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• W1980167330 abstract "Increased triglyceride synthesis resulting from enhanced flux of fatty acids into liver is frequently associated with VLDL overproduction. This has led to the common belief that hepatic triglyceride synthesis can directly modulate VLDL production. We used adenoviral vectors containing either murine acyl-coenzyme A:diacylglycerol transferase 1 (DGAT1) or DGAT2 cDNA to determine the effect of a short-term increase in hepatic triglyceride synthesis on VLDL triglyceride and apolipoprotein B (apoB) production in female wild-type mice. Hepatic DGAT1 and DGAT2 overexpression resulted in 2.0-fold and 2.4-fold increases in the triglyceride content of liver, respectively. However, the increase in hepatic triglyceride content had no effect on the production rate of VLDL triglyceride or apoB in either case. Liver subfractionation showed that DGAT1 and DGAT2 overexpression significantly increased the content of triglyceride within the cytoplasmic lipid fraction, with no change in the triglyceride content of the microsomal membrane or microsomal VLDL. The increased cytoplasmic triglyceride content was observed in electron micrographs of liver sections from mice overexpressing DGAT1 or DGAT2. Overexpression of DGAT1 or DGAT2 resulted in enhanced [3H]glycerol tracer incorporation into triglyceride within cytoplasmic lipids. These results suggest that increasing the cytoplasmic triglyceride pool in hepatocytes does not directly influence VLDL triglyceride or apoB production. In the presence of adequate cytoplasmic lipid stores, factors other than triglyceride synthesis are rate-limiting for VLDL production. Increased triglyceride synthesis resulting from enhanced flux of fatty acids into liver is frequently associated with VLDL overproduction. This has led to the common belief that hepatic triglyceride synthesis can directly modulate VLDL production. We used adenoviral vectors containing either murine acyl-coenzyme A:diacylglycerol transferase 1 (DGAT1) or DGAT2 cDNA to determine the effect of a short-term increase in hepatic triglyceride synthesis on VLDL triglyceride and apolipoprotein B (apoB) production in female wild-type mice. Hepatic DGAT1 and DGAT2 overexpression resulted in 2.0-fold and 2.4-fold increases in the triglyceride content of liver, respectively. However, the increase in hepatic triglyceride content had no effect on the production rate of VLDL triglyceride or apoB in either case. Liver subfractionation showed that DGAT1 and DGAT2 overexpression significantly increased the content of triglyceride within the cytoplasmic lipid fraction, with no change in the triglyceride content of the microsomal membrane or microsomal VLDL. The increased cytoplasmic triglyceride content was observed in electron micrographs of liver sections from mice overexpressing DGAT1 or DGAT2. Overexpression of DGAT1 or DGAT2 resulted in enhanced [3H]glycerol tracer incorporation into triglyceride within cytoplasmic lipids. These results suggest that increasing the cytoplasmic triglyceride pool in hepatocytes does not directly influence VLDL triglyceride or apoB production. In the presence of adequate cytoplasmic lipid stores, factors other than triglyceride synthesis are rate-limiting for VLDL production. Abbreviations AdmDGAT1 or AdmDGAT2recombinant adenovirus containing the murine DGAT1 or DGAT2 cDNAAdNullrecombinant adenovirus containing no transgeneALTalanine aminotransferaseapoBapolipoprotein BDGATacyl-coenzyme A:diacylglycerol transferaseERendoplasmic reticulumGFPgreen fluorescent proteinMTPmicrosomal triglyceride transfer protein recombinant adenovirus containing the murine DGAT1 or DGAT2 cDNA recombinant adenovirus containing no transgene alanine