ into C57BL/6J OHDQPLFHVLJQL¿FDQWO\LQFUHDVHGDGLSRVHWLVVXH$60DVH160DVHDQG637P51$ expression.32 7KH FRQWULEXWLRQ RI 71)_ to obesity mediated increase in plasma ceramide was also directly investigated in ob/ob mice that lack both the p55 and p75 TNF-_ receptors. Compared with wild type ob/ob mice, plasma ceramide levels were VLJQL¿FDQWO\GHFUHDVHGLQREREPLFHWKDWODFNERWKWKHSDQGS71)UHFHSWRUV (Samad, et al, unpublished observations). These results suggest that TNF-_ is upstream RIWKHSDWKZD\OHDGLQJWRFHUDPLGHJHQHUDWLRQLQREHVLW\2EHVLW\LVDVVRFLDWHGZLWK insulin resistance and hyperinsulinemia and, insulin dramatically induced ASMase, 160DVHDQG637P51$JHQHH[SUHVVLRQLQDGLSRVHWLVVXHVRIOHDQDQGLQVXOLQUHVLVWDQW ob/ob mice.327KHPDJQLWXGHRILQGXFWLRQRIWKHVHJHQHVZDVVLJQL¿FDQWO\KLJKHULQ insulin-treated ob/ob mice compared with insulin-treated lean mice, suggesting that WKHK\SHULQVXOLQHPLDWKDWIUHTXHQWO\DFFRPSDQLHVREHVLW\DQGLQVXOLQUHVLVWDQFHPD\ SURPRWHWKHDEQRUPDOH[SUHVVLRQRIJHQHVLQYROYHGLQWKHDFWLYDWLRQRIWKHFHUDPLGH pathway in the obese adipose tissue. Obesity is additionally characterized by elevated plasma FFA and oxidative stress. Increased adiposity and associated insulin resistance, particularly in abdominal adipose WLVVXHOHDGVWRLQFUHDVHGOLSRO\VLVDQGUHOHDVHRI))$WRWKHV\VWHPLFFLUFXODWLRQ7KLV LQFUHDVHGDYDLODELOLW\RI))$VGULYHVWKHGHQRYRJHQHUDWLRQRIFHUDPLGHV\QWKHVLVLQ tissues such as the skeletal muscle, liver and pancreas thus causing metabolic derangements LQWKHVHWLVVXHV+RZHYHUSDOPLWDWHDQGROHDWHERWKIDLOHGWRLQGXFHFHUDPLGHV\QWKHVLV in cultured 3T3-L1 adipocytes.40 Whether FFAs directly contributes to the increase in adipose ceramide levels in obesity has not been conclusively demonstrated.

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SPHINGOLIPIDS AND METABOLIC DISEASE

*OXFRFRUWLFRLGVDUHNQRZQWRFRQWULEXWHWRDGLSRVHG\VIXQFWLRQDQGWKHPHWDEROLF V\QGURPH /HYHOV RI WKH JOXFRFRUWLFRLG DFWLYDWLQJ HQ]\PH EHWDK\GUR[\VWHURLG dehydrogenase Type 1 (11beta-HSD1), are increased in adipose tissues in obesity.41 0LFH ODFNLQJ EHWD+6' DUH SURWHFWHG IURP REHVLW\ LQGXFHG GLDEHWHV ZKHUHDV LWV RYHUH[SUHVVLRQOHDGVWRWKHPDQLIHVWDWLRQRIIHDWXUHVRIWKHPHWDEROLFV\QGURPHLQFOXGLQJ obesity, insulin resistance and hypertension.42-447KHH[SUHVVLRQRIEHWD+6'LQ7/ SUHDGLSRF\WHVZHUHVLJQL¿FDQWO\LQGXFHGLQUHVSRQVHWRFHOOSHUPHDEOHFHUDPLGHDQDORJXH &FHUDPLGHEDFWHULDOVSKLQJRP\HOLQDVHDQG63VXJJHVWLQJDGLUHFWUROHIRUFHUDPLGHLQ WKHUHJXODWLRQRIDGLSRVHJOXFRFRUWLFRLGV45 Glucocorticoids in turn induce sphingolipid ELRV\QWKHVLVFHUDPLGHVSKLQJRVLQHDQGVSKLQJRP\HOLQ LQDYDULHW\RIFHOOW\SHV46-48 In YLYRGH[DPDWKDVRQHWUHDWPHQWRIUDWVGUDPDWLFDOO\LQGXFHGFHUDPLGHOHYHOVLQWKHSRUWDO FLUFXODWLRQVXJJHVWLQJLQFUHDVHGLQGXFWLRQDQGVHFUHWLRQIURPDGLSRVHWLVVXHVVWRUHV33 2EHVLW\LQGXFHVDFRQGLWLRQRIV\VWHPLFR[LGDWLYHVWUHVVDQGLQFUHDVHGR[LGDWLYHVWUHVV in tissues such as the adipose tissue, may at last in part contribute to the dysregulated H[SUHVVLRQ RI DGLSRF\WRNLQHV DQG WKH GHYHORSPHQW RI WKH PHWDEROLF V\QGURPH49-52 :KLOHR[LGDWLYHVWUHVVDQGPLWRFKRQGULDOG\VIXQFWLRQSHUVHFRXOGSURPRWHFHUDPLGH DFFXPXODWLRQWKLVKDVQRWEHHQGLUHFWO\GHPRQVWUDWHGIRUDGLSRVHFHUDPLGHLQREHVLW\ $QXPEHURIVWXGLHVDOVRGHPRQVWUDWHWKDWFHUDPLGHGLUHFWO\LQFUHDVHVPLWRFKRQGULDO G\VIXQFWLRQR[LGDWLYH VWUHVV )RU H[DPSOH FHUDPLGH WULJJHUV 526 SURGXFWLRQ53 and regulates mitochondrial membrane permeability.54,555HFHQWVWXGLHVVXJJHVWDUROHIRU PLWRFKRQGULDO FHUDPLGH LQ WKH UHFUXLWPHQW DQG DFWLYDWLRQ RI %D[56,57 a pro-apoptotic PHPEHURIWKH%FOIDPLO\WKDWUHJXODWHVPLWRFKRQGULDOSHUPHDELOLW\58 While potentially important mechanistic links are present between obesity, ceramide and oxidative stress, it still remains unclear however, whether aberrant ceramide accumulation is a cause or FRQVHTXHQFHRIR[LGDWLYHVWUHVVDVVRFLDWHGZLWKREHVLW\ NOVEL LINKS BETWEEN SPHINGOLIPID METABOLISM AND PLASMINOGEN ACTIVATOR INHIBITOR 1 (PAI-1) 3$, LV WKH SULPDU\ SK\VLRORJLFDO LQKLELWRU RI SODVPLQRJHQ DFWLYDWLRQ LQ YLYR DQGLQFUHDVHG3$,FRPSURPLVHVQRUPDO¿EULQFOHDUDQFHPHFKDQLVPVDQGSURPRWHV thrombosis.12 PAI-1 levels are consistently increased in the adipose tissues and plasma LQREHVLW\DQGFRUUHODWHVWURQJO\ZLWKSDUDPHWHUVRIWKHPHWDEROLFV\QGURPHLQFOXGLQJ body mass index, insulin resistance and hyperinsulinemia.18 Increasing evidence suggests WKDW 3$, PD\ FRQWULEXWH GLUHFWO\ WR WKH FRPSOLFDWLRQV RI REHVLW\ LQFOXGLQJ LQVXOLQ resistance, Type 2 diabetes and atherothrombosis18 and thus central to increased adiposity and its metabolic consequences. Interestingly, the increase in adipose and plasma ceramide observed in diet induced obsess (DIO) wild type C57BL/6J mice was attenuated in mice lacking PAI-1.34+)'IHG 3$,GH¿FLHQWPLFHZHUHSURWHFWHGIURPWKHGLHWLQGXFHGLQFUHDVHLQ637$60DVHDQG 160DVHP51$SURYLGLQJDPHFKDQLVWLFOLQNIRUGHFUHDVHGFHUDPLGHLQ3$,
ADIPOSE TISSUE AND CERAMIDE BIOSYNTHESIS IN OBESITY

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GHFUHDVHLQFHUDPLGHDQLQWHUPHGLDU\PROHFXOHOLQNLQJH[FHVVQXWULHQWVLQÀDPPDWRU\ cytokines and insulin resistance. CONTRIBUTION OF CERAMIDE BIOSYNTHESIS TO BODY WEIGHT REGULATION AND ENERGY METABOLISM 5HJXODWLRQRIERG\ZHLJKWLVJRYHUQHGE\PXOWLSOHSDWKZD\VRQHRIZKLFKLVOHSWLQ signaling. Leptin, the hormone secreted primarily by adipocytes regulates central and SHULSKHUDOVLJQDOLQJSDWKZD\VWKDWXOWLPDWHO\OHDGWRGHFUHDVHGIRRGLQWDNHDQGRULQFUHDVHG metabolism/energy expenditure.59-61 However, most obese humans are resistant to the HIIHFWVRIOHSWLQRQERG\ZHLJKWUHJXODWLRQ62-64 DIO in mice is a physiologically relevant PRGHORIKXPDQREHVLW\VLQFHREHVLW\LQWKHVHPLFHLVDVVRFLDWHGZLWKWKHKDOOPDUNVRI OHSWLQUHVLVWDQFHK\SHUOHSWLQHPLDLQFUHDVHGIRRGLQWDNHDQGGHFUHDVHGPHWDEROLVP63,65 5HFHQWVWXGLHVSURYLGHFRQYLQFLQJHYLGHQFHWKDWWKHGHYHORSPHQWRIOHSWLQUHVLVWDQFH LVDSUHUHTXLVLWHIRU+)'LQGXFHGLQFUHDVHLQIDWVWRUDJHLQDGLSRF\WHVDQGWKHUHE\WR weight gain/obesity.66 :HGHWHUPLQHGZKHWKHUDFFXPXODWLRQRIFHUDPLGHLQUHVSRQVHWRD+)'FRQWULEXWHV to weight gain and leptin resistance. De novo ceramide synthesis was inhibited by treating mice with myriocin, which inhibits SPT, the rate limiting enzyme in de novo ceramide V\QWKHVLV&%/-PLFHZHHNROGPDOHV ZHUHIHGD+)'NFDOIURPIDW DQG treated with vehicle or myriocin as previously described67,68 (IP, O.3mg/kg body weight) HYHU\RWKHUGD\IRUZHHNV/LSLGRPLFVSUR¿OLQJVKRZHGWKDWP\ULRFLQWUHDWPHQWRI PLFHRQWKH+)'UHVXOWVLQVLJQL¿FDQWGHFUHDVHVLQDQXPEHURIFHUDPLGHVSHFLHVWKH ODUJHVWGHFUHDVHZDVREVHUYHGIRU&DQG&FHUDPLGH69:KLOHWKHERG\ZHLJKWVRI YHKLFOHWUHDWHGPLFHLQFUHDVHGUDSLGO\P\ULRFLQWUHDWHGPLFHJDLQHGVLJQL¿FDQWO\OHVV weight (Fig. 3A). Inhibiting de novo ceramide synthesis also resulted in increased oxygen consumption and CO2RXWSXW)LJ%& LQGLFDWLYHRILQFUHDVHGPHWDEROLVPDQGHQHUJ\ expenditure. Myriocin treatment decreased the respiratory exchange ratio (RER), a ratio RIFDUERK\GUDWHR[LGDWLRQWROLSLGR[LGDWLRQLQGLFDWLQJDVKLIWWRZDUGVIDWR[LGDWLRQLQ WKHVHPLFH)LJ' 7KHZHLJKWUHGXFWLRQGHFUHDVHGIDWSDGZHLJKWDGLSRF\WHVL]H LQFUHDVHGPHWDEROLVPDQGIDWR[LGDWLRQLQP\ULRFLQWUHDWHGPLFHDUHDOOKDOOPDUNVRI D OHSWLQ VHQVLWLYH VWDWH 7KHVH VWXGLHV KHQFH LPSOLFDWH WKDW DEHUUDQW DFFXPXODWLRQ RI ceramide via increased in vivo ceramide biosynthesis may potentially contribute to the GHYHORSPHQWRIOHSWLQUHVLVWDQFHDSUHUHTXLVLWHWRZHLJKWJDLQDQGDVVRFLDWHGPHWDEROLF and cardiovascular disorders. +LJKIDWIHHGLQJDOVROHDGVWRDODUJHLQFUHDVHLQWKHVXSSUHVVRURIF\WRNLQHVLJQDOLQJ 62&6 DSRVWUHFHSWRULQKLELWRURIOHSWLQVLJQDOLQJLPSRUWDQWLQWKHGHYHORSPHQWRI leptin resistance.66,70,71-74,QKLELWLRQRIGHQRYRFHUDPLGHV\QWKHVLVGUDPDWLFDOO\UHGXFHGWKH DGLSRVHH[SUHVVLRQRI62&6LQ+)'IHGPLFH690RUHRYHUGLUHFWWUHDWPHQWRI7/ adipocytes with the short chain ceramide analogue C6 increased SOCS3 mRNA.69 Thus SOCS3, may mechanistically link aberrant ceramide accumulation to peripheral leptin resistance in adipocytes. /HSWLQUHJXODWHVDQXPEHURIWDUJHWJHQHVDQGVLJQDOLQJSDWKZD\VLQYROYHGLQ energy homeostasis including the mitochondrial uncoupling proteins (UCP), whose expression is decreased in leptin resistant states.74-78 UCPs are mitochondrial inner PHPEUDQH SURWHLQV WKDW SURPRWH PLWRFKRQGULDO HQHUJ\ H[SHQGLWXUH YLD IDWW\ DFLG oxidation and thereby play important roles in whole body energy expenditure.79 Enzymes

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involved in ceramide biosynthesis are expressed in mitochondria and mitochondria DUHFDSDEOHRIFHUDPLGHJHQHUDWLRQ54,80-82 Mitochondrial ceramide was shown to be induced by TNF-_, a cytokine whose expression is elevated in obesity.83 In parallel ZLWKWKHGHFUHDVHLQ62&6H[SUHVVLRQLQKLELWLRQRIGHQRYRFHUDPLGHV\QWKHVLV VLJQL¿FDQWO\LQFUHDVHGWKHH[SUHVVLRQRIWKHGRZQVWUHDPOHSWLQWDUJHWJHQH8&3LQ DGLSRVHWLVVXHVRIPLFHRQWKH+)'69 Furthermore, UCP-3 mRNA expression was DOVRVLJQL¿FDQWO\UHGXFHGLQ7/DGLSRF\WHVWUHDWHGZLWKWKHFHUDPLGHDQDORJXH C6.69 While the proposition that ceramide accumulation may be involved in the SDWKRJHQHVLVRIREHVLW\PHGLDWHGOHSWLQUHVLVWDQFHLVLQWULJXLQJIXUWKHUVWXGLHVDUH QHHGHGWRGH¿QLWLYHO\SURYHWKLVK\SRWKHVLV SPHINGOLIPIDS AS MEDIATORS OF ADIPOSE INFLAMMATION IN OBESITY 2EHVLW\LVDVVRFLDWHGZLWKFKDQJHVLQDGLSRVHWLVVXHH[SUHVVLRQRIFKHPRNLQHV cytokines, hormones and other adipokines that is thought to underlie the cardiovascular and metabolic risk associated with obesity. These adipokines secreted by adipocytes and other cell types such as the macrophages that accumulate in the adipose tissue during ZHLJKWJDLQKDYHORFDODQGV\VWHPLFHIIHFWVRQLQVXOLQVLJQDOLQJSDWKZD\VLQPXVFOH DQGOLYHUDQGFRQWULEXWHVWRFKURQLFV\VWHPLFLQÀDPPDWLRQWKDWLQFUHDVHVFDUGLRYDVFXODU

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ULVN7UHDWPHQWRI7/DGLSRF\WHVZLWKVKRUWFKDLQFHUDPLGHVSKLQJRVLQHRU63 LQGXFHGWKHH[SUHVVLRQRI3$,DQGWKHSURLQÀDPPDWRU\PROHFXOHV71)_, IL-6, MCP-1 and KC to various extents.32 Ceramide also increased PAI-1 expression in cultured endothelial cells84 and astrocytes.85,QKLELWLRQRIGHQRYRFHUDPLGHJHQHUDWLRQ in DIO mice, reduced adipose tissue PAI-1 and MCP-1 expression, providing evidence that ceramide accumulation contributes to adipose tissue PAI-1 and MCP-1 expression in vivo.69 Ceramide however can be readily converted to sphingosine, which in turn can EHSKRVSKRU\ODWHGWR63DQGDOORIWKHVHUHDFWLRQVDUHUHYHUVLEOH$SSURDFKHVXVLQJ VSHFL¿FLQKLELWRUVRUVPDOOLQWHUIHULQJ51$RIHQ]\PHVLQYROYHGLQWKHVHFRQYHUVLRQV DUHQHHGHGWRVSHFL¿FDOO\LGHQWLI\WKHUROHVRILQGLYLGXDOVSKLQJROLSLGVLQWKHUHJXODWLRQ RIWKHVHSURLQÀDPPDWRU\DGLSRNLQHVLQDGLSRVHWLVVXHVLQREHVLW\ CONTRIBUTION OF CERAMIDE BIOSYNTHESIS TO OBESITY MEDIATED INSULIN RESISTANCE 2EHVLW\LVDVVRFLDWHGZLWKDQLQFUHDVHGULVNIRULQVXOLQUHVLVWDQFHFKDUDFWHUL]HG E\ DQ LPSDLUHG UHVSRQVLYHQHVV RI WKH SULPDU\ LQVXOLQ VHQVLWLYH WLVVXH WKH DGLSRVH PXVFOHDQGOLYHUWRWKHDQDEROLFUHVSRQVHVRIDQRUPDOSK\VLRORJLFDOGRVHRILQVXOLQ Insulin maintains glucose hemostasis by promoting glucose uptake by the muscle DQGDGLSRVHWLVVXHVDQGLQKLELWLQJJOXFRVHHIÀX[IURPWKHOLYHU$GGLWLRQDOO\LQVXOLQ LQGXFHVIDWW\DFLGXSWDNHDQGVWRUDJHDVWULJO\FHULGHVLQDGLSRVHWLVVXHVDQGLQVXOLQ UHVLVWDQFHLVDVVRFLDWHGZLWKLQFUHDVHGDGLSRVHWLVVXHOLSRO\VLVUHVXOWLQJLQVHFUHWLRQRI ))$OHDGLQJWRLPSDLUPHQWRILQVXOLQVLJQDOLQJLQWKHPXVFOHDQGOLYHUDQGWRSDQFUHDWLF G\VIXQFWLRQ7KXVLQVXOLQUHVLVWDQFHLQWKHDGLSRVHWLVVXHPD\EHWKHSULPDU\HYHQW WKDW SUHFLSLWDWHV ZKROH ERG\ LQVXOLQ UHVLVWDQFH DQG WKH VXEVHTXHQW GHYHORSPHQW RI Type 2 diabetes. %RWKLQYLWURDQGLQYLYRVWXGLHVXQHTXLYRFDOO\GHPRQVWUDWHDUROHIRUVSKLQJROLSLGV VSHFL¿FDOO\FHUDPLGHLQWKHGHYHORSPHQWRILQVXOLQUHVLVWDQFHUHYLHZHGLQUHIV  and ceramide appears to be a putative intermediate linking both excess nutrients such DVVDWXUDWHG))$DQGLQÀDPPDWRU\F\WRNLQHVVXFKDV71)_WRWKHLQGXFWLRQRILQVXOLQ resistance. Ceramide and sphingosine inhibits insulin action and signaling in various cell culture systems.25,87-91 In vivo studies by Holland et al demonstrated that inhibition RI FHUDPLGH V\QWKHVLV E\ SUHYHQWLQJ GH QRYR FHUDPLGH V\QWKHVLV XVLQJ WKH VSHFL¿F 637LQKLELWRUP\ULRFLQDPHOLRUDWHGJOXFRFRUWLFRLGVDWXUDWHGIDWDQGREHVLW\LQGXFHG insulin resistance.33 Heterozygous dihydroceramide desaturase mice exhibited enhanced insulin sensitivity and protection against dexamathasone-induced insulin resistance.33 ,QKLELWLRQ RI FHUDPLGH ELRV\QWKHVLV XVLQJ P\ULRFLQ DOVR LPSURYHG JOXFRVH KHPRVWDVLV LQ KLJK IDW GLHW LQGXFHG REHVH PLFH DV LQGLFDWHG E\ WKH VLJQL¿FDQW improvement in both the glucose and insulin tolerance tests (Fig. 4A,B).69 While the DERYHVWXGLHVFOHDUO\GHPRQVWUDWHWKDWFHUDPLGHPD\SOD\DUROHLQWKHGHYHORSPHQWRI insulin resistance, the molecular mechanisms by which it does so remain controversial DQGPD\GHSHQGRQWKHPRGHOV\VWHPXVHGIRUVWXG\$QXPEHURIUHSRUWVGHPRQVWUDWH that ceramide inhibits insulin-stimulated glucose uptake, GLUT4 translocation and/or glycogen synthesis.87-93 Ceramide has been shown to inhibit several distinct intermediates in the insulin signaling pathway including insulin receptor substrate (IRS)-1, PI3-kinase and Akt/PKB.94,95 However, these results have been controversial. While some studies have shown that in cultured cells, ceramide inhibited insulin mediated tyrosine

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Figure 4.*OXFRVHKHPRVWDVLVLQ',2PLFHLQUHVSRQVHWRLQKLELWLRQRIGHQRYRFHUDPLGHELRV\QWKHVLV Panel A: Insulin tolerance test. Panel B: Glucose tolerance test. Bottom panel: Western blot: Basal and ,QVXOLQPHGLDWHG $NW SKRVSKRU\ODWLRQ LQ /LYHU 0XVFOH DQG $GLSRVH WLVVXHV RI YHKLFOHWUHDWHG FRQWUROV &  P\ULRFLQ WUHDWHG 0  ',2 PLFH $GDSWHG IURP UHIHUHQFH 

SKRVSKRU\ODWLRQRI,56DQGVXEVHTXHQWDFWLYDWLRQRI3,NLQDVHRWKHUVKDYHIDLOHGWR REVHUYHWKLVHIIHFW87,89,96-103$OOVWXGLHVWKXVIDUKRZHYHUKDYHFRQVLVWHQWO\GHPRQVWUDWHG WKDWFHUDPLGHLQKLELWVSKRVSKRU\ODWLRQDQGDFWLYDWLRQRI$NW3.%DQGWKLVHIIHFWRI FHUDPLGHFRXOGSRWHQWLDOO\RFFXULQGHSHQGHQWO\RI,5687,98-100,103 Ceramide directly activates protein phosphatases 2A (PP2A),104 the primary phosphatases responsible IRU GHSKRVSKRU\ODWLQJ $NW3.%105 Ceramide also activates PKCc, an enzyme that inhibits Akt/PKB translocation to the membrane.106,107,QKLELWLRQRIGHQRYRFHUDPLGH ELRV\QWKHVLVVLJQL¿FDQWO\LQGXFHGEDVDO$NWSKRVSKRU\ODWLRQLQDGLSRVHWLVVXHZKHUHDV both basal and insulin-mediated Akt phosphorylation was induced in the liver and PXVFOH)LJ 7KXVWKHDPHOLRUDWLRQRIZKROHERG\LQVXOLQUHVLVWDQFHLQUHVSRQVH to decreased ceramide biosynthesis in obese mice can be attributed to the combined UHVWRUDWLRQRI$NWDFWLYLW\DQGLQVXOLQVHQVLWLYLW\LQDOOWKUHHLQVXOLQVHQVLWLYHWLVVXHV 0RUHUHFHQWO\SRWHQWLDOO\LPSRUWDQWUROHVIRUF-XQ1WHUPLQDONLQDVH-1. DQG LQKLELWRURIɄ%NLQDVH `,Ʉ.`) in insulin signaling have been recognized.108,109 JNK activity is increased in obesity and obese mice lacking JNK showed improved glucose homeostasis.109 6LPLODUO\ SKDUPDFRORJLFDO LQKLELWLRQ RI ,Ʉ.` RU PLFH GH¿FLHQW KHWHUR]\JRXV RU WLVVXH VSHFL¿F  IRU ,Ʉ.` ZHUH SURWHFWHG IURP LQVXOLQ UHVLVWDQFH108 &HUDPLGHDFWLYDWHVERWK-1.DQG,Ʉ.` , mechanisms that may additionally contribute

