Role of Acyl-Coenzyme A:Cholesterol Acyltransferase-1 in the Control of Hepatic Very Low Density Lipoprotein Secretion and Low Density Lipoprotein Receptor Expression in the Mouse and Hamster

Cholesteryl esters present in nascent very low density lipoproteins are generated in a reaction catalyzed by acyl CoA:cholesterol acyltransferase (ACAT). To examine the effect of cholesteryl esters on the secretion of apoB-containing lipoproteins, we transiently overexpressed human (h) ACAT-1 in the livers of low density lipoprotein (LDL) receptor−/− mice using adenovirus-mediated gene transfer. Overexpression of hACAT-1 increased hepatic total and esterified cholesterol but did not reduce hepatic free cholesterol due to a compensatory increase in the rate of de novo cholesterol synthesis. Overexpression of hACAT-1 markedly increased the plasma concentration and hepatic secretion of apoB-containing lipoproteins but had no effect on the clearance of very low density lipoprotein-apoB from plasma indicating that cholesteryl esters play an important role in regulating the assembly and secretion of apoB-containing lipoproteins. ACAT activity has been implicated in the regulation of the LDL receptor pathway by dietary fatty acids. It has been hypothesized that unsaturated fatty acids, by enhancing ACAT activity, reduce the amount of free cholesterol in a putative regulatory pool that feeds back on LDL receptor expression. We directly tested this hypothesis in hamsters by transiently overexpressing hACAT-1 in the liver. Enhanced cholesterol esterification in the liver resulted in a compensatory increase inde novo cholesterol synthesis but no induction of LDL receptor expression suggesting that fatty acids regulate LDL receptor expression via a mechanism independent of ACAT. Cholesteryl esters present in nascent very low density lipoproteins are generated in a reaction catalyzed by acyl CoA:cholesterol acyltransferase (ACAT). To examine the effect of cholesteryl esters on the secretion of apoB-containing lipoproteins, we transiently overexpressed human (h) ACAT-1 in the livers of low density lipoprotein (LDL) receptor−/− mice using adenovirus-mediated gene transfer. Overexpression of hACAT-1 increased hepatic total and esterified cholesterol but did not reduce hepatic free cholesterol due to a compensatory increase in the rate of de novo cholesterol synthesis. Overexpression of hACAT-1 markedly increased the plasma concentration and hepatic secretion of apoB-containing lipoproteins but had no effect on the clearance of very low density lipoprotein-apoB from plasma indicating that cholesteryl esters play an important role in regulating the assembly and secretion of apoB-containing lipoproteins. ACAT activity has been implicated in the regulation of the LDL receptor pathway by dietary fatty acids. It has been hypothesized that unsaturated fatty acids, by enhancing ACAT activity, reduce the amount of free cholesterol in a putative regulatory pool that feeds back on LDL receptor expression. We directly tested this hypothesis in hamsters by transiently overexpressing hACAT-1 in the liver. Enhanced cholesterol esterification in the liver resulted in a compensatory increase inde novo cholesterol synthesis but no induction of LDL receptor expression suggesting that fatty acids regulate LDL receptor expression via a mechanism independent of ACAT. intermediate density lipoprotein(s) acyl-coenzyme A:cholesterol acyltransferase human ACAT low density lipoprotein(s) high density lipoprotein(s) very low density lipoprotein apolipoprotein AI microsomal triglyceride transfer protein glyceraldehyde-3-phosphate dehydrogenase plaque-forming unit fast protein liquid chromatography The rate at which the liver secretes