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Hepatic overexpression of microsomal triglyceride transfer protein (MTP) results in increased in vivo secretion of VLDL triglycerides and apolipoprotein B

1999; Elsevier BV; Volume: 40; Issue: 11 Linguagem: Inglês

10.1016/s0022-2275(20)32437-8

ISSN

1539-7262

Autores

Uwe J.F. Tietge, Ahmed Bakillah, Cyrille Maugeais, Kazuhisa Tsukamoto, M. Mahmood Hussain, Daniel J. Rader,

Tópico(s)

Lipid metabolism and disorders

Resumo

The microsomal triglyceride transfer protein (MTP) is essential for the hepatic secretion of apolipoprotein (apo) B-containing lipoproteins. Previous studies have indicated that inhibition of MTP results in decreased apoB plasma levels and decreased hepatic triglyceride secretion. However, the metabolic effects of overexpression of MTP have not been investigated. We constructed a recombinant adenovirus expressing MTP (AdhMTP) and used it to assess the effects of hepatic overexpression of MTP in mice. Injection of AdhMTP into C57BL/6 mice resulted in a 3-fold increase in hepatic microsomal triglyceride transfer activity compared to mice injected with Adnull. On day 4 after virus injection, AdhMTP-injected mice had significantly elevated plasma TG levels as compared to control virus (Adnull)-injected mice. Hepatic TG secretion rates were significantly greater in AdhMTP-injected mice (184 ± 12 mg/kg/h) compared with Adnull-injected mice (65 ± 9 mg/kg/h, P < 0.001). In addition, hepatic very low density lipoprotein (VLDL) apoB secretion in the AdhMTP-injected group was 74% higher than in the control virus group. Hepatic secretion of apoB-48 and apoB-100 contributed equally to this increase. These results provide the first data that hepatic overexpression of MTP results in increased secretion of VLDL-triglycerides as well as VLDL-apoB in vivo. These results suggest that MTP is rate-limiting for VLDL apoB secretion in wild-type mice under basal chow-fed conditions.— Tietge, U. J. F., A. Bakillah, C. Maugeais, K. Tsukamoto, M. Hussain, and D. J. Rader. Hepatic overexpression of microsomal triglyceride transfer protein (MTP) results in increased in vivo secretion of VLDL triglycerides and apolipoprotein B. J. Lipid Res. 1999. 40: 2134–2139. The microsomal triglyceride transfer protein (MTP) is essential for the hepatic secretion of apolipoprotein (apo) B-containing lipoproteins. Previous studies have indicated that inhibition of MTP results in decreased apoB plasma levels and decreased hepatic triglyceride secretion. However, the metabolic effects of overexpression of MTP have not been investigated. We constructed a recombinant adenovirus expressing MTP (AdhMTP) and used it to assess the effects of hepatic overexpression of MTP in mice. Injection of AdhMTP into C57BL/6 mice resulted in a 3-fold increase in hepatic microsomal triglyceride transfer activity compared to mice injected with Adnull. On day 4 after virus injection, AdhMTP-injected mice had significantly elevated plasma TG levels as compared to control virus (Adnull)-injected mice. Hepatic TG secretion rates were significantly greater in AdhMTP-injected mice (184 ± 12 mg/kg/h) compared with Adnull-injected mice (65 ± 9 mg/kg/h, P < 0.001). In addition, hepatic very low density lipoprotein (VLDL) apoB secretion in the AdhMTP-injected group was 74% higher than in the control virus group. Hepatic secretion of apoB-48 and apoB-100 contributed equally to this increase. These results provide the first data that hepatic overexpression of MTP results in increased secretion of VLDL-triglycerides as well as VLDL-apoB in vivo. These results suggest that MTP is rate-limiting for VLDL apoB secretion in wild-type mice under basal chow-fed conditions.— Tietge, U. J. F., A. Bakillah, C. Maugeais, K. Tsukamoto, M. Hussain, and D. J. Rader. Hepatic overexpression of microsomal triglyceride transfer protein (MTP) results in increased in vivo secretion of VLDL triglycerides and apolipoprotein B. J. Lipid Res. 1999. 40: 2134–2139. Apolipoprotein B-100 (apoB-100) is the major structural apolipoprotein of liver-derived very low density lipoproteins (VLDL) and low density lipoproteins (LDL) (1Havel R.J. Kane J.P. Introduction. Structure and metabolism of plasma lipoproteins.in: Scriver C.R. Beaudet A.L. Sly W.S. Valle D. 7th edition. The Metabolic and Molecular Basis of Inherited Disease. 2. McGraw-Hill, New York1995: 1841-1851Google Scholar, 2Young S. Recent progress in understanding apolipoprotein B.Circulation. 1990; 82: 1574-1594Google Scholar). The rate of secretion of apoB-containing VLDL by the liver is a major determinant of plasma triglycerides, LDL cholesterol, and apoB levels and therefore substantially influences cardiovascular risk (3Griffin B.A. Zampelas A. Influence of dietary fatty acids on the atherogenic lipoprotein phenotype.Nutr. Res. Rev. 1995; 8: 1-26Google Scholar, 4Wetterau J.R. Gregg R.E. Harrity T.W. Arbeeny C. Cap M. Connolly F. Chu C. George R.J. Gordon D.A. Jamil H. Jolibois K.G. Kunselman L.K. Lan S. Maccagnan T.J. Ricci B. Yan M. Young D. Chen Y. Fryszman O.M. Logan J.V.H. Musial C.L. Poss M.A. Robl J.A. Simpkins L.M. Slusarchyk W.A. Sulsky R. Taunk P. Magnin D.R. Tino J.A. Lawrence R.M. Dickson J.K. Biller S.A. An MTP inhibitor that normalizes atherogenic lipoprotein levels in WHHL rabbits.Science. 