Hepatic monoacylglycerol acyltransferase 1 is induced by prolonged food deprivation to modulate the hepatic fasting response
2019; Elsevier BV; Volume: 60; Issue: 3 Linguagem: Inglês
10.1194/jlr.m089722
ISSN1539-7262
AutoresAndrew J. Lutkewitte, Kyle S. McCommis, George G. Schweitzer, Kari T. Chambers, Mark J. Graham, Lingjue Wang, Gary J. Patti, Angela Hall, Brian N. Finck,
Tópico(s)Peroxisome Proliferator-Activated Receptors
ResumoDuring prolonged fasting, the liver plays a central role in maintaining systemic energy homeostasis by producing glucose and ketones in processes fueled by oxidation of fatty acids liberated from adipose tissue. In mice, this is accompanied by transient hepatic accumulation of glycerolipids. We found that the hepatic expression of monoacylglycerol acyltransferase 1 (Mogat1), an enzyme with monoacylglycerol acyltransferase (MGAT) activity that produces diacylglycerol from monoacylglycerol, was significantly increased in the liver of fasted mice compared with mice given ad libitum access to food. Basal and fasting-induced expression of Mogat1 was markedly diminished in the liver of mice lacking the transcription factor PPARα. Suppressing Mogat1 expression in liver and adipose tissue with antisense oligonucleotides (ASOs) reduced hepatic MGAT activity and triglyceride content compared with fasted controls. Surprisingly, the expression of many other PPARα target genes and PPARα activity was also decreased in mice given Mogat1 ASOs. When mice treated with control or Mogat1 ASOs were gavaged with the PPARα ligand, WY-14643, and then fasted for 18 h, WY-14643 administration reversed the effects of Mogat1 ASOs on PPARα target gene expression and liver triglyceride content. In conclusion, Mogat1 is a fasting-induced PPARα target gene that may feed forward to regulate liver PPARα activity during food deprivation. During prolonged fasting, the liver plays a central role in maintaining systemic energy homeostasis by producing glucose and ketones in processes fueled by oxidation of fatty acids liberated from adipose tissue. In mice, this is accompanied by transient hepatic accumulation of glycerolipids. We found that the hepatic expression of monoacylglycerol acyltransferase 1 (Mogat1), an enzyme with monoacylglycerol acyltransferase (MGAT) activity that produces diacylglycerol from monoacylglycerol, was significantly increased in the liver of fasted mice compared with mice given ad libitum access to food. Basal and fasting-induced expression of Mogat1 was markedly diminished in the liver of mice lacking the transcription factor PPARα. Suppressing Mogat1 expression in liver and adipose tissue with antisense oligonucleotides (ASOs) reduced hepatic MGAT activity and triglyceride content compared with fasted controls. Surprisingly, the expression of many other PPARα target genes and PPARα activity was also decreased in mice given Mogat1 ASOs. When mice treated with control or Mogat1 ASOs were gavaged with the PPARα ligand, WY-14643, and then fasted for 18 h, WY-14643 administration reversed the effects of Mogat1 ASOs on PPARα target gene expression and liver triglyceride content. In conclusion, Mogat1 is a fasting-induced PPARα target gene that may feed forward to regulate liver PPARα activity during food deprivation. During prolonged fasting, the liver receives an influx of carbon substrates from adipose tissue lipolysis and skeletal muscle lactate production and proteolysis. The liver must convert these substrates into glucose and ketones for energy use in extrahepatic tissues, most notably the brain, via processes that are fueled by high level fatty acid oxidation. In mice, the marked influx