apoB-containing lipoproteins is a major determinant of the plasma concentration of atherogenic intermediate (IDL)1 and low (LDL) density lipoproteins. Secretion of apoB-containing lipoproteins by the liver requires the coordinated synthesis and assembly of apoB, triglyceride, cholesterol, cholesteryl ester, and phospholipid. Synthesis of apoB is regulated primarily at the translational and posttranslational levels (1Davis R.A. Biochim. Biophys. Acta. 1999; 1440: 1-31Crossref PubMed Scopus (163) Google Scholar). The proportion of apoB that is translocated across the endoplasmic reticulum membrane relative to the proportion that undergoes intracellular degradation determines, to a large extent, the rate of apoB secretion by the liver. A number of hormonal and nutritional factors may influence this process (2Pullinger C.R. North J.D. Teng B.B. Rifici V.A. Ronhild de Brito A.E. Scott J. J. Lipid Res. 1989; 30: 1065-1077Abstract Full Text PDF PubMed Google Scholar, 3Dixon J.L. Ginsberg H.N. J. Lipid Res. 1993; 34: 167-179Abstract Full Text PDF PubMed Google Scholar). Of these, the availability of lipid, in particular triglyceride (2Pullinger C.R. North J.D. Teng B.B. Rifici V.A. Ronhild de Brito A.E. Scott J. J. Lipid Res. 1989; 30: 1065-1077Abstract Full Text PDF PubMed Google Scholar, 3Dixon J.L. Ginsberg H.N. J. Lipid Res. 1993; 34: 167-179Abstract Full Text PDF PubMed Google Scholar), is thought to be the most important. Association of triglyceride with apoB, which reduces the proportion of apoB degraded intracellularly and thus increases secretion of apoB-containing lipoproteins (3Dixon J.L. Ginsberg H.N. J. Lipid Res. 1993; 34: 167-179Abstract Full Text PDF PubMed Google Scholar), is facilitated by the abetalipoproteinemia gene product, microsomal triglyceride transfer protein (MTP) (4Wetterau J.R. Aggerbeck L.P. Bouma M.-E. Eisenberg C. Munck A. Hermier M. Schmitz J. Gay G. Rader D.J. Gregg R.E. Science. 1992; 258: 999-1001Crossref PubMed Scopus (634) Google Scholar, 5Gordon D.A. Curr. Opin. Lipidol. 1997; 8: 131-137Crossref PubMed Scopus (81) Google Scholar). Although usually not limiting, reduced availability of phospholipid, the major surface component of VLDL, also decreases VLDL secretion from rat hepatocytes (6Yao Z. Vance D.E. J. Biol. Chem. 1988; 263: 2998-3004Abstract Full Text PDF PubMed Google Scholar). The availability of cholesterol and/or cholesteryl esters may also influence apoB synthesis and secretion by the liver. Inhibition of hepatic cholesterol synthesis or cholesterol esterification has been shown to reduce apoB secretion in some studies (7Cianflone K. Yasruel Z. Rodriguez M.A. Vas D. Sniderman A.D. J. Lipid Res. 1990; 31: 2045-2055Abstract Full Text PDF PubMed Google Scholar, 8Dashti N. J. Biol. Chem. 1992; 267: 7160-7169Abstract Full Text PDF PubMed Google Scholar, 9Tanaka M. Jingami H. Otani H. Cho M. Ueda Y. Arai H. Nagano Y. Doi T. Yokode M. Kita T. J. Biol. Chem. 1993; 268: 12713-12718Abstract Full Text PDF PubMed Google Scholar, 10Carr T.P. Hamilton J.R.L. Rudel L.L. J. Lipid Res. 1995; 36: 25-36Abstract Full Text PDF PubMed Google Scholar, 11Musanti R. Giorgini L. Lovisolo P. Pirillo A. Chiari A. Ghiselli G. J. Lipid Res. 1996; 37: 1-14Abstract Full Text PDF PubMed Google Scholar, 12Zhang Z. Cianflone K. Sniderman A.D. Arterioscler. Thromb. Vasc. Biol. 1999; 19: 743-752Crossref PubMed Scopus (38) Google Scholar, 13Burnett J.R. Wilcox L.J. Telford D.E. Kleinstiver S.J. Barrett P.H.R. Newton R.S. Huff M.W. J. Lipid Res. 1999; 40: 1317-1327Abstract Full Text Full Text PDF PubMed Google Scholar) but not in others (14Sato R. Imanaka T. Takano T. Biochim. Biophys. Acta. 1990; 1042: 36-41Crossref PubMed Scopus (33) Google Scholar, 15Wu X. Sakata N. Ginsberg H.N. J. Biol. Chem. 1994; 269: 12375-12382Abstract Full Text PDF PubMed Google Scholar, 16Graham A. Wood J.L. Russell L.J. Biochim. Biophys. Acta. 1996; 1302: 46-54Crossref PubMed Scopus (23) Google Scholar, 17Ooyen C. Zecca A. Zanelli T. Catapano A.L. Atherosclerosis. 