1998; 282: 751-754Google Scholar). In general, apoB gene transcription is constitutive and the major mechanisms of regulation of apoB secretion occur at the posttranscriptional level (5Yao Z. Tran K. McLeod R.S. Intracellular degradation of newly synthesized apolipoprotein B.J. Lipid Res. 1987; 38: 1937-1953Google 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: 5080-5086Google Scholar, 7Pullinger C.R. North J.D. Teng B.B. Rifici V.A. Ronhild de Brito A.E. Scott J. The apolipoprotein B gene is constitutively expressed in HepG2 cells: regulation of secretion by oleic acid, albumin, and insulin, and measurement of the mRNA half-life.J. Lipid Res. 1989; 30: 1065-1077Google Scholar). These involve mainly the variation of the proportion of apoB that is intracellularly degraded versus the proportion that is lipidated and finally secreted (8Shelness G.S. Ingram M.F. Huang X.F. DeLozier J.A. Apolipoprotein B in the rough endoplasmic reticulum: translation, translocation and the initiation of lipoprotein assembly.J. Nutr. 1999; 129: 456S-462SGoogle Scholar, 9White A.L. Graham D.L. LeGros J. Pease R.J. Scott J. Oleate-mediated stimulation of apolipoprotein B secretion from rat hepatoma cells. A function of the ability of apolipoprotein B to direct lipoprotein assembly and escape presecretory degradation.J.Biol. Chem. 1992; 267: 15657-15664Google Scholar, 10Sato R. Imanaka T. Takatsuki A. Takano T. Degradation of newly synthesized apolipoprotein B-100 in a pre-Golgi compartment.J. Biol. Chem. 1990; 265: 11880-11884Google Scholar, 11Rustaeus S. Lindberg K. Stillemark P. Claesson C. Asp L. Larsson T. Boren J. Olofsson S.O. Assembly of very low density lipoprotein: a two-step process of apolipoprotein B core lipidation.J. Nutr. 1999; 129: 463S-466SGoogle Scholar, 12Hussain M.M. Kancha R.K. Zhou Z. Luchoomun J. Zu H. Bakillah A. Chylomicron assembly and catabolism: role of apolipoproteins and receptors.Biochim. Biophys. Acta. 1996; 1300: 151-170Google Scholar). Availability of adequate lipid substrate for lipoprotein assembly is one important factor in regulating apoB secretion rates (5Yao Z. Tran K. McLeod R.S. Intracellular degradation of newly synthesized apolipoprotein B.J. Lipid Res. 1987; 38: 1937-1953Google 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: 5080-5086Google Scholar, 12Hussain M.M. Kancha R.K. Zhou Z. Luchoomun J. Zu H. Bakillah A. Chylomicron assembly and catabolism: role of apolipoproteins and receptors.Biochim. Biophys. Acta. 1996; 1300: 151-170Google Scholar, 13Ginsberg H.N. Role of lipid synthesis, chaperone proteins and proteasomes in the assembly and secretion of apoprotein B-containing lipoproteins from cultured liver cells.Clin. Exp. Pharmacol. Physiol. 1997; 24: A29-A32Google Scholar). Another required factor for VLDL assembly and secretion is the microsomal triglyceride transfer protein (MTP) (14White D.A. Bennett A.J. Billett M.A. Salter A.M. The assembly of triacylglycerol-rich lipoproteins: an essential role for the microsomal triglyceride transfer protein.Br. J. Nutr. 1998; 80: 219-229Google Scholar, 15Gordon D.A. Recent advances in elucidating the role of the microsomal triglyceride transfer protein in apolipoprotein B lipoprotein assembly.Curr. Opin. Lipidol. 1997; 8: 131-137Google Scholar). MTP is a heterodimeric protein consisting of a catalytic 97 kD subunit (further referred to as MTP in this study) and protein disulfide isomerase, a multifunctional ubiquitously expressed protein (16Wetterau J.R. Lin M.C. Jamil H. Microsomal triglyceride transfer protein.Biochim. Biophys. Acta. 1997; 1345: 136-150Google Scholar). MTP expression has been localized mainly to the endoplasmic reticulum (ER) of hepatocytes as well as enterocytes (14White D.A. Bennett A.J. Billett M.A. Salter A.M. The assembly of triacylglycerol-rich lipoproteins: an essential role for the microsomal triglyceride transfer protein.Br. J. Nutr. 1998; 80: 219-229Google Scholar, 16Wetterau J.R. Lin M.C. Jamil H. Microsomal triglyceride transfer protein.Biochim. Biophys. Acta. 1997; 1345: 136-150Google Scholar, 17Sharp D. Blinderman L. Combs K.A. Kienzle B. Ricci B. Wager-Smith K. Gil C.M. Turck C.W. Bouna M.E. Rader D.J. Aggerbeck L.P. Gregg R.E. Gordon D.A. Wetterau J.R. Cloning and gene defects in microsomal triglyceride transfer protein associated with abetalipoproteinemia.Nature. 1993; 365: 65-69Google Scholar). Mutations in the MTP gene are the molecular defect in abetalipoproteinemia, an autosomal recessive disorder characterized by no detectable apoB-containing lipoproteins in plasma, indicating the requirement for MTP in apoB-lipoprotein secretion (15Gordon D.A. Recent advances in elucidating the role of the microsomal triglyceride transfer protein in apolipoprotein B lipoprotein assembly.Curr. Opin. Lipidol. 