of fatty acids leads to intrahepatic triglyceride accumulation (steatosis) despite high rates of fatty acid oxidation. Although a number of studies have examined the effects of fasting on hepatic intermediary metabolism, some metabolic pathways remain understudied. Hepatic triglyceride synthesis is generally believed to use primarily glycerol-3-phosphate as an initial substrate. Fatty acyl chains are esterified to glycerol-3-phosphate by acyltransferases to form lysophosphatidic acid and then phosphatidic acid (PA), which is converted into diacylglycerol (DAG) by the PA phosphohydrolases (1.Coleman R.A. Lee D.P. Enzymes of triacylglycerol synthesis and their regulation.Prog. Lipid Res. 2004; 43: 134-176Crossref PubMed Scopus (708) Google Scholar). Alternatively, monoacylglycerol (MAG), which is composed of one fatty acid and a glycerol backbone (unphosphorylated), can be directly converted into DAG through acylation by MAG acyltransferases (MGATs, gene symbol Mogats) (1.Coleman R.A. Lee D.P. Enzymes of triacylglycerol synthesis and their regulation.Prog. Lipid Res. 2004; 43: 134-176Crossref PubMed Scopus (708) Google Scholar, 2.Shi Y. Cheng D. Beyond triglyceride synthesis: the dynamic functional roles of MGAT and DGAT enzymes in energy metabolism.Am. J. Physiol. Endocrinol. Metab. 2009; 297: E10-E18Crossref PubMed Scopus (155) Google Scholar). The MGAT pathway is highly active in the intestine, where it is involved in dietary fat absorption (3.Chon S-H. Zhou Y.X. Dixon J.L. Storch J. Intestinal monoacylglycerol metabolism: developmental and nutritional regulation or monoacylglycerol lipase and monoacylglycerol acyltransferase.J. Biol. Chem. 2007; 282: 33346-33357Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar, 5.Yen C-L. E. Stone S.J. Cases S. Zhou P. Farese R.V. Identification of a gene encoding MGAT1, a monoacylglycerol acyltransferase.Proc. Natl. Acad. Sci. USA. 2002; 99: 8512-8517Crossref PubMed Scopus (151) Google Scholar). However, much less is known about the MGAT enzymes in other tissues with high rates of triglyceride synthesis, such as adipose tissue and liver. Mice have two functional MGAT isoforms, while humans have three (4.Hall A.M. Kou K. Chen Z. Pietka T.A. Kumar M. Korenblat K.M. Lee K. Ahn K. Fabbrini E. Klein S. et al.Evidence for regulated monoacylglycerol acyltransferase expression and activity in human liver.J. Lipid Res. 2012; 53: 990-999Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar, 7.Yue Y.G. Chen Y.Q. Zhang Y. Wang H. Qian Y-W. Arnold J.S. Calley J.N. Li S.D. Perry W.L. Zhang H.Y. et al.The acyl coenzymeA:monoacylglycerol acyltransferase 3 (MGAT3) gene is a pseudogene in mice but encodes a functional enzyme in rats.Lipids. 2011; 46: 513-520Crossref PubMed Scopus (23) Google Scholar). In mice, Mogat1 is highly expressed in liver, stomach, and adipose tissue, while Mogat2 is the intestinal isoform (4.Hall A.M. Kou K. Chen Z. Pietka T.A. Kumar M. Korenblat K.M. Lee K. Ahn K. Fabbrini E. Klein S. et al.Evidence for regulated monoacylglycerol acyltransferase expression and activity in human liver.J. Lipid Res. 2012; 53: 990-999Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar, 6.Cao J. Lockwood J. Burn P. Shi Y. 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Selective retention of essential fatty acids: the role of hepatic monoacylglycerol acyltransferase.Am. J. Physiol. 