Lack of monoacylglycerol lipase prevents hepatic steatosis by favoring lipid storage in adipose tissue and intestinal malabsorption
2019; Elsevier BV; Volume: 60; Issue: 7 Linguagem: Inglês
10.1194/jlr.m093369
ISSN1539-7262
AutoresMatteo Tardelli, Francesca Virginia Bruschi, Thierry Claudel, Claudia Fuchs, Nicole Auer, Victoria Kunczer, Tatjana Stojaković, Hubert Scharnagl, Aı̈da Habib, G Grabner, Robert Zimmermann, Sophie Lotersztajn, Michael Trauner,
Tópico(s)Lipid metabolism and biosynthesis
ResumoMonoacylglycerol lipase (MGL) is the rate-limiting enzyme in the degradation of monoacylglycerols. To examine the role of MGL in hepatic steatosis, WT and MGL KO (MGL−/−) mice were challenged with a Western diet (WD) over 12 weeks. Lipid metabolism, inflammation, and fibrosis were assessed by serum biochemistry, histology, and gene-expression profiling of liver and adipose depots. Intestinal fat absorption was measured by gas chromatography. Primary adipocyte and 3T3-L1 cells were analyzed by flow cytometry and Western blot. Human hepatocytes were treated with MGL inhibitor JZL184. The absence of MGL protected mice from hepatic steatosis by repressing key lipogenic enzymes in liver (Srebp1c, Pparγ2, and diacylglycerol O-acyltransferase 1), while promoting FA oxidation. Liver inflammation was diminished in MGL−/− mice fed a WD, as evidenced by diminished epidermal growth factor-like module-containing mucin-like hormone receptor-like 1 (F4/80) staining and C-C motif chemokine ligand 2 gene expression, whereas fibrosis remained unchanged. Absence of MGL promoted fat storage in gonadal white adipose tissue (gWAT) with increased lipogenesis and unchanged lipolysis, diminished inflammation in gWAT, and subcutaneous AT. Intestinal fat malabsorption prevented ectopic lipid accumulation in livers of MGL−/− mice fed a WD. In vitro experiments demonstrated increased adipocyte size/lipid content driven by PPARγ. In conclusion, our data uncover that MGL deletion improves some aspects of nonalcoholic fatty liver disease by promoting lipid storage in gWAT and fat malabsorption. Monoacylglycerol lipase (MGL) is the rate-limiting enzyme in the degradation of monoacylglycerols. To examine the role of MGL in hepatic steatosis, WT and MGL KO (MGL−/−) mice were challenged with a Western diet (WD) over 12 weeks. Lipid metabolism, inflammation, and fibrosis were assessed by serum biochemistry, histology, and gene-expression profiling of liver and adipose depots. Intestinal fat absorption was measured by gas chromatography. Primary adipocyte and 3T3-L1 cells were analyzed by flow cytometry and Western blot. Human hepatocytes were treated with MGL inhibitor JZL184. The absence of MGL protected mice from hepatic steatosis by repressing key lipogenic enzymes in liver (Srebp1c, Pparγ2, and diacylglycerol O-acyltransferase 1), while promoting FA oxidation. Liver inflammation was diminished in MGL−/− mice fed a WD, as evidenced by diminished epidermal growth factor-like module-containing mucin-like hormone receptor-like 1 (F4/80) staining and C-C motif chemokine ligand 2 gene expression, whereas fibrosis remained unchanged. Absence of MGL promoted fat storage in gonadal white adipose tissue (gWAT) with increased lipogenesis and unchanged lipolysis, diminished inflammation in gWAT, and subcutaneous AT. Intestinal fat malabsorption prevented ectopic lipid accumulation in livers of MGL−/− mice fed a WD. In vitro experiments demonstrated increased adipocyte size/lipid content driven by PPARγ. In conclusion, our data uncover that MGL deletion improves some aspects of nonalcoholic fatty liver disease by promoting lipid storage in gWAT and fat malabsorption. Nonalcoholic fatty liver disease (NAFLD) has become an important public health issue because of its high prevalence and association with adverse cardio-metabolic and hepatic outcomes (1Younossi Z. Anstee Q.M. Marietti M. Hardy T. Henry L. Eslam M. George J. Bugianesi E. Global burden of NAFLD and NASH: trends, predictions, risk factors and prevention.Nat. Rev. Gastroenterol. Hepatol. 