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• W2016286787 abstract "Conventional knockout of the microsomal triglyceride transfer protein large subunit (ℓMTP) gene is embryonic lethal in the homozygous state in mice. We have produced a conditional ℓMTP knockout mouse by inserting loxP sequences flanking exons 5 and 6 by gene targeting. Homozygous floxed mice were born live with normal plasma lipids. Intravenous injection of an adenovirus harboring Cre recombinase (AdCre1) produced deletion of exons 5 and 6 and disappearance of ℓMTP mRNA and immunoreactive protein in a liver-specific manner. There was also disappearance of plasma apolipoprotein (apo) B-100 and marked reduction in apoB-48 levels. Wild-type mice showed no response, and heterozygous mice, an intermediate response, to AdCre1. Wild-type mice doubled their plasma cholesterol level following a high cholesterol diet. This hypercholesterolemia was abolished in AdCre1-treated ℓMTP−/− mice, the result of a complete absence of very low/intermediate/low density lipoproteins and a slight reduction in high density lipoprotein. Heterozygous mice showed an intermediate lipoprotein phenotype. The rate of accumulation of plasma triglyceride following Triton WR1339 treatment in ℓMTP−/− mice was <10% that in wild-type animals, indicating a failure of triglyceride-rich lipoprotein production. Pulse-chase experiments using hepatocytes isolated from wild-type and ℓMTP−/− mice revealed a failure of apoB secretion in ℓMTP−/−animals. Therefore, the liver-specific inactivation of the ℓMTP gene completely abrogates apoB-100 and very low/intermediate/low density lipoprotein production. These conditional knockout mice are a usefulin vivo model for studying the role of MTP in apoB biosynthesis and the biogenesis of apoB-containing lipoproteins. Conventional knockout of the microsomal triglyceride transfer protein large subunit (ℓMTP) gene is embryonic lethal in the homozygous state in mice. We have produced a conditional ℓMTP knockout mouse by inserting loxP sequences flanking exons 5 and 6 by gene targeting. Homozygous floxed mice were born live with normal plasma lipids. Intravenous injection of an adenovirus harboring Cre recombinase (AdCre1) produced deletion of exons 5 and 6 and disappearance of ℓMTP mRNA and immunoreactive protein in a liver-specific manner. There was also disappearance of plasma apolipoprotein (apo) B-100 and marked reduction in apoB-48 levels. Wild-type mice showed no response, and heterozygous mice, an intermediate response, to AdCre1. Wild-type mice doubled their plasma cholesterol level following a high cholesterol diet. This hypercholesterolemia was abolished in AdCre1-treated ℓMTP−/− mice, the result of a complete absence of very low/intermediate/low density lipoproteins and a slight reduction in high density lipoprotein. Heterozygous mice showed an intermediate lipoprotein phenotype. The rate of accumulation of plasma triglyceride following Triton WR1339 treatment in ℓMTP−/− mice was <10% that in wild-type animals, indicating a failure of triglyceride-rich lipoprotein production. Pulse-chase experiments using hepatocytes isolated from wild-type and ℓMTP−/− mice revealed a failure of apoB secretion in ℓMTP−/−animals. Therefore, the liver-specific inactivation of the ℓMTP gene completely abrogates apoB-100 and very low/intermediate/low density lipoprotein production. These conditional knockout mice are a usefulin vivo model for studying the role of MTP in apoB biosynthesis and the biogenesis of apoB-containing lipoproteins. Abetalipoproteinemia is an autosomal recessive disorder characterized by the almost complete absence of circulating apolipoprotein (apo) 1The abbreviations used are: apo, apolipoprotein; MTP, microsomal triglyceride transfer protein; ℓMTP, MTP large subunit; VLDL, very low density lipoproteins; IDL, intermediate density lipoproteins; LDL, low density lipoproteins; HDL, high density lipoproteins; kb, kilobase(s); PAGE, polyacrylamide gel electrophoresis; FPLC, fast protein liquid chromatography; FIAU, 1-(2′-deoxy-2′-fluoro-β-d-arabinofuranosyl)-5-iodouracil. B-containing lipoproteins (1Kane J.P. Havel R.J. Scriver C.R. Beaudet A.L. Sly W.S. Valle D. The Metabolic and Molecular Bases of Inherited Disease. McGraw-Hill, Inc., New York1995: 1853-1885Google Scholar). In addition to the lipoprotein abnormalities, patients with abetalipoproteinemia also suffer from severe anemia with acanthocytosis, fat malabsorption, and progressive neurodegenerative syndromes. Abetalipoproteinemia is caused by mutations in the gene for the large subunit of the microsomal triglyceride transfer protein (MTP) (2Wetterau 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 (644) Google Scholar, 3Sharp D. Blinderman L. Combs K.A. Kienzie B. Ricci B. Wager-Smith K. Gil C.M. Turck C.W. Bouma M.