DGAT1 deficiency decreases PPAR expression and does not lead to lipotoxicity in cardiac and skeletal muscle
2011; Elsevier BV; Volume: 52; Issue: 4 Linguagem: Inglês
10.1194/jlr.m011395
ISSN1539-7262
AutoresLi Liu, Shuiqing Yu, Raffay Khan, Gene P. Ables, Kalyani G. Bharadwaj, Yunying Hu, Lesley A. Huggins, Jan W. Eriksson, Linda K. Buckett, Andrew V. Turnbull, Henry N. Ginsberg, William S. Blaner, Li‐Shin Huang, Ira J. Goldberg,
Tópico(s)Peroxisome Proliferator-Activated Receptors
ResumoDiacylglycerol (DAG) acyl transferase 1 (Dgat1) knockout (−/−) mice are resistant to high-fat-induced obesity and insulin resistance, but the reasons are unclear. Dgat1−/− mice had reduced mRNA levels of all three Ppar genes and genes involved in fatty acid oxidation in the myocardium of Dgat1−/− mice. Although DGAT1 converts DAG to triglyceride (TG), tissue levels of DAG were not increased in Dgat1−/− mice. Hearts of chow-diet Dgat1−/− mice were larger than those of wild-type (WT) mice, but cardiac function was normal. Skeletal muscles from Dgat1−/− mice were also larger. Muscle hypertrophy factors phospho-AKT and phospho-mTOR were increased in Dgat1−/− cardiac and skeletal muscle. In contrast to muscle, liver from Dgat1−/− mice had no reduction in mRNA levels of genes mediating fatty acid oxidation. Glucose uptake was increased in cardiac and skeletal muscle in Dgat1−/− mice. Treatment with an inhibitor specific for DGAT1 led to similarly striking reductions in mRNA levels of genes mediating fatty acid oxidation in cardiac and skeletal muscle. These changes were reproduced in cultured myocytes with the DGAT1 inhibitor, which also blocked the increase in mRNA levels of Ppar genes and their targets induced by palmitic acid. Thus, loss of DGAT1 activity in muscles decreases mRNA levels of genes involved in lipid uptake and oxidation. Diacylglycerol (DAG) acyl transferase 1 (Dgat1) knockout (−/−) mice are resistant to high-fat-induced obesity and insulin resistance, but the reasons are unclear. Dgat1−/− mice had reduced mRNA levels of all three Ppar genes and genes involved in fatty acid oxidation in the myocardium of Dgat1−/− mice. Although DGAT1 converts DAG to triglyceride (TG), tissue levels of DAG were not increased in Dgat1−/− mice. Hearts of chow-diet Dgat1−/− mice were larger than those of wild-type (WT) mice, but cardiac function was normal. Skeletal muscles from Dgat1−/− mice were also larger. Muscle hypertrophy factors phospho-AKT and phospho-mTOR were increased in Dgat1−/− cardiac and skeletal muscle. In contrast to muscle, liver from Dgat1−/− mice had no reduction in mRNA levels of genes mediating fatty acid oxidation. Glucose uptake was increased in cardiac and skeletal muscle in Dgat1−/− mice. Treatment with an inhibitor specific for DGAT1 led to similarly striking reductions in mRNA levels of genes mediating fatty acid oxidation in cardiac and skeletal muscle. These changes were reproduced in cultured myocytes with the DGAT1 inhibitor, which also blocked the increase in mRNA levels of Ppar genes and their targets induced by palmitic acid. Thus, loss of DGAT1 activity in muscles decreases mRNA levels of genes involved in lipid uptake and oxidation. Fight fat with DGATJournal of Lipid ResearchVol. 