1997; 130: 143-152Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar). The cholesteryl esters present in liver-derived nascent VLDL are generated in a reaction catalyzed by acyl CoA:cholesterol acyltransferase (ACAT). ACAT is an integral membrane protein located in the endoplasmic reticulum that catalyzes the formation of cholesteryl esters from cholesterol and long chain fatty acyl CoA, thus modulating the potential toxic effects of excess cholesterol in cell membranes (18Chang T.Y. Chang C.C.Y. Cheng D. Annu. Rev. Biochem. 1997; 66: 613-638Crossref PubMed Scopus (439) Google Scholar). Because of their limited solubility in cell membranes, cholesteryl esters accumulate as lipid droplets in the cytoplasm, from which they can be incorporated directly into VLDL without hydrolysis and re-esterification in the endoplasmic reticulum. At least two proteins contribute to ACAT activity in mammalian tissues. ACAT-1, which was first cloned from a macrophage cDNA library (19Chang C.C.Y. Huh H.Y. Cadigan K.M. Chang T.Y. J. Biol. Chem. 1993; 268: 1353-1356Google Scholar), is expressed ubiquitously, with highest levels in the preputial gland, adrenal gland, and macrophages (20Uelmen P.J. Oka K. Sullivan M. Chang C.C.Y. Chang T.Y. Chan L. J. Biol. Chem. 1995; 270: 26192-26201Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar, 21Anderson R.A. Joyce C. Davis M. Reagan J.W. Clark M. Shelness G.S. Rudel L.L. J. Biol. Chem. 1998; 273: 26747-26754Abstract Full Text Full Text PDF PubMed Scopus (257) Google Scholar). ACAT-1 accounts for the majority of ACAT activity in HepG2 cells (22Chang C.C.Y. Chen J. Thomas M.A. Cheng D. Del Priore V.A. Newton R.S. Pape M.E. Chang T.Y. J. Biol. Chem. 1995; 270: 29532-29540Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar) and human hepatocytes (23Lee O. Chang C.C.Y. Lee W. Chang T.Y. J. Lipid Res. 1998; 39: 1722-1727Abstract Full Text Full Text PDF PubMed Google Scholar). Mice with targeted disruption of the Acact1gene, however, show no reduction in hepatic ACAT activity or cholesteryl ester levels (24Meiner V.L. Cases S. Myers H.M. Sande E.R. Bellosta S. Schambelan M. Pitas R.E. McGuire J. Herz J. Farese Jr., R.V. Proc. Natl. Acad. Sci. U. S. A. 1996; 92: 14041-14046Crossref Scopus (239) Google Scholar). Moreover, in rabbit liver ACAT-1 mRNA appeared to be present mainly in nonparenchymal cells (25Pape M.E. Schultz P.A. Rea T.J. DeMattos R.B. Kieft K. Bisgaier C.L. Newton R.S. Krause B.R. J. Lipid Res. 1995; 36: 823-838Abstract Full Text PDF PubMed Google Scholar). Thus, there may be species differences in the relative importance of ACAT-1 to total ACAT activity in the liver. ACAT-2 was cloned based on its homology to ACAT-1 and is expressed primarily in the liver and intestine (21Anderson R.A. Joyce C. Davis M. Reagan J.W. Clark M. Shelness G.S. Rudel L.L. J. Biol. Chem. 1998; 273: 26747-26754Abstract Full Text Full Text PDF PubMed Scopus (257) Google Scholar, 26Cases S. Novak S. Zheng Y.-W. Myers H.M. Lear S.R. Sande E. Welch C.B. Lusis A.J. Spencer T.A. Krause B.R. Erickson S.K. Farese Jr., R.V. J. Biol. Chem. 1998; 273: 26755-26764Abstract Full Text Full Text PDF PubMed Scopus (330) Google Scholar). ACAT activity has been implicated in the regulation of the LDL receptor pathway by dietary fatty acids (27Spady D.K. Woollett L.A. Dietschy J.M. Annu. Rev. Nutr. 1993; 13: 355-381Crossref PubMed Scopus (239) Google Scholar, 28Rumsey S.C. Galeano N.F. Lipschitz B. Deckelbaum R.J. J. Biol. Chem. 1995; 270: 10008-10016Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar). In the hamster, polyunsaturated fatty acids up-regulate hepatic LDL receptor expression and lower plasma LDL concentrations when compared with saturated fatty acids (29Horton J.D. Cuthbert J.A. Spady D.K. J. Clin. Invest. 