Hepatic secretion of apoB-100 is impaired in hypobetalipoproteinemic mice with an apoB-38.9-specifying allele
2004; Elsevier BV; Volume: 45; Issue: 1 Linguagem: Inglês
10.1194/jlr.m300275-jlr200
ISSN1539-7262
AutoresZhouji Chen, Robin L. Fitzgerald, Gang Li, Nicholas O. Davidson, Gustav Schonfeld,
Tópico(s)Lipid metabolism and disorders
ResumoApolipoprotein B (apoB) truncation-specifying mutations cause familial hypobetalipoproteinemia (FHBL). Lipoprotein kinetics studies have shown that production rates of apoB-100 are reduced by 70–80% in heterozygous FHBL humans, instead of the expected 50%. To develop suitable mouse models to study the underlying mechanism, apoB-38.9-only (Apob38.9/38.9) mice were crossbred with Apobec-1 knockout (Apobec-1−/−) mice or apoB-100-only (Apob100/100) mice to produce two lines of apoB-38.9 heterozygous mice that produce only apoB-38.9 and apoB-100, namely Apobec-1−/−/Apob38.9/+ and Apob38.9/100 mice. In vivo rates of apoB-100 secretion were measured using [35S]Met/Cys to label proteins and Triton WR-1339 to block apoB-100 VLDL lipolysis/uptake. Rates of secretion were reduced by 80%, rather than the expected 50%, in both Apobec-1−/−/Apob38.9/+ and Apob38.9/100 mice compared with those of the respective Apobec-1−/−/Apob+/+ and Apob100/100 control mice. Continuous labeling and pulse-chase experiments in primary hepatocyte cultures revealed that rates of apoB-100 synthesis by Apobec-1−/−/Apob38.9/+ and Apob38.9/100 hepatocytes were reduced to the expected 50% of those of the respective controls, but the efficiency of secretion of apoB-100 was significantly lower in apoB-38.9 heterozygous hepatocytes.The greater-than-expected decreases in apoB-100 production rates of FHBL heterozygous humans appear to be attributable to a defect in secretion rather than in the synthesis of apoB-100 from the unaffected apoB allele. Apolipoprotein B (apoB) truncation-specifying mutations cause familial hypobetalipoproteinemia (FHBL). Lipoprotein kinetics studies have shown that production rates of apoB-100 are reduced by 70–80% in heterozygous FHBL humans, instead of the expected 50%. To develop suitable mouse models to study the underlying mechanism, apoB-38.9-only (Apob38.9/38.9) mice were crossbred with Apobec-1 knockout (Apobec-1−/−) mice or apoB-100-only (Apob100/100) mice to produce two lines of apoB-38.9 heterozygous mice that produce only apoB-38.9 and apoB-100, namely Apobec-1−/−/Apob38.9/+ and Apob38.9/100 mice. In vivo rates of apoB-100 secretion were measured using [35S]Met/Cys to label proteins and Triton WR-1339 to block apoB-100 VLDL lipolysis/uptake. Rates of secretion were reduced by 80%, rather than the expected 50%, in both Apobec-1−/−/Apob38.9/+ and Apob38.9/100 mice compared with those of the respective Apobec-1−/−/Apob+/+ and Apob100/100 control mice. Continuous labeling and pulse-chase experiments in primary hepatocyte cultures revealed that rates of apoB-100 synthesis by Apobec-1−/−/Apob38.9/+ and Apob38.9/100 hepatocytes were reduced to the expected 50% of those of the respective controls, but the efficiency of secretion of apoB-100 was significantly lower in apoB-38.9 heterozygous hepatocytes. The greater-than-expected decreases in apoB-100 production rates of FHBL heterozygous humans appear to be attributable to a defect in secretion rather than in the synthesis of apoB-100 from the unaffected apoB allele. Mutations in the apolipoprotein B (apoB) gene (Apob) that lead to carboxyl-terminal truncation of apoB-100 cause familial hypobetalipoproteinemia (FHBL) in humans (1Schonfeld G. The hypobetalipoproteinemias.Annu. Rev. Nutr. 1995; 15: 23-34Crossref PubMed Scopus (78) Google Scholar, 2Wu J. Kim J. Li Q. Kwok P-Y. Cole T.G. Cefalu B. Averna M. Schonfeld G. Known