52Issue 4PreviewThe triglyceride synthesis pathway is active in virtually every cell type. Conversion of fatty acids into triglycerides (TGs) serves two main purposes. First, it allows for storage of fuel in a very energy dense form and second, it neutralizes free fatty acids and other lipotoxic derivates. Synthesis of TGs is governed by the glycerol phosphate and the monoacylglycerol pathway (1). Most cells use the glycerol phosphate pathway, whereas the monoacylglycerol pathway is utilized specifically by adipocytes, enterocytes, and hepatocytes (2). Full-Text PDF Open Access Triglyceride (TG) is the major energy storage form in tissues. The final step of TG synthesis, the conversion of diacylglycerol (DAG) to TG, is catalyzed by DAG acyltransferases (DGAT). Dgat1 belongs to a family of membrane-bound O-acyltranferase (MBOAT), which includes acyl CoA:cholesterol acyltransferase (Acat) 1 and Acat2 (1Cases S. Smith S.J. Zheng Y.W. Myers H.M. Lear S.R. Sande E. Novak S. Collins C. Welch C.B. Lusis A.J. Identification of a gene encoding an acyl CoA:diacylglycerol acyltransferase, a key enzyme in triacylglycerol synthesis.Proc. Natl. Acad. Sci. USA. 1998; 95: 13018-13023Crossref PubMed Scopus (886) Google Scholar, 2Yen C.L. Monetti M. Burri B.J. Farese Jr, R.V. The triacylglycerol synthesis enzyme DGAT1 also catalyzes the synthesis of diacylglycerols, waxes, and retinyl esters.J. Lipid Res. 2005; 46: 1502-1511Abstract Full Text Full Text PDF PubMed Scopus (222) Google Scholar, 3Oelkers P. Behari A. Cromley D. Billheimer J.T. Sturley S.L. Characterization of two human genes encoding acyl coenzyme A:cholesterol acyltransferase-related enzymes.J. Biol. Chem. 1998; 273: 26765-26771Abstract Full Text Full Text PDF PubMed Scopus (336) Google Scholar). Dgat1 is widely expressed in all tissues, with high expression in white adipose tissue, skeletal muscle, heart, and intestine (1Cases S. Smith S.J. Zheng Y.W. Myers H.M. Lear S.R. Sande E. Novak S. Collins C. Welch C.B. Lusis A.J. Identification of a gene encoding an acyl CoA:diacylglycerol acyltransferase, a key enzyme in triacylglycerol synthesis.Proc. Natl. Acad. Sci. USA. 1998; 95: 13018-13023Crossref PubMed Scopus (886) Google Scholar, 2Yen C.L. Monetti M. Burri B.J. Farese Jr, R.V. The triacylglycerol synthesis enzyme DGAT1 also catalyzes the synthesis of diacylglycerols, waxes, and retinyl esters.J. Lipid Res. 2005; 46: 1502-1511Abstract Full Text Full Text PDF PubMed Scopus (222) Google Scholar). Dgat2 is a member of the monoacylglycerol acyltransferase family and is primarily expressed in the liver and adipose tissue (2Yen C.L. Monetti M. Burri B.J. Farese Jr, R.V. The triacylglycerol synthesis enzyme DGAT1 also catalyzes the synthesis of diacylglycerols, waxes, and retinyl esters.J. Lipid Res. 2005; 46: 1502-1511Abstract Full Text Full Text PDF PubMed Scopus (222) Google Scholar); DGAT2 is thought to be the primary source of DGAT activity in the liver (4Choi C.S. Savage D.B. Kulkarni A. Yu X.X. Liu Z.X. Morino K. Kim S. Distefano A. Samuel V.T. Neschen S. Suppression of diacylglycerol acyltransferase-2 (DGAT2), but not DGAT1, with antisense oligonucleotides reverses diet-induced hepatic steatosis and insulin resistance.J. Biol. Chem. 2007; 282: 22678-22688Abstract Full Text Full Text PDF PubMed Scopus (293) Google Scholar). The effects of Dgat1 overexpression in tissues have suggested that conversion of DAG to TG can be a detoxifying process (5Liu L. Zhang Y. Chen N. Shi X. Tsang B. Yu Y.H. Upregulation of myocellular DGAT1 augments triglyceride synthesis in skeletal muscle and protects against fat-induced insulin resistance.J. Clin. Invest. 2007; 117: 1679-1689Crossref PubMed Scopus (269) Google Scholar, 6Liu L. Shi X. Bharadwaj K.G. Ikeda S. Yamashita H. Yagyu H. Schaffer J.E. Yu Y.H. Goldberg I.J. DGAT1 expression increases heart triglyceride content but ameliorates lipotoxicity.J. Biol. Chem. 2009; 284: 36312-36323Abstract Full Text Full Text PDF PubMed Scopus (179) Google Scholar). Overexpression of Dgat1 in skeletal muscle increased tissue content of TG but improved insulin sensitivity (5Liu L. Zhang Y. Chen N. Shi X. Tsang B. Yu Y.H. Upregulation of myocellular DGAT1 augments triglyceride synthesis in skeletal muscle and protects against fat-induced insulin resistance.J. Clin. Invest. 