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to ceramide mediated insulin resistance.110 Thus, therapeutic strategies that lower in YLYRFHUDPLGHJHQHUDWLRQDUHSURYLQJWREHXVHIXOWRFRPEDWREHVLW\PHGLDWHGLQVXOLQ resistance and cardiovascular risk. Although there is compelling evidence in the literature pointing to a direct or indirect UROHIRUFHUDPLGHLQWKHLQKLELWLRQRILQVXOLQVLJQDOLQJDUROHIRUJO\FRVSKLQJROLSLG PHWDEROLWHVRIFHUDPLGHLQWKHGHYHORSPHQWRILQVXOLQUHVLVWDQFHLVDOVRLQFUHDVLQJO\ being recognized. 111-113 $GGLWLRQ RI *0 WR FXOWXUHG DGLSRF\WHV VXSSUHVVHG SKRVSKRU\ODWLRQRIWKHLQVXOLQUHFHSWRUDQG,56DQGGHFUHDVHGJOXFRVHWUDQVSRUW111 3KDUPDFRORJLFDOGHSOHWLRQRI*0LQDGLSRF\WHVXVLQJDJOXFRV\OFHUDPLGHV\QWKDVH LQKLELWRUSUHYHQWHGWKH71)LQGXFHGLQKLELWLRQRI,56VLJQDOLQJ111 Mutant mice that ODFN*0GHPRQVWUDWHLQFUHDVHGVHQVLWLYLW\WRLQVXOLQDQGZHUHSURWHFWHGIURPKLJK IDWGLHWLQGXFHGLQVXOLQUHVLVWDQFH1147UHDWPHQWRIREREPLFHZLWKDKLJKO\VSHFL¿F VPDOO PROHFXOH LQKLELWRU RI JOXFRV\OFHUDPLGH V\QWKDVH QRUPDOL]HG WKHLU HOHYDWHG WLVVXH JOXFRV\OFHUDPLGH OHYHOV DQG VLJQL¿FDQWO\ LPSURYHG JOXFRVH KRPHRVWDVLV113 6LPLODULPSURYHGLQVXOLQUHVLVWDQFHZDVREVHUYHGLQKLJKIDWIHGPLFHDQGLQ=') rats in response to inhibiting glucosylceramide synthase.113 Thus, glycosphingolipid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pool.1157KXVWKHRYHUSURGXFWLRQRIIDWW\DFLGVLQDGLSRVHWLVVXHGXHWRLQVXOLQUHVLVWDQFH LQDGLSRVHWLVVXHOHDGLQJWRHQKDQFHGOLSRO\VLV WKDWÀRZWRWKHOLYHUYLDWKHFLUFXODWLQJ ))$ SRRO FRQWULEXWHV VLJQL¿FDQWO\ WR WKH H[FHVV 7$* DFFXPXODWLRQ LQ 1$)/' ,Q LQVXOLQUHVLVWDQWVWDWHVLQVXOLQGRHVQRWIXOO\VXSSUHVVWKHDFWLYLW\RIKRUPRQHVHQVLWLYH OLSDVHZKLFKFDWDO\VHVWKHUHOHDVHRIIDWW\DFLGVIURP7$*VDQGUHVXOWVLQHQKDQFHG OLSRO\VLVDQGÀX[RIIDWW\DFLGVLQWRWKHSODVPD,QDGGLWLRQUHGXFHGJOXFRVHXSWDNHGXH to insulin resistance reduces glycerol 3 phosphate levels, thereby reducing the reutilization RIIDWW\DFLGVIRU7$*V\QWKHVLV7KXVLQVXOLQUHVLVWDQFHLQWKHDGLSRVHWLVVXHDSSHDUVWR EHLPSRUWDQWLQWKHSDWKRJHQHVLVRI1$)/',QWKLVFRQWH[WDGLSRVHWLVVXHFHUDPLGHV ZHUH LQFUHDVHG LQ VXEMHFWV ZLWK IDWW\ OLYHU FRPSDUHG WR HTXDOO\ REHVH VXEMHFWV ZLWK QRUPDO OLYHU IDW FRQWHQW37 *HQH DUUD\ VWXGLHV RI KXPDQ OLYHU VDPSOHV UHYHDOHG WKDW genes involved in ceramide signaling and metabolism were positively correlated with OLYHUIDW116%LRLQIRUPDWLFVDQDO\VLVGHPRQVWUDWHGVWURQJDVVRFLDWLRQVEHWZHHQKHSDWLF FHUDPLGHFRQWHQWDQGWKHH[WHQWRIVWHDWRVLVLQWKHJHQHWLFDOO\REHVHREREPLFH117 A recent study however demonstrated that diacylglycerols but not ceramides are increased LQQRQDOFRKROLFKXPDQIDWW\OLYHU1180LFHGH¿FLHQWLQERWKDFLGVSKLQJRP\HOLQDVHDQG ORZGHQVLW\OLSRSURWHLQUHFHSWRUZHUHSURWHFWHGIURPKLJKIDWGLHWLQGXFHGLQFUHDVHLQ hepatic triglyceride accumulation, body weight, hyperglycemia and insulin resistance in

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VSLWHRIHOHYDWHGVSKLQJRP\HOLQDQGRWKHUVSKLQJROLSLGV119 These results implicate that KHSDWLFVSKLQJROLSLGVPD\QRWGLUHFWO\FRQWULEXWHWRIDWW\OLYHU$QDOWHUQDWHSRVVLELOLW\ is that improved adipose insulin resistance resulting in reduced total plasma FFA may FRQWULEXWHWRSURWHFWLRQIURPKHSDWLFVWHDWRVLV 7KHFRQWULEXWLRQRIFHUDPLGHELRV\QWKHVLVWRWKHSDWKRJHQHVLVRIKHSDWLFVWHDWRVLV was directly addressed in HFD induced obese mice, where de novo ceramide biosynthesis was inhibited using myriocin.69 Hematoxylin and eosin staining showed pronounced VWHDWRVLVZLWKPDFURYHVLFXODUIDWDFFXPXODWLRQLQ',2PLFHZKLFKZDVVLJQL¿FDQWO\ UHGXFHGDIWHUP\ULRFLQWUHDWPHQW)LJ$% +HSDWLFWULJO\FHULGHVRQHRIWKHPDMRU VWRUDJHIRUPVRIOLSLGVLQWKHOLYHUZHUHDOVRUHGXFHGLQP\ULRFLQWUHDWHGREHVHPLFH (Fig. 5C). The reduced hepatic steatosis observed in myriocin treated obese mice ZDV DFFRPSDQLHG E\ D VLJQL¿FDQW UHGXFWLRQ LQ 62&6 JHQH H[SUHVVLRQ )LJ '  DPROHFXOHWKDWSOD\VDFHQWUDOUROHLQWKHSDWKRORJ\RIKHSDWLFVWHDWRVLV120 Thus the mechanism by which ceramide contributes to hepatic steatosis may at least in part be UHODWHGWRLWVHIIHFWVRQ62&6H[SUHVVLRQ:KLOHWKHVHVWXGLHVVXJJHVWWKDWDEHUUDQW ceramide accumulation does contribute to hepatic steatosis, whether hepatic ceramide GLUHFWO\FRQWULEXWHVWRIDWW\OLYHUDQGRUZKHWKHUFHUDPLGHPHGLDWHGLQVXOLQUHVLVWDQFH LQWKHIDWOHDGLQJWRLQFUHDVHGSODVPD))$VLVWKHPDLQFRQWULEXWLQJIDFWRULVFXUUHQWO\ under investigation.

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CERAMIDE AND CARDIOVASCULAR DISEASE 7KHHWLRORJ\RIREHVLW\DVVRFLDWHGLQFUHDVHLQFDUGLRYDVFXODUGLVHDVHLVREYLRXVO\ FRPSOH[DQGPD\LQYROYHDFRPELQDWLRQRIK\SHUOLSLGHPLDLQVXOLQUHVLVWDQFHLQÀDPPDWLRQ and an increased prothrombotic state.121-123$FODVVLFDOSHUVSHFWLYHRIFDUGLRYDVFXODUULVN KRZHYHUGRHVQRWDGHTXDWHO\DFFRXQWIRUDOORIWKHFDUGLRYDVFXODUHYHQWVDVVRFLDWHGZLWK obesity and exciting new studies have demonstrated that ceramide and/or other sphingolipids PD\SOD\DFULWLFDOUROHLQWKHSDWKRJHQHVLVRIFDUGLRYDVFXODUGLVHDVH67,124-128 The increase in cardiovascular risk in obesity is associated with a systemic prothrombotic and pro LQÀDPPDWRU\VWDWHSULPDULO\YLDLQFUHDVHGV\QWKHVLVDQGVHFUHWLRQRIWKHVHPROHFXOHV IURPWKHDGLSRVHWLVVXH,QFUHDVHGH[SUHVVLRQRIJHQHVHQFRGLQJSURWKURPERWLFSURWHLQV VXFKDV3$,DQGLQÀDPPDWRU\SURWHLQVHJ71)_, IL-6, MCP-1 and KC) has been FRQVLVWHQWO\GHPRQVWUDWHGLQDGLSRVHWLVVXHVIURPREHVHDQLPDOVDQGKXPDQV,PSRUWDQWO\ FHUDPLGHKDVEHHQVKRZQWRLQGXFHWKHH[SUHVVLRQRI3$,IURPHQGRWKHOLDOFHOOV84 and adipocytes32 DQG LQFUHDVH SURLQÀDPPDWRU\ F\WRNLQH 71)_, IL-6) and chemokine 0&3.& SURGXFWLRQIURPDGLSRF\WHV32 Animal and human studies have shown that plasma sphingomyelin and ceramide OHYHOVDUHFORVHO\UHODWHGWRWKHGHYHORSPHQWRIDWKHURVFOHURVLV67,68,129,130 For example, LQ/'/UHFHSWRUGH¿FLHQWPLFHDPXULQHPRGHORIDWKHURVFOHURVLV DWKHURVFOHURWLF OHVLRQIRUPDWLRQZDVVLJQL¿FDQWO\LQFUHDVHGLQUHVSRQVHWRVSKLQJROLSLGULFKGLHW129 6LPLODUO\LQKLELWLRQRIGHQRYRFHUDPLGHV\QWKHVLVVLJQL¿FDQWO\UHGXFHGDWKHURURVORURWLF OHVLRQVLQWKHVHPLFHWKHUHE\LGHQWLI\LQJDGH¿QLWLYHUROHIRUFHUDPLGHELRV\QWKHVLVLQ WKHSDWKRJHQHVLVRIDWKHURVFOHURVLV67,68 Ceramide may also play a role in cardiomyocyte DSRSWRVLVDQGFDUGLDFIDLOXUH7UHDWPHQWRIFDUGLRP\RF\WHVZLWK))$VWKDWVWLPXODWH ceramide synthesis (e.g., palmitate and stearate) induced apoptosis in these cells.131-133 ,Q UDW OHIW YHQWULFXODU P\RF\WHV FHUDPLGH FRQWULEXWHG WR OHSWLQPHGLDWHG FDUGLDF FRQWUDFWLOHG\VIXQFWLRQ134 Ceramide may increase atherosclerosis via several mechanisms. Sphingomyelin carried into the arterial wall on atherogenic lipoproteins may be locally hydrolyzed to ceramide by sphingomyelinase, promoting lipoprotein aggregation and macrophage IRDPFHOOIRUPDWLRQ135&HUDPLGHOHYHOVRIDJJUHJDWHG/'/LVDOPRVWIROGKLJKHU WKDQWKDWRISODVPD/'/DQGH[SRVLQJ/'/WREDFWHULDOVSKLQJRP\HOLQDVHSURPRWHV its aggregation.136 Moreover, ceramide may contribute to the instability and rupture RIDWKHURVFOHURWLFSODTXHVEHFDXVHRILWVSURDSRSWRWLFSRWHQWLDORQPDFURSKDJHVDQG smooth muscle cells.137,1387KHUROHRIWKHFHUDPLGHPHWDEROLWH63LQWKHSDWKRJHQHVLV RI DWKHURVFOHURVLV LV OHVV FOHDU DQG DSSHDUV WR EH VRPHZKDW FRQWURYHUVLDO139,140 Platelets store and release S1P141DQGRIWKH63LQVHUXPLVERXQGWR+'/142-144 :KLOHVRPHIXQFWLRQVRI63PD\SRLQWWRDSURDWKHURJHQLFHIIHFWRWKHUVVXJJHVW DQDUWKHURSURWHFWLYHHIIHFW7KLVRXWFRPHZLWKUHVSHFWWRDFKURQLFGLVHDVHVXFKDV DUWKURVFOHURVLV LQ DOO SUREDELOLW\ GHSHQGV RQ WKH H[SUHVVLRQ SDWWHUQ RI WKH VSHFL¿F S1P receptors (S1P1-5 RQVSHFL¿FFHOOVRIWKHYHVVHOZDOODWDQ\JLYHQWLPHGXULQJWKH SURJUHVVLRQRIWKHDWKHURVFOHURWLFOHVLRQ63VWLPXODWHVWKHSUROLIHUDWLRQRIHQGRWKHOLDO DQG VPRRWK PXVFOH FHOOV VXJJHVWLQJ D UROH IRU 63 LQ OHVLRQ IRUPDWLRQ DQG SODTXH stabilization.14563KDVDOVREHHQVKRZQWRLQGXFHWKHH[SUHVVLRQRIDGKHVLRQPROHFXOHV including E-selectin, ICAM-1 and VCAM-1 on endothelial cells, leading to enhanced adhesion to monocytic cells, a process expected to enhance arthrosclerosis.146,147 Other VWXGLHVUHYHDODQWLDWKHURJHQLFIXQFWLRQVIRU63148 In endothelial cells, S1P stimulates VHYHUDOIXQFWLRQVVXFKDVVXUYLYDOPLJUDWLRQDQGQLWULFR[LGHV\QWKHVLVLQDPDQQHU

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REFERENCES 2JGHQ&/&DUUROO0'&XUWLQ/5HWDO3UHYDOHQFHRIRYHUZHLJKWDQGREHVLW\LQWKH8QLWHG6WDWHV JAMA 2006; 295(13):1549-55. +RWDPLVOLJLO*6,QÀDPPDWLRQDQGPHWDEROLFGLVRUGHUV1DWXUH  +RWDPLVOLJLO*60ROHFXODUPHFKDQLVPVRILQVXOLQUHVLVWDQFHDQGWKHUROHRIWKHDGLSRF\WH,QW-2EHV5HODW Metab Disord 2000; 24(Suppl 4):S23-S27. 4. Mohamed-Ali V, Goodrick S, Rawesh A et al. Subcutaneous adipose tissue releases interleukin-6, but not WXPRUQHFURVLVIDFWRUDOSKDLQYLYR-&OLQ(QGRFULQRO0HWDE 5. Christiansen T, Richelsen B, Bruun JM. Monocyte chemoattractant protein-1 is produced in isolated DGLSRF\WHVDVVRFLDWHGZLWKDGLSRVLW\DQGUHGXFHGDIWHUZHLJKWORVVLQPRUELGREHVHVXEMHFWV,QW-2EHV (Lond) 2005; 29(1):146-50. %UXXQ-03HGHUVHQ6%5LFKHOVHQ%5HJXODWLRQRILQWHUOHXNLQSURGXFWLRQDQGJHQHH[SUHVVLRQLQKXPDQ adipose tissue in vitro. J Clin Endocrinol Metab 2001; 86(3):1267-73. :HOOHQ.(+RWDPLVOLJLO*6,QÀDPPDWLRQVWUHVVDQGGLDEHWHV-&OLQ,QYHVW  )DQWX]]L*$GLSRVHWLVVXHDGLSRNLQHVDQGLQÀDPPDWLRQ-$OOHUJ\&OLQ,PPXQRO  'DQGRQD3$OMDGD$%DQG\RSDGK\D\$,QÀDPPDWLRQWKHOLQNEHWZHHQLQVXOLQUHVLVWDQFHREHVLW\DQG diabetes. Trends Immunol 2004; 25(1):4-7. +RWDPLVOLJLO *6 ,QÀDPPDWRU\ SDWKZD\V DQG LQVXOLQ DFWLRQ ,QW - 2EHV 5HODW 0HWDE 'LVRUG  27(Suppl 3):S53-S55. 11. Neels JG, Badeanlou L, Hester KD et al. Keratinocyte-derived Chemokine in Obesity: Expression and role LQDGLSRVHPDFURSKDJHLQ¿OWUDWLRQDQGJOXFRVHKRPHRVWDVLV-%LRO&KHP 'HOODV&/RVNXWRII'-+LVWRULFDODQDO\VLVRI3$,IURPLWVGLVFRYHU\WRLWVSRWHQWLDOUROHLQFHOOPRWLOLW\ and disease. Thromb Haemost 2005; 93(4):631-40. 6DPDG)/RVNXWRII'-7LVVXHGLVWULEXWLRQDQGUHJXODWLRQRISODVPLQRJHQDFWLYDWRULQKLELWRULQREHVH mice. Mol Med 1996; 2:568-82. -XKDQ9DJXH,$OHVVL0&0DYUL$HWDO3ODVPLQRJHQDFWLYDWRULQKLELWRULQÀDPPDWLRQREHVLW\LQVXOLQ resistance and vascular risk. J Thromb Haemost 2003; 1:1575-9. /RVNXWRII'-6DPDG)7KHDGLSRF\WHDQGKHPRVWDWLFEDODQFHLQREHVLW\6WXGLHVRI3$,$UWHULRVFOHU Thromb Vasc Biol 1998; 18:1-6. (ULNVVRQ35H\QLVGRWWLU6/|QQTYLVW)HWDO$GLSRVHWLVVXHVHFUHWLRQRISODVPLQRJHQDFWLYDWRULQKLELWRU in non-obese and obese individuals. Diabetologia 1998; 41:65-71. /RVNXWRII'-,KDUD+
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118. Kotronen A, Seppanen-Laakso T, Westerbacka J et al. Hepatic stearoyl-CoA desaturase (SCD)-1 activity DQGGLDF\OJO\FHUROEXWQRWFHUDPLGHFRQFHQWUDWLRQVDUHLQFUHDVHGLQWKHQRQDOFRKROLFKXPDQIDWW\OLYHU Diabetes 2009; 58(1):203-8. 'HHYVND*05R]HQRYD.$*LOWLD\19HWDO$FLG6SKLQJRP\HOLQDVH'H¿FLHQF\3UHYHQWV'LHWLQGXFHG Hepatic Triacylglycerol Accumulation and Hyperglycemia in Mice. J Biol Chem 2009; 284(13):8359-68. 8HNL..RQGR77VHQJ<+HWDO&HQWUDOUROHRIVXSSUHVVRUVRIF\WRNLQHVLJQDOLQJSURWHLQVLQKHSDWLF steatosis, insulin resistance and the metabolic syndrome in the mouse. Proc Natl Acad Sci USA 2004; 101(28):10422-7. .UDXVV50:LQVWRQ0)OHWFKHU51HWDO2EHVLW\LPSDFWRIFDUGLRYDVFXODUGLVHDVH&LUFXODWLRQ 98(14):1472-6. /DYLH&-0LODQL599HQWXUD+22EHVLW\DQGFDUGLRYDVFXODUGLVHDVHULVNIDFWRUSDUDGR[DQGLPSDFW RIZHLJKWORVV-$P&ROO&DUGLRO  123. Vaughan DE. Plasminogen activator inhibitor-1: A common denominator in cardiovascular disease. J Invest Med 1998; 46:370-6. &KDWWHUMHH66SKLQJROLSLGVLQDWKHURVFOHURVLVDQGYDVFXODUELRORJ\$UWHULRVFOHU7KURPE9DVF%LRO 18(10):1523-33. 125. Auge N, Negre-Salvayre A, Salvayre R et al. Sphingomyelin metabolites in vascular cell signaling and atherogenesis. Prog Lipid Res 2000; 39(3):207-29. /L+-XQN3+XZLOHU$HWDO'XDOHIIHFWRIFHUDPLGHRQKXPDQHQGRWKHOLDOFHOOVLQGXFWLRQRIR[LGDWLYH VWUHVVDQGWUDQVFULSWLRQDOXSUHJXODWLRQRIHQGRWKHOLDOQLWULFR[LGHV\QWKDVH&LUFXODWLRQ  $XJH10DXSDV6FKZDOP)(OED]0HWDO5ROHIRUPDWUL[PHWDOORSURWHLQDVHLQR[LGL]HGORZGHQVLW\ OLSRSURWHLQLQGXFHGDFWLYDWLRQRIWKHVSKLQJRP\HOLQFHUDPLGHSDWKZD\DQGVPRRWKPXVFOHFHOOSUROLIHUDWLRQ Circulation 2004; 110(5):571-8. 1L[RQ *) 0DWKLHVRQ )$ +XQWHU , 7KH SRWHQWLDO UROHV RI VSKLQJROLSLGV LQ YDVFXODU VPRRWKPXVFOH IXQFWLRQ%LRFKHP6RF7UDQV3W  /L=%DVWHUU0-+DLOHPDULDP7.HWDO7KHHIIHFWRIGLHWDU\VSKLQJROLSLGVRQSODVPDVSKLQJRP\HOLQ metabolism and atherosclerosis. Biochim Biophys Acta 2005; 1735(2):130-4. -LDQJ;&3DXOWUH)3HDUVRQ7$HWDO3ODVPDVSKLQJRP\HOLQOHYHODVDULVNIDFWRUIRUFRURQDU\DUWHU\ disease. Arterioscler Thromb Vasc Biol 2000; 20:2614-8. 3DXPHQ 0% ,VKLGD < 0XUDPDWVX 0 HW DO ,QKLELWLRQ RI FDUQLWLQH SDOPLWR\OWUDQVIHUDVH , DXJPHQWV sphingolipid synthesis and palmitate-induced apoptosis. J Biol Chem 1997; 272(6):3324-9. 'H9ULHV-(9RUN005RHPHQ7+HWDO6DWXUDWHGEXWQRWPRQRXQVDWXUDWHGIDWW\DFLGVLQGXFHDSRSWRWLF cell death in neonatal rat ventricular myocytes. J Lipid Res 1997; 38(7):1384-94. '\QWDU'(SSHQEHUJHU(EHUKDUGW00DHGOHU.HWDO*OXFRVHDQGSDOPLWLFDFLGLQGXFHGHJHQHUDWLRQRI P\R¿EULOVDQGPRGXODWHDSRSWRVLVLQUDWDGXOWFDUGLRP\RF\WHV'LDEHWHV  5HQ-5HOOLQJ'3/HSWLQLQGXFHGVXSSUHVVLRQRIFDUGLRP\RF\WHFRQWUDFWLRQLVDPSOL¿HGE\FHUDPLGH Peptides 2006; 27(6):1415-9. 0DUDWKH6&KRL</HYHQWKDO$5HWDO6SKLQJRP\HOLQDVHFRQYHUWVOLSRSURWHLQVIURPDSROLSRSURWHLQ( NQRFNRXWPLFHLQWRSRWHQWLGXFHUVRIPDFURSKDJHIRDPFHOOIRUPDWLRQ$UWHULRVFOHU7KURPE9DVF%LRO 2000; 20:2607-13. 136. Schissel SL, Tweedie-Hardman J, Rapp JH et al. Tabas I. Rabbit aorta and human atherosclerotic OHVLRQVK\GURO\]HWKHVSKLQJRP\HOLQRIUHWDLQHGORZGHQVLW\OLSRSURWHLQ3URSRVHGUROHIRUDUWHULDOZDOO VSKLQJRP\HOLQDVHLQVXEHQGRWKHOLDOUHWHQWLRQDQGDJJUHJDWLRQRIDWKHURJHQLFOLSRSURWHLQV-&OLQ,QYHVW 1996; 98(6):1455-64. 137. Mitchinson MJ, Hardwick SJ, Bennett MR. Cell death in atherosclerotic plaques. Curr Opin Lipidol 1996; 7(5):324-9. 0DOODW=7HGJXL$$SRSWRVLVLQWKHYDVFXODWXUHPHFKDQLVPVDQGIXQFWLRQDOLPSRUWDQFH%U-3KDUPDFRO 2000; 130(5):947-62. 139. Spiegel S, Milstien S. Sphingosine-1-phosphate: an enigmatic signalling lipid. Nat Rev Mol Cell Biol 2003; 4(5):397-407. $OHZLMQVH $( 3HWHUV 6/ 0LFKHO 0& &DUGLRYDVFXODU HIIHFWV RI VSKLQJRVLQHSKRVSKDWH DQG RWKHU sphingomyelin metabolites. Br J Pharmacol 2004; 143(6):666-84. 
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$XJH11LNRORYD.DUDNDVKLDQ0&DUSHQWLHU6HWDO5ROHRIVSKLQJRVLQHSKRVSKDWHLQWKHPLWRJHQHVLV LQGXFHGE\R[LGL]HGORZGHQVLW\OLSRSURWHLQLQVPRRWKPXVFOHFHOOVYLDDFWLYDWLRQRIVSKLQJRP\HOLQDVH ceramidase and sphingosine kinase. J Biol Chem 1999; 274(31):21533-8. ,SDWRYD207RUNKRYVND\D7,=DNKDURYD76HWDO6SKLQJROLSLGVDQGFHOOVLJQDOLQJLQYROYHPHQWLQ apoptosis and atherogenesis. Biochemistry (Mosc) 2006; 71(7):713-22. ;LD3*DPEOH-55\H.$HWDO7XPRUQHFURVLVIDFWRUDOSKDLQGXFHVDGKHVLRQPROHFXOHH[SUHVVLRQ through the sphingosine kinase pathway. Proc Natl Acad Sci USA 1998; 95(24):14196-201. %RW01RIHU-5YDQ%HUNHO7-&HWDO/\VRSKRVSKROLSLGVWZRIDFHGPHGLDWRUVLQDWKHURVFOHURVLV)XWXUH Lipidol 2007; 2(3): 341-56. 149. Karliner JS, Honbo N, Summers K et al. The lysophospholipids sphingosine-1-phosphate and lysophosphatidic acid enhance survival during hypoxia in neonatal rat cardiac myocytes. J Mol Cell Cardiol 2001; 33(9):1713-7. -LQ=4=KRX+==KX3HWDO&DUGLRSURWHFWLRQPHGLDWHGE\VSKLQJRVLQHSKRVSKDWHDQGJDQJOLRVLGH GM-1 in wild-type and PKC epsilon knockout mouse hearts. Am J Physiol Heart Circ Physiol 2002; 282(6):H1970-H1977. /HFRXU66PLWK50:RRGZDUG%HWDO,GHQWL¿FDWLRQRIDQRYHOUROHIRUVSKLQJROLSLGVLJQDOLQJLQ71) alpha and ischemic preconditioning mediated cardioprotection. J Mol Cell Cardiol 2002; 34(5):509-18. 1RIHU-5$VVPDQQ*$WKHURSURWHFWLYHHIIHFWVRIKLJKGHQVLW\OLSRSURWHLQDVVRFLDWHGO\VRVSKLQJROLSLGV Trends Cardiovasc Med 2005; 15(7):265-71. .LPXUD76DWR..XZDEDUD$HWDO6SKLQJRVLQHSKRVSKDWHPD\EHDPDMRUFRPSRQHQWRISODVPD OLSRSURWHLQVUHVSRQVLEOHIRUWKHF\WRSURWHFWLYHDFWLRQVLQKXPDQXPELOLFDOYHLQHQGRWKHOLDOFHOOV-%LRO Chem 2001; 276(34):31780-5. 154. Kimura T, Sato K, Malchinkhuu E et al. High-density lipoprotein stimulates endothelial cell migration and survival through sphingosine 1-phosphate and its receptors. Arterioscler Thromb Vasc Biol 2003; 23(7):1283-8. 1RIHU-5%RW0%URGGH0HWDO)7<DV\QWKHWLFVSKLQJRVLQHSKRVSKDWHDQDORJXHLQKLELWVGHYHORSPHQW RIDWKHURVFOHURVLVLQORZGHQVLW\OLSRSURWHLQUHFHSWRUGH¿FLHQWPLFH&LUFXODWLRQ  156. Keul P, Tolle M, Lucke S et al. The sphingosine-1-phosphate analogue FTY720 reduces atherosclerosis LQDSROLSRSURWHLQ(GH¿FLHQWPLFH$UWHULRVFOHU7KURPE9DVF%LRO  157. Kahan BD. Update on pharmacokinetic/pharmacodynamic studies with FTY720 and sirolimus. Ther Drug Monit 2002; 24(1):47-52. .RYDULN-06FKPRXGHU5/6ODGH$-2YHUYLHZRI)7<FOLQLFDOSKDUPDFRNLQHWLFVDQGSKDUPDFRORJ\ Ther Drug Monit 2004; 26(6):585-7. 159. Pettus BJ, Bielawski J, Porcelli AM et al. The sphingosine kinase 1/sphingosine-1-phospate pathway mediates COX-2 induction and PGE2 production in response to TNF-_. FASEB J 2003; 17:1411-21. 3HWWXV %- .LWDWDQL . &KDOIDQW &( HW DO 7KH FRRUGLQDWLRQ RI SURVWDJODQGLQ ( SURGXFWLRQ E\ sphingosine-1-phosphate and ceramide-1-phosphate. Mol Pharmacol 2005; 68(2):330-5. 161. Cipollone F, Fazia ML. COX-2 and atherosclerosis. J Cardiovasc Pharmacol 2006; 47(Suppl 1):S26-S36.