2018; 15: 11-20Crossref PubMed Scopus (2385) Google Scholar). NAFLD spans a spectrum from simple hepatic steatosis through nonalcoholic steatohepatitis, ultimately leading to liver fibrosis, cirrhosis, and cancer (1Younossi Z. Anstee Q.M. Marietti M. Hardy T. Henry L. Eslam M. George J. Bugianesi E. Global burden of NAFLD and NASH: trends, predictions, risk factors and prevention.Nat. Rev. Gastroenterol. Hepatol. 2018; 15: 11-20Crossref PubMed Scopus (2385) Google Scholar, 2Benedict M. Zhang X. Non-alcoholic fatty liver disease: an expanded review.World J. Hepatol. 2017; 9: 715-732Crossref PubMed Scopus (416) Google Scholar, 3Estes C. Razavi H. Loomba R. Younossi Z. Sanyal A.J. Modeling the epidemic of nonalcoholic fatty liver disease demonstrates an exponential increase in burden of disease.Hepatology. 2018; 67: 123-133Crossref PubMed Scopus (959) Google Scholar). Hepatic triglyceride (TG) homeostasis is regulated by a complex interplay between plasma NEFA uptake and de novo lipogenesis, as well as FA oxidation and TG export by VLDLs. Hepatic steatosis develops when the rate of hepatic FA input (uptake and synthesis) exceeds the rate of FA output (oxidation and secretion) (4Musso G. Gambino R. Cassader M. Recent insights into hepatic lipid metabolism in non-alcoholic fatty liver disease (NAFLD).Prog. Lipid Res. 2009; 48: 1-26Crossref PubMed Scopus (516) Google Scholar). In addition, steatosis is associated with hepatic insulin resistance and thereby reduced sensitivity of the liver to the suppressive effects of insulin on the hepatic output of glucose. Monoacylglycerol lipase (MGL) is the final enzymatic step of the TG degradation pathway and hydrolyzes monoglycerides (MGs) deriving from phospholipids or TGs into glycerol and FAs (5Poursharifi P. Madiraju S.R.M. Prentki M. Monoacylglycerol signalling and ABHD6 in health and disease.Diabetes Obes. Metab. 2017; 19: 76-89Crossref PubMed Scopus (44) Google Scholar). Moreover, MGL notably hydrolyzes 2-arachidonoylglycerol, which is a potent ligand within the endocannabinoid system (6Long J.Z. Li W. Booker L. Burston J.J. Kinsey S.G. Schlosburg J.E. Pavón F.J. Serrano A.M. Selley D.E. Parsons L.H. et al.Selective blockade of 2-arachidonoylglycerol hydrolysis produces cannabinoid behavioral effects.Nat. Chem. Biol. 2009; 5: 37-44Crossref PubMed Scopus (746) Google Scholar). Metabolic studies highlighted that hepatic levels of saturated and unsaturated species of MGs were highly increased in MGL−/− mice, showing improved insulin sensitivity and glucose tolerance under a high-fat diet (HFD) (7Douglass J.D. Zhou Y.X. Wu A. Zadrogra J.A. Gajda A.M. Lackey A.I. Lang W. Chevalier K.M. Sutton S.W. Zhang S-P. et al.Global deletion of MGL in mice delays lipid absorption and alters energy homeostasis and diet-induced obesity.J. Lipid Res. 2015; 56: 1153-1171Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar). Although previous reports demonstrated that the absence of MGL protects from adipose tissue (AT) inflammation and insulin resistance, resulting in a leaner phenotype (7Douglass J.D. Zhou Y.X. Wu A. Zadrogra J.A. Gajda A.M. Lackey A.I. Lang W. Chevalier K.M. Sutton S.W. Zhang S-P. et al.Global deletion of MGL in mice delays lipid absorption and alters energy homeostasis and diet-induced obesity.J. Lipid Res. 2015; 56: 1153-1171Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar, 8Taschler U. Radner F.P.W. Heier C. Schreiber R. Schweiger M. Schoiswohl G. Preiss-Landl K. Jaeger D. Reiter B. Koefeler H.C. et al.Monoglyceride