1997; 8: 131-137Google Scholar, 18Kane J.P. Havel R.J. Disorders of the biogenesis and secretion of lipoproteins containing the B apolipoproteins.in: Scriver C.R. Beaudet A.L. Sly W.S. Valle D. 7th edition. The Metabolic and Molecular Basis of Inherited Disease. 2. McGraw-Hill, New York1995: 1853-1885Google Scholar). Transfection of a MTP cDNA into cells that normally do not secrete lipoproteins permits secretion of stably transfected apoB (19Gordon D.A. Jamil H. Sharp D. Mullaney D. Yao Z. Gregg R.E. Wetterau J. The secretion of apolipoprotein B-containing lipoproteins from HeLa-cells is dependent on expression of the microsomal triglyceride transfer protein and is regulated by lipid availability.Proc. Natl. Acad. Sci. USA. 1994; 91: 7628-7632Google Scholar, 20Patel S.B. Grundy S.M. Interactions between microsomal triglyceride transfer protein and apolipoprotein B within the endoplasmic reticulum in a heterologous expression system.J. Biol. Chem. 1996; 271: 18686-18694Google Scholar, 21Leiper J.M. Bayliss J.D. Pease R.J. Brett D.J. Scott J. Shoulders C.C. Microsomal triglyceride transfer protein, the abetalipoproteinemia gene product, mediates the secretion of apolipoprotein B-containing lipoproteins from heterologous cells.J. Biol. Chem. 1994; 269: 21951-21954Google Scholar, 22Gretch D.G. Sturley S.L. Wang L. Lipton B.A. Dunning A. Grunwald K.A. Wetterau J.R. Yao Z. Talmud P. Attie A.D. The amino-terminus of apolipoprotein B is necessary but not sufficient for microsomal triglyceride transfer protein responsiveness.J. Biol. Chem. 1996; 271: 8682-8691Google Scholar). A variety of data indicate that reduction in MTP activity results in reduced secretion of apoB and apoB-containing lipoproteins. Pharmacologic inhibition of MTP in vitro resulted in a dose-dependent decrease of secretion of apoB-containing lipoproteins by HepG2 and CaCo2 cells (23Jamil H. Gordon D.A. Eustice D.C. Brooks C.M. Dickson J.K.J. Chen Y. Ricci B. Chu C.H. Harrity T.W. Ciosek C.P.J. Biller S.A. Gregg R.E. Wetterau J.R. An inhibitor of the microsomal triglyceride transfer protein inhibits apoB secretion from HepG2 cells.Proc. Natl. Acad. Sci. USA. 1996; 93: 11991-11995Google Scholar – 26van Greevenbroek M.M. Robertus-Teunissen M.G. Erkelens D.W. de Bruin T.W. Participation of the microsomal triglyceride transfer protein in lipoprotein assembly in CaCo-2 cells: interaction with saturated and unsaturated fatty acids.J. Lipid Res. 1998; 39: 173-185Google Scholar), and reduced the oleate-induced secretion of apoB-48–VLDL from transfected McA-RH7777 cells (27Wang Y. McLeod R.S. Yao Z.M. Normal activity of microsomal triglyceride protein is required for the oleate-induced secretion of very low density lipoproteins containing apoprotein B from McA-RH7777 cells.J. Biol. Chem. 1997; 272: 12272-12278Google Scholar). Administration of an MTP inhibitor to WHHL rabbits (which lack functional LDL receptors) resulted in a reduction of plasma triglycerides, total cholesterol, and VLDL/LDL cholesterol levels in a dose-dependent manner (4Wetterau J.R. Gregg R.E. Harrity T.W. Arbeeny C. Cap M. Connolly F. Chu C. George R.J. Gordon D.A. Jamil H. Jolibois K.G. Kunselman L.K. Lan S. Maccagnan T.J. Ricci B. Yan M. Young D. Chen Y. Fryszman O.M. Logan J.V.H. Musial C.L. Poss M.A. Robl J.A. Simpkins L.M. Slusarchyk W.A. Sulsky R. Taunk P. Magnin D.R. Tino J.A. Lawrence R.M. Dickson J.K. Biller S.A. An MTP inhibitor that normalizes atherogenic lipoprotein levels in WHHL rabbits.Science. 1998; 282: 751-754Google Scholar). Rats administered the MTP inhibitor were demonstrated to have significantly reduced secretion rates of VLDL-TG (4Wetterau J.R. Gregg R.E. Harrity T.W. Arbeeny C. Cap M. Connolly F. Chu C. George R.J. Gordon D.A. Jamil H. Jolibois K.G. Kunselman L.K. Lan S. Maccagnan T.J. Ricci B. Yan M. Young D. Chen Y. Fryszman O.M. Logan J.V.H. Musial C.L. Poss M.A. Robl J.A. Simpkins L.M. Slusarchyk W.A. Sulsky R. Taunk P. Magnin D.R. Tino J.A. Lawrence R.M. Dickson J.K. Biller S.A. An MTP inhibitor that normalizes atherogenic lipoprotein levels in WHHL rabbits.Science. 1998; 282: 751-754Google Scholar). Homozygosity for the MTP gene knockout is lethal in mice (28Raabe M. Flynn L.M. Zlot C.H. Wong J.S. Veniant M.M. Hamilton R.L. Young S.G. Knockout of the abetalipoproteinemia gene in mice: reduced lipoprotein secretion in heterozygotes and embryonic lethality in homozygotes.Proc. Natl. Acad. Sci. USA. 1998; 95: 8686-8691Google Scholar). Heterozygous MTP-deficient mice have plasma apoB levels that are ∼70% of those in wild-type mice and primary hepatocytes from heterozygous MTP knockout mice have reduced rates of apoB secretion (28Raabe M. Flynn L.M. Zlot C.H. Wong J.S. Veniant M.M. Hamilton R.L. Young S.G. Knockout of the abetalipoproteinemia gene in mice: reduced lipoprotein secretion in heterozygotes and embryonic lethality in homozygotes.Proc. Natl. Acad. Sci. USA. 1998; 95: 8686-8691Google Scholar). These results indicate that MTP is necessary for secretion of apoB-containing lipoproteins and that substantial inhibition of MTP reduces the rate of apoB secretion. However, the question of whether increased MTP activity over basal levels of expression results in increased VLDL