2007; 117: 1679-1689Crossref PubMed Scopus (269) Google Scholar). These data support the hypothesis that conversion of intermediary lipids to TG via DGAT1 is beneficial. In fact, this alteration in lipid metabolism appears to mimic that found with athletic training, in which TG stores increase, fatty acid oxidation increases, and insulin sensitivity improves (7Goodpaster B.H. He J. Watkins S. Kelley D.E. Skeletal muscle lipid content and insulin resistance: evidence for a paradox in endurance-trained athletes.J. Clin. Endocrinol. 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When a cardiomyocyte-expressing human DGAT1 transgene was crossed onto a heart failure model overexpressing acyl CoA synthetase 1, heart function and survival rate were significantly improved (6Liu L. Shi X. Bharadwaj K.G. Ikeda S. Yamashita H. Yagyu H. Schaffer J.E. Yu Y.H. Goldberg I.J. DGAT1 expression increases heart triglyceride content but ameliorates lipotoxicity.J. Biol. Chem. 2009; 284: 36312-36323Abstract Full Text Full Text PDF PubMed Scopus (179) Google Scholar). Similarly, overexpression of DGAT1 in macrophages alleviated fatty acid-induced inflammation and high-fat feeding-induced insulin resistance (9Koliwad S.K. Streeper R.S. Monetti M. Cornelissen I. Chan L. Terayama K. Naylor S. Rao M. Hubbard B. Farese Jr, R.V. DGAT1-dependent triacylglycerol storage by macrophages protects mice from diet-induced insulin resistance and inflammation.J. Clin. Invest. 2010; 120: 756-767Crossref PubMed Scopus (156) Google Scholar). Despite these seemingly beneficial actions of DGAT1 overexpression, its deletion does not lead to an opposite phenotype. Dgat1 knockout (Dgat1−/−) mice have reduced diet-induced obesity and less insulin resistance (10Smith S.J. Cases S. Jensen D.R. Chen H.C. Sande E. Tow B. Sanan D.A. Raber J. Eckel R.H. Farese Jr, R.V. Obesity resistance and multiple mechanisms of triglyceride synthesis in mice lacking Dgat.Nat. Genet. 2000; 25: 87-90Crossref PubMed Scopus (743) Google Scholar). Greater insulin sensitivity was also found in skeletal muscle in these mice (22Barger P.M. Kelly D.P. PPAR signaling in the control of cardiac energy metabolism.Trends Cardiovasc. Med. 2000; 10: 238-245Crossref PubMed Scopus (407) Google Scholar); however, the reasons remain unclear. Other experiments showed that the transplantation of Dgat1−/− white adipose tissue decreases adiposity and enhances glucose disposal in wild-type (WT) mice; adiponectin secreted by adipose tissue was postulated to contribute to the increased energy expenditure and insulin sensitivity in Dgat1−/− mice (11Chen H.C. Jensen D.R. Myers H.M. Eckel R.H. Farese Jr, R.V. Obesity resistance and enhanced glucose metabolism in mice transplanted with white adipose tissue lacking acyl CoA:diacylglycerol acyltransferase 1.J. Clin. Invest. 2003; 111: 1715-1722Crossref PubMed Scopus (82) Google Scholar, 12Chen H.C. Smith S.J. Ladha Z. Jensen D.R. Ferreira L.D. Pulawa L.K. McGuire J.G. Pitas R.E. Eckel R.H. Farese Jr, R.V. Increased insulin and leptin sensitivity in mice lacking acyl CoA:diacylglycerol acyltransferase 1.J. Clin. Invest. 