1993; 265: R414-R419PubMed Google Scholar). Recently, several groups have shown an important function of Mogat1 in obesity-related nonalcoholic fatty liver disease (9.Mostafa N. Bhat B.G. Coleman R.A. Increased hepatic monoacylglycerol acyltransferase activity in streptozotocin-induced diabetes: characterization and comparison with activities from adult and neonatal rat liver.Biochim. Biophys. Acta. 1993; 1169: 189-195Crossref PubMed Scopus (24) Google Scholar, 11.Cortés V.A. Curtis D.E. Sukumaran S. Shao X. Parameswara V. Rashid S. Smith A.R. Ren J. Esser V. Hammer R.E. et al.Molecular mechanisms of hepatic steatosis and insulin resistance in the AGPAT2-deficient mouse model of congenital generalized lipodystrophy.Cell Metab. 2009; 9: 165-176Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar, 14.Soufi N. Hall A.M. Chen Z. Yoshino J. Collier S.L. Mathews J.C. Brunt E.M. Albert C.J. Graham M.J. Ford D.A. et al.Inhibiting monoacylglycerol acyltransferase 1 ameliorates hepatic metabolic abnormalities but not inflammation and injury in mice.J. Biol. Chem. 2014; 289: 30177-30188Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar). Mogat1 expression was induced in liver of obese mice with hepatic steatosis and the expression of Mogat1 in this context was driven by PPARγ. Strategies to suppress Mogat1 with RNAi methodology markedly improved hepatic insulin resistance (12.Hall A.M. Soufi N. Chambers K.T. Chen Z. Schweitzer G.G. McCommis K.S. Erion D.M. Graham M.J. Su X. Finck B.N. Abrogating monoacylglycerol acyltransferase activity in liver improves glucose tolerance and hepatic insulin signaling in obese mice.Diabetes. 2014; 63: 2284-2296Crossref PubMed Scopus (52) Google Scholar, 13.Lee Y.J. Ko E.H. Kim J.E. Kim E. Lee H. Choi H. Yu J.H. Kim H.J. Seong J-K. Kim K-S. et al.Nuclear receptor PPARγ-regulated monoacylglycerol O-acyltransferase 1 (MGAT1) expression is responsible for the lipid accumulation in diet-induced hepatic steatosis.Proc. Natl. Acad. Sci. USA. 2012; 109: 13656-13661Crossref PubMed Scopus (112) Google Scholar) and reduced hepatic steatosis in some models (13.Lee Y.J. Ko E.H. Kim J.E. Kim E. Lee H. Choi H. Yu J.H. Kim H.J. Seong J-K. Kim K-S. et al.Nuclear receptor PPARγ-regulated monoacylglycerol O-acyltransferase 1 (MGAT1) expression is responsible for the lipid accumulation in diet-induced hepatic steatosis.Proc. Natl. Acad. Sci. USA. 2012; 109: 13656-13661Crossref PubMed Scopus (112) Google Scholar). While recent work with whole-body Mogat1 knockout mice has questioned the contribution of this gene to tissue MGAT activity (15.Agarwal A.K. Tunison K. Dalal J.S. Yen C-L. E. Farese R.V. Horton J.D. Garg A. Mogat1 deletion does not ameliorate hepatic steatosis in lipodystrophic (Agpat2-/-) or obese (ob/ob) mice.J. Lipid Res. 2016; 57: 616-630Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar), acute suppression of Mogat1 expression markedly reduced MGAT activity in steatotic liver (12.Hall A.M. Soufi N. Chambers K.T. Chen Z. Schweitzer G.G. McCommis K.S. Erion D.M. Graham M.J. Su X. Finck B.N. Abrogating monoacylglycerol acyltransferase activity in liver improves glucose tolerance and hepatic insulin signaling in obese mice.Diabetes. 2014; 63: 2284-2296Crossref PubMed Scopus (52) Google Scholar), suggesting chronic compensation in knockout mice. Herein, we demonstrate that the hepatic expression of Mogat1 and MGAT activity is increased in the liver of mice undergoing prolonged fasting. Furthermore, hepatic Mogat1 expression is dependent on PPARα, which is a master regulator of the hepatic fasting response (16.Kersten S. Seydoux J. Peters J.M. Gonzalez F.J. Desvergne B. Wahli W. Peroxisome proliferator-activated receptor alpha mediates the adaptive response to fasting.J. Clin. Invest. 