and apoB secretion in vivo has not yet been answered. Interestingly, a recent in vitro study suggested that MTP might be limiting for the secretion of apoB-containing lipoproteins (29Jamil H. Chu C.H. Dickson J.K.J. Chen Y. Yan M. Biller S.A. Gregg R.E. Wetterau J.R. Gordon D.A. Evidence that microsomal triglyceride transfer protein is limiting in the production of apolipoprotein B-containing lipoproteins in hepatic cells.J. Lipid Res. 1998; 39: 1448-1454Google Scholar). The same study reported that attempts to increase MTP activity by transfecting hepatoma cell lines with an expression plasmid encoding MTP were unsuccessful (29Jamil H. Chu C.H. Dickson J.K.J. Chen Y. Yan M. Biller S.A. Gregg R.E. Wetterau J.R. Gordon D.A. Evidence that microsomal triglyceride transfer protein is limiting in the production of apolipoprotein B-containing lipoproteins in hepatic cells.J. Lipid Res. 1998; 39: 1448-1454Google Scholar). Recently, adenovirus-mediated overexpression of MTP was shown to increase apoB secretion in HepG2 cells (30Liao W. Kobayashi K. Chan L. Adenovirus-mediated overexpression of microsomal triglyceride transfer protein (MTP): mechanistic studies on the role of MTP in apolipoprotein B-100 biogenesis.Biochemistry. 1999; 38: 7532-7544Google Scholar). The purpose of the present study was to test the hypothesis that hepatic overexpression of MTP using a recombinant adenovirus would increase VLDL-TG and apoB secretion by the liver in vivo. Our results demonstrate that increased MTP expression in the liver resulted in increased secretion of VLDL apoB and TG in wild-type mice on a chow diet. These data provide the first evidence that MTP is rate-limiting for hepatic VLDL assembly and secretion in vivo. The plasmid pRChMTP containing the full-length cDNA of the large subunit of human MTP (kindly provided by Dr. D. Gordon and Dr. J. Wetterau, Bristol-Myers-Squibb Pharmaceutical Research Institute, Princeton, NJ) was subcloned into the shuttle plasmid vector pAdCMVlink (31Ye X. Robinson M. Batshaw M. Furth E. Smith I. Wilson J.M. Prolonged metabolic correction in adult ornithine transcarbamylase-deficient mice with adenoviral vectors.J. Biol. Chem. 1996; 271: 3639-3646Google Scholar) generating the plasmid pAdCMVhMTP. Recombinant adenoviruses were produced with established methods (32Kozarsky K.F. Wilson J.M. Gene therapy: adenovirus vectors.Curr. Opin. Genet. Dev. 1993; 3: 499-503Google Scholar) using adenoviral DNA containing a temperature-sensitive mutation (ts125) in the E2A region that renders the adenovirus incapable of replicating at 39°C (33Engelhardt J.F. Ye X. Doranz B. Wilson J.M. Ablation of E2A in recombinant adenoviruses improves transgene persistence and decreases inflammatory response in mouse liver.Proc. Natl. Acad. Sci. USA. 1994; 91: 6196-6200Google Scholar). In this way two recombinant adenoviruses containing hMTP (AdhMTP) as well as no transgene (Adnull) were generated. Recombinant adenovirus was grown, screened by PCR, subjected to two subsequent rounds of plaque purification, purified, and stored as described (34Tsukamoto 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: 107-114Google Scholar). COS-7 cells were obtained from the American Type Culture Collection (Rockville, MD) and grown in DMEM with 10% fetal bovine serum, and 1% antibiotic–antimycotic mixture (all reagents from Life Sciences). COS cells were infected with recombinant adenoviruses at a multiplicity of infection, of 750. At 48 h after infection, MTP activity was determined by measuring the transfer rate of [14C]triglycerides from donor to acceptor small unilamellar vesicles using a preparation of microsomal proteins as described previously (23Jamil H. Gordon D.A. Eustice D.C. Brooks C.M. Dickson J.K.J. Chen Y. Ricci B. Chu C.H. Harrity T.W. Ciosek C.P.J. Biller S.A. Gregg R.E. Wetterau J.R. An inhibitor of the microsomal triglyceride transfer protein inhibits apoB secretion from HepG2 cells.Proc. Natl. Acad. Sci. USA. 1996; 93: 11991-11995Google Scholar, 35Luchoomun J. Zhou Z. Bakillah A. Jamil H. Hussain M.M. Assembly and secretion of VLDL in nondifferentiated Caco-2 cells stably transfected with human recombinant apoB48 cDNA.Arterioscler. Thromb. Vasc. Biol. 1997; 17: 2955-2963Google Scholar). Assessment of MTP activity in the livers of mice injected with either AdhMTP or Adnull was performed using lumenal proteins released by the treatment of microsomes with 100 mm sodium carbonate, pH 11.5, with the same triglyceride transfer assay (23Jamil H. Gordon D.A. Eustice D.C. Brooks C.M. Dickson J.K.J. Chen Y. Ricci B. Chu C.H. Harrity T.W. Ciosek C.P.J. Biller S.A. Gregg R.E. Wetterau J.R. An inhibitor of the microsomal triglyceride transfer protein inhibits apoB secretion from HepG2 cells.Proc. Natl. Acad. Sci. USA. 1996; 93: 11991-11995Google Scholar, 35Luchoomun J. Zhou Z. Bakillah A. Jamil H. Hussain M.M. Assembly and secretion of VLDL in nondifferentiated Caco-2 cells stably transfected with human recombinant apoB48 cDNA.Arterioscler. Thromb. Vasc. Biol. 1997; 17: 2955-2963Google Scholar). For Western blot analysis of hepatic MTP