1993; 92: 743-749Crossref PubMed Scopus (80) Google Scholar). Based on the observation that hepatic ACAT activity is higher in animals fed unsaturated fatty acids than in those fed saturated fatty acids (30Spector A.A. Kaduce T.L. Dane R.W. J. Lipid Res. 1980; 21: 169-179Abstract Full Text PDF PubMed Google Scholar), it has been hypothesized that unsaturated fatty acids, by enhancing ACAT activity, may reduce the amount of free cholesterol in a putative regulatory pool that feeds back on LDL receptor expression (27Spady D.K. Woollett L.A. Dietschy J.M. Annu. Rev. Nutr. 1993; 13: 355-381Crossref PubMed Scopus (239) Google Scholar, 28Rumsey S.C. Galeano N.F. Lipschitz B. Deckelbaum R.J. J. Biol. Chem. 1995; 270: 10008-10016Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar, 29Horton J.D. Cuthbert J.A. Spady D.K. J. Clin. Invest. 1993; 92: 743-749Crossref PubMed Scopus (80) Google Scholar, 31Salter A.M. Ekins N. Al-Seeni M. Brindley D.N. Middleton B. Biochem. J. 1989; 263: 255-260Crossref PubMed Scopus (11) Google Scholar, 32Tabas I. Weiland D.A. Tall A.R. J. Biol. Chem. 1986; 261: 3147-3155Abstract Full Text PDF PubMed Google Scholar). The current studies were carried out to determine the role of esterified cholesterol in regulating VLDL output and hepatic sterol balance. Hepatic cholesteryl ester concentrations were raised by overexpressing hACAT-1 in the liver using adenovirus-mediated gene transfer. Most studies were performed in mice with targeted disruption of the LDL receptor gene (Ldlr −/−), where plasma VLDL concentrations reflect the rate at which these particles are secreted by the liver. Studies to determine the effect of ACAT overexpression on hepatic LDL receptor expression were carried out in the Syrian hamster. Studies were performed in maleLdlr −/− mice (33Ishibashi S. Brown M.S. Goldstein J.L. Gerard R.D. Hammer R.E. Herz J. J. Clin. Invest. 1993; 92: 883-893Crossref PubMed Scopus (1268) Google Scholar) or in male Syrian hamsters. Animals were housed in colony cages in a room with temperature and humidity control and subjected to light cycling for at least 2 weeks prior to use in studies. All experiments were performed during the mid-dark phase of the light cycle. The animals were maintained on a low fat (50 mg per g of diet), low cholesterol (0.23 mg per g of diet) cereal-based diet (Wayne Lab Blox, Allied Mills, Chicago). All experiments were approved by the Institutional Animal Care and Research Advisory Committee of the University of Texas Southwestern Medical Center, Dallas. The recombinant adenovirus AdCMV-ACAT, carrying a gene encoding hACAT-1 expressed from a human cytomegalovirus promoter, was generated by homologous recombination in 293 cells as described previously (34Spady D.K. Cuthbert J.A. Willard M. Meidell R.S. J. Clin. Invest. 1995; 96: 700-709Crossref PubMed Scopus (104) Google Scholar). The cDNA encoding hACAT-1 was generously provided by Dr. T. Y. Chang (Dartmouth Medical School, Hanover, NH). Generation of the recombinant adenovirus AdCMV-Luc, carrying a gene encoding firefly luciferase, was described previously (35Herz J. Gerard R.D. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 2812-2816Crossref PubMed Scopus (504) Google Scholar). Large scale production of recombinant adenovirus was performed by infecting confluent monolayers of 293 cells grown in 15-cm tissue culture plates with primary stock at a multiplicity of infection of 0.1–1.0 (36Willard J.E. Landqu C. Glamann D.B. Burns D. Jessen M.E. Pirwitz M.J. Gerard R.D. Meidell R.S. Circulation. 