mutations of apoB account for only a small minority of hypobetalipoproteinemia.J. Lipid Res. 1999; 40: 955-959Abstract Full Text Full Text PDF PubMed Google Scholar, 3Linton M.F. Farese Jr., R.V. Young S.G. Familial hypobetalipoproteinemia.J. Lipid Res. 1993; 34: 521-541Abstract Full Text PDF PubMed Google Scholar), an autosomal codominant disorder characterized by low levels (<5th percentile) of plasma apoB and LDL cholesterol (1Schonfeld G. The hypobetalipoproteinemias.Annu. Rev. Nutr. 1995; 15: 23-34Crossref PubMed Scopus (78) Google Scholar, 2Wu J. Kim J. Li Q. Kwok P-Y. Cole T.G. Cefalu B. Averna M. Schonfeld G. Known mutations of apoB account for only a small minority of hypobetalipoproteinemia.J. Lipid Res. 1999; 40: 955-959Abstract Full Text Full Text PDF PubMed Google Scholar, 3Linton M.F. Farese Jr., R.V. Young S.G. Familial hypobetalipoproteinemia.J. Lipid Res. 1993; 34: 521-541Abstract Full Text PDF PubMed Google Scholar). In these subjects, the truncated apoB variants usually circulate in the plasma at very low levels as a result of their enhanced catabolism (4Parhofer K.G. Barrett P.H. Bier D.M. Schonfeld G. Lipoproteins containing the truncated apolipoprotein, Apo B-89, are cleared from human plasma more rapidly than Apo B-100-containing lipoproteins in vivo.J. Clin. Invest. 1992; 89: 1931-1937Crossref PubMed Scopus (61) Google Scholar, 5Krul E.S. Parhofer K.G. Barrett P.H.R. Wagner R.D. Schonfeld G. ApoB-75, a truncation of apolipoprotein B associated with familial hypobetalipoproteinemia: genetic and kinetic studies.J. Lipid Res. 1992; 33: 1037-1050Abstract Full Text PDF PubMed Google Scholar, 6Zhu X. Noto D. Seip R. Shaish A. Schonfeld G. Organ loci of catabolism of short truncations of ApoB.Arterioscler. Thromb. Vasc. Biol. 1997; 17: 1032-1038Crossref PubMed Scopus (23) Google Scholar, 7Chen Z. Saffitz J.E. Latour M.A. Schonfeld G. Truncated apo B-70.5-containing lipoproteins bind to megalin but not the LDL receptor.J. Clin. Invest. 1999; 103: 1419-1430Crossref PubMed Scopus (28) Google Scholar) and/or reduced secretion rates (8Parhofer K.G. Barrett P.H. Aguilar-Salinas C.A. Schonfeld G. Positive linear correlation between the length of truncated apolipoprotein B and its secretion rate: in vivo studies in human apoB-89, apoB-75, apoB-54.8, and apoB-31 heterozygotes.J. Lipid Res. 1996; 37: 844-852Abstract Full Text PDF PubMed Google Scholar, 9Welty F.K. Lichtenstein A.H. Barrett P.H. Dolnikowski G.G. Ordovas J.M. Schaefer E.J. Production of apolipoprotein B-67 in apolipoprotein B-67/B-100 heterozygotes: technical problems associated with leucine contamination in stable isotope studies.J. Lipid Res. 1997; 38: 1535-1543Abstract Full Text PDF PubMed Google Scholar). One might expect humans heterozygous for FHBL attributable to apoB truncation-specifying mutations to have half-normal plasma levels of apoB-100, because they possess one normal apoB-100 allele. However, this is not usually the case. Instead, the typical plasma concentrations of apoB-100 are only 20–30% of normal (1Schonfeld G. The hypobetalipoproteinemias.Annu. Rev. Nutr. 1995; 15: 23-34Crossref PubMed Scopus (78) Google Scholar, 3Linton M.F. Farese Jr., R.V. Young S.G. Familial hypobetalipoproteinemia.J. Lipid Res. 1993; 34: 521-541Abstract Full Text PDF PubMed Google Scholar). Likewise, lipoprotein kinetic studies of heterozygous FHBL humans indicate that production rates of apoB-100, the sole protein product of the unaffected apoB allele in human livers, are reduced by 70–80%, instead of the expected 50% (10Aguilar-Salina C.A. Barrett P.H.R. Parhofer K.G. Young S.G. Tessereau D. Bateman J. Quinn C. Schonfeld G. Apoprotein B-100 production is decreased in subjects heterozygous for truncations in apoprotein