expression in the different experimental groups of mice, 5 μg of microsomal protein was electrophoresed under reducing conditions on a 10% polyacrylamide gel (UltraPure, Gibco BRL, Gaithersburg, MD) and subsequently blotted to nitrocellulose (Schleicher & Schuell, Keene, NH). MTP was identified with a polyclonal rabbit anti-bovine MTP antibody (19Gordon D.A. Jamil H. Sharp D. Mullaney D. Yao Z. Gregg R.E. Wetterau J. The secretion of apolipoprotein B-containing lipoproteins from HeLa-cells is dependent on expression of the microsomal triglyceride transfer protein and is regulated by lipid availability.Proc. Natl. Acad. Sci. USA. 1994; 91: 7628-7632Google Scholar) in a dilution of 1:1000 (JW1 F, a kind gift from Dr. D. Gordon and Dr. J. Wetterau, Bristol-Myers-Squibb Pharmaceutical Research Institute, Princeton, NJ) and an appropriate second antibody (Jackson ImmunoResearch Laboratories, West Grove, PA) using the ECL (enhanced chemoluminescence) detection system (Amersham Corp., Arlington Heights, IL). This antibody cross-reacts with murine and human MTP (19Gordon D.A. Jamil H. Sharp D. Mullaney D. Yao Z. Gregg R.E. Wetterau J. The secretion of apolipoprotein B-containing lipoproteins from HeLa-cells is dependent on expression of the microsomal triglyceride transfer protein and is regulated by lipid availability.Proc. Natl. Acad. Sci. USA. 1994; 91: 7628-7632Google Scholar, 28Raabe M. Flynn L.M. Zlot C.H. Wong J.S. Veniant M.M. Hamilton R.L. Young S.G. Knockout of the abetalipoproteinemia gene in mice: reduced lipoprotein secretion in heterozygotes and embryonic lethality in homozygotes.Proc. Natl. Acad. Sci. USA. 1998; 95: 8686-8691Google Scholar, 36Chang B. Liao W. Li L. Nakamuta M. Mack D. Chan L. Liver-specific inactivation of the abetalipoproteinemia gene completely abrogates very low density lipoprotein/low density lipoprotein production in a viable conditional knockout mouse.J. Biol. Chem. 1999; 274: 6051-6055Google Scholar). Plasma total cholesterol, triglycerides, phospholipids, and HDL cholesterol levels were measured enzymatically on a Cobas Fara (Roche Diagnostics Systems Inc., Nutley, NJ) using Sigma Diagnostics reagents (Sigma Diagnostics, St. Louis, MO). Pooled plasma samples from 4 mice (120 μl total volume) were subjected to fast protein liquid chromatography (FPLC) gel filtration using two Superose 6 columns (Pharmacia LKB Biotechnology, Uppsala, Sweden) as described (34Tsukamoto 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: 107-114Google Scholar). Lipoprotein fractions of 500 μl each were collected. Individual fractions were assayed for cholesterol concentrations using commercially available assay kits (Wako Pure Chemical Industries, Ltd., Osaka, Japan). Female C57BL/6 mice, 6–8 weeks old, were purchased from Jackson Laboratory (Bar Harbor, ME) and fed a regular mouse chow diet (Diet 5010, PMI Nutrition International, Richmond, IN). Mice were bled from the retroorbital plexus after a 4-h fast using heparinized capillary tubes. Blood was drawn into tubes containing 2 mm EDTA, 0.2% NaN3, and 1 mm benzamidine and placed immediately on ice. Aliquots of plasma were stored at –20°C until analysis. For the adenovirus studies, mice were injected with 1.5 × 1011 particles (4.5–6 × 109 pfu) of AdhMTP, Adnull, or saline as indicated. To assess MTP expression in AdhMTP- and Adnull-injected mice, experimental animals were killed on day 3 after virus injection, perfused with ice-cold PBS, and livers were immediately frozen in liquid nitrogen. Tissues were then processed as described above. Mice (C57BL/6) that had been injected with either AdhMTP, Adnull, or PBS 3 days prior to the experiment were allowed to eat fat-free food for 2 h and were then injected via tail vein at 9 am with 20 mg of Triton WR1339 solution (Tyloxapol, Sigma, St. Louis, MO) in a total volume of 200 μl sterile PBS (37Borensztajn J. Rone M.S. Kotlar T.J. The inhibition in vivo of lipoprotein lipase (clearing-factor lipase) activity by Triton WR-1339.Biochem. J. 1976; 156: 539-543Google Scholar, 38Ishikawa T. Fidge N. Changes in the concentration of plasma lipoproteins and apoproteins following the administration of Triton WR 1339 to rats.J. Lipid Res. 1979; 20: 254-264Google Scholar). Prior to injection and at 30 min, 1 h, and 2 h after Triton WR1339 injection, 40 μl of blood was drawn by retroorbital bleeding, and plasma was separated and assayed for triglycerides. Based on pilot experiments that were performed to determine the linearity of the plasma triglyceride curve, we decided to use the 30-min time point for calculation of hepatic TG secretion. Therefore baseline TG levels were subtracted from the 30-min values in each of the mice and TG secretion rates expressed as mg/kg per h assuming the plasma volume to be 3.5% of the body weight. The measurement of hepatic VLDL apoB secretion was carried out by endogenous labeling under conditions of blocking VLDL catabolism with Triton WR1339. Under these conditions the rate of accumulation of endogenously labeled VLDL apoB is directly related to the rate of apoB-VLDL secretion. Therefore mice