The Serine Hydrolase Abhd6 Is a Critical Regulator of the Metabolic Syndrome

THE SERINE HYDROLASE ABHD6 IS A CRITICAL REGULATOR OF THE METABOLIC SYNDROME

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

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

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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

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

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

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

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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

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.

CHAPTER I

INTRODUCTION

Significance

Lipids are essential for life as molecules of energy storage, membrane structure, and

signal transduction. Aberrations in lipid signaling and metabolism underlie many human

diseases, such as obesity, which is considered the most serious public health problem of the

21st century. Therefore, it is imperative to understand the proteins that regulate these

processes.

The ABHD Protein Family

In recent years, the mammalian α/β-hydrolase domain (ABHD) proteins have emerged

as novel potential regulators of glycerophospholipid metabolism and signal transduction.

Mutations in several members have also been implicated in disease states of altered lipid

metabolism. Most notably is ABHD5/CGI-58, which is the causative gene mutated in human

Chanarin-Dorfman Syndrome, also known as Neutral Lipid Storage Disease with Ichthyosis

(NLSDI), a rare, autosomal recessive non-lysosomal disorder of ectopic triacylglycerol (TAG)

accumulation [1, 2]. The ABHD family contains at least 19 proteins and is part of a

superfamily of proteins that possess an α/β-hydrolase fold [3]. The α/β-hydrolase fold

superfamily, which includes proteases, lipases, esterases, dehalogenases, peroxidases, and

epoxide hydrolases, is now one of the largest and most diverse protein families known [4].

The canonical α/β hydrolase fold is made up of 8 β-strands, with the second strand being

antiparallel. These β-strands form a core β-sheet which is surrounded by helices and loops

connecting the β-strands. Hydrolase activity arises from a catalytic triad composed of

nucleophile-acid-histidine residues which are located on loop regions. The nucleophile (Ser,

Cys, or Asp) is always located in a very tight loop, known as the “nucleophilic elbow,”

following strand β5. The nucleophilic elbow can be identified by a consensus motif Sm-X-Nu-

X-Sm, where Sm is a small residue, X is any residue, and Nu is the nucleophile [4]. The

corresponding motif in most members of the ABHD family is GXSXG. The acid residue of

the catalytic triad can be either a glutamate or aspartate, usually located after strand β7.

Finally, the histidine residue is absolutely conserved [4] and is located in a variable loop

following the final β strand. Interestingly, the majority of the ABHD proteins also have a

conserved His-XXXX-Asp motif (where X is any residue), which has previously been

associated with acyltransferase activity [5, 6]. Thus, the ABHD proteins are predicted to

possess both hydrolase and acyltransferase activities. Together, these conserved motifs predict

an important role of ABHD proteins in the hydrolysis or synthesis of small molecules involved

in lipid metabolism and signal transduction. Although the biochemical substrates and

physiological functions of the majority of the ABHD proteins are unknown, there is a growing

recognition of the physiological significance of this family in metabolism and disease.

The ABHD Family and Lipid Metabolism

2 phosphatidylethanolamine (NAPE), and 2-arachidonoylglycerol, respectively [7, 8, 12] while

ABHD5 has recently been shown to acylate lysophosphatidic acid to form phosphatidic acid [10

11]. In further support of the ABHD’s role in glycerophospholipid metabolism, there is a significant decrease in phosphatidylcholine in the bronchoalveolar lavage of ABHD2 knockout mice [14], and more recently ABHD3 reached genome-wide significance as a predictor of plasma phospholipid levels in humans [15]. Although much additional work is needed to identify physiological substrates for the remaining ABHD family members, it will be important to consider glycerophospholipids as clear potential substrates. It is well known that glycerophospholipids serve as key structural elements for the cell membrane, but many also act as key intermediates in neurotransmission and cellular signaling cascades. In line with this, another shared feature of the ABHD family is that several members have been directly implicated in signaling pathways that involve enzymatic synthesis or degradation of key signaling lipids. For instance, both ABHD6 and ABHD12 have been identified and 2-AG hydrolases [12], linking these enzymes to the termination of acute endocannabinoid signaling

[12, 13, 16]. In contrast, ABHD4 has been implicated as a NAPE and lysoNAPE lipase, implicating ABHD4 in endocannabinoid synthesis [8, 9]. Further, ABHD5 has recently been shown to be necessary for the generation of phosphatidic acid and other signaling lipids in response to inflammatory stimuli [17], which then have many secondary effects on both insulin signaling and energy metabolism. Although this still needs to be experimentally tested,

ABHD3’s ability to hydrolyze C14-lysophosphatidylcholine is likely to alter cellular signaling, given that lysophosphatidylcholine species are potent regulators of GPCR and TRPC5 signaling

[18, 19]. Collectively, several members of the ABHD protein family share the ability to metabolize diverse glycerophospholipid species that are intimately involved in cellular signaling.

These shared features of the known ABHDs will likely provide important clues as progress is made with the characterization of the rest of the ABHD protein family.

General Information and Proposed Functions of ABHD6

Human ABHD6, also known as 2-arachidonylglycerol hydrolase, is a 337 residue protein

(38 kDa) encoded by 10 exons on chromosome 3p21.1. It is ubiquitously expressed in mouse with highest expression in brown adipose tissue (BAT), small intestine and brain. ABHD6 is predicted to have a single N-terminal transmembrane region, and based on N-linked glycosidase digestion studies ABHD6 is predicted to have a cytosolic facing orientation. Currently, there are no known mutations in ABHD6 associated with human disease. ABHD6 has been proposed as a possible target gene for Epstein-Barr virus (EBV) nuclear antigen 2 [20]. Furthermore, ABHD6 expression may be linked to the pathogenesis of EBV-related disorders such as: endemic

Burkitt’s lymphoma, Hodgkin’s Lymphoma, and Post-Transplant Lymphoma [20, 21]. The first physiological substrate identified for ABHD6 was 2-arachidonylglycerol (2-AG) [12, 16, 22], an endocannabinoid signaling lipid that plays key roles in neurotransmission and metabolic disease.

Although monoacylglycerol lipase (MAGL) was long thought to be the only mechanism of 2-AG hydrolysis, recently both ABHD6 and ABHD12 have also been shown to hydrolyze this key signaling lipid [12, 13, 16, 22, 23]. Site-directed mutagenesis of amino acids forming the catalytic triad abolishes the enzymatic activity of ABHD6 as well as the labeling of the active serine [12, 13, 16, 23]. When ABHD6 is inhibited it does not affect MAGL activity, the primary enzyme associated with 2-AG hydrolysis in the brain, and ABHD6 also control the stimulated accumulation of 2-AG in intact neurons independently of MAGL [12, 13, 23, 24]. In BV-2 cell homogenates and intact BV-2 cells, inhibition of ABHD6 reduces 2-AG hydrolysis by ~50%

[13]. It has also been shown to control the efficacy of 2-AG at CB receptors in neurons, suggesting ABHD6 enzymatic activity is key in controlling the bioactivity of this lipid transmitter [13]. Given that MAGL, ABHD6, and ABHD12 display distinct subcellular localization, it has been postulated that each enzyme acts to control specific subcellular pools of

2-AG [12, 13]. Since ABHD6 is found post-synaptically as opposed to MAGL, which is found presynaptically, it does not appear to play an active role in depolarization-induced suppression of excitation (DSE) [22, 23]. As well as its role in endocannabinoid metabolism, ABDH6 is differentially expressed among 7 tumor cell lines with highest expression in bone, prostate, and leukocyte tumor cell lines [20]. ABHD6 displayed lowest expression in liver and ovary tumor cells lines, but was not expressed in brain or cervical tumor cell lines [20]. ABHD6 shows exceptional high expression levels in Ewing family tumors (EFT), but not in other related sarcomas, suggesting that ABHD6 may be regulated by onco-fusion proteins in EFTs [54].

ABHD6 expression has been shown to be highly correlated with an EFT-associated gene, aristaless [25]. However, tumor growth velocity, apoptosis rate and cell morphology are unaffected with ABHD6 knockdown [25]. Although one physiological substrate for ABHD6 has been successfully identified as 2-AG, additional studies are needed to fully understand whether

ABHD6 regulates endocannabinoid signaling in peripheral tissues, and whether this signaling role is involved in cancer pathogenesis.

Newly Identified Functions of ABHD6

Previously, ABHD6 has been described several times as a monoacylglycerol lipase

hydrolyzing the key endocannabinoid lipid 2-arachidonylglycerol in the brain [12, 13, 26].

However, ABHD6’s biochemical and physiological function in peripheral tissues had not been

5 characterized. Our group was able to identify physiologically relevant substrates and products for ABHD6 using a metabolic profiling approach. Based on the expectation that physiologically relevant substrates should accumulate when ABHD6 enzyme function is inhibited, we performed both unbiased and targeted lipidomics to identify potential substrate lipids. Interestingly, a number of phospholipids and lysophospholipids accumulated to varying degrees in ABHD6 ASO-treated livers [27]. The most prominent accumulation was seen in nearly all molecular species of phosphatidylglycerol and lysophosphatidylglycerol [27]. We subsequently tested whether ABHD6 can hydrolyze phospholipid and lysophospholipid substrates in vitro using recombinant ABHD6. Recombinant ABHD6 showed considerable lipase activity toward several lysophospholipids including lysophosphatidylglycerol, lysophosphatidylserine, lysophosphatidic acid, and lysophosphatidylethanolamine, but not lysophosphatidylcholine [27]. This newly identified lysophospholipase activity for ABHD6 was indeed intrinsic to the recombinant enzyme, given that mutation of the active site serine abolished all lysophospholipase activity [27]. Taken together, these observations suggest that

ABHD6 can act both as a monoacylglycerol lipase and lysophospholipase exhibiting a preference for LPG among the tested lysophospholipids. Given ABHD6’s dual role of hydrolyzing the endocannabinoid lipid 2-arachdidonylglycerol in the brain and hydrolyzing key lysophospholipids in the liver, tissue-selective inhibitors may be needed to avoid hyper activation of the central endocannabinoid system while still providing metabolic benefit in the periphery. Collectively, these studies using combined in vivo lipidomic identification and in vitro enzymology approaches show that ABHD6 can hydrolyze both monoacylglycerols as well as lysophospholipids, positioning ABHD6 at the interface of glycerophospholipid metabolism and lipid signal transduction.

References

[1] R.A. Igal, J.M. Rhoads, R.A. Coleman, Neutral lipid storage disease with fatty liver and cholestasis, J Pediatr Gastroenterol Nutr 25 (1997) 541-547.

[2] C. Lefevre, F. Jobard, F. Caux, B. Bouadjar, A. Karaduman, R. Heilig, H. Lakhdar, A.

Wollenberg, J.L. Verret, J. Weissenbach, M. Ozguc, M. Lathrop, J.F. Prud'homme, J. Fischer,

Mutations in CGI-58, the gene encoding a new protein of the esterase/lipase/thioesterase subfamily, in Chanarin-Dorfman syndrome, Am J Hum Genet 69 (2001) 1002-1012.

[3] D.L. Ollis, E. Cheah, M. Cygler, B. Dijkstra, F. Frolow, S.M. Franken, M. Harel, S.J.

Remington, I. Silman, J. Schrag, et al., The alpha/beta hydrolase fold, Protein Eng 5 (1992) 197-

211.

[4] M. Nardini, B.W. Dijkstra, Alpha/beta hydrolase fold enzymes: the family keeps growing,

Curr Opin Struct Biol 9 (1999) 732-737.

[5] A.K. Ghosh, G. Ramakrishnan, C. Chandramohan, R. Rajasekharan, CGI-58, the causative gene for Chanarin-Dorfman syndrome, mediates acylation of lysophosphatidic acid, J Biol Chem

283 (2008) 24525-24533.

[6] G. Montero-Moran, J.M. Caviglia, D. McMahon, A. Rothenberg, V. Subramanian, Z. Xu, S.

Lara-Gonzalez, J. Storch, G.M. Carman, D.L. Brasaemle, CGI-58/ABHD5 is a coenzyme A- dependent lysophosphatidic acid acyltransferase, J Lipid Res (2009).

[7] J.Z. Long, J.S. Cisar, D. Milliken, S. Niessen, C. Wang, S.A. Trauger, G. Siuzdak, B.F.

Cravatt, Metabolomics annotates ABHD3 as a physiologic regulator of medium-chain phospholipids, Nat Chem Biol 7 (2011) 763-765.

[8] G.M. Simon, Cravatt, B.F, Endocannabinoid biosynthesis proceeding through glycerophospho-N-acyl ethanolamine and a role for alpha/beta-hydrolase 4 in this pathway, J

Biol Chem 281 (2006) 26465-26472.

[9] J. Liu, L. Wang, J. Harvey-White, B.X. Huang, H.Y. Kim, S. Luquet, R.D. Palmiter, G.

Krystal, R. Rai, A. Mahadevan, R.K. Razdan, G. Kunos, Multiple pathways involved in the biosynthesis of anandamide, Neuropharmacology 54 (2008) 1-7.

[10] A.K. Ghosh, G. Ramakrishnan, C. Chandramohan, R. Rajasekharan, CGI-58, the causative gene for Chanarin-Dorfman syndrome, mediates acylation of lysophosphatidic acid, J Biol Chem

283 (2008) 24525-24533.

[11] G. Montero-Moran, J.M. Caviglia, D. McMahon, A. Rothenberg, V. Subramanian, Z. Xu, S.

Lara-Gonzalez, J. Storch, G.M. Carman, D.L. Brasaemle, CGI-58/ABHD5 is a coenzyme A- dependent lysophosphatidic acid acyltransferase, J Lipid Res (2009).

[12] J.L. Blankman, G.M. Simon, B.F. Cravatt, A comprehensive profile of brain enzymes that hydrolyze the endocannabinoid 2-arachidonoylglycerol, Chem Biol 14 (2007) 1347-1356.

[13] W.R. Marrs, J.L. Blankman, E.A. Horne, A. Thomazeau, Y.H. Lin, J. Coy, A.L. Bodor,

G.G. Muccioli, S.S. Hu, G. Woodruff, S. Fung, M. Lafourcade, J.P. Alexander, J.Z. Long, W. Li,

C. Xu, T. Moller, K. Mackie, O.J. Manzoni, B.F. Cravatt, N. Stella, The serine hydrolase

ABHD6 controls the accumulation and efficacy of 2-AG at cannabinoid receptors, Nat Neurosci

13 (2010) 951-957.

[14] S. Jin, G. Zhao, Z. Li, Y. Nishimoto, Y. Isohama, J. Shen, T. Ito, M. Takeya, K. Araki, P.

He, K. Yamamura, Age-related pulmonary emphysema in mice lacking alpha/beta hydrolase domain containing 2 gene, Biochem Biophys Res Commun 380 (2009) 419-424.

[15] A. Demirkan, C.M. van Duijn, P. Ugocsai, A. Isaacs, P.P. Pramstaller, G. Liebisch, J.F.

Wilson, A. Johansson, I. Rudan, Y.S. Aulchenko, A.V. Kirichenko, A.C. Janssens, R.C. Jansen,

C. Gnewuch, F.S. Domingues, C. Pattaro, S.H. Wild, I. Jonasson, O. Polasek, I.V. Zorkoltseva,

A. Hofman, L.C. Karssen, M. Struchalin, J. Floyd, W. Igl, Z. Biloglav, L. Broer, A. Pfeufer, I.

Pichler, S. Campbell, G. Zaboli, I. Kolcic, F. Rivadeneira, J. Huffman, N.D. Hastie, A.

Uitterlinden, L. Franke, C.S. Franklin, V. Vitart, C.P. Nelson, M. Preuss, J.C. Bis, C.J.

O'Donnell, N. Franceschini, J.C. Witteman, T. Axenovich, B.A. Oostra, T. Meitinger, A.A.

Hicks, C. Hayward, A.F. Wright, U. Gyllensten, H. Campbell, G. Schmitz, Genome-wide association study identifies novel loci associated with circulating phospho- and sphingolipid concentrations, PLoS Genet 8 (2012) e1002490.

[16] J.R. Savinainen, S.M. Saario, J.T. Laitinen, The serine hydrolases MAGL, ABHD6 and

ABHD12 as guardians of 2-arachidonoylglycerol signalling through cannabinoid receptors, Acta

Physiol (Oxf) 204 (2012) 267-276.

[17] C.C. Lord, J.L. Betters, P.T. Ivanova, S.B. Milne, D.S. Myers, J. Madenspacher, G.

Thomas, S. Chung, M. Liu, M.A. Davis, R.G. Lee, R.M. Crooke, M.J. Graham, J.S. Parks, D.L.

Brasaemle, M.B. Fessler, H.A. Brown, J.M. Brown, CGI-58/ABHD5-Derived Signaling Lipids

Regulate Systemic Inflammation and Insulin Action, Diabetes 61 (2012) 355-363.

[18] P.K. Fleming, A.M. Dedman, S.Z. Xu, J. Li, F. Zeng, J. Naylor, C.D. Benham, A.N.

Bateson, K. Muraki, D.J. Beech, Sensing of lysophospholipids by TRPC4 calcium channel. J

Biol Chem 281 (2006) 4977-4982.

[19] N. Murakami, T. Yokomizo, T. Okuno, T. Shimizu, G2A is a proton-sensing G-protein- coupled receptor antagonized by lysophosphatidylcholine. J Biol Chem 279 (2004) 42484-

42491.

[20] F. Li, X. Fei, J. Xu, C. Ji, An unannotated alpha/beta hydrolase superfamily member,

ABHD6 differentially expressed among cancer cell lines, Mol Biol Rep 36 (2009) 691-696.

[21] S. Maier, G. Staffler, A. Hartmann, J. Hock, K. Henning, K. Grabusic, R. Mailhammer, R.

Hoffmann, M. Wilmanns, R. Lang, J. Mages, B. Kempkes, Cellular target genes of Epstein-Barr virus nuclear antigen 2, J Virol 80 (2006) 9761-9771.

[22] A. Straiker, J. Wager-Miller, S.S. Hu, J.L. Blankman, B.F. Cravatt, K. Mackie, COX-2 and fatty acid amide hydrolase can regulate the time course of depolarization-induced suppression of excitation, Br J Pharmacol 164 (2011) 1672-1683.

[23] P. Zhong, B. Pan, X.P. Gao, J.L. Blankman, B.F. Cravatt, Q.S. Liu, Genetic deletion of monoacylglycerol lipase alters endocannabinoid-mediated retrograde synaptic depression in the cerebellum, J Physiol 589 (2011) 4847-4855.

[24] W.R. Marrs, E.A. Horne, S. Ortega-Gutierrez, J.A. Cisneros, C. Xu, Y.H. Lin, G.G.

Muccioli, M.L. Lopez-Rodriguez, N. Stella, Dual inhibition of alpha/beta-hydrolase domain 6 and fatty acid amide hydrolase increases endocannabinoid levels in neurons, J Biol Chem 286

(2011) 28723-28728.

[25] D. Max, M. Hesse, I. Volkmer, M.S. Staege, High expression of the evolutionarily conserved alpha/beta hydrolase domain containing 6 (ABHD6) in Ewing tumors, Cancer Sci 100

(2009) 2383-2389.

[26] D. Navia-Paldanius, J.R. Savinainen, J.T. Laitinen. Biochemical and pharmacological characterization of human α/β-hydrolase domain containing 6 (ABHD6) and 12 (ABHD12). J

Lipid Res 53 (2012) 2413-2424.

[27] G. Thomas, J.L. Betters, C.C. Lord, A.L. Brown, S. Marshall, D. Ferguson, J. Sawyer, M.A.

Davis, J. Melchoir, L.C. Blume, A.C. Howlett, P.T. Ivanova, S.B. Milne, D.S Myers, I. Mrak, V.

Leber, C. Heier, U. Taschler, J. Blankman, B.F. Cravatt, R.G. Lee, R.M. Crooke, M.J. Graham,

R. Zimmermann, H.A. Brown, J.M. Brown, The Serine Hydrolase ABHD6 is a Critical

Regulator of the Metabolic Syndrome, Cell Rep (2013) (In Press) PMID: 24095738.

CHAPTER II

THE SERINE HYDROLASE ABHD6 IS A CRITICAL REGULATOR OF THE

METABOLIC SYNDROME

Gwynneth Thomas, Jenna L. Betters, Caleb C. Lord, Amanda L. Brown, Stephanie

Marshall, Daniel Ferguson, Janet Sawyer, Matthew A. Davis, John T. Melchoir, Lawrence C.

Blume, Allyn C. Howlett, Pavlina T. Ivanova, Stephen B. Milne, David S. Meyers, Irina Mrak,

Vera Leber, Christoph Heier, Ulrike Taschler, Jacqueline L. Blankman, Benjamin F. Cravatt,

Richard G. Lee, Rosanne M. Crooke, Mark J. Graham, Robert Zimmermann, H. Alex Brown,

and J. Mark Brown

Published in Cell Reports 5, 508-520 on October 31, 2013

*The stylistic variations result from the demands of the journal to which the paper was

published

I was responsible for parts of project and experimental design, carrying out the

experiments, analysis, and writing and editing the manuscript

SUMMARY

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, the biochemical and physiological function for ABHD6 outside of the central nervous system has not been established. To address this 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. Here we show that selective knockdown of

ABHD6 in metabolic tissues protects mice from high fat diet-induced obesity, hepatic steatosis, and systemic insulin resistance. Using combined in vivo lipidomic identification and in vitro enzymology approaches we show that ABHD6 can hydrolyze several lipid substrates, positioning ABHD6 at the interface of glycerophospholipid metabolism and lipid signal transduction. Collectively, these data suggest that ABHD6 inhibitors may serve as novel therapeutics for obesity, nonalcoholic fatty liver disease, and type II diabetes.