B.Arterioscler. Thromb. Vasc. Biol. 1995; 15: 71-80Crossref PubMed Scopus (67) Google Scholar, 11Welty F.K. Lichtenstein A.H. Barret P.H.R. Dolnikowski G.G. Ordovas J.M. Schaefer E.J. Decreased production and increased catabolism of apolipoprotein B-100 in apolipoprotein B-67/B-100 heterozygotes.Arterioscler. Thromb. Vasc. Biol. 1997; 17: 881-888Crossref PubMed Scopus (44) Google Scholar, 12Elias N. Patterson B.W. Schonfeld G. Decreased production rates of VLDL triglycerides and apoB-100 in subjects heterozygous for familial hypobetalipoproteinemia.Arterioscler. Thromb. Vasc. Biol. 1999; 19: 2714-2721Crossref PubMed Scopus (60) Google Scholar). Thus, the presence of a premature-termination codon-specifying mutation in one apoB allele may unexpectedly impair the synthesis and/or secretion of apoB-100, the gene product of the unaffected apoB-100 allele. FHBL patients are usually asymptomatic (1Schonfeld G. The hypobetalipoproteinemias.Annu. Rev. Nutr. 1995; 15: 23-34Crossref PubMed Scopus (78) Google Scholar, 2Wu J. Kim J. Li Q. Kwok P-Y. Cole T.G. Cefalu B. Averna M. Schonfeld G. Known mutations of apoB account for only a small minority of hypobetalipoproteinemia.J. Lipid Res. 1999; 40: 955-959Abstract Full Text Full Text PDF PubMed Google Scholar, 3Linton M.F. Farese Jr., R.V. Young S.G. Familial hypobetalipoproteinemia.J. Lipid Res. 1993; 34: 521-541Abstract Full Text PDF PubMed Google Scholar), posing ethical barriers to obtaining liver biopsies for direct biochemical studies to determine rates of apoB-100 synthesis and secretion.In recent years, we (13Chen Z. Fitzgerald R.L. Averna M.R. Schonfeld G. A targeted apolipoprotein B-38.9-producing mutation causes fatty livers in mice due to the reduced ability of apolipoprotein B-38.9 to transport triglycerides.J. Biol. Chem. 2000; 275: 32807-32815Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar, 14Chen Z. Fitzgerald R.L. Schonfeld G. Hypobetalipoproteinemic mice with a targeted apolipoprotein (Apo) B-27.6-specifying mutation. In vivo evidence for an important role of amino acids 1254–1774 of apoB in lipid transport and metabolism of the apoB-containing lipoproteins.J. Biol. Chem. 2002; 277: 14135-14145Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar) and other investigators (15Kim E. Cham C.M. Veniant M.M. Ambroziak P. Young S.G. Dual mechanisms for the low plasma levels of truncated apolipoprotein B proteins in familial hypobetalipoproteinemia. Analysis of a new mouse model with a nonsense mutation in the Apob gene.J. Clin. Invest. 1998; 101: 1468-1477Crossref PubMed Google Scholar, 16Kim E. Ambroziak P. Véniant M.M. Hamilton R.L. Young S.G. A gene-targeted mouse model for familial hypobetalipoproteinemia. Low levels of apolipoprotein B mRNA in association with a nonsense mutation in exon 26 of the apolipoprotein B gene.J. Biol. Chem. 1998; 273: 33977-33984Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar, 17Homanics G.E. Smith T.J. Zhang S.H. Lee D. Young S.G. Maeda N. Targeted modification of the apolipoprotein B gene results in hypobetalipoproteinemia and developmental abnormalities in mice.Proc. Natl. Acad. Sci. USA. 1993; 90: 2389-2393Crossref PubMed Scopus (110) Google Scholar) have developed several lines of Apob-modified mice to model the FHBL condition. These mice provided new insights into the FHBL syndrome and the structure-function relationship of apoB-100 (13Chen Z. Fitzgerald R.L. Averna M.R. Schonfeld G. A targeted apolipoprotein B-38.9-producing mutation causes fatty livers in mice due to the reduced ability of apolipoprotein B-38.9 to transport triglycerides.J. Biol. Chem. 2000; 275: 32807-32815Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar, 14Chen Z. Fitzgerald R.L. Schonfeld G. Hypobetalipoproteinemic mice