lipase deficiency in mice impairs lipolysis and attenuates diet-induced insulin resistance.J. Biol. Chem. 2011; 286: 17467-17477Abstract Full Text Full Text PDF PubMed Scopus (201) Google Scholar), the molecular link between lipid products generated by MGL deficiency and observed metabolic changes remains largely unexplored. Importantly, it is unclear whether MGL has a functional role in the AT-liver axis in the background of obesity-associated hepatic steatosis. Therefore, we explored the role of MGL in hepatic steatosis induced by a Western diet (WD) challenge. Here, we demonstrate that MGL deficiency prevented hepatic lipid accumulation induced by WD feeding. Mechanistically, increased gonadal white AT (gWAT) lipid storage in MGL−/− mice prevented ectopic lipid accumulation in the liver, whereas intestinal malabsorption of FA species diminished body weight. In vitro, MGL-deficient adipocyte progenitor cells (APCs) showed increased lipid accumulation, and MGL was identified as a PPARγ target in 3T3-L1 cells. Thus, targeting MGL action in vivo could provide a novel therapeutic strategy to combat obesity-associated hepatic steatosis and related metabolic complications. Experiments were performed in 2-month-old male MGL−/− mice and WT littermates (C57BL/6 background, n = 7 per group unless otherwise stated) weighing 20–25 g, generated by R. Zimmermann (8Taschler U. Radner F.P.W. Heier C. Schreiber R. Schweiger M. Schoiswohl G. Preiss-Landl K. Jaeger D. Reiter B. Koefeler H.C. et al.Monoglyceride lipase deficiency in mice impairs lipolysis and attenuates diet-induced insulin resistance.J. Biol. Chem. 2011; 286: 17467-17477Abstract Full Text Full Text PDF PubMed Scopus (201) Google Scholar). Animals were housed in a 12 h light/dark house facility and fed ad libitum either a WD (Research Diet: D12079B, protein 17%, fat 40%, and carbohydrate 43%) or standard chow for 12 weeks; organ and blood collection were performed in the morning. The experimental protocols were approved by the local Animal Care and Use Committee (BMWF.66.009/0117-II/3b/2013), and routine serum biochemical analyses were performed as previously described (9Fickert P. Zollner G. Fuchsbichler A. Stumptner C. Weiglein A.H. Lammert F. Marschall H.U. Tsybrovskyy O. Zatloukal K. Denk H. et al.Ursodeoxycholic acid aggravates bile infarcts in bile duct-ligated and Mdr2 knockout mice via disruption of cholangioles.Gastroenterology. 2002; 123: 1238-1251Abstract Full Text Full Text PDF PubMed Scopus (253) Google Scholar). Mouse liver samples were fixed with neutral buffered 4% paraformaldehyde and subsequently paraffin embedded. Hematoxylin-eosin (H&E) staining and Sirius Red were performed in liver as described (10Fickert P. Fuchsbichler A. Wagner M. Zollner G. Kaser A. Tilg H. Krause R. Lammert F. Langner C. Zatloukal K. et al.Regurgitation of bile acids from leaky bile ducts causes sclerosing cholangitis in Mdr2 (Abcb4) knockout mice.Gastroenterology. 2004; 127: 261-274Abstract Full Text Full Text PDF PubMed Scopus (364) Google Scholar). Detection of the epidermal growth factor-like module-containing mucin-like hormone receptor-like 1 (F4/80) and Mac-2 were performed as previously described (11Fuchs C.D. Paumgartner G. Mlitz V. Kunczer V. Halilbasic E. Leditznig N. Wahlström A. Ståhlman M. Th÷ringer A. Kashofer K. et al.Colesevelam attenuates cholestatic liver and bile duct injury in Mdr2 −/− mice by modulating composition, signalling and excretion of faecal bile acids.Gut. 2018; 67: 1683-1691Crossref PubMed Scopus (41) Google Scholar). Hepatocytes were isolated from normal liver tissue from surgical resections unsuitable for transplantation, approved by the ethics committee of the Medical University of Vienna (EK 2032/2013) using a protocol described elsewhere (12Bruschi F.V. Claudel T. Tardelli M. Caligiuri A. Stulnig T.M. Marra F. Trauner M. The PNPLA3 I148M variant modulates the fibrogenic phenotype of human hepatic stellate cells.Hepatology. 