INTRODUCTION

A major challenge for drug discovery in the post genomic era is the functional characterization of unannotated genes identified by sequencing efforts. Although many unannotated gene products belong to structurally-related gene or protein families, which may provide important functional clues, membership to such families do not always accurately predict the true biochemical and physiological role of proteins. Genes encoding the α/β hydrolase fold domain

(ABHD) protein family are present in all reported genomes (Nardini and Dijkstra, 1999; Hotelier et al., 2004), and conserved structural motifs shared by these proteins predict common roles in lipid metabolism and signal transduction (Lefevre et al., 2001; Fiskerstrand et al., 2010; Long et al., 2011; Simon and Cravatt, 2006; Montero-Moran et al., 2009; Blankman et al., 2007; Lord et al., 2011; Brown et al., 2010). Furthermore, mutations in several members of the ABHD protein

13 family have been implicated in inherited inborn errors of lipid metabolism (Lefevre et al., 2001;

Fiskerstrand et al., 2010). Most recently, studies in cell and animal models have revealed important roles for ABHD proteins in glycerophospholipid metabolism, lipid signal transduction, and metabolic disease (Long et al., 2011; Simon and Cravatt 2006; Montero-Moran et al., 2009;

Blankman et al., 2007; Lord et al., 2011; Brown et al., 2010). However, the physiological substrates and products for these lipid metabolizing enzymes and their broader role in metabolic pathways remain largely uncharacterized. Given this, functional annotation of ABHD enzymes holds clear promise for drug discovery targeting diseases of altered lipid metabolism and lipid signaling.

ABHD5, also known as CGI-58, is the most well-characterized member of the ABHD family due to its key role in triacylglycerol (TAG) metabolism, lipid signaling, and genetic association with the human disease Chanarin-Dorfman Syndrome (CDS) (Lefevre et al., 2001; Montero-

Moran et al., 2009; Lord et al., 2011; Brown et al., 2010; Lass et al., 2006; Schweiger et al.,

2009). Given ABHD5’s clear role in nutrient metabolism and lipid signal transduction, we set out to test whether the closely related enzyme ABHD6 might play a similar role in lipid signaling and metabolic disease. ABHD6 has recently been described as an enzymatic regulator of endocannabinoid (ECB) signaling in the brain (Blankman et al., 2007; Marrs et al., 2010; Marrs et al., 2011). However, ABHD6 is ubiquitously expressed, and the biochemical and physiological functions of ABHD6 outside of the central nervous system has not been studied. Furthermore, unbiased identification of ABHD6 substrates in vivo has not been reported. To address this we selectively knocked down ABHD6 in peripheral tissues, allowing us to identify novel substrates in vivo and to uncover a previously underappreciated role for ABHD6 in promoting the metabolic syndrome. This tissue-selective loss of function approach has unmasked a previously underappreciated role for ABHD6 in promoting the metabolic syndrome, and has allowed for identification of novel substrates and products in mouse liver. These studies demonstrate that

ABHD6 plays a non-redundant enzymatic role in promoting the metabolic disorders induced by high-fat feeding, and suggest that ABHD6 inhibition may be effective in preventing obesity, nonalcoholic fatty liver disease, and type II diabetes.

RESULTS

ABHD6 is Ubiquitously Expressed and Upregulated by High Fat Diet Feeding

Mouse ABHD6 is a 336 amino acid protein that shares high sequence identity with its human

(94%), macaque (94%), and rat (97%) orthologues (Figure 1A). A highly conserved active site serine nucleophile is found at residue 148 (Figure 1A), which is predicted to be necessary for enzyme catalysis. ABHD6 mRNA is ubiquitously expressed (Figure 1B), with highest expression in small intestine, liver, and brown adipose tissue in mice fed standard rodent chow.

Additionally, high fat diet feeding increases ABHD6 mRNA expression in the small intestine and the liver (Figure 1B). This transcriptional regulation of ABHD6 in metabolic tissue prompted us to examine whether ABHD6 may be an important mediator of high fat diet induced metabolic disease.

ABHD6 Knockdown Protects Against High Fat Diet-Induced Obesity

To examine the role of ABHD6 in lipid metabolism in peripheral tissues without altering expression in the brain, we used antisense oligonucleotide (ASO) targeting (Crooke et al.,

1997). Initially, we tested four separate ASOs targeting the knockdown of murine ABHD6, and found that all reduced hepatic ABHD6 mRNA by >80% after 4 weeks of injection (Figure S1).

Following 12 weeks of ASO knockdown with our two ABHD6 ASOs (ASOα and ASOβ), we observed tissue selective knockdown of ABHD6 protein with the following rank order: liver

(>90%) > white adipose tissue (>90%) > kidney (~50%) (Figure 2A & Figure S1B). Whereas,

ABHD6 protein expression in the brain, spleen, and brown adipose tissue was relatively unaffected by ASO treatment (Figure S2). Given that ABHD6 has been described as a key

15 enzymatic regulator or endocannabinoid signaling in the brain (Blankman et al., 2007; Marrs et al., 2010; Marrs et al., 2011), we carefully examined whether ASO treatment reduced ABHD6 in brain regions relevant to metabolic disease. Importantly, ABHD6 was not altered in the hippocampus (Figure S1D), whereas livers from the same mice showed marked reductions in

ABHD6 expression in the liver (Figure S1C). Furthermore, the mRNA expression of ABHD6 in the hypothalamus was likewise not altered by ABHD6 ASO treatment (Figure S1E).

ABHD6 ASO treatment did not alter body weight, adiposity, or food intake in mice fed a standard rodent chow diet (Figure 2B, 2D, and 2E). However, when challenged with a high fat diet, ABHD6 ASO-treated mice were protected from diet induced body weight gain (Figure 2B and 2C), which was in large part due to a reduction in adipose tissue (Figure 2D). Magnetic resonance imaging (MRI) showed that ABHD6 ASO treatment significantly reduced body fat mass (Figure 2E), without altering lean body mass (Figure 2F). Furthermore, ABHD6 ASO treatment did not result in general growth retardation, given that snout to anus length was unaffected (Figure 2G). The protection from high fat diet-induced obesity in ABHD6 ASO-treated mice could not be explained by reductions in food intake (Figure 2H) or by reductions in intestinal fat absorption (Figure 2I). In fact the absorption of several long chain fatty acids was actually slightly increased in ABHD6 ASO treated mice (Table SI). Gene expression analysis in epididymal white adipose tissue revealed that ABHD6 knockdown caused increased expression of lipolytic genes such as HSL and MAGL (Figure 2J), while knockdown of ABHD6 resulted in very minor changes in lipogenic gene expression such as SREBP1c and SCD1 in white adipose tissue (Figure 2J). The ability of ABHD6 ASO treatment to protect against high fat diet-induced obesity could be explained in part by increases in physical activity during the dark cycle (Figure

2K) and increases in energy expenditure during both the diurnal and nocturnal phases (Figure

2L). There were no detectable difference in the respiratory exchange ratio (RER) in control and

ABHD6 ASO-treated mice (Figure 2M).

ABHD6 Knockdown Protects Against Metabolic Disorders Induced by High Fat Feeding

ABHD6 knockdown completely prevented high fat diet-induced accumulation of total hepatic triacylglycerol (TAG) (Figure 3A), without altering total hepatic levels of diacylglycerols (DAG) or monoacylglycerols (MAG) in either dietary setting (Figure 3B). This reduction in neutral lipids was seen in almost all molecular species of TAG, with the exception of 56:6 TAG (Figure S3A).

ABHD6 ASO treatment significantly blunted high fat diet-driven increases in some hepatic DAG species (32:2, 32:1, 34:3, 34:2, 34:1, and 38:6), but not in others (36:2) (Figure S3B). In parallel,

ABHD6 knockdown protected mice from high fat diet induced hyperglycemia (Figure 3C), hyperinsulinemia (Figure 3D), and improved both glucose and insulin tolerance (Figure 3H and

3I). Interestingly, ABHD6 knockdown did not alter plasma TAG levels on either diet (Figure 3E), yet increased plasma non-esterified fatty acid (NEFA) levels specifically in high fat diet fed mice

(Figure 3G). Also, ABHD6 ASO treatment did not alter VLDL triacylglycerol secretion rates

(Figure 3J). However, ABHD6 knockdown protected against high fat-diet induced hypercholesterolemia (Figure 3F), which was reflected as a significant decrease in LDL and a modest increase in HDL (data not shown). Importantly, ABHD6 knockdown significantly decreased hepatic short chain TAG species (50:4, 52:5, 52:4, and 54:7) in chow fed mice, where systemic insulin sensitivity and plasma triacylglycerol levels were similar to control mice

(Figure S7).

ABHD6 is a Critical Regulator of De Novo Fatty Acid Synthesis

When comparing global hepatic gene expression between high fat diet fed control vs. ABHD6

ASO treated mice, we found a surprisingly small number of genes that were differentially expressed by greater than 2-fold (167 upregulated genes and 138 downregulated genes; p

<0.005) (Figure 4A). The most highly enriched gene ontology (-log [p-value] = 10) regulated by

ABHD6 knockdown was fatty acid metabolism (Figure 4A). ABHD6 ASO treatment resulted in a

50% reduction in hepatic ABHD5 expression, which is unlikely to be from direct ASO-mediated silencing due to lack of sequence homology between the two mRNAs. Knockdown of ABHD6 also increased ATGL expression, while reducing HSL expression in the liver (Figure 4B). More consistently, we observed coordinate downregulation of genes involved in de novo fatty acid synthesis and lipogenesis (SREBP1c, FAS, ACC-1, and SCD-1) in ABHD6 ASO treated mouse liver (Figure 4B and 4C). In agreement, the in vivo rate of de novo fatty acid synthesis was reduced by 62% in ABHD6 ASO treated mice (Figure 4D). In agreement, primary hepatocytes isolated from ABHD6 ASO-treated livers showed decreased rates of 3H-oleate esterification into triacylglycerol (Figure 4E) as well as decreased de novo lipogenesis rates as measured by the kinetic conversion of 14C-acetate into 14C-triacylglycerol (Figure 4F). It is important to note that all four ASOs targeting the knockdown of ABHD6 at different sites consistently decreased hepatic lipogenic gene expression (Figure S1B, S1C, and S1D), without altering adipose tissue

(Figure 2J) or hypothalamic (Figure S1E) lipogenic gene expression. We also examined the phosphorylation state of both AMPKα and AMPKβ in mouse liver, but noted no obvious differences in activation state (Figure S2E).

ABHD6 is a Minor Monoacylglycerol Lipase in Mouse Liver, and Knockdown Does Not

Alter Hepatic Endocannabinoid Levels or Acute Cannabinoid Receptor 1 (CB1) Signaling

Given that ABHD6 was previously described as a monoacylglycerol lipase in the brain

(Blankman et al., 2007; Marrs et al., 2010; Marrs et al., 2011), we carefully examined whether

ABHD6 knockdown resulted in accumulation of hepatic MAG species, and whether ABHD6 knockdown resulted in hyper activation of the ECB system in mouse liver. To our surprise,

ABHD6 knockdown did not alter the total hepatic levels of MAG in mice fed either chow or a high fat diet (Figure 3B), although two species of MAG containing oleate (18:1) or linoleate

(18:2) did modestly increase (Figure 5A). In contrast, hepatic levels of endocannabinoid lipids

(2-AG and anandamide) were not changed by ABHD6 ASO treatment (Figure 5B and 5C).

There was also no apparent difference in total hepatic MAG lipase activity with ABHD6 knockdown (data not shown). Given that previous studies have demonstrated that the CB1- dependent signaling is largely desensitized when 2-AG builds up as a result of monoacylglycerol lipase (MAGL) inhibition or genetic deficiency (Schlosburg et al., 2010;

Taschler, et al., 2012), we carefully examined acute CB1 signaling in ABHD6 ASO treated mice.

To examine the effect of ABHD6 knockdown on CB1 receptor desensitization, we administered the cannabinoid receptor (CB1) agonist CP-55,940 or vehicle directly into the portal vein in

ASO-treated mice and followed downstream signaling. ABHD6 knockdown did not alter CP-

55,940-induced ERK-MAPK activation compared to control ASO treated mice (Figure 5D).

However, basal activation of ERK-MAPK was lower in ABHD6 ASO treated mice (Figure 5D), indicating that ABHD6 may be involved in regulating other inputs into ERK-MAPK activation.

ASO-Mediated Knockdown of ABHD6 Uncovers a Role for ABHD6 in Lysophospholipid

Metabolism

Interestingly, a number of phospholipids and lysophospholipids accumulated to varying degrees in ABHD6 ASO treated livers (Figure 6), implicating them as potential physiologically relevant substrates in mouse liver. ABHD6 knockdown significantly increased total hepatic levels of phosphatidylcholine (PC), lysophosphatidylcholine (LPC), phosphatidylethanolamine (PE), lysophosphatidylethanolamine (LPE), phosphatidylglycerol (PG), lysophosphatidylglycerol

(LPG), phosphatidylinositol (PI), lysophosphatidylinositol (LPI), and phosphatidylserine (PS)

(Figure 6B, 6C, 6D, 6E, 6F, 6H, 6I, 6J, 6K, and 6L), without altering hepatic levels of phosphatidic acid or lysophosphatidic acid species (Figure 6A, 6G, and Figure S4C). Of interest, the most prominent accumulation was observed for nearly all species of LPG (Figure 6J, Figure

S6A) and PG (Figure 6D, Figure S5C) in ABHD6 knockdown mice. ABHD6 knockdown also promoted the accumulation of several ether-linked glycerophospholipids (plasmalogens) including all detected molecular species of plasmenylcholine (Figure S4A) and

19 plasmenylethanolamine (Figure S4B). In parallel, nearly all species of LPE, LPG, LPI, and LPS were increased with ABHD6 knockdown regardless of diet (Figure S6). We subsequently tested whether ABHD6 can hydrolyze phospholipid and neutral lipid substrates in vitro. To accomplish this, we expressed a GST-tagged ABHD6 in S. cerevisiae (Figure 6M), and the purified protein was incubated in the presence of a panel of lipid substrates (Figure 6M-Q). As previously postulated (Blankman et al., 2007; Marrs et al., 2010; Marrs et al., 2011) ABHD6 does have the ability to hydrolyze MAG substrates including 1,(3) rac oleoylglycerol and 2-oleoylglycerol, and its ability to hydrolyze MAG substrates is lost when the active site serine was mutated to alanine

(S148A) (Figure 6N). However, in saturation kinetic experiments recombinant MAGL exhibited an 11-fold higher specific activity (Vmax = 5359.3 μmol/h*mg) than ABHD6 (Vmax = 478.6

μmol/h*mg) (Figure 6O), and under the applied conditions the apparent Km was lower for MAGL

(Km = 1.2) compared to ABHD6 (Km = 1.9), indicating that ABHD6 has lower affinity for MAG.

Next, we investigated whether affinity-purified ABHD6 hydrolyzes the other glycerophospholipid substrates identified by in vivo lipidomics (Figure 6A-6L). Recombinant

ABHD6 showed considerable lipase activity towards several lysophospholipids including LPG,

LPA, and LPE but not LPC (Figure 6P). In contrast, ABHD6 exhibited no lipase activity against major phospholipid classes including PG, PE, PA, PS, and PC (Figure 6P). Highest activity was observed using LPG as a substrate (Figure 6P and 6Q), and this was even higher in a detergent-containing (5 mM CHAPS) buffer system. Under these conditions, we determined a

Vmax of 93.2 μmol/h*mg and a Km of 0.75 (Figure 6Q). Furthermore, purified ABHD6 exhibited low activity against retinyl palmitate (RP) and rac-dioleoylglycerol, whereas no activity was observed in the presence of trioleoylglycerol or cholesteryl oleate (data not shown). Notably,

ABHD6’s activity against 1, 3-diacylglycerol was 5-fold higher in comparison to 1, 2(2, 3)- diacylglycerol (data not shown). The ability of ABHD6 to hydrolyze neutral lipid and lysophospholipid substrates was completely lost when the active site serine was mutated to

20 alanine (Figure 6 and data not shown). It is important to note that MAGL did not hydrolyze any of the tested phospholipid substrates (data not shown) annotating MAGL, but not ABHD6, as a specific MAG hydrolase. Taken together, these observations suggest that ABHD6 can act both as a monoacylglycerol lipase and lysophospholipase exhibiting a clear preference for LPG.

Small Molecule Inhibition of ABHD6 Protects Against High Fat Diet-Induced Glucose

Intolerance and Obesity

ASO-mediated inhibition is a tissue-restricted therapeutic approach, targeting knockdown in metabolic tissues (liver, white adipose tissue, and kidney) without altering ABHD6 expression or activity in many other tissues (brain, spleen, brown adipose tissue). However, ABHD6 is ubiquitously expressed (Figure 1B), begging the question whether systemic inhibition would likewise protect against metabolic disease. Therefore, we determined whether a small molecule inhibitor of ABHD6, which would be predicted to target all tissues including the brain, also provides protection against the metabolic disorders driven by high fat diet feeding. Treatment with the small molecule inhibitor of ABHD6 (WWL-70) protected mice from high fat diet-induced body weight gain (Figure 7A), which was largely due to a reduction in adipose tissue mass

(Figure 7B). WWL-70 treatment also protected mice against high fat diet-induce glucose intolerance. Although there was a trend towards a decrease, WWL-70 did not significantly reduce hepatic triacylglycerol levels in high fat diet fed mice (Figure 7D), which contrast to what is seen with ASO-mediated inhibition (Figure 3). These results show that inhibition of ABHD6 using a systemic inhibitor improves some aspects of the metabolic syndrome, but does not protect against hepatic steatosis to the same degree that ASO-mediated inhibition does (Figure

3).

DISCUSSION

Although other members of the ABHD protein family have been clearly linked to lipid

21 signaling and metabolic disease (Lefevre et al., 2001; Fiskerstrand et al., 2010; Long et al.,

2011; Simon and Cravatt 2006; Montero-Moran et al., 2009; Blankman et al., 2007; Lord et al.,

2011; Brown et al., 2010), this is the first study to document a role for ABHD6 in promoting the metabolic disorders driven by high fat diet feeding. The major findings of this work demonstrate that: 1) peripheral knockdown of ABHD6 protects mice from high fat diet-induced obesity, 2)

ABHD6 knockdown protects mice from high fat diet-induced hepatic steatosis and associated insulin resistance, 3) ABHD6 is a critical regulator of hepatic de novo lipogenesis, and 4)

ABHD6 possesses the intrinsic ability to hydrolyze several lipid substrates in vitro, and functions as a lysophospholipid hydrolase with preference for LPG. Accordingly, we show that knockdown of ABHD6 results in the accumulation of LPG and PG in vivo. Taken together, our results reveal that ABHD6 plays a role in the development of the metabolic syndrome.

It is generally accepted that ABHD6 is critical regulator of the ECB system in the brain due to its ability to hydrolyze specific pools of 2-AG (Blankman et al., 2007; Marrs et al., 2010; Marrs et al., 2011). We also confirmed that ABHD6 has intrinsic MAG lipase activity in vitro (Figure 6N and 6O), yet our data suggest that unlike its role in the brain, ABHD6 is a minor contributor to

MAG lipolysis and ECB signaling in mouse liver. This finding is supported by the fact that total

MAG levels do not change (Figure 3B), 2-AG and anandamide levels are not elevated (Figure

5B and 5C), and acute CB1-driven activation of ERK-MAPK is unaltered with ABHD6 knockdown (Figure 5D), arguing against CB1 receptor desensitization. It is important to contrast these findings with the very striking accumulation of hepatic 2-AG, marked reduction in hepatic

MAG lipase activity, and CB1 receptor desensitization seen in both MAGL-/- mice and mice treated chronically with a specific MAGL inhibitor (Taschler et al., 2011; Schlosburg et al.,

2010). It is also important to note that if ABHD6 was a major regulator of hepatic 2-AG levels, one would anticipate that ABHD6 knockdown would cause hyperactivation of hepatic CB1 signaling, which has been reported to promote hepatic steatosis through increasing de novo

22 lipogenesis (Osei-Hyiaman et al., 2005, Osei-Hyiaman et al., 2008). In contrast to this, we observe that ABHD6 knockdown protected mice from high fat diet induced hepatic steatosis and suppressed de novo lipogenesis (Figure 4), further refuting a major role for ABHD6 in hepatic

ECB signaling. Collectively, these results support the notion that MAGL is the predominant MAG lipase in mouse liver. However, we did see minor elevations in 18:1 and 18:2 MAGS (Figure

5A); making it possible that ABDH6’s ability to hydrolyze these lipids may affect other signaling processes regulating hepatic de novo lipogenesis.