-E. Rader D.J. Aggerbeck L.P. Gregg R.E. Gordon D.A. Wetterau J.R. Nature. 1993; 365: 65-69Crossref PubMed Scopus (408) Google Scholar, 4Shoulders C.C. Brett D.J. Bayliss J.D. Narcisi T.M.E. Jarmuz A. Grantham T.T. Leoni P.R.D. Bhattacharya S. Pease R.J. Cullen P.M. Levi S. Byfield P.G.H. Purkiss P. Scott J. Hum. Mol. Genet. 1993; 2: 2109-2116Crossref PubMed Scopus (228) Google Scholar, 5Narcisi T.M.E. Shoulders C.C. Chester S.A. Read J. Brett D.J. Harrison G.B. Grantham T.T. Fox M.F. Povey S. de Bruin T.W.A. Erkelens D.W. Muller D.P.R. Lloyd J.K. Scott J. Am. J. Hum. Genet. 1995; 57: 1298-1310PubMed Google Scholar). MTP is an integral protein in the endoplasmic reticulum. It was originally described as a protein that accelerates the transport of lipids (triglycerides, cholesteryl ester, and phosphatidylcholine) between synthetic membranes (6Wetterau J.R. Zilversmitt D.B. J. Biol. Chem. 1984; 259: 10863-10866Abstract Full Text PDF PubMed Google Scholar). MTP is a heterodimer composed of a 97-kDa subunit (designated ℓMTP) and a 58-kDa subunit, which turned out to be protein-disulfide isomerase (7Wetterau J.R. Combs K.A. Spinner S.N. Joiner B.J. J. Biol. Chem. 1990; 265: 9800-9807Abstract Full Text PDF PubMed Google Scholar). ApoB-100 and apoB-48 secretion from the cell requires the presence of functional MTP. In its absence, all the newly synthesized intracellular apoB is degraded, and little of it is secreted (8Leiper J.M. Bayliss J.D. Pease R.J. Brett D.J. Scott J. Shoulders C.C. J. Biol. Chem. 1994; 269: 21951-21954Abstract Full Text PDF PubMed Google Scholar, 9Gordon D.A. Jamil H. Sharp D. Mullaney D. Yao Z. Greeg R.E. Wetterau J. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 7628-7632Crossref PubMed Scopus (189) Google Scholar). It is believed that MTP facilitates the stabilization of newly synthesized apoB-100 (10Rustaeus S. Stillemark P. Lindberg K. Gordon D. Olofsson S.-O. J. Biol. Chem. 1998; 273: 5196-5203Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar) and apoB-48 (11Gordon D.A. Jamil H. Gregg R.E. Olofsson S.-O. Borén J. J. Biol. Chem. 1996; 271: 33047-33053Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar) in a narrow “window” shortly after completion of apoB translation but before the addition of the major amount of lipids to the lipoprotein particle (reviewed in Refs. 12Gordon D.A. Curr. Opin. Lipidol. 1997; 8: 131-137Crossref PubMed Scopus (81) Google Scholar, 13Yao Z. Tran K. McLeod R.S. J. Lipid Res. 1997; 38: 1937-1953Abstract Full Text PDF PubMed Google Scholar, 14Wetterau J.R. Lin M.C.M. Jamil H. Biochim. Biophys. Acta. 1997; 1345: 136-150Crossref PubMed Scopus (286) Google Scholar). The pathophysiological basis of abetalipoproteinemia is postulated to be an almost complete failure of apoB secretion from the liver and small intestine because of the absence of functional MTP in affected patients. An animal model would be valuable in studying the molecular basis of the lipoprotein abnormalities and elucidating the pathophysiological basis of the other non-lipoprotein complications found in abetalipoproteinemia. Unfortunately, the genetic inactivation of the ℓMTP locus in mice is embryonic lethal in the homozygous state ((15) and see below). In this communication, we report the production and characterization of a viable mouse model of abetalipoproteinemia using a conditional knockout strategy. A mouse 129 strain λ genomic library was purchased (Stratagene) and screened with mouse ℓMTP cDNA (16Nakamuta M. Chang B.H.J. Hoogeveen R. Li W.-H. Chan L. Genomics. 