aminotransferase apolipoprotein B acyl-coenzyme A:diacylglycerol transferase endoplasmic reticulum green fluorescent protein microsomal triglyceride transfer protein The assembly of VLDL within the endoplasmic reticulum (ER) is dependent on an adequate supply of neutral lipids, triglyceride and cholesteryl ester, which occupy the core of nascent VLDL. Studies in cultured cells have shown that low availability of neutral lipids for lipoprotein assembly, attributable either to reduced rates of triglyceride and cholesteryl ester synthesis or to a lack of the lipid transfer protein microsomal triglyceride transfer protein (MTP), results in a large proportion of translated apolipoprotein B (apoB) being misfolded and targeted for degradation within the ER (1Fisher E.A. Ginsberg H.N. Complexity in the secretory pathway: the assembly and secretion of apolipoprotein B-containing lipoproteins..J. Biol. Chem. 2002; 277: 17377-17380Abstract Full Text Full Text PDF PubMed Scopus (376) Google Scholar). Consequently, there is decreased secretion of VLDL and apoB from the hepatocyte under these conditions. When the supply of neutral lipids for lipoprotein synthesis is abundant, a greater proportion of translated apoB is correctly folded and secreted as VLDL (1Fisher E.A. Ginsberg H.N. Complexity in the secretory pathway: the assembly and secretion of apolipoprotein B-containing lipoproteins..J. Biol. Chem. 2002; 277: 17377-17380Abstract Full Text Full Text PDF PubMed Scopus (376) Google Scholar, 2Dixon J.L. Ginsberg H.N. Regulation of hepatic secretion of apolipoprotein B-containing lipoproteins: information obtained from cultured liver cells..J. Lipid Res. 1993; 34: 167-179Abstract Full Text PDF PubMed Google Scholar, 3Hussain M.M. Shi J. Dreizen P. Microsomal triglyceride transfer protein and its role in apoB-lipoprotein assembly..J. Lipid Res. 2003; 44: 22-32Abstract Full Text Full Text PDF PubMed Scopus (445) Google Scholar). Whereas a relatively low availability of neutral lipids, such as triglycerides, clearly decreases the secretion of apoB from liver, an overabundance of triglycerides in liver is conventionally thought to increase hepatic apoB secretion. Although the type of fat consumed can influence VLDL secretion after nascent VLDL formation in the ER (4Pan M. Cederbaum A.I. Zhang Y.L. Ginsberg H.N. Williams K.J. Fisher E.A. Lipid peroxidation and oxidant stress regulate hepatic apolipoprotein B degradation and VLDL production..J. Clin. Invest. 2004; 113: 1277-1287Crossref PubMed Scopus (232) Google Scholar), in general, conditions in which there is an increased availability of triglyceride or triglyceride precursors to the hepatocyte have resulted in enhanced VLDL triglyceride and apoB secretion (5Craig W.Y. Nutik R. Cooper A.D. Regulation of apoprotein synthesis and secretion in the human hepatoma Hep G2. The effect of exogenous lipoprotein..J. Biol. Chem. 1988; 263: 13880-13890Abstract Full Text PDF PubMed Google Scholar, 6Dixon J.L. Furukawa S. Ginsberg H.N. Oleate stimulates secretion of apolipoprotein B-containing lipoproteins from Hep G2 cells by inhibiting early intracellular degradation of apolipoprotein B..J. Biol. Chem. 1991; 266: 5080-5086Abstract Full Text PDF PubMed Google Scholar). Similarly, an overproduction of VLDL triglyceride and apoB is seen in humans and animals consuming high-fat or lipogenic diets and under conditions, such as insulin resistance, in which triglyceride precursors are abundant in plasma (7Julve J. Escola-Gil J.C. Marzal-Casacuberta A. Ordonez-Llanos J. Gonzalez-Sastre F. Blanco-Vaca F. Increased production of very-low-density lipoproteins in transgenic mice overexpressing human apolipoprotein A-II and fed with a high-fat diet..Biochim. Biophys. Acta. 2000; 1488: 233-244Crossref