ABHD6 clearly plays a specific role in 2-AG hydrolysis and ECB signaling in the brain

(Blankman et al., 2007; Marrs et al., 2010; Marrs et al., 2011). However, MAGL accounts for the vast majority (>80%) of 2-AG hydrolysis in the brain, whereas ABHD6 only accounts for less than 5% of the total 2-AG hydrolase activity (Blankman et al., 2007). Our studies suggest that

ABHD6’s additional ability to hydrolyze other lipid substrates may be more important in linking lipid mediator signaling to the metabolic adaptations to high fat diet feeding. In this study we utilized a targeted lipidomics approach to identify novel substrates of ABHD6 in mouse liver, and verified LPG as bona fide substrate in vitro and in vivo. LPG is considered a bioactive lipid, although its receptor remains to be identified (Makide et al., 2009; Jo et al., 2008).

Lysophospholipids have previously been implicated in promoting metabolic disease via their ability to dictate membrane dynamics and initiate cell signaling, particularly in immune cells

(Wymann et al., 2008; Skoura and Hla, 2009). We assume that defective LPG degradation favors the reesterification of LPG to PG, which may explain the elevated PG levels in the

ABHD6-knockdown liver. PG is also an important precursor for other complex lipids including cardiolipin and bis (monoacylglycero) phosphate. Therefore, changes in ABHD6-driven LPG metabolism could alter many mitochondrial or lysosomal metabolic processes. Interestingly, a recent genome-wide association study (GWAS) in Pima Indians identified the lysophosphatidylglycerol acyltransferase 1 (LPGAT1) loci as being predictive of body mass

23 index (BMI), providing additional genetic evidence that LPG metabolism may influence BMI and adiposity (Traurig et al., 2012). Additionally, the in vitro hydrolysis of diacylglycerols and retinyl esters by ABHD6 has clear implications in membrane signaling, nuclear hormone receptor signaling, and neutral lipid accumulation in cells (Berridge et al., 1984; Erion et al., 2010;

Schreiber et al., 2012; Shirakami et al., 2012). It is important to note that we did not see accumulation of diacylglycerols (Figure 3B, Figure S7) or retinyl esters (data not shown) in the liver of ABHD6 ASO-treated mice. Moreover, RE and DAG hydrolase activities for ABHD6 are very low in comparison to LPG degradation, making it difficult to know if these lipids are indeed physiological substrates of ABHD6.

Projecting forward, there are several important factors to consider for developing ABHD6 as a drug target. It will be important to determine how ABHD6 expression and subcellular distribution are regulated in diverse cell and tissue contexts. Furthermore, it will be essential to determine whether ABHD6 has other physiological substrates in vivo, and whether tissue- selective inhibitors will be necessary for safe and effective prevention of metabolic disease.

Another important consideration will be whether chronic ABHD6 inhibition results in CB1 receptor desensitization in the brain or other receptor signaling abnormalities. For comparison purposes, inhibition of MAG hydrolysis results in many beneficial effects with respect to analgesia, nociception, inflammatory disorders, and cancer (Hohmann et al., 2005; Nomura et al., 2011a; Nomura et al., 2011b; Nomura et al., 2010; Long et al., 2009). However, chronic

MAGL inhibition clearly causes CB1 receptor desensitization (Taschler et al., 2011; Schlosburg et al., 2010), which potentially limits the usefulness for MAGL inhibitors against chronic diseases. Our data suggests that chronic ABHD6 inhibition with ASOs does not cause CB1 receptor desensitization in mouse liver (Figure 5D). However, whether CB1 receptor desensitization occurred in other tissues was not examined here. As previously documented

(Bachovchin et al., 2010), we show that ABHD6 is ubiquitously expressed in mice (Figure 1B).

Additional studies will be required to assess the risk versus benefit of inhibiting ABHD6 in specific tissues. Our studies provide novel information in regards to ABHD6’s biochemical and physiological role in certain peripheral tissues (liver, adipose, kidney) given the tissue-selective inhibition seen with ASOs (Figure S1). However, our studies do not address ABHD6’s primary role in other tissues where it is abundantly expressed (brain, small intestine, pancreas, and skeletal muscle), all of which are key sites regulatory sites for energy metabolism.

It is important to contrast the results of inhibiting ABHD6 with systemic small molecule inhibitors versus a more selective approach using ASO technology. Although both ASO- mediated and WWL-70-mediated inhibition of ABHD6 protected against high fat diet induced obesity and glucose intolerance, only ABHD6 ASO treatment improved hepatic steatosis. The mechanism underlying this difference is unclear at this point, but this most likely stems from systemic versus tissue-restricted inhibition. Interestingly, ASO-mediated inhibition of ABHD6 was not associated with alteration in food intake at any time point examined (Figure 2H).

However, WWL-70 treatment caused a 20% reduction in food intake (data not shown), indicating that the mechanism by which WWL-70 protected against obesity and glucose intolerance is likely driven by hypophagia, while ASO-mediated inhibition more specifically causes dampened hepatic lipogenesis and increases in energy expenditure.

Recently, progress has been made in the development of highly specific small molecular inhibitors selectively targeting ABHD6 (Bachovchin et al., 2010; Li et al., 2007). These tools will become instrumental in defining additional in vivo substrates, and for defining the biology surrounding acute and chronic ABHD6 inhibition systemically. Furthermore, generation of global and conditional knockout mouse models is necessary to define the tissue-specific role for

ABHD6 in chronic diseases of altered lipid metabolism. Future studies using small molecule inhibitors and tissue-specific knockout of ABHD6 will further define the potential for ABHD6 as a drug target. In summary, our data identify ABHD6 as a key determinant of in the pathogenesis

25 of HFD-induced obesity, insulin resistance, and hepatic steatosis. Furthermore, we have uncovered novel lipid substrates of ABHD6 by coupling in vivo lipidomic and in vitro biochemical approaches. Looking forward, we believe that utilizing a similar in vivo loss of function approach may prove useful for mapping natural enzyme-substrate relationships for the other uncharacterized enzymes in the ABHD family. It is interesting to note that during the preparation of this manuscript that a structurally similar enzyme ABHD12 was likewise identified as a dual monoacylglycerol lipase and lysophospholipase with preference towards lysophosphatidylserine species using a similar in vivo substrate identification approach (Blankman et al., 2013). Taken together, our findings suggest that ABHD6 is a key lipase involved in monoacylglycerol and lysophospholipid hydrolysis, and that ABHD6 inhibitors hold promise as novel therapeutics for obesity, non-alcoholic fatty liver disease, and type II diabetes.

EXPERIMENTAL PROCEDURES

Mice

Intraperitoneal glucose tolerance tests and insulin tolerance tests were performed essentially as previously described (Brown et al., 2008a, Brown et al., 2010) in mice treated with diet and ASO for 10-11 weeks. Metabolic measurements were conducted in the comprehensive lab animal monitoring systems (CLAMS) from Columbus Instruments. Body composition was determined by magnetic resonance imaging (MRI). A detailed description of all mouse experiments is

26 provided in the online supplementary methods section.

In Vivo Cannabinoid Receptor (CB1) Receptor Signaling Analyses

Mice were injected with control ASO or ABHD6 ASOβ and maintained on a HFD for a period of

8 weeks prior to experiment. After an overnight fast (9:00 p.m. - 9:00 a.m.), mice were anesthetized with isoflurane (4% for induction, 2% for maintenance), and were maintained on a

37oC heating pad to control body temperature. A minimal midline laparotomy was performed and the portal vein was visualized. Thereafter, mice received either vehicle (cremophor EL: ethanol: saline at a 1:1:18 ratio) or the CB1 agonist (CP-55,490, Cayman Chemical # 13241) at a dose of 0.1 mg/kg body weight directly into the portal vein. Exactly 5 minutes later, the livers were excised and immediately snap frozen in liquid nitrogen. Protein extracts from tissues were analyzed by Western blotting to examine CB1 signaling as described below.

In Vivo Determination of Very Low Density Lipoprotein (VLDL) Secretion, Intestinal Fat

Absorption, and De Novo Fatty Acid Synthesis Rates

VLDL secretion rates were determined using the detergent block method (Li et al., 1996).

Dietary fat absorption was measured using the Olestra® method (Jandacek et al., 2004), and de novo fatty acid synthesis was measured using the 3H-water method (Shimano et al., 1996).

These methods are described in detail in the online supplementary methods section.

Primary Hepatocyte Studies

Hepatocytes were isolated from chow fed ASO treated mice using the collagenase perfusion method, and the rate de novo lipogenesis was determined following the conversion of 14C- acetate and 3H-oleate into newly synthesized triacylglycerol in the presence of lipase inhibitors to block lipolysis/re-esterification. A detailed method is provided in the online supplementary methods.

Generation and Purification of a Polyclonal Antibody Against ABHD6

A maltose binding protein (MBP) ABHD6 fusion protein construct was generated to create affinity purified rabbit polyclonal antibodies against murine ABHD6. A detailed description of cloning, expression, and purification are included in the online supplementary methods section.

Immunoblotting

Whole tissue homogenates were made from multiple tissues in a modified RIPA buffer, and

Western blotting was conducted as previously described (Brown et al., 2004).

Microarray and Quantitative Real-Time PCR Analysis of Gene Expression

Tissue RNA extraction was performed as previously described for all mRNA analyses (Lord et al., 2011; Brown et al., 2010; Brown et al., 2008a; Brown et al., 2008b). Microarray analyses were performed by the Wake Forest School of Medicine Microarray Shared Resource Core using standard operating procedures, and quantitative real time PCR (Q-PCR) analyses were conducted as previously described (Lord et al., 2011; Brown et al., 2010; Brown et al., 2008a;

Brown et al., 2008b). A detailed description of RNA methods is available in the online supplementary methods section.

Hepatic Lipid Analyses

Extraction of liver lipids and quantification of molecular species by mass spectrometry was performed as previously described (Lord et al., 2011; Ivanova et al., 2007; Myers et al., 2011;

Callendar et al., 2007; Saghatelian et al., 2004).

Purification of GST-tagged murine ABHD6 for Enzymology Studies

The coding sequence of murine ABHD6 was cloned into the yeast expression vector pYEX4T-1.

The resulting protein was purified using glutathione-sepharose beads, and used for enzymology

28 studies as described in the online supplementary methods section.

Statistical Analysis

All data are expressed as the mean ± S.E.M., and were analyzed using either a one-way or two- way analysis of variance (ANOVA) followed by Student’s t tests for post hoc analysis using JMP version 5.0.12 software (SAS Institute, Cary, NC).

SUPPLEMENTAL INFORMATION

Supplemental information includes Extended Experimental Procedures, 7 Supplemental

Figures, and 1 Supplemental Table can be found with this article online.

ACKNOWLEDGEMENTS

We thank Larry Rudel, Paul Dawson, and Ryan Temel (Wake Forest School of Medicine) for insightful comments and suggestions. We also sincerely thank Marc Prentki and Murthy

Madiraju (Montreal Diabetes Research Center) for critical review of this work. This work was supported by the Department of Pathology at Wake Forest School of Medicine, a pilot grant from the Wake Forest School of Medicine Venture Fund, and a pilot grant awarded under the

Wake Forest and Harvard Center for Botanical Lipids (P50-AT002782). Lipidomics studies were funded by the National Institute of General Medical Sciences LIPID MAPS (U54-GM069338 to

H.A.B.). Other than Richard Lee, Rosanne Crooke, and Mark Graham, who are employees at

ISIS pharmaceuticals, Inc. (Carlsbad, CA), all other authors report that they have no conflicts of interest.

REFERENCES

Bachovchin, D.A., Ji, T., Li, W., Simon, G.M., Blankman, J.L., Adibekian, A., Hoover, H.,

Niessen, S., Cravatt, B.F., (2010). Superfamily-wide portrait of serine hydrolase inhibition

29 achieved by library-versus-library screening. Proc. Natl. Acad. Sci. USA 107, 20941-20946.

Berridge, M.M., and Irvine, R.F. (1984). Inositol-trisphosphate, a novel second messenger in cellular signal transduction. Nature 312, 315-321.

Blankman, J.L., Simon, G.M., and Cravatt, B.F. (2007). A comprehensive profile of brain enzymes that hydrolyze the endocannabinoid 2-arachidonoylglycerol. Chem. Biol. 14, 1347-

1356.

Blankman, J.L., Long, J.Z., Trauger, S.A., Siuzdak, G., and Cravatt, B.F. (2013) ABHD12 controls brain lysophosphatidylserine pathways that are deregulated in a murine model of the neurodegenerative disease PHARC. Proc. Natl. Acad. Sci. USA 110, 1500-1505.

Brown, J.M., Boysen, M.S., Chung, S., Fabiyi, F., Morrison, R.F., Mandrup, S., and McIntosh,

M.K. (2004). Conjugated linoleic acid induces human adipocyte delipidation: autocrine/paracrine regulation of MEK/ERK signaling by adipocytokines. J. Biol. Chem. 279, 26735-26747.

Brown, J.M., Chung, S., Sawyer, J.K., Degirolamo, C., Alger, H.M., Nguyen, T., Zhu, X., Duong,

M.N., Wibley, A.L., Shah, R., et al. (2008a). Inhibition of stearoyl coenzyme A desaturase 1 dissociates insulin resistance and obesity from atherosclerosis. Circulation 118, 1467-1475.

Brown, J.M., Bell, T.A. 3rd, Alger, H.M., Sawyer, J.K., Smith, T.L., Kelley, K., Shah, R., Wilson,

M.D., Davis, M.A., Lee, R.G., et al. (2008b). Targeted depletion of hepatic ACAT2-driven cholesterol esterification reveals a non-biliary route for fecal neutral sterol loss. J. Biol. Chem.

283, 10522-10534.

Brown, J.M., Betters, J.L., Lord, C., Ma, Y., Han, X., Yang, K., Alger, H.M., Melchior, J., Sawyer,

J.K., Shah, R., et al. (2010). CGI-58 knockdown in mice causes hepatic steatosis but prevents diet-induced obesity and glucose intolerance. J. Lipid Res. 51, 3306-3316.

Callender, H.L., Forrester, J.S., Ivanova, P., Preininger, A., Milne, S., and Brown, H.A. (2007).

Quantification of diacylglycerol species from cellular extracts by electrospray ionization mass spectrometry using a linear regression algorithm. Anal. Chem. 79, 263-272.

Crooke, S.T. (1997). Advances in understanding the pharmacological properties of antisense oligonucleotides. Adv. Pharmacol. 40, 1-49.

Erion, D.M., and Shulman, G.I. (2010). Diacylglycerol-mediated insulin resistance. Nat. Med. 16,

400-402.

Fiskerstrand, T., H’mida-Ben Brahim, D., Johansson, S., M’zahem, A., Haukanes, B.I., Drouot,

N., Zimmermann, J., Cole, A.J., Vedeler, C., Bredrup, C., et al. (2010). Mutations in ABHD12 cause the neurodegenerative disease PHARC: An inborn error of endocannabinoid metabolism.

Am. J. Hum. Genet. 87, 410-417.

Hohmann, A.G., Suplita, R.L., Bolton, N.M., Neely, M.H., Fegley, D., Mangieri, R., Krey, J.F.,

Walker, J.M., Holmes, P.V., Crystal, J.D., et al. (2005). An endocannabinoid mechanism for stress-induced analgesia. Nature 435, 1108-1112.

Hotelier, T., Renault, L., Cousin, X., Negre, V., Marchot, P., and Chatonnet, A. (2004).

ESTHER, the database of the alpha/beta-hydrolase fold superfamily of proteins. Nucleic Acids

Res. 32, D145-147.

Ivanova, P.T., Milne, S.B., Byrne, M.O., Xiang, Y., and Brown H.A. (2007). Glycerophospholipid identification and quantitation by electrospray ionization mass spectrometry. Meth. Enzymol.

432, 21-57.

Jandacek, R., Heubi, J.E., and Tso, P. (2004). A novel, noninvasive method for the measurement of intestinal fat absorption. Gastroenterology 127, 139-144 (2004).

Jo, S.H., Kim, S.D., Kim, J.M., Lee, H.Y., Lee, S.Y., Shim, J.W., Yun, J., Im, D.S., and Bae, Y.S.

(2008). Lysophosphatidylglycerol stimulates chemotactic migration in human natural killer cells.

Biochem. Biophys. Res. Commun. 372, 147-151.

Lefevre, F., Jobard, F., Caux, F., Bouadjar, B., Karaduman, A., Helig, R., Lakhdar, H.,

Wollenberg, A., Verret, J.L., Weissenbach, J., et al. (2001). Mutations in CGI-58, the gene encoding a new protein of the esterase/lipase/thioesterase subfamily, in Chanarin-Dorfman syndrome. Am. J. Hum. Genet. 69, 1002-1012.

Lass, A., Zimmermann, R., Haemmerle, G., Riederer, M., Schoiswohl, G., Schweiger, M.,

Kienesberger, P., Strauss, J.G., Gorkiewicz, G., and Zechner, R. (2006). Adipose triglyceride lipase-mediated lipolysis of cellular fat stores is activated by CGI-58 and defective in Chanarin-

Dorfman Syndrome. Cell Metab. 3, 309-319.

Li, X., Catalina, F., Grundy, S.M., and Patel, S. (1996). Method to measure apolipoprotein B-48 and B-100 secretion rates in an individual mouse: evidence for a very rapid turnover of VLDL and preferential removal of B-48- relative to B-100- containing lipoproteins. J. Lipid Res. 37,

210-220.

Li, W., Blankman, J.L., and Cravatt, B.F. (2007). A functional proteomic strategy to discover inhibitors for uncharacterized hydrolases. J. Am. Chem. Soc. 129, 9594-9595.

Long, J.Z., Nomura, D.K., and Cravatt, B.F. (2009). Characterization of monoacylglycerol lipase inhibition reveals differences in central and peripheral endocannabinoid metabolism. Chem.

Biol. 16, 744-753.

Long, J.Z., Cisar, J.S., Milliken, D., Niessen, S., Wang, C., Trauger, S.A., Sludzdak, G., and

Cravatt, B.F. (2011) Metabolomics annotates ABHD3 as a physiologic regulator of medium- chain phospholipids, Nat. Chem. Biol. 7, 763-765.

Lord, C.C., Betters, J.L., Ivanova, P.T., Milne, S.B., Myers, D.S., Madenspacher, J., Thomas,

G., Chung, S., Liu, M., Davis, M.A., et al. (2012) CGI-58/ABHD5-derived signaling lipids regulate systemic inflammation and insulin action. Diabetes 61, 355-363.

Makide, K., Kitamura, H., Sato, Y., Okutani, M., and Aoki, J. (2009). Emerging lysophospholipid mediators. Lysophosphatidylserine, lysophosphatidylthreonine, lysophosphatidylethanolamine, and lysophosphatidylglycerol. Prostaglandins Other Lipid Mediat. 89, 135-139.

Marrs, W.R., Blankman, J.L., Horne, E.A., Thomazeau, A., Lin, Y.H., Coy, J., Bodor, A.L.,

Muccioli, G.G., Hu, S.S. Woodruff, G., et al. (2010). The serine hydrolase ABHD6 controls the accumulation and efficacy of 2-AG at cannabinoid receptors, Nat. Neurosci. 13, 951-957.

Marrs, W.R., Horne, E.A., Ortega-Gutierrez, S., Cisneros, J.A., Xu, C., Lin, Y.H., Muccioli, G.G.,

Lopez-Rodriguez, M.L., and Stella, N. (2011). Dual inhibition of alpha/beta-hydrolase domain 6 and fatty acid amide hydrolase increases endocannabinoid levels in neurons, J. Biol. Chem.

286, 28723-28728.

Montero-Moran, G., Caviglia, J.M., McMahon, D., Rothenberg, A., Subramanian, V., Xu, Z.,

Lara-Gonzalez, S., Storch, J., Carman, G.M., and Brasaemle, D.L. (2009). CGI-58/ABHD5 is a coenzyme A-dependent lysophosphatidic acid acyltransferase, J. Lipid Res. 51, 709-719.

Myers, D.S., Ivanova, P.T., Milne, S.B., and Brown, H.A. (2011). Quantitative analysis of glycerophospholipids by LC-MS: Acquisition, data handling and interpretation. Biochim.

Biophys. Acta 1811, 748-757.

Nagan, N., and Zoeller, R.A. (2001). Plasmalogens: biosynthesis and functions. Prog. Lipid Res.

40, 199-229.

Nardini, M., and Dijkstra, B.W. (1999). Alpha/beta hydrolase fold enzymes: the family keeps growing. Curr. Opin. Struct. Biol. 9, 732-737.

Nomura, D.K., Long, J.Z., Niessen, S., Hoover, H.S., Ng, S.W., and Cravatt, B.F. (2010).

Monoacylglycerol lipase regulates a fatty acid network that promotes cancer pathogenesis. Cell

140, 49-61.

Nomura, D.K., Lombardi, D.P., Chang, J.W., Niessen, S., Ward, A.M., Long, J.Z., Hoover, H.H., and Cravatt, B.F. (2011a). Monoacylglycerol lipase exerts dual control over endocannabinoid and fatty acid pathways to support prostate cancer. Chem. Biol. 18, 846-856.

Nomura, D.K., Morrison, B.E., Blankman, J.L., Long, J.Z., Kinsey, S.G., Marcondes, M.C.,

Ward, A.M., Hahn, Y.K., Lichtman, A.H., Conti, B., and Cravatt, B.F. (2011b). Endocannabinoid hydrolysis generates brain prostaglandins that promote neuroinflammation. Science 334, 809-

813.