2017; 65: 1875-1890Crossref PubMed Scopus (142) Google Scholar, 13Bhogal R.H. Hodson J. Bartlett D.C. Weston C.J. Curbishley S.M. Haughton E. Williams K.T. Reynolds G.M. Newsome P.N. Adams D.H. et al.Isolation of primary human hepatocytes from normal and diseased liver tissue: a one hundred liver experience.PLoS One. 2011; 6: e18222Crossref PubMed Scopus (100) Google Scholar). Hepatocytes were seeded on uncoated plastic dishes and cultivated with DMEM supplemented with 10% FBS and antibiotic (Gibco Life Technologies, CA). Cells were treated with an MGL inhibitor (JZL184, 1 µM) for 24 h. gWAT tissue obtained from MGL−/− mice and WT littermates (n = 4 per group) was minced with scissors and digested in adipocyte isolation buffer as previously described (14Moreno-Viedma V. Tardelli M. Zeyda M. Sibilia M. Burks J.D. Stulnig M.T. Osteopontin-deficient progenitor cells display enhanced differentiation to adipocytes.Obes. Res. Clin. Pract. 2018; 12: 277-285Crossref PubMed Scopus (8) Google Scholar). Briefly, samples were incubated for 40 min at 37°C on a shaker, and the digested tissue was filtered and centrifuged. The pellet was resuspended and filtered through a 70 µm and then 40 µm cell strainer (14Moreno-Viedma V. Tardelli M. Zeyda M. Sibilia M. Burks J.D. Stulnig M.T. Osteopontin-deficient progenitor cells display enhanced differentiation to adipocytes.Obes. Res. Clin. Pract. 2018; 12: 277-285Crossref PubMed Scopus (8) Google Scholar). The resulting mouse APCs were cultured in DMEM supplemented with 10 ng/ml recombinant basic human fibroblast growth factor (FGFβ; Gibco, CA) to maintain their self-renewal capacity and inhibit differentiation (15Rodeheffer M.S. Birsoy K. Friedman J.M. Identification of white adipocyte progenitor cells in vivo.Cell. 2008; 135: 240-249Abstract Full Text Full Text PDF PubMed Scopus (697) Google Scholar). To assess the adipogenic potential of APCs, cells were seeded to reach confluence and differentiated as follows: 3 days treatment with adipogenic medium (advanced DMEM, 5% heat-inactivated FBS supplemented with 20 µg/ml insulin, 10 µM dexamethasone, and 0.5 mM isobutilmethylxantine) followed by 3 days of DMEM with 5% FBS and 20 µg/ml insulin and 4 days of DMEM with 5% FBS. The 3T3-L1 cells were differentiated according to previously published works (16Madsen L. Petersen R.K. Sørensen M.B. Jørgensen C. Hallenborg P. Pridal L. Fleckner J. Amri E.Z. Krieg P. Furstenberger G. et al.Adipocyte differentiation of 3T3–L1 preadipocytes is dependent on lipoxygenase activity during the initial stages of the differentiation process.Biochem. J. 2003; 375: 539-549Crossref PubMed Scopus (114) Google Scholar, 17Neal J.W. Clipstone N.A. Calcineurin mediates the calcium-dependent inhibition of adipocyte differentiation in 3T3–L1 cells.J. Biol. Chem. 2002; 277: 49776-49781Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar). Cells were treated with an MGL inhibitor (JZL184, 1 µM), rosiglitazone (rosi) alone, or in combination with GW9662 or pioglitazone (pio) for 24 h prior to harvesting. Whole-cell extracts were obtained using RIPA buffer (150 mM NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, and 50 mM Tris, pH 8.0) containing complete EDTA-free protease inhibitor cocktail tablets (Roche Diagnostics GmbH, Germany) and phosphatase inhibitors (20 mM b-glycerophosphate, 10 mM 4-nitrophenylphosphate, and 50 mM sodium vanadate; all Sigma-Aldrich). Primary Abs were diluted in 3% BSA TBS-Tween 1× solution at different concentrations, and SDS-PAGE was run as previously reported (12Bruschi F.V. Claudel T. Tardelli M. Caligiuri A. Stulnig T.M. Marra F. Trauner M. The PNPLA3 I148M variant modulates the fibrogenic phenotype of human hepatic stellate cells.Hepatology. 