PubMed Scopus (30) Google Scholar, 8Siri P. Candela N. Zhang Y.L. Ko C. Eusufzai S. Ginsberg H.N. Huang L.S. Post-transcriptional stimulation of the assembly and secretion of triglyceride-rich apolipoprotein B lipoproteins in a mouse with selective deficiency of brown adipose tissue, obesity, and insulin resistance..J. Biol. Chem. 2001; 276: 46064-46072Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). Acyl-coenzyme A:diacylglycerol transferases (DGATs) are enzymes that mediate the final and only committed step in triglyceride synthesis from acyl-CoA and diacylglycerol within the ER (9Chen H.C. Farese Jr., R.V. DGAT and triglyceride synthesis: a new target for obesity treatment?.Trends Cardiovasc. Med. 2000; 10: 188-192Crossref PubMed Scopus (90) Google Scholar). To date, two mammalian DGATs (DGAT1 and DGAT2) have been cloned (10Cases 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. et al.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 (879) Google Scholar, 11Cases S. Stone S.J. Zhou P. Yen E. Tow B. Lardizabal K.D. Voelker T. Farese Jr., R.V. Cloning of DGAT2, a second mammalian diacylglycerol acyltransferase, and related family members..J. Biol. Chem. 2001; 276: 38870-38876Abstract Full Text Full Text PDF PubMed Scopus (638) Google Scholar). DGAT1 and DGAT2 are structurally unrelated and have different but overlapping tissue expression patterns (10Cases 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. et al.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 (879) Google Scholar, 11Cases S. Stone S.J. Zhou P. Yen E. Tow B. Lardizabal K.D. Voelker T. Farese Jr., R.V. Cloning of DGAT2, a second mammalian diacylglycerol acyltransferase, and related family members..J. Biol. Chem. 2001; 276: 38870-38876Abstract Full Text Full Text PDF PubMed Scopus (638) Google Scholar). The DGAT enzymes are membrane-associated enzymes whose activity has been detected on the cytosolic and luminal sides of the ER (12Owen M.R. Corstorphine C.C. Zammit V.A. Overt and latent activities of diacylglycerol acytransferase in rat liver microsomes: possible roles in very-low-density lipoprotein triacylglycerol secretion..Biochem. J. 1997; 323: 17-21Crossref PubMed Scopus (110) Google Scholar, 13Abo-Hashema K.A. Cake M.H. Power G.W. Clarke D. Evidence for triacylglycerol synthesis in the lumen of microsomes via a lipolysis-esterification pathway involving carnitine acyltransferases..J. Biol. Chem. 1999; 274: 35577-35582Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). The presence of DGAT activity within the ER, where VLDL assembly occurs, suggests that one or both DGATs may play a direct role in synthesizing triglyceride for use in the assembly of apoB-containing lipoproteins. Both DGAT1 and DGAT2 are expressed in mouse liver (10Cases 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. et al.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 (879) Google Scholar, 11Cases S. Stone S.J. Zhou P. Yen E. Tow B. Lardizabal K.D. Voelker T. Farese Jr., R.V. Cloning of DGAT2, a second mammalian diacylglycerol acyltransferase, and related family members..J. Biol. Chem. 2001; 276: 38870-38876Abstract Full Text Full Text PDF PubMed Scopus (638) Google Scholar). DGAT1 knockout mice have normal plasma triglyceride levels, suggesting that this enzyme may not play a major role in modulating hepatic lipoprotein production (14Smith 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 (739) Google Scholar). However, lipoprotein synthesis and secretion in DGAT1 knockout mice have not been studied extensively. Liang et al. (15Liang J.J. Oelkers P. Guo C. Chu P.C. Dixon J.L. Ginsberg H.N. Sturley S.L. Overexpression of human diacylglycerol acyltransferase 1, acyl-CoA:cholesterol acyltransferase 1, or acyl-CoA:cholesterol