CHAPTER 6 SPHINGOLIPIDS AND HEPATIC STEATOSIS %HQMDPLQ7%LNPDQ1 and Scott A. Summers*,1,2 Program in Cardiovascular and Metabolic Disorders, Duke-NUS Graduate Medical School, Singapore; The Stedman Center for Nutrition and Metabolism Research, Duke University Medical Center, Durham, North Carolina, USA *Corresponding Author: Scott A. Summers—Email: [email protected] 1 2

Abstract:

7KH GHYHORSPHQW RI D IDWW\ OLYHU SUHGLVSRVHV LQGLYLGXDOV WR DQ DUUD\ RI KHDOWK SUREOHPVLQFOXGLQJGLDEHWHVFDUGLRYDVFXODUGLVHDVHDQGFHUWDLQIRUPVRIFDQFHU ,QKLELWLRQRUJHQHWLFDEODWLRQRIJHQHVFRQWUROOLQJVSKLQJROLSLGV\QWKHVLVLQURGHQWV UHVROYHVKHSDWLFVWHDWRVLVDQGLQPDQ\FDVHVZDUGVRIIWKHKHDOWKFRPSOLFDWLRQV associated with excessive hepatic triglyceride accumulation. Examples include WKHSKDUPDFRORJLFDOLQKLELWLRQRIVHULQHSDOPLWR\OWUDQVIHUDVHRUJOXFRV\OFHUDPLGH V\QWKDVHRUWKHJHQHWLFGHSOHWLRQRIDFLGVSKLQJRP\HOLQDVHZKLFKGUDPDWLFDOO\UHGXFH KHSDWLFWULJO\FHULGHOHYHOVLQPLFHVXVFHSWLEOHWRWKHGHYHORSPHQWRIDIDWW\OLYHU7KH PDJQLWXGHRIWKHHIIHFWVRQWULJO\FHULGHGHSOHWLRQLQWKHVHPRGHOVLVLPSUHVVLYHEXW WKHUHOHYDQFHWRKXPDQVDQGWKHPHFKDQLVPRIDFWLRQLVXQFOHDU+HUHLQZHSUREH into the connections between sphingolipids and triglyceride synthesis in an attempt WRLGHQWLI\FDXVDOUHODWLRQVKLSVDQGRSSRUWXQLWLHVIRUWKHUDSHXWLFLQWHUYHQWLRQ

INTRODUCTION &ORVHO\PLUURULQJWKHPHWHRULFULVHLQREHVLW\OHYHOVLVDQDUUD\RIKHDOWKSUREOHPV NQRZQFROOHFWLYHO\DVWKHPHWDEROLFV\QGURPH7KHIHDWXUHVRIWKLVV\QGURPHLQFOXGH abdominal obesity, dyslipidemia, hypertension, insulin resistance and chronic low-grade LQÀDPPDWLRQ1:KLOHWKHPHWDEROLFV\QGURPHLVQRWLQLWVHOIDGLVHDVHWKHSUHVHQFHRI WKLVFOXVWHURIULVNIDFWRUVSUHGLVSRVHVLQGLYLGXDOVWRKHDUWGLVHDVHDQG7\SHGLDEHWHV ZKLFKDFFRXQWIRUURXJKO\SHUFHQWRIDOOGHDWKVZRUOGZLGH2-4 Given the liver’s place in OLSRSURWHLQPHWDEROLVPDQGWKHFRQWURORIQXWULHQWKRPHRVWDVLV5 it is noteworthy that these ULVNIDFWRUVKDYHDWUHPHQGRXVLPSDFWRQKHSDWLFOLSLGLQ¿OWUDWLRQDQGWKHSDWKRJHQHVLVRI QRQDOFRKROLFIDWW\OLYHUGLVHDVH1$)/' 2QRQHKDQGWKHOLYHUVXIIHUVIURPWKHIDFWRUV

Sphingolipids and Metabolic Disease, edited by L. Ashley Cowart. ©2011 Landes Bioscience and Springer Science+Business Media, LLC 87

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SPHINGOLIPID LEVELS IN THE STEATOTIC LIVER 6HYHUDODQLPDOPRGHOVRIREHVLW\RUYDULRXVPHWDEROLFGLVHDVHVGHPRQVWUDWHHOHYDWHG hepatic sphingolipid levels.11-14 Employing an agnostic lipidomic methodology to FKDUDFWHUL]HWKHOLYHUVRIWKHob/ob mouse, Yetukuri et al15 determined that hepatic levels RIFHUDPLGHZKLFKLVDELRV\QWKHWLFLQWHUPHGLDWHLQWKHVSKLQJROLSLGV\QWKHVLVSDWKZD\ DQGDSUHFXUVRURIDOOFRPSOH[VSKLQJROLSLGVHJVSKLQJRP\HOLQDQGJOXFRV\OFHUDPLGHV  VWURQJO\ FRUUHODWHG ZLWK WKH GHJUHH RI VWHDWRVLV 6WXGLHV LQ KXPDQV KDYH QRW EHHQ DV H[KDXVWLYHRUGH¿QLWLYH/LYHUIDWFRUUHODWHVVWURQJO\ZLWKWUDQVFULSWVHQFRGLQJJHQHVWKDW drive sphingolipid metabolism, but not with ceramides themselves.16-17 However, Kolak et al18IRXQGDFRUUHODWLRQEHWZHHQadipose ceramide content and liver triglyceride in KXPDQVLQGHSHQGHQWRIREHVLW\VXJJHVWLQJDSRVVLEOHHQWHURKHSDWLFUHODWLRQVKLSWKURXJK which adipose ceramides could contribute to NAFLD. MODULATION OF SPHINGOLIPID SYNTHESIS IMPACTS HEPATIC STEATOSIS 'HVSLWHDUHFHQWVXUJHLQDWWHQWLRQWRWKHHQ]\PHVUHJXODWLQJWKHUDWHRIVSKLQJROLSLG synthesis and degradation (Fig. 2),19 relatively little interest has been devoted to the WLVVXHVSHFL¿FFKDUDFWHULVWLFVRIWKHHQ]\PHVLQWKHOLYHU7KRVHWKDWKDYHEHHQVWXGLHG have yielded noteworthy results. Serine Palmitoyltransferase 6HULQH SDOPLWR\OWUDQVIHUDVH 637  LV WKH LQLWLDO DQG UDWHOLPLWLQJ VWHS LQ GH QRYR sphingolipid synthesis, condensing palmitoyl-CoA and serine to produce 3-ketosphinganine (Fig. 2). Memon et al20 H[SORUHG WKH HIIHFWV RI LQÀDPPDWRU\ PHGLDWRUV LQFOXGLQJ OLSRSRO\VDFFKDULGHLQWHUOHXNLQDQGWXPRUQHFURVLVIDFWRU_, on SPT activity and gene expression in hamsters and HepG2 cells. LPS treatment increased hepatic sphingolipid levels (i.e., sphingomyelin (up 75%) and ceramide (up 200%) and doubled hepatic SPT JHQHH[SUHVVLRQDQGHQ]\PHDFWLYLW\6LPLODUO\,/LQMHFWLRQLQFUHDVHGKHSDWLF637 transcript levels and activity in vivo and both IL-1 and TNF_ induced SPT transcript levels in HepG2 cells. In these studies, cytokine and LPS treatment increased sphingolipid UHOHDVHLHFHUDPLGHVVSKLQJRP\HOLQDQGJOXFRV\OFHUDPLGHV IURPWKHOLYHULQWRWKH circulation via lipoproteins. 0\ULRFLQLVDSRWHQW637LQKLELWRUDQGKDVEHHQXVHGIUHTXHQWO\LQURGHQWVWXGLHV WRVORZUDWHVRIVSKLQJROLSLGV\QWKHVLVD ob/ob PLFH E  &%/- PLFH WUHDWHG ZLWK P\ULRFLQ DW WKH RQVHW RI KLJKIDW GLHW DQG F  &%/-PLFHWUHDWHGZLWKP\ULRFLQDIWHUHLJKWZHHNVRIKLJKIDWGLHW@WUHDWPHQWZLWK P\ULRFLQVLJQL¿FDQWO\UHGXFHGKHSDWLFWULJO\FHULGHV21 Glucosylceramide Synthase *O\FRVSKLQJROLSLGV DUH SURGXFHG E\ WKH DGGLWLRQ RI FDUERK\GUDWH PRLHWLHV to ceramide. The initial product is glucosylceramide, which is produced by

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SPHINGOLIPIDS AND HEPATIC STEATOSIS

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whole-body glucose homeostasis as evident by reduced blood glucose and HbA1c levels. ,QH[SORULQJWKHHIIHFWRIWUHDWPHQWRQOLYHUSDWKRORJ\WKHOLYHUVRIWKHDQLPDOVWUHDWHG ZLWK*HQ]KDGGHFUHDVHGVL]HDQGQXPEHURIOLSLG¿OOHGYDFXROHVDQGDVXEVWDQWLDO UHGXFWLRQLQKHSDWLFWULJO\FHULGHOHYHOV&RPSDUDEOH¿QGLQJVZHUHREVHUYHGLQDQRWKHU PRGHORIVWHDWRVLV²&%/PLFHIHGDKLJKIDWGLHWIRUZHHNV)LQDOO\WRGHWHUPLQH whether GCS inhibition could reverse preexisting steatosis, older mice that had been KLJKIDWIHGIRUZHHNVZHUHWUHDWHGZLWKYHKLFOHRU*HQ]IRUZHHNV$W WKHFRQFOXVLRQRIWKHWUHDWPHQWSHULRGWKHOLYHUVRIWKHROGHUKLJKIDWIHGPLFHUHFHLYLQJ *HQ]H[KLELWHGDUHGXFHGUDWLRRIIDWWROHDQPDVVFRPSDUHGZLWKFRQWUROV Sphingomyelinase 6SKLQJRP\HOLQ60 LVWKHPRVWSUHYDOHQWVSKLQJROLSLGLQPDPPDOLDQFHOOVIRXQG abundantly in plasma membranes and lipoproteins. The sphingolipid can be converted EDFNLQWRFHUDPLGHE\DIDPLO\RIVSLQJRPH\OLQDVHV60DVH GLVWLQJXLVKHGE\WKHLUS+ optima.24 Deevska et al25UHSRUWHGWKHGHOHWLRQRIDFLGVSKLQJRPH\OLQDVHIURP/'/UHFHSWRU knockout mice resolved diet-induced hepatic steatosis and improved insulin sensitivity. The improvement in steatosis and insulin sensitivity was associated with a paradoxical elevation in hepatic ceramides and sphingomyelin and a marked increase in de novo synthesis. Numerous cell stimuli regulate SMase activity and the enzyme is implicated in UHVSRQVHVWRDGLYHUVHQXPEHURIFHOOXODUDJRQLVWVHJF\WRNLQHVDQGR[LGDWLYHVWUHVV  TNF_ and IL-1`LQFUHDVHFHUDPLGHOHYHOVE\VWLPXODWLQJDWOHDVWWZRGLIIHUHQW60DVH FODVVHV²QHXWUDODQGDFLGLF26-320RUHRYHUWKHDELOLW\RI71)WRLQGXFH60DVHKDVEHHQ established across various human and rodent cell lines.33-36 Among its many activities, TNF_ induces SMase by binding the p55 TNFR.377KHVH¿QGLQJVDUHLQWHUHVWLQJLQOLJKW RIWKHHYLGHQFHWKDWPLFHJHQHWLFDOO\GH¿FLHQWLQWKHS71)_ receptor (also known as 71)7\SHUHFHSWRU DUHUHVLVWDQWWRGLHWLQGXFHGVWHDWRVLVDQGOLYHULQMXU\38 Oxidative stress also regulates SMase activity.24 Alessenko et al39 demonstrated that a SMase-mediated elevation in hepatic ceramides positively correlated with peroxide products in response to endotoxic stress. Additionally, they observed that administration RI QLWULF R[LGH UHOHDVLQJ FRPSRXQGV UHVXOWHG LQ UHGXFHG KHSDWLF 60DVH DFWLYLW\ DQG ceramide levels and lipid peroxide oxidation.39 In exploring the mechanisms behind bile-salt induced hepatocyte apoptosis, Reinehr et al40 treated primary rat hepatocytes ZLWKWDXUROLWKRFKRODWHVXOIDWH7/&6 DVXEVWDQFHNQRZQWRLQGXFHDSRWHQWR[LGDWLYH stress response. In addition to rapidly inducing oxidative stress, TLCS treatment was also shown to stimulate ceramide synthesis via elevated SMase activity. When cells were treated with desipramine, a SMase inhibitor, prior to TLCS exposure, ceramide levels and downstream oxidative stress were both reduced.40 FACTORS ASSOCIATED WITH NAFLD INDUCE SPHINGOLIPID SYNTHESIS 7KHSDWKRJHQHVLVRI1$)/'KDVEHHQUHIHUUHGWRDVDµGRXEOHKLW¶SURFHVVZLWK KHSDWRFHOOXODUOLSLGDFFXPXODWLRQSUHVHQWLQJWKH¿UVWLQVXOWDQGWKHVHFRQGDVDUHVXOW RILQÀDPPDWLRQLQGXFHGKHSDWLFLQMXU\41-43%RWKOLSLGRYHUVXSSO\DQGLQÀDPPDWLRQDUH likely to drive sphingolipid production (Fig. 3).

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Saturated Fatty Acids Induce Hepatic Sphingolipids )DWW\DFLGVXVHGLQWKHSURGXFWLRQRIKHSDWLFWULJO\FHULGHFDQGHULYHIURPDQXPEHU RIGLVWLQFWVRXUFHVLQFOXGLQJSODVPD))$VDFFRXQWLQJIRU¾RIKHSDWLFWULJO\FHULGH  IDWW\DFLGVPDGHGHQRYRZLWKLQWKHOLYHU¾ DQGGLHWDU\IDWW\DFLGV¾15%), which can enter the liver by either spillover into plasma FFA or through chylomicron remnants.44-45 :HSUHYLRXVO\GHPRQVWUDWHGWKDWVDWXUDWHGIDWW\DFLGVGURYHVSKLQJROLSLGV\QWKHVLVLQ WKHOLYHUDVKHSDWLFFHUDPLGHOHYHOVZHUHHOHYDWHGE\URXJKO\LQUDWVIROORZLQJ DVL[KRXULQIXVLRQRIODUGRLOZKHQFRPSDUHGZLWKDQLPDOVUHFHLYLQJDFRQWUROLH JO\FHURO LQIXVDWH127RKLJKOLJKWWKHQHFHVVLW\RIVDWXUDWHGIDWW\DFLGVLQWKLVHIIHFWWKHUH ZDVQRLQFUHDVHLQKHSDWLFFHUDPLGHOHYHOVLQWKHDQLPDOVUHFHLYLQJDVR\EDVHGLQIXVDWH FRPSULVHGSUHGRPLQDQWO\RIXQVDWXUDWHGIDWW\DFLGV12 ,QÀDPPDWRU\&\WRNLQHV,QGXFH+HSDWLF6SKLQJROLSLGV 7KHGLVFRYHU\RIWKHLQWHUDFWLRQRILQÀDPPDWRU\DQGPHWDEROLFSDWKZD\VRYHU \HDUVDJRSURYLGHGDQRYHODQGLPSRUWDQWSHUVSHFWLYHLQXQGHUVWDQGLQJWKHRULJLQRIPDQ\ metabolic disorders.46:KLOHWKHGH¿QLQJFKDUDFWHULVWLFRI1$)/'LVH[FHVVLYHKHSDWLF GHSRVLWLRQRIIDWW\DFLGVWKHODWWHUDQGPRUHDGYDQFHGVWDJHVDUHGLVWLQJXLVKHGE\DQLQFUHDVLQJ LQÀDPPDWRU\WRQH47-48 Several groups provide evidence supporting a correlation between YDU\LQJGHJUHHVRI1$)/'DQGFLUFXODWLQJDQGWLVVXHF\WRNLQHOHYHOVDQGH[SUHVVLRQ38,49-57 In particular, Jarrar et al58 observed both elevated TNF_OHYHOVSURLQÀDPPDWRU\F\WRNLQH  DQG UHGXFHG DGLSRQHFWLQ DQWLLQÀDPPDWRU\  LQ SDWLHQWV ZLWK 1$)/' FRPSDUHG ZLWK REHVHDQGQRQREHVHFRQWUROV0RUHRYHUZLWKLQWKH1$)/'JURXSDIXUWKHUFRQWUDVWZDV REVHUYHG²WKRVHVXIIHULQJIURPDGYDQFHGVWDJHVRI1$)/'LQFOXGLQJ1$6+GLVSOD\HG VLPLODUVLJQL¿FDQWGLIIHUHQFHVZKHQFRPSDUHGZLWKWKRVHVXIIHULQJIURPVLPSOHVWHDWRVLV Similar trends are observed with hepatic TNF_JHQHH[SUHVVLRQDQG71)UHFHSWRUV²ERWK DUHLQFUHDVHGZLWK1$)/'ZKHQFRPSDUHGZLWKKHDOWK\OLYHUVDQGWKHH[SUHVVLRQLVIXUWKHU elevated in those with advanced NASH.59-61

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7KHUHODWLRQVKLSEHWZHHQF\WRNLQHVDQGVSKLQJROLSLGVKDVEHHQH[SORUHGIRURYHU years and evidence attesting to the liver’s ability to produce sphingolipids in response to dyslipidemia is even older.62-64 Since those early discoveries, emerging evidence suggests a noteworthy relationship between sphingolipids and cytokines in various tissues.65-68 Further, LQVXSSRUWRIWKHDGLSRVHOLYHUUHODWLRQVKLSVXEMHFWVZLWK1$)/'H[KLELWVLJQL¿FDQWO\JUHDWHU adipose-derived cytokines and ceramides.18 Unsurprisingly, many studies have revealed a positive correlation between TNF_ and sphingolipids.19,21,695RGHQWPRGHOVRIREHVLW\KDYH VKRZQHOHYDWHGOHYHOVRIERWKKHSDWLFVSKLQJROLSLGVDQGSURLQÀDPPDWRU\F\WRNLQHV13-14,51,70 &HQWUDOWRLQÀDPPDWLRQDQGF\WRNLQHUHOHDVHWKURXJKRXWWKHERG\LVWKHPDFURSKDJH DQGWKHUHVLGHQWKHSDWLFPDFURSKDJHVNQRZQDV.XSIIHUFHOOVUHSUHVHQWWKHPDMRULW\RI DOOWLVVXHPDFURSKDJHVLQWKHERG\DQGDVPXFKDVRIWKHWRWDOFHOOSRSXODWLRQZLWKLQ WKHOLYHULWVHOI71-72.XSIIHUFHOOVERWKUHVSRQGWRDQGUHOHDVHF\WRNLQHVLQWRWKHOLYHUDQG FLUFXODWLRQDQGWKHLUQXPEHUVYDU\JUHDWO\LQUHVSRQVHWRLQÀDPPDWRU\VWLPXOLDVDUHVXOW RIK\SHUSODVLDDQGLQ¿OWUDWLRQRIERQHPDUURZSURJHQLWRUV730HGLDWLQJWKH.XSIIHUFHOOV response to pathogens are the pattern recognition receptors (PRR),74-75 among which are WKHIDPLO\RIWROOOLNHUHFHSWRUV7/5V )LJ 75-77 In particular, TLR4 plays a key role in PHGLDWLQJLQÀDPPDWRU\VLJQDOVWKURXJKRXWWKHOLYHUE\WULJJHULQJ.XSIIHUFHOOVWRSURGXFH DQ DUUD\ RI SURLQÀDPPDWRU\ F\WRNLQHV 71)_, IL-1`, IL-6, etc.), which are known to VWLPXODWHFHUWDLQHQ]\PHVRIVSKLQJROLSLGPHWDEROLVPVHHDERYH 77-80 7KHEHVWFKDUDFWHUL]HGDJRQLVWRI7/5LVOLSRSRO\VDFFKDULGH/36 ZKLFKLVSUHVHQWRQ Gram-negative bacteria. The liver’s ability to respond to LPS via TLR4 plays an important UROHLQPDLQWDLQLQJKRPHRVWDVLVE\DFWLYDWLQJKRVWLPPXQLW\DQGDFWLQJDVD¿QDOEDUULHUWR WR[LQVEHIRUHWKH\HQWHUWKHV\VWHPLFEORRGVWUHDP81-82 Indeed, due to the liver’s place as D¿UVWUHVSRQGHUWRWR[LQVDQGDELOLW\WRDFWLYDWHDSRWHQWLPPXQHUHVSRQVHLWLVWHPSWLQJ WR YLHZ WKH .XSIIHU FHOOV DV D FHQWUDO UHJXODWRU RI LQÀDPPDWLRQLQGXFHG VSKLQJROLSLG synthesis. In addition to eliciting an immune response, LPS treatment has been shown to LQGXFHVSKLQJROLSLGDFFUXDOLQKHSDWLFDQGH[WUDKHSDWLFWLVVXHVWKRXJKZKHWKHUWKLVHIIHFW is mediated by TLR4 is unknown.20,83 Adiponectin :KLOHWKHWKUXVWRIF\WRNLQHLQGXFHGVWHDWRVLVUHVHDUFKKDVIRFXVHGRQ71)_ and other SURLQÀDPPDWRU\F\WRNLQHVDQHPHUJLQJDUHDRIUHVHDUFKKDVH[SORUHGWKHUHODWLRQVKLS EHWZHHQ DGLSRQHFWLQ WKH PRVW SURPLQHQW DQG DEXQGDQW DQWLLQÀDPPDWRU\ F\WRNLQH LQ UHJXODWLQJKHSDWLFVSKLQJROLSLGPHWDEROLVP,QDQDVVHVVPHQWRISDWLHQWVZLWKVLPSOHVWHDWRVLV and NASH, Jarrar et al58VDZWKDW1$6+SDWLHQWVKDYHVLJQL¿FDQWO\ORZHUDGLSRQHFWLQ levels than those with simple steatosis and obese controls without NAFLD. Moreover, in DQDO\]LQJWKHOLYHUVRIREHVHQRQGLDEHWLFVXEMHFWV.RODNHWDO18 revealed that adiponectin WUDQVFULSWH[SUHVVLRQLQKLJKHUIDWOLYHUVZDVQHJDWLYHO\DVVRFLDWHGZLWKKHSDWLFFHUDPLGHV DQGVSKLQJRP\HOLQDVHWUDQVFULSWOHYHOV,QDUHFHQWVWXG\RIDGLSRQHFWLQDQGLWVUHFHSWRUV Peng et al84VKRZHGWKDWVHUXPDGLSRQHFWLQGHFUHDVHGLQPLFHIHGDKLJKIDWGLHWZKHQ FRPSDUHGZLWKFRQWUROIHGDQLPDOVDQGWKDWWKLVFKDQJHZDVDVVRFLDWHGZLWKGUDPDWLFDOO\ UHGXFHGKHSDWLFH[SUHVVLRQRIDGLSR5WKHSUHGRPLQDQWDGLSRQHFWLQUHFHSWRULQWKHOLYHU 6LPLODUH[DPLQDWLRQVLQKXPDQVKDYHUHYHDOHGFRPSDUDEOHUHVXOWV²DUHGXFWLRQLQKHSDWLF adiponectin and adipoR2 expression with advancing steatosis.56,85 Interventional studies support the idea that adiponectin may deplete hepatic triglycerides. In particular, Yamauchi et al86 discovered that obese mice treated with adiponectin had UHGXFHGLQWUDKHSDWLFWULJO\FHULGHV0RUHYHU6WHIDQHWDO87 demonstrated that adiponectin