2017; 65: 1875-1890Crossref PubMed Scopus (142) Google Scholar, 18Tardelli M. Claudel T. Bruschi F.V. Moreno-Viedma V. Trauner M. Adiponectin regulates AQP3 via PPARα in human hepatic stellate cells.Biochem. Biophys. Res. Commun. 2017; 490: 51-54Crossref PubMed Scopus (17) Google Scholar). Proteins were detected by ECL chemiluminescence (GE Amersham, Arlington Heights, IL). AT and liver were homogenized in TRIzol reagent (Invitrogen, Carlsbad, CA), and RNA was isolated and transcribed to cDNA according to the manufacturer's protocol. Gene expression was normalized to 18S and assessed by quantitative real-time RT-PCR on an ABI Prism 7000 cycler using commercial Assays-on-Demand kits (all Applied Biosystems, Foster City, CA). For BODIPY lipid staining, 3T3-L1 cells and APCs were trypsinized, collected, and incubated with a working solution of 1 μg/ml BODIPY 493/503 in PBS for 30 min at 37°C. Afterward, 5 μg/ml propidium iodide (Sigma-Aldrich) was added to each tube for 5 min before flow cytometric analysis. The side scatter was used to detect changes in the cell granularity and the green fluorescence (525 nm) of BODIPY to measure the intracellular lipid droplet formation (19Spangenburg E.E. Pratt S.J.P. Wohlers L.M. Lovering R.M. Use of BODIPY (493/503) to visualize intramuscular lipid droplets in skeletal muscle.J. Biomed. Biotechnol. 2011; 2011: 598358Crossref PubMed Scopus (82) Google Scholar, 20Tardelli M. Moreno-Viedma V. Zeyda M. Itariu B.K. Langer F.B. Prager G. Stulnig T.M. Adiponectin regulates aquaglyceroporin expression in hepatic stellate cells altering their functional state.J. Gastroenterol. Hepatol. 2017; 32: 253-260Crossref PubMed Scopus (18) Google Scholar). Flow cytometry was performed using BD FACSCANTO™ and BD FACSDIVA™ software (Becton Dickinson, NJ). For determination of lipid absorption, diets containing sucrose polybehenate (5% of total dietary fat content; wt/wt) were prepared and fed to mice for three consecutive days. Fresh fecal pellets were collected, extracted, and analyzed by gas chromatography of FA methyl esters as previously described (21Jandacek R.J. Heubi J.E. Tso P. A novel, noninvasive method for the measurement of intestinal fat absorption.Gastroenterology. 2004; 127: 139-144Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar). In brief, 10 mg feces and diet were extracted using chloroform/methanol (2/1), 1% acetic acid, and 500 mM butylated hydroxytoluene (BHT) for 1 h at room temperature. The aqueous phase was reextracted using chloroform and 500 mM BHT for 15 min at room temperature. The combined organic phases were evaporated under a stream of nitrogen and FA methyl ester where generated from whole lipid extracts using methanolic hydrogen chloride (22Adlof, R. O., 2003. Advances in Lipid Methodology, Oily Press Lipid Library Series. R. O. Adlof, editor. Vol. 5, Chapter 2. Elsevier. 69–111.Google Scholar). Samples were reconstituted in 100 ml hexane and measured by gas chromatography-flame ionization detection. A wall-coated fused silica 25 m, 0.32 mm ID column (FFAB-CB for free FAs, Varian) was used. A total of 1 µl of the sample was injected at an injector temperature of 230°C; column A: temperature gradient from 150°C (hold for 0 min) to 250°C (hold for 2 min) with 5°C·min−1, and a second ramp to increase the temperature to 260°C (hold for 5 min) with 10°C·min−1 was applied. Detector settings: base temperature, 200°C; ignition threshold, 0.2 pA; air flow rate, 200 ml·min−1; H2 flow rate, 30 ml·min−1; and makeup, 20 ml·min−1. The absorption of FAs was calculated from the ratios of behenic acid to other FAs in diet and feces (23Schweiger M. Romauch M. Schreiber R. Grabner G.F. H÷tter S. Kotzbeck P. Benedikt P. Eichmann T.O. Yamada S. Knittelfelder O. et al.Pharmacological inhibition of adipose triglyceride lipase corrects high-fat diet-induced insulin resistance and hepatosteatosis in mice.Nat. Commun. 