acyltransferase 2 stimulates secretion of apolipoprotein B-containing lipoproteins in McA-RH7777 cells..J. Biol. Chem. 2004; 279: 44938-44944Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar) have shown that overexpression of DGAT1 and DGAT2 in a rat hepatoma cell line results in increased secretion of triglyceride and apoB. DGAT2 knockout mice, although not viable for more than 8 h after birth, have significantly lower plasma triglyceride levels than heterozygous knockout or wild-type littermates, suggesting that triglyceride synthesis by DGAT2 may modulate the production of apoB-containing lipoproteins (16Stone S.J. Myers H.M. Watkins S.M. Brown B.E. Feingold K.R. Elias P.M. Farese Jr., R.V. Lipopenia and skin barrier abnormalities in DGAT2-deficient mice..J. Biol. Chem. 2003; 279: 11767-11776Abstract Full Text Full Text PDF PubMed Scopus (473) Google Scholar). In this study, we created recombinant adenoviruses containing the cDNA of either murine DGAT1 (AdmDGAT1) or DGAT2 (AdmDGAT2) and used these to overexpress DGAT1 and DGAT2 in mouse liver. Our goal was to determine the effect of increased hepatic triglyceride synthesis by these enzymes on triglyceride and apoB production rates. Our results show that DGAT1 or DGAT2 overexpression in wild-type mouse liver results in increased hepatic triglyceride content, with no effect on the VLDL triglyceride or apoB production rate. Female C57BL/6 mice (6–8 weeks old) were obtained from Jackson Laboratories (Bar Harbor, ME). Female human apoB transgenic mice on a C57BL/6 background (17Linton M.F. Farese Jr., R.V. Chiesa G. Grass D.S. Chin P. Hammer R.E. Hobbs H.H. Young S.G. Transgenic mice expressing high plasma concentrations of human apolipoprotein B100 and lipoprotein(a)..J. Clin. Invest. 1993; 92: 3029-3037Crossref PubMed Scopus (211) Google Scholar), bred at the University of Pennsylvania, were also used in some experiments to measure triglyceride and the production VLDL apoB involving DGAT1. All mice were maintained on a chow diet (No. 5001; LabDiet, St. Louis, MO) containing 4.5% fat by weight (1.5% saturated, 1.6% monounsaturated, and 1.4% polyunsaturated). Blood samples were collected from mice fasted for 4 h and bled from the retro-orbital plexus using heparinized capillary tubes. Unless indicated otherwise, data presented are from experiments that included control [injected with recombinant adenovirus containing no transgene (AdNull)], DGAT1 (injected with AdmDGAT1), and DGAT2 (injected with AdmDGAT2) groups. Mice were injected intravenously (tail vein) with adenoviral preparations at a dose of 1 × 1011 viral particles in 100 μl of PBS per mouse. Blood samples were collected at baseline (day 0) and at day 4 after adenoviral injection. Serum was assayed for total cholesterol, triglycerides, and HDL as described previously (18Tsukamoto K. Smith P. Glick J.M. Rader D.J. Liver-directed gene transfer and prolonged expression of three major human apoE isoforms in apoE-deficient mice..J. Clin. Invest. 1997; 100: 107-114Crossref PubMed Scopus (87) Google Scholar). All experiments were terminated on day 4 after adenoviral injection. All procedures conducted in mice were in accordance with University of Pennsylvania Institutional Animal Care and Use Committee guidelines. Plasmids containing the full-length cDNA of either murine DGAT1 (10Cases 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. et al.