1999; 103: 1489-1498Crossref PubMed Scopus (1360) Google Scholar, 17.Leone T.C. Weinheimer C.J. Kelly D.P. A critical role for the peroxisome proliferator- activated receptor alpha (PPARalpha) in the cellular fasting response: the PPARalpha-null mouse as a model of fatty acid oxidation disorders.Proc. Natl. Acad. Sci. USA. 1999; 96: 7473-7478Crossref PubMed Scopus (819) Google Scholar). We demonstrate that mice with antisense oligonucleotide (ASO)-mediated acute knockdown of Mogat1 have decreased MGAT activity and lower liver triglyceride content. Interestingly, Mogat1 suppression during fasting also led to reduced expression of PPARα target genes; an effect that was reversed by the synthetic PPARα ligand, WY-14643. These data suggest that Mogat1 is a fasting-induced PPARα target gene that may feed-forward to enhance PPARα activity and the systemic response to food deprivation. All mouse studies were approved by the Institutional Animal Care and Use Committee of Washington University. All mice were in the pure C57BL/6J background. Male whole-body PPARα-null mice were obtained from Taconic. Mice were group housed and maintained on standard laboratory chow on a 12 h light/dark cycle. On the day of the experiment, 10- to 12-week-old male and female mice were randomly assigned to receive either ad libitum (fed) access to standard laboratory chow or food deprived (fasted) for the times indicated. Fasts were timed such that all mice were euthanized at 0900. Liver tissue was harvested and immediately frozen in liquid nitrogen until further use. Blood was collected by venipuncture of the inferior vena cava in EDTA-coated tubes and plasma was frozen after collection by centrifugation. ASO treatments were performed as previously described (12.Hall A.M. Soufi N. Chambers K.T. Chen Z. Schweitzer G.G. McCommis K.S. Erion D.M. Graham M.J. Su X. Finck B.N. Abrogating monoacylglycerol acyltransferase activity in liver improves glucose tolerance and hepatic insulin signaling in obese mice.Diabetes. 2014; 63: 2284-2296Crossref PubMed Scopus (52) Google Scholar). Briefly, 8-week-old male C57BL/6J mice (Jackson Laboratory, Bar Harbor, ME) were given twice weekly intraperitoneal injections of ASO directed against Mogat1 [5′-GATCTTGGCCACGTGGAGAT-3′ (20-mer)] or scramble control (Ionis, Pharmaceuticals, Inc., Carlsbad, CA; 25 mg/kg body weight) for 3 weeks. Mice were then fasted for 24 h or continued to receive ad libitum access to food. Knockdown was confirmed by the quantitative (q)PCR methods described below using the following sequences against Mogat1: forward 5′- TGGTGCCAGTTTGGTTCCAG-3′ and reverse 5′-TGCTCTGAGGTCGGGTTCA-3′. For studies involving PPARα ligand administration, mice were treated with ASO as before and given a one-time oral gavage of vehicle alone (0.5% carboxymethyl cellulose) or WY-14643 (100 mg/kg body weight; Cayman Chemical, Ann Arbor, MI) (18.Rusli F. Deelen J. Andriyani E. Boekschoten M.V. Lute C. van den Akker E.B. Müller M. Beekman M. Steegenga W.T. Fibroblast growth factor 21 reflects liver fat accumulation and dysregulation of signalling pathways in the liver of C57BL/6J mice.Sci. Rep. 2016; 6: 30484Crossref PubMed Scopus (52) Google Scholar). Mice were then immediately fasted for 18 h and harvested as before. Total liver RNA was isolated using RNA-BEE (Iso-Tex Diagnostics, Friendswood, TX) according to manufacturer's instructions. RNA was reverse transcribed into cDNA using TaqMan high-capacity reverse transcriptase (Life Technologies, Woolston, WA). Quantitative RT-PCR was performed using Power SYBR green and measured on an ABI PRISM 7500 sequence detection system (Applied Biosystems, Foster City, CA). Results were normalized to 36B4 expression and calculated using the 2−ΔΔct method and shown as arbitrary units relative to control groups. Primer sequences are listed in Table 