Osei-Hyiaman, D., DePetrillo, M., Pacher, P., Liu, J., Radaeva, S., Batkai, S., Harvey-White, J.,

Mackie, K., Offertaier, L., Wang, L., and Kunos, G. (2005). Endocannabinoid activation at hepatic CB1 receptors stimulates fatty acid synthesis and contributes to diet-induced obesity. J.

Clin. Invest. 115, 1298-1305.

Osei-Hyiaman, D., Liu, J., Zhou, L., Godlewski, G., Harvey-White, J., Jeong, W.I., Batkai, S.,

Marsicano, G., Lutz, B., Buettner, C., and Kunos, G. (2008). Hepatic CB1 receptor is required for development of diet-induced steatosis, dyslipidemia, and insulin and leptin resistance in mice. J. Clin. Invest. 118, 3160-3169.

Saghatelian, A., Trauger, S.A., Want, E.J., Hawkins, E.G., Siuzdak, G., and Cravatt, B.F. (2004)

Assignment of endogenous substrates to enzymes by global metabolite profiling. Biochemistry

43, 14332-14339.

Schlosburg, J.E., Blankman, J.L., Long, J.Z., Nomura, D.K., Pan, B., Kinsey, S.G., Nguyen,

P.T., Ramesh, D., Booker, L., Burston, J.J., et al. (2010) Chronic monoacylglycerol lipase

34 blockade causes functional antagonism of the endocannabinoid system. Nat. Neurosci. 13,

1113-1119.

Schreiber, R., Taschler, U., Preiss-Landl, K., Wongsiriroj, N., Zimmermann, R., and Lass, A.

(2012). Retinyl ester hydrolases and their roles in vitamin A homeostasis. Biochim. Biophys.

Acta. 182, 113-123.

Schweiger, M., Lass, A., Zimmermann, R., Eichmann, T.O., and Zechner, R. (2009) Neutral lipid storage disease: genetic disorders caused by mutations in adipose triglyceride lipase/PNPLA2 or CGI-58/ABHD5. Am. J. Physiol. Endocrinol. Metab. 297, E289-E296.

Shimano, H., Horton, J.D., Hammer, R.E., Shimomura, I., Brown, M.S., and Goldstein, J.L.

(1996). Overproduction of cholesterol and fatty acids causes massive liver enlargement in transgenic mice expressing truncated SREBP-1a, J. Clin. Invest. 98, 1575-1584.

Shirakami, Y., Lee, S.A., Clugston, R.D., and Blaner, W.S. (2012). Hepatic metabolism of retinoids and disease associations. Biochim. Biophys. Acta 182, 124-136.

Simon, G.M. and Cravatt, B.F. (2006) Endocannabinoid biosynthesis proceeding through glycerophospho-N-acyl ethanolamine and a role for alpha/beta-hydrolase 4 in this pathway, J.

Biol. Chem. 281, 26465-26472.

Skoura, A., and Hla, T. (2009). Lysophospholipid receptors in vertebrate development, physiology, and pathology. J. Lipid Res. 50 Suppl, S293-S298.

Taschler, U., Radner, F.P., Heier, C., Schreiber, R., Schweiger, M., Schoiswohl, G., Preiss-

Landl, K., Jaeger, D., Reiter, B., Koefeler, H.C., et al. (2011). Monoacylglyceride lipase deficiency in mice impairs lipolysis and attenuates diet induced insulin resistance. J. Biol. Chem.

286, 17467-17477.

Traurig, M.T., Orczewska, J.I., Ortiz, D.J., Blan, L., Marinelerena, A.M., Kobes, S., Malhotra, A.,

Hanson, R.L., Mason, C.C., Knowler, W.C., et al. (2013). Evidence for a role of LPGAT1 in influencing BMI and percent body fat in Native Americans. Obesity (Silver Spring) 21, 193-202.

Wymann P., and Schneiter, R. (2008). Lipid signaling in disease. Nature Rev. Mol. Cell. Biol. 9,

162-176.

Figure 1. ABHD6 is Ubiquitously Expressed and is Regulated by High Fat Diet

(A) Alignment of human (Hu), macaque (Ma), rat (Ra), and mouse (Mu) ABHD6 orthologues showing conserved (gray) and divergent residues (white); C = consensus sequence. The underlined letters represent the consensus GXSXG “nucleophile elbow” containing the predicted serine nucleophile S148 (black box). Alignment done by Matthew Davis

(B) mRNA expression analysis of ABHD6 in C57BL/6 mouse tissues following 10 weeks of chow or high fat diet (HF) feeding. Data represent the mean ± SEM (n = 4); * = P < 0.05 (vs. chow fed group within each tissue). AU = arbitrary units; Liv = liver; He = heart; Lu = lung; BAT = brown adipose tissue; Kid = kidney; Spl = spleen; Small intestine segments proximal to distal are labeled SI-1, SI-2, and SI-3; Te = testes; Br = brain; Mu = skeletal muscle. Work done by Gwynneth Thomas

Figure 2. ASO-Mediated Knockdown of ABHD6 Protects Against High Fat Diet-Induced Obesity

(A) ABHD6 protein levels in the liver, epididymal adipose tissue (WAT), and brain of mice treated with either saline, a control non-targeting ASO or two independent ABHD6 ASOs for 12 weeks. Work done by Gwynneth Thomas.

(B) Body weight in chow-fed or high fat diet (HFD)-fed mice; data represent the mean ± SEM (n = 6-9); * = P < 0.05 (vs. control ASO group within each time point). Work done by Jenna Betters

38 and Mark Brown

(D) Gross appearance of epididymal fat pads in HFD-fed mice, and total epididymal fat pad weight in both chow-fed and HFD-fed mice; data represent the mean ± SEM (n = 6-9); * = P < 0.05 (vs. control ASO within each diet). Work done by Jenna Betters and Mark Brown

(E) Body fat % as measured by magnetic resonance imaging (n=4). Work done by Gwynneth Thomas

(F) Lean body mass as measured by magnetic resonance imaging (n=4). Work done by Gwynneth Thomas

(G) Snout to anus length (n=10). Work done by Gwynneth Thomas

(H) Cumulative food intake in ASO-treated mice (n=10). Work done by Gwynneth Thomas

(I) Total intestinal fat absorption in mice treated with ASOs and fed a HFD for 9 weeks (n=14- 15). Work done by Gwynneth Thomas

(J) qPCR analyses of epididymal adipose tissue (WAT) gene expression in HFD fed mice; data represent the mean ± SEM (n = 4); * = P < 0.05 (vs. control ASO group). ATGL, adipose triglyceride lipase; HSL, hormone-sensitive lipase, MAGL, monoacylglycerol lipase; SREBP1c, sterol-response element binding protein 1c; FAS, fatty acid synthase; ACC1, acetyl-CoA carboxylase 1; SCD1, stearoyl-CoA desaturase 1; AU = arbitrary units. Work done by Gwynneth Thomas and Jenna Betters???

(K) Diurnal and nocturnal quantification of physical activity (total beam break counts). Work done by Gwynneth Thomas

(L) Diurnal and nocturnal quantification of oxygen consumption (VO2). Work done by Gwynneth Thomas

(M) Diurnal and nocturnal quantification of respiratory exchange ratio (RER; VCO2/VO2). For metabolic cage studies, mice were weight matched and acclimated for at least 48 h prior to measurement. Data represent the mean ± SEM (n = 4); * = P < 0.05 (vs. control ASO). Work done by Gwynneth Thomas

Figure 3. ASO-Mediated Knockdown of ABHD6 Protects Against Metabolic Disorders Induced by High Fat Feeding

Mice were fed a chow or high fat diet (HFD) and treated with a control non targeting ASO or an ASO targeting the knockdown of ABHD6 for 12 weeks.

(A) Gross appearance of livers and microscopic examination (H&E staining at 40x magnification) in HFD-fed mice. Work done by Jenna Betters and Mark Brown

(B) Total hepatic levels of triacylglycerols (TAG), diacylglycerols (DAG), and monoacylglycerols (MAG) in mice fed diets for 12 weeks. Work done by the laboratory of Dr. H. Alex Brown

(D) Plasma insulin levels in mice treated with ASOs and diets for 12 weeks. Work done by Jenna Betters and Caleb Lord

(E) Plasma TAG levels in mice treated with ASOs and diets for 12 weeks. Work done by Jenna Betters and Caleb Lord

(F) Plasma cholesterol levels in mice treated with ASOs and diets for 12 weeks. Work done by Jenna Betters and Caleb Lord

(G) Plasma non-esterified fatty acids (NEFA) in in mice treated with ASOs and diets for 12 weeks. Work done by Jenna Betters and Caleb Lord

(H) Glucose tolerance tests in mice treated with ASOs and diets for 10-11 weeks. Work done by Jenna Betters and Mark Brown

(I) Insulin tolerance tests in mice treated with ASOs and diets for 10-11 weeks. Work done by Jenna Betters and Mark Brown

(J) Hepatic VLDL-TAG secretion rates in mice treated with ASOs and high fat diet for 11 weeks.

All data represent the mean ± SEM (n = 4-6), * = P < 0.05 (vs. control ASO within each diet). Work done by Gwynneth Thomas and Stephanie Marshall

Figure 4. ABHD6 is a Critical Regulator of De Novo Lipogenesis

(A) Biologic processes overrepresented among up-regulated and down-regulated genes identified by microarray analysis from livers of ABHD6 ASO-treated mice compared with control ASO-treated mice fed a high fat diet. Work done by Jenna Betters and Mark Brown

(B) qPCR confirmation of hepatic genes identified by microarray analyses in HFD-fed mice; data represent the mean ± SEM (n = 4); * = P < 0.05 (vs. control ASO group). ATGL, adipose triglyceride lipase; HSL, hormone-sensitive lipase, MAGL, monoacylglycerol lipase; SREBP1c, sterol response element-binding protein 1c; FAS, fatty acid synthase; ACC1, acetyl-CoA carboxylase 1; SCD1, stearoyl-CoA desaturase 1; SREBP2, sterol response element-binding protein 2; HMG-Red, 3-hydroxy-3-methylglutaryl-CoA reductase; LDLr, low density lipoprotein receptor; PPARα, peroxisome proliferator activated receptor alpha; CPT-1α, carnitine palmitoyltransferase 1; AOX, acyl-CoA oxidase; AU = arbitrary units. Work done by Gwynneth Thomas and Jenna Betters

(C) Hepatic lipogenic protein expression in mice treated with a control non targeting ASO or two independent ABHD6 ASOs for 12 weeks. Work done by Gwynneth Thomas and Jenna Betters

(D) In vivo synthesis rates of fatty acids in livers of control and ABHD6 ASO treated mice. HFD- fed male mice (6 weeks of diet and ASO) were injected intraperitoneally with 3H-labeled water, and one hour later liver was removed for measurement of 3H-labeled fatty acids as described in methods. Work done by ISIS Pharmaceuticals

(E) Esterification rates in primary hepatocytes isolated from ASO treated mice. Hepatocytes were kinetically labeled with 3H-oleate to follow the conversion into 3H-triacylglycerol in the presence of lipase inhibitor to block lipolysis/re-esterification. Data represent the mean ± SEM (n = 3) from a representative experiment, which was repeated twice in pooled hepatocytes isolated from ASO treated mice; * = P < 0.05 (vs. control ASO group). Work done by Mark Brown and Gwynneth Thomas

(F) De novo lipogenesis rates in primary hepatocyte isolated from ASO treated mice. Hepatocytes were kinetically labeled with 14C-acetate to follow the conversion into 14C- triacylglycerol in the presence of lipase inhibitor to block lipolysis/re-esterification. Data represent the mean ± SEM (n = 3) from a representative experiment, which was repeated twice in pooled hepatocytes isolated from ASO treated mice; * = P < 0.05 (vs. control ASO group). Work done by Mark Brown and Gwynneth Thomas

Figure 5. ABHD6 Knockdown Results in Modest Alterations in Hepatic Monoacylglycerol Levels, Yet Does Not Alter Hepatic Endocannabinoid Levels or Acute Cannabinoid Receptor 1 (CB1) Signaling in Mouse Liver

(A) Male C57BL/6 mice were fed a chow or high fat diet (HFD) and treated with a control non- targeting ASO or an ASO targeting the knockdown of ABHD6 for 12 weeks. The hepatic levels of monoacylglycerol (MAG) species were measured by mass spectrometry as described in the methods section. Data represent the mean ± SEM (n = 6), * = P < 0.05 (vs. control ASO within each diet). Work done by Jacqueline Blankman and Ben Cravatt

(B) Male C57BL/6 mice were fed a high fat diet (HFD) and treated with a control non-targeting ASO or an ASO targeting the knockdown of ABHD6 for 12 weeks. The hepatic levels of 2- arachidonylglycerol (2-AG) and anandamide were measured by mass spectrometry as described in the methods section. Data represent the mean ± SEM (n = 6), and no significant differences were found. Work done by Jacqueline Blankman and Ben Cravatt

(C) Male C57BL/6 mice were fed a high fat diet (HFD) and treated with a control non-targeting ASO or an ASO targeting the knockdown of ABHD6 for 8 weeks. Following 8 weeks of ASO treatment, mice were fasted for 12 h and subsequently injected with either vehicle or CP-55,940 (0.1 mg/kg) directly into the portal vein. Exactly 5 min later, livers were excised and immediately snap frozen in liquid nitrogen. Protein extracts from the liver were analyzed by Western blotting for ABHD6, phospho-ERK (Thr202/Tyr204), total ERK MAPK, monoacylglycerol lipase (MAGL), or beta actin (β-actin); three representative animals are shown for each group. Work done by Gwynneth Thomas and Mark Brown

Figure 6. ASO-Mediated Knockdown Annotates ABHD6 as a Physiological Lysophospholipase in Mouse Liver

Mice were fed a chow or high fat diet and treated with a control non-targeting ASO or an ASO targeting the knockdown of ABHD6 for 12 weeks (panel’s a-l).

(A) Total hepatic phosphatidic acid (PA) levels.

(B) Total hepatic phosphatidylcholine (PC) levels.

(D) Total hepatic phosphatidylglycerol (PG) levels.

(E) Total hepatic phosphatidylinositol (PI) levels.

(F) Total hepatic phosphatidylserine (PS) levels.

(G) Hepatic levels of 16:0 lysophosphatidic acid (LPA).

(H) Hepatic levels of 18:2 lysophosphatidylcholine (LPC).

(I) Hepatic levels of 18:2 lysophosphatidylethanolamine (LPE).

(J) Hepatic levels of 18:2 lysophosphatidylglycerol (LPG).

(K) Hepatic levels of 18:0 lysophosphatidylinositol (LPI).

(L) Hepatic levels of 18:0 lysophosphatidylserine (LPS). All data in panels A-L represent the mean ± SEM (n = 6), * = P < 0.05 (vs. control ASO within each diet). In panels M-Q recombinant ABHD6 or monoacylglycerol lipase (MAGL) were used to test in vitro substrate specificity of both enzymes.

(M) Coomassie stain of purified GST-tagged murine ABHD6, which was expressed in S. cerevisiae and purified by affinity chromatography; lane 1 = molecular weight ladder, lane 2 = affinity purified ABHD6.

(N) Degradation of 1(3)-oleoylglycerol [1,(3) rac OG; 3mM] and 2-oleoylglycerol [2-OG; 3mM] by wild type (WT) GST-ABHD6 and a mutant variant of ABHD6 lacking the putative active serine (S148G).

(O) Saturation kinetics of ABHD6 and monoacylglycerol lipase (MAGL) using 1(3)-monoolein as substrate. Data are presented as mean ± S.D. and representative for at least two independent experiments.

(P) Purified GST-ABHD6 was incubated in the presence of a panel of potential glycerophospholipid substrates and the release of fatty acids was determined. Data are presented as mean ± S.D. and are representative for at least two independent experiments. * = P < 0.05 (lysophospholipid vs. phospholipid)

(Q) Saturation kinetics of ABHD6 using 1-oleoyl lysophosphatidylglycerol (LPG) as substrate. Data are presented as mean ± S.D. and representative for at least two independent experiments.

All glycerophospholipid levels were measured by the laboratory of Dr. H. Alex Brown. All enzymology studies were performed in the laboratory of Robert Zimmermann

Figure 7. Small Molecule Inhibition of ABHD6 Protects Against High Fat Diet-Induced Glucose Intolerance and Obesity

Male C57BL/6 mice were fed a high fat diet (HFD) and treated with a vehicle or 10 mg/kg of the ABHD6 inhibitor WWL-70 for 8 weeks.

(A) Body weight.

(B) Epididymal white adipose tissue (WAT) weight.

(D) Total hepatic triacylglycerol levels in mice treated with inhibitor and diet for 8 weeks.

Data represent the mean ± SEM (n = 10), * = P < 0.05 (vs. vehicle). Work done by Gwynneth Thomas

Supplemental Data

Figure S1

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. (A) C57Bl/6 mice were maintained on chow diet in conjunction with biweekly injections (25mg/kg) of either saline, a non-targeting control ASO, or four different ASOs targeting knockdown of ABHD6 for 4 weeks. Relative quantification of ABHD6 mRNA levels in the liver was conducted by real-time qPCR, and normalized to cyclophilin. Data Represents pooled RNA samples with n=5 mice per group. ABHD6 ASO α and ABHD6 ASOβ were chosen for further characterization in high fat diet fed mice. Work done by Matthew Davis and Mark Brown

(B) ASO-mediated knockdown of ABHD6 is tissue-specific. C57bl/6 mice wer maintained on high fat diet in conjunction with biweekly injections of either saline, a non-targeting control ASO (Control), or one of two independent ASOs targeting the knockdown of ABHD6 for 12 weeks.

Western blotting was performed as described in materials and methods; 3 representative mice are shown per group. Work done by Gwynneth Thomas and Jenna Betters

(C) IHC determination of effective knockdown in mouse liver. C57Bl/6 mice wer maintained on high fat diet in conjunction with injection of 50mg/kg per week of either non-targeting control ASO (Control) or an ASO targeting knockdown nof ABHD6 for 21 weeks. Immunohistochemical (IHC) detection using affinity purified ABHD6 antibody was performed. Work done by ISIS Pharmaceuticals

(D) ASO treatment does not reduce ABHD6 expression in hippocampal neurons. Hippocampal neurons wer subjected to IHC staining from the same experiment shown in panel C. Work done by ISIS Pharmaceuticals

(E) ASO treatment does not reduce ABHD6 or lipogenic gene expression in the hypothalamus. C57b/6 mice were fed a high fat diet and treated with either Control ASO or ABHD6 ASO for a period of 8 weeks. At necropsy, the whole brain was removed and the hypothalamus isolated, homogenized and analyzed for mRNA expression. Data is expressed as fold change from control ASO treated group and no significant differences were detected. Eno2, enolase 2 is a neuron specific marker; Npy, neuropeptide 1 is a hypothalamus markers; SREBP-1c, sterol response element binding protein 1c; SCD-1, stereoyl-CoA desaturase 1; FAS, fatty acid synthase; ACC!, acetyl-CoA carboxylase 1. Work done by Gwynneth Thomas and Lawrence Blume

Figure S2

Figure S2. ABHD6 Knockdown Consistently Reduces Hepatic Lipogenic Gene Expression, Without Altering AMPK Activation, Related to Figure 3. (A-D) C57bl/6 mice were maintained on chow diet in conjunction with biweekly injections (25mg/kg) of either saline, a non-targeting control ASO, or four different ASOs targeting knockdown of ABHD6 for 4 weeks. Relative quantification of lipogenic gene expression in the liver was conduction by realtime qPCR, and normalized to cyclophilin. Data represents pooled RNA samples with n=5 mice per group. FAS, fatty acid synthase; ACC1, acetyl-CoA carboxylase; SCD1, stearoyl-CoA desaturase 1. Work done by Matthew Davis and Mark Brown (E) Mice were fed a high fat diet (HFD) and treated with a control non-targeting ASO or two independent ASOs targeting the knockdown of ABHD6 for 12 weeks. Livers were excised and immediately snap-frozen in liquid nitrogen. Protein extracts from the liver were analyzed by Western blotting for phospho-AMPKα (Thr172), total AMPα, phospho-AMPKβ (Ser108), total AMPKβ, and phospho-ACC (ser79); tree representative animals are shown for each group

Figure S3

Figure S3. Hepatic Triacylglycerol (TAG) and Diacylglycerol (DAG) Levels in ABHD6 Knockdown Mice, Related to Figure 3. All lipid data here are by H. Alex Brown (update each figure) Male C57Bl/6 mice were fed a chow or high fat diet (HFD) and treated with a control non- targeting ASO or an ASO targeting the knockdown of ABHD6 for 12 weeks. (A) Hepatic levels of TAG species were measured by mass spectrometry as described in the methods section (B) Hepatic levels of DAG species were measured by mass spectrometry as described in the methods section Data represent the mean + SEM (n=6), *=P < 0.05 (vs. control ASO within each diet)