2002; 109: 1049-1055Crossref PubMed Scopus (284) Google Scholar). This observation has spurred pharmaceutical development of DGAT1 inhibitors. However, the effect of loss of DGAT1 activity in other important organs, such as the heart, has not been reported. We hypothesized that Dgat1−/− mice would have reduced conversion of DAG to triglyceride. This might lead to accumulation of DAG, ceramide, and fatty acids in cardiac muscle, which could lead to cardiomyopathy, especially in the setting of high-fat diets. In this study, we describe the effects of acute and chronic loss of DGAT1 activity in muscles. We show that loss of this enzyme modulates counter-regulatory pathways that reduce muscle lipid and increase glucose uptake. This finding suggests that DGAT inhibitors may be of benefit in the treatment of reperfusion injury. Animal protocols were in compliance with accepted standards of animal care and were approved by the Columbia University Institutional Animal Care and Use Committee. Male mice were used in experiments unless otherwise indicated. WT, C57BL/6J, and Dgat1−/− C57BL/6J mice (10Smith S.J. Cases S. Jensen D.R. Chen H.C. Sande E. Tow B. Sanan D.A. Raber J. Eckel R.H. Farese Jr, R.V. Obesity resistance and multiple mechanisms of triglyceride synthesis in mice lacking Dgat.Nat. Genet. 2000; 25: 87-90Crossref PubMed Scopus (743) Google Scholar) were purchased from the Jackson Laboratory. Mice were housed in a barrier facility with 12-h light/12-h dark cycles and had ad libitum access to either chow diet (5053 PicoLab Rodent Diet 20; Purina Mills) or high-fat diet (45% fat primarily from lard, 20% protein, 35% carbohydrates, catalog no. D12451: Research Diets, Inc.). To measure TG, DAG, and ceramide, lipids were first extracted from muscles using chloroform/methanol/HCl (v/v/v, 2:1:0.01) (5Liu L. Zhang Y. Chen N. Shi X. Tsang B. Yu Y.H. Upregulation of myocellular DGAT1 augments triglyceride synthesis in skeletal muscle and protects against fat-induced insulin resistance.J. Clin. Invest. 2007; 117: 1679-1689Crossref PubMed Scopus (269) Google Scholar). Butylated hydroxytoluene (0.01%) was included in the extraction solution as an antioxidant, and [3H]triolein (0.25 μCi) was used as an internal control for TG recovery. TG and fatty acid concentrations in lipid extracts were determined enzymatically with colorimetric kits (Sigma-Aldrich). DAG and ceramide levels were measured using a DAG kinase-based method. Lipids extracted from muscle specimens were dried under a stream of N2, redissolved in 7.5% octyl-β-D-glucoside containing 5 mM cardiolipin and 1 mM diethylenetriamine pentaacetate, in which DAG and ceramide are quantitatively phosphorylated to form 32P-labeled phosphatidic acid and ceramide-1-phosphate, respectively, which were then quantified. The reaction was carried out at room temperature for 30 min in 100 mM imidazole HCl, 100 mM NaCl, 25 mM MgCl2, 2 mM EGTA (pH 6.6), 2 mM DTT, 10 μg/100 μl DAG kinase (Sigma-Aldrich), 1 mM ATP, and 1 μCi/100 μl [γ-32P]ATP. The reaction was stopped by addition of chloroform/methanol (v/v, 2:1) and 1% HClO4, and lipids were extracted and washed twice with 1% HClO4. Lipids were resolved by TLC (Partisil K6 adsorption TLC plates, Whatman catalog no. LK6D); mobile phase contained chloroform/methanol/acetic acid (v/v/v, 65:15:5). The bands corresponding to phosphatidic acid and ceramide-1-phosphate were identified with known standards, and silicon was scraped into a scintillation vial for radioactivity measurement. [3H]triolein bands from the same TLC plates were identified and quantified in the same way and were used as controls for lipid recovery. DGAT activity was measured in membrane fractions isolated from muscle specimens using [14C]labeled palmitoyl-CoA as previously described (5Liu L. Zhang Y. Chen N. Shi X. Tsang B. Yu Y.H. Upregulation of myocellular DGAT1 augments triglyceride synthesis in skeletal muscle and protects against fat-induced insulin resistance.J. Clin. Invest. 