with a targeted apolipoprotein (Apo) B-27.6-specifying mutation. In vivo evidence for an important role of amino acids 1254–1774 of apoB in lipid transport and metabolism of the apoB-containing lipoproteins.J. Biol. Chem. 2002; 277: 14135-14145Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar, 15Kim E. Cham C.M. Veniant M.M. Ambroziak P. Young S.G. Dual mechanisms for the low plasma levels of truncated apolipoprotein B proteins in familial hypobetalipoproteinemia. Analysis of a new mouse model with a nonsense mutation in the Apob gene.J. Clin. Invest. 1998; 101: 1468-1477Crossref PubMed Google Scholar, 16Kim E. Ambroziak P. Véniant M.M. Hamilton R.L. Young S.G. A gene-targeted mouse model for familial hypobetalipoproteinemia. Low levels of apolipoprotein B mRNA in association with a nonsense mutation in exon 26 of the apolipoprotein B gene.J. Biol. Chem. 1998; 273: 33977-33984Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar). However, apoB-48, instead of apoB-100, is the major protein product of the normal mouse apoB allele as a result of the presence of apoB mRNA-editing machinery in the mouse liver (18Davidson N.O. Shelness G.S. Apolipoprotein B: mRNA editing, lipoprotein assembly, and presecretory degradation.Annu. Rev. Nutr. 2000; 20: 169-193Crossref PubMed Scopus (231) Google Scholar, 19Greeve J. Altkemper I. Diesterich J.H. Greten H. Windler E. Apolipoprotein B mRNA editing in 12 different mammalian species: hepatic expression is reflected in low concentrations of apoB-containing plasma lipoproteins.J. Lipid Res. 1993; 34: 1367-1383Abstract Full Text PDF PubMed Google Scholar). Thus, the currently available FHBL mouse models are not suitable for studying the effect of apoB-truncation mutations on the synthesis and secretion of apoB-100 from the normal apoB allele in the liver.In this study, we bred our previously generated apoB-38.9-only mice (Apob38.9/38.9) (13Chen Z. Fitzgerald R.L. Averna M.R. Schonfeld G. A targeted apolipoprotein B-38.9-producing mutation causes fatty livers in mice due to the reduced ability of apolipoprotein B-38.9 to transport triglycerides.J. Biol. Chem. 2000; 275: 32807-32815Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar) with apoB mRNA-editing catalytic enzyme-1 (Apobec-1) knockout mice (Apobec-1−/−) (20Hirano K-I. Young S.G. Farese Jr., R.V. Ng J. Sande E. Warburton C. Powell-Braxton L-M. Davidson N.O. Targeted disruption of the mouse apobec-1 gene abolishes apolipoprotein B mRNA editing and eliminates apolipoprotein B48.J. Biol. Chem. 1996; 271: 9887-9890Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar) and with apoB-100-only mice (Apob100/100) (21Farese Jr., R.V. Véniant M.M. Cham C.M. Flynn L.M. Pierotti V. Loring J.F. Traber M. Ruland S. Stokowski R.S. Huszar D. Young S.G. Phenotypic analysis of mice expressing exclusively apolipoprotein B48 or apolipoprotein B100.Proc. Natl. Acad. Sci. USA. 1996; 93: 6393-6398Crossref PubMed Scopus (114) Google Scholar) to generate two different lines of apoB-38.9 heterozygous mice in which only apoB-38.9 and apoB-100 are produced by the liver. These mice provided us an opportunity to determine directly the effects of the apoB-38.9 mutation on the synthesis and secretion of apoB-100 and to explore the underlying mechanisms at the cellular level.METHODSMaterials[35S]Promix (530 MBq/ml), an l-[35S]Met and l-[35S]Cys metabolic labeling solution, was from Amersham Bioscience Corp. (Piscataway, NJ). Triton WR-1339, rabbit anti-human albumin antisera, N-acetyl-l-leucyl-l-leucyl-l-norleucinal (ALLN), (2S,3S)-trans-epoxysuccinyl-l-leucylamido-3-methylbutane ethyl ester (EST), and heparin (from porcine intestinal mucosa) were from Sigma Chemical Co. (St. Louis, MO). Glutathione-S-transferase (GST)-LDL receptor-associated protein (RAP) fusion protein (GST-RAP) was produced and purified as described (7Chen Z. Saffitz J.E. Latour M.A. Schonfeld G. Truncated apo B-70.5-containing lipoproteins bind to megalin but not the LDL receptor.J. Clin. Invest. 