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receptor polymorphisms, which prevent normal adiponectin signal transduction, are DVVRFLDWHGZLWKLQVXOLQUHVLVWDQFHDQGKLJKOLYHUIDWLQREHVHPLFH:KLOHWKHGLUHFWHIIHFWV RIDGLSRQHFWLQRQhepatic sphingolipids have yet to be explored, recent evidence suggesting that adiponectin receptors contain intrinsic ceramidase activity88-90 provides exciting insight into a possible mechanism. POSSIBLE MECHANISMS 7KH SUHFLVH PHFKDQLVP RI KRZ VSKLQJROLSLGV FRQWULEXWH WR VWHDWRVLV UHPDLQV XQGH¿QHG 7KH PRVW FHQWUDO TXHVWLRQ LV ZKHWKHU WKH VSKLQJROLSLG HIIHFW LV GXH WR D FHOODXWRQRPRXVUROHLQWKHUHJXODWLRQRIWULJO\FHULGHV\QWKHVLVDVLVVXJJHVWHGE\WKH VWXGLHVVKRZLQJWKDWLQKLELWLRQRI$60DVHLQFXOWXUHGKHSDWRF\WHVLQKLELWVWULJO\FHULGH synthesis.25+RZHYHUVLQFHVSKLQJROLSLGOHYHOVZHUHHOHYDWHGLQOLYHUVIROORZLQJ$60DVH GHSOHWLRQWKHVWXG\VHHPVLQFRQJUXRXVZLWKWKH¿QGLQJWKDW637LQKLELWLRQZKLFKORZHUV hepatic sphingolipids, also depletes hepatic triglycerides.217KXVWKHGHWDLOVRIVXFKD FHOODXWRQRPRXVPHFKDQLVPDUHGLI¿FXOWWRHQYLVLRQ$QDOWHUQDWLYHSRVVLELOLW\LVWKDW depleting ceramides improves peripheral insulin sensitivity, leading to the appropriate GHSRVLWLRQ RI QXWULHQWV LQ SHULSKHUDO WLVVXHV LH VNHOHWDO PXVFOH  DQG D SUHVHUYDWLRQ RIWKHOLYHU:KHWKHULQVXOLQUHVLVWDQFHSUHFHGHVVWHDWRVLVRUZKHWKHUVWHDWRVLVOHDGVWR LQVXOLQUHVLVWDQFHLVDQDUHDRIFRQVLGHUDEOHLQWHUHVWDQGGHEDWH CONCLUSION 7KH PHWDEROLF V\QGURPH LV D PDMRU KHDOWK FRQFHUQ WKURXJKRXW WKH ZRUOG DQG evidence indicates the liver occupies an increasingly prominent role in the etiology RI WKLV PXOWLIDFHWHG FRQGLWLRQ $V ZRUOGZLGH SUHYDOHQFH FRQWLQXHV WR ULVH LQ SDUDOOHO ZLWKREHVLW\UDWHVUHVHDUFKLQYROYLQJQRQDOFRKROLFIDWW\OLYHUGLVHDVHDQGLWVYDULRXV PDQLIHVWDWLRQVKDVSURGXFHGLUUHIXWDEOHHYLGHQFHFRQQHFWLQJWKHGLVHDVHZLWKWKHOLSRWR[LF PLOLHXJHQHUDWHGLQDKLJKOLSLGLQVXOLQUHVLVWDQWVWDWH'HVSLWH\HDUVRIFRUUHODWLRQDO GDWDRQO\UHFHQWO\KDVUHVHDUFKEHJXQWRGLUHFWO\H[SORUHWKHUROHRIVSKLQJROLSLGVLQ DJJUDYDWLQJ 1$)/' *LYHQ WKH SURPLVLQJ UHVXOWV WKXV IDU IXWXUH HIIRUWV ZLOO OLNHO\ FRQWLQXHWRSURYLGHHYLGHQFHLPSOLFDWLQJVSKLQJROLSLGVLQWKHSDWKRJHQHVLVRI1$)/' DQGDVZHFRPHWRDEHWWHUXQGHUVWDQGLQJRILWVRULJLQVZHZLOOEHLQDEHWWHUSRVLWLRQ to uncover better treatments. ACKNOWLEDGEMENTS 7KHDXWKRULVJUDWHIXOWRWKHPHPEHUVRIKLVODERUDWRU\IRUKHOSIXOGLVFXVVLRQVDQG IHHGEDFN0XFKRIWKHZRUNWKDWZDVGLVFXVVHGKHUHLQLVEDVHGRQUHVHDUFK¿QGLQJVREWDLQHG XVLQJ VXSSRUW IURP WKH 1DWLRQDO ,QVWLWXWHV RI +HDOWK '.  DQG WKH $JHQF\ IRU 6FLHQFH7HFKQRORJ\DQG5HVHDUFK$ 67$5 RI6LQJDSRUH

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REFERENCES *UXQG\60%UHZHU+%-U&OHHPDQ-,HWDO'H¿QLWLRQRIPHWDEROLFV\QGURPHUHSRUWRIWKH1DWLRQDO+HDUW /XQJDQG%ORRG,QVWLWXWH$PHULFDQ+HDUW$VVRFLDWLRQFRQIHUHQFHRQVFLHQWL¿FLVVXHVUHODWHGWRGH¿QLWLRQ Arterioscler Thromb Vasc Biol 2004; 24:e13-18. (FRQRPLFFRVWVRIGLDEHWHVLQWKH86,Q'LDEHWHV&DUH 3. Cardiovascular Disease Statistics 2009. American Heart Assocation. Available at: http://www.americanheart. RUJSUHVHQWHUMKWPO"LGHQWL¿HU4478. 4. Global Strategy on Diet and Physical Activity 2009. World Health Organization. Available at: http://www. ZKRLQWGLHWSK\VLFDODFWLYLW\SXEOLFDWLRQVIDFWVGLDEHWHVHQLQGH[KWPO &ROHPDQ5$/HH'3(Q]\PHVRIWULDF\OJO\FHUROV\QWKHVLVDQGWKHLUUHJXODWLRQ3URJ/LSLG5HV 1HXVFKZDQGHU7HWUL%$&DOGZHOO6+1RQDOFRKROLFVWHDWRKHSDWLWLVVXPPDU\RIDQ$$6/'6LQJOH7RSLF &RQIHUHQFH+HSDWRORJ\ .LP&+
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&UHVSR - HW DO *HQH H[SUHVVLRQ RI WXPRU QHFURVLV IDFWRU DOSKD DQG 71)UHFHSWRUV S DQG S LQ nonalcoholic steatohepatitis patients. Hepatology 2001; 34:1158-1163. 61. Abiru S et al. Serum cytokine and soluble cytokine receptor levels in patients with non-alcoholic steatohepatitis. Liver Int 2006; 26:39-45. 62. Hannun YA, Bell RM. The sphingomyelin cycle: a prototypic sphingolipid signaling pathway. Adv Lipid Res 1993; 25:27-41. .ROHVQLFN5*ROGH':7KHVSKLQJRP\HOLQSDWKZD\LQWXPRUQHFURVLVIDFWRUDQGLQWHUOHXNLQVLJQDOLQJ Cell 1994; 77:325-328. 0HVVPHU72:DQJ(6WHYHQV9/HWDO6SKLQJROLSLGELRV\QWKHVLVE\UDWOLYHUFHOOVHIIHFWVRIVHULQHIDWW\ acids and lipoproteins. J Nutr 1989; 119:534-538. *LOO-06DWWDU1&HUDPLGHV$QHZSOD\HULQWKHLQÀDPPDWLRQLQVXOLQUHVLVWDQFHSDUDGLJP"'LDEHWRORJLD 66. Samad F, Hester KD, Yang G et al. Altered adipose and plasma sphingolipid metabolism in obesity: a SRWHQWLDOPHFKDQLVPIRUFDUGLRYDVFXODUDQGPHWDEROLFULVN'LDEHWHV 6KDK&
CHAPTER 7 GLYCOSPHINGOLIPIDS AND INSULIN RESISTANCE Johannes M. Aerts,*,15ROI*%RRW10DUFRYDQ(LMN1 Johanna Groener,11RUD%LMO1 Elisa Lombardo,1 Florence M. Bietrix,1 Nick Dekker,1 Albert K. Groen,15RHORI2WWHQKRII1 Cindy van Roomen,1 Jan Aten,2 Mireille Serlie,30LUMDP/DQJHYHOG3 Tom Wennekes4DQG+HUPHQ62YHUNOHHIW4 1 Department of Medical Biochemistry, 2Department of Pathology, 3Department of Internal Medicine, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands; 4Leiden Institute of Chemistry, Department of Bioorganic Synthesis, Gorlaeus Laboratories, Leiden University, Leiden, The Netherlands *Corresponding Author: Johannes M. Aerts—Email: [email protected]

Abstract:

Glycosphingolipids are structural membrane components, residing largely in the SODVPDPHPEUDQHZLWKWKHLUVXJDUPRLHWLHVH[SRVHGDWWKHFHOO¶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¿QGLQJLQDQLPDOPRGHOV WKDWSKDUPDFRORJLFDOUHGXFWLRQRIJO\FRVSKLQJROLSLGELRV\QWKHVLVDPHOLRUDWHVLQVXOLQ UHVLVWDQFHDQGSUHYHQWVVRPHPDQLIHVWDWLRQVRIPHWDEROLFV\QGURPHVXSSRUWVWKH view that somehow glycosphingolipids act as critical regulators, Importantly, since UHGXFWLRQVLQJO\FRVSKLQJROLSLGELRV\QWKHVLVKDYHEHHQIRXQGWREHZHOOWROHUDWHG such approaches may have a therapeutic potential.

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IRUSURWHLQVFRQWDLQLQJSOHFNVWULQKRPRORJ\3+ GRPDLQV7KH3+GRPDLQFRQWDLQLQJ serine/threonine kinases PKB and phosphatidylinositol-3-phosphate dependent kinase 1 (PDK1) are brought into close proximity with each other by their interactions with 3,3$GGLWLRQDOO\3,3KHOSVWRDFWLYDWH3.%E\LQGXFLQJFRQIRUPDWLRQDOFKDQJHVWKDW H[SRVHWZRUHJXODWRU\SKRVSKRU\ODWLRQVLWHV)LUVWWKHPDPPDOLDQWDUJHWRIUDSDP\FLQ (mTOR)-RICTOR protein complex phosphorylates the exposed regulatory serine (S473) LQWKH&WHUPLQXVRI3.%1H[W3'.SKRVSKRU\ODWHVWKHUHJXODWRU\WKUHRQLQHUHVLGXH 7 RI3.%WKDWLVUHTXLVLWHIRUHQ]\PHDFWLYLW\2-4 All these early signaling steps WDNHSODFHDWWKHSODVPDPHPEUDQH$FWLYDWLRQRI3.%DOORZVLWVUHORFDOL]DWLRQWRWKH F\WRVRO3DUWRIWKHDFWLYDWHG3.%HQWHUVWKHQXFOHXVZHUHLWDIIHFWVWUDQVFULSWLRQRI VHYHUDOWDUJHWJHQHV7KHSUHFLVHSDWKZD\IURP3,NLQDVH3.%WR*/87WUDQVORFDWLRQ is still not known.5 PKB substrates are glycogen synthase kinase 3 or GSK3,6 the WUDQVFULSWLRQIDFWRU)R[27,8 the Rheb GAP TSC29 the phosphodiesterase PDE3b10 and the RabGAP TBC1D4/AS160.11 Only TBC1D4 is shown to play an important role in insulin-stimulated GLUT4 translocation. It has recently been argued by Hoehn and FRZRUNHUVWKDWGHIHFWVLQXSVWUHDPHOHPHQWVRIWKHLQVXOLQFDVFDGHOLNH,5DQG,56 SURWHLQVDUHDFWXDOO\DQXQOLNHO\FDXVHIRULQVXOLQUHVLVWDQFH5 This statement sharply contrasts with the existing dogma. The revolutionary view by Hoehn and colleagues is EDVHGRQWKHLU¿QGLQJLQYDULRXVPRGHOVRILQVXOLQUHVLVWDQFHRIGLVFRUGDQFHEHWZHHQ upstream insulin signaling and GLUT4 translocation, most strikingly in palmitate WUHDWHG/P\RWXEHVDQGKLJKIDWIHGPLFH57KH\FRQFOXGHIURPWKHVH¿QGLQJVWKDW ZKLOHGHIHFWVLQ,563,NLQDVH3.%PD\RFFXULQLQVXOLQUHVLVWDQFHLWLVXQOLNHO\WKDW VXFKGHIHFWVFRQWULEXWHWRLWVHDUO\GHYHORSPHQW7KH\IXUWKHUUDLVHWKHSRVVLELOLW\WKDW XSVWUHDPVLJQDOLQJGHIHFWVDUHHLWKHUFRUROODU\RUDFRQVHTXHQFHUDWKHUWKDQDFDXVHRI insulin resistance possibly exacerbating the insulin resistance state once it has become established and promoting progression to metabolic disease. The apparent present lack RILQVLJKWLQWKHSUHFLVHUROHRIWKH3,NLQDVH3.%SDWKZD\LQ*/87WUDQVORFDWLRQ ZDUUDQWVIXUWKHULQYHVWLJDWLRQ2IQRWHDQDGGLWLRQDO3,NLQDVHLQGHSHQGHQWSDWKZD\ IRU LQVXOLQVWLPXODWHG */87 WUDQVORFDWLRQ LQYROYLQJ WKH $36±&$3±&EO SURWHLQ complex has been postulated.12 In this pathway, the APS–CAP–Cbl protein complex activates the small GTPase TC10, which in turn signals the cytoskeleton to promote */87FHOOVXUIDFHUHFUXLWPHQW12 ,WVKRXOGDOVREHQRWHGWKDWLQDGGLWLRQWRWKHDFWLYDWLRQRIWKH3,NLQDVH3.%DQG *73DVH7&SDWKZD\VELQGLQJRILQVXOLQWR,5LQGXFHVDGLVWLQFWVLJQDOLQJSDWKZD\ YLD *URZWK IDFWRU ELQGLQJ SURWHLQ  *UE 6RQ RI VHYHQOHVV 6RV  DQG 5DV OHDGLQJ WR DFWLYDWLRQ RI WKH PLWRJHQ DFWLYDWHG SURWHLQ NLQDVH 0$3.  LVRIRUPV H[WUDFHOOXODU signal-regulated kinases (ERK)1 and ERK2. The MAPK cascade is not involved in insulin-stimulated glucose transport or glycogen synthesis but may relate to regulation RIFHOOVXUYLYDODQGSUROLIHUDWLRQ13 Insulin, together with other stimuli, is also involved LQ3,NLQDVHGHSHQGHQW UHJXODWLRQRIDXWRSKDJ\14,15 Insulin Resistance and Obesity: A Remaining Riddle 7KHLQFLGHQFHRIREHVLW\LVUDSLGO\LQFUHDVLQJLQ:HVWHUQDQGGHYHORSLQJFRXQWULHVGXH WRLQFUHDVHGFRQVXPSWLRQRIHQHUJ\ULFKIRRGDQGGLPLQLVKLQJSK\VLFDODFWLYLW\>66@ $VWDJJHULQJELOOLRQDGXOWVDUHRYHUZHLJKWRIZKLFKPLOOLRQDUHREHVHDQG PLOOLRQVXIIHUIURP7\SHGLDEHWHVPHOOLWXV16 Obesity is not without negative health FRQVHTXHQFHV,WLVVWURQJO\DVVRFLDWHGZLWKDQLQFUHDVHGULVNIRUFKURQLFGLPLQLVKHG

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responsiveness to insulin, so-called insulin resistance. In insulin-resistant individuals, QRUPDOOHYHOVRILQVXOLQIDLOWRLOOLFLWWKHDGHTXDWHUHVSRQVHVLQSHULSKHUDOWLVVXHVUHTXLUHG IRUVZLIWQRUPDOL]DWLRQRISODVPDJOXFRVHFRQFHQWUDWLRQ1,177RFRPSHQVDWHIRUWKLV the pancreas is stimulated to release more insulin. This leads to high circulating levels RILQVXOLQDFRQGLWLRQFDOOHGFRPSHQVDWRU\K\SHULQVXOLQHPLD7KHLPSDLUHGUHVSRQVH to glucose administration in insulin-resistant individuals is designated as glucose intolerance. Temporary insulin resistance is most likely not pathological, but rather a XVHIXOSK\VLRORJLFDOUHVSRQVHIRUH[DPSOHWRH[FHVVLYHVXSSO\RIHQHUJ\RUWRVSHFL¿F ERG\GHPDQGV$WHPSRUDU\VWDWHRILQVXOLQUHVLVWDQFHPD\EHLQGXFHGE\DYDULRXV IDFWRUVIRUH[DPSOHLQÀDPPDWLRQ+RZHYHULQREHVHLQGLYLGXDOVLQVXOLQUHVLVWDQFH becomes gradually chronic and more prominent, constituting a pathological trait. In REHVH LQVXOLQUHVLVWDQW LQGLYLGXDOV WKH FRPSHQVDWRU\ RYHU SURGXFWLRQ RI LQVXOLQ XOWLPDWHO\ EHFRPHV LUUHYHUVLEO\ LPSDLUHG GXH WR GDPDJH RI SDQFUHDWLF EHWDFHOOV Subsequently, chronic hyperglycemia (Type 2 diabetes mellitus) develops which is usually accompanied by dyslipidemia.18 It is still an enigma why obese individuals are so prone to develop chronic LQVXOLQUHVLVWDQFH2EHVHLQGLYLGXDOVDOVRW\SLFDOO\VKRZORZJUDGHLQÀDPPDWLRQ19 PHGLDWHGE\F\WRNLQHVVHFUHWHGE\PDFURSKDJHVLQWKHLUDGLSRVHWLVVXHZKLFKIXUWKHU promotes insulin resistance.20-24 It is attractive to speculate that in this manner a vicious VHOIUHLQIRUFLQJ F\FOH RI REHVLW\ LQVXOLQ UHVLVWDQFH DQG LQÀDPPDWLRQ LV IRUPHG ,PSRUWDQWO\WKHHIIHFWRILQVXOLQRQOLSLGPHWDEROLVPPD\IXUWKHUDGGWRWKLV,QWKH liver insulin stimulates lipogenesis by activating SREBP-1c, enhancing its transcription DQGLQFUHDVLQJWKHDPRXQWRIQXFOHDU65(%3F25 SREBO-1c promotes transcription RIJHQHVUHTXLUHGIRUIDWW\DFLGDQGWULJO\FHULGHELRV\QWKHVLVVXFKDVDFHW\OFRHQ]\PH $FDUER[\ODVH$&& DQGIDWW\DFLGV\QWKDVH)$6 26 Intriguingly, insulin resistance is VHOHFWLYH,QREHVHLQVXOLQUHVLVWDQWLQGLYLGXDOVGHVSLWHFOHDUUHVLVWDQFHIRUWKHLQKLELWRU\ HIIHFWRILQVXOLQRQKHSDWLFJOXFRVHRXWSXWWKHVHQVLWLYLW\IRUWKHVWLPXODWRU\HIIHFW RI LQVXOLQ RQ OLSRJHQHVLV LV PDLQWDLQHG27 This selective insulin resistance explains WKH SDUDGR[LFDO FRPELQDWLRQ RI K\SHULQVXOLQHPLD DQG K\SHUOLSLGHPLD LQ 7\SH ,, diabetes.18 Partial postreceptor hepatic insulin resistance seems a key element in the GHYHORSPHQWRIG\VOLSLGHPLDDQGKHSDWLFVWHDWRVLV$YHU\UHFHQWSDSHUE\6DMDQHWDO sheds some new light on the puzzling phenomenon.28 Evidence is presented indicating WKDWLQWKHOLYHURILQVXOLQUHVLVWDQWREHVHUDWVDQGPLFH3.%DFWLYDWLRQLVLPSDLUHG but concomitantly atypical protein kinase C (aPKC) activation by insulin is conserved DQGWKDWWKHODWWHUFDXVHVH[FHVVLYHH[SUHVVLRQDQGDFWLYDWLRQRI65(%3FDVZHOODV DFWLYDWLRQRI,..`/NFg%6DMDQHWDOSURSRVHWKDWLQGLDEHWLFURGHQWOLYHUGLPLQLVKHG 3.%DFWLYDWLRQPD\ODUJHO\UHÀHFWLPSDLUHG,563,.DFWLYDWLRQZKLOHFRQVHUYHG D3.&DFWLYDWLRQUHÀHFWVUHWDLQHG,563,.DFWLYLW\FDXVLQJH[FHVVLYH65(3%F and NFgB activities.28 In hyperinsulinemic individuals, the increased hepatic lipogenesis and the FRUUHVSRQGLQJO\LQFUHDVHGVXSSO\RISHULSKHUDOWLVVXHVZLWKOLSLGIURPWKHOLYHULVOLNHO\ WRDGGWRWKHYLFLRXVVHOIUHLQIRUFLQJF\FOHRIREHVLW\LQVXOLQUHVLVWDQFHLQÀDPPDWLRQ ,QGHHGWKHUHLVJURZLQJHYLGHQFHSRLQWLQJWRDNH\UROHIRUH[FHVVLYHOLSLGVLQWKHHWLRORJ\ RIREHVLW\LQGXFHGLQVXOLQUHVLVWDQFH29 Triglycerides are normally predominantly stored LQDGLSRVHWLVVXH,QREHVLW\WKLVVWRUDJHFDSDFLW\LVRIWHQH[FHHGHGKHSDWLFOLSRJHQHVLV is increased and triglycerides accumulate in nonadipose tissues such as liver and muscle. Lipid excess in these tissues is associated with impaired insulin signaling and action.30,31 A particular strong inverse correlation exists between the intramyocellular lipid content