1.TABLE 1Primer sequences for qPCRMouseForwardReverse36B4gca gac aac gtg ggc tcc aag cag atggt cct cct tgg tga aca cga agc ccMogat1tgg tgc cag ttt ggt tcc agtgc tct gag gtc ggg ttc aMogat2tgg gag cgc agg tta cag aCag gtg gca tac agg aca gaMgllcgg act tcc aag ttt ttg tca gagca gcc act agg atg gag atgAtglgga cac ctc aat aat gtt ggcctt gag cag cta gaa caa tgDgat1tgc agt ttg gag acc gcg agt tcac cca ttt gct gct gcc atg tNapepldagc gcc aag cta tca gta tcctca gcc atc tga gca cat tcgLpin1ccc tcg att tca agg cac ctgca gcc tgt ggc aat tcaLpin2gaa gtg gcg gct ctc tat ttcaga ggg tta cat cag gca agtAngptl4cat cct ggg acg aga tga acttga caa gcg tta cca cag gcApoc3ggt cca gga tgc gct aag tatgc tcc agt agc ctt tca ggAcox1gga tgg tag tcc gga gaa caagt ctg gat cgt tca gaa tca agCpt1agga cgc gcc cat cgcca ctg tag cct ggt ggg tAcadmgga aat gat caa caa aaa aag tat ttatg gcc gcc aca tca gaAcadlcct ccg ccc gat gtt gtc att cgct gtc cac aaa agc tct ggt gac acBdh1ttc ccc ttc tcc gaa gag cccc aga ggg tgc atc tca tagHmgcs2acc tgc ggg cct tgg atggt gaa aag gct ggt tgt ttc cPck1ggg tgc aga atc tcg agt tgcac cat cac ctc ctg gaa gaPparaact acg gag ttc acg cat gtgttg tcg tac acc agc ttc agcPparg1gga aga cca ctc gca ttc cttgta atc agc aac cat tgg gtc aPparg2tcg ctg atg cac tgc cta tggag agg tcc aca gag ctg attCrebhcct gtt tga tcg gca gga ccgg ggg acc ata atg gag aPpargc1acgg aaa tca tat cca acc agtga gga ccg cta gca agt ttg Open table in a new tab Frozen liver tissue (100 mg/ml) was homogenized in ice-cold PBS and lipids were solubilized in 1% sodium deoxycholate via vortexing and heating at 37°C for 5 min. Hepatic triglyceride content was measured using a commercially available triglyceride colorimetric assay kit as previously described (19.Chen Z. Gropler M.C. Norris J. Lawrence J.C. Harris T.E. Finck B.N. Alterations in hepatic metabolism in fld mice reveal a role for lipin 1 in regulating VLDL-triacylglyceride secretion.Arterioscler. Thromb. Vasc. Biol. 2008; 28: 1738-1744Crossref PubMed Scopus (69) Google Scholar). Plasma samples were analyzed as previously described (12.Hall A.M. Soufi N. Chambers K.T. Chen Z. Schweitzer G.G. McCommis K.S. Erion D.M. Graham M.J. Su X. Finck B.N. Abrogating monoacylglycerol acyltransferase activity in liver improves glucose tolerance and hepatic insulin signaling in obese mice.Diabetes. 2014; 63: 2284-2296Crossref PubMed Scopus (52) Google Scholar). Briefly, plasma NEFAs were measured using a colorimetric assay from WAKO (Wako Diagnostics, Richmond, VA). Plasma triglyceride was measured using the Thermo Fisher kit and plasma ketone bodies were measured using a 3-hydroxybutyrate enzymatic assay (WAKO). For blood glucose measurements, blood was procured from the tail just prior to euthanization, and glucose was measured using a OneTouch Ultra glucometer (LifeScan Inc.). Plasma insulin and glucagon were measured by Singulex immunoassay and mouse Mercodia glucagon ELISA – 10 μL, respectively, by the Washington University Core Laboratory for Clinical Studies. MGAT activity was determined as described (12.Hall A.M. Soufi N. Chambers K.T. Chen Z. Schweitzer G.G. McCommis K.S. Erion D.M. Graham M.J. Su X. Finck B.N. Abrogating monoacylglycerol acyltransferase activity in liver improves glucose tolerance and hepatic insulin signaling in obese mice.Diabetes. 