1999; 103: 1419-1430Crossref PubMed Scopus (28) Google Scholar).MiceThe generation of the apoB-38.9-producing mouse has been described (13Chen Z. Fitzgerald R.L. Averna M.R. Schonfeld G. A targeted apolipoprotein B-38.9-producing mutation causes fatty livers in mice due to the reduced ability of apolipoprotein B-38.9 to transport triglycerides.J. Biol. Chem. 2000; 275: 32807-32815Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). This mouse carries a single nucleotide deletion in its Apob, leading to the formation of a premature stop codon at residue 1,767. The apoB-100-only mouse was purchased from Jackson Laboratories (Bar Harbor, ME). This mouse was generated by Farese et al. (21Farese Jr., R.V. Véniant M.M. Cham C.M. Flynn L.M. Pierotti V. Loring J.F. Traber M. Ruland S. Stokowski R.S. Huszar D. Young S.G. Phenotypic analysis of mice expressing exclusively apolipoprotein B48 or apolipoprotein B100.Proc. Natl. Acad. Sci. USA. 1996; 93: 6393-6398Crossref PubMed Scopus (114) Google Scholar). It carries a targeted missense mutation at codon 2,153 that prevents the formation of apoB-48 resulting from apoB mRNA editing. Both lines of mice have a mixed genetic background, with 50% C57BL/6 and 50% 129/SvJ (13Chen Z. Fitzgerald R.L. Averna M.R. Schonfeld G. A targeted apolipoprotein B-38.9-producing mutation causes fatty livers in mice due to the reduced ability of apolipoprotein B-38.9 to transport triglycerides.J. Biol. Chem. 2000; 275: 32807-32815Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar, 21Farese Jr., R.V. Véniant M.M. Cham C.M. Flynn L.M. Pierotti V. Loring J.F. Traber M. Ruland S. Stokowski R.S. Huszar D. Young S.G. Phenotypic analysis of mice expressing exclusively apolipoprotein B48 or apolipoprotein B100.Proc. Natl. Acad. Sci. USA. 1996; 93: 6393-6398Crossref PubMed Scopus (114) Google Scholar). The mice doubly heterozygous for the apoB-38.9- and apoB-100-specifying alleles (Apob100/38.9) were generated by crossbreeding of Apob38.9/38.9 and Apob100/100 mice. The Apob100/38.9 and Apob 100/100 mice obtained from offspring of the intercross of Apob100/38.9 mice were used in this study. All of these offspring had a mixed genetic background, with 50% C57BL/6 and 50% 129/SvJ. The Apob100/38.9 mice were also crossed with LDL-receptor knockout (Ldlr−/−) and apoE knockout (Apoe−/−) mice (Jackson Laboratories) to produce Apob100/100/Ldlr−/−, Apob100/38.9/Ldlr−/−, Apob100/100/Apoe−/−, and Apob100/38.9/Apoe−/− mice. The Ldlr−/− and Apoe−/− mice had a C57BL/6 genetic background; thus, the resulting Apob100/100/Ldlr−/−, Apob100/38.9/Ldlr−/−, Apob100/100/Apoe−/−, and Apob100/38.9/Apoe−/− mice were predicted to have a mixed genetic background, with 75% C57BL/6 and 25% 129/SvJ.The Apobec-1−/− mouse was generated by Hirano et al. (20Hirano K-I. Young S.G. Farese Jr., R.V. Ng J. Sande E. Warburton C. Powell-Braxton L-M. Davidson N.O. Targeted disruption of the mouse apobec-1 gene abolishes apolipoprotein B mRNA editing and eliminates apolipoprotein B48.J. Biol. Chem. 1996; 271: 9887-9890Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar). It has a C57BL/6 genetic background. Thus, offspring (Apobec-1−/+/Apob38.9/+) produced from crossbreeding of Apob38.9/38.9 with Apobec-1−/− mice had a mixed genetic background, with 75% C57BL/6 and 25% 129/SvJ. Apobec-1−/−/Apob+/+ and Apobec-1−/−/Apob38.9/+ mice generated from intercrossing of Apobec-1−/+/Apob38.9/+ mice were used for this study.Only offspring from intercross breeding were used in this study, and