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and whole body insulin-stimulated glucose uptake.32 It is generally thought that in obese VXEMHFWVWKHVXUSOXVRIOLSLGGHOLYHUHGWRWLVVXHVOLNHPXVFOHLVSDWKRJHQLFLHOLSRWR[LFLW\ GULYLQJIRUZDUGWKHSDWKRORJ\RILQVXOLQUHVLVWDQFH+RZHYHUWKHSUHFLVHQDWXUHRIWKH OLSRWR[LFIDFWRUVLQYROYHGDVZHOODVWKHVHTXHQFHRISDWKRORJLFDOHYHQWVDUHVWLOOYHU\ poorly understood. In a very recent, elegant review Summers and colleagues discuss the present insight in the molecular mechanisms through which glycerolipids contribute to selective insulin resistance and lipotoxicity.33 They conclude that triglyceride (TAG) LVOLNHO\WREHMXVWDQLQHUWE\VWDQGHU,QFRQWUDVWGLDF\OJO\FHURO'$* LVFRQVLGHUHG WREHDVWURQJFDQGLGDWHDVOLSRWR[LFIDFWRU'$*LVNQRZQWREHDEOHWRDFWLYDWH3.& LVRIRUPVUHVXOWLQJLQSKRVSKRU\ODWLRQRILQVXOLQUHFHSWRUVXEVWUDWHVDQGWKHLQKLELWLRQ RISKRVSKDWLG\OLQRVLWRONLQDVH34,35 Phosphatidic acid, an intermediate in the synthesis RI'$*KDVDOVREHHQSURSRVHGDVDQWDJRQLVWRILQVXOLQDFWLRQ36 $FRPSOHWHO\GLIIHUHQWFODVVRIOLSLGVWKHVRFDOOHGVSKLQJROLSLGVKDVUHFHQWO\DOVREHHQ LPSOLFDWHGLQWKHHWLRORJ\RILQVXOLQUHVLVWDQFH6SKLQJROLSLGVKDYHDOVREHHQFRQVLGHUHG as primary lipotoxic agents, as will be discussed in detail in the sections below. 1H[W WR VSHFL¿F OLSRWR[LF OLSLG VSHFLHV PDQ\ LQYHVWLJDWRUV KDYH SURSRVHG WKDW H[FHVVLYHOLSLGVXSSO\WRWLVVXHVLQREHVHLQGLYLGXDOVLQJHQHUDOIRUFHVWKHPLWRFKRQGULDWR SURGXFHHYHULQFUHDVLQJDPRXQWVRI526LPSDLULQJPLWRFKRQGULDOIXQFWLRQDQGLQGXFLQJ DFRQFRPLWDQWGHYHORSPHQWRILQVXOLQUHVLVWDQFH+RXVWLVHWDO37 observed that chronic treatment with an antioxidant agent (MnTBAP) improved insulin sensitivity and glucose homeostasis in insulin-resistant ob/ob mice. More recently, Hoehn and colleagues38,39 IRXQG WKDW 526 VFDYHQJHUV SUHYHQWHG LQVXOLQ UHVLVWDQFH LQ DQLPDO PRGHOV DV ZHOO DV /P\RWXEHVH[SRVHGWRDQXPEHURIIDFWRUVZKLFKLQGXFHLQVXOLQUHVLVWDQFHLQFOXGLQJ YDULRXVH[RJHQRXVIDWW\DFLGV GLYCOSPHINGOLIPIDS Sphingolipids (SLs) and glycosphingolipids (GSLs) are structural components RI PDPPDOLDQ FHOO PHPEUDQHV DQG ODUJHO\ UHVLGH DW WKH FHOO VXUIDFH 6SKLQJROLSLGV DUHFRPSRVHGRIDFHUDPLGHPRLHW\ZLWKDQ1DF\ODWHGVSKLQJRVLQHJURXS40-42 Either JOXFRVHRUJDODFWRVHLVOLQNHGWRWKHSULPDU\K\GUR[\JURXSRIWKHVSKLQJRVLQHPRLHW\ through a beta-glycosidic bond, thereby giving rise to the simplest glycosphingolipids: JOXFRV\OFHUDPLGHDQGJDODFWRV\OFHUDPLGH/LQNDJHRIDSKRVSKRU\OFKROLQHPRLHW\WR glucosylceramide results in sphingomyelin, a very abundant membrane lipid. Further DGGLWLRQVRIROLJRVDFFKDULGHVDQGVXOIDWHJURXSVWRJO\FRVSKLQJROLSLGVJLYHULVHWRDEURDG UDQJHRIFRPSOH[JO\FRVSKLQJROLSLGV43,44 Those with a capping N-acetylneuraminic acid are known as gangliosides. 7KH GLVFRYHU\ RI WKH JO\FRVSKLQJROLSLGV LV JHQHUDOO\ DWWULEXWHG WR -RKDQ /: 7KXGLFKXPZKRLQSXEOLVKHGRQWKHFKHPLFDOFRPSRVLWLRQRIWKHEUDLQ7KXGLFXP LVRODWHGVHYHUDOFRPSRXQGVIURPHWKDQROLFEUDLQH[WUDFWVZKLFKKHFRLQHGFHUHEURVLGHV+H VXEMHFWHGRQHRIWKHVHSKUHQRVLQQRZNQRZQDVJDODFWRV\OFHUDPLGH WRDFLGK\GURO\VLV DQGWKLVSURGXFHGWKUHHGLVWLQFWFRPSRQHQWV2QHKHLGHQWL¿HGDVDIDWW\DFLGDQGDQRWKHU SURYHG WR EH DQ LVRPHU RI D-glucose, which is now known as D-galactose. The third component, with an “alkaloidal nature”, presented “many enigmas” to Thudichum and WKHUHIRUHKHQDPHGLWVSKLQJRVLQHDIWHUWKHP\WKRORJLFDOULGGOHRIWKH6SKLQ[

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epoxide. The enzyme has a pH optimum in the neutral region, as opposed to the acidic RSWLPXPRI*%$*%$LVQRWORFDWHGLQWKHO\VRVRPHVEXWSUREDEO\FORVHWRWKH FHOOVXUIDFH7KHIXQFWLRQRI*%$LVFXUUHQWO\QRWNQRZQEXWLWVLQKLELWLRQLQPLFHLV DVVRFLDWHGZLWKLPSDLUHGVSHUPDWRJHQHVLVDUHVXOWWKDWLVFRQ¿UPHGLQVWXGLHVZLWKD GBA2 knock-out mouse model.78-80 7KH LPSRUWDQFH RI HQGRO\VRVRPDO FDWDEROLVP RI VSKLQJROLSLGV LV EHVW LOOXVWUDWHG E\WKHH[LVWHQFHRIDJURXSRILQKHULWHGGLVRUGHUVLQKXPDQVFDXVHGE\GH¿FLHQF\LQ lysosomal catabolic pathways, the sphingolipidoses.68,QKHULWHGGH¿FLHQFLHVLQDVSHFL¿F O\VRVRPDO HQ]\PH RU DFWLYDWRU SURWHLQ UHVXOW LQ DFFXPXODWLRQ RI WKH FRUUHVSRQGLQJ JO\FR VSKLQJROLSLGV7KHPRVWFRPPRQRIWKHVSKLQJROLSLGRVHVLV*DXFKHUGLVHDVH81 DQ DXWRVRPDO UHFHVVLYH GLVRUGHU FDXVHG E\ GH¿FLHQW JOXFRFHUHEURVLGDVH DFWLYLW\82,83 7KHPDQLIHVWDWLRQRI*DXFKHUGLVHDVHLVUHPDUNDEO\KHWHURJHQHRXVLWVRQVHWFDQRFFXU IURPELUWKXSWRDQDOPRVWDV\PSWRPDWLFFRXUVHDWROGDJH7KHXQGHUO\LQJPXWDWLRQV in the GBA1 JHQH VKRZ VRPH FRUUHODWLRQ ZLWK WKH VHYHULW\ RI GLVHDVH PDQLIHVWDWLRQ DQGLQSDUWLFXODUWKHGHYHORSPHQWRIQHXURORJLFDOV\PSWRPV$ORZUHVLGXDOHQ]\PH DFWLYLW\LQOHXNRF\WHVRU¿EUREODVWVLVDVVRFLDWHGZLWKDPRUHVHYHUHSURJUHVVLRQRIWKH disease.84,85 The most common mutation in the GBA1 gene, which encodes the amino acid VXEVWDWLRQ16LVXVXDOO\DVVRFLDWHGZLWKDUHODWLYHO\EHQLJQFRXUVHRIWKHGLVHDVH with no neuropathology involved. N370S-GBA1 is normally synthesized and delivered to lysosomes, but shows catalytic abnormalities.86,87 In sharp contrast, the other common /3PXWDWLRQUHVXOWVLQDSRO\SHSWLGHWKDWIROGVSRRUO\LQWKH(586+RPR]\JRWHVIRU /3*%$GHYHORSDVHYHUHQHXURSDWKLFFRXUVHRIWKHGLVHDVH,QFRQWUDVWWRRWKHU lysosomal glycosidases, GBA1 does not acquire mannose-6-phosphate moieties, but is sorted and transported to lysosomes by interaction with the integral membrane-protein LIMP-2.88-90'H¿FLHQF\LQ/,03PD\WKHUHIRUHDOVRUHVXOWLQUHGXFHGFHOOXODU*%$ activity.916LQFH*%$UHTXLUHVWKHDFWLYDWRUSURWHLQVDSRVLQ&IRUHI¿FLHQWLQWUDO\VRVRPDO GHJUDGDWLRQRIJOXFRV\OFHUDPLGHGH¿FLHQF\LQWKLVDFFHVVRU\SURWHLQDOVRUHVXOWVLQWKH DFFXPXODWLRQ RI JOXFRV\OFHUDPLGH LQ FHOOV92 7KH PDMRULW\ RI *DXFKHU SDWLHQWV KDYH one N370S-GBA1 allele and develop a nonneuropathic, so-called Type 1, disease. In WKHVH SDWLHQWV DFFXPXODWLRQ RI WKH VXEVWUDWH JOXFRV\OFHUDPLGH LV UHVWULFWHG WR WLVVXH macrophages. These heavily lipid-laden macrophages, named Gaucher cells, have a characteristic appearance. Gaucher cells are viable and secrete characteristic proteins such as chitotriosidase and CCL-18.93,94(OHYDWHGOHYHOVRIWKHVHSURWHLQVDUHIRXQGLQ*DXFKHU patients and their measurement is currently used to monitor disease progression as well DVHI¿FDF\RIWKHUDSHXWLFLQWHUYHQWLRQV957KHSUHVHQFHRIODUJHQXPEHUVRI*DXFKHUFHOOV in various tissues results in characteristic clinical signs such as hepatosplenomegaly, SDQF\WRSHQLDDQGVNHOHWDOGHWHULRUDWLRQ7KHFRQVWDQWUHOHDVHRIK\GURODVHVDQGF\WRNLQHV by Gaucher cells and surrounding phagocytes is thought to underlie the pathological IHDWXUHVRIWKHGLVRUGHU96 (GLYCO)SPHINGOLIPIDS AND INSULIN RESISTANCE A Role for Ceramide &HUDPLGH IXQFWLRQV DV D PHGLDWRU LQ VLJQDOLQJ FDVFDGHV WKDW UHJXODWH DSRSWRVLV GLIIHUHQWLDWLRQDQGFHOOF\FOHDUUHVW977KHVHPLQDOZRUNE\8QJHULGHQWL¿HGWKHVSKLQJROLSLG ceramide as candidate lipotoxic agent in obesity-induced insulin resistance.98 The role

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GLYCOSPHINGOLIPIDS AND INSULIN RESISTANCE

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1DON\ODWHGGHR[\QRMLULP\FLQVRIZKLFKVRPHSURYHGWREHYHU\SRWHQWLQKLELWRUV RI *&6142-145 2QH RI WKH PRVW DSSHDOLQJ FRPSRXQGV LV WKH LPLQRVXJDU GHULYDWLYH NDGDPDQWDQH\OPHWKR[\  SHQW\OGHR[\QRMLULP\FLQ $03'10  WKDW VKRZVDWWUDFWLYHSKDUPDFRNLQHWLFVDQGLVKDUGO\PHWDEROL]HG2UDODGPLQLVWUDWLRQRI AMP-DNM at high concentrations is well tolerated in rodents and dogs. The IC50 value RI$03'10IRU*&6LVDURXQGQ0143 Such a steady-state plasma concentration RIWKHFRPSRXQGFDQEHREWDLQHGE\DGPLQLVWUDWLRQRIPJNJEZGD\LQPLFHDQG  PJNJ EZGD\ LQ UDWV ,QFXEDWLRQ RI FXOWXUHG FHOOV ZLWK $03'10 UHVXOWV LQ GRVHGHSHQGHQWUHGXFWLRQVRIJOXFRV\OFHUDPLGHDQGJDQJOLRVLGHVZLWKRXWDFRQFRPLWDQW increase in ceramide levels. The same is observed in drug-treated mice and rodents. 7KH LPSDFW RI $03'10 RQ LQVXOLQ UHVLVWDQFH ZDV LQYHVWLJDWHG 7UHDWPHQW ZLWK$03'10RIREHVHLQVXOLQUHVLVWDQWREREPLFHFRUUHFWHGWKHLUHOHYDWHGWLVVXH glucosylceramide levels, markedly lowered circulating glucose levels, improved oral glucose tolerance, reduced HbA1C and improved insulin sensitivity in muscle and liver as measured by euglycemic clamps.143 6LPLODUO\ EHQH¿FLDO PHWDEROLF HIIHFWV ZHUHVHHQLQKLJKIDW±IHG',2 PLFHDQG=')UDWV143 In cultured 3T3-L1 adipocytes, $03'10 FRXQWHUDFWHG WXPRU QHFURVLV IDFWRUDOID±LQGXFHG DEQRUPDOLWLHV LQ glycosphingolipid concentrations and concomitantly reversed abnormalities in insulin signal transduction. Thus, AMP-DNM rendered improvements in upstream elements RIWKHLQVXOLQVLJQDOLQJSDWKZD\OLNH,5DQG,56SURWHLQERWKLQWKH7/FHOO PRGHODVLQYDULRXVWLVVXHVRIWUHDWHGDQLPDOV9HU\VLPLODU¿QGLQJVZHUHPDGHE\=KDR DQGFRZRUNHUVZLWKDFKHPLFDOO\XQUHODWHGLQKLELWRURI*&6QDPHG*(1= (1R,2R)-nonanoic acid[2-(2v ,3v -dihydro-benzo [1,4] dioxin-6v -yl)-2-hydroxy-1pyrrolidin-1-ylmethyl-ethyl]-amide.114,146,QKLELWLRQRIJO\FRVSKLQJROLSLGIRUPDWLRQLQ =')UDWVDQGGLHWLQGXFHGLQVXOLQUHVLVWDQWPLFHZLWK*HQ]KDGFRPSDUDEOH EHQH¿FLDOHIIHFWVRQJOXFRVHKRPHRVWDVLVFRUUHFWLRQRIK\SHULQVXOLQHPLDUHGXFWLRQ RIQRQIDVWHGEORRGJOXFRVHOHYHOVORZHULQJRIJO\FDWHGKHPRJORELQDQGLPSURYHPHQW RI JOXFRVH WROHUDQFH 7UHDWPHQW ZLWK *HQ] OLNH $03'10 SURPRWHG LQVXOLQVWLPXODWHG SKRVSKRU\ODWLRQ RI WKH LQVXOLQ UHFHSWRU LQ PXVFOH114 2I LQWHUHVW ERWK$03'10DQG*(1=WUHDWPHQWZHUHIRXQGWRRIIHUSURWHFWLRQDJDLQVW EHWDFHOOGDPDJHLQWKHSDQFUHDVRI=')UDWV Mode of Action of Inhibitors *(1= EHLQJ D YHU\ VSHFL¿F LQKLELWRU RI *&6 KDV D FOHDU WDUJHW glucosylceramide synthase.146 In sharp contrast, AMP-DNM not only inhibits GCS (IC50 ¾ 150 nM), but also GBA2 (IC50 ¾ 1 nM) and GBA, the lysosomal glucocerebrosidase (IC50 220 nM).143,QKLELWLRQRI*%$VHHPVWRKDYHOLPLWHGFRQVHTXHQFHVDVVXJJHVWHG E\WKHQRUPDOSKHQRW\SHRI*%$GH¿FLHQWPLFH:HRQO\QRWHGDVSHUPDWRJHQHVLV LPSDLUPHQWLQVRPHPRXVHVWUDLQVDQHIIHFWDOVRREVHUYHGZLWK=DYHVFDDQGORZGRVH AMP-DNM (see section Glycosphingolipid degradation ,QUDEELWVDQGGRJVWKLVHIIHFW GRHVQRWRFFXU3DUWLDOLQKLELWLRQRI*%$DWWKHSODVPDFRQFHQWUDWLRQVRI$03'10 reached in conducted animal studies (20-200 nM) can not be excluded. However, even at DVWHDG\VWDWHSODVPDFRQFHQWUDWLRQVRIQ0$03'10WKHUHVKRXOGEHVXI¿FLHQW UHVLGXDO*%$HQ]\PHFDSDFLW\WRSUHYHQWO\VRVRPDODFFXPXODWLRQRIJOXFRV\OFHUDPLGH ,QGHHGQRHOHYDWLRQVLQWKHOLSLGZHUHQRWHGLQPXVFOHRUOLYHURI$03'10WUHDWHG DQLPDOV$QRWKHUSRWHQWLDOWDUJHWRI$03'10DUHLQWHVWLQDOJO\FRVLGDVHV3DUWLFXODUO\

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119. Straczkowski M, Kowalska I, Baranowski M et al. Increased skeletal muscle ceramide level in men at ULVNRIGHYHORSLQJW\SHGLDEHWHV'LDEHWRORJLD 6NRYEUR0%DUDQRZVNL06NRY-HQVHQ&HWDO+XPDQVNHOHWDOPXVFOHFHUDPLGHFRQWHQWLVQRWDPDMRU IDFWRULQPXVFOHLQVXOLQVHQVLWLYLW\'LDEHWRORJLD 6WUDF]NRZVNL0.RZDOVND,1LNRODMXN$HWDO5HODWLRQVKLSEHWZHHQLQVXOLQVHQVLWLYLW\DQGVSKLQJRP\HOLQ signaling pathway in human skeletal muscle. Diabetes 2004; 53:1215-21. 6HUOLH0-0HLMHU$-*URHQHU-(HWDO6KRUWWHUPPDQLSXODWLRQRISODVPDIUHHIDWW\DFLGVGRHVQRWFKDQJH VNHOHWDOPXVFOHFRQFHQWUDWLRQVRIFHUDPLGHDQGJOXFRV\OFHUDPLGHLQOHDQDQGRYHUZHLJKWVXEMHFWV-&OLQ Endocrinol Metab 2007; 92:1524-9. +RMMDWL05/L=-LDQJ;&6HULQHSDOPLWR\O&R$WUDQVIHUDVH637 GH¿FLHQF\DQGVSKLQJROLSLGOHYHOV in mice. Biochim Biophys Acta 2005; 1737:44-51. 1RMLUL+6WURXG0+DNRPRUL6$VSHFL¿FW\SHRIJDQJOLRVLGHDVDPRGXODWRURILQVXOLQGHSHQGHQW FHOOJURZWKDQGLQVXOLQUHFHSWRUW\URVLQHNLQDVHDFWLYLW\3RVVLEOHDVVRFLDWLRQRIJDQJOLRVLGHLQGXFHG LQKLELWLRQRILQVXOLQUHFHSWRUIXQFWLRQDQGPRQRF\WLFGLIIHUHQWLDWLRQLQGXFWLRQLQ+/FHOOV-%LRO Chem 1991; 266:4531-7. 125. Tagami S, Inokuchi JJ, Kabayama K et al. Ganglioside GM3 participates in the pathological conditions RILQVXOLQUHVLVWDQFH-%LRO&KHP 126. Kabayama K, Sato T, Kitamura F et al. TNFalpha-induced insulin resistance in adipocytes as a membrane PLFURGRPDLQGLVRUGHULQYROYHPHQWRIJDQJOLRVLGH*0*O\FRELRORJ\ 127. Yamashita T, Hashiramoto A, Haluzik M et al. Enhanced insulin sensitivity in mice lacking ganglioside GM3. Proc Natl Acad Sci USA 2003; 100:3445-9. 
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CHAPTER 8 GLYCOSPHINGOLIPIDS AND KIDNEY DISEASE Andrew R. Mather and Leah J. Siskind* Department of Medicine, Division of General Internal Medicine/Geriatrics, Medical University of South Carolina, Charleston, South Carolina, USA *Corresponding Author: Leah J. Siskind—Email: [email protected]

Abstract:

*O\FRVSKLQJROLSLGVGHULYHGIURPWKHDGGLWLRQRIVXJDUPRLHWLHVWRWKHVSKLQJROLSLG ceramide, are highly abundant in the kidney. Glycosphingolipids are known to SOD\DQLPSRUWDQWUROHLQRUJDQIXQFWLRQDWOHDVWLQSDUWIURPLQKHULWHGOLSLGVWRUDJH GLVHDVHVVXFKDV$QGHUVRQ)DEU\GLVHDVH)DEU\¶VGLVHDVH)' WKDWUHVXOWVIURP a mutation in alpha-galactosidase A (_-GLA or _-Gal A), the enzyme responsible IRUFDWDO\]LQJWKHUHPRYDORIWHUPLQDOJDODFWRVHUHVLGXHVIURPJO\FRVSKLQJROLSLGV The inactivation in _*/$LQ)'UHVXOWVLQWKHDFFXPXODWLRQRIJO\FRVSKLQJROLSLGV LQFOXGLQJJORERVLGHVDQGODFWRV\OFHUDPLGHVZKLFKPDQLIHVWVDVVHYHUDOFRPPRQ pathologies including end-stage kidney disease. More recently, glycosphingolipids DQGRWKHUVSKLQJROLSLGVKDYHEHFRPHLQFUHDVLQJO\UHFRJQL]HGIRUWKHLUUROHVLQDYDULHW\ RIRWKHUNLGQH\GLVHDVHVLQFOXGLQJSRO\F\VWLFNLGQH\GLVHDVHDFXWHNLGQH\LQMXU\ glomerulonephritis, diabetic nephropathy and kidney cancer. This chapter reviews HYLGHQFHVXSSRUWLQJDPHFKDQLVWLFUROHIRUJO\FRVSKLQJROLSLGVLQNLGQH\GLVHDVHDQG GLVFXVVHVGDWDLPSOLFDWLQJDUROHIRUWKHVHOLSLGVLQNLGQH\GLVHDVHUHVXOWLQJIURP PHWDEROLFV\QGURPH,PSRUWDQWO\LQKLELWRUVRIJO\FRVSKLQJROLSLGV\QWKHVLVDUHZHOO tolerated in animal models as well as in humans. Thus, an increased understanding RIWKHPHFKDQLVPVE\ZKLFKDOWHUHGUHQDOJO\FRVSKLQJROLSLGPHWDEROLVPOHDGVWR kidney disease has great therapeutic potential.

INTRODUCTION *O\FRVSKLQJROLSLGVDUHDYHU\KHWHURJHQHRXVFODVVRIOLSLGVYDU\LQJGUDPDWLFDOO\ LQ ERWK WKH K\GURSKLOLF DQG K\GURSKRELF UHJLRQ RI WKH PROHFXOH 7KH K\GURSKLOLF SRUWLRQRIJO\FRVSKLQJROLSLGVFRQVLVWVRIDVXJDUKHDGJURXSDQGFDQFRQWDLQQHXWUDORU FKDUJHGJURXSV7KHK\GURSKRELFSRUWLRQFDQYDU\LQWKHOHQJWKRIWKHNOLQNHGIDWW\ DF\OFKDLQDQGVSKLQJRLGEDVHDVZHOODVWKHGHJUHHRIXQVDWXUDWLRQK\GUR[\ODWLRQDQG branching. Glycosphingolipids have numerous roles in regulating cellular processes, Sphingolipids and Metabolic Disease, edited by L. Ashley Cowart. ©2011 Landes Bioscience and Springer Science+Business Media, LLC 121

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LQFOXGLQJFHOOSUROLIHUDWLRQDSRSWRVLVLQÀDPPDWLRQDQGFHOOXODUVLJQDOLQJ'\VUHJXODWLRQ RI JO\FRVSKLQJROLSLG PHWDEROLVP OHDGV WR WKH DFFXPXODWLRQ RI SDUWLFXODU VSHFLHV RI JO\FRVSKLQJROLSLGV DQG LQGXFHV VHYHUDO GLIIHUHQW SDWKRORJLHV DQG GHYHORSPHQWDO DEQRUPDOLWLHV DV HYLGHQFHG IURP NQRFNRXW VWXGLHV LQ PLFH 7KXV LW LV QRW VXUSULVLQJ that glycosphingolipids have been implicated in numerous diseases and that mutations in several enzymes involved in glycosphingolipid metabolism occur in a wide variety RIKXPDQGLVHDVHV*O\FRVSKLQJROLSLGVDUHSDUWLFXODUO\DEXQGDQWLQWKHNLGQH\DQGDUH WKRXJKWWRSOD\DQLPSRUWDQWUROHLQNLGQH\IXQFWLRQ,QGHHGDOWHUHGJO\FRVSKLQJROLSLG PHWDEROLVPRFFXUVLQDYDULHW\RINLGQH\GLVHDVHV7KLVFKDSWHUUHYLHZVFXUUHQWOLWHUDWXUH RQWKHUROHRIJO\FRVSKLQJROLSLGVLQNLGQH\GLVHDVHDQGGLVFXVVHVLPSOLFDWLRQVIRUWKHLU potential role in kidney pathologies associated with metabolic disease. METABOLISM OF SPHINGOLIPIDS *O\FRVSKLQJROLSLGVEHORQJWRWKHVSKLQJROLSLGIDPLO\ZKLFKDOOVKDUHDFRPPRQ sphingoid base backbone. Sphingolipid metabolism is a complex process involving many GLIIHUHQWVSKLQJROLSLGVDQGHQ]\PHVHDFKZLWKLPSRUWDQWDQGGLVWLQFWFHOOXODUIXQFWLRQV1 At WKHKHDUWRIVSKLQJROLSLGPHWDEROLVPLVFHUDPLGHZKLFKLVFRPSULVHGRIDN-acylated (14-26 carbons) sphingosine (16 or 18 carbons). Ceramide can be generated by multiple pathways in cells, namely sphingomyelin hydrolysis, de novo synthesis, the salvage pathway, or EUHDNGRZQRIPRUHFRPSOH[VSKLQJROLSLGVVHH)LJ 6SKLQJRP\HOLQDVHV60DVHV  FDWDO\]HWKHK\GURO\VLVRIVSKLQJRP\HOLQ60 WRIRUPFHUDPLGHDQGSKRVSKRU\OFKROLQH DQGDUHFODVVL¿HGE\WKHLUS+RSWLPD2,32IWKHVHWKHDFLGDQG0J 2-dependent neutral sphingomyelinases have been implicated in stress-induced ceramide generation.2,3

