The Serine Hydrolase Abhd6 Is a Critical Regulator of the Metabolic Syndrome
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THE SERINE HYDROLASE ABHD6 IS A CRITICAL REGULATOR OF THE METABOLIC SYNDROME BY GWYNNETH SARAH THOMAS A Dissertation Submitted to the Graduate Faculty of WAKE FOREST UNIVERSITY GRADUATE SCHOOL OF ARTS AND SCIENCES in Partial Fulfillment of the Requirements for the Degree of DOCTOR OF PHILOSOPHY Molecular Pathology May 2014 Winston-Salem, North Carolina Approved By: John S. Parks, Ph.D., Advisor Paul A. Dawson, Ph.D., Chair J. Mark Brown, Ph.D. Ryan E. Temel, Ph.D. Greg S. Shelness, Ph.D. ACKNOWLEDGEMENTS I first and foremost want to thank my mentor, Dr. J. Mark Brown for everything he has done to develop me into the scientist I am today. I appreciate the opportunity he gave me to work on such an interesting and novel project as well as trusting me to take on a variety of other projects. I will forever be thankful for his kindness, patience and encouragement and glad that I not only gained a mentor but also a friend. I wold also like to thank other members of my committee, Drs John Parks, Gregory Shelness, Paul Dawson and Ryan Temel who were always available to discuss and give feedback while providing continued support towards my goals, even from a distance. I would like to give a special thanks to Dr. Parks for inviting me into his lab when Mark took a new position at Cleveland Clinic. He was instrumental in finishing up my work at Wake Forest while providing new perspective and continued mentorship. This work would not have been possible without the help of the Brown/Rudel/Temel and Parks labs. I would especially like to thank Jenna Betters and Caleb Lord who were very patient with me as I learned about the world of lipids and taught me so much about science. I would also like to thank the Parks lab for graciously accepting me into their lab so late in my time here at Wake Forest. Thank you to everyone else in the Lipid Sciences Department who provided me with thoughtful feedback, advice and friendship. Finally, I would like to thank the unconditional support of my family and friends. My fiance, Neil Ballentine, who always makes me laugh and has loved me through this crazy time, enough to propose and marry me a week before I graduate! I am thankful I got to share this time of my life with him. And of course, my parents, Malcolm and Susan ii Thomas, who even though I changed my mind a million times about the career I wanted, supported me without hesitation and with endless encouragement. I can never thank them enough for everything they have done for me, but I am lucky to have them iii TABLE OF CONTENTS Page LIST OF ILLUSTRATIONS AND TABLES…………………………………………….v LIST OF ABBREVIATIONS…………………………………………………………...vii ABSTRACT……………………………………………………………………………...ix CHAPTER I. INTRODUCTION………………………………………………………………...1 II. THE SERINE HYDROLASE ABHD6 IS A CRITICAL REGULATOR OF THE METABOLIC SYNDROME…………………………………………………….12 III. IMPACT OF OTHER SERINE HYDROLASES ON LIPID METABOLISM…………………………………...……………………………..70 IV. SUMMARY AND CONCLUSIONS……………………………………………90 CURRICULUM VITAE………………………………………………………………..101 iv LIST OF ILLUSTRATIONS AND TABLES CHAPTER II Page Figure 1: ABHD6 is Ubiquitously Expressed and is Regulated by High Fat Diet……....37 Figure 2: ASO-Mediated Knockdown of ABHD6 Protects Against High Fat Diet-Induced Obesity…………………………………………………………………………………...38 Figure 3: ASO-Mediated Knockdown of ABHD6 Protects Against Metabolic Disorders Induced by High Fat Feeding…………………………………………………………….40 Figure 4: ABHD6 is a Critical Regulator of De Novo Lipogenesis……………………..42 Figure 5: ABHD6 Knockdown Results in Modest Alterations in Hepatic Monoacylglycerol Levels yet Does Not Alter Hepatic Endocannabinoid Levels or Acute Cannabinoid Receptor 1 Signaling in Mouse Liver……………………………………...44 Figure 6: ASO-Mediated Knockdown Annotates ABHD6 as a Physiological Lysophospholipase in Mouse Liver………………………………………………….......46 Figure 7: Small Molecular Inhibition of ABHD6 Protects against High-Fat-Diet-Induced Glucose Intolerance and Obesity…………………………………………………….......48 Figure S1: Identification of Antisense Oligonucleotides Efficiently Targeting Knockdown of ABHD6 Specifically in Peripheral Metabolic Tissues and Not in the Central Nervous System, Related to Figure 2……………………………………………………………...49 v Figure S2: ABHD6 Knockdown Consistently Reduces Hepatic Lipogenic Gene Expression, Without Altering AMPK Activation, Related to Figure 3………………….51 Figure S3: Hepatic Triacylglycerol (TAG) and Diacylglycerol (DAG) Levels in ABHD6 Knockdown Mice, Related to Figure 3…………………………………………………..52 Figure S4: Hepatic Phosphatidylcholine (PC), Phosphatidylethanolamine (PE) and Phosphatidic Acid (PA) Levels in ABHD6 Knockdown Mice, Related to Figure 6…….53 Figure S5: Hepatic Phosphatidylserine (PS), Phosphatidylinositiol (PI), and Phosphatidylglycerol (PG) Levels in ABHD6 Knockdown Mice, Related to Figure 6....54 Figure S6: Hepatic Lysophosphatidylglycerol (LPG), Lysophosphatidylcholine (LPC), Lysophosphatidylethanolamine (LPE), Lysophosphatidylinositol (LPI), Lysophosphatidylserine (LPS), and Lysophosphatidic Acid (LPA) Levels in ABHD6 Knockdown Mice, Related to Figure 6…………………………………………………..55 Figure S7: ABHD6 Knockdown Does Not Significantly Alter Plasma Lipid or Glucose Homeostasis in Chow-Fed Mice, but Significantly Reduces Certain Species of Hepatic Triacylglycerol (TAG), Related to Figure 3……………………………………………..56 Table S1: Intestinal fatty acid absorption, related to Figure 2…………………………...57 vi CHAPTER III Page Figure 1: Canonical structure of the α/β hydrolase fold…………………………………71 Figure 2: The Human ABHD Family……………………………………………………72 Figure 3: Tissue Distribution of the ABHD Family of Enzymes………………………..73 Figure 4: ABHD2-/- Measurements………………………………………………………75 Figure 5: Saline, Control and ABHD4 ASO Treatments………………………………...77 Figure 6: ABHD4-/- Measurements………………………………………………………78 Figure 7: Saline, Control and ABHD12 ASO Treatments………………………….........82 vii LIST OF ABBREVIATIONS 2-AG – 2-arachidonylglycerol ABHD – alpha beta hydrolase domain AEA - anandamide ASO – antisense oligonucleotide BAT – brown adipose tissue CB – cannabinoid CDS – Chanarin-Dorman Syndrome DAG – diacylglycerol ECB – endocannabinoid HFD – high fat diet LABH2 – lung alpha beta hydrolase 2 LPA – lysophosphatidic acid LPC – lysophosphatidyl choline LPE - lysophosphatidylethanolamine LPG – lysophosphatidylglycerol LPI – lysophosphatidylinositol viii LPS - lipopolysaccharide MAG – monoacylglycerol MAGL – monoacylglycerol lipase NAPE – N-acylphosphatidylethanolamine PG – phosphatidylglycerol PHARC – polyneuropathy, hearing loss, ataxia, retinitis pigmentosa and cataract qPCR – quantitative polymerase chain reaction SMC – smooth muscle cells TAG/TG – triacylglycerol WAT – white adipose tissue ix ABSTRACT Gwynneth S. Thomas THE SERINE HYDROLASE ABHD6 PLAYS A CRITICAL ROLE IN ENERGY EXPENDITURE AND THE METABOLIC SYNDROME Dissertation Under the Direction of J. Mark Brown, Ph.D. Professor of Pathology Dysregulation of lipid metabolism underlies many chronic diseases such as obesity, diabetes, cardiovascular disease, and cancer. Genes encoding α/β hydrolase fold domain (ABHD) proteins are present in all reported genomes, and conserved structural motifs shared by these proteins predict common roles in lipid synthesis and degradation. Recently, mutations in several members of the ABHD family have been implicated in inherited inborn errors of lipid metabolism. Furthermore, studies in cell and animal models have revealed important roles for ABHD proteins in glycerophospholipid metabolism, lipid signal transduction, and metabolic disease. The serine hydrolase α/β hydrolase domain 6 (ABHD6) has recently been implicated as a key lipase for the endocannabinoid 2-arachidonylglycerol (2-AG) in the brain. However, ABHD6 is ubiquitously expressed, and the biochemical and physiological function for ABHD6 outside of the central nervous system has not been established. Furthermore, unbiased identification of ABHD6 substrates in vivo has not been reported. Therefore, we utilized targeted antisense oligonucleotides (ASO) to selectively knock down ABHD6 in peripheral tissues to identify in vivo substrates and to understand ABHD6’s role in energy metabolism. We found that selective knockdown of ABHD6 in metabolic tissues protects mice from high fat-diet induced obesity, hepatic steatosis, and systemic insulin resistance. We confirmed ABHD6 has intrinsic monoacylglycerol (MAG) lipase activity in vitro, yet x unlike its role in the brain, ABHD6 is a minor contributor to MAG lipolysis and endocannabinoid (ECB) signaling in mouse liver. Using combined in vivo lipidomic identification and in vitro enzymology approaches we show that ABHD6 can hydrolyze several lipid substrates, with strongest activity towards lysophosphatidylglycerol (LPG), a bioactive lipid important in cellular signaling. Results from using ASO technology to knockdown ABHD6 were then contrasted with the use of a small molecular inhibitor, WWL-70. Although inhibition of ABHD6 using WWL-70 also protected against high-fat diet induced obesity and glucose intolerance, it did not improve hepatic steatosis and consequently altered food intake, suggesting a difference in mechanism between the two treatments. Our findings suggest ABHD6 is a key lipase involved in monoacylglycerol and lysophospholipid hydrolysis and that ABHD6 inhibitors may serve as a novel therapeutic for obesity, liver disease and diabetes. xi CHAPTER I INTRODUCTION Significance Lipids are essential for life as molecules of energy storage, membrane structure, and signal transduction. Aberrations in lipid signaling and