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 (879) Google Scholar) or murine DGAT2 (11Cases S. Stone S.J. Zhou P. Yen E. Tow B. Lardizabal K.D. Voelker T. Farese Jr., R.V. Cloning of DGAT2, a second mammalian diacylglycerol acyltransferase, and related family members..J. Biol. Chem. 2001; 276: 38870-38876Abstract Full Text Full Text PDF PubMed Scopus (638) Google Scholar), both of which include a FLAG epitope, or containing green fluorescent protein (GFP) were subcloned into the shuttle plasmid vector pAdCMVlink (18Tsukamoto K. Smith P. Glick J.M. Rader D.J. Liver-directed gene transfer and prolonged expression of three major human apoE isoforms in apoE-deficient mice..J. Clin. Invest. 1997; 100: 107-114Crossref PubMed Scopus (87) Google Scholar), generating the plasmids pAdCMVmDGAT1, pAdCMVmDGAT2, and pAdCMVGFP. Recombinant adenoviruses were produced with established methods (18Tsukamoto K. Smith P. Glick J.M. Rader D.J. Liver-directed gene transfer and prolonged expression of three major human apoE isoforms in apoE-deficient mice..J. Clin. Invest. 1997; 100: 107-114Crossref PubMed Scopus (87) Google Scholar) using adenoviral DNA containing a temperature-sensitive mutation (ts125) in the E2A region that renders the adenovirus incapable of replicating at 39°C. In this way, four recombinant adenoviruses containing mDGAT1 (AdmDGAT1), mDGAT2 (AdmDGAT2), GFP (AdGFP), or no transgene (AdNull), the latter two being controls, were generated. Recombinant adenovirus was grown, screened by PCR, subjected to two subsequent rounds of plaque purification, purified, and stored as described (18Tsukamoto K. Smith P. Glick J.M. Rader D.J. Liver-directed gene transfer and prolonged expression of three major human apoE isoforms in apoE-deficient mice..J. Clin. Invest. 1997; 100: 107-114Crossref PubMed Scopus (87) Google Scholar). DGAT activity assays were performed by measuring activity under apparent Vmax conditions in tissue membranes (10–50 μg of protein) as described (11Cases S. Stone S.J. Zhou P. Yen E. Tow B. Lardizabal K.D. Voelker T. Farese Jr., R.V. Cloning of DGAT2, a second mammalian diacylglycerol acyltransferase, and related family members..J. Biol. Chem. 2001; 276: 38870-38876Abstract Full Text Full Text PDF PubMed Scopus (638) Google Scholar). In brief, these assays measured the incorporation of [14C]oleoyl-CoA (specific activity, ∼20,000 dpm/nmol) into triglycerides in a 5 min assay using 0.4 mM diacylglycerol and 25 μM oleoyl-CoA as substrates. Lipids were extracted with chloroform-methanol (2:1, v:v), dried under N2, and separated by TLC in hexane-ethyl ether-acetic acid (80:20:1, v:v:v). Radioactivity in the triglyceride band was measured by scintillation counting. Livers were perfused with ice-cold PBS, dissected, patted dry, weighed, and sectioned. A section was immediately homogenized in the presence of protease inhibitors and frozen in liquid nitrogen. Western blotting for DGAT1 and DGAT2 in mouse liver was performed on mouse liver homogenates using anti-FLAG M2 antibody (Sigma-Aldrich). Western blotting of liver subfractions was done using anti-adipophilin (American Research Products, Inc.), which cross-reacts with the mouse homolog adipose differentiation-related protein, anti-calnexin (Sigma-Aldrich), and anti-mouse apoB (Biodesign International). Hepatic triglyceride production rates were determined using the detergent P-407 as described previously (19Millar J.S. Cromley D.A. McCoy M.G. Rader D.J. Billheimer J.T. Determining hepatic triglyceride production in mice: comparison of poloxamer 407 with Triton WR-1339..J. Lipid Res. 2005; 46: 2023-2028Abstract Full Text Full Text PDF PubMed Scopus (201) Google Scholar). To determine apoB production rates, mice were injected via tail vein with 500 μCi of [35S]methionine (Perkin-Elmer, Wellesley, MA) in 100 μl of PBS 5 min after the intraperitoneal injection of P-407. Blood was sampled before injection (0 h) and at 1 h and 2 h after injection. Similar results were obtained using the detergent Triton WR1339 (20Millar J.S. Maugeais C. Fuki I.V. Rader D.J. Normal production rate of apolipoprotein