THE SERINE HYDROLASE ABHD6 IS A CRITICAL REGULATOR OF THE

METABOLIC SYNDROME

BY

GWYNNETH SARAH THOMAS

A Dissertation Submitted to the Graduate Faculty of

WAKE FOREST UNIVERSITY GRADUATE SCHOOL OF ARTS AND SCIENCES

in Partial Fulfillment of the Requirements

for the Degree of

DOCTOR OF PHILOSOPHY

Molecular Pathology

May 2014

Winston-Salem, North Carolina

Approved By:

John S. Parks, Ph.D., Advisor

Paul A. Dawson, Ph.D., Chair

J. Mark Brown, Ph.D.

Ryan E. Temel, Ph.D.

Greg S. Shelness, Ph.D.

ii

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

iii

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

iv

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

v

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

vi

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

vii

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

viii

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

ix

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

x

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

xi

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

2

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

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 [7, 8, 9, 10, 11, 12, 13]. For example ABHD3, ABHD4,

and ABHD6 hydrolyze the glycerophospholipids C14-lysophosphatidylcholine, N-acyl

3

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.

4

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%

5

[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

6

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.

7

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[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.

12

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

13

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

14

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

15

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, non-

alcoholic 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

16

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).

17

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

18

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).

19

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

20

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

21

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

22

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

23

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

24

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).

25

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

26

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

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 (Lord et al., 2011; Brown et al., 2010; Brown et

al., 2008a; Brown et al., 2008b). For small molecule inhibitor studies, mice were fed a high fat

diet and simultaneously treated with either a vehicle or 10 mg/kg WWL-70 for 8 weeks.

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

27

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.

28

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

29

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.

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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

38

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

39

and Mark Brown

(C) Gross appearance of HFD-fed mice. Work done by Jenna Betters 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

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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

(C) Plasma glucose levels in mice treated with ASOs and diets for 10-11 weeks. Work done by Jenna Betters and Mark 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

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(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

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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

43

(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

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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

45

(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

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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.

(C) Total hepatic phosphatidylethanolamine (PE) levels.

(D) Total hepatic phosphatidylglycerol (PG) levels.

(E) Total hepatic phosphatidylinositol (PI) levels.

47

(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

48

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.

(C) Glucose tolerance tests in mice treated with inhibitor and diet for 6 weeks.

(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

49

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.

50

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

51

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

52

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)

53

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)

54

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)

55

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)

56

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

57

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

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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

59

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).

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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

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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 80C 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

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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

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(brownm5@ccf.org).

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-AMPKThr172 rabbit

monoclonal (Cell Signaling # 2535), anti-total AMPK rabbit monoclonal (Cell Signaling

Technologies # 2603), anti-phospho-AMPKSer108 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.

64

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.

65

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

cold CHCl3 for 1 min. The extraction proceeded with centrifugation (5 min, 4oC, 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).

66

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,

67

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

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).

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.

68

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.

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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.

70

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.

71

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.

72

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.

73

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

74

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

75

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

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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

77

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

resistance (Fig 5).

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

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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).

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

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79

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

80

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

81

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).

82

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

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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

84

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).

85

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

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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

91

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

92

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

93

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

94

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

95

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.

96

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101

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

gthomas@wakehealth.edu

thomgs12@wfu.edu

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)