2017; 8: 14859Crossref PubMed Scopus (120) Google Scholar). Liver and plasma TG/glycerol content was determined according to manufacturer protocol and measured by using a commercially available enzymatic reagent, TGs FS (DiaSys, Diagnostic Systems, Germany). Mouse plasma insulin (Mercodia, Sweden) and β-hydroxybutyrate (BOB; Sigma-Aldrich) were measured with commercially available ELISA kits. FFAs were measured in stools; approximately 10 mg of feces was homogenized in a chloroform solution, and the organic phase was dried at 50°C and resuspended for colorimetric measurement according to manufacturer protocols (FFA quantification assay kit, Abcam, UK). Serum adiponectin levels were evaluated using a mouse adiponectin ELISA kit (Bio Vendor, Czech Republic). Data are shown as mean ± SD. Statistical analysis was performed between two groups by Student's two-tailed t-test. For analysis of multiple measurements, we performed two-way ANOVA followed by Tukey post hoc test using GraphPad Prism version 7.00 for Windows (GraphPad Software, La Jolla, CA). A p value of 0.05 or less was considered to be statistically significant. In order to assess the role of MGL in hepatic steatosis, WT and MGL−/− mice were fed either chow or a WD for 12 weeks to induce obesity and steatosis. Notably, MGL deletion protected from weight gain (Fig. 1A, supplemental Fig. S1A) and hepatomegaly (as demonstrated by a decreased liver/body weight ratio; Fig. 1B) despite an increased gWAT/BW ratio (Fig. 1B) and unchanged food intake (data not shown). This resulted in protective effects against hepatic steatosis upon WD challenge, as evidenced by reduced macrovesicular steatosis in H&E and diminished serum transaminases [alanine transaminase (ALT) and aspartate transaminase (AST)] and plasma TGs (Fig. 1C, D, Table 1). Moreover, fasting plasma NEFAs were significantly diminished in MGL−/− animals (Table 1), in line with diminished lipolysis from adipose depots (24Scherer P.E. Adipose tissue: from lipid storage compartment to endocrine organ.Diabetes. 2006; 55: 1537-1545Crossref PubMed Scopus (828) Google Scholar). Accordingly, total hepatic TG content showed decreased accumulation of lipids in MGL−/− mice fed a WD (Fig. 1E). Next, we measured FFA amounts in stool, showing a slight increase already at baseline in MGL−/− mice compared with WT, which was further exacerbated by the dietary challenge (Fig. 1F). To investigate this difference in FA output, we determined intestinal absorption of dietary fat using the nonhydrolyzable and nonabsorbable sucrose polybehenate as internal standard in the WD (21Jandacek R.J. Heubi J.E. Tso P. A novel, noninvasive method for the measurement of intestinal fat absorption.Gastroenterology. 2004; 127: 139-144Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar) as previously performed (23Schweiger M. Romauch M. Schreiber R. Grabner G.F. H÷tter S. Kotzbeck P. Benedikt P. Eichmann T.O. Yamada S. Knittelfelder O. et al.Pharmacological inhibition of adipose triglyceride lipase corrects high-fat diet-induced insulin resistance and hepatosteatosis in mice.Nat. Commun. 2017; 8: 14859Crossref PubMed Scopus (120) Google Scholar). We found significantly reduced lipid absorption in MGL−/− animals compared with controls (Fig. 1G) with increased intestinal cholesterol (Chol) excretion (Abcg5) but unchanged Abcg8 and other lipid transporters Npc1l1, Srb1, and cluster of differentiation 36 (Cd36) (supplemental Fig. S1C) in accordance with a previous report (25Vujic N. Korbelius M. Leopold C. Duta-Mare M. Rainer S. Schlager S. Goeritzer M. Kolb D. Eichmann T.O. Diwoky C. et al.Monoglyceride lipase deficiency affects hepatic cholesterol metabolism and lipid-dependent gut transit in ApoE−/− mice.Oncotarget. 