Figure S4

Figure S4. Hepatic Phosphatidylcholine (PC), Phosphatidylethanolamine (PE) and Phosphatidic Acid (PA) Levels in ABHD6 Knockdown Mice, Related to Figure 6. Male C57Bl/6 mice were fed a chow or high fat diet (HFD) and treated with a control non- targeting ASO or an ASO targeting the knockdown of ABHD6 for 12 weeks. (A) Hepatic levels of PC species were measured by mass spectrometry as described in the methods section. (B) Hepatic levels of PE species were measured by mass spectrometry as described in the methods section. (C) Hepatic levels of PA species were measured by mass spectrometry as described in the methods section. Data represent the mean + SEM (n=6), *=P < 0.05 (vs. control ASO within each diet)

Figure S5

Figure S5. Hepatic Phosphatidylserine (PS), Phosphatidylinositiol (PI), and Phosphatidylglycerol (PG) Levels in ABHD6 Knockdown Mice, Related to Figure 6. Male C57Bl/6 mice were fed a chow or high fat diet (HFD) and treated with a control non- targeting ASO or an ASO targeting the knockdown of ABHD6 for 12 weeks. (A) Hepatic levels of PS species were measured by mass spectrometry as described in the methods section. (B) Hepatic levels of PI species were measured by mass spectrometry as described in the methods section. (C) Hepatic levels of PG species were measured by mass spectrometry as described in the methods section. Data represent the mean + SEM (n=6), *=P < 0.05 (vs. control ASO within each diet)

Figure S6

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. Male C57Bl/6 mice were fed a chow or high fat diet (HFD) and treated with a control non- targeting ASO or an ASO targeting the knockdown of ABHD6 for 12 weeks. Hepatic levels of LPG (A), LPC (B), LPI (C), LPE (D), LPS (E), and LPA (F) species were measured by mass spectrometry as described in the methods section. Data represent the mean + SEM (n=6), *=P < 0.05 (vs. control ASO within each diet)

Figure S7

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 A-D done by Jenna Betters, Caleb Lord, and Mark Brown Male C57Bl/6 mice were maintained on a standard chow diet, and starting at 8 weeks of age began receiving biweekly treatments with a control non-targeting ASO or an ASO targeting the knockdown of ABHD6 for 12 weeks. (A) Plasma TAG levels (B) Plasma insulin levels (C) Glucose tolerance tests in mice treated with ASOs for 10-11 weeks (D) Insulin tolerance tests in mice treated with ASOs for 10-11 weeks (E) ABHD6 knockdown causes reduction of certain short chain TAG species in mouse liver All data represent the mean + SEM (n=5-6), * = P < 0.05 (vs. control ASO group). Work done by H. Alex Brown

Table S1. Intestinal fatty acid absorption, related to Figure 2, was determined by the sucrose polybehenate method as described in methods. Measurements were taken in mice treated with either Control ASO or ABHD6 ASOβ, and fed a high fat diet for 9 weeks. Data shown are % absorption values and represent the mean + SEM (n = 14-15), * = P < 0.05 (vs. control ASO within each diet group). Work done by Gwynneth Thomas

EXTENDED EXPERIMENTAL PROCEDURES

Mice

For ABHD6 knockdown studies, at 6-8 weeks of age Male C57BL/6N mice (Harlan) were either maintained on standard rodent chow or switched to a high fat diet for a period of 4-12 weeks, and simultaneously injected with antisense oligonucleotides biweekly (25 mg/kg BW) targeting knockdown of ABHD6 as previously described (Brown et al., 2008a; Brown et al., 2008b; Brown et al., 2010; Lord et al., 2011). The high fat diet (HFD) was prepared by the Wake Forest School of Medicine institutional diet core, and contains ~45% of energy as lard (16:0 = 23.3%, 18:0 = 15.9%, 18:1 = 34.8%, 18:2 = 18.7%), and has been previously described (Brown et al., 2010; Lord et al., 2011). The 20-mer phosphorothioate ASOs were designed to contain 2'-0- methoxyethyl groups at positions 1 to 5 and 15 to 20, and were synthesized, screened, and purified as described previously (Crooke et al., 2005) by ISIS Pharmaceuticals, Inc. (Carlsbad, CA). For tissue distribution studies, at 6-8 weeks of age Male C57BL/6N mice (Harlan) were either maintained on standard rodent chow or switched to a high fat diet for a period of 10 weeks without ASO treatment. For WWL-70 treatment studies, 6 week old male C57BL/6N mice were switched to a high fat diet for a period of 8 weeks, and simultaneously injected intraperitoneally with either a vehicle (9:1 dimethylsulfoxide:saline) or WWL-70 (10 mg/kg BW) once daily. All mice were maintained in an American Association for Accreditation of Laboratory Animal Care (AALAC) approved specific pathogen-free environment on a 12:12 light:dark cycle and allowed free access to food and water. All experiments were performed with the approval of the institutional animal care and use committee at Wake Forest School of Medicine.

Necropsy, Tissue Histology, and Plasma Analyses

At necropsy, all mice were terminally anesthetized with ketamine/xylazine (100-160mg/kg ketamine-20-32mg/kg xylazine), and a midline laparotomy was performed. Blood was collected by heart puncture. Following blood collection, a whole body perfusion was conducted by puncturing the inferior vena cava and slowly delivering 10 ml of saline into the heart to remove blood from tissues. Tissues were collected and snap frozen in liquid nitrogen for most analyses. For histology, tissue samples were fixed in 10% neutral buffered formalin, embedded in paraffin, sectioned, and stained with hematoxylin and eosin. Plasma glucose levels were measured using a glucometer (Ascensia Countour, Bayer Healthcare, Leverkusen, Germany). Plasma

58 insulin levels were measured by ELISA (Crystal Chem. Inc., Downers Grove, IL, USA). Plasma non-esterified fatty acids (NEFA) were quantified enzymatically (NEFA-HR; Wako Diagnostics, Richmond, VA, USA). Plasma triacylglycerol levels were quantified enzymatically (L-Type TG M, Wako Diagnostics, Richmond, VA, USA). A detailed description of total plasma cholesterol and lipoprotein cholesterol distribution analyses has been previously described (Brown et al., 2008a; Brown et al., 2008b; Brown, et al., 2010).

Body Composition Analysis by Magnetic Resonance Imaging

A subset of mice were treated with ASO for 8 weeks prior to magnetic resonance imaging. Isoflurane anesthetized mice were imaged in a Bruker 7T MRI used for small animals as previously described (Olson et al., 2012). 1mm2 slices were analyzed using ImageJ software using the threshold adjustment method to calculate adipose mass and lean body mass.

Food Intake Analysis

At 6 weeks of age mice were switched from rodent chow to a high fat diet and injected with either a control or ABHD6 ASO biweekly (25 mg/kg BW) for 2 weeks to initiate knockdown. Following 2 weeks of diet/ASO induction, mice were moved into individual cages fitted with wire bottom inserts to avoid coprophagy. These mice continued to receive high fat diet and biweekly ASO injections, and quantitative food recovery was determined once daily for 28 consecutive days. Food intake is expressed as cumulative food intake for this 4 week period.

Glucose and Insulin Tolerance Tests

Intraperitoneal glucose tolerance tests and insulin tolerance tests were performed essentially as previously described (Brown et al., 2008a) in mice treated with diet and ASO for 10-11 weeks. Briefly, glucose tolerance tests (GTT) were performed after an overnight (10 hour, 11:00 pm – 9:00 am) fast by injecting 1 mg/kg body weight of glucose into the peritoneal cavity. Insulin tolerance tests (ITT) were performed after a short term fast (4 hour, 9:00 am – 1:00 pm) by injecting 0.75 U/kg body weight into the peritoneal cavity. Plasma glucose levels were measured using a commercial glucometer (Ascensia Countour, Bayer).

In Vivo Cannabinoid Receptor (CB1) Receptor Signaling Analyses

Mice were injected with control ASO or ABHD6 ASOβ and maintained on a HFD for a period of 8 weeks. After an overnight fast (9:00 p.m. - 9:00 a.m.), mice were anesthetized with isoflurane (4% for induction, 2% for maintenance), and were maintained on a 37oC heating pad to control body temperature. Mice received either vehicle (cremophor EL:ethanol:saline at a 1:1:18 ratio) or the CB1 agonist (CP-55,490, Cayman Chemical # 13241) at a dose of 0.1 mg/kg body weight directly into the portal vein. Exactly 5 minutes later, the liver was excised and snap frozen in liquid nitrogen. Protein extracts were analyzed by Western blotting to examine CB1 signaling.

In Vivo Determination of Very Low Density Lipoprotein (VLDL) Secretion

Mice were injected with control ASO or ABHD6 ASOβ and maintained on a HFD for a period of 11 weeks prior to experiment. After a 4 hour fast, male mice were anesthetized with isoflurane (4% for induction, 2% for maintenance) and Triton WR 1339 (500 mg/kg body weight; Sigma) was delivered via retro-orbital injection, to block lipolysis. Thereafter, blood samples were collected from anesthetized mice by retro-orbital bleeding at 0, 0.5, 1, 2, and 3 h after injection. Plasma was harvested from the blood samples and used to quantify TG mass by enzymatic assay.

Quantification of the Absorption of Dietary Fat Mice were injected with control ASO or ABHD6 ASOβ and maintained on a HFD for a period of 9 weeks prior to experiment. Thereafter, fatty acid absorption was measured using the Olestra® method (Jandacek et al., 2004). Briefly, mice were fed the same HFD containing the nonabsorbable fat sucrose poly behenate (1.2%; wt/wt) for 4 consecutive days. Feces were collected from mice housed individually in cages with wire mesh floors to avoid coprophagy. Weighed samples of the synthetic diet and feces were saponified with methanolic NaOH, extracted with hexane, converted to methyl esters, and analyzed by gas chromatography4 to quantitate behenic acid (BA, C22:0) and saturated (14:0, 16:0, 18:0), monounsaturated (C18:1), and polyunsaturated (18:1, 18:2, 18:3ω3, 20:5ω3, 22:6ω3) fatty acids. The coefficient of absorption for each FA was computed as {1 − (FA/BA)feces / (FA/BA)diet} × 100.

Indirect Calorimetry and Metabolic Cage Measurements

Mice were injected with control ASO or ABHD6 ASO and maintained on a HFD for a period of 7-8 weeks prior to the experiment. Mice were acclimated to metabolic cages for 24 h, and on day 2 injected with ASO. Thereafter, physical activity, oxygen consumption (VO2), respiratory exchange ratio (RER) were continually monitored for an additional 72 hours using the Oxymax CLAMS system (Columbus Instruments) at a temperature of 22oC. Data represent the last 6 a.m. to 6 a.m. period after adequate acclimation.

In Vivo Quantification of De Novo Fatty Acid Synthesis Rates Quantification of hepatic lipogenesis was carried out as described previously (Shimano et al., 1996). Briefly, non-fasted mice were intraperitoneally injected with 10 mCi of [3H] water (Moravek Biochemicals and Radiochemicals, Brea, CA, USA). One hour post-injection liver and plasma were collected. Plasma was used to determine the plasma [3H] water specific activity. A portion of the liver (200-300 mg) was digested in potassium hydroxide. Neutral sterols were extracted with petroleum ether and discarded. Fatty acids were acidified by adding hydrochloric acid to the aqueous phase, and extracted with hexane. Aliquots of the hexane extract were dried down, and residual water was removed by incubating at 80C in a vacuum oven. Methanol and scintillation fluid were added and cpm were counted. The rates of fatty acid synthesis were calculated as mol of [3H]-water incorporated in fatty acids per hour per gram of tissue.

Primary Hepatocyte Isolation and Determination of Lipogenic Rates

Chow fed C57BL/6 male mice were injected with control ASO or ABHD6 ASO for a period of 8 weeks prior to the experiment. After 8 weeks of ASO treatment, mouse primary hepatocytes were isolated by collagenase perfusion as previously described (Lord et al., 2011). Hepatocytes were pooled from 2 donor mice from each ASO group each day for quantification of lipogenic rates. Briefly, freshly isolated hepatocytes were initially seeded on collagen coated 6 well plates at a seeding density of 1x106 cells per well. Thereafter, cells were cultured for 3 h in serum free William’s E Medium, and during the last hour (hour 2-3) all cells were supplemented with a lipase inhibitor cocktail (diethylumbelliferyl phosphate = DEUP + diethyl-p-nitrophenyl phosphate = E-600) to measure triacylglycerol synthesis without the complications of the simultaneous lipolysis/re-esterification reactions. To generate lipase inhibitor stocks E600 was dissolved in water (2.5 mg/ml) and DEUP was solubilized in Me2SO (50 mg/ml). These inhibitors were added a final concentration of 50 μg/ml DEUP and 500 μM E-600 for one hour prior to radiotracer

61 addition and were present throughout the labeling time course. Following lipase inhibition, cells were labeled over a time course with 5 μCi/ml [3H]oleic acid and 0.5 μCi/ml [14C]acetate. Following labeling, cells were gently washed 2x with PBS and total lipid extracts were made from remaining cells using the method of Bligh and Dyer (1959). Thereafter, carrier standards were added to samples and lipid classes were separated by thin layer chromatography (TLC) using Silica Gel 60 plates and a solvent system containing hexane-diethyl ether-acetic acid (70:30:1). Lipids were visualized by exposure to iodine vapor, and bands corresponding to CE, TG, and PL were scraped and counted. Protein concentration was determined using the bicinchoninic acid assay.

Generation and Purification of a Polyclonal Antibody Against ABHD6

A maltose binding protein (MBP) ABHD6 fusion protein construct was produced by inserting the DNA sequence encoding amino acids 119-315 of mouse ABHD6 into the pMAL-C2 vector (New England Biolabs) at BamHI and HindIII restriction sites. For PCR cloning an expression vector containing murine ABHD6 cDNA (pCMV-SPORT6-mABHD6) was purchased from Open Biosystems (#MMM1013-7512548) and used as a template for PCR cloning using the following primers: forward (5’-gcggatccgacctgtccatagtggggcaag-3’) and reverse (5’- gcaagcttctatgtcttcctcggtctctccastcac-3’). The resulting pMAL-mABHD6119-315 vector was sequence confirmed, and then used then to drive fusion protein expression in Escherichia coli using an Isopropyl-β-D-1-thiogalactopyranoside (IPTG) inducible expression system. Recombinant MBP-mABHD6119-315 fusion proteins were purified by affinity chromatography using an amylose resin. Given that some degradation products were detected after amylose affinity purification, the full length fusion protein was further purified by passing over an 8% agarose column. Three milligrams of the resulting affinity purified MBP-mABHD6119-315 fusion protein was sent to Lampire (Pipersville, PA) for antibody production in 3 independent rabbits according to standard protocol. To obtain affinity purified antibodies, approximately 20mg of MBP- mABHD6119-315 fusion protein was coupled to 4ml Amino link coupling gel (Pierce) using the manufacturer’s protocol to generate an affinity column. Thereafter, 10 ml of rabbit antisera was placed on this affinity column and rocked overnight at 4oC. The column was drained, washed, and antibody was eluted with 0.01 M glycine, 10% (v/v) ethylene glycol, pH 2.8. The eluted antibody was further purified by passing it over a maltose binding protein affinity column and collecting the flow-thru. To obtain this antibody for research purposes contact Dr. J. Mark Brown

([email protected]).

Immunoblotting and Immunohistochemistry

Whole tissue homogenates were made from multiple tissues in a modified RIPA buffer as previously described8. Proteins were separated by 4–12% SDS-PAGE, transferred to polyvinylidene difluoride (PVDF) membranes, and proteins were detected after incubation with specific antibodies as previously described (Brown et al., 2004). Antibodies used include: affinity purified anti-ABHD6 rabbit polyclonal (described above), anti-histone 3 rabbit monoclonal (Cell Signaling Technologies #4499), anti- actin rabbit monoclonal (Cell Signaling Technologies #4970), anti-BIP rabbit monoclonal (Cell Signaling Technologies #3177), anti-FAS rabbit monoclonal, anti-SCD1 rabbit monoclonal (Cell Signaling Technologies # 2794), anti-phospho- p44/p42 MAPKThr202/Tyr204 rabbit monoclonal (Cell Signaling Technologies # 4370), anti-total- p44/p42 MAPK (Cell Signaling Technologies # 4695), anti-phospho-AMPKThr172 rabbit monoclonal (Cell Signaling # 2535), anti-total AMPK rabbit monoclonal (Cell Signaling Technologies # 2603), anti-phospho-AMPKSer108 rabbit polyclonal (Cell Signaling Technologies # 4181), anti-total AMPK rabbit monoclonal (Cell Signaling Technologies # 4150), anti-phospho-ACCSer79 rabbit polyclonal (Cell Signaling Technologies # 3661), anti-total ACC rabbit monoclonal (Cell Signaling Technologies # 3676), Anti-rabbit IgG-HRP linked secondary (Cell Signaling Technologies #7074), and Anti-mouse IgG-HRP linked secondary (Cell Signaling Technologies #7076). For immunohistochemical (IHC) detection of ABHD6 in liver and brain section, tissues were mounted in OCT and frozen in dry ice and methylbutane. 6 μm sections were prepared using a cryostat and air dried overnight. Slides were subsequently fixed in cold acetone for 5 minutes, rinsed 3x for 2 minutes with TBS. Thereafter, endogenous peroxidase was quenched by the addition of 3% hydrogen peroxide for 10 minutes, followed by 3x rinsing with TBS for 2 minutes a piece. Slides were then blocked with Cyto-Q Background Buster (Innovex Biosciences), and subsequently incubated with affinity purified ABHD6 rabbit polyclonal antibody for 1 hour at room temperature (1:100 dilution). Following 3x rinsing with TBS for 2 minutes each, a goat anti-rabbit HRP secondary antibody was added (1:500) and incubated for 30 minutes at room temperature, followed by sequential rinsing and application of the chromagen DAB.

Microarray and Quantitative Real-Time PCR Analysis of Hepatic Gene Expression

Tissue RNA extraction was performed as previously described for all mRNA analyses (Brown et al., 2008a; Brown et al., 2008b; Brown, et al., 2010; Lord et al., 2012). For microarray analyses, hepatic total RNA samples were further purified using RNeasy MinElute Cleanup Kit (Qiagen # 74204) followed by quality assessment using an Agilent 2100 bioanalyzer. Samples with RIN values > 8.0 were carried forward for cRNA synthesis and hybridization to GeneAtlas MG-430 PM Array Strips (Affymetrix, Santa Clara, CA) following the manufacturer’s recommended protocol. Microarray analyses were performed by the Wake Forest School of Medicine Microarray Shared Resource Core using standard operating procedures. The raw data generated were normalized using the robust multi-array average (RMA) method (Irizarry et al., 2003), and functional annotation to gene ontology was performed using Ingenuity-IPA software (Ingenuity Systems, Inc., Redwood City, CA). Quantitative real time PCR (Q-PCR) analyses were conducted as previously described (Brown et al., 2008a; Brown et al., 2008b; Brown, et al., 2010; Lord et al., 2011). mRNA expression levels were calculated based on the -CT method. Q-PCR was conducted using the Applied Biosystems 7500 Real-Time PCR System. Primers used for Q-PCR are available on request.

Analysis of Hypothalamic Gene Expression in ABHD6 ASO Treated Mice

Mice were sacrificed at 8 weeks post-injection of control versus ABHD6 ASO, and brains were dissected and placed in an ice-cold brain matrix. Hypothalamus was isolated, placed on a chilled dissecting block, and 1 mm-diameter circular brain punches were collected from the basal lateral hypothalamus. Total RNA was isolated from each punch and purified using an RNeasy Mini Kit (Qiagen). RNA quantification and purity was determined with purity set at 260/280 and 260/230 ≥ 2.0 (NanoDrop 1000 Spectrophotometer, Thermo Scientific, DE, USA). Total RNA (1μg) was reverse transcribed into cDNA using a High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, CA, USA). Real-time qPCR was performed using FastStart Universal SYBR Green Master Mix (ROX) and specific Forward and Reverse primer probe assay sets. Data were analyzed using the ΔΔCT method with eno2 serving as the reference standard.