B in LDL receptor-deficient mice..Arterioscler. Thromb. Vasc. Biol. 2002; 22: 989-994Crossref PubMed Scopus (38) Google Scholar). Triglycerides were measured enzymatically using Wako reagents (Wako Chemicals USA, Inc., Richmond, VA). Total and VLDL triglyceride concentrations were expressed as mg/kg, assuming a plasma volume of 3.5% of body weight. Triglyceride production rates, in mg/kg/h, were calculated by subtracting the baseline value from the 2 h value and then expressed per hour (19Millar J.S. Cromley D.A. McCoy M.G. Rader D.J. Billheimer J.T. Determining hepatic triglyceride production in mice: comparison of poloxamer 407 with Triton WR-1339..J. Lipid Res. 2005; 46: 2023-2028Abstract Full Text Full Text PDF PubMed Scopus (201) Google Scholar). Plasma for the baseline, 1 h, and 2 h samples was subjected to ultracentrifugation to isolate the VLDL fraction for the calculation of VLDL triglyceride production rate and VLDL apoB production rate. VLDL (d < 1.006 g/ml) was separated from plasma by ultracentrifugation at 90,000 rpm for 3 h in a TL 100 ultracentrifuge (Beckman) using a TLA 100.2 rotor (Beckman). VLDL was subjected to SDS-PAGE, and protein bands were visualized with Coomassie blue staining. ApoB was cut from the gel, eluted with Solvable (Packard, Meriden, CT) according to the manufacturer's instructions, and subjected to scintillation counting. ApoB production rates, expressed as cpm/μl plasma/h, were calculated as the background-corrected counts at the 2 h time point, adjusted for plasma volume, and expressed per hour. Primary hepatocytes were isolated after liver perfusion with Liver Perfusion Medium (Invitrogen, Carlsbad, CA) followed by Liver Digest Medium (Invitrogen). Digested livers were minced, strained through a 40 μm filter, washed twice in DMEM, and purified in a Percoll density gradient. Cells were resuspended in DMEM containing 10% FBS supplemented with insulin (20 mU/ml), dexamethasone (25 nM), and antibiotics and were plated at a density of 400,000 cells/ml onto type I collagen-coated plates and left to attach for 4 h in a 5% CO2 incubator at 37°C. After 4 h, medium was replaced with adenovirus-containing medium at a multiplicity of infection of 3,000 and incubated for 18 h. After 18 h, medium was replaced with DMEM containing 10% FBS supplemented with antibiotics containing 10 μCi/ml [3H]glycerol (38.9 μCi/nmol) and 1 μCi/ml of [14C]oleic acid (54 μCi/μmol). Cells were labeled for 4 h and washed three times, and a portion from each group was harvested for the determination of initial cellular triglyceride radioactivity. The remaining cells were incubated in DMEM containing 10% FBS supplemented with antibiotics for 18 h, at the end of which they were washed three times and harvested for the determination of final radioactivity. Lipids were extracted with chloroform-methanol (2:1, v/v), dried under N2, and separated by TLC in hexane-diethyl ether-acetic acid (170:30:1, v/v/v). Radioactivity in the triglyceride band was measured by scintillation counting. Pooled plasma samples from six mice of the same experimental group were subjected to fast-protein liquid chromatography gel filtration using two Superose 6 columns (Pharmacia LKB Biotechnology) as described (21Tietge U.J.F. Maugeais C. Cain W. Grass D. Glick J.M. deBeer F.C. Rader D.J. Overexpression of secretory phospholipase A2 causes rapid catabolism and altered tissue uptake of high density lipoprotein cholesteryl ester and apolipoprotein A-I..J. Biol. Chem. 2000; 275: 10077-10084Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar). Samples were chromatographed at a flow rate of 0.5 ml/min, and fractions of 500 μl each were collected. Individual fractions were assayed for cholesterol concentration using a commercially