2007; 117: 1679-1689Crossref PubMed Scopus (269) Google Scholar). The amounts of both DAG and fatty acyl-CoA in the reaction mixture were in excess, and the reaction rate was of the first order with respect to the amount of input DGAT activity to be assayed. Total RNA was extracted using a Trizol kit from Invitrogen. An amount of 1 μg of RNA was initially treated with DNase I (Invitrogen). The RNA samples were then reverse-transcribed using the SuperScript III First-Strand Synthesis System for RT-PCR (Invitrogen). Real-time amplification was performed using iQ SYBR Green Supermix (Bio-Rad). Primers used for PCR amplification are listed in supplementary Table I. Analysis was performed using iCycler iQ Real-Time Detection System software (Bio-Rad). The dual energy X-ray absorptiometry (DEXA) machine was calibrated before use. WT, Dgat1+/−, and Dgat1−/− mice were anesthetized with intraperitoneal injection of ketamine/xylazine (86.98 mg/kg and 13.4 mg/kg, respectively), and whole body measurements, excluding the head, were made (DEXA, PIXImus Lunar-GE). Measurements included body weight, lean mass, fat mass, and percentage of fat/body weight. One person performed all scans, and mice were always in the prostrate position on the imaging-positioning tray. Mice were forced to swim in tanks. The swimming protocol was a modification of the procedure used by Ryder et al. (13Ryder J.W. Kawano Y. Galuska D. Fahlman R. Wallberg-Henriksson H. Charron M.J. Zierath J.R. Postexercise glucose uptake and glycogen synthesis in skeletal muscle from GLUT4-deficient mice.FASEB J. 1999; 13: 2246-2256Crossref PubMed Scopus (60) Google Scholar). Water temperature was maintained at 34-35°C. Mice swam for 10 min sessions twice a day separated by a 10 min break. Sessions were increased by 10 min each day until the fourth day; mice were then allowed to swim for six 30 min intervals separated by 10-15 min rest periods. After the last swim interval, mice were dried and put back in their cages for 18-20 h. An amount of 10-30 mg of tissue was homogenized in lysis buffer containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% Nonidet P40, 0.5% sodium deoxycholate, and 1 protease inhibitor cocktail tablet (Roche)/25-50 ml. Cell lysates (50 μg per sample) obtained after centrifugation at 15,000 g for 10 min at 4°C were resolved by SDS-PAGE and then transferred onto nitrocellulose membranes. Immunoblotting was carried out using the following primary antibodies: phospho-AKT (p-AKT), phospho-mTOR (p-mTOR), total AKT, total mTOR, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Cell Signaling), and PPARα, β, and γ (Santa Cruz). Two-dimensional echocardiography was performed using a high-resolution imaging system with a 30-MHz imaging transducer (Vevo 770; VisualSonics) in unconscious 3-4-month-old mice. The mice were anesthetized with 1.5-2% isoflurane and thereafter maintained on 1-1.5% isoflurane throughout the procedure. Two-dimensional echocardiographic images were obtained and recorded in a digital format. Images were analyzed offline by a researcher blinded to the murine genotype. Left ventricular end-diastolic dimension (LVDd) and left ventricular end-systolic dimension (LVDs) were measured. Percentage fractional shortening, which quantifies contraction of the ventricular wall and is an indication of muscle function, was calculated as % FS = ([LVDd − LVDs] / LVDd) × 100. Cardiac muscle fatty acid oxidation was measured with heart slices as described previously (6Liu L. Shi X. Bharadwaj K.G. Ikeda S. Yamashita H. Yagyu H. Schaffer J.E. Yu Y.H. Goldberg I.J. DGAT1 expression increases heart triglyceride content but ameliorates lipotoxicity.J. Biol. Chem. 2009; 284: 36312-36323Abstract Full Text Full Text PDF PubMed Scopus (179) Google Scholar). Hearts were removed and immediately sliced across