1994; 89: 2190-2197Crossref PubMed Scopus (110) Google Scholar). Infected monolayers were lysed with Nonidet P-40 when >90% of the cells showed cytopathic changes, and recombinant virus was purified by precipitation with polyethylene glycol 8000, centrifugation on a discontinuous CsCl density gradient, and desalting by chromatography on Sepharose CL4B. Purified virus eluting in the void volume was collected, snap-frozen in liquid N2, and stored at −80 °C until used (37Gerard R.D. Meidell R.S. Glover D.M. Hames B.D. DNA Cloning: A Practical Approach, Mammalian Systems. IRL Press at Oxford University Press, Oxford1995Google Scholar). Virus titer was determined by plaque assay in monolayer cultures of 293 cells. Hepatic hACAT-1, apoB, apoE, MTP, and 7α-hydroxylase mRNA levels were determined using a ribonuclease protection assay. GAPDH or β-actin was used as an internal control. Species-specific 32P-labeled riboprobes were synthesized using MAXIscript in vitro transcription kits (Ambion Inc, Austin, TX) in the presence of 3 (7α-hydroxylase, LDL receptor), 15 (hACAT-1, apoB, MTP, apoE), or 100 μm(GAPDH and β-actin) labeled nucleotide. Samples of liver were homogenized in RNA STAT-60 (Tel-Test, Friendswood, TX). Total RNA (40 μg) was hybridized with 32P-labeled riboprobes simultaneously at 68 °C using the HybSpeed RPA protocol (Ambion Inc). Following RNase digestion, the mRNA-protected32P-labeled probes were separated on 8 m urea, 5% polyacrylamide gels together with 32P-labeledMspI-digested pBR322 size standards. The radioactivity in each band, as well as background radioactivity, was quantified using a phosphorimaging system (Molecular Dynamics, Sunnyvale, CA). Rates of hepatic cholesterol and fatty acid synthesis were measured in vivo using [3H]water. As described previously (38Spady D.K. Cuthbert J.A. Willard M.N. Meidell R.S. J. Biol. Chem. 1998; 273: 126-132Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar), the animals were administered ∼25 mCi of [3H]water intravenously and killed 1 h later by exsanguination through the inferior vena cava. Aliquots of plasma were taken for the determination of body water-specific activity, and samples of liver were saponified in alcoholic KOH. Saponified liver samples were first extracted with petroleum ether and aliquots used for the isolation of digitonin-precipitable sterols. The saponified liver samples were then acidified with concentrated HCl and the fatty acids extracted into hexane. Rates of sterol and fatty acid synthesis are expressed as the nanomoles or micromoles of [3H]water incorporated into digitonin-precipitable sterols or fatty acids per h per g of liver. Liver cholesterol was quantified by capillary gas-liquid chromatography. The cholesterol distribution in plasma was determined by FPLC using a Superose 6 HR 10/30 column (Amersham Pharmacia Biotech). Aliquots were collected, and enzymatic kits were used to assay for total cholesterol (Sigma), free cholesterol (Wako Chemicals Inc., Richmond, VA), and triglyceride (Sigma). Hepatic 7α-hydroxylase activity was determined using a high pressure liquid chromatography-spectrophotometric assay that quantifies the mass of 7α-hydroxycholesterol formed from endogenous microsomal cholesterol after enzymatic conversion to 7α-hydroxy-cholesten-3-one using cholesterol oxidase (39Chiang J.Y.L. Methods Enzymol. 