that had been injected with AdhMTP, Adnull, or PBS 3 days before were placed on a fat-free diet 2 h prior to a 40 μl baseline bleed at 9 am. Then 200 μl of a 10% Triton WR1339 solution containing 500 μCi of [35S]methionine (NEN) was admininstered by tail vein injection. Blood was drawn by retroorbital bleeding at 5 min (10 μl), 30 min, 1 h, and 2 h (40 μl) after injection of the tracer solution. Based on previous experiments we used a 1 h time point for further processing of the samples in the experimental mice. To isolate VLDL, 20 μl of mouse plasma was layered under 750 μl of KBr solution (d 1.006 g/ml) and centrifuged at 90000 rpm at 10°C for 3 h (Beckman TL100 Ultracentrifuge). VLDL was recovered in the top fraction by tube slicing in a volume of 250 μl. To 200 μl of VLDL, 100 μl of 3.6 mm deoxycholate and 100 μl of TCA (4.9 m) were added, and the tubes were vortexed and placed on ice for 15 min to precipitate VLDL proteins. After centrifugation in a table-top centrifuge at top speed for 20 min, the supernatant was decanted and 90 μl of Laemmli sample buffer (BioRad) and 10 μl of 1 m NaOH were added. Samples were heated at 80°C for 10 min to dissolve the precipitate and 45 μl was subjected to linear gradient SDS-PAGE (3–20%) (UltraPure, Gibco BRL). The gel was fixed and incubated in Amplify solution (Amersham Life Sciences) under gentle agitation for 30 min. After that the gel was dried under vacuum and exposed to an X-ray film (Fuji Medical Systems, Stamford, CT) at –80°C. From the autoradiography, relative positions of apoB-48 and apoB-100 were identified and the respective bands were cut out of the gel. The gel pieces were then placed in glass scintillation vials (Fisher Scientific) and 500 μl of H2O was added to rehydrate the gel. Then 500 μl of Solvable (Packard, Meriden, CT) was added and the samples were incubated for 3 h at 50°C. Samples were cooled, 5 ml of Scintiverse BD (Fisher Scientific) was added, and the vials were vortexed and then counted on a scintillation counter (Beckman LS6500). The apoB counts were corrected for the injected dose of tracer in each mouse assessed as the plasma counts at 5 min after tracer injection. Therefore, individual counts in apoB were divided by the ratio of the respective 5 min plasma count and the highest 5 min plasma count in the experiment. Values are presented as mean ± SEM unless otherwise indicated. Results were analyzed by ANOVA and Student's t test for independent samples (two-tailed) using the GraphPad Prism software (GraphPad, San Diego, CA). Statistical significance for all comparisons was assigned at P < 0.05. COS-7 cells infected with AdhMTP exhibited significantly higher MTP lipid transfer activity than Adnull infected cells (1.45 ± 0.08 vs. 0.3 ± 0.01% TG transfer/100 μg protein/h). We then injected the recombinant viruses AdhMTP or Adnull via the tail vein into C57BL/6 mice fed a chow diet. On day 3 after adenovirus injection, livers harvested from AdhMTP-injected mice had increased expression of MTP protein (Fig. 1A) and significantly higher microsomal triglyceride transfer activity than control virus-injected mice (6.5 ± 2.2 vs. 2.2 ± 0.3% TG transfer/h/50 μg protein, resp., P < 0.01, Fig. 1B). Next we assessed the effect of AdhMTP and Adnull on plasma lipids. Baseline lipid profiles in the two groups of experimental mice were not different. On day 4 after virus injection, triglycerides in the Adnull-injected mice were 79 ± 7 mg/dl as compared to 63 ± 3 mg/dl on day 0 (P = n.s.) demonstrating that virus injection itself had no effect on plasma triglyceride levels. In contrast, in the AdhMTP group, plasma TG levels increased from 59 ± 3 mg/dl at baseline to 110 ± 12 mg/dl on day 4 after injection (P < 0.05). This represents a mean 86% increase over baseline triglycerides (Fig. 2). The plasma levels of total cholesterol and HDL-cholesterol were not different between the experimental groups of mice (data not shown). At baseline, FPLC gel filtration separation of lipoproteins showed that the lipoprotein cholesterol profiles were identical with HDL being the major lipoprotein (data not shown). Separation of lipoproteins by FPLC on day 4 after virus injection revealed an increase in the small VLDL cholesterol peak in the AdhMTP-injected group while there was no change in this peak in the Adnull-injected mice (data not shown). The LDL and HDL cholesterol peaks were not different between the experimental groups. To further investigate the metabolic basis for the changes in plasma triglycerides, we assessed the rate of hepatic triglyceride secretion in AdhMTP- and Adnull-injected mice. Hepatic triglyceride secretion rates in AdhMTP-, Adnull-, and vehicle (PBS)-injected mice were determined after blocking VLDL TG catabolism with Triton WR1339. The TG secretion rate in AdhMTP-injected mice was 184 ± 12 mg/kg per h (Fig. 3). This was significantly increased as compared with the control virus-injected group (65 ± 9 mg/kg/h, P < 0.001) and the PBS-injected control group (96 ± 8 mg/kg/h, P < 0.05). Hepatic TG secretion rates between the control virus and PBS-injected groups