2017; 8: 33122-33136Crossref PubMed Scopus (8) Google Scholar). This indicates that reduced lipid absorption also contributes to decreased obesity in MGL−/− mice.TABLE 1Plasma metabolites and insulin levels in WT versus MGL−/− mice fed a WDWTMGL −/−NEFA (mmol/l)1.6 ± 0.10.86 ± 0.1*TG (mg/dl)51 ± 1223 ± 2*Chol (mg/dl)159 ± 20110 ± 12Glycerol (mM)0.3 ± 0.20.1 ± 0.1*Insulin (µg/l)4 ± 0.62.6 ± 0.2*BOB (µmol/l)0.1 ± 0.10.1 ± 0.2Values were obtained from male mice receiving a WD for 12 weeks. Data are presented as mean ± SD (n = 5).* P < 0.05. Open table in a new tab Values were obtained from male mice receiving a WD for 12 weeks. Data are presented as mean ± SD (n = 5). * P < 0.05. In line with reduced hepatic TG content, gene expression of key markers of FA synthesis such as Srebp1c, Fasn, and FA storage Pparγ2 was down-regulated (Fig. 2A). Given the downregulation of hepatic Pparγ2 mRNA expression, we next analyzed its transcriptional target diacylglycerol O-acyltransferase 1 (Dgat1), which catalyzes the formation of TGs from diacylglycerol and acyl-CoA (26Harris C.A. Haas J.T. Streeper R.S. Stone S.J. Kumari M. Yang K. Han X. Brownell N. Gross R.W. Zechner R. et al.DGAT enzymes are required for triacylglycerol synthesis and lipid droplets in adipocytes.J. Lipid Res. 2011; 52: 657-667Abstract Full Text Full Text PDF PubMed Scopus (213) Google Scholar). Notably, Dgat1 expression was significantly downregulated together with Cd36 in obese MGL−/− mice (Fig. 2A). Western blot analysis, however, showed no statistically significant changes in PPARγ2 protein expression (Fig. 2B). mRNA levels of Pparα, carnitine palmitoyltransferase 1A (Cpt1α), PPARG coactivator 1 Alpha, and aldehyde oxidase (Aox) increased (Fig. 2C), whereas adipose triglyceride lipase (Atgl), abhydrolase domain containing 5 (Cgi-58), and hormone-sensitive lipase (Hsl) diminished (Fig. 2D), suggesting an effect on FA metabolism. Genes involved in Chol synthesis were decreased for sterol regulatory element binding transcription factor 2 (Srebf2) and in trend for 3-hydroxy-3-methylglutaryl-CoA reductase (Hmgcr) in MGL−/− mice fed a WD (Fig. 2D). Plasma glycerol and insulin level significantly diminished, whereas ketone bodies remained unchanged, as shown by BOB measurement (Table 1). mRNA levels of Foxo1, as key regulators of hepatic gluconeogenesis, were significantly enhanced in obese WT livers, whereas the absence of MGL protected from WD-induced hepatic Foxo1 upregulation (supplemental Fig. S1D). Expression of Pepck, a gene involved in gluconeogenesis, was decreased in obese MGL−/− mice compared with WT (supplemental Fig. S1D). Intriguingly, primary hepatocytes treated with the MGL inhibitor JZL184 confirmed in vivo data, with a significant downregulation of Pparγ2, Fasn, and Dgat1 gene expression (Fig. 2E). Collectively, these results suggest that the reduced hepatic TGs and intestinal loss of FAs could contribute to the diminished hepatic steatosis in MGL−/− mice. Moreover, downregulation of hepatic gluconeogenic enzymes translates into beneficial effects of MGL deficiency on lipid homeostasis in obesity. In order to evaluate the potential impact on hepatic inflammation, gene expression of proinflammatory markers was analyzed together with immunohistochemistry (IHC) for F4/80 in liver. Interestingly, hepatic gene expression of C-C motif chemokine ligand 2 (Ccl2) and F4/80 were markedly increased in WD-fed WT, but not in MGL−/−, animals (Fig. 2F). Furthermore, IHC analysis of liver sections showed a higher accumulation of macrophages in obese WT mice, but not in MGL−/− mice (supplemental Fig. S1B). Fibrosis