the ventricles forming rings ∼0.1-0.2 mm thick and usually 20-30 mg wet weight. The cardiac muscles were first washed for 1 h at 37°C in Krebs-Henseleit bicarbonate buffer [10 mM HEPES (pH 7.35), 24 mM NaHCO3, 114 mM NaCl, 5 mM KCl, 1 mM MgCl2, and 2.2 mM CaCl2] containing 8 mM glucose, 32 mM mannitol, and 0.1% BSA. The reaction was initiated by adding to the mix preconjugated BSA/fatty acid solution (final concentrations: 0.2 mM palmitate with 1 μCi/ml of 9,10-[3H]palmitate and 1.25% BSA). The fatty acid oxidation product 3H2O was determined in muscles incubated for 2 h at 37°C. An aliquot of the completed reaction mixture was transferred into an open microtube, which was placed inside a scintillation vial containing 0.5 ml of distilled water. The scintillation vial was then capped and incubated at 50°C for 18-24 h to reach vapor-phase equilibrium. After the transfer of 3H2O from the reaction mixture to the scintillation vial (by water equilibration), the 3H2O in the scintillation vial was scintillation-counted. The transfer and counting efficiency was calibrated using a standard solution containing 0.1 μCi of 3H2O for re-equilibration. Glucose uptake was measured as described (12Chen H.C. Smith S.J. Ladha Z. Jensen D.R. Ferreira L.D. Pulawa L.K. McGuire J.G. Pitas R.E. Eckel R.H. Farese Jr, R.V. Increased insulin and leptin sensitivity in mice lacking acyl CoA:diacylglycerol acyltransferase 1.J. Clin. Invest. 2002; 109: 1049-1055Crossref PubMed Scopus (284) Google Scholar). Basal glucose uptake was measured following an intravenous administration of 3 μCi of 2-deoxy-D-[1-3H]glucose (PerkinElmer Life Sciences) in 4 h fasted mice. Blood was collected 0.5, 15, 30, and 60 min following injection. At 60 min, tissues were excised after the body cavity was perfused with 10 ml of PBS by cardiac puncture. VLDL was labeled using cholesteryl ester transfer protein to incorporate [14C]triolein into VLDL as described previously (14Augustus A.S. Buchanan J. Park T.S. Hirata K. Noh H.L. Sun J. Homma S. D'Armiento J. Abel E.D. Goldberg I.J. Loss of lipoprotein lipase-derived fatty acids leads to increased cardiac glucose metabolism and heart dysfunction.J. Biol. Chem. 2006; 281: 8716-8723Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar, 15Pillutla P. Hwang Y.C. Augustus A. Yokoyama M. Yagyu H. Johnston T.P. Kaneko M. Ramasamy R. Goldberg I.J. Perfusion of hearts with triglyceride-rich particles reproduces the metabolic abnormalities in lipotoxic cardiomyopathy.Am. J. Physiol. Endocrinol. Metab. 2005; 288: E1229-E1235Crossref PubMed Scopus (41) Google Scholar). After intravenous injection, plasma counts at 0.5, 5, 10, and 15 min were measured, and the mice were perfused with PBS as above. Radioactivity in the tissues was measured in a scintillation counter, and tissue uptake was normalized to recovered counts in the liver. Male 8-week-old mice fed a chow diet or male 14-week-old mice after 6 weeks of high-fat (Harlan TD 88137) feeding were gavaged with 200 μl of vehicle (0.5% hydroxypropylmethyl cellulose/0.1% polysorbate 80) or DGAT1i (5 mg/kg, Compound 2 (10Smith S.J. Cases S. Jensen D.R. Chen H.C. Sande E. Tow B. Sanan D.A. Raber J. Eckel R.H. Farese Jr, R.V. Obesity resistance and multiple mechanisms of triglyceride synthesis in mice lacking Dgat.Nat. Genet. 2000; 25: 87-90Crossref PubMed Scopus (743) Google Scholar, 16Birch A.M. Birtles S. Buckett L.K. Kemmitt P.D. Smith G.J. Smith T.J. Turnbull A.V. Wang S.J. Discovery of a potent, selective, and orally efficacious pyrimidinooxazinyl bicyclooctaneacetic acid diacylglycerol acyltransferase-1 inhibitor.J. Med. Chem. 2009; 52: 1558-1568Crossref PubMed Scopus (65) Google Scholar, 17Birch A.M. Buckett L.K. Turnbull A.V. DGAT1 inhibitors as anti-obesity and anti-diabetic agents.Curr. Opin. Drug Discov. Devel. 