2014; 63: 2284-2296Crossref PubMed Scopus (52) Google Scholar) with slight modification. Membrane fractions were isolated from 50 mg of liver tissue by homogenization in membrane buffer [50 mM Tris-HCl (pH 7.4), 1 mM EDTA, 250 mM sucrose, and complete protease inhibitors tablets (Roche Diagnostics, Indianapolis, IN)]. Homogenates were precleared of whole cell debris at 500 g for 10 min at 4°C followed by ultracentrifugation at 21,000 g for 1 h at 4°C. The resultant pellet was suspended by vigorous pipetting in membrane buffer without protease inhibitors. Fifty micrograms of membrane were incubated in 5 mM MgCl2, 1.25 mg/ml BSA, 200 mM sucrose, 100 mM Tris-HCl (pH 7.4), 20 μM 14C-oleoyl-CoA (American Radiolabeled Chemicals), and 200 μM sn-2-oleoylglycerol for 5 min. The reaction was terminated with 50 μl of 1% phosphoric acid. Lipids were extracted in 2:1 v/v% CHCl3:methanol and separated by thin-layer chromatography in hexane/ethyl ether/acetic acid (80:20:1, v/v/v %). Samples were run against standards for FFA, DAG, and triglyceride and corresponding spots were scraped from the plate and 14C-radioactivity was measured via scintillation counter. Backgrounds were calculated from reaction mixtures without membrane fractions. Mice were treated with ASO for 3 weeks as described above and then fasted overnight. Nuclear fractions were isolated from fresh liver samples according to the manufacture's protocol (Nuclear Extraction Kit 10009277; Cayman Chemical). Briefly, 250 mg of liver tissue were homogenized in 750 μl of ice-cold hypotonic buffer supplemented with 1 mM DTT and 0.01% NP-40 using a Dounce homogenizer. After a 15 min incubation on ice, the cytosol was removed via a 300 g spin at 4°C for 10 min. The pellets were resuspended in 500 μl of hypotonic buffer for an additional 15 min on ice. Cells were lysed with the addition of 1% NP-40 and cytosolic fractions removed after a 14,000 g spin at 4°C for 30 s. Nuclei were lysed in 100 μl of nuclear extraction buffer for 30 min. The nuclear fractions were removed following a 14,000 g spin at 4°C for 10 min and then immediately flash-frozen in liquid nitrogen. PPARα activity was measured according to manufacturer's protocol (PPARα Transcription Factor Assay Kit 10006915; Cayman Chemical). Briefly, 25 μg of nuclear lysates were added in duplicate to each well of the ELISA plate and incubated overnight at 4°C. The plate was read at 450 nm and the results were normalized to control ASO-treated fasted mice following the subtraction of nonspecific binding wells as the blank. Liver and serum samples were treated with 3 ml of 2:1 chloroform:methanol. Liver samples were homogenized in the presence of the following internal standards: U-13C-labeled palmitate (100 pmol/mg), C17:0 MAG (10 pmol/mg), and C17:0 DAG (10 pmol/mg). For serum samples, all internal standards were adjusted to 10 pmol/μl serum. Lipid extraction was performed by vortexing for 1 min and bath sonication for 30 min. After adding 1 ml of 0.1 M Tris buffer, samples were vortexed (1 min) and bath sonicated (30 min). The organic and aqueous layers were then separated by centrifugation at 3,000 g for 20 min. The organic layer was collected. Chloroform (1 ml) was mixed with the aqueous layer followed by vortexing, bath sonication, and centrifugation as described above. The organic layers were combined, dried by N2 gas, and reconstituted in 200 μl of 9:1 methanol:chloroform. The extract was transferred to an LC vial for MS analysis. LC/MS analysis of liver and serum extracts was performed by using an Agilent 6530 or 6540 quadrupole TOF mass spectrometer with an ESI source. Separations were achieved by using an Agilent 1290 capillary UPLC system. Samples were either analyzed in positive ionization mode with a Cortex T3 column (2.7 μm, 150 × 2.1 mm inner diameter; Waters) or in negative ionization mode with a Luna aminopropyl column (3 μm, 150 × 2.0 mm internal diameter; Phenomenex). The T3 column was used with the following mobile phases: A = 95% water, 