available assay kit (Wako Pure Chemical Industries, Ltd.). Livers were subfractionated into cytoplasmic, microsomal membrane, and microsomal VLDL fractions according to Chao, Stiers, and Ontko (22Chao F.F. Stiers D.L. Ontko J.A. Hepatocellular triglyceride synthesis and transfer to lipid droplets and nascent very low density lipoproteins..J. Lipid Res. 1986; 27: 1174-1181Abstract Full Text PDF PubMed Google Scholar). Briefly, freshly perfused liver sections were weighed and minced with a scalpel. Buffer (0.25 M sucrose and 10 mM HEPES, pH 7.4) was then added, and livers were homogenized using a PowerGen 125 homogenizer (Fisher Scientific) for 30 s. Homogenates were spun at low speed (9,000 g) to remove cellular debris followed by high-speed centrifugation (105,000 g) to separate the cytoplasmic lipid fraction from the intact microsomal membrane fraction. The pelleted microsomal membranes were then ruptured by sonication followed by separation of the microsomal membrane and microsomal VLDL by high-speed centrifugation. Mice were perfused through the left ventricle with a flush of 0.1 M sodium cacodylate buffer, pH 7.4, followed by a fixative of 1.5% glutaraldehyde, 4% polyvinylpyrrolidone, 0.05% calcium chloride, and 0.1 M sodium cacodylate, pH 7.4. Tissues were stained for lipid by the imidazole-buffered osmium tetroxide procedure (23Angermuller S. Fahimi H.D. Imidazole-buffered osmium tetroxide: an excellent stain for visualization of lipids in transmission electron microscopy..Histochem. J. 1982; 14: 823-835Crossref PubMed Scopus (198) Google Scholar), en bloc stained in 2% aqueous uranyl acetate for 1 h at 4°C, processed, and embedded in Epon 812. Ultrathin sections were stained for 5 min with 0.8% lead citrate and photographed with the Siemens Elmiskop 101 (Siemens/CTI Corp., Knoxville, TN). Values are presented as means ± SD. Results were analyzed by Student's t-test for independent samples (two-tailed). Data for alanine aminotransferase (ALT), triglyceride mass, and radioactivity within the cytoplasmic lipid fraction were log-transformed before statistical analysis. Statistical significance for comparisons was assigned at P < 0.05. Mice injected with AdmDGAT1 expressed a protein with a molecular mass of ∼47 kDa in liver homogenates that reacted with anti-FLAG (Fig. 1). Expression of DGAT1 was associated with a 3.4-fold increase in total DGAT activity in liver compared with mice injected with control adenovirus (Fig. 1). Biochemical analysis showed significantly higher triglyceride content in livers from mice overexpressing DGAT1, the hepatic triglyceride being ∼2-fold that found in control mice (Table 1). The content of total cholesterol or phospholipid (data not shown) in liver was unchanged as a result of DGAT1 overexpression. Although control mice had normal ALT levels (Table 1), indicating no hepatotoxic effect of the viral load, ALT levels were increased in plasma of mice overexpressing DGAT1, likely in response to increased hepatic lipid content.TABLE 1.Plasma and liver lipid levels in female control mice and those overexpressing murine DGAT1 and DGAT2 4 days after adenoviral infectionAdenovirusTC (n = 8)TG (n = 8)HDL (n = 8)Alanine Aminotransferase (n = 4)Liver TG (n = 6)Liver TC (n = 6)mg/dlU/lmg/g liverAdNull58 ± 1090 ± 1244 ± 744 ± 418.8 ± 5.55.4 ± 0.7DGAT157 ± 988 ± 2145 ± 6102 ± 57 aP < 0.05 versus AdNull.35.2 ± 12.2 aP < 0.05 versus AdNull.5.5 ± 0.9DGAT264 ± 984 ± 1347 ± 5132 ± 60 bP < 0.01 versus AdNull.42.6 ± 10.1 aP < 0.05 versus AdNull.5.5 ± 0.8AdNull, recombinant adenovirus containing no transgene; DGAT, acyl-coenzyme A:diacylglycerol transferase; TC, total cholesterol; TG, triglyceride.a P < 0.05 versus AdNull.b P < 0.01 versus AdNull. Open table in a new tab AdNull, recombinant