in most cases, littermates were used to minimize variation in the strain percentage.All mice were weaned at 3 weeks of age, housed in a specific-pathogen-free barrier facility with a 12 h light/dark cycle, and fed a regular mouse chow diet (Ralston Purina, St. Louis, MO).Fast-protein liquid chromatography fractionation and Western blot analysis of plasma apoBFor Western blot analysis, mouse plasma was subjected to electrophoresis on 3–12% gradient SDS-PAGE gels under reducing conditions and electrotransferred onto Immobilon-P (Millipore Corp., Bedford, MA). Western blot analyses were carried out using rabbit antisera raised against a GST fusion protein containing amino acids 26 to 289 of mouse apoB (13Chen Z. Fitzgerald R.L. Averna M.R. Schonfeld G. A targeted apolipoprotein B-38.9-producing mutation causes fatty livers in mice due to the reduced ability of apolipoprotein B-38.9 to transport triglycerides.J. Biol. Chem. 2000; 275: 32807-32815Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar, 14Chen Z. Fitzgerald R.L. Schonfeld G. Hypobetalipoproteinemic mice with a targeted apolipoprotein (Apo) B-27.6-specifying mutation. In vivo evidence for an important role of amino acids 1254–1774 of apoB in lipid transport and metabolism of the apoB-containing lipoproteins.J. Biol. Chem. 2002; 277: 14135-14145Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar) and an enhanced chemiluminescence (ECL) Western blot detection kit (Amersham Pharmacia Biotech, Inc.). The ECL signals were quantified by analyzing the density of the protein bands on Kodak X-Omat film using SigmaGel computer software (SPPS Science Corp., Chicago, IL).Fast-protein liquid chromatography (FPLC) fractionation of plasma lipoproteins was carried out as described (13Chen Z. Fitzgerald R.L. Averna M.R. Schonfeld G. A targeted apolipoprotein B-38.9-producing mutation causes fatty livers in mice due to the reduced ability of apolipoprotein B-38.9 to transport triglycerides.J. Biol. Chem. 2000; 275: 32807-32815Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar, 14Chen Z. Fitzgerald R.L. Schonfeld G. Hypobetalipoproteinemic mice with a targeted apolipoprotein (Apo) B-27.6-specifying mutation. In vivo evidence for an important role of amino acids 1254–1774 of apoB in lipid transport and metabolism of the apoB-containing lipoproteins.J. Biol. Chem. 2002; 277: 14135-14145Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar) using 200 μl of plasma obtained from Apob100/100/Ldlr−/− or Apob100/38.9/Ldlr−/− mice. Cholesterol contents in each fraction were determined enzymatically, and apoB contents were determined by Western blot analysis.In vivo studyTo determine the in vivo secretion rates of apoB and triglycerides, mice (12 weeks old) were fasted for 4 h and administered Triton WR-1339 (Sigma) (500 mg/kg body weight) and [35S]Promix (300 μCi/g body weight) via intravenous injection. Blood samples (∼80 μl each) were taken using heparinized capillary tubes at 0 h and at 0.5, 1, 2, and 3 h thereafter. VLDL was isolated from 20 μl of plasma in 200 μl of EDTA-saline-KBr (d = 1.009) using 250 μl Polyallomer Centrifuge tubes (Beckman) and a 42.2 Ti rotor (Beckman). Aliquots of VLDL and infranatant were electrophoresed on SDS-PAGE gels. 35S activity in apoB-100 of VLDL samples and albumin of infranatant were quantified on a GS-525 PhosphorImager system using a low β-screen (Bio-Rad). The relative amount of [35S]apoB-100 in each plasma sample was expressed as the ratio of [35S]apoB-100 to [35S]albumin.Triglyceride concentrations in the plasma samples were also determined. The values obtained at the 0 time point were subtracted from the values obtained at later times.Hepatocyte culture studyPrimary hepatocytes were isolated from mouse