2010; 13: 489-496PubMed Google Scholar)) upon food removal at 8 AM. This DGAT1i is a potent inhibitor of human DGAT1 and is selective over DGAT2 and ACAT1 (16Birch A.M. Birtles S. Buckett L.K. Kemmitt P.D. Smith G.J. Smith T.J. Turnbull A.V. Wang S.J. Discovery of a potent, selective, and orally efficacious pyrimidinooxazinyl bicyclooctaneacetic acid diacylglycerol acyltransferase-1 inhibitor.J. Med. Chem. 2009; 52: 1558-1568Crossref PubMed Scopus (65) Google Scholar, 17Birch A.M. Buckett L.K. Turnbull A.V. DGAT1 inhibitors as anti-obesity and anti-diabetic agents.Curr. Opin. Drug Discov. Devel. 2010; 13: 489-496PubMed Google Scholar). Importantly, this DGAT1i potently inhibits mouse microsomal DGAT1 activity (16Birch A.M. Birtles S. Buckett L.K. Kemmitt P.D. Smith G.J. Smith T.J. Turnbull A.V. Wang S.J. Discovery of a potent, selective, and orally efficacious pyrimidinooxazinyl bicyclooctaneacetic acid diacylglycerol acyltransferase-1 inhibitor.J. Med. Chem. 2009; 52: 1558-1568Crossref PubMed Scopus (65) Google Scholar). At the doses utilized in this study, this DGAT1i produces good blood levels in C57BL/6 mice. At 8 h postgavage, mice were sacrificed, and tissues collected for lipid measurement and RNA isolation. AC16 human cardiomyocytes and C2C12 myoblasts were grown in DMEM supplemented with 10% FBS and 1% penicillin/streptomycin at 37°C. C2C12 myoblasts were maintained below 60% confluence to prevent confluence-induced differentiation. For experiments, cells were allowed to become confluent, and the media were supplemented with 2% horse serum instead of FBS for myotube formation. After 4-5 days of differentiation, myotubes were formed and used for experiments. AC16 human cardiomyocytes were changed to 2% FBS 24 h before experiments. Each experiment was performed on three separate occasions. Palmitic acid was administered to cells as a conjugate with 1.25% (w/v) fatty acid-free BSA at the final concentration of 0.5 mM for 12 h followed by 4 h of incubation in the presence or absence of DGAT1i (5 mM). Both cells were washed two times with ice-cold PBS prior to RNA isolation and quantitative real-time PCR. To determine palmitic acid uptake, AC16 cardiomyocytes were pretreated with 5 mM DGAT1i for 4 h and then incubated for 15 min with 0.5 mM palmitic acid with 1 μCi/ml of 9,10-[3H]palmitic acid and 1.25% BSA. Cells were washed three times with ice-cold PBS prior to 0.1N Na2OH addition. An aliquot of homogenate was taken for radioactivity measurement. All data are presented as mean ± SD, unless indicated otherwise. Comparisons between two groups were performed using unpaired two-tailed Student's t-test with Statistica version 6.0 (StatSoft). A P value less than 0.05 was used to determine statistical significance. To determine if loss of Dgat1 in heart and skeletal muscle reduces or eliminates DGAT function, we measured DGAT activity in the heart and skeletal muscle tissue of Dgat1+/− and Dgat1 −/−mice. DGAT activity was decreased ∼20% and ∼65% in hearts of Dgat1+/− and Dgat1−/− mice, respectively (Fig. 1A). Dgat2 mRNA did not change in these tissues (Fig. 1B). In skeletal muscle, DGAT activity was also reduced (Fig. 1C), but Dgat2 mRNA did not change significantly (Fig. 1D). Dgat1−/− mouse liver had a ∼57% decrease in total DGAT activity, but DGAT activity was 4-fold greater than skeletal muscle both under WT and Dgat1−/− conditions. Therefore, Dgat1−/− liver had an activity level that was greater than that found in WT soleus muscle (Fig. 1E). These animals also had no compensatory increase in Dgat2 mRNA (Fig. 1F). Plasma