Hepatic Lipid Analyses

Extraction of liver lipids and quantification of molecular species by mass spectrometry were performed essentially as previously described (Lord et al., 2011; Myers et al., 2011; Ivanova et al., 2007; Callendar et al., 2007). For glycerophospholipid analyses, liver tissue was extracted using a modified Bligh and Dyer procedure (Bligh and Dyer 1959). Approximately 10 mg of frozen mouse liver was homogenized in 800 µl of ice-cold 0.1 N HCl:CH3OH (1:1) using a tight- fit glass homogenizer (Kimble/Kontes Glass Co, Vineland, NJ) for about 1 min on ice. Suspension was then transferred to cold 1.5 ml Eppendorf tubes and vortexed with 400 µl of o cold CHCl3 for 1 min. The extraction proceeded with centrifugation (5 min, 4 C, 18,000 x g) to separate the two phases. Lower organic layer was collected and solvent evaporated. The resulting lipid film was dissolved in 100 µl of isopropanol:hexane:100 mM NH4COOH(aq) 58:40:2 (mobile phase A). Quantification of glycerophospholipids was achieved by the use of an LC-MS technique employing synthetic odd-carbon diacyl- and lysophospholipid standards. Typically, 200 ng of each odd-carbon standard was added per 10-20 mg tissue. Glycerophospholipids were analyzed on an Applied Biosystems/MDS SCIEX 4000 Q TRAP hybrid triple quadrupole/linear ion trap mass spectrometer (Applied Biosystems, Foster City, CA, USA) and a Shimadzu high pressure liquid chromatography system with a Phenomenex Luna Silica column (2 × 250 mm, 5-μm particle size) using a gradient elution as previously described (Ivanova et al., 2007; Myers et al. 2011). The identification of the individual species, achieved by LC/MS/MS, was based on their chromatographic and mass spectral characteristics. This analysis allows identification of the two fatty acid moieties but does not determine their position on the glycerol backbone (sn-1 versus sn-2). Neutral lipids from frozen mouse liver tissue (10- 15 mg) were extracted by homogenizing tissue in the presence of internal standards (14:0 monoacylglycerol, 24:0 diacylglycerol , and 42:0 triacylglycerol) in 2 ml 1X PBS and extracting with 2 ml ethyl acetate:trimethylpentane (25:75). After drying the extracts the lipid film was dissolved in 1 ml hexane:isopropanol (4:1) and passed through a bed of Silica gel 60 Å to remove remaining polar phospholipids. Solvent from the collected fractions was evaporated and lipid film was redissolved in 100 µl 9:1 CH3OH:CHCl3, containing 10µl of 100mM CH3COONa for MS analysis essentially as described (Callendar et al., 2007). The specific analysis of endocannabinoid lipids such as 2-arachidonylglycerol and anandamide were conducted as previously described (Saghatelian, et al. 2004).

Cloning and Purification of GST-tagged murine ABHD6

The coding sequence of mABHD6 was cloned into the yeast expression vector pYEX4T-1 (Clontech Laboratories Inc.; Mountain View, USA) creating an N-terminal GST-fusion protein under a copper inducible promoter. The construct was verified by sequencing (Eurofins MWG Operon; Ebersberg, Germany) and transformed into S.cerevisiae wildtype BY4742 (MATa ; his3Δ 1; leu2Δ 0; lys2Δ 0; ura3Δ 0) obtained from Euroscarf, Frankfurt, Germany. Expression of

GST-mABHD6 was induced in mid-log phase at 30°C using 0.5 mM CuSO4. Thereafter, cells were harvested and protoplasts were obtained by zymolyase treatment (Zymolyase ® 20T; AMS Biotechnology Limited, Germany). Cells were disrupted by sonication in 10 mM PBS (pH 7.4) and 0.2 % NP-40. Subsequently, the lysate was centrifuged (10.000g, 15min) and incubated with Glutathione-Sepharose beads (Glutathione-Sepharose 4B; GE Healthcare, Piscataway, NJ) for 30 minutes at room temperature. After extensive washing with PBS/0.2 % NP-40, the purified protein was eluted using a buffer containing 16 mM GSH, 20 mM Tris-HCl (pH 8.0), 100 mM NaCl, and 0.2% NP-40 and dialyzed overnight against a buffer containing 20 mM Tris-HCl (pH 8.0), 100 mM NaCl, 0.05 % NP-40, 250 mM sucrose, 1 mM EDTA and 20 µM DTT at 4°C. To generate the S148A mutant protein, site-directed mutagenesis was performed using the the GeneArt® Site-Directed Mutagenesis System (Invitrogen; Carlsbad, CA, USA) according to the manufacturer’s instructions, and the protein was purified as described for the wild type protein.

ABHD6 Enzyme Activity Assays For determination of monoacylglycerol hydrolase (MGH) activity, monoacylglycerol (MG) substrates [rac-1(3)-oleoylglycerol (rac-1(3)-OG) or 2-oleoylglycerol (Sigma Aldrich, St. Louis, USA)] were dried under nitrogen and dispersed by brief sonication in 50 mM Tris-HCl (pH 8.0), sodium acetate buffer (pH 4.5-5.5), MES buffer (pH 5.5-6.5) or bistrispropane buffer (pH 6.5-9), 100 mM NaCl, 0.1% NP-40, 1 mM defatted BSA, and various concentrations of MG. Then, 10 µl of purified ABHD6 (approx. 0.2 µg) was incubated with 90 µl of substrate for 20 minutes at

37°C. Thereafter, the reaction was terminated by addition of 100 µl CHCl3 and glycerol release was quantified in the aqueous supernatant using a commercially available kit (Free glycerol kit; Sigma Aldrich, St.Louis, USA). Phospholipids and lysophospholipids were solubilized by brief sonication (5 sec). Neutral lipids were emulsified on ice by sonication in the presence of PC at a molar ratio PC:RP (TO, CO, DO) of 1:7 (4x 1 min, 1 min break). The reaction buffer contained 1 mM substrate, 20 mM Tris-HCl (pH 8.0), 100 mM NaCl, and 1 mM defatted BSA. Additionally,

10 µl of purified ABHD6 (approx. 0.2 µg) was incubated with 40 µl of substrate for 20 minutes at 37°C. Alternatively, LPG hydrolase activity was determined in a buffer containing 50 mM Tris/HCl pH 8, 100 mM NaCl, 1 mM EDTA and 4.8 mM CHAPS. All reactions were terminated by heat inactivation of the enzyme at 70°C for 1 min. The release of free fatty acids was quantified enzymatically (Nefa C kit; Wako Chemicals, Germany).

Statistical Analysis

Extended Experimental Procedures References:

Bligh E.G., and Dyer W.J. (1959). A rapid method for total lipid extraction and purification. Can. J. Biochem. Physiol. 37, 911-917.

Brown, J.M., Boysen, M.S., Chung, S., Fabiyi, O., Morrison, R.F., and McIntosh, M.K. (2004). Conjugated linoleic acid induces human adipocyte delipidation: autocrine/paracrine regulation of MEK/ERK signaling by adipocytokines. J. Biol. Chem. 279, 26735-26747.

Brown, J.M. Betters, J.L., Lord, C., Ma, Y., Han, X., Yang, K., Alger, H.M., Melchior, J., Sawyer, J.K., Shah, R., et al. (2010) CGI-58 knockdown in mice causes hepatic steatosis but prevents diet-induced obesity and glucose intolerance. J. Lipid Res. 51, 3306-3316.

Brown, J.M., Chung, S., Sawyer, J.K., Degirolamo, C., Alger, H.M., Nguyen, T., Zhu, X., Duong, M.N., Wibley, A.L., Shah, R., et al. (2008a) Inhibition of stearoyl-coenzyme A desaturase 1 dissociates insulin resistance and obesity from atherosclerosis. Circulation 118, 1467-1475.

Brown, J.M., Bell, T.A. 3rd, Alger, H.M., Sawyer, J.K., Smith, T.L., Kelley, K., Shah, R., Wilson, M.D., Davis, M.A., Lee, R.G., et al. (2008b) Targeted depletion of hepatic ACAT2-driven cholesterol esterification reveals a non-biliary route for fecal sterol loss. J. Biol. Chem. 16, 10522-10534.

Callender, H.L., Forrester, J.S., Ivanova, P., Preininger, A., Milne, S., and Brown, H.A. (2007). Quantification of diacylglycerol species from cellular extracts by electrospray ionization mass spectrometry using a linear regression algorithm. Anal. Chem. 79, 263-272.

Crooke, R.M., Graham, M.J., Lemonidis, K.M., Whipple, C.P., Koo, S., and Perera, R.J. (2005). An apolipoprotein B antisense oligonucleotide lowers LDL cholesterol in hyperlipidemic mice without causing hepatic steatosis. J. Lipid Res. 46, 872-884.

Ivanova, P.T., Milne, S.B., Byrne, M.O., Xiang, Y., and Brown H.A. (2007). Glycerophospholipid identification and quantitation by electrospray ionization mass spectrometry. Meth. Enzymol. 432, 21-57.

Irizarry, R.A., Hobbs, B., Collin, F., Beazer-Barclay, Y.D., Antonellis, K.J., Scherf, U., and Speed, T.P. (2003). Exploration, normalization and summaries of high density oligonucleotide array probe level data. Biostatistics 4, 249-264.

Jandacek, R., Heubi, J.E., and Tso, P. (2004). A novel, noninvasive method for the measurement of intestinal fat aborption. Gastroenterology 127, 139-144.

Lord, C.C., Betters, J.L., Ivanova, P.T., Milne, S.B., Myers, D.S., Madenspacher, J, Thomas, G., Chung, S., Liu, M., Davis, M.A. et al. (2012). CGI-58/ABHD5-derived signaling lipids regulate systemic inflammation and insulin action. Diabetes 61, 355-363.

Myers, D.S., Ivanova, P.T., Milne, S.B., and Brown, H.A. (2011). Quantitative analysis of glycerophospholipids by LC-MS: Acquisition, data handling and interpretation. Biochim. Biophys. Acta 1811, 748-757.

Olson, J.D., Walb, M.C., Moore, J.E., Attia, A., Sawyer, H.L., McBride, J.E., Wheeler, K.T., Miller, M.S., and Munley, M.T. (2012) A gated-7T MRI technique for tracking lung tumor development and progression in mice after exposure to low doses of ionizing radiation. Radiat. Res. 178, 321-327.

Saghatelian, A., Trauger, S.A., Want, E.J., Hawkins, E.G., Siuzdak, G., and Cravatt, B.F. (2004) Assignment of endogenous substrates to enzymes by global metabolite profiling. Biochemistry 43, 14332-14339.

Shimano, H., Horton, J.D., Hammer, R.E., Shimomura, I., Brown, M.S., and Goldstein, J.L. (1996). Overproduction of cholesterol and fatty acids causes massive liver enlargement in transgenic mice expressing truncated SREBP-1a, J. Clin. Invest. 98, 1575-1584.

CHAPTER III

IMPACT OF OTHER SERINE HYDROLASES ON LIPID METABOLISM

Gwynneth Thomas, Caleb Lord, J. Mark Brown

Portions of the following chapter were published in Biochimica Biophysica Acta. Gwynneth

Thomas performed experiments and assisted in writing both manuscripts this chapter encompasses. Caleb Lord assisted with writing and Mark Brown assisted with study design and writing.

Introduction

The α/β hydrolase fold domain (ABHD) family is part of a superfamily of proteins. This superfamily contains proteases, lipases, esterases, dehalogenases, peroxidases and epoxide hydrolases. The conserved structural motifs and presentation in all reported genomes, predicts common roles for the ABHD proteins in lipid metabolism. The canonical α/β hydrolase fold is made up of 8 mostly parallel β-strands (Fig 1). There is also a catalytic triad located in the loop region which in several family members contains the active nucleophile (Ser, Cys, or Asp).

Figure 1. Canonical structure of the α/β hydrolase fold (adapted from Nardini and Dijkstra [1]). The α/β Fold is an 8 stranded mostly parallel structure; β-sheets denoted as arrows and α- helices denoted as cylinders. Highly conserved catalytic triad containing a histidine, an acid, and a nucleophile (typically a serine residue in mammalian proteins) are represented by spheres.

The presence of both a catalytic serine and an acyltransferase domain in several members gives potential ability of these proteins to both hydrolyze and acylate complex lipids (Fig 2). Even though the functions and substrates of many members of this family remain unknown, there remains a growing interest in the physiological significance of this family in lipid metabolism and disease.

Fig 2.The Human ABHD Family. A. Phylogenetic relationship of the human ABHD proteins based on Clustal W alignment with Posson correction. The numbers at the right indicate the number of amino acid residues in the full-length human protein. Red lines represent the predicted active site nucleophile, and blue lines indicate the predicted acyltransferase motif (HXXXD) when present. B. The conserved nucleophilic elbow and acyltransferase motifs “-“ indicates that the motif is not present in the human proteins.

ABHD2: A suppressor of smooth muscle cell migration and pulmonary emphysema

Human ABHD2, previously known as lung alpha/beta hydrolase 2 (LABH2), is a 425 residue protein (48 kDa) encoded by 11 exons located on chromosome 15q26.1. ABHD2 is expressed in multiple tissues in mice, with highest expression in the lung, adrenal gland, and brain (Fig. 3).

ABHD2 is predicted to be a single-pass type II membrane protein, although this has never been experimentally determined. ABHD2 is predicted to possess hydratase catalytic activity [2-5], but its true biochemical function remains unclear.

Fig 3. Tissue Distribution of the ABHD family of enzymes in liver (Li), small intestine (SI1- 3), white adipose tissue (WA), brown adipose tissue (BA), heart (He), lung (Lu), spleen (Sp), kidney (Ki), testes (Te), muscle (Mu), brian (Br), and adrenal (Ad). C57/bl6N male mice were maintained on standard rodent chow until 18 weeks of age. Total RNA was extracted from individual mouse tissues, and an equal amount of total RNA from each sample in each group (n=4) was pooled for each tissue, and relative mRNA abundance was measured using quantitative real-time PCR (normalized to cyclophilin B). The range of threshold cycle (CT) values for each ABHD across tissues is listed above each panel. All PCR amplicons were verified to correct size by gel electrophoresis and specificity of primer pairs was confirmed by DNA sequencing. Work done by Gwynneth Thomas

ABHD2 has been previously linked to the migration of vascular smooth muscle cells (SMC), where it is initially expressed in endothelial cells with expression shifting to SMCs to suppress cellular migration and development of intimal hyperplasia [2-4]. ABHD2 is downregulated by lamivudine, a drug used to treat HIV patients, while the level of ABHD2 mRNA increases during monocyte to macrophage differentiation [3, 5]. Suppression of ABHD2 function in mice has been achieved in vivo using both antisense oligonucleotides (ASO) [5] and gene trapping techniques [2]. Knockdown of ABHD2 using ASO treatment in mice has been suggested to block hepatitis B virus propagation in a dose dependent manner without inducing apoptosis, suggesting it plays an important role in the virus’s replication process [5]. Global deletion of

ABHD2 by gene trapping results in a reduction in the number of alveolar type II cells in the lung and a striking accumulation of macrophages in the lungs in aged mice [2]. ABHD2 deficiency is also accompanied by an increase in inflammatory cytokines, increased apoptotic cells, reduced surfactant phospholipids, and a protease/anti-protease imbalance in the lung causing age-related emphysema [2]. In addition to its role in the lung, ABHD2 appears to play an important role in macrophage infiltration to atherosclerotic lesions [3]. ABHD2 expression is increased in patients with unstable angina in atherosclerotic lesions, specifically in neointimal lesions where it colocalizes with CD68+ cells [3]. To examine a potential role of ABHD2 in the metabolic syndrome, a knockout mouse model was used and compared to age-matched wild type (WT) and heterozygote mice. After being fed a high fat diet (45% of energy as lard, no added cholesterol) for 12 weeks, ABHD2 knockout mice show no difference in body weight, hepatic steatosis or insulin resistance compared to WT and heterozygotes (Fig 4). Collectively, ABHD2 seems to play an important role in chronic disease that involve monocyte/macrophage recruitment (i.e. atherosclerosis and emphysema), but may not play a significant part in control of disease states

A Body Weight B Liver Weight C GTT - HFD 500 ABHD2+/ ABHD2+/+ 45 + 1.8 ABHD2+/- 450 ABHD2-/- ABHD2+/ 400

40 - 1.6

35 1.4 350 30 1.2 300 250 25 1 200 20 0.8 Glucose (mg/dL) Glucose 150 ABHD2+/

15 0.6 + Body Weight (g) Weight Body Liver Weight (g) Weight Liver 100 ABHD2+/ 10 0.4 - 50 ABHD2-/- 5 0.2 0 0 0 0 30 60 120 Chow HFD Chow HFD Time (minutes)

Fig 4. ABHD2 -/- Measurements. A. Terminal body weights of WT, heterozygote and knockout ABHD2 mice after 12 weeks on chow or high-fat diet. B. Terminal liver weights of WT, heterozygote and knockout ABHD2 mice after 12 weeks on chow or high-fat diet. C. Glucose tolerance test for WT, heterozygote and knockout ABHD2 mice after 11 weeks n chow or high- fat diet. Data represent the mean + the SEM (n=6-8 per group); *p <0.05 (versus WT group). Work done by Gwynneth Thomas

associated with the metabolic syndrome. Still, the true physiological substrates and products of

ABHD2 remain unidentified.

ABHD4: An enzymatic regulator of endocannabinoid signaling and suppressor of tumor growth

Human ABHD4 is a 342 residue protein (39 kDa) encoded by 7 exons located on chromosome

14q11.2. Murine ABHD4 is ubiquitously expressed in multiple tissues, with highest expression in brain, small intestine, kidney and testis (Fig. 3), yet the subcellular localization of ABHD4 remains uncharacterized. Although vastly understudied, ABHD4 has recently been suggested to play a role in tumor suppression through limiting cell proliferation and cell cycling [6]. ABHD4

75 has also been described as a lyso-N-acyl phosphatidylethanolamine lipase, capable of hydrolyzing both N-acyl phosphatidylethanolamine (NAPE) and lysoNAPE to generate glycerophospho-arachidonyl ethanolamine (Gp-AEA) [6-8]. ABHD4 shows no enzymatic activity towards other lysophospholipid substrates tested, but rather with just a wide range of lyso-NAPE substrates, including the anandamide (AEA) precursor N-arachidonoyl lyso-NAPE

[6, 7]. Lipopolysaccharide (LPS) has previously been shown to induce AEA synthesis, yet knockdown of ABHD4 does not affect LPS-induced AEA synthesis [8]. Cells treated with LPS for 90 minutes results in only a modest 27% increase in ABHD4 mRNA, suggesting that

ABHD4 may be dominant in only the long term synthesis of AEA rather than the acute generation of the endocannabinoid in response to a LPS stimulus [8]. More recently, ABHD4 has also been suggested to play an important role in cancer metastasis through a mechanism that does not involve its role in AEA synthesis [9]. ABHD4 knockdown through the use of short- hairpin RNA (shRNA) has been shown to render cells resistant to anoikis, a process in which anchorage-dependent cells undergo cell death upon detachment from the extracellular matrix [9].

Knockdown of ABHD4 using the short-hairpin RNA also results in more moderate tumor growth compared to controls, suggesting it may be a target gene of the tumor suppressor gene, p53 [6].

ABHD4 has also been shown to be upregulated in in vitro matured (IVM) oocytes, again supporting its proposed role in tumor growth suppression [10]. Since ABHD4 plays a significant role in endocannabinoid signaling we wanted to examine the effects of knockdown in metabolically relevant tissues, such as the liver and adipose tissue. Utilizing antisense oligonucleotides (ASOs) and high-fat diet feeding (45% of energy as lard, no added cholesterol), we found similar protection from the metabolic syndrome to that of the ABHD6 ASO. After 12

weeks, the mice were protected from high fat-diet induced obesity, hepatic steatosis and insulin

resistance (Fig 5).

A B C Body Weight - HFD WAT Weight - HFD GTT - HFD 45 Saline 3 500 40 Control ASO 450 ABHD4 ASO * 2.5

400 30 2 350 25 300 1.5 20 * 250 15 1 200

Glucose (mg/dl) Glucose *

10 150 Body Weight (g) Weight Body WAT Weight (g) Weight WAT 0.5 100 5 * 50 0 0 0 * 0 30 60 120 Time (mins) D E Liver Weight - HFD Liver TG - HFD 2 200 1.8 180

1.6 160

1.4 140 1.2 120 1 100 0.8 80

0.6 60 mg/mg protein mg/mg Liver Weight (g) Weight Liver 0.4 40 * 0.2 20 0 0

Fig 5. Saline, Control and ABHD4 ASO treatments; IP injection 2x/week (25mg/kg BW) for 12 weeks. A. Terminal body weights of Saline, Control and ABHD4 ASO treated mice after 12 weeks on high-fat diet. B. Terminal white adipose weights of Saline, Control and ABHD4 ASO treated mice after 12 weeks on high-fat diet. C. Glucose tolerance test for Saline, Control and ABHD4 ASO treated mice after 12 weeks on high-fat diet. D. Terminal liver weights of Saline, Control and ABHD4 ASO treated mice after 12 weeks on high-fat diet. E. Terminal liver triglycerides (TG) of Saline, Control and ABHD4 ASO treated mice after 12 weeks on high-fat diet. Data represent the mean + the SEM (n=8-10 per group); *p<0.05 (versus saline group). Work done by Gwynneth Thomas

The ABHD4 ASO treated mice also displayed increases in activity and energy expenditure (data

not shown), which may be contributing causes to the improvements in the metabolic syndrome.

Interestingly, in comparison to an ASO treated mouse, an ABHD4 knockout animal is not

protected from obesity, hepatic steatosis or insulin resistance after 12 weeks of high-fat diet

feeding (Fig 6). A B C Body Weight - HFD Liver Weight - HFD WAT Weight - HFD 50 3 3 45

40 2 3

35 2 30 2 25 2 20 1 1 15

10 1 1

Body Weight (g) Weight Body Body Weight (g) Weight Body 5 (g) Weight Body 0 0 0

D GTT - HFD WT Het 600 KO 500 400 300 200 100 0 0 30 60 120 Plasma Glucose (mg/dl) Glucose Plasma Time (minutes)

Fig 6. ABHD4-/- Measurements. A. Terminal body weights of WT (+/+), heterozygotes (+/-) and ABHD4 knockout (-/-) mice after 12 weeks of high-fat diet feeding. B. Terminal liver weights of WT, heterozygotes and ABHD4 knockout mice after 12 weeks of high-fat diet feeding. C. Terminal white adipose weight of WT, heterozygotes and ABHD4 knockout mice after 12 weeks of high-fat diet feeding. D. Glucose tolerance test for WT, heterozygotes, and ABHD4 knockout mice after 11 weeks of high-fat diet feeding. Date represent the mean + the

SEM (n=6-8 per group); *p <0.05 (versus WT group). Work done by Gwynneth Thomas and Kathryn Kelley

This may be in part due to contributions of ABHD4 to the endocannabinoid pathway and possible alterations in anandamide synthesis in multiple tissues.