liver by perfusion of a collagenase solution via the portal vein (13Chen Z. Fitzgerald R.L. Averna M.R. Schonfeld G. A targeted apolipoprotein B-38.9-producing mutation causes fatty livers in mice due to the reduced ability of apolipoprotein B-38.9 to transport triglycerides.J. Biol. Chem. 2000; 275: 32807-32815Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar, 14Chen Z. Fitzgerald R.L. Schonfeld G. Hypobetalipoproteinemic mice with a targeted apolipoprotein (Apo) B-27.6-specifying mutation. In vivo evidence for an important role of amino acids 1254–1774 of apoB in lipid transport and metabolism of the apoB-containing lipoproteins.J. Biol. Chem. 2002; 277: 14135-14145Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar). Viability of the cell was ∼80% as determined by Trypan exclusion. Cells were plated onto six-well plates (0.6 × 106 cells/well) coated with poly-d-lysine (Sigma) and incubated at 37°C under 5% CO2 in 10% FBS/DMEM. After 1 h of attachment, cell monolayers were washed twice and incubated in 10% FBS/DMEM until use. All experiments involving cultured hepatocytes were commenced at 7–8 h after the cells were cultured. After this initial culture period, cells were washed three times with PBS and incubated in Met- and Cys-free DMEM for 30 min to deplete the cellular pool of Met and Cys. Thereafter, the medium was replaced with 1 ml of Met- and Cys-free DMEM containing 200 μCi of [35S]Promix with or without oleic acid (OA) (0.5 mM; to give anOA/BSA ratio of 3.6) (14Chen Z. Fitzgerald R.L. Schonfeld G. Hypobetalipoproteinemic mice with a targeted apolipoprotein (Apo) B-27.6-specifying mutation. In vivo evidence for an important role of amino acids 1254–1774 of apoB in lipid transport and metabolism of the apoB-containing lipoproteins.J. Biol. Chem. 2002; 277: 14135-14145Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar), and cells were labeled for the specified time period to determine the rates of apoB secretion.For pulse-chase experiments, the cells were pulsed with [35S]Promix for 45 min as described above. Thereafter, they were washed twice with PBS and incubated in 1 ml of DMEM containing 10 mM Met and 3 mM Cys for the specified time period with or without the specified additives.Immunoprecipitation and quantification of 35S-labeled apoBImmunoprecipitation of the labeled apoB in cell lysate and cultured media was carried out using rabbit anti-mouse apoB antisera as described (13Chen Z. Fitzgerald R.L. Averna M.R. Schonfeld G. A targeted apolipoprotein B-38.9-producing mutation causes fatty livers in mice due to the reduced ability of apolipoprotein B-38.9 to transport triglycerides.J. Biol. Chem. 2000; 275: 32807-32815Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar, 14Chen Z. Fitzgerald R.L. Schonfeld G. Hypobetalipoproteinemic mice with a targeted apolipoprotein (Apo) B-27.6-specifying mutation. In vivo evidence for an important role of amino acids 1254–1774 of apoB in lipid transport and metabolism of the apoB-containing lipoproteins.J. Biol. Chem. 2002; 277: 14135-14145Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar). Rabbit anti-human albumin antisera (Sigma) were also included in the immunoprecipitation mixture to immunoprecipitate mouse albumin simultaneously. The immunoprecipitated proteins (apoB and albumin) were resolved on a 3–12% gradient gel under reducing conditions. The gels were dried, and the radioactivity associated with apoB and albumin was quantified on a GS-525 PhosphorImager system using a low β-screen (Bio-Rad). Our initial studies found no difference in the rates of synthesis and secretion of albumin between the Apobec-1−/−/Apob+/+ and Apobec-1−/−/Apob+/38.9 and between the Apob100/100 and Apob100/38.9 hepatocytes. Thus, the 