1991; 206: 483-491Crossref PubMed Scopus (43) Google Scholar). Plasma apoproteins were separated by SDS-polyacrylamide gel electrophoresis. Total plasma lipoproteins were isolated by ultracentrifugation at d = 1.21 g/ml and size-fractionated by FPLC using a Superose 6 HR column. Fractions corresponding to VLDL and IDL/LDL were pooled, delipidated, loaded onto 2–15% gradient polyacrylamide gels (Owl Separation Systems, Portsmouth, NH), and subjected to electrophoresis. Apoproteins were visualized using Gelcode Blue Coomassie stain (Pierce), and gels were scanned using a densitometer (Molecular Dynamics). VLDL (d < 1.006 g/ml) was isolated from fasted LDL receptor−/− mice by preparative ultracentrifugation and radioiodinated using iodine monochloride (40Bilheimer D.W. Eisenberg S. Levy R.I. Biochim. Biophys. Acta. 1972; 260: 212-221Crossref PubMed Scopus (1185) Google Scholar). VLDL turnover studies were performed in mice as described by Ishibashi et al. (33Ishibashi S. Brown M.S. Goldstein J.L. Gerard R.D. Hammer R.E. Herz J. J. Clin. Invest. 1993; 92: 883-893Crossref PubMed Scopus (1268) Google Scholar). After a 2-h fast, LDL receptor−/− mice were administered 20 mg of Triton WR1339 (Sigma) in 100 μl of sterile saline by intravenous injection into a femoral vein. Blood was obtained from the retro-orbital sinus at 3 min and 1 h later. (Preliminary studies showed a linear accumulation of triglyceride in plasma for at least 1 h after Triton WR1339 administration.) The increase in plasma triglyceride during this 1-h interval was taken as the triglyceride production rate (41Li X. Catalina F. Grundy S.M. Patel S. J. Lipid Res. 1996; 37: 210-220Abstract Full Text PDF PubMed Google Scholar). In some studies animals were exsanguinated from the inferior vena cava 1 h after the intravenous administration of Triton WR1339 or vehicle. Plasma lipoproteins were size-fractionated by FPLC using a Superose 6 HR column, and enzymatic kits were used to quantify total and unesterified cholesterol and triglyceride in fractions corresponding to VLDL. VLDL-apoB100 and -apoB48 were separated on gradient polyacrylamide gels, stained, and quantified by densitometry as described above. The difference in these lipid and apoprotein animals with Triton WR1339 and was taken as the hepatic secretion rate for these of VLDL. Plasma was obtained from −/− and LDL was isolated by preparative ultracentrifugation in the density of Because the plasma −/− mice also this density studies were carried out to turnover for −/− = g/ml lipoproteins and after by to the were at the rate from the plasma −/− and studies were performed using the IDL/LDL IDL/LDL were labeled or as described previously D. J. Biol. Chem. 258: Full Text PDF PubMed Google Scholar), and of hepatic LDL were measured using of The of LDL were for h at which each was administered a of LDL as a of plasma volume and killed min later by exsanguination through the inferior vena cava. samples with aliquots of plasma were for radioactivity in a Inc., The tissue by the labeled LDL at min of tissue by of and at h of tissue by of were then and the of The increase in tissue the the rate of LDL into each and is expressed as the of plasma of its LDL per h per g of tissue or per The are as 1 were used to determine differences Studies to the ACAT overexpression and hepatic VLDL secretion were carried out −/− where in the plasma concentration of VLDL reflect the rate of hepatic VLDL To that animals the hACAT-1 adenovirus expressed hACAT-1 mRNA in the total RNA was from livers 3 after the administration of 1 or intravenously −/− and mRNA encoding hACAT-1 and GAPDH an internal were measured using an RNase protection assay. In this assay the hACAT-1 and GAPDH probes are and in As shown in mice administered expressed mRNA for hACAT-1 in the liver. studies showed a linear increase in hepatic hACAT-1 mRNA levels with of and studies showed that hepatic hACAT-1 mRNA levels were 3 after administration of We determined the effect of hACAT-1 