5% methanol, 5 mM ammonium acetate, 0.1% formic acid (pH 3.14); B = 90% isopropanol and 10% methanol. The following linear gradient was applied: 0 min, 30% B; 10 min, 70% B; 30 min, 87.5% B; 31 min, 100% B; 40 min, 100% B. The aminopropyl column was used with following mobile phases: A = 95% water, 5% acetonitrile, 10 mM ammonium acetate, and 10 mM ammonium hydroxide; B = 95% acetonitrile and 5% water. The following linear gradient was applied: 0 min, 95% B; 2 min, 95% B; 15 min, 50% B; 20 min, 0% B; 25 min, 0% B. The injection volumes were 2 μl. The flow rate was 200 μl/min. The column temperature was maintained at 37°C. Mass detection was set from m/z 100 to 1,700 with a scan rate of 1 spectra/s. The ESI source parameters were: nebulizer pressure, 30 psi; nozzle voltage, 1,000 V; sheath gas temperature, 300°C; flow rate, 11 liters/min; drying gas, 12 liters/min; gas temperature, 300°C; capillary voltage, 3,000 V; fragmentor voltage, 100 V. Data analysis was performed with the Agilent Profinder software. The absolute quantitation of lipids in picomoles per milligram of liver or picomoles per microliter of serum was achieved by measuring the area under the extracted ion chromatogram peak. Data were normalized to an internal standard (U-13C palmitate for FFAs, C17:0 MAG for MAGs) to correct for extraction efficiency. Data were analyzed via SPSS software. Independent samples t-tests, one-way ANOVA, or factorial ANOVAs with Bonferonni corrections were performed. Prior to analysis, normality and equal variance of samples was determined by Kolmogorov-Smirnov testing and Levene testing, respectively. If these conditions were violated logarithmic transformation intended to produce data to satisfy normality and equal variance assumptions were used. Otherwise, nonparametric methods (e.g., Kruskal-Wallis for one-way ANOVAs with Mann-Whitney U post hoc analysis and Friedman's test for two-way ANOVAs) were used as an alternative to the more standard analyses. P < 0.05 was considered significant. Fasting is a physiologic stimulus associated with high rates of hepatic fatty acid oxidation and increased accumulation of hepatic triglyceride. We fasted mice for 4, 18, or 24 h and found that intrahepatic triglyceride content was increased after 18 and 24 h of fasting (Fig. 1A). Given our interest in the MGAT enzymes that are involved in glycerolipid synthesis, we also quantified their gene expression and found that fasting induced the expression of Mogat1, but not Mogat2 (Fig. 1B). Total hepatic MGAT activity was also increased after 18 and 24 h fasting (Fig. 1C). Previous work has shown that Mogat1 is target gene of the PPAR family of nuclear receptors, particularly PPARγ (12.Hall A.M. Soufi N. Chambers K.T. Chen Z. Schweitzer G.G. McCommis K.S. Erion D.M. Graham M.J. Su X. Finck B.N. Abrogating monoacylglycerol acyltransferase activity in liver improves glucose tolerance and hepatic insulin signaling in obese mice.Diabetes. 2014; 63: 2284-2296Crossref PubMed Scopus (52) Google Scholar, 13.Lee Y.J. Ko E.H. Kim J.E. Kim E. Lee H. Choi H. Yu J.H. Kim H.J. Seong J-K. Kim K-S. et al.Nuclear receptor PPARγ-regulated monoacylglycerol O-acyltransferase 1 (MGAT1) expression is responsible for the lipid accumulation in diet-induced hepatic steatosis.Proc. Natl. Acad. Sci. USA. 2012; 109: 13656-13661Crossref PubMed Scopus (112) Google Scholar, 20.Yu J.H. Song S.J. Kim A. Choi Y. Seok J.W. Kim H.J. Lee Y.J. Lee K.S. Kim J. Suppression of PPARγ-mediated monoacylglycerol O-acyltransferase 1 expression ameliorates alcoholic hepatic steatosis.Sci. Rep. 2016; 6: 29352Crossref PubMed Scopus (28) Google Scholar). However, in this experiment, the