Although physiological substrates for ABHD4 have been successfully identified as NAPE and lysoNAPE, and ABHD4 seems to play a role in cancer pathogenesis, additional studies are needed to understand the physiological function of ABHD4 in metabolic disease. This may include identification of alternative substrates, as well as pathways other than endocannabinoid pathway to which it is known to function in.

ABHD12: Regulator of endocannabinoid signaling and protector against PHARC development

Human ABHD12, also known as ABHD12A, c20orf22, and 2-arachidonoylglerol hydrolase, is a 398 residue protein (45 kDa) encoded by 13 exons on chromosome 20p11.21. ABHD12 is predicted to be a single-pass integral membrane protein with its active site facing the ER lumen or extracellular space based on N-linked glycosidase digestion studies [11, 12]. ABHD12 is ubiquitously expressed in mouse tissue with highest expression in the brain (Fig. 3), where it accounts for ~9% of total hydrolase activity toward the endocannabinoid 2-arachidonylglycerol, along with monoacylglycerol lipase and ABHD6 [11, 12, 13]. In vivo, inhibitor profiling demonstrates substrate specificity differences between ABHD12 and ABHD6, where ABHD12 prefers 1 (3) - and 2-isomers of arachidonoylglycerol [14]. In various brain cell types, specifically the microglia, ABHD12 transcripts are highly expressed [12]. ABHD12 is also abundant in other cell types such as macrophages and osteoclasts [12], and it is important to note

79 that ABHD12 mRNA is very abundant and ubiquitiously expressed in all mouse tissue examined

(Fig. 3). Much like ABHD6, ABHD12 also has no effect of the recovery of DSE however it does appears to traffic throughout the neuron [15]. Interestingly, mutations in ABHD12 have been shown to be causative of a neurodegenerative disease called polyneuropathy, hearing loss, ataxia, retinitis pigmentosa and cataract (PHARC) in humans [16]. This disorder is a slow, progressive neurologic disorder that displays a phenotype resembling Refsum disease, which is characterized by a biochemical defect in the degradation of phytanic acid and plasmalogen lipids [16]. Four different ABHD12 mutations have been associated with PHARC development suggesting a causal genotype-phenotype relationship [16]. It has been suggested that ABHD12 defects leads to PHARC based on its enzymatic ability to hydrolyze 2-AG, since the endocannabinoid has important functions in synaptic plasticity and neuroinflammation [16]. It was originally thought

ABHD12’s enzymatic ability to hydrolyze to 2-arachidonylglycerol may contribute to the development of PHARC, but a recent metabolic profiling study has identified a novel role for

ABHD12 acting as a lysophospholipase to regulate the progression of PHARC [17]. By coupling both untargeted and targeted lipidomic approaches in ABHD12 knockout (ABHD12-/-) mice,

Blankman and colleagues uncovered a massive accumulation of very long chain lysophosphatidylserine lipids in the brain [17]. Using a complimentary biochemical approach, the authors showed that recombinant ABHD12 has robust intrinsic lysophosphatidylserine lipase activity [17]. Interestingly, the accumulation of lysophosphatidylserine species in the brains of

ABHD12-/- mice preceded the development of age-dependent microglial activation and behavior phenotypes that resemble phenotypes seen in PHARC patients [17]. Collectively, this important set of studies demonstrates that ABHD12’s ability to hydrolyze lysophosphatidylserine lipids in brain lies at the center of the human disease PHARC [17].Collectively, the accumulation of

80 lysophosphatidylserine seen with ABHD12 mutations subsequently causes hyperactivation of proinflammatory signals [17]. A nonsense mutation in ABHD12 has also been associated with an autosomal recessive genetically heterogeneous disorder called Usher Syndrome, specifically

Usher Syndrome 3 [18]. Usher syndrome 3 is associated with congenital sensorineural hearing impairment, retinitis pigmentosa, hearing loss and cataracts representing a variant of PHARC

[18]. In addition, a genome-wide association study identified ABHD12 as a candidate gene associated with concentrations of liver enzymes in plasma, a widely used indicator of liver disease [19]. Finally, ABHD12 gene expression has also been associated with colorectal cancer development [20]. We sought to examine the role ABHD12 may play in lipid metabolism because of its important and similar roles to ABHD6 in endocannabinoid signaling. Therefore, we again utilized targeted antisense oligonucleotides (ASOs) to knockdown ABHD12 in metabolically relevant tissues while simultaneously feeding a high-fat diet (45% of energy as lard, no added cholesterol). Much like what we saw with ABHD6 and ABHD4 knockdown,

ABHD12 ASO treatment protects mice from diet induced obesity, hepatic steatosis and insulin resistance (Fig 7).

A B C Body Weight - HFD WAT Weight - HFD GTT Saline 50 Control ASO 3 ABHD12 ASO 40 500 * 2.5 450 30 2 * 400 1.5 350 20 300 1 10 250 0.5 200 0 0 150 *

Body Weight (g) Weight Body 100 WAT Weight (g) Weight WAT Glucose (mg/dl) Glucose 50 * 0 * 0 30 60 120 Time (mins) E D Liver Weight - HFD Liver TG - HFD 2

200

1.5 150

1 100

0.5 50

mg/mg protein mg/mg Liver Weight (g) * 0 0 Saline Control ABHD12 Saline Control ABHD12

Fig 7. Saline, Control and ABHD12 ASO Treatments; IP injection 2x/week (25mg/kg BW) for 12 weeks. A. Terminal body weights of Saline, Control and ABHD12 ASO treated mice after 12 weeks on high-fat diet. B. Terminal white adipose weights of Saline, Control and ABHD12 ASO treated mice after 12 weeks on high-fat diet. C. Glucose tolerance test for Saline, Control and ABHD12 ASO treated mice after 12 weeks on high-fat diet. D. Terminal liver weights of Saline, Control and ABHD12 ASO treated mice after 12 weeks on high-fat diet. E. Terminal liver triglycerides (TG) of Saline, Control and ABHD12 ASO treated mice after 12 weeks on high-fat diet. Data represent the mean + the SEM (n=8-10 per group); *p<0.05 (versus saline group). Work done by Gwynneth Thomas.

These mice also display similar increases in energy expenditure and activity (data not shown).

ABHD12 ASO treatment also increases several genes associated with fatty acid metabolism

(data not shown). Therefore, ABHD12’s effects may truly be regulated through the endocannabinoid pathway or through another mechanism to regulate lipid metabolism. Although one physiological substrate for ABHD12 has been successfully identified as 2-AG, the

82 physiological role of this activity remains unclear. A deeper look into ABHD12’s shared activity with ABHD6 as a 2-AG hydrolase in the brain and in peripheral tissues may uncover 2-AG or alternative substrates of ABHD12 contributions to metabolic disease.

EXPERIMENTAL PROCEDURES

Mice

For ABHD4 and ABHD12 knockdown studies, at 6-8 weeks of age Male C57BL/6N mice

(Harlan) were either maintained on standard rodent chow or switched to a high fat diet for a period of 12 weeks, and simultaneously injected with antisense oligonucleotides biweekly (25 mg/kg BW) targeting knockdown of ABHD4 and ABHD12 as previously described (Lord et al.,

2011; Brown et al., 2010; Brown et al., 2008a; Brown et al., 2008b). Intraperitoneal glucose tolerance tests were performed essentially as previously described (Brown et al., 2008a, Brown et al., 2010) in mice treated with diet and ASO for 10-11 weeks. Metabolic measurements were conducted in the comprehensive lab animal monitoring systems (CLAMS) from Columbus

Instruments. For ABHD2 and ABHD4 knockout studies, mice were genotyped and grouped according to age match. They were then either maintained on standard rodent chow or switched to a high fat diet for a period of 12 weeks. Intraperitoneal glucose tolerance tests were performed essentially as previously described (Brown et al., 2008a, Brown et al., 2010) in all groups after

10-11 weeks on diet.

Microarray and Quantitative Real-Time PCR Analysis of Gene Expression

Tissue RNA extraction was performed as previously described for all mRNA analyses (Lord et

83 al., 2011; Brown et al., 2010; Brown et al., 2008a; Brown et al., 2008b). Quantitative real time

PCR (Q-PCR) analyses were conducted as previously described (Lord et al., 2011; Brown et al.,

2010; Brown et al., 2008a; Brown et al., 2008b).

Hepatic Lipid Analyses

Extraction of liver lipids and quantification of molecular species by mass spectrometry was performed as previously described (Lord et al., 2011; Ivanova et al., 2007; Myers et al., 2011;

Callendar et al., 2007; Saghatelian et al., 2004).

REFERENCES

[1] M. Nardini, B.W. Dijkstra, Alpha/beta hydrolase fold enzymes the family keeps growing,

Curr. Opin. Struct. Biol. 9 (1999) 732-737

[2] S. Jin, G. Zhao, Z. Li, Y. Nishimoto, Y. Isohama, J. Shen, T. Ito, M. Takeya, K. Araki, P. He,

K. Yamamura, Age-related pulmonary emphysema in mice lacking alpha/beta hydrolase domain containing 2 gene, Biochem. Biophys. Res. Commun. 380 (2009) 419-424

[3] K. Miyata, M. Nakayama, S. Mizuta, S. Hokimoto, K. Sugamura, S. Oshima, Y. Oike, S.

Sugiyama, H. Ogawa, K. Yamamura, Elevated mature macrophage expression of human

ABHD2 gene in vulnerable plaque, Biochem. Biophys. Res. Commun. 365 (2008) 207-213

[4] K. Miyata, Y. Oike, T. Hoshii, H. Maekawa, H. Ogawa, T. Suda, K. Araki, K. Yamamura,

Increase of smooth muscle cell migration and of intimal hyperplasia in mice lacking the alpha/beta hydrolase domain containing 2 gene, Biochem. Biophys. Res. Commun 329 (2005)

296-304

[5] X. Ding, J. Yang, S. Wang, Antisense oligonucleotides targeting a/b-hydrolase domain containing 2 block human hepatitis B virus propagation, Oligonucleotides 21 (2011) 77-84

[6] C.A. Brady, D. Jiang, S.S. Mello, T.M. Johnson, L.A. Jarvis, M.M. Kozak, D. Kenzelmann

Broz, S. Basak, E.J. Park, M.E. McLaughlin, A.N. Karnezis, L.D. Attardi, Distinct p53 transcriptional programs dictate acute DNA-damage responses and tumor suppresion, Cell 145

(2011) 571-583

[7] G.M. Simon, B.F. Cravatt, Endocannabinoid biosynthesis proceeding through glycerophospho-N-acyl ethanolamine and a role for alpha/beta hydrolase 4 in this pathway, J.

Biol. Chem. 281 (2006) 26465-26472

[8] J. Liu, L. Wang, J. Harbey-White, B.X. Huang, H.Y. Kim, S. Luquet, R.D. Palmiter, G.

Krystal, R. Rai, A. Mahadevan, R.K. Razdan, G. Kunos, Multiple pathways involved in the biosynthesis of anandamide, Neuropharmacology 54 (2008) 1-7

[9] C.D. Simpson, R. Hurren, D. Kasimer, N. MacLean, Y. Eberhard, T. Ketela, J. Moffat, A.D.

Schimmer, A genome wide shRNA screen identifies alpha/beta hydrolase domain containing 4

(ABHD4) as a novel regulator of anoikis resistance, Apoptosis 17 (2012) 666-678

[10] M.G. Katz-Jaffe, B.R. McCallie, K.A. Preis, J. Filipovits, D.K. Gardner, Transcriptome analysis of in vivo and in vitro matured bovine MII oocytes, Theriogenology 71 (2009) 939-946

[11] J.L .Blankman, G.M. Simon, B.F .Cravatt, A comprehensive profile of brain enzymes that hydrolyze the endocannabinoid 2-arachidonylglycerol, Chem. Biol. 14 (2007) 1347-1356

[12] J.R. Savinainen, S.M. Saario, J.T. Laitinen, The serine hydrolases MAGL, ABHD6 and

ABHD12 as guardians of 2-arachidonoylglycerol signalling through cannabinoid receptors. Acta.

Physiol. (Oxf.) 204 (2012) 267-276

[13] W.R. Marrs, J.L. Blankman, E.A. Horne, A. Thomazeau, Y.H. Lin, J. Coy, A.L. Bodor,

G.G. Muccioli, S.S. Hu, G. Woodruff, S. Fung, M. Lafourcade, J.P. Alexander, J.Z. Long, W. Li,

C. Xu, T. Moller, K. Mackie, O.J. Manzoni, B.F. Cravatt, N. Stella, The serine hydrolase

ABHD6 controls the accumulation and efficacy of 2-AG at cannabinoid receptors. Nat.

Neurosci. 13 (2010) 951-957

[14] D. Navia-Paldanius, J.R. Savinainen, J.T. Latinen, Biochemical and pharmacological characterization of human a/b-hydrolase domain containing 6 (ABHD6) and 12 (ABHD12), J.

Lipid Res. 53 (2012) 2413-2424

[15] A. Straiker, J. Wager-Miller, S.S. Hu, J.L. Blankman, B.F. Cravatt, K. Mackie, COX-2 and fatty acid amide hydrolase can regulate the time course of depolarization-induced suppression of excitation, Br.J. Pharmacol. 164 (2011) 1672-1683

[16] T. Fiskerstrand, D. H’Mida-Ben Brahim, S. Johansson, A. M’Zahem, B.I. Haukanes, N.

Drouot, J. Zimmermann, A.J. Cole, C. Vedeler, C. Bredrup, M. Assoum, M. Tazir, T.

Klockgether, A. Hamri, V.M. Steen, H. Boman, L.A. Blindoff, M. Koenig, P.M. Knappskog,

Mutations in ABHD12 cause the neurodegenerative disease PHARC: An inborn error of endocannabinoid metabolism. Am. J. Hum. Genet. 87 (2010) 410-417

[17] J.L. Blankman, J.Z. Long, S. A. Trauger, G. Siuzdak, B.F. Cravatt, ABHD12 controls brain lysophosphatidylserine pathways that are deregulated in a murine model of the neurodegenerative disease PHARC. PNAS 110 (2013) 1500-1505

[18] T. Eisenberger, R. Slim, A. Mansour, M. Nauck, G. Numberg, C. Decker, C. Dafinger, I.

Ebermann, C. Bergmann, H.J. Boltz, Targeted next-generation sequencing identifies a homozygous nonsense mutation in ABHD12, the gene underlying PHARC in a family clinically diagnosed with Usher syndrome type 3. Orphanet J. Rare Dis. 7 (2012) 59

[19] Chambers JC1, Zhang W, Sehmi J, Li X, Wass MN, Van der Harst P, Holm H, Sanna S,

Kavousi M, Baumeister SE, Coin LJ, Deng G, Gieger C, Heard-Costa NL, Hottenga JJ, Kühnel

B, Kumar V, Lagou V, Liang L, Luan J, Vidal PM, Mateo Leach I, O'Reilly PF, Peden JF,

Rahmioglu N, Soininen P, Speliotes EK, Yuan X, Thorleifsson G, Alizadeh BZ, Atwood LD,

Borecki IB, Brown MJ, Charoen P, Cucca F, Das D, de Geus EJ, Dixon AL, Döring A, Ehret G,

Eyjolfsson GI, Farrall M, Forouhi NG, Friedrich N, Goessling W, Gudbjartsson DF, Harris TB,

Hartikainen AL, Heath S, Hirschfield GM, Hofman A, Homuth G, Hyppönen E, Janssen HL,

Johnson T, Kangas AJ, Kema IP, Kühn JP, Lai S, Lathrop M, Lerch MM, Li Y, Liang TJ, Lin

JP, Loos RJ, Martin NG, Moffatt MF, Montgomery GW, Munroe PB, Musunuru K, Nakamura

Y, O'Donnell CJ, Olafsson I, Penninx BW, Pouta A, Prins BP, Prokopenko I, Puls R, Ruokonen

A, Savolainen MJ, Schlessinger D, Schouten JN, Seedorf U, Sen-Chowdhry S, Siminovitch KA,

Smit JH, Spector TD, Tan W, Teslovich TM, Tukiainen T, Uitterlinden AG, Van der Klauw

MM, Vasan RS, Wallace C, Wallaschofski H, Wichmann HE, Willemsen G, Würtz P, Xu C,

Yerges-Armstrong LM; Alcohol Genome-wide Association (AlcGen) Consortium; Diabetes

Genetics Replication and Meta-analyses (DIAGRAM+) Study; Genetic Investigation of

Anthropometric Traits (GIANT) Consortium; Global Lipids Genetics Consortium; Genetics of

Liver Disease (GOLD) Consortium; International Consortium for Blood Pressure (ICBP-

GWAS); Meta-analyses of Glucose and Insulin-Related Traits Consortium (MAGIC), Abecasis

GR, Ahmadi KR, Boomsma DI, Caulfield M, Cookson WO, van Duijn CM, Froguel P, Matsuda

K, McCarthy MI, Meisinger C, Mooser V, Pietiläinen KH, Schumann G, Snieder H, Sternberg

MJ, Stolk RP, Thomas HC, Thorsteinsdottir U, Uda M, Waeber G, Wareham NJ, Waterworth

DM, Watkins H, Whitfield JB, Witteman JC, Wolffenbuttel BH, Fox CS, Ala-Korpela M,

Stefansson K, Vollenweider P, Völzke H, Schadt EE, Scott J, Järvelin MR, Elliott P, Kooner JS.

Genome-wide association study identifies loci influencing concentrations of liver enzymes in plasma. Nat. Genet. 43 (2011) 1131-1138

[20] T. Yosida, T. Kobayashi, M. Itoda, T. Muto, K. Miyaguchi, K. Mogushi, S. Shoji, K.

Shimokawa, S. Iida, H. Uetake, T. Ishikawa, K. Sugihara, H. Mizushima, H. Tanaka. Clinical

88 omics analysis of colorectal cancer incorporating copy number aberrations and gene expression data. Cancer Inform. 9 (2010) 147-161

CHAPTER IV

SUMMARY AND CONCLUSIONS

Introduction

Although it is unlikely that all members of the ABHD protein family share a common biological role, there is early evidence to support this concept. In particular, of the ABHD enzymes with known substrates, most of them share the ability to either hydrolyze or synthesize different glycerophospholipid species [1-7]. For example, ABHD3, ABHD4, and ABHD6 hydrolyze the glycerophospholipids C14-lysophosphatidylcholine, N-acyl phosphatidylethanolamine (NAPE), and 2-arachidonylglycerol, respectively [1, 2, 8]. Also, ABHD5 has recently been shown to acylate lysophosphatidic acid to form phosphatidic acid [4, 5]. In further support of the ABHD’s role in glycerophospholipid metabolism, there is a significant decrease in phosphatidylcholine in the bronchoalveolar lavage of ABHD2 knockout mice [9], and more recently ABHD3 reached genome-wide significance as a predictor of plasma phospholipid levels in humans [10].

Although, much additional work is needed to identify physiological substrates for the remaining

ABHD family memebers, it will be important to consider glycerophospholipids as clear potential substrates. It is well known that glycerophospholipids serve as key structural elements for the cell membrane, but many aslo act as key intermediates in neurotransmission and cellular signaling cascades. In line with this, another shared feature of the ABHD family is that several members have been directly implicated in signaling pathways that involve enzymatic synthesis or degradation of key signaling lipids. For instance, both ABHD6 and ABHD12 have been identified as 2-AG hydrolases [6], linking these enzymes to the termination of acute endocannabinoid signalling [6, 7, 11, 12]. In contrast, ABHD4 has been implicated as a NAPE

90 and lysoNAPE lipase, implicating ABHD4 in endocannabinoid synthesis [2, 3]. Further, ABHD5 has recently been shown to be necessary for the generation of phosphatidic acid and other signaling lipids in response to inflammatory stimuli [13], which then have many secondary effects on both insulin signaling and energy metabolism. Although this still needs to be experimentally tested, ABHD3’s ability to hydrolyze C-14 lysophosphatidylcholine is likely to alter cellular signaling, given that lysophosphatidylcholine species are potent regulators of GPCR and TRPC5 signaling [14, 15]. Collectively, several members of the ABHD protein family share the ability to metabolize diverse glycerophospholipid species that are intimately involved in cellular signaling. These shared features of the known ABHD’s will likely provide important clues as progress is made with the characterization of the rest of the ABHD protein family. Also, along with the ABHD family, there are more than 200 serine hydrolases present in the human genome. Within the groups of small-molecule hydrolases, extracellular neutral lipid lipases, phospholipase A2 enzymes, peptidases, protein- and glycan-modifying hydrolases, and other phospholipases several remain understudied [16]. Members of these groups may share similar abilities to hydrolyze or synthesize specific substrates that play important roles in human diseases.