1996; 33: 313-316Crossref PubMed Scopus (21) Google Scholar). Two overlapping clones encompassing exons 3 to 8 were used to construct two types of targeting vectors. For straight replacement-type targeting construct, a neo cassette was inserted into exon 7 of the ℓMTP gene between a SmaI and aXhoI site (data available from L. C. upon request). The conditional targeting construct was designed by inserting a neo-loxP cassette in the XbaI site of intron 6 and a loxP fragment in the BamHI site of intron 4 of the ℓMTP (Fig.1 A). A thymidine kinase cassette was ligated to the 5′ end of the construct. An R1 ES cell line was obtained from Dr. Andras Nagy at the University of Toronto. The cells were expanded to passage 14 and used to generate knockout mice as described previously (17Nakamuta M. Chang B.H.J. Zsigmond E. Kobayashi K. Lei H. Ishida B.Y. Oka K. Li E. Chan L. J. Biol. Chem. 1996; 271: 25981-25988Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). Three positive ES cell clones were injected into blastocysts of C57BL/6J, and chimeric mice were obtained. They were mated with C57BL/6J mice, and germ line transmission was confirmed by Southern blot analysis. Most experiments were conducted on siblings of F2 or F3 mice. The mice were weaned at 21 days and fed either a chow diet (Teklad 7001) or a high fat cholesterol diet (ICN 960393) containing 1.23% cholesterol and 17.84% fat. Total RNA from the liver and small intestine were isolated using TriZOL (Life Technologies, Inc.). A polymerase chain reaction product containing the first 350 base pairs of the mouse ℓMTP cDNA were cloned into pBluescript KS vector and used as a probe for RNase protection assay. The authenticity of the clone was verified by sequencing, and the antisense strand RNA was transcribed by using MAXIscript (Ambion). The assay was done using the RPAII kit (Ambion) with 5 μg of total RNA following the instructions of the vendor's manual. A β-actin probe was used as an internal control in the assay. Liver and small intestine were removed, and total proteins were extracted by a Wheaton No. 6 hand-held homogenizer in buffer B (10 mm Hepes pH 7.4, 2.5 mm sodium phosphate monobasic, 250 mm sucrose, 5 mm EDTA, and 0.5 mm phenylmethylsulfonyl fluoride). Microsomal fractions were isolated from extracts of liver and small intestine by ultracentrifugation at 100,000 ×g for 1 h. The pellet was resuspended in buffer B, and the protein concentration was determined by a DC Protein Assay Kit from Bio-Rad. Ten μg of microsomal protein was loaded onto a 4–15% gradient polyacrylamide gel, and a rabbit anti-bovine ℓMTP antiserum (a gift from Dr. David Gordon of Bristol-Myers Squibb Pharmaceutical Research Institute) was used for Western blot analysis. One hundred μg of microsomal protein was used to measure the MTP-mediated [14C]triglyceride transfer activity as described in Jamilet al. (18Jamil H. Dickson Jr., J.K. Chu C.-H. Lago M.W. Rinehart J.K. Biller S.A. Gregg R.E. Wetterau J.R. J. Biol. Chem. 1995; 270: 6549-6554Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar). Blood was collected after a 4–5-h fast, and total plasma cholesterol and triglyceride concentrations were measured by enzymatic kits (Sigma Diagnostics). Two hundred μl of pooled plasma from 3–4 animals was loaded on a FPLC system with 2 Superose 6 columns connected in series (Pharmacia FPLC System, Amersham Pharmacia Biotech). 0.5-ml fractions were collected using an elution buffer (1 mm EDTA, 154 mmNaCl, and 0.02% NaN3, pH 8.2) (19Cole T. Kitchens R.T. Daugherty A. Schonfeld G. FLPC BioCommunique. 1988; 4: 4-6Google Scholar). Lipid contents in individual fractions were determined with enzymatic assay kits (Sigma Diagnostics). Very low density (VLDL), intermediate density (IDL), low density (LDL), and high density (HDL) lipoproteins are well separated by this technique (19Cole T. Kitchens R.T. Daugherty A. Schonfeld G. FLPC BioCommunique. 1988; 4: 4-6Google Scholar). A replication-defective adenovirus containing recombinant Cre recombinase, AdCre1, was a gift from Dr. Frank Graham (McMaster University, Hamilton, Ontario, Canada) (20Anton M. Graham F.L. J. Virol. 1995; 69: 4600-4606Crossref PubMed Google Scholar). It was amplified in 293 cells and purified as described previously (21Kobayashi K. Oka K. Forte T. Ishida B.Y. Teng B.-B. Ishimura-Oka K. Nakamuta M. Chan L. J. Biol. Chem. 1996; 271: 6851-6860Google Scholar). Eight-week-old male mice were injected with 3 × 109 plaque-forming units of AdCre1 through a jugular vein. The AdCre1-treated mice were fed either a normal chow or a high cholesterol diet before and after injection. At day 10–21 after adenovirus administration, the mice were sacrificed and studied. Hepatocytes were isolated from AdCre1-treated mice by White's method (22White A. Clin. Genet. 