expression of Mogat1 was highly dependent on PPARα. Indeed, the fed and fasted expression of Mogat1 was markedly diminished in whole-body PPARα-null mice compared with wild-type controls, whereas Mogat2 expression tended to be increased in whole-body PPARα-null mice in both fed and fasted conditions (Fig. 1D). These data demonstrate that Mogat1, but not Mogat2, is induced in liver by fasting and that its expression is highly dependent on PPARα. We utilized an acute model of Mogat1 knockdown using ASOs to determine Mogat1 function during fasting (12.Hall A.M. Soufi N. Chambers K.T. Chen Z. Schweitzer G.G. McCommis K.S. Erion D.M. Graham M.J. Su X. Finck B.N. Abrogating monoacylglycerol acyltransferase activity in liver improves glucose tolerance and hepatic insulin signaling in obese mice.Diabetes. 2014; 63: 2284-2296Crossref PubMed Scopus (52) Google Scholar). Mice were treated for 3 weeks with ASOs directed against Mogat1 or scramble control, and then fasted for 24 h or continued receiving ad libitum access to food. As expected, Mogat1 ASO suppressed hepatic Mogat1 gene expression (Fig. 2A). Mogat1 knockdown did not lead to a compensatory increase in Mogat2 expression in liver (Fig. 2A). Furthermore, 24 h fasting increased total hepatic MGAT activity, while Mogat1 knockdown led to reduced MGAT activity during fasting, indicating the contribution of Mogat1 to total hepatic MGAT activity in fasting (Fig. 2B). Previous work has suggested that ASOs administered by intraperitoneal injection may affect the expression of the targeted gene in adipose tissue. Indeed, epididymal white adipose tissue (eWAT) Mogat1 expression was significantly reduced following ASO treatment (Fig. 2C). However, eWAT tissue MGAT activity was decreased by ASO treatment only in the fed state (Fig. 2D). Furthermore, Mogat1 ASO treatment did not affect adipose tissue weight (Fig. 2E). Fasting reduced blood glucose levels in both fasting conditions, but was not significantly different among ASO treatment groups (Table 2). Interestingly, plasma glucagon levels were increased by Mogat1 ASO treatment in both the fed and fasted states when compared with control ASO treatment (Table 2). However, plasma insulin and glucose levels were unaffected by Mogat1 ASO treatments, suggesting that glucagon levels do not effect glucose homeostasis in these mice (Table 2).TABLE 2Plasma parameters of ASO-treated miceControl ASO: FedMogat1 ASO: FedControl ASO: FastedMogat1 ASO: FastedBlood glucose (mg/dl)192.4 ± 6.0175.0 ± 9.38105.2 ± 12.3aP < 0.05 versus fed state.97.6 ± 10.9aP < 0.05 versus fed state.Plasma Insulin (pg/ml)699.4 ± 41.6709.6 ± 41.1234.4 ± 38.5329.2 ± 36.4Plasma Glucagon (pg/ml)9.6 ± 1.535.4 ± 6.4bP < 0.05 versus control littermates in the same feeding condition.10.9 ± 4.933.0 ± 6.6 (P = 0.09)Blood was procured from the tail vein at the time of euthanization after fasting (24 h) or ad libitum feeding (Fed) for blood glucose measurements. Plasma was collected after euthanization. Insulin and glucagon levels were measured using ELISAs. Data are shown as mean ± SE (n = 5).a P < 0.05 versus fed state.b P < 0.05 versus control littermates in the same feeding condition. Open table in a new tab Blood was procured from the tail vein at the time of euthanization after fasting (24 h) or ad libitum feeding (Fed) for blood glucose measurements. Plasma was collected after euthanization. Insulin and glucagon levels were measured using ELISAs. Data are shown as mean ± SE (n = 5). We assessed the effects of Mogat1 ASO on hepatic steatosis and found that fasting increased the intrahepatic content of trigly
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