levels of lipids and glucose in these mice are shown in supplementary Table II. Surprisingly the content of TG was not significantly decreased in cardiac muscle in Dgat1−/− mice (Table 1). Also unlike what we expected, the contents of TG precursors DAG and ceramide were not increased but, in fact, were decreased ∼20% in Dgat1−/− hearts (Table 1). We then fed the mice a high-fat diet. High-fat-diet control mice had an increase in TG levels (∼15%), DAG levels (26%), and FFA levels (37%) in the heart (Table 1). As with chow, the contents of DAG and ceramide were reduced ∼16% and ∼22%, respectively, in hearts of high-fat-diet Dgat1−/− mice.TABLE 1Lipid content in chow diet and high-fat diet WT and Dgat1 hearts (n=5–7)Chow DietHigh-Fat DietLipidsWTDgat1Dgat1WTDgat1Dgat1TG (nmol/mg)20.3 ± 2.119.3 ± 1.618 ± 2.523.3 ± 1.6aP < 0.05 versus chow diet WT.21.9 ± 2.321.1 ± 1.8FFA (nmol/mg)7.4 ± 0.97 ± 0.98.3 ± 0.810.2 ± 1.3bP < 0.01 versus chow diet WT.9.7 ± 1.79.3 ± 2.4DAG (pmol/mg)293 ± 42250 ± 38224 ± 58aP < 0.05 versus chow diet WT.371 ± 57aP < 0.05 versus chow diet WT.342 ± 78310 ± 66Ceramid (pmol/mg)287 ± 53244 ± 32215 ± 49aP < 0.05 versus chow diet WT.345 ± 70323 ± 44267 ± 41cP < 0.05 versus high fat diet WTDAG, diacylglycerol; DGAT, diacylglycerol acyl transferase; TG, triglyceride; WT, wild type.a P < 0.05 versus chow diet WT.b P < 0.01 versus chow diet WT.c P < 0.05 versus high fat diet WT Open table in a new tab DAG, diacylglycerol; DGAT, diacylglycerol acyl transferase; TG, triglyceride; WT, wild type. Heart weight was increased 29% (P < 0.01) in chow diet and 7% (P < 0.05) in high-fat diet Dgat1−/− mice. Soleus and extensor digitorum longus (EDL) muscle weight increased 28% and 21%, respectively, in chow diet Dgat1−/− mice (P < 0.05) and 10% and 9%, respectively, in high-fat diet Dgat1−/− mice (P < 0.05) (Fig. 2A, B). Soleus and EDL muscle weights also were increased 13% (P < 0.05) and 20% (P < 0.01) in chow diet Dgat1+/− mice (Fig. 2A). Thus, Dgat1 deficiency increased muscle weight, especially on a chow diet. DEXA showed that lean tissue weight was increased 8% in chow diet Dgat1−/− mice (Fig. 2C). No significant change was observed for lean weight in high-fat diet mice with DEXA. However, as has been reported (10Smith S.J. Cases S. Jensen D.R. Chen H.C. Sande E. Tow B. Sanan D.A. Raber J. Eckel R.H. Farese Jr, R.V. Obesity resistance and multiple mechanisms of triglyceride synthesis in mice lacking Dgat.Nat. Genet. 2000; 25: 87-90Crossref PubMed Scopus (743) Google Scholar), fat tissue was reduced 56% in high-fat diet Dgat1−/− mice (Fig. 2D). Photographs of DEXA are shown in supplementary Fig. I. There are several signaling pathways that can mediate cardiac hypertrophy (18Dorn 2nd, G.W. The fuzzy logic of physiological cardiac hypertrophy.Hypertension. 2007; 49: 962-970Crossref PubMed Scopus (250) Google Scholar, 19Spangenburg E.E. Changes in muscle mass with mechanical load: possible cellular mechanisms.Appl. Physiol. Nutr. Metab. 2009; 34: 328-335Crossref PubMed Scopus (49) Google Scholar). We assessed markers for both pathological and physiologic hypertrophy. mRNA levels of heart failure markers brain-type natriuretic peptide (BNP) and atrial natriuretic factor (ANF) were not increased in the heart (supplementary Fig. IC, D). However, protein levels of p-AKT, a mediator of physiologic hypertrophy (18Dorn 2nd, G.W. The fuzzy logic of physiological cardiac hypertrophy.Hypertension. 2007; 49: 962-970Crossref PubMed Scopus (250) Google Scholar, 19Spangenburg E.E. Changes in muscle mass with mechanical load: possible cellular mechanisms.Appl. Physiol. Nutr. Metab. 2009; 34: 328-335Crossref
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