The serine hydrolase ABHD6 Is a critical regulator of the metabolic syndrome

Abstract

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 (ASOs) to selectively knock down ABHD6 in peripheral tissues in order to identify in vivo substrates and 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 therapeutics for obesity, nonalcoholic fatty liver disease, and type II diabetes.

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). (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.

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

(A) ABHD6 protein levels in the liver, epididymal white adipose tissue (WAT), and brain of mice treated with either saline, a control non-targeting ASO, or two separate ABHD6 ASOs for 12 weeks. (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). (C) Gross appearance of HFD-fed mice. (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). (E) Body fat % as measured by magnetic resonance imaging (n=4). (F) Lean body mass as measured by magnetic resonance imaging (n=4). (G) Snout to anus length (n=10). (H) Cumulative food intake in ASO-treated mice (n=10). (I) Total intestinal fat absorption in mice treated with ASOs and fed a HFD for 9 weeks (n=14-15). (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. (K) Diurnal and nocturnal quantification of physical activity (total beam break counts). (L) Diurnal and nocturnal quantification of oxygen consumption (VO2). (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).

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 ASO or an ASO targeting ABHD6 for 12 weeks. (A) Gross appearance of livers and microscopic examination (H&E staining at 40x magnification) in HFD-fed mice. (B) Total hepatic levels of triacylglycerols (TAG), diacylglycerols (DAG), and monoacylglycerols (MAG) in mice fed diets for 12 weeks. (C) Plasma glucose levels in mice treated with ASOs and diets for 10-11 weeks. (D) Plasma insulin levels in mice treated with ASOs and diets for 12 weeks. (E) Plasma TAG levels in mice treated with ASOs and diets for 12 weeks. (F) Plasma cholesterol levels in mice treated with ASOs and diets for 12 weeks. (G) Plasma non-esterified fatty acids (NEFAs) in mice treated with ASOs and diets for 12 weeks. (H) Glucose tolerance tests in mice treated with ASOs and diets for 10-11 weeks. (I) Insulin tolerance tests in mice treated with ASOs and diets for 10-11 weeks. (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).

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. (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. (C) Hepatic lipogenic protein expression in mice treated with a control non targeting ASO, or two independent ABHD6 ASOs for 12 weeks. (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 1 hour later livers were removed for measurement of ³H-labeled fatty acids as described in the methods section. (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 inhibitors 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). (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 inhibitors 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).

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

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 ABHD6 for 12 weeks (panels 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. (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 lysophosphatylserine (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; 3 mM] and 2-oleoylglycerol [2-OG; 3 mM] 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 the mean ± S.D. and representative of 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 the mean ± S.D. and are representative of 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 the mean ± S.D. and representative of at least two independent experiments.

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).