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De novo ceramide synthesis4,5 occurs in the ER.6 The enzyme serine palmitoyl WUDQVIHUDVH637 FDWDO\]HVWKH¿UVWDQGUDWHOLPLWLQJVWHSLQGHQRYRV\QWKHVLVQDPHO\ WKHFRQGHQVDWLRQRIVHULQHDQGSDOPLWR\O&R$WRIRUPNHWRVSKLQJDQLQH7KHHQ]\PH NHWRVSKLQJDQLQH UHGXFWDVH FDWDO\]HV WKH UHGXFWLRQ RI NHWRVSKLQJDQLQH WR IRUP sphinganine. Ceramide synthases catalyze the NDF\ODWLRQ RI VSKLQJDQLQH WR IRUP GLK\GURFHUDPLGH'+ $GHVDWXUDVHFDWDO\]HVWKHFRQYHUVLRQRIGLK\GURFHUDPLGHWR FHUDPLGHWKURXJKWKHLQVHUWLRQRIDWUDQVGRXEOHERQGDWWKHSRVLWLRQRIWKHVSKLQJRLG base backbone. Once generated, ceramide’s NOLQNHGIDWW\DF\OFKDLQFDQEHUHPRYHGWRJHQHUDWH VSKLQJRVLQHDVFDWDO\]HGE\FHUDPLGDVHV&'DVH 6HYHUDO&'DVHVKDYHEHHQLGHQWL¿HGLQ PDPPDOVDQGDUHFODVVL¿HGE\WKHLUS+RSWLPD7 The sphingosine generated by the action RI&'DVHVFDQEHXVHGE\FHUDPLGHV\QWKDVHVWRUHJHQHUDWHFHUDPLGHLQWKHUHF\FOLQJ salvage pathway. Thus, ceramide synthases are thought to occupy a central position in WKHVSKLQJROLSLGPHWDEROLFSDWKZD\DVWKH\FDWDO\]HWKHIRUPDWLRQRIFHUDPLGHLQWZR distinct pathways: de novo synthesis and the recycling/salvage pathway (see Fig. 1). ,QPDPPDOVWKHUHDUHVL[FHUDPLGHV\QWKDVHLVRIRUPV&HU6 WKDWKDYHSUHIHUHQFHV IRUIDWW\DF\O&R$VRISDUWLFXODUFKDLQOHQJWKV8-10WRIRUPWKHFRUUHVSRQGLQJFHUDPLGHV ,WLVEHFRPLQJLQFUHDVLQJO\UHFRJQL]HGWKDWVSHFL¿F&HU6DQGWKHLUUHVXOWLQJFHUDPLGH SURGXFWVFRQWULEXWHGLIIHUHQWO\WRFHOOXODUSURFHVVHV11-23 Once generated, ceramides can serve as a metabolic precursor to complex sphingolipids, such as SM and glycosphingolipids. 2IQRWHV\QWKHVLVRIFRPSOH[VSKLQJROLSLGVFDQLQÀXHQFHRWKHUOLSLGPHWDEROLFSDWKZD\V IRU H[DPSOH VSKLQJRP\HOLQ V\QWKDVH FDQ FDWDO\]H WKH WUDQVIHU RI D SKRVSKRFKROLQH KHDGJURXSIURPSKRVSKDWLG\OFKROLQHWRFHUDPLGHWRJHQHUDWH60DQGGLDF\OJO\FHUROLQ the Golgi apparatus. Ceramide can also be phosphorylated by ceramide kinase to generate ceramide-1-phosphate (C1P). Ceramide is broken down when its NOLQNHG IDWW\ DF\O FKDLQ LV UHPRYHG WR liberate sphingosine (catalyzed by CDase), which in turn is phosphorylated to generate VSKLQJRVLQHSKRVSKDWH63 DVFDWDO\]HGE\VSKLQJRVLQHNLQDVHV6. 7ZRLVRIRUPV RI6.KDYHEHHQFORQHGDQGFKDUDFWHUL]HGLQPDPPDOV6.DQG6.7KHVHHQ]\PHVDUH HPHUJLQJDVLPSRUWDQWDQGUHJXODWHGHQ]\PHVWKDWQRWRQO\PRGXODWHWKHOHYHOVRI63 EXWDOVRWKRVHRIVSKLQJRVLQHDQGFHUDPLGH24,25 S1P is broken down to yield hexadecenal DQGHWKDQRODPLQHSKRVSKDWHDVFDWDO\]HGE\VSKLQJRVLQHO\DVH63LVDSURLQÀDPPDWRU\ SURSUROLIHUDWLYHDQGDQWLDSRSWRWLFVSKLQJROLSLG25 The dynamic balance between the OHYHOV RI GLIIHUHQW VSKLQJROLSLGV LV WLJKWO\ UHJXODWHG E\ WKH DFWLYLWLHV RI WKH HQ]\PHV WKDW FDWDO\]H WKHLU IRUPDWLRQ DQG EUHDNGRZQ 7KH UHODWLYH FHOOXODU OHYHOV RI GLIIHUHQW VSKLQJROLSLGVDUHSURSRVHGWRLQÀXHQFHFHOOXODUIDWHEHFDXVHRIWKHLURSSRVLQJHIIHFWV and their ability to be interconverted. METABOLISM OF GLYCOSPHINGOLIPIDS ,QJHQHUDOJO\FRVSKLQJROLSLGVDUHLQLWLDOO\GLYLGHGLQWRRQHRIWZRPDLQFODVVHVEDVHG RQWKH¿UVWVXJDUPRLHW\OLQNHGWRWKHFHUDPLGHEDFNERQHQDPHO\JDODFWRVHRUJOXFRVHRI galactosylceramide (GalCer) and glucosylceramide (GluCer), respectively. The GalCer LVIRUPHGLQWKH(5YLDWKHHQ]\PH8'3*DOFHUDPLGHJDODFWRV\OWUDQVIHUDVHRU*DO&HU V\QWKDVH&*DO7 YLDWKHWUDQVIHURIJDODFWRVHIURP8'3JDODFWRVHWRFHUDPLGHVHH)LJ  *DO&HULVWKHQWUDQVSRUWHGWRWKH*ROJLZKHUHVXOIDWLGHRUJDODFWRVHJURXSVFDQEH added, processes which mainly occur in the kidney.

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*O\FRVSKLQJROLSLGV FDQ DOVR EH IRUPHG E\ GHJUDGDWLRQ RI PRUH FRPSOH[ glycosphingolipids, which occurs mainly in the lysosome, but is known to occur at the plasma membrane and mitochondrion as well.33-41 Indeed, several lysosomal VWRUDJHGLVHDVHVUHVXOWIURPPXWDWLRQVLQHQ]\PHVUHVSRQVLEOHIRUWKHGHJUDGDWLRQRI glycosphingolipids.32,42-49'HIHFWLYHGHJUDGDWLRQRIJO\FRVSKLQJROLSLGVFDQUHVXOWLQVHULRXV SDWKRORJLHVDVLVGHVFULEHGEHORZIRU)DEU\¶VNLGQH\GLVHDVH GLYCOSPHINGOLIPID SUBCELLULAR LOCALIZATION AND TRANSPORT *O\FRVSKLQJROLSLGV DUH IRXQG LQ VHYHUDO FHOOXODU ORFDWLRQV 2Q WKH RXWVLGH RI WKH SODVPD PHPEUDQH JO\FRVSKLQJROLSLGV DUH D PLQRU FRPSRQHQW H[FHSW IRU WKH DSLFDOSODVPDPHPEUDQHRIHSLWKHOLDOFHOOVLQWKHXULQDU\DQGLQWHVWLQDOWUDFWVZKHUH they are highly enriched in the apical plasma membrane, representing 30-40 mole SHUFHQWRIWKHWRWDOOLSLGV50-53 The myelin is enriched in galactosylceramide derived glycosphingolipids and the neuronal plasma membranes have particularly high levels RIJDQJOLRVLGHV,QWUDFHOOXODUO\JO\FRVSKLQJROLSLGVDUHDOVRIRXQGLQWKHH[RF\WLFDQG endocytic pathways. Mitochondria and mitochondrial associated ER derived membranes (MAMs) have been reported to contain particular glycosphingolipids, namely GD1 DQG*'JDQJOLRVLGHVDQGSDUWLFXODUHQ]\PHVRIJO\FRVSKLQJROLSLGPHWDEROLVPKDYH EHHQ ORFDOL]HG WR PLWRFKRQGULD DQGRU 0$0V LQFOXGLQJ QHXUDPLQLGDVH LVRIRUP  which is a sialidase and can act on both gangliosides and glycoproteins.40,41 Enzymes that breakdown glycosphingolipids are also localized to lysosomes with inactivating PXWDWLRQVOHDGLQJWRWKHDFFXPXODWLRQRISDUWLFXODUVSHFLHVRIJO\FRVSKLQJROLSLGVDV LVWKHFDVHZLWK)DEU\¶VGLVHDVHDQGQXPHURXVRWKHUV,QDGGLWLRQWKHUHDUHDOVRDIHZ HQ]\PHVUHVSRQVLEOHIRUEUHDNGRZQRIJO\FRVSKLQJROLSLGVWKDWORFDOL]HWRWKHF\WRVRO PLWRFKRQGULDDQGWKHSODVPDPHPEUDQHDVLVWKHFDVHIRUVLDOLGDVHLVRIRUPVDQG 4.36,38-41,54-56$VWKHPDMRULW\RIJO\FRVSKLQJROLSLGVDUHV\QWKHVL]HGLQWKH*ROJLEXWDUH ORFDOL]HGLQRWKHUVXEFHOOXODUORFDWLRQVDV\VWHPIRUWKHLUWUDQVSRUWPXVWH[LVW 7KHPDLQV\VWHPIRUWUDQVSRUWRIJO\FRVSKLQJROLSLGVLVYHVLFXODUDORQJWKHH[RF\WLF and endocytic recycling pathways, which is consistent with their synthesis on the luminal VLGHRIWKH*ROJLDQGHQULFKPHQWLQOXPLQDOVXUIDFHRIWKHHQGRF\WLFYHVLFOHVDQGWKHRXWHU VXUIDFHRIWKHSODVPDPHPEUDQHDVGHVFULEHGLQGHWDLOHOVHZKHUH33,57-59 It is interesting WKDW GHVSLWH WKH IDFW WKDW YHVLFXODU WUDQVSRUW LV ELGLUHFWLRQDO JO\FRVSKLQJROLSLGV DUH SUHIHUHQWLDOO\PDLQWDLQHGLQFHUWDLQVXEFHOOXODUFRPSDUWPHQWVRYHURWKHUV'DWDLQWKH OLWHUDWXUHVXJJHVWWKDWLWLVDKLJKO\UHJXODWHGSURFHVVDQGLVFHOOW\SHVSHFL¿F33,57-59 Lipid UDIWVDQGFDYHROLQDUHWKRXJKWWRSOD\DQLPSRUWDQWUROHLQWKHSUHIHUHQWLDOVRUWLQJRI glycosphingolipids.58,60-62 In addition to vesicular transport, there is evidence to suggest WKDWJOXFRV\OFHUDPLGHWUDQVSRUWWRWKHSODVPDPHPEUDQHRFFXUVLQGHSHQGHQWO\RIWKH *ROJLSDWKZD\DQGDJO\FRVSKLQJROLSLGWUDQVSRUWSURWHLQKDVEHHQLGHQWL¿HG63,64 FUNCTIONS OF GLYCOSPHINGOLIPIDS *O\FRVSKLQJROLSLGV UHJXODWH VHYHUDO GLIIHUHQW FHOOXODU SURFHVVHV LQFOXGLQJ FHOO SUROLIHUDWLRQFHOOVLJQDOLQJDSRSWRVLVDQGFHOOUHFRJQLWLRQ2QO\DQRYHUYLHZRIWKH IXQFWLRQRIJO\FRVSKLQJROLSLGVLVJLYHQEHORZDQGLVQRWWKHSXUSRVHRIWKLVFKDSWHU

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polycystin-1 and -2 and has been mapped to human chromosome 6.995HFHVVLYHIRUPVRI WKHGLVHDVHUHVXOWLQFKLOGKRRGRQVHWDQGHDUO\HQGVWDJHUHQDOIDLOXUH(65) 140 Patients QRUPDOO\SUHVHQWZLWKHQODUJHGNLGQH\VGXHWRWKHSUHVHQFHRIF\VWVLQWKHFROOHFWLQJGXFW RIWKHQHSKURQ&\VWVJURZLQVL]HDQGQXPEHUZLWKLQFUHDVLQJDJH$OWKRXJKWKHUHDUH PDQ\IRUPVRI3.'PRVWDUHFDXVHGE\XQFRQWUROOHGWXEXODUHSLWKHOLDOFHOOSUROLIHUDWLRQ DOWHUHGUHJXODWLRQRIDSRSWRVLVDQGWKHDFWLYDWLRQRIJURZWKIDFWRUV141 Glycosphingolipids such as glucosylceramide (GluCer), lactosylceramide (LacCer) and GM3 have shown to regulate these processes. 7KHUROHRIJO\FRVSKLQJROLSLGPHWDEROLVPLQ3.'KDVEHHQVWXGLHGLQVHYHUDOPRXVH PRGHOVRI3.'7KHUHQDOOHYHOVRI*0DQG*OX&HUZHUHIRXQGWREHKLJKHULQ3.'PRXVH models than in the corresponding control mice, suggesting that these glycosphingolipids might play a role in PKD. Renal glycosphingolipids are elevated in both the recessive DQGGRPLQDQWIRUPVRI3.'KDYHDFFXPXODWLRQVRIUHQDOJO\FRVSKLQJROLSLGV7KH3.' PRXVHPRGHOVMFN3NGF.2DQGSF\DOOVKRZHOHYDWHGOHYHOVRI*0*OX&HUZLWK VOLJKWO\LQFUHDVHGEXWQRWVWDWLVWLFDOO\VLJQL¿FDQWLQFUHDVHVLQFHUDPLGH7KHVHVSKLQJROLSLG SUR¿OHFKDQJHVDUHFRQVLVWHQWZLWKWKRVHSUHVHQWLQKXPDQ3.'NLGQH\V141 7KHLQKLELWLRQRIJO\FRVSKLQJROLSLGV\QWKHVLVDQGWKHVXEVHTXHQWUHGXFWLRQLQWKHLU FRQFHQWUDWLRQVKDVVKRZQSURPLVHIRUWUDQVODWLQJWRQHZWUHDWPHQWVIRU3.'7KH*OX&HU inhibitor Genz-123346 inhibits the glucosylceramide synthase and depletes GluCer without inducing accumulations in ceramide.141 Studies have shown that administration RI*HQ]WRPRXVHPRGHOVRI3.'GHFUHDVHGUHQDOOHYHOVRI*OX&HUDQG*0141 ,PSRUWDQWO\*HQ]QRWRQO\UHGXFHGF\VWYROXPHDQG¿EURVLVEXWDOVRGHOD\HG WKHSURJUHVVLRQRQVHWRI3.'LQPRXVHPRGHOV141 7KHUROHRIJO\FRVSKLQJROLSLGVLQWKHUHJXODWLRQRIFHOOF\FOHDVZHOODVDFWLYDWLRQRI JURZWKIDFWRUVLVWKRXJKWWREHPHFKDQLVPVE\ZKLFKWKH\PHGLDWH3.'GHYHORSPHQW The improvement in PKD with Genz-123346 is thought to be due to decreased cell SUROLIHUDWLRQWKURXJKUHJXODWLRQRIFHOOF\FOHSURJUHVVLRQDVLWVXVHRQNLGQH\FHOOVJURZQ LQFXOWXUHGHOD\HGSURJUHVVLRQRIWKHFHOOF\FOHDW*01417KHDFWLYDWLRQRIJURZWK IDFWRUV SDUWLFXODUO\ HSLGHUPDO JURZWK IDFWRUV (*)  WKURXJK WKH SKRVSKRU\ODWLRQ RI PLWRJHQDFWLYDWHGSURWHLQNLQDVHV0$3. KDVEHHQOLQNHGWRWKHIRUPDWLRQRIF\VWV DORQJ ZLWK LQFUHDVHG LQÀDPPDWLRQ GXH WR WKH XSUHJXODWHG DFWLYLW\ RI F\FOLF$0399 Importantly, in vitro lactosylceramide addition to human kidney proximal tubule cells LQGXFHV SKRVSKRU\ODWLRQ RI 0$3.99 7KH UHFHQW GHYHORSPHQW RI QRYHO LQKLELWRUV RI glucosylceramide synthase that are well tolerated in mice as well as in humans shows PXFKSURPLVHIRUWUHDWLQJ3.'LQKXPDQV KIDNEY CANCER 6HYHUDO W\SHV RI JO\FRVSKLQJROLSLGV DUH HOHYDWHGLQ D YDULHW\ RIWXPRUV,QUHQDO cancer, glycosphingolipids reported to be elevated include GM1, GM2, GD2, GD3 and NeuGc-GM3.142-1452WKHUJO\FRVSKLQJROLSLGVKDYHEHHQIRXQGDWPXFKORZHUOHYHOVLQ kidney cancers. For example, the glycosphingolipid disialylgalactosylgloboside and WKH VLDO\OWUDQVIHUDVH 67*DO1$F 9, UHVSRQVLEOH IRU LWV V\QWKHVLV ZHUH IRXQG WR EH downregulated in renal cancer cell lines and cancer tissues.146 Indeed, human renal cell FDUFLQRPDV5&& DUHFKDUDFWHUL]HGE\VLJQL¿FDQWFKDQJHVLQJDQJOLRVLGHFRPSRVLWLRQ ZKLFKLVVXSSRUWHGE\GRFXPHQWHGFKDQJHVLQH[SUHVVLRQRIHQ]\PHVLQYROYHGLQWKHLU metabolism in RCC.87,147,148 )RU H[DPSOH WKH VLDOLGDVH 1(8 VSHFL¿FDOO\ GHJUDGHV

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gangliosides and is markedly upregulated in many renal carcinomas.87 In addition, the glycosphingolipid GalNAc disialosyl lactotetraosylceramide is abundant in metastatic renal cell carcinomas in humans.149 Thus, altered glycosphingolipid metabolism may be NH\WRWKHJURZWKDQGPHWDVWDVLVRIUHQDOWXPRUV 9DULRXVJO\FRVSKLQJROLSLGVKDYHEHHQLPSOLFDWHGLQFHOOXODUSUROLIHUDWLRQDVGLVFXVVHG above and this may be one mechanism by which they play a role in renal cancers. In DGGLWLRQ WR UHJXODWLQJ FHOO SUROLIHUDWLRQ FHUWDLQ JDQJOLRVLGHV KDYH VKRZQ WR LQKLELW the immune response mounted by the body against tumor cells. Gangliosides such as *0*'DQG*'DFFXPXODWHLQUHQDOWXPRUVDQGDUHVKHGIURPWKHVHWXPRUVZKHUH they are taken up by activated T cells, inducing their apoptosis and causing immune G\VIXQFWLRQ142,144,150 Understanding how glycosphingolipid metabolism is altered in renal FDQFHUVDQGWKHPHFKDQLVPE\ZKLFKJO\FRVSKLQJROLSLGVFRQWULEXWHWRWKHIRUPDWLRQDQG PHWDVWDVLVRIUHQDOWXPRUVZLOODOORZWKLVSDWKZD\WREHWDUJHWHGIRUWKHGHYHORSPHQWRI QRYHOWKHUDSHXWLFV,QGHHGLQKLELWRUVRIJO\FRVSKLQJROLSLGV\QWKHVLVDUHZHOOWROHUDWHG LQURGHQWVDVZHOODVKXPDQVDQGFRXOGSRWHQWLDOO\EHXVHIXOLQWKHWUHDWPHQWRIUHQDO cell carcinoma. GLOMERULONEPHRITIS Glomerulonephritis encompasses several disorders grouped according to varying V\PSWRPVVXFKDVSURWHLQXULDUHQDOK\SHUWURSK\DQGLQÀDPPDWLRQZLWKVRPHYDULDWLRQV culminating in end-stage renal disease.151,152 Although no single mechanism has been DFFHSWHGUHFHQWVWXGLHVLQGLFDWHWKDWWKHORVVRIDQHJDWLYHFKDUJHRQSURWHLQVJO\FROLSLGV DQGFDUERK\GUDWHVLQWKHSRGRF\WHVLVDWOHDVWLQSDUWUHVSRQVLEOHIRUWKHGHFUHDVHG¿OWUDWLRQ RIWKHJORPHUXODUFDSLOODULHV153-1556LDOLFDFLGLVORFDWHGRQWKHRXWVLGHRIWKHSRGRF\WHDQG LWLVUHVSRQVLEOHIRUWKH¿OWUDWLRQGXHWRWKHFKDUJHUHSXOVLRQRIDGMDFHQWQHJDWLYHO\FKDUJHG VLDOLFDFLGV7KLVUHSXOVLRQLVDWOHDVWLQSDUWUHVSRQVLEOHIRUWKHFUHDWLRQRI¿OWUDWLRQJDSV151 7KHORVVRIVLDOLFDFLGRQWKHVXUIDFHRIWKHSRGRF\WHVKDVEHHQOLQNHGWRSURWHLQXULDDPDMRU LQGLFDWRURIKXPDQJORPHUXODUGLVHDVH151 Sialic acid residues are cleaved by sialidases (neuraminidases) that are present in the plasma membrane, cytosol, mitochondria and lysosomes.155,QGHHGUHPRYDORIQHJDWLYHO\FKDUJHGVLDOLFDFLGUHVLGXHVIURPWKHDSLFDO SODVPDPHPEUDQHRISRGRF\WHVYLDLQYLYRWUHDWPHQWZLWKEDFWHULDOVLDOLGDVHLVVXI¿FLHQW WRLQGXFHIRRWSURFHVVHIIDFHPHQWDQGSURWHLQXULD156-159,PSRUWDQWO\WKHNLGQH\VRIPLFH FDUU\LQJDPXWDWHGIRUPRIDNH\HQ]\PHLQYROYHGLQWKHSURGXFWLRQRIVLDOLFDFLGKDYH SRGRF\WHHIIDFHPHQWDQGVSOLWWLQJRIWKHJORPHUXODUEDVHPHQWPHPEUDQHUHVXOWLQJLQVHYHUH proteinuria that is partially rescued with dietary supplementation,160,161IXUWKHULOOXVWUDWLQJ WKHLPSRUWDQFHRIVLDOLFDFLGUHVLGXHVLQSRGRF\WHIXQFWLRQ Sialidases do indeed desialylate the gangliosides GM3, GM2 and GM1.155 The plasma membrane sialidase NEU3, acts exclusively on gangliosides and in renal cancers has been shown to be upregulated.1627KLVXSUHJXODWLRQOHDGVWRDQDFFXPXODWLRQRIODFWRV\OFHUDPLGH ZLWKDFRQFRPLWDQWGHFUHDVHLQ*0LQÀXHQFLQJWKHUDWLREHWZHHQQHJDWLYHO\FKDUJHG DQGQHXWUDOJO\FRVSKLQJROLSLGVZKLFKDOWHUVWKHEDODQFHEHWZHHQFHOOSUROLIHUDWLRQDQGFHOO death.162/LNHZLVHLQSXURP\FLQLQGXFHGPRXVHPRGHOVRIPLQLPDOFKDQJHQHSKURSDWK\ DGLVDSSHDUDQFHRI*'ZDVQRWHGDWGD\163 Around day 30, the gangliosides were PHDVXUHGDJDLQDQGIRXQGWREHFORVHWRWKRVHRIWKHFRQWUROPLFHZLWKGLVHDVHV\PSWRPV subsiding.1637KHVHGDWDVXJJHVWWKDWWKHORVVRIUHQDOJDQJOLRVLGHVSOD\VDPDMRUUROHLQ minimal change nephropathy.163

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synthase inhibitors improved glucose tolerance and insulin sensitivity in diet-induced as ZHOODV67=LQGXFHGGLDEHWLFPLFH180,181 Reduced glycosphingolipid levels in cells induces */87WUDQVORFDWLRQWRWKHSODVPDPHPEUDQHYLDHQKDQFHGIRUPDWLRQRI*/87VWRUDJH vesicles.1837KLVUHFHQWGDWDIURPWKH3DJDQRODERUDWRU\SURYLGHVPHFKDQLVWLFLQVLJKWIRU in vivo studies utilizing glucosylceramide synthase inhibitors to improve glycemic control DQGLQVXOLQVHQVLWLYLW\LQURGHQWPRGHOVRIGLDEHWHV183 &KURQLFK\SHUJO\FHPLDDQGLQVXOLQUHVLVWDQFHGUDPDWLFDOO\DOWHUNLGQH\IXQFWLRQDQG lead to diabetic nephropathy. Indeed, complex glycosphingolipids have been implicated in diabetic nephropathy where changes in glomerular sialic acids and/or sialidase activity FRUUHODWHZLWKRQVHWRISURWHLQXULD184-186,QDGGLWLRQVWXGLHVLQGLFDWHDUROHIRUFHUDPLGH LQUHQDOLQMXU\UHVXOWLQJIURPKLJKIDWGLHWLQGXFHGQHSKURSDWK\1871RPHDVXUHPHQWVRI glycosphingolipids were reported in these studies, but given that ceramides are required IRUWKHIRUPDWLRQRIJO\FRVSKLQJROLSLGVLWLVSRVVLEOHWKDWWKH\DOVRDUHDOVRHOHYDWHGDQG play a role.187 A link between glucose metabolism, insulin resistance and glycosphingolipid PHWDEROLVPLVFOHDUIURPQXPHURXVUHSRUWVLQWKHOLWHUDWXUH180,181,183,188-190 suggesting a UROHIRUJO\FRVSKLQJROLSLGVLQGLDEHWLFQHSKURSDWK\DQGNLGQH\GLVHDVHUHVXOWLQJIURP metabolic syndrome. CONCLUSION 'DWDLQWKHOLWHUDWXUHFOHDUO\LQGLFDWHDUROHIRUJO\FRVSKLQJROLSLGVLQDYDULHW\RI kidney diseases, including Fabry’s kidney disease, polycystic disease, kidney cancers, VHYHUDOW\SHVRIJORPHUXORQHSKULWLVDQGGLDEHWLFQHSKURSDWK\'DWDVWURQJO\VXJJHVWD UROHIRUJO\FRVSKLQJROLSLGVLQNLGQH\GLVHDVHUHVXOWLQJIURPPHWDEROLFV\QGURPHDQG IXWXUHVWXGLHVDUHDLPHGDWXQFRYHULQJWKHVHVSHFL¿FUROHV'DWDZLWKQRYHOLQKLELWRUV RIJOXFRV\OFHUDPLGHV\QWKDVHWKDWGHSOHWHNLGQH\JO\FRVSKLQJROLSLGVZLWKRXWHOHYDWLQJ FHUDPLGHVVKRZSURPLVHQRWRQO\IRUWKHWUDQVODWLRQWRWUHDWPHQWRISRO\F\VWLFNLGQH\ disease in humans, but also the other kidney diseases mentioned above. .LGQH\JO\FRVSKLQJROLSLGVHQFRPSDVVDFRPSOH[DQGKLJKO\GLYHUVHIDPLO\RIOLSLGV ZLWK GLYHUVH FHOOXODU UROHV 7KXV WKH LGHQWL¿FDWLRQ RI WKH VSHFL¿F JO\FRVSKLQJROLSLG VSHFLHV LQYROYHG LQ VSHFL¿F NLGQH\ SDWKRORJLHV LV HVVHQWLDO 7KH UHFHQW GHYHORSPHQW RI QRYHO PDVV VSHFWURPHWU\ WHFKQLTXHV WKDW DOORZ IRU WKH TXDQWL¿FDWLRQ RI FRPSOH[ glycosphingolipids will help with this challenging and essential endeavor. Inhibitors RI JOXFRV\OFHUDPLGH V\QWKDVH GHSOHWH D SOHWKRUD RI GLIIHUHQW JO\FRVSKLQJROLSLGV DQG GHYHORSPHQWRIDGGLWLRQDOLQKLELWRUVWKDWWDUJHWVSHFL¿FSRLQWVLQWKHJO\FRVSKLQJROLSLG metabolic pathway is essential. In addition, deciphering the mechanism by which the DFFXPXODWLRQRIVSHFL¿FJO\FRVSKLQJROLSLGVSHFLHVOHDGVWRVSHFL¿FNLGQH\SDWKRORJLHV ZLOODOORZIRUWKHLGHQWL¿FDWLRQRIQRYHOWKHUDSHXWLFWDUJHWVIRUWKHGHYHORSPHQWRIGUXJV DLPHGDWLQWHUIHULQJZLWKWKHLUDFWLRQVLQDGGLWLRQWRWKHLUPHWDEROLVP ACKNOWLEDGEMENTS /-6JUDWHIXOO\DFNQRZOHGJHVUHVHDUFKVXSSRUWIURPD9$&'$$ZDUGSLORWSURMHFW IXQGLQJIURPWKH9$5($3D$&6,5*,5*VXEDZDUGDQGSLORWSURMHFW IXQGLQJIURPWKH1,+1&55355&2%5(LQ/LSLGRPLFVDQG3DWKRELRORJ\