adenovirus containing no transgene; DGAT, acyl-coenzyme A:diacylglycerol transferase; TC, total cholesterol; TG, triglyceride. Although overexpression of DGAT1 in liver increased hepatic triglyceride content, there was no effect on plasma lipid levels (Table 1) or lipoprotein profile (Fig. 2). This was consistent with the results from metabolic studies in which there were no differences in the hepatic production rate of the triglyceride or VLDL apoB DGAT1 overexpression (Fig. 3). Similar results were obtained when studies were conducted in female human apoB transgenic mice overexpressing DGAT1 compared with GFP-expressing controls (data not shown).Fig. 3.Triglyceride and apolipoprotein B (apoB) production rates in mice overexpressing DGAT1 or DGAT2. Mice overexpressing DGAT1 or DGAT2 showed no differences in plasma triglyceride (TG) production rate (n = 11 per group) (A) or in production rates of apoB-48, apoB-100, or total apoB (B). Results shown are from production studies conducted using P-407 (n = 11 for the AdNull group; n = 10 for the DGAT1 and DGAT2 groups). Values are presented as means ± SD.View Large Image Figure ViewerDownload Hi-res image Download (PPT) We next studied mice overexpressing DGAT2 after injection with adenovirus containing the cDNA for DGAT2. Mice injected with AdmDGAT2 expressed a protein with a molecular mass of ∼44.5 kDa that reacted with anti-FLAG (Fig. 1). The levels of DGAT2 protein overexpression were similar to those for DGAT1. This was associated with a 1.4-fold increase in total DGAT activity in liver compared with mice injected with control adenovirus (Fig. 3). There was a significantly greater triglyceride content of livers from mice overexpressing DGAT2, the hepatic triglyceride content being ∼2.5-fold higher than that found in control mice (Table 1). There were no significant changes in the total cholesterol or phospholipid (data not shown) contents in mice overexpressing DGAT2 in liver. Compared with control mice, ALT levels were also increased in plasma of mice overexpressing DGAT2 (Table 1), likely in response to increased hepatic lipid content. Similar to what was observed with DGAT1, the increased hepatic triglyceride content attributable to DGAT2 overexpression had no effect on plasma lipid levels (Table 1) or lipoprotein profile (Fig. 2). Lipoprotein production studies showed no changes in the hepatic production rate of triglyceride or VLDL apoB (Fig. 3). To determine the subcellular loc" @default.
• W1980167330 created "2016-06-24" @default.
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• W1980167330 date "2006-10-01" @default.
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• W1980167330 title "Short-term overexpression of DGAT1 or DGAT2 increases hepatic triglyceride but not VLDL triglyceride or apoB production" @default.
• W1980167330 cites W1486210853 @default.
• W1980167330 cites W1523235079 @default.
• W1980167330 cites W1599366995 @default.
• W1980167330 cites W1898264975 @default.
• W1980167330 cites W1898694844 @default.
• W1980167330 cites W1964392625 @default.
• W1980167330 cites W1973186908 @default.
• W1980167330 cites W1974115865 @default.
• W1980167330 cites W1976791053 @default.
• W1980167330 cites W1978868053 @default.
• W1980167330 cites W1980093649 @default.
• W1980167330 cites W1981135463 @default.
• W1980167330 cites W1981293724 @default.
• W1980167330 cites W1991696239 @default.
• W1980167330 cites W1991735443 @default.
• W1980167330 cites W2022801887 @default.
• W1980167330 cites W2025382042 @default.
• W1980167330 cites W2035187068 @default.
• W1980167330 cites W2037748881 @default.
• W1980167330 cites W2039481955 @default.
• W1980167330 cites W2039543531 @default.
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