were not significantly different. Because there was a significant increase in hepatic TG secretion in the AdhMTP overexpressing mice, we next determined hepatic VLDL apoB secretion rates using endogenous labeling with [35S]methionine. Total hepatic apoB secretion in the AdhMTP-injected group was 74% higher than in the Adnull-injected and 101% higher than in the vehicle-injected groups of mice (Fig. 4A). The observed increase in total hepatic apoB secretion rates was due to both increased apoB-48 as well as increased apoB-100 secretion in the mice injected with the AdhMTP adenovirus (Fig. 4B). ApoB-48 secretion in the MTP overexpressing mice was 70% higher than in the Adnull group and 123% higher than in the group injected with vehicle. ApoB-100 secretion was 81% higher in the AdhMTP-injected mice as compared with the control virus-injected mice and 73% higher as compared with the PBS-injected group. These data demonstrate for the first time that hepatic overexpression of MTP in vivo results in increased secretion of VLDL triglycerides and apoB by the liver. This finding is particularly interesting as our experimental model was the wild-type C57BL/6 mouse on normal chow without addition of lipid to the diet. Our results suggest that MTP is rate-limiting for hepatic VLDL secretion, as recently suggested by Jamil et al. (29Jamil H. Chu C.H. Dickson J.K.J. Chen Y. Yan M. Biller S.A. Gregg R.E. Wetterau J.R. Gordon D.A. Evidence that microsomal triglyceride transfer protein is limiting in the production of apolipoprotein B-containing lipoproteins in hepatic cells.J. Lipid Res. 1998; 39: 1448-1454Google Scholar) and Liao, Kobayashi, and Chan (30Liao W. Kobayashi K. Chan L. Adenovirus-mediated overexpression of microsomal triglyceride transfer protein (MTP): mechanistic studies on the role of MTP in apolipoprotein B-100 biogenesis.Biochemistry. 1999; 38: 7532-7544Google Scholar) based on in vitro studies. In vitro studies using pharmacological MTP inhibitors have established that decreased MTP activity results in a decrease of TG and apoB secretion (23Jamil H. Gordon D.A. Eustice D.C. Brooks C.M. Dickson J.K.J. Chen Y. Ricci B. Chu C.H. Harrity T.W. Ciosek C.P.J. Biller S.A. Gregg R.E. Wetterau J.R. An inhibitor of the microsomal triglyceride transfer protein inhibits apoB secretion from HepG2 cells.Proc. Natl. Acad. Sci. USA. 1996; 93: 11991-11995Google Scholar, 24Benoist F. Nicodeme E. Grand-Perret T. Microsomal triacylglycerol transfer protein prevents presecretory degradation of apolipoprotein B-100. A dithiothreitol-sensitive protease is involved.Eur. J. Biochem. 1996; 240: 713-720Google Scholar, 25Haghpassand M. Wilder D. Moberly J.B. Inhibition of apolipoprotein B and triglyceride secretion in human hepatoma cells (HepG2).J. Lipid Res. 1996; 37: 1468-1480Google Scholar, 26van Greevenbroek M.M. Robertus-Teunissen M.G. Erkelens D.W. de Bruin T.W. Participation of the microsomal triglyceride transfer protein in lipoprotein assembly in CaCo-2 cells: interaction with saturated and unsaturated fatty acids.J. Lipid Res. 1998; 39: 173-185Google Scholar). Reduction of MTP activity in vivo also results in reduced TG and apoB secretion (4Wetterau J.R. Gregg R.E. Harrity T.W. Arbeeny C. Cap M. Connolly F. Chu C. George R.J. Gordon D.A. Jamil H. Jolibois K.G. Kunselman L.K. Lan S. Maccagnan T.J. Ricci B. Yan M. Young D. Chen Y. Fryszman O.M. Logan J.V.H. Musial C.L. Poss M.A. Robl J.A. Simpkins L.M. Slusarchyk W.A. Sulsky R. Taunk P. Magnin D.R. Tino J.A. Lawrence R.M. Dickson J.K. Biller S.A. An MTP inhibitor that normalizes atherogenic lipoprotein levels in WHHL rabbits.Science. 1998; 282: 751-754Google Scholar, 28Raabe M. Flynn L.M. Zlot C.H. Wong J.S. Veniant M.M. Hamilton R.L. Young S.G. Knockout of the abetalipoproteinemia gene in mice: reduced lipoprotein secretion in heterozygotes and embryonic lethality in homozygotes.Proc. Natl. Acad. Sci. USA. 1998; 95: 8686-8691Google Scholar, 36Chang B. Liao W. Li L. Nakamuta M. Mack D. Chan L. Liver-specific inactivation of the abetalipoproteinemia gene completely abrogates very low density lipoprotein/low density lipoprotein production in a viable conditional knockout mouse.J. Biol. Chem. 1999; 274: 6051-6055Google Scholar). However, the physiologic effect of MTP overexpression on TG and apoB secretion in vivo had not been previously determined. Current concepts on hepatic assembly and secretion of apoB-containing lipoproteins suggest a two-step process of apoB core lipidation, although the details of this mechanism remain incompletely understood (8Shelness G.S. Ingram M.F. Huang X.F. DeLozier J.A. Apolipoprotein B in the rough endoplasmic reticulum: translation, translocation and the initiation of lipoprotein assembly.J. Nutr. 1999; 129: 456S-462SGoogle Scholar, 14White D.A. Bennett A.J. Billett M.A. Salter A.M. The assembly of triacylglycerol-rich lipoproteins: an essential role for the microsomal triglyceride transfer protein.Br. J. Nutr. 1998; 80: 219-229Google Scholar, 15Gordon D.A. Recent advances in elucidating the role of the microsomal triglyceride transfer protein in apolipoprotein B lipoprotein assembly.Curr. Opin. Lipidol. 1997; 8: 131-137Google Scholar). ApoB is transcribed constitutively, and transcriptional rates are apparently not regulated by dietary manipulations (1Havel R.J. Kane J.P. Introduction. Structure and metabolism of plasma lipoproteins.in: Scriver C.R. Beaudet A.L. Sly W.S. Valle D. 7th edition. The Metabolic and Molecular Basis of Inherited Disease. 2. McGraw-Hill, New York1995: 1841-1851Google Scholar, 7Pullinger C.R. North J.D. Teng B.B. Rifici V.A. Ronhild de Brito A.E. Scott J. The apolipoprotein B gene is constitutively expressed in HepG2 cells: regulation of secretion by oleic acid, albumin, and insulin, and measurement of the mRNA half-life.J. Lipid Res. 1989; 30: 1065-1077Google Scholar). ApoB secretion appears to be primarily regulated at the posttranscriptional level. A significant portion of newly synthesized apoB is targeted for intracellular degradation (5Yao Z. Tran K. McLeod R.S. Intracellular degradation of newly synthesized apolipoprotein B.J. Lipid Res. 1987; 38: 1937-1953Google Scholar, 12Hussain M.M. Kancha R.K. Zhou Z. Luchoomun J. Zu H. Bakillah A. Chylomicron assembly and catabolism: role of apolipoproteins and receptors.Biochim. Biophys. Acta. 1996; 1300: 151-170Google Scholar, 13Ginsberg H.N. Role of lipid synthesis, chaperone proteins and proteasomes in the assembly and secretion of apoprotein B-containing lipoproteins from cultured liver cells.Clin. Exp. Pharmacol. Physiol. 1997; 24: A29-A32Google Scholar). Lipidation of the newly synthesized apoB appears to prevent its degradation. In the presence of MTP and a sufficient intracellular lipid supply, cotranslational lipoprotein assembly is initiated (9White A.L. Graham D.L. LeGros J. Pease R.J. Scott J. Oleate-mediated stimulation of apolipoprotein B secretion from rat hepatoma cells. A function of the ability of apolipoprotein B to direct lipoprotein assembly and escape presecretory degradation.J.Biol. Chem. 1992; 267: 15657-15664Google Scholar, 10Sato R. Imanaka T. Takatsuki A. Takano T. Degradation of newly synthesized apolipoprotein B-100 in a pre-Golgi compartment.J. Biol. Chem. 1990; 265: 11880-11884Google Scholar, 39Wu X. Shang A. Jiang H. Ginsberg H.N. Low rates of apoB secretion from HepG2 cells result from reduced delivery of newly synthesized triglyceride to a "secretion-coupled" pool.J. Lipid Res. 1996; 37: 1198-1206Google Scholar). MTP is believed to be necessary for adding triglycerides as well as cholesteryl ester to the nascent apoB in a process that is incompletely understood. MTP may also promote the translocation of apoB across the ER membrane and have other effects in early VLDL assembly not directly related to lipid transfer (15Gordon D.A. Recent advances in elucidating the role of the microsomal triglyceride transfer protein in apolipoprotein B lipoprotein assembly.Curr. Opin. Lipidol. 1997; 8: 131-137Google Scholar, 16Wetterau J.R. Lin M.C. Jamil H. Microsomal triglyceride transfer protein.Biochim. Biophys. Acta. 1997; 1345: 136-150Google Scholar). Our finding that increased VLDL TG and apoB secretion is induced by overexpressing MTP in normal mice on a chow diet indicates that even under basal conditions MTP is rate-limiting in the process of VLDL assembly and secretion. Further studies will be necessary to elucidate the molecular mechanisms by which overexpression of MTP results in increased VLDL apoB secretion. In summary, our results provide the first data on the effects of MTP overexpression in vivo and demonstrate that it causes increased secretion of VLDL apoB as well as VLDL triglycerides in normal mice on a chow diet. These results suggest the possibility that some forms of hepatic VLDL apoB overproduction, a common cause of familial combined hyperlipidemia (40Grundy S.M. Chait A. Brunzell J.D. Familial combined hyperlipidemia workshop.Arteriosclerosis. 1987; 7: 203-207Google Scholar), could be related to increased MTP activity. Further studies will be required to test this hypothesis. Adenovirus-mediated overexpression of MTP will provide a useful tool to gain further insights in the role of MTP in the secretion of apoB-containing lipoproteins in various experimental settings. We would like to thank Robert Hughes, Anna Lillethun, and Anthony Secreto for excellent technical assistance, Dorothea Maddox for assistance in preparing the manuscript, and Dr. Jane Glick and Dr. Stephen Young for helpful discussions. MMH is an Established Investigator of the American Heart Association. This work was partially supported by NIH grant HL57811 and a national grant-in-aid from the American Heart Association (DJR), and NIH grant DK-46900 and a grant-in-aid from the American Heart Association (MMH). UJFT is a recipient of a research fellowship from the Deutsche Forschungsgemeinschaft (DFG). CM is a recipient of a research fellowship from ARCOL (France). apolipoprotein B very low density lipoprotein low density lipoprotein microsomal triglyceride transfer protein endoplasmic reticulum triglycerides fast protein liquid chromatography.

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