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CHAPTER 9 SPHINGOLIPID SYNTHETIC PATHWAYS ARE MAJOR REGULATORS OF LIPID HOMEOSTASIS Tilla S. Worgall Department of Pathology, Columbia University, New York, New York, USA Email: [email protected]

Abstract:

7KLVFKDSWHUIRFXVHVRQWKHUROHRIVSKLQJROLSLGVLQWKHUHJXODWLRQRIVWHUROUHJXODWRU\ element binding protein (SREBP) dependent lipid synthesis and ATP-binding cassette SURWHLQ$%&$DQG$%&*PHGLDWHGOLSLGHIÀX[NH\UHJXODWRUVRIFHOOXODUOLSLG KRPHRVWDVLV 6SKLQJROLSLG V\QWKHVLV DFWLYDWHV 65(%3V LQGHSHQGHQWO\ RI ZKHWKHU sphingolipid synthesis occurs through recycling or de novo pathways. SREBPs are PDMRUWUDQVFULSWLRQIDFWRUVRIOLSLGPHWDEROLVPWKDWUHJXODWHPRUHWKDQJHQHVRI FKROHVWHUROIDWW\DFLGDQGSKRVSKROLSLGV\QWKHWLFHQ]\PHVDQGWKH\UHTXLUHG1$'3+ FRIDFWRUV65(%3VDUHGRZQVWUHDPRIVSKLQJROLSLGV\QWKHVLVDQGGRQRWUHJXODWHDFWLYLW\ RIVSKLQJROLSLGV\QWKHWLFHQ]\PHV&HOOVWKDWFDQQRWV\QWKHVL]HVSKLQJROLSLGVIDLOWR LQFUHDVH65(%3LQUHVSRQVHWROLSLGGHSOHWLRQ6LPLODUPHFKDQLVPVDUHIRXQGLQD. melanogasterLQZKLFK65(%3DFWLYLW\GHSHQGVRQH[SUHVVLRQRIDFHUDPLGHV\QWKDVH DQDORJ65(%3LVLQKLELWHGE\LWVHQGSURGXFWVFKROHVWHURODQGXQVDWXUDWHGIDWW\DFLGV Ceramide decreases SREBP by inhibiting sphingolipid synthesis. Molecular mechanisms RIUHJXODWLRQDUHUHODWHGWRWKHHIIHFWRIVSKLQJROLSLGVRQLQWUDFHOOXODUWUDI¿FNLQJEXW DUHRYHUDOOQRWFOHDU6HYHUDOJURXSVKDYHLQYHVWLJDWHGWKHHIIHFWRIVSKLQJROLSLGVLQ WKHUHJXODWLRQRIFKROHVWHUROHIÀX[UHFHSWRUV$%&$DQG$%&*PDMRUUHJXODWRUVRI plasma high-density lipoprotein (HDL) concentration, an important anti-atherogenic lipoprotein. Data indicate an inverse relationship between sphingolipid de novo synthesis DQGFKROHVWHUROHIÀX[,QKLELWLRQRIVSKLQJROLSLGGHQRYRV\QWKHVLVLQFUHDVHV$%&$ PHGLDWHG FKROHVWHURO HIÀX[ LQGHSHQGHQW RI VSKLQJRP\HOLQ 3RWHQWLDO PHFKDQLVPV LQFOXGHWKHSK\VLFDOLQWHUDFWLRQRIVXEXQLWRIVHULQHSDOPLWR\OWUDQVIHUDVH637  WKHUDWHOLPLWLQJHQ]\PHRIGHQRYRVSKLQJROLSLGV\QWKHVLVZLWK$%&$$%&* PHGLDWHGHIÀX[LQFRQWUDVWLVGHSHQGHQWRQVSKLQJRP\HOLQPDVV$QLPDOVWXGLHV VXSSRUWWKH¿QGLQJVPDGHLQFXOWXUHGFHOOV,QKLELWLRQRIVSKLQJROLSLGGHQRYRV\QWKHVLV increases anti-atherogenic lipoproteins and decreases atherosclerosis in mouse models. 7RJHWKHUPDQLSXODWLRQRIVSKLQJROLSLGV\QWKHWLFSDWKZD\VLVDSRWHQWLDOO\SURPLVLQJ WKHUDSHXWLFWDUJHWIRUWUHDWPHQWRIORZ+'/G\VOLSLGHPLDDQGDWKHURVFOHURVLV

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INTRODUCTION 6SKLQJROLSLGVDUHNH\FRPSRQHQWVRIFHOOXODUDQGVXEFHOOXODUHXNDU\RWLFPHPEUDQHV DQGUHJXODWHHVVHQWLDOFHOOIXQFWLRQVWKDWLQFOXGHJURZWKDSRSWRVLVDQGLPPXQHIXQFWLRQ1,2 7KLVFKDSWHUIRFXVHVRQWKHUROHRIVSKLQJROLSLGVLQWKHUHJXODWLRQRIVWHUROUHJXODWRU\ element binding protein (SREBP) dependent lipid synthesis and ATP-binding cassette WUDQVSRUWHUV$%&$DQG$%&*PHGLDWHGFKROHVWHUROHIÀX[LPSRUWDQWFRQWULEXWRUV RIFHOOXODUOLSLGKRPHRVWDVLV SREBPs ARE KEY TRANSCRIPTION FACTORS OF PROTEIN THAT SYNTHESIZE CHOLESTEROL, FATTY ACIDS AND PHOSPHOLIPIDS 7KH 65(%3V DUH WUDQVFULSWLRQ IDFWRUV WKDW ZHUH GLVFRYHUHG LQ 3,4 SREBPs GLUHFWO\ DFWLYDWH WKH H[SUHVVLRQ RI PRUH WKDQ WKLUW\ JHQHV GHGLFDWHG WR WKH V\QWKHVLV DQG XSWDNH RI FKROHVWHURO IDWW\ DFLGV WULJO\FHULGH DQG SKRVSKROLSLGV DV ZHOO DV WKH 1$'3+FRIDFWRUUHTXLUHGWRV\QWKHVL]HWKHVHPROHFXOHV57KUHH65(%3LVRIRUPVDUH IRXQGLQPDPPDOLDQFHOOVRQH65(%3LVRIRUPLVIRXQGLQGURVRSKLOD5-75HJXODWLRQRI 65(%3VRFFXUVWUDQVFULSWLRQDOO\DQGSRVWWUDQVFULSWLRQDOO\,QVXOLQPHGLDWHGLQFUHDVHRI WKHOLYHU;UHFHSWRU/;5 IDFWRULVWKHPDMRUWUDQVFULSWLRQDODFWLYDWRURI65(%38 The SRVWWUDQVFULSWLRQDODFWLYDWLRQRI65(%3LVPRUHFRPSOH[DQGFDQRFFXUDWGLIIHUHQWOHYHOV )LJ 7KHLQDFWLYHSUHFXUVRUIRUPRI65(%3S65(%3 LVORFDWHGLQWKHHQGRSODVPLF reticulum where it is bound to a sterol-sensing protein, SCAP. Cholesterol promotes the DVVRFLDWLRQRIWKH,QVLJSURWHLQWRWKH65(%36&$3FRPSOH[7KLVDVVRFLDWLRQDQFKRUV SREBP in the endoplasmic reticulum and inhibits SRE-mediated gene transcription. :KHQFHOOVDUHOLSLGGHSOHWHG65(%3DQG6&$3DUHWUDI¿FNHGE\YHVLFXODUWUDQVSRUW to the Golgi apparatus. SCAP is recycled to the endoplasmic reticulum. In the Golgi, pSREBP undergoes proteolysis by two distinct enzymes. These steps generate mature 65(%3P65(%3 EDVLFKHOL[ORRSKHOL[OHXFLQH]LSSHUE+/+ WUDQVFULSWLRQIDFWRUV9 P65(%3DFWLYDWHGJHQHVLQFOXGHWKHUDWHOLPLWLQJHQ]\PHVRIIDWW\DFLGV\QWKHVLVDV well as 6GHVDWXUDVHWKDWJHQHUDWHVORQJFKDLQXQVDWXUDWHGIDWW\DFLGVIURPHVVHQWLDO IDWW\DFLGVDQGIDWW\DFLG&R$OLJDVHDQHQ]\PHWKDWHQDEOHVWKHLQFRUSRUDWLRQRIIDWW\ acids into triglycerides, phospholipids and ceramides. mSREBP-2 activates the LDL UHFHSWRUJHQHDQGHVVHQWLDOO\DOOJHQHVUHTXLUHGIRUFKROHVWHUROV\QWKHVLV6 Sphingolipid synthetic enzymes are not regulated by SREBPs. SPHINGOLIPIDS SYNTHESIS IS REQUIRED TO INCREASE SREBP IN LIPID DEPLETION SREBPs are activated by lipid depletion. This observation had led to the discovery RI WKLV FODVV RI WUDQVFULSWLRQ IDFWRUV3,4 Consecutive studies in mammalian cells and GURVRSKLOD GHPRQVWUDWHG WKDW VSKLQJROLSLG V\QWKHVLV LV UHTXLUHG IRU WKH DFWLYDWLRQ RI SREBP.7,10-126\QWKHVLVRIVSKLQJROLSLGVRFFXUVWKURXJKUHF\FOLQJDQGGHQRYRSDWKZD\V The de-novo pathway is increased when the recycling pathway cannot supply enough VSKLQJRLG EDVHV /RQJ FKDLQ VSKLQJRLG EDVHV DUH GHULYHG IURP VSKLQJRP\HOLQ RU glucosylceramides. Sphingolipid de-novo synthesis is increased in rapidly dividing cells and in the epidermis. The initial step in de-novo sphingolipid synthesis is the condensation

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Figure 1. 6SKLQJROLSLG V\QWKHVLV DFWLYDWHV 65(%3 WUDQVFULSWLRQ IDFWRUV 6WHUROUHJXODWRU\HOHPHQW ELQGLQJSURWHLQV65(%3V DUHPHPEUDQHSURWHLQVRIWKHHQGRSODVPLFUHWLFXOXP7KHLQDFWLYHSUHFXUVRU SREBP (pSREBP) resides in the endoplasmic reticulum. When cells are lipid depleted pSREBP translocates to the Golgi apparatus. In the Golgi, two distinct proteases cleave pSREBP and release WKH WUDQVFULSWLRQDO DFWLYH PDWXUH 65(%3 P65(%3  65(%3V UHJXODWH D WRWDO RI WKLUW\WKUHH JHQHV RI FKROHVWHURO WULJO\FHULGH IDWW\ DFLG DQG SKRVSKROLSLG PHWDEROLVP 6SKLQJROLSLG V\QWKHVLV LV QHFHVVDU\ WR DFWLYDWH 65(%3 &HOOV WKDW DUH GHIHFWLYH LQ VSKLQJROLSLG V\QWKHVLV IDLO WR LQFUHDVH 65(%3 LQ OLSLG GHSOHWLRQ 8QVDWXUDWHG IDWW\ DFLGV FKROHVWHURO DQG FHUDPLGH LQKLELW 65(%3 &HUDPLGH LQKLELWV 65(%3 by inhibiting sphingolipid synthesis.

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We showed that sphingolipid synthesis is necessary to activate SREBP.10 These studies were carried out in Chinese-hamster ovary (CHO) cells and in LY-B cells. LY-B cells are CHO cells in which the SPTLC1 subunit is mutated. LY-B cells are unable to SURGXFH VSKLQJROLSLGV GHQRYR DQG GHSHQG RQ H[RJHQRXV VSKLQJROLSLGV IRU VXUYLYDO When LY-B cells are incubated in lipid depleted medium, SREBP is not increased and cells die. Cells increase SREBP and SRE-mediated gene expression when sphingosine RU VSKLQJDQLQH VXEVWUDWHV RI VSKLQJROLSLG V\QWKHVLV DUH VXSSOLHG LQ WKH LQFXEDWLRQ PHGLXP6SKLQJROLSLGPHGLDWHGUHJXODWLRQRI65(%3RFFXUVLQGHSHQGHQWO\RIFHOOXODU FKROHVWHURO FRQFHQWUDWLRQ 7KLV PHFKDQLVP RI UHJXODWLRQ LV DOVR REVHUYHG DOEHLW WR D smaller degree in wild type CHO cells. Sphingosine or sphinganine increase SREBP and SRE-mediated gene transcription in CHO cells. Similar mechanisms that demonstrated a VSKLQJROLSLGV\QWKHVLVGHSHQGHQF\RI65(%3ZHUHUHFHQWO\IRXQGLQD. melanogaster.11 2QH65(%3LVIRXQGLQGURVRSKLOD'URVRSKLODGRQRWSURGXFHVWHUROVEXWLQWKLVPRGHO 65(%3FRQWUROVIDWW\DFLGPHWDEROLVPDQGLVUHJXODWHGE\SKRVSKDWLG\OHWKDQRODPLQH LWVPDMRUSKRVSKROLSLG7,25,26,QGURVRSKLODDFWLYDWLRQRI65(%3GHSHQGVRQIXQFWLRQDO µVFKODQN¶ D FHUDPLGH V\QWKDVH DQDORJXH 0XWDWLRQV LQ µVFKODQN¶ RU VL51$ PHGLDWHG GRZQUHJXODWLRQRIVFKODQNGHFUHDVHVGLK\GUR FHUDPLGHVDQG65(%3'HFUHDVHG65(%3 GHFUHDVHVWULDF\OJO\FHUROV\QWKHVLVDQGPDVVRIVWRUDJHIDW11 A compensatory increase RI VSKLQJROLSLG V\QWKHVLV FRXOG DOVR XQGHUOLH WKH REVHUYDWLRQ WKDW JO\FRVSKLQJROLSLG GH¿FLHQF\FDXVHGLQKLELWLRQRIFHUDPLGHJOXFRV\OWUDQVIHUDVHLQFUHDVHV65(%312 Animal PRGHOVKDYHFRQ¿UPHGWKHUHOHYDQFHRIVSKLQJROLSLGV\QWKHVLVDVDUHJXODWRURIOLSLG metabolism. Two independent groups demonstrated decreased atherosclerotic lesions in apolipoprotein E knock-out mice treated with the SPT inhibitor myriocin.27-31 One JURXSUHODWHGWKHEHQH¿FLDOHIIHFWRIVSKLQJROLSLGLQKLELWLRQWRDUHGXFWLRQLQ65(%3 DFWLYLW\DQGVKRZHGWKDWORZHULQJRIVSKLQJROLSLGVDQGDWKHURJHQLFSODVPDOLSLGVOHG WRUHJUHVVLRQRISUHH[LVWLQJDWKHURVFOHURWLFOHVLRQV29,31)XUWKHUVXSSRUWIRUWKHUROHRI VSKLQJROLSLGVDVUHJXODWRUVRIOLSLGPHWDEROLVPFRPHVIURPVWXGLHVWKDWHYDOXDWHGWDUJHWV RIVSKLQJRVLQHSKRVSKDWHDQHQGSURGXFWRIVSKLQJROLSLGV\QWKHVLVDQGLPSRUWDQWOLSLG messenger. It was shown that sphingosine-1-phosphate increases Cyp-17, an important VWHURLGRJHQLFJHQHRIWKHDGUHQDOFRUWH[LVPHGLDWHGE\DFWLYDWLQJ65(%30HFKDQLVPV by which sphingosine-1-phosphate increases SREBP are currently not known.32 CHOLESTEROL, UNSATURATED FATTY ACIDS AND CERAMIDES INHIBIT SREBP 65(%3LVLQKLELWHGE\FKROHVWHURODQGSRO\XQVDWXUDWHGIDWW\DFLGVHQGSURGXFWVRI SREBP-dependent lipid synthesis and ceramide.3,4,33 Mechanisms by which cholesterol LQKLELWV 65(%3 DUH ZHOO NQRZQ &KROHVWHURO SURPRWHV UHVLGHQFH RI 65(%3 LQ WKH HQGRSODVPLFUHWLFXOXPE\SURPRWLQJWKHLQWHUDFWLRQRI6&$3ZLWK,QVLJ7KLVLQWHUDFWLRQ SUHYHQWVWUDQVORFDWLRQRIS65(%3WRWKH*ROJL0HFKDQLVPVKRZSRO\XQVDWXUDWHGIDWW\ DFLGVLQKLELW65(%3DUHOHVVFOHDU&KDUDFWHULVWLFIRUIDWW\DFLGPHGLDWHGLQKLELWLRQRI 65(%3LVWKDWLQKLELWLRQLQFUHDVHVZLWKFKDLQOHQJWKDQGGHJUHHRIXQVDWXUDWLRQ1RWDEO\ XQVDWXUDWHGIDWW\DFLGPHGLDWHGLQKLELWLRQRI65(%3LVFKROHVWHUROLQGHSHQGHQWDVLWSHUVLVWV in sterol depletion.332QHPHFKDQLVPE\ZKLFKIDWW\DFLGVGHFUHDVH65(%3LVOLQNHGWR WKHLUDELOLW\WRDIIHFWWKHELRSK\VLFDOLQWHUDFWLRQEHWZHHQFKROHVWHURODQGVSKLQJRP\HOLQ LQPHPEUDQHV8QVDWXUDWHGIDWW\DFLGVPRELOL]HWUDQVIHURIFKROHVWHURODQGVSKLQJRP\HOLQ between model membranes.34,35,QKLELWLRQRI65(%3FRXOGEHDUHVXOWRIIDWW\DFLGPHGLDWHG

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GLVSODFHPHQWRIFKROHVWHUROWRLQWUDFHOOXODUSRROV$QRWKHUPHFKDQLVPLVOLQNHGWRWKH DELOLW\RIXQVDWXUDWHGIDWW\DFLGVWRLQFUHDVHVSKLQJRP\HOLQDVHDFWLYLW\6SKLQJRP\HOLQDVH GHFUHDVHV VSKLQJRP\HOLQ PDVV DQG IRUFHV WKH DVVRFLDWHG FKROHVWHURO WR WUDQVORFDWH WR extracellular acceptors or to translocate to intracellular sites. Increased intracellular cholesterol, in turn, inhibits SREBP.34,36,37 Another, cholesterol-independent mechanism RIUHJXODWLRQRFFXUVWKRXJKWKHJHQHUDWLRQRIFHUDPLGHDSURGXFWRIVSKLQJRP\HOLQDVH We showed that ceramides inhibit SREBP, independent whether ceramides are generated HQGRJHQRXVO\RUH[RJHQRXVO\7KHHIIHFWRIFHUDPLGHVLVDGGLWLYHWRSRO\XQVDWXUDWHGIDWW\ DFLGVDQGRFFXUVDOVRLQOLSLGGHSOHWLRQPHGLDWHGE\LQFXEDWLRQLQOLSLGIUHHPHGLXPRU FDWLRQLFDPSKLSKLOHVWKDWVHTXHVWHUFKROHVWHURO&HUDPLGHVLQFUHDVHOHYHOVRIS65(%3DQG GHFUHDVHOHYHOVRIP65(%37KLV¿QGLQJLQGLFDWHVWKDWFHUDPLGHVLQKLELWWUDQVORFDWLRQ RIS65(%3IURPWKHHQGRSODVPLFUHWLFXOXPWRWKH*ROJL10 More recent studies indicate WKDW WKH FHUDPLGH PHGLDWHG LQKLELWLRQ RI 65(%3 LV PRUH VLJQL¿FDQW WKDQ FKROHVWHURO WUDQVORFDWLRQPHGLDWHGSDWKZD\V([SHULPHQWVLQFXOWXUHGFHOOVFRPSDUHGWKHHIIHFWRI sphingomyelinase C and sphingomyelinase D on cholesterol synthesis. Both treatments GHFUHDVH VSKLQJRP\HOLQ PDVV WKH GLIIHUHQFH LV WKDW VSKLQJRP\HOLQDVH & JHQHUDWHV ceramides and phosphocholine while sphingomyelinase D generates ceramide phosphate. Results demonstrated that sphingomyelinase C decreased HMG-CoA reductase by 90% in comparison to sphingomyelinase D, which decreased HMG-CoA reductase by 10%.38 ([WUDSRODWLRQ RI WKHVH UHVXOWV LQGLFDWHV WKDW VSKLQJRP\HOLQDVHPHGLDWHG LQKLELWLRQ RI 65(%3LVPDLQO\PHGLDWHGE\JHQHUDWLRQRIFHUDPLGHV2XUVWXGLHVGHPRQVWUDWHGWKDW ceramide decreased SREBP by inhibiting sphingolipid synthesis.10 Together, the data are consistent with a model in which sphingolipid synthesis and sphingolipids coordinate the WUDQVFULSWLRQDOFRQWURORIOLSLGVWKDWPDLQWDLQPHPEUDQHFRPSRVLWLRQ ABCA1 AND ABCG1 ARE KEY LIPID EFFLUX RECEPTORS THAT DETERMINE ANTI-ATHEROGENIC HIGH-DENSITY LIPOPROTEIN (HDL) LEVELS Extensive epidemiological data have shown an inverse relationship between high density lipoprotein (HDL) levels and cardiovascular disease.39,40 Notably, plasma HDL OHYHOVUHPDLQDQLQGHSHQGHQWSUHGLFWRUIRUVXIIHULQJFDUGLRYDVFXODUHYHQWVHYHQLQSDWLHQWV with low low-density lipoprotein (LDL) levels.41 Plasma HDL levels are determined by FKROHVWHUROHIÀX[6WXGLHVIURPVHYHUDOODERUDWRULHVKDYHGHPRQVWUDWHGDPDMRUUROHIRU WKH$73ELQGLQJFDVVHWWHUHFHSWRUV$$%&$ DQG$%&*DVUHJXODWRUVRIFKROHVWHURO HIÀX[DQGSODVPD+'/OHYHOV$%&$WKHPXWDQWPROHFXOHLQ7DQJLHUGLVHDVHSURPRWHV QHWFKROHVWHUROHIÀX[WROLSLGSRRU$SR$ZKLOH$%&*IDFLOLWDWHVQHWFKROHVWHUROHIÀX[ to HDL particles.42-45 SPHINGOLIPIDS AFFECT ABCA1 AND ABCG1 BY DIFFERENT MECHANISMS 6HYHUDO JURXSV KDYH LQYHVWLJDWHG WKH HIIHFW RI VSKLQJROLSLGV LQ WKH UHJXODWLRQ RI FKROHVWHUROHIÀX[UHFHSWRUV$%&$DQG$%&*)LJ 6SKLQJROLSLGVDIIHFWFKROHVWHURO HIÀX[ WKURXJK PHFKDQLVPV UHODWHG WR VSKLQJROLSLG GHQRYR V\QWKHVLV DQG UHODWHG WR VSKLQJRP\HOLQ PDVV LQ WKH SODVPD PHPEUDQH $%&$PHGLDWHG FKROHVWHURO HIÀX[