1997; 52: 326-337Crossref PubMed Scopus (11) Google Scholar) except that the perfusion was done on an anesthetized animal through the portal vein instead of on excised liver. A pulse-chase experiment using [35S]methionine was done to determine apoB degradation and secretion in these cells (23Yeung S.J. Chen S.-H. Chan L. Biochemistry. 1996; 35: 13843-13848Crossref PubMed Scopus (170) Google Scholar). Triglyceride secretion in vivo was quantified by the intravenous administration of Triton WR1339 (24Li X. Catalina F. Grundy S.M. Patel S. J. Lipid Res. 1996; 37: 210-220Abstract Full Text PDF PubMed Google Scholar). Plasma triglycerides were measured at 1, 2, 3, and 4 h after treatment; the triglyceride accumulation remained linear during this time. As reported by Raabe et al. (15Raabe M. Flynn L.M. Zlot C.H. Wong J.S. Véniant M.M. Hamilton R.L. Young S.G. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 8686-8691Crossref PubMed Scopus (223) Google Scholar), we produced ℓMTP knockout mice by gene targeting in ES cells and found that inactivation of the ℓMTP locus in mice is embryonic lethal in the homozygous state. 2B. H.-J. Chang, M. Nakamuta, and L. Chan, unpublished observations. We have therefore produced a conditional knockout construct shown in Fig.1 A. It would insert two loxP sequences encompassing exons 5 and 6. Deletion of these exons would be predicted to inactivate the ℓMTP protein because it causes a shift in the translation frame of the mRNA if the remaining exons are correctly spliced into a mutant mRNA. Homologous recombination was verified by digestion of genomic ES cell DNA with BamHI and Southern blot analysis using a probe outside the targeting vector (Fig.1 A). The presence of a diagnostic 4-kb fragment instead of the wild-type 6-kb fragment indicates insertion of the targeted vector by homologous recombination. 14 of 124 clones (11%) that were G418- and FIAU-resistant exhibited a pattern consistent with homologous recombination. 3 of 14 ES cell clones were injected into C57BL/6J blastocysts, yielding 8 chimeric mice with agouti coat color indicating essentially 100% contribution of ES cells. Chimeric males were bred with C57BL/6J females, and germ line transmission was observed in 5% (2/40) of the progeny by Southern blot analysis of tail DNA (Fig.1 B). Heterozygous and homozygous floxed ℓMTP−/− mice were obtained by cross-breeding. These animals were fertile and produced normal-sized litters. The birth weights, growth, and development of wild-type, ℓMTP+/−, and ℓMTP−/− mice were indistinguishable. The basal plasma cholesterol and triglyceride levels are similar in the three types of animals while they were on a regular chow diet (TableI). Therefore, insertion of the loxP sequences and the neo cassette did not appear to disrupt the function of ℓMTP under basal conditions. Two weeks after these animals were put on a high cholesterol diet, there was an approximate doubling of the plasma cholesterol but little change in plasma triglyceride concentrations. Again, the levels were not significantly different among the three groups of animals (Table I).Table IPlasma lipids in wild-type and floxed MTP knockout miceGenotypeBefore AdCre1 administration (normal chow)Before AdCre1 administration (high cholesterol diet for 2 weeks)3 weeks after AdCre1 administration (high cholesterol diet)TGaTG, triglyceride; CHOL, cholesterol.CHOLaTG, triglyceride; CHOL, cholesterol.TGCHOLTGCHOL+/+34.90 ± 1.3190.39 ± 5.0342.89 ± 3.76191.21 ± 11.5268.62 ± 7.23227.58 ± 12.48+/−35.41 ± 2.4895.37 ± 3.4543.32 ± 2.73207.22 ± 18.8471.52 ± 18.38180.69 ± 12.38bSignificant difference between +/+ and +/− (p < 0.05).−/−40.67 ± 3.5285.18 ± 13.4743.12 ± 4.61174.53 ± 11.9154.95 ± 7.20119.60 ± 23.97cSignificant difference between +/+ and −/− (p < 0.01).The number of mice studied before AdCre1 administration were 6 each from +/+ and +/− and 4 from −/−; after AdCre1 treatment there were 4 for each genotype.a TG, triglyceride; CHOL, cholesterol.b Significant difference between +/+ and +/− (p < 0.05).c Significant difference between +/+ and −/− (p < 0.01). Open table in a new tab The number of mice studied before AdCre1 administration were 6 each from +/+ and +/− and 4 from −/−; after AdCre1 treatment there were 4 for each genotype. We examined the effect of the introduction of Cre recombinase on the ℓMTP gene and its expression in wild-type and knockout animals. Induction of Cre recombinase expression was effected by the intravenous administration of a purified recombinant adenovirus carrying the Cre1 recombinase (AdCre1) (20Anton M. Graham F.L. J