remained unchanged as evidenced by Sirius Red staining (supplemental Fig. S1B) and hepatic mRNA expression of collagen type I alpha 1 chain and transforming growth factor beta 1 (Tgfβ) (Fig. 2F). Taken together, these results reveal that MGL is a mediator of hepatic steatosis and inflammation, but not fibrosis, in obesity-driven inflammation. To further explore fat storage in adipose depots as a potential mechanism preventing ectopic fat accumulation, we measured gWAT weight, which was increased in MGL−/− compared with WT animals, as already shown in Fig. 1B. Adipocyte expansion was further evident from increased mGPES1a mRNA levels (supplemental Fig. S2A). Gene expression involved in, respectively, FA storage, lipolysis, and synthesis was increased in MGL−/− mice as demonstrated by Pparγ2, Cd36, Atgl, Hsl, Fasn, and Dgat1 (Fig. 3A), together with leptin and adiponectin (Fig. 3B). Adiponectin serum levels were increased in MGL−/− mice fed a WD (Fig. 3C), whereas genes involved in mitochondrial β-oxidation such as Pparα remained unchanged, and Cpt1α decreased in gWAT (Fig. 3D). Importantly, inflammation was diminished in gWAT from MGL−/− mice, as reflected by Ccl2 and Tnfa gene expression (Fig. 3E) and diminished crown-like structures on Mac-2 staining (Fig. 3F). Notably, subcutaneous white AT (sWAT) weight increased in MGL−/− mice (supplemental Fig. S2B), and FA synthesis pathways increased for Dgat1 and remained unchanged for Pparγ2 and Cd36, whereas inflammation strongly diminished in the MGL−/− sWAT depot (supplemental Fig. S2C). Furthermore, PPARα signature remained unremarkable in brown AT (BAT), whereas uncoupling protein 1 gene expression increased after WD feeding in MGL−/− mice (supplemental Fig. S2D). Because our data suggested that global MGL deficiency may promote adipogenesis and TG storage capacity in gWAT in vivo, we next used the 3T3-L1 cell line and differentiated them into full adipocytes during a time course of 13 days (16Madsen L. Petersen R.K. Sørensen M.B. Jørgensen C. Hallenborg P. Pridal L. Fleckner J. Amri E.Z. Krieg P. Furstenberger G. et al.Adipocyte differentiation of 3T3–L1 preadipocytes is dependent on lipoxygenase activity during the initial stages of the differentiation process.Biochem. J. 2003; 375: 539-549Crossref PubMed Scopus (114) Google Scholar, 17Neal J.W. Clipstone N.A. Calcineurin mediates the calcium-dependent inhibition of adipocyte differentiation in 3T3–L1 cells.J. Biol. Chem. 2002; 277: 49776-49781Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar). Interestingly, Mgl expression peaked at day 9 (Fig. 4A), whereas Pparγ peaked at day 13. We next used 3T3-L1 cells to perform MGL inhibition with JZL184, which strongly upregulated Pparγ2, leptin, and adiponectin (Fig. 4B) and therefore augmenting lipid storage and cell size as shown in the flow cytometry analysis (Fig. 4C). Furthermore, we verified this observation in APCs isolated from the stroma-vascular fraction of WT (Fig. 4D) and MGL−/− mice (Fig. 4E). Interestingly, progenitor cells from MGL−/− mice displayed higher potential to differentiate into mature adipocytes as evidenced by enhanced expression of adipogenic markers (in MGL−/− preadipocytes such as Dgat1, Pparγ2, and adiponectin; Fig. 4E) together with increased lipid content and cell size in the BODIPY flow cytometric analysis (supplemental Fig. S2E). Characterization of APCs from WT and MGL−/− mice further showed a nonsignificant reduction for PPARγ (Fig. 4F). Because Pparγ2 gene expression upregulated in 3T3-L1 cells upon JZL184 treatment and in APC differentiation, we aimed at investigating the ability of PPARγ to regulate MGL expression. Fully differentiated 3T3-L1 adipocytes were treated with known PPARγ agonists s
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