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CONCLUSION 6SKLQJROLSLGV UHJXODWH OLSLG KRPHRVWDVLV E\ DIIHFWLQJ 65(%3 GHSHQGHQW OLSLG V\QWKHVLVDQG$%&$DQG$%&*GHSHQGHQWFKROHVWHUROHIÀX[PDMRUUHJXODWRUVRI plasma high-density lipoprotein (HDL) concentration, an important anti-atherogenic OLSRSURWHLQ6SKLQJROLSLGV\QWKHVLVLVDQREOLJDWHDFWLYDWRURI65(%3VNH\WUDQVFULSWLRQ IDFWRUVRIFKROHVWHUROIDWW\DFLGDQGSKRVSKROLSLGV\QWKHVLV,QKLELWLRQRIVSKLQJROLSLG V\QWKHVLV GHFUHDVHV 65(%3 RQ WKH SRVW WUDQVFULSWLRQDO OHYHO 7KH SUHFXUVRU IRUP RI SREBP is not processed to the transcriptional active mSREBP. Sphingolipid de-novo V\QWKHVLVLVLQYHUVHO\UHODWHGWRFKROHVWHUROHIÀX[E\$%&$DQG$%&*0HFKDQLVPV IRUGHFUHDVHG$%&$PHGLDWHGFKROHVWHUROHIÀX[LQFOXGHWKHSK\VLFDOLQWHUDFWLRQRI WKH637VXEXQLW637/&ZLWK$%&$$%&*PHGLDWHGFKROHVWHUROHIÀX[GHSHQGV RQVSKLQJRP\HOLQ&KROHVWHUROHIÀX[E\$%&*DQG$%&$LVUHVWRUHGLQFRQGLWLRQV WKDWLQKLELWVSKLQJROLSLGGHQRYRV\QWKHVLV$QRWKHUDVSHFWRIWKHHIIHFWRIVSKLQJROLSLG synthesis on cardiovascular disease is that sphingomyelin increases pro-atherogenic lipoprotein patterns and susceptibility to particle aggregation. Animal studies support the ¿QGLQJVPDGHLQFXOWXUHGFHOOV,QKLELWLRQRIVSKLQJROLSLGGHQRYRV\QWKHVLVLQFUHDVHV anti-atherogenic lipoproteins and decreases atherosclerosis in mouse models. Together, PDQLSXODWLRQRIVSKLQJROLSLGV\QWKHWLFSDWKZD\VLVDSRWHQWLDOO\SURPLVLQJWKHUDSHXWLF WDUJHWIRUWUHDWPHQWRIORZ+'/G\VOLSLGHPLDDQGDWKHURVFOHURVLV ACKNOWLEDGEMENT T.S. Worgall is supported by NIH Grant NIA KO8 AG025833 and Cystic Fibrosis Pilot and Feasibility Award 07010. REFERENCES )XWHUPDQ$+5LH]PDQ+7KHLQVDQGRXWVRIVSKLQJROLSLGV\QWKHVLV7UHQGV&HOO%LRO +DQQXQ<$2EHLG/07KH&HUDPLGHFHQWULFXQLYHUVHRIOLSLGPHGLDWHGFHOOUHJXODWLRQVWUHVVHQFRXQWHUV RIWKHOLSLGNLQG-%LRO&KHP 3. Hua X, Yokoyama C, Wu J et al. SREBP-2, a second basic-helix-loop-helix-leucine zipper protein that stimulates transcription by binding to a sterol regulatory element. Proc Natl Acad Sci USA 1993; 90:11603-7. :DQJ;6DWR5%URZQ06HWDO65(%3DPHPEUDQHERXQGWUDQVFULSWLRQIDFWRUUHOHDVHGE\VWHUROUHJXODWHG proteolysis. Cell 1994; 77:53-62. +RUWRQ-'*ROGVWHLQ-/%URZQ0665(%3VDFWLYDWRUVRIWKHFRPSOHWHSURJUDPRIFKROHVWHURODQGIDWW\ acid synthesis in the liver. J Clin Invest 2002; 109:1125-31. +RUWRQ-'6KDK1$:DUULQJWRQ-$HWDO&RPELQHGDQDO\VLVRIROLJRQXFOHRWLGHPLFURDUUD\GDWDIURP WUDQVJHQLF DQG NQRFNRXW PLFH LGHQWL¿HV GLUHFW 65(%3 WDUJHW JHQHV 3URF 1DWO $FDG 6FL 86$  100:12027-32. 'REURVRWVND\D,<6HHJPLOOHU$&%URZQ06HWDO5HJXODWLRQRI65(%3SURFHVVLQJDQGPHPEUDQHOLSLG production by phospholipids in Drosophila. Science 2002; 296:879-83. 5HSD -- /LDQJ * 2X - HW DO 5HJXODWLRQ RI PRXVH VWHURO UHJXODWRU\ HOHPHQWELQGLQJ SURWHLQF JHQH (SREBP-1c) by oxysterol receptors, LXRalpha and LXRbeta. Genes Dev 2000; 14:2819-30. 6DNDL-'XQFDQ($5DZVRQ5%HWDO6WHUROUHJXODWHGUHOHDVHRI65(%3IURPFHOOPHPEUDQHVUHTXLUHV two sequential cleavages, one within a transmembrane segment. Cell 1996; 85:1037-46. 10. Worgall TS, Juliano RA, Seo T et al. Ceramide synthesis correlates with the posttranscriptional regulation RIWKHVWHUROUHJXODWRU\HOHPHQWELQGLQJSURWHLQ$UWHULRVFOHU7KURPE9DVF%LRO %DXHU59RHO]PDQQ$%UHLGHQ%HWDO6FKODQNDPHPEHURIWKHFHUDPLGHV\QWKDVHIDPLO\FRQWUROV JURZWKDQGERG\IDWLQ'URVRSKLOD(0%2- 

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,VKLWVXND5+LUDED\DVKL<.RED\DVKL7*O\FRVSKLQJROLSLGGH¿FLHQF\LQFUHDVHVWKHVWHUROUHJXODWRU\ element-mediated gene transcription. Biochem Biophys Res Commun 2009; 378:240-3. *DEOH.6OLIH+%DFLNRYD'HWDO7VFSLVDQDPLQRDFLGSURWHLQDVVRFLDWHGZLWKVHULQHSDOPLWR\OWUDQVIHUDVH DQGUHTXLUHGIRURSWLPDOHQ]\PHDFWLYLW\-%LRO&KHP +RUQHPDQQ75LFKDUG65WWL0)HWDO&ORQLQJDQGLQLWLDOFKDUDFWHUL]DWLRQRIDQHZVXEXQLWIRUPDPPDOLDQ VHULQHSDOPLWR\OWUDQVIHUDVH-%LRO&KHP +DQ**XSWD6'*DEOH.HWDO,GHQWL¿FDWLRQRIVPDOOVXEXQLWVRIPDPPDOLDQVHULQHSDOPLWR\OWUDQVIHUDVH WKDWFRQIHUGLVWLQFWDF\O&R$VXEVWUDWHVSHFL¿FLWLHV3URF1DWO$FDG6FL86$ +RUQHPDQQ73HQQR$5WWL0)HWDO7KH637/&VXEXQLWRIVHULQHSDOPLWR\OWUDQVIHUDVHJHQHUDWHVVKRUW chain sphingoid bases. J Biol Chem 2009; 284:26322-30. 0HUULOO$+-U6XOODUGV0&$OOHJRRG-&HWDO6SKLQJROLSLGRPLFVKLJKWKURXJKSXWVWUXFWXUHVSHFL¿FDQG TXDQWLWDWLYHDQDO\VLVRIVSKLQJROLSLGVE\OLTXLGFKURPDWRJUDSK\WDQGHPPDVVVSHFWURPHWU\0HWKRGV 2005; 36:207-24. 9HQNDWDUDPDQ.5LHEHOLQJ&%RGHQQHF-HWDO8SVWUHDPRIJURZWKDQGGLIIHUHQWLDWLRQIDFWRUXRJ D PDPPDOLDQKRPRORJRIWKH\HDVWORQJHYLW\DVVXUDQFHJHQH/$* UHJXODWHV1VWHDUR\OVSKLQJDQLQH &GLK\GUR FHUDPLGH V\QWKHVLVLQDIXPRQLVLQ%LQGHSHQGHQWPDQQHULQPDPPDOLDQFHOOV-%LRO Chem 2002; 277:35642-9. 19. Pewzner-Jung Y, Ben-Dor S, Futerman AH. When do Lasses (longevity assurance genes) become CerS FHUDPLGHV\QWKDVHV ",QVLJKWVLQWRWKHUHJXODWLRQRIFHUDPLGHV\QWKHVLV-%LRO&KHP +DQDGD..XPDJDL.
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6FKDHIHU(-/DPRQ)DYD62UGRYDV-0HWDO)DFWRUVDVVRFLDWHGZLWKORZDQGHOHYDWHGSODVPDKLJK GHQVLW\ OLSRSURWHLQ FKROHVWHURO DQG DSROLSRSURWHLQ $, OHYHOV LQ WKH )UDPLQJKDP 2IIVSULQJ 6WXG\ - Lipid Res 1994; 35:871-82. 40. Lewington S, Whitlock G, Clarke R et al. Blood cholesterol and vascular mortality by age, sex and blood SUHVVXUHDPHWDDQDO\VLVRILQGLYLGXDOGDWDIURPSURVSHFWLYHVWXGLHVZLWKYDVFXODUGHDWKV Lancet 2007; 370:1829-39. %DUWHU3*RWWR$0/D5RVD-&HWDO+'/FKROHVWHUROYHU\ORZOHYHOVRI/'/FKROHVWHURODQGFDUGLRYDVFXODU events. N Engl J Med 2007; 357:1301-10. 42. Bodzioch M, Orsó E, Klucken J et al. The gene encoding ATP-binding cassette transporter 1 is mutated in Tangier disease. Nat Genet 1999; 22:347-51. %URRNV:LOVRQ$0DUFLO0&OHH60HWDO0XWDWLRQVLQ$%&LQ7DQJLHUGLVHDVHDQGIDPLOLDOKLJKGHQVLW\ OLSRSURWHLQGH¿FLHQF\1DW*HQHW .HQQHG\0$%DUUHUD*&1DNDPXUD.HWDO$%&*KDVDFULWLFDOUROHLQPHGLDWLQJFKROHVWHUROHIÀX[ to HDL and preventing cellular lipid accumulation. Cell Metab 2005; 1:121-31. 45. Wang N, Lan D, Chen W et al. ATP-binding cassette transporters G1 and G4 mediate cellular cholesterol HIÀX[WRKLJKGHQVLW\OLSRSURWHLQV3URF1DWO$FDG6FL86$ *ODURV(1.LP:64XLQQ&0HWDO*O\FRVSKLQJROLSLGDFFXPXODWLRQLQKLELWVFKROHVWHUROHIÀX[YLDWKH ABCA1/apolipoprotein A-I pathway: 1-phenyl-2-decanoylamino-3-morpholino-1-propanol is a novel FKROHVWHUROHIÀX[DFFHOHUDWRU-%LRO&KHP :LWWLQJ650DLRUDQR-1'DYLGVRQ:6&HUDPLGHHQKDQFHVFKROHVWHUROHIÀX[WRDSROLSRSURWHLQ$,E\ LQFUHDVLQJWKHFHOOVXUIDFHSUHVHQFHRI$73ELQGLQJFDVVHWWHWUDQVSRUWHU$-%LRO&KHP *KHULQJ$%'DYLGVRQ:6&HUDPLGHVWUXFWXUDOIHDWXUHVUHTXLUHGWRVWLPXODWH$%&$PHGLDWHGFKROHVWHURO HIÀX[WRDSROLSRSURWHLQ$,-/LSLG5HV 6KD\PDQ-$/HH/$EH$6KX/,QKLELWRUVRIJOXFRV\OFHUDPLGHV\QWKDVH0HWKRGV(Q]\PRO 311:373-87. 7DPHKLUR 1 =KRX 6 2NXKLUD . HW DO 637/& ELQGV $%&$ WR QHJDWLYHO\ UHJXODWH WUDI¿FNLQJ DQG FKROHVWHUROHIÀX[DFWLYLW\RIWKHWUDQVSRUWHU%LRFKHPLVWU\ 6DQR2.RED\DVKL$1DJDR.HWDO6SKLQJRP\HOLQGHSHQGHQFHRIFKROHVWHUROHIÀX[PHGLDWHGE\$%&* J Lipid Res 2007; 48:2377-84. -LDQJ;&3DXOWUH)3HDUVRQ7$HWDO3ODVPDVSKLQJRP\HOLQOHYHODVDULVNIDFWRUIRUFRURQDU\DUWHU\ disease. Arterioscler Thromb Vasc Biol 2000; 20:2614-2618. 1HOVRQ-&-LDQJ;&7DEDV,HWDO3ODVPDVSKLQJRP\HOLQDQGVXEFOLQLFDODWKHURVFOHURVLV¿QGLQJVIURP WKHPXOWLHWKQLFVWXG\RIDWKHURVFOHURVLV$P-(SLGHPLRO 'RQJ - /LX - /RX % HW DO $GHQRYLUXVPHGLDWHG RYHUH[SUHVVLRQ RI VSKLQJRP\HOLQ V\QWKDVHV  DQG  increases the atherogenic potential in mice. J Lipid Res 2006; 47:1307-14. 55. Schissel SL, Tweedie-Hardman J, Rapp JH et al. Rabbit aorta and human atherosclerotic lesions hydrolyze WKHVSKLQJRP\HOLQRIUHWDLQHGORZGHQVLW\OLSRSURWHLQ3URSRVHGUROHIRUDUWHULDOZDOOVSKLQJRP\HOLQDVH LQVXEHQGRWKHOLDOUHWHQWLRQDQGDJJUHJDWLRQRIDWKHURJHQLFOLSRSURWHLQV-&OLQ,QYHVW -HRQJ76FKLVVHO6/7DEDV,HWDO,QFUHDVHGVSKLQJRP\HOLQFRQWHQWRISODVPDOLSRSURWHLQVLQDSROLSRSURWHLQ ( NQRFNRXW PLFH UHÀHFWV FRPELQHG SURGXFWLRQ DQG FDWDEROLF GHIHFWV DQG HQKDQFHV UHDFWLYLW\ ZLWK mammalian sphingomyelinase. J Clin Invest 1998; 101:905-12. $ULPRWR,6DLWR+.DZDVKLPD<HWDO(IIHFWVRIVSKLQJRP\HOLQDQGFKROHVWHURORQOLSRSURWHLQOLSDVHPHGLDWHG lipolysis in lipid emulsions. J Lipid Res 1998; 39:143-51. %ROLQ'--RQDV$6SKLQJRP\HOLQLQKLELWVWKHOHFLWKLQFKROHVWHURODF\OWUDQVIHUDVHUHDFWLRQZLWKUHFRQVWLWXWHG high density lipoproteins by decreasing enzyme binding. J Biol Chem 1996; 271:19152-8. 59. Lee CY, Lesimple A, Denis M et al. Increased sphingomyelin content impairs HDL biogenesis and maturation in human Niemann-Pick disease type B. J Lipid Res 2006; 47:622-32. 6FKXFKPDQQ(+'HVQLFN5-1LHPDQQ3LFNGLVHDVHV7\SH$DQG%$FLGVSKLQJRP\HOLQDVHGH¿FLHQFLHV,Q 6FULYHU&5HG7KH0HWDEROLF%DVHVRI,QKHULWHG'LVHDVH9RO1HZ
INDEX

A ABCA1 25-27, 139, 140, 143-146 ABCG1 25, 26, 139, 140, 143-146 Adipocyte 59, 68, 70-75, 77, 79, 108, 109, 111, 112 Adipose tissue 3, 31, 67-72, 74-77, 79, 80, 102, 112, 126 Alpha-galactosidase a (a-GLA or a-Gal A) 61, 121, 127 Anderson-Fabry disease (Fabry’s disease or FD) 61, 121, 125, 127 Apoptosis 4, 7, 11, 29, 31, 42, 48, 51-53, 58, 69, 79, 80, 91, 107, 122, 125, 126, 128, 129, 140, 141 Atherosclerosis 19, 20, 24, 26-28, 30-33, 57, 61, 62, 67, 79, 139, 140, 146

C Cancer 10, 57, 67, 87, 121, 126, 128, 129, 131 Cardiomyocyte 42, 44, 45, 48-53, 79 Cardiomyopathy 19, 20, 31, 32, 41, 50-52 Cardioprotection 49, 50 Cardiovascular risk 67-69, 74, 77, 79, 80, 145

Ceramide 4-11, 20, 21, 23, 25, 28-32, 41-48, 50-54, 57, 58, 61, 67, 69-80, 89, 91-94, 103, 104, 106-109, 111, 121-124, 126, 128, 131, 139-145 1-phosphate (Cer1P or C1P) 6, 10, 58, 69, 80, 123 Ceramidase (CDase) 4, 5, 8, 10, 42-46, 48, 53, 57, 61, 69, 70, 94, 106, 108, 123 Cholesterol 19, 20, 22-32, 57, 59-63, 113, 126, 139-146 Coronary artery disease (CAD) 19, 20, 26, 59-62

D De novo ceramide synthesis 7, 44, 46, 52, 73-75, 79, 80, 108 Diabetes 3, 8, 19, 20, 31, 41, 42, 51-54, 67, 68, 71, 72, 75, 80, 87, 99, 100-102, 108, 110, 112, 127, 131 Diabetic nephropathy 121, 130, 131 Diacylglycerol kinase 10 Diet-induced obese mice 70-75, 78 Dihydroceramide (DH or dhCer) 4, 10, 20, 58, 75, 104, 123, 141 desaturase (DES or DES1 or DEGS1) 4, 7, 20, 75, 104, 108 synthase (CerS) 4, 104 Dihydrosphingosine (dhSph) 4, 9, 58, 59 1-phosphate (dhS1P) 58, 59 149

150

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E

I

(QGVWDJHUHQDOIDLOXUH(65)  127, 128 Exercise 41, 45-48

Farber disease 61 Fatty acid 1, 3, 4, 7-9, 11, 20, 28, 32, 44, 46, 50-52, 58, 69, 73, 75, 77, 88, 90, 92, 102-104, 106, 108, 113, 139-143, 146 Fatty liver 77, 78, 87, 88, 94, 99 )UHHIDWW\DFLG))$  3, 7, 8, 31, 32, 69, 71, 72, 75, 77-80, 92, 108

Iminosugar 100, 111, 114 ,QÀDPPDWLRQ 30, 60, 67-69, 74, 79, 80, 87, 88, 91, 93, 102, 112, 113, 122, 126-130 Insulin 1, 3, 6-8, 20, 27, 33, 42, 45, 52, 67-69, 71-80, 87, 88, 90, 91, 94, 99-103, 107-114, 130, 131, 140 resistance 1, 3, 6-8, 20, 33, 42, 67-69, 71-73, 75-80, 87, 88, 94, 99-103, 107-111, 113, 114, 130, 131 ,VFKHPLDUHSHUIXVLRQLQMXU\ 41, 42, 48-50, 54 Ischemic preconditioning (IPC) 48, 50

G

K

Galactosylceramide (GalCer) 5, 103, 104, 123, 125, 126 Ganglioside 5, 49, 90, 103, 105, 106, 109-111, 113, 124-126, 128-130 Gaucher disease 61, 107, 110 Genz 123346 90, 91, 111, 113, 114, 128 Globotriosylceramide 124 Glomerulonephritis 121, 129, 131 Glucosylceramide (GluCer) 5, 61, 69, 77, 87, 89, 90, 103-107, 110, 111, 113, 123-128, 130, 131, 141, 144 Glycosphingolipid (GSL) 5, 6, 61, 77, 80, 89, 90, 99, 100, 103-106, 109-113, 121-131, 142 metabolism 100, 121, 122, 124-126, 128-131

Kidney 5, 6, 28, 121-131 cancer 121, 128, 131 disease 121, 122, 125-127, 131

F

H Heart 3, 5-8, 19, 24, 31, 32, 41, 43-54, 67, 80, 87, 122, 127, 145 Hepatic steatosis 77, 78, 87, 89-91, 102, 113 Hexosylceramide (HexCer) 58 High density lipoprotein (HDL) 22-26, 49, 57-60, 79, 80, 139, 140, 143-146 +LJKSHUIRUPDQFHOLTXLGFKURPDWRJUDSK\ (HPLC) 58, 59, 62 coupled with tandem mass spectrometry (HPLC-MS/MS) 58, 59 Hyperglycemia 77, 102, 110, 112, 131

L Lactosylceramide (LacCer) 58, 105, 121, 124, 126, 128-130 synthase 124, 130 /DFWRV\OVXOIDWLGH 124 Leptin 8, 28, 69, 70, 73, 74, 79, 126 resistance 70, 73, 74 Lipidomics 63, 69, 70, 73, 131 /LSLGUDIW 21, 25, 26, 29, 30, 125, 126 Lipoprotein 7, 19, 22-26, 28, 31, 32, 49-51, 57-63, 77, 79, 87, 89, 91, 106, 139, 143, 145, 146 metabolism 19, 22, 23, 28, 32, 60, 63, 87 Lipotoxic cardiomyopathy 31, 41, 50-52 Lipotoxicity 6, 9, 20, 41, 42, 50, 103 Low density lipoprotein (LDL) 6, 22-24, 31, 32, 57, 59, 60, 77, 79, 80, 88, 91, 106, 140, 143 Lupus nephritis 130 LY-B 142

INDEX

M Membranous glomerulopathy 130 Metabolic syndrome 1, 3, 6-8, 67, 71, 72, 77, 87, 88, 94, 99, 114, 121, 130, 131 Monocyte chemoattractant protein 1 (MCP-1) 68, 75, 79 Myriocin 3, 7, 26, 27, 31, 32, 52, 73, 75, 76, 78, 80, 89, 108, 142, 145

N N-acetylneuraminic acid (sialic acid) 103, 105, 124, 129 Niemann Pick disease 25, 61, 145, 146

O Obesity 3, 8, 31, 33, 42, 51, 53, 54, 57, 67-77, 79, 80, 87, 89, 93, 94, 99, 101, 102, 107, 108

P 3HUR[LVRPHSUROLIHUDWRUDFWLYDWHG receptor (PPAR) 41, 44-46, 112 Plasminogen activator inhibitor 1 (PAI-1) 59, 68, 72 Polycystic kidney disease (PKD) 121, 127, 128, 131 3ULPDU\DQGVHFRQGDU\IRFDOVHJPHQWDO glomerulosclerosis (FSGS) 130 3UROLIHUDWLRQ 42, 57, 79, 101, 122, 125, 126, 128-130

S 6HULQHSDOPLWR\OWUDQVIHUDVH637  3, 4, 7-9, 20, 21, 26-28, 32, 42-47, 52, 53, 69-73, 75, 87, 89, 94, 104, 108, 123, 139, 141, 142, 144-146 Sialidase 109, 125, 126, 128-131 Skeletal muscle 3, 7-9, 20, 43, 46, 48, 71, 80, 94, 108, 109

151

Sphinganine 43, 44, 46, 48, 62, 104, 123, 141, 142 Sphingoid base 1, 3, 9, 43, 46, 58, 59, 62, 121-123, 141 Sphingolipid 1-11, 19-21, 26, 33, 41-46, 48, 53, 54, 57-63, 67, 69, 71, 72, 74, 75, 77-80, 87-94, 103-109, 121-123, 126, 128, 139-146 biosynthesis 10, 20, 21, 33, 58, 72 metabolism 1, 3, 4, 6, 8-11, 20, 41, 43-46, 57, 62, 67, 69, 71, 72, 89, 93, 109, 122 Sphingolipidomics 9, 57, 63 Sphingomyelin (SM) 5, 6, 9, 20-30, 32, 33, 42-44, 46, 48, 57, 58-62, 69, 71, 72, 77, 79, 89, 91, 103, 104, 108, 109, 122, 123, 126, 139, 141-146 Sphingomyelinase (SMase) 6, 8, 23-25, 28, 42-44, 46, 48, 53, 57, 61, 69, 70, 72, 77, 79, 87, 91, 93, 122, 143, 145 synthase (SMS) 5, 20, 21, 28-30, 104, 123, 142, 145 Sphingosine (Sph) 4-6, 8, 9, 11, 21, 27, 41-46, 48, 49, 53, 57-62, 69, 71, 72, 75, 103, 104, 106, 122, 123, 141, 142 -1-phosphate (S1P or S1Ps) 5-9, 11, 21, 41-44, 48-50, 54, 57-61, 69, 71, 72, 75, 79, 80, 106, 123, 142 -1-phosphate receptor 48, 49, 59 kinase (SK or SPHK) 5, 6, 8, 9, 42, 44, 49, 53, 57, 69, 123 Sterol-regulatory element binding protein (SREBP) 102, 113, 139-146, 157 Subclass 59, 60 6XSSUHVVRURIF\WRNLQHVLJQDOLQJ (SOCS3 or SOCS-3) 73, 74, 78

T 7XPRUQHFURVLVIDFWRUDOSKD71)DRU TNFa) 6, 29, 30, 58, 61, 68, 69, 71, 72, 74, 75, 79, 89, 91-93, 126 Type 2 diabetes 20, 41, 42, 53, 67, 68, 71, 72, 75, 87, 99-102, 112

152

U 8'3*DOFHUDPLGHJDODFWRV\OWUDQVIHUDVH (GalCer synthase or CGalT) 104, 123 8'3*OFFHUDPLGHJOXFRV\OWUDQVIHUDVH (glucosylceramide synthase) 69, 77, 87, 90, 91, 104, 110-113, 124, 128, 130, 131, 144 Uncoupling protein 3 (UCP) 73, 74

V Very low density lipoprotein (VLDL) 22, 24, 57, 59

SPHINGOLIPIDS AND METABOLIC DISEASE