ABHD6 Protects from the Metabolic Syndrome

Some of the major findings surrounding ABHD6 demonstrate the following: (1) peripheral knockdown of ABHD6 protects mice from high-fat-diet induced obesity; (2) ABHD6 knockdown protects mice from high-fat-diet-induced hepatic steatosis and associated insulin resistance. ABHD6 has been shown to regulate the ECB system in the brain due to its ability to hydrolyze specific pools of 2-AG [6, 7, 17]. Our work confirmed that ABHD6 exhibits MAG lipase activity in vitro also but it is a minor contributor to MAG lipolysis and ECB signaling in

91 mouse liver. This finding is supported by the fact that total MAG levels do not change, 2-AG and anandamide levels are not elevated, and acute CB1-driven activation of ERK-MAPK is unaltered with ABHD6 knockdown, arguing against CB1 receptor desensitization. It is important to contrast these findings with the very striking accumulation of hepatic 2-AG, marked reduction in hepatic MAG lipase activity, and CB1 Receptor desensitization seen in MAGL-/- mice and mice treated chronically with a specific MAGL inhibitor [18, 19].

Possible Mechanisms for ABHD6

We observed that with ABHD6 ASO treatment, mice were protected from high-fat-diet-induced hepatic steatosis. This appears to be largely attributable to suppression of de novo lipogenesis. It appears that MAGL lipase is the predominant MAG lipase in mouse liver. However, we did observe minor elevations in 18:1 and 18:2 MAGS, making it possible that ABHD6’s ability to hydrolyze these lipids may affect other signaling processes regulating hepatic de novo lipogenesis.

ABHD6 is a Promiscuous Lysophospholipid Lipase

ABHD6 only contributes <5% to the total 2-AG hydrolase activity, whereas MAGL accounts for the vast majority [6]. Our studies suggest that ABHD6’s additional ability to hydrolyze a variety of lipid substrates may be important in linking lipid mediator signaling to the metabolic adaptations resulting from high-fat-diet feeding. By utilizing a targeted lipidomics approach to identify substrates of ABHD6 in mouse liver, we found and verified LPG as a bona fide substrate in vitro and in vivo. LPG is considered a bioactive lipid, although its receptor remains to be identified [20, 21]. Lysophospholipids have previously been implicated in promoting metabolic disease via their ability to dictate membrane dynamics and initiate cell signaling, particularly in

92 immune cells [22, 23]. We assume that when we knockdown ABHD6, this initiates defective degradation of LPG. This may favor the re-esterification of LPG to PG, which may explain why we see elevated levels of PG in the ABHD6-knockdown liver. PG is also an important precursor for other complex lipids including cardiolipin and bis (monoacylglycerol) phosphate [24].

Therefore, changes in ABHD6-driven LPG metabolism could alter many mitochondrial or lysosomal metabolic processes. In fact, it was recently found in a genome-wide association study

(GWAS) in Pima Indians, that the lysophosphatidylglycerol acyltransferase 1 (LPGAT1) loci was predictive of body mass index (BMI) [25]. This provides even greater evidence that LPG metabolism and its possible regulation by ABHD6 may influence BMI and adiposity.

ABHD6 as a Drug Target

There are several important factors to consider for developing ABHD6 as a drug target. For example, it will be important to determine how ABHD6 expression and subcellular distribution are regulated in diverse cell and tissue contexts. ABHD6 has been predicted to be a single-pass membrane protein with a cytosolic orientation [6]; however this has never been experimentally tested and concluded. Furthermore, it will be essential to evaluate whether sue-selective inhibitors will be necessary for safe and effective prevention of metabolic disease. Another important consideration will be whether chronic ABHD6 inhibition results in CB1 receptor desensitization in the brain or other receptor signaling abnormalities. We only examined CB1 receptor sensitivity in the liver, where ABHD6 inhibition did not cause CB1 receptor desensitization, however other tissues also need to be examined. We have provided key information in regards to ABHD6’s biochemical and physiological role in certain peripheral tissues (liver, adipose and kidney) given that the ASO exhibits tissue-selective inhibition. It remains important to examine ABHD6’s primary role in other tissues where it is abundantly

93 expressed (brain, small intestine, pancreas, and skeletal muscle) being that these tissues are also key regulatory sites for energy metabolism.

The Small Molecule Approach

It is important to compare and contrast the results of inhibiting ABHD6 with systemic small molecule inhibitors versus a more selective approach using ASO technology. Although both

ASO-mediated and WWL-70-mediated inhibitor of ABHD6 protected against high-fat-diet induced obesity and glucose intolerance, only ABHD6 ASO treatment improved hepatic steatosis. The mechanism underlying this difference is unclear, but most likely stems from systemic versus tissue-restricted inhibition. Interestingly, ASO-mediated inhibition of ABHD6 was not associated with alterations in food intake at any time point, whereas WWL-70 treatment caused a 20% reduction in food intake. This would suggest that the mechanism by which WWL-

70 protected against obesity and glucose intolerance is likely drive by hypophagia, whereas

ASO-mediated inhibition more specifically dampened hepatic lipogenesis and increased activity and energy expenditure. This could be due in large part to the fact that the small molecule,

WWL-70 has the ability to cross the blood brain barrier, whereas the ASO does not.

Other Considerations

In addition to further studies with selective ABHD6 inhibitors, the generation of global and conditional knockout mousse models is necessary to define the tissue-specific role for ABHD6 in chronic disease of altered lipid metabolism. We have identified ABHD6 as a key determinant in the pathogenesis of high-fat-diet-induced obesity, insulin resistance, and hepatic steatosis.

Furthermore, we have uncovered lipid substrates of ABHD6 by coupling in vivo lipidomic and in vitro biochemical approaches. The use of this multidisciplinary approach will prove useful for

94 mapping natural enzyme-substrate relationships for other enzymes that have not yet been characterized. In order to support the process of interrogation, it remains critical that good enzyme inhibitors or knockout mouse models continue to be developed. Our findings suggest that ABHD6 is a key lipase involved in monoacylglycerol and lysophospholipid hydrolysis. The development of an ABHD6 small-molecular inhibitor that is peripherally restricted holds great promise as a therapeutic for obesity, nonalcoholic fatty liver disease, and type II diabetes.

Future of the ABHD Family

Although the functional annotation the ABHD protein family is in its infancy, ongoing studies have great promise in advancing our fundamental understanding of lipid metabolism in human disease. The ABHD enzyme family shares common features that predict roles in lipid metabolism, signal transduction, and metabolic disease. More importantly, several ABHD family members have been associated with several human diseases of altered lipid metabolism. Given that serine hydrolases are easily amenable to chemical inhibition, development of small molecular inhibitors will become instrumental in defining the biology associated with each

ABHD enzyme. Furthermore, the generation of global and conditional knockout mouse models will be the key in defining biochemical and physiological roles of ABHD enzymes in vivo.

Progress on these fronts has important implications for targeting ABHD enzymes for the treatment or, more importantly, the prevention of lipid metabolic disease and diseases of altered signal transduction.

REFERENCES

[1] J.Z. Long, J.S. Cisar, D. Milliken S. Niessen, C. Wang, S.A. Trauger, G. Siuzdak, B.F.

Cravatt, Metabolomics annotates ABHD3 as a physiologic regulator of medium-chain phospholipids, Nat. Chem. Biol. 7 (2011) 763-765

[2] G.M Simon, B.F. Cravatt, Endocannabinoid biosynthesis proceeding through glycerophospho-N-acyl ethanolamineand a role for alpha/beta-hydrolase 4 in this pathway, J.

Biol. Chem. 281 (2006) 26465-26472

[3] J. Liu, L. Wang, J. Harvey-White, B.X. Huang, H.Y. Kim, S. Luquet, R.D. Palmiter, G.

Krystal, R. Rai, A. Mahadevan, R.K. Razdan, G. Kunos, Multiple pathways involved in the biosynthesis of anandamide, Neuropharmacology 54 (2008) 1-7

[4] A.K. Ghosh, G. Ramakrishnan, C. Chandramohan, R. Rajeskharan, CGI-58, the causative gene for Chanarin-Dorfman syndrome, mediates acylation of lysophosphatidic acid, J. Biol.

Chem. 283 (2008) 24525-24533

[5] G. Montero-Moran, J.M. Caviglia, D. McMahon, A. Rothenberg, V. Subramanian, Z. Xu, S.

Lara-Gonzalex, J. Storch, G.M. Carman, D.L. Brasaemle, CGI-58/ABHD5 is a coenzyme A- dependent lysophosphatidic acid acyltransferase, J. Lipid. Res. 51 (2010) 709-719

[6] J.L. Blankman, G.M. Simon, B.F. Cravatt, A comprehensive profile of brain enzymes that hydrolyze the endocannabinoid 2-arachidonylglycerol, Chem. Biol. 14 (2007) 1347-1356

[7] W.R. Marrs, J.L. Blankman, E.A. Horne, A. Thomazeau, Y.H. Lin, J. Coy, A.L. Bodor, G.G.

Muccioli, S.S> Hu, G. Woodruff, S. Fung, M. Lafourcade, J.P. Alexander, J.Z. Long, W. Li, C.

Xu, T. Moller, K. Mackie, O.J. Manzoni, B.F. Cravatt, N. Stella, The serine hydrolase ABHD6

96 controls the accumulation and efficacy of 2-AG at cannabinoid receptors, Nat. Neurosci. 13

(2010) 951-957

[8] R.A. Igal, J.M. Rhoads, R.A. Coleman, Neutral lipid storage disease with fatty liver and cholestasis, J. Pediatr. Gastronterol. Nutr. 25 (1997) 541-547

[9] S. Jin, G. Zhao, Z. Li, Y. Nishimoto, Y. Isohama, J. Shen, T. Ito, M. Takeya, K. Araki, P. He,

K. Yamamura, Age-related pulmonary emphysema in mice lacking alpha/beta hydrolase domain containing 2 gene, Biochem. Biophys. Res. Commun. 380 (2009) 419-424

[10] A. Demirkan, C.M. van Duijn, P. Ugocsai, A. Isaacs, P.P. Pramstaller, G. Liebisch, J.F.

Wilson, A. Johansson, I. Rudan, Y.S. Aulchenko, A.V. Kirichenko, A.C. Janssens, R.C. Jansen,

C. Gnewuch, F.S. Domingues, C. Pattaro, S.H. Wild, I. Jonasson, O. Polasek, I.V. Zorkoltseva,

A. Hofman, L.C. Karssen, M. Struchalin, J. Floyd, W. Igl, Z. Biloglav, L. Broer, A. Pfeufer, I.

Pichler, S. Campbell, G. Zaboli, I. Kolcic, F. Rivadeneira, J. Huffman, N.D. Hastie, A.

Uitterlinden, L. Franke, C.S. Franklin, V. Vitart, C.P. Nelson, M. Preuss, J.C. Bis, C.J.

O'Donnell, N. Franceschini, J.C. Witteman, T. Axenovich, B.A. Oostra, T. Meitinger, A.A.

[11] J.R. Savinainen, S.M. Saario, J.T. Laitinen, The serine hydrolases MAGL, ABHD6 and

ABHD12 as guardians of 2-arachidonoylglycerol signalling through cannabinoid receptors, Acta

Physiol (Oxf) 204 (2012) 267-276.

[12] D. Navia-Paldanius, J.R. Savinainen, J.T. Laitinen, Biochemical and pharmacological characterization of human a/b-hydrolase domain containing 6 (ABHD6) and 12 (ABHD12), J.

Lipid. Res. 53 (2012) 2413-2424

[13] C.C. Lord, J.L. Betters, P.T. Ivanova, S.B. Milne, D.S. Myers, J. Madenspacher, G.

Thomas, S. Chung, M. Liu, M.A. Davis, R.G. Lee, R.M. Crooke, M.J. Graham, J.S. Parks, D.L.

Brasaemle, M.B. Fessler, H.A. Brown, J.M. Brown, CGI-58/ABHD5-Derived Signaling Lipids

Regulate Systemic Inflammation and Insulin Action, Diabetes 61 (2012) 355-363

[14] P.K. Fleming, A.M. Dedman, S.Z. Xu, J. Li, F. Zeng, J. Naylor, C.D. Benham, A.N.

Bateson, K. Muraki, D.J. Beech, Sensing of lysophospholipids by TRPC4 calcium channel. J

Biol Chem 281 (2006) 4977-4982

[15] N. Murakami, T. Yokomizo, T. Okuno, T. Shimizu, G2A is a proton-sensing G-protein- coupled receptor antagonized by lysophosphatidylcholine. J Biol Chem 279 (2004) 42484-

42491.

[16] J.Z. Long, B.F. Cravatt, The Metabolic Serine Hydrolases and Their Functions in

Mammalian Physiology and Disease, Chem. Rev. 111 (2011) 1131-1138

[17] W. R. Marrs, E.A. Horne, S. Ortega-Guitierrez, J.A. Cisneros, C. Xu, Y.H. Lin, G.G.

Muccioli, M.L. Lopez-Rodriguez, N. Stella, Dual inhibition of alpha/beta-hydrolase domain 6 and fatty acid amide hydrolase increases endocannabinoid levels in neurons, J. Biol. Chem. 286

(2011) 28723-28728

[18] U. Taschler, F.P. Radner, C. Heier, R. Schreiber, M. Schweriger, G. Schoiswohl, K. Preiss-

Landi, D. Jaeger, B. Reiter, H.C. Koefeler, Monoglyceride lipase deficiency in mice impairs lipolysis and attenuates diet-induced insulin resistance, J. Biol. Chem. 286 (2011) 17467-17477

[19] J.E. Scholsburg, J.L. Blankman, J.Z. Long, D.K. Nomura, B. Pan, S.G. Kinsey, P.T.

Nguyen, D. Ramesh, L. Booker, J.J. Burston, Chronic monoacylglycerol lipase blockade causes functional antagonism of the endocannabinoid system, Nat Neurosci. 13 (2010) 1113-1119

[20] K. Makide, H. Kitamura, Y. Sato, M. Okutani, J. Aoki, Energing lysophospholipid mediators, lysophosphatidylserine, lysophosphatidylthreonine, lysophosphatidylethanolamine and lysophosphatidylglycerol, Prostaglandins Other Lipid Mediat. 89 (2009) 135-139

[21] S.H. Jo, S.D. Kim, J.M. Kim, H.Y. Lee, S.Y. Lee, J.W Shim, J. Yun, D.S. Im, Y.S. Bae,

Lysophosphatidylglycerol stimulates chemotactic migration in human natural killer cells,

Biochem. Biophys. Res. Commun. 372 (2008) 147-151

[22] M.P. Wymann, R. Schneiter, Lipid signalling in disease, Nat. Rev. Mol. Cell Biol. 9 (2008)

162-176

[23] A. Skoura, T. Hla, Lysophospholipid receptors in vertebrate development, physiology and pathology, J. Lipid Res. Suppl. 50 (2009) S293-S298

[24] F. Hullin-Matsuda, K. Kawasaki, I. Delton-Vandenbroucke, Y. Xu, M. Nishijima, M.

Lagarde, M. Schlame, T. Kobayashi, De novo biosynthesis of the late endosome lipid, bis(monoacylglycero)phosphate, J. Lipid. Res. 48 (2007) 1997-2008

[25] M.T. Traurig, J.I. Orczewska, D.J. Ortiz, L. Bian, A.M. Marinelarena, S. Kobes, A.

Malhotra, R.L. Hanson, C.C> Mason, W.C> Knowler, Evidence for a role of LPGAT1 in

99 influencing BMI and percent body fat in Native Americans, Obesity (Silver Spring) 21 (2013)

193-202

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Gwynneth Sarah Thomas

Wake Forest University School of Medicine Department of Molecular Pathology Section on Lipid Sciences 391 Technology Way Winston Salem, NC 27157 (919) 345-8113 [email protected] [email protected]

Education Bachelors of Science Degree in Biochemistry North Carolina State University, Raleigh, NC 2006 – 2010

PhD Candidate in Molecular Pathology Wake Forest University, Winston Salem, NC 2010 - 2014

MBA Candidate Wake Forest University, Winston Salem, NC 2012 – 2014

Career History

Laboratory Research Associate, Arbovax Inc. 2008 – 2010  Worked with animal models using novel technology to develop a tetravalent vaccine for dengue fever  Worked on vaccine development for other arboviruses  Attended meetings with angel groups for early round funding

PhD Student, Wake Forest University Department 2010 - 2014 of Molecular Pathology  Current Thesis Research: “The Serine Hydrolase ABHD6 is a Critical Regulator of the Metabolic Syndrome”

MBA Student, Wake Forest University School of Business 2012 – 2014  Student in Executive Evening Program

Accomplishments & Affiliations

Appointed to NIH T-32 Training Grant, Wake Forest University Department of Pathology 2011, 2012, 2013  Integrative Lipid Metabolism, Inflammation and Chronic Diseases  The goal of this institutional training grant is to train graduate Student from four different graduate programs at Wake Forest University in lipid metabolism, inflammation and chronic diseases

Taught for NC DNA Day, Wake Forest University Graduate 2011 School of Arts and Sciences  Visited a local high school for a day and gave an interactive lecture on DNA, my own research and why science is fun!

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Appointed to Chair of Evening Program for Wake Graduate 2012 Women in Business (WGWIB), Wake Forest University School of Business  Platform: “Helping Women Understand the Business of Science”

Member of Wake Forest Graduate Student Association (GSA), 2011 - 2012 Wake Forest University Graduate School of Arts and Sciences  Social Chair; helped organize student run functions like tail-gates, holiday socials, etc.

Travel Award for Kern Lipid Conference, Wake Forest University 2013 Graduate School of Arts and Sciences  Early Career Investigator travel stipend award to attend Kern Lipid Conference

Invitation to Speak at South East Lipid Research Conference, 2013 Wake Forest University Graduate School of Arts and Sciences  1 hour presentation discussing “The Serine Hydrolase ABHD6 is a Critical Regulator of the Metabolic Syndrome”

Appointed as 1 of the 2 members to the Wake Forest Business 2012 - 2014 School Honor Council, Wake Forest University School of Business  Sit on honor council meetings, hearings and appeals as necessary

Publications

 Smith K.M., Nanda K., Spears C.J., Ribiero M., Vancini R., Piper A., Thomas G.S., Thomas M.E., Brown D.T., Hernandex R. (2011) Structural Mutants of Dengue Virus 2 Transmembrane Domains Exhibit Host-Range Phenotype. Virology Journal. 8:289  Lord CC, Betters JL, Ivanova PT, Milne SB, Myers DS, Madenspacher J, Thomas G, Chung S, Liu M, Davis MA, Lee RG, Crooke RM, Graham MJ, Parks JS, Brasaemle DL, Fessler MB, Brown HA, Brown JM. (2012) CGI-58/ABHD5-Derived Signaling Lipids Regulate Systemic Inflammation and Insulin Action. Diabetes. 2:355-63  Nanda K., Smith K.M., Spears C.J., Piper A., Ribiero M., Quiles M., Thomas G.S., Thomas M.E., Brown D.T., Hernandez R. (2012) Novel Dengue Virus-2 Vaccines in the African Green Monkey: Safety, Immunogenecity and Efficacy. American Journal of Tropical Medicine and Hygiene. 2:743-53  Lord CC, Thomas GS, Brown JM. (2013) Mammalian alpha beta hydrolase domain (ABHD) proteins: Lipid metabolizing enzymes at the interface of cell signaling and energy metabolism. Biochemica et Biophysica Acta 1831(4):792-802  Gwynneth Thomas, Jenna L. Betters, Caleb C. Lord, Pavlina T. Ivanova, David S. Myers, Irina Mrak, Vera Leber, Christoph Heier, Ulrike Taschler, Daniel Ferguson, Janet Sawyer, Matthew A. Davis, Stephanie Marshall, John Melchior, Richard G. Lee, Rosanne M. Crooke, Mark J. Graham, Robert Zimmermann, H. Alex Brown, & J. Mark Brown. (2013) The Serine Hydrolase ABHD6 is a Critical Regulator of the Metabolic Syndrome. Cell Reports 5(2): 508-520  Caleb C. Lord, Gwynneth Thomas, Stephanie Marshall, Amanda L. Brown, Richard G. Lee, Rosanne M. Crooke, Mark J. Graham, Martina Schweiger, Guenter Haemmerle, Rudolf Zechner, & J. Mark Brown. CGI-58 Controls Hepatic Triglyceride Storage and Inflammation Separate from the Co-activation of ATGL-mediated Hydrolysis. Cell Metabolism (in review)  Kanwardeep S. Bura, Caleb Lord, Stephanie Marshall, Allison McDaniel, Gwynneth Thomas, Manya Warrier, Jun Zhang, Matthew A. Davis, Janet K. Sawyer, Ramesh Shah, Martha D. Wilson, Arne Dikkers, Uwe J. Tietge, Xavier Collet, Lawrence L. Rudel, Ryan E. Temel, and J.

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Mark Brown (2013) Intestinal SR-BI Does Not Impact Cholesterol Absorption or Trans intestinal Cholesterol Efflux (TICE) Rates in Mice. Journal of Lipid Research 54(6):1567-77  Thomas GS, Brown AL, Brown JM. (2014) In vivo metabolite profiling as a means to identify uncharacterized lipase function: recent success stories within the alpha beta hydrolase domain (ABHD) enzyme family. Biochemica et Biophysica Acta (in press – January 2014)

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