overexpression on liver cholesterol −/− mice were 3 after the intravenous administration of 1 or As shown in hepatic cholesteryl ester concentrations were higher in animals that than in In the of compensatory changes, an increase in the rate of cholesterol esterification liver of unesterified however, the unesterified cholesterol of the liver was not by overexpression of hACAT-1 suggesting that compensatory in sterol across the liver had To this we quantified of hepatic cholesterol synthesis in −/− mice 3 after the administration of 1 or As shown in overexpression of hACAT-1 was with an increase in the rate of hepatic cholesterol synthesis. The major pathway for cholesterol from the body is through conversion to acids, and the in the acid pathway is catalyzed by cholesterol To determine hACAT-1 overexpression this pathway of cholesterol we measured the activity of hepatic 7α-hydroxylase −/− mice 3 after the administration of 1 or overexpression of hACAT-1 had no effect on hepatic 7α-hydroxylase activity or on hepatic 7α-hydroxylase mRNA levels using an RNase protection assay of hACAT-1 gene transfer on the concentration of cholesterol and triglyceride in liver and plasma and on the synthesis of cholesterol and fatty acids in total cholesterol concentration from the control < cholesterol synthesis per from the control < 7α-hydroxylase activity per triglyceride concentration fatty acid synthesis per from the control < cholesterol concentration from the control < triglyceride concentration from the control < experiments were performed 3 after the administration of 1 of or The from the control < in a All experiments were performed 3 after the administration of 1 of or fatty acyl is also a in the ACAT we measured hepatic triglyceride concentrations and fatty acid synthesis in −/− mice 3 after the administration of 1 or Hepatic triglyceride did not the two of but hepatic fatty acid synthesis was increased by hACAT-1 gene transfer We determined the effect of hACAT-1 overexpression on plasma lipoprotein concentrations in mice LDL −/− mice were administered 1 or intravenously and killed 3 later after a As shown in plasma cholesterol concentrations were higher −/− mice administered than in animals administered control virus Plasma triglyceride concentrations were also higher in animals administered than in animals administered control virus The lipoprotein distribution of plasma total and esterified cholesterol and triglyceride was determined in these of plasma animals per were pooled, and lipoproteins were size-fractionated on a Superose 6 HR column. As shown in the total and esterified cholesterol of apoB-containing lipoproteins and was markedly higher −/− mice administered than in control In the plasma concentration of total and esterified cholesterol in VLDL was and As shown in 3 the triglyceride of VLDL and IDL/LDL was also increased by hACAT-1 gene transfer. The plasma concentration of triglyceride in VLDL was higher in animals administered than in control were in studies each animals per The effect of hACAT-1 overexpression on the apoprotein of apoB-containing lipoproteins was determined by SDS-polyacrylamide gel electrophoresis. of plasma obtained from fasted −/− mice 3 after administration of 1 or were pooled, the d = 1.21 isolated by and the lipoproteins size-fractionated by of FPLC fractions corresponding to VLDL and IDL/LDL were pooled, delipidated, and separated on 2–15% gradient polyacrylamide As shown in the of and was higher in VLDL from animals administered than in the control The of and was also higher in the IDL/LDL from in the IDL/LDL was not increased by hACAT-1 gene transfer and in some experiments as shown in appeared to be were in studies each animals per In these the apoB