35S-labeled albumin signal was used to correct the [35S]apoB-100 signal in each lane.Miscellaneous proceduresLiver triglyceride contents were determined as described (13Chen Z. Fitzgerald R.L. Averna M.R. Schonfeld G. A targeted apolipoprotein B-38.9-producing mutation causes fatty livers in mice due to the reduced ability of apolipoprotein B-38.9 to transport triglycerides.J. Biol. Chem. 2000; 275: 32807-32815Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar, 14Chen Z. Fitzgerald R.L. Schonfeld G. Hypobetalipoproteinemic mice with a targeted apolipoprotein (Apo) B-27.6-specifying mutation. In vivo evidence for an important role of amino acids 1254–1774 of apoB in lipid transport and metabolism of the apoB-containing lipoproteins.J. Biol. Chem. 2002; 277: 14135-14145Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar). Cellular protein contents were determined using a modified Lowry method (22Markwell M.A. Hass S.M. Bieber L.L. Tolbert N.E. A modification of the Lowry procedure to simplify protein determination in membrane and lipoprotein samples.Anal. Biochem. 1978; 87: 206-210Crossref PubMed Scopus (5290) Google Scholar). Plasma triglyceride concentrations were determined using an enzymatic kit (WAKO Chemicals USA, Inc., Richmond, VA). Student's t-test and ANOVA analysis were performed to determine the significance of the differences.RESULTSLow plasma apoB-100 in Apobec-1−/−/Apob+/38.9 and Apob100/38.9 miceAs expected, apoB-100 was the sole apoB in Apobec-1−/−/Apob+/+ and Apob100/100 mouse plasma, whereas both apoB-100 and apoB-38.9 were present in Apobec-1−/−/Apob+/38.9 and Apob100/38.9 mouse plasma (Fig. 1). The Apobec-1−/−/Apob+/+, Apobec-1−/−/Apob+/38.9, Apob100/100, and Apob100/38.9 mice all appeared healthy and gained weight at normal rates.Compared with those of their respective Apobec-1−/−/Apob+/+ and Apob100/100 littermates, plasma apoB-100 levels of the Apobec-1−/−/Apob+/38.9 and Apob100/38.9 mice were reduced by 70% (Fig. 1). In Apobec-1−/−/Apob+/38.9 and Apob100/38.9 mice, plasma apoB-38.9 concentrations were approximately one-sixth of those of apoB-100 (Fig. 1).To determine whether the low levels of plasma apoB-100 in Apobec-1−/−/Apob+/38.9 and Apob100/38.9 mice were caused by enhanced clearance or reduced secretion rates of apoB-100-containing lipoproteins, we crossed the Apob100/38.9 mice with Ldlr−/− and Apoe−/− mice, thereby blocking apoE-mediated plasma VLDL remnant and LDL receptor-mediated LDL catabolism, respectively. As shown in Fig. 2, apoB-100 levels in Apob100/38.9/Apoe−/− mice amounted to only 15–20% of those of Apob100/100/Apoe−/− mice (Fig. 2A), similar to the results comparing Apob100/38.9/Apoe+/+ to Apob100/100/Apoe+/+ mice. This suggests that apoE in the apoB-38.9 mice did not affect either the secretion of VLDL from the liver or its catabolism. Disruption of LDL receptor function yielded similar results (Fig. 2B). FPLC analysis showed that plasma LDL cholesterol concentrations in Apob100/38.9/Ldlr−/− mice were also ∼25% of those the Apob100/100/Ldlr−/− mice (Fig. 3). This suggested that LDL receptor-mediated uptake and catabolism of VLDL did not account for the apparent low rates of VLDL secretion in the apoB-38.9 mice. Together, these results provided physiological evidence indicating that low rates of hepatic apoB-100 VLDL secretion may be the major mechanism for the lower than expected levels of plasma apoB-100 in the Apobec-1−/−/Apob+/38.9 and Apob100/38.9 mice.Fig. 2Disruption of apoE (A) or the LDL receptor gene (B) did not diminish the larger than expected difference in plasma apoB-100 levels between Apob100/100 and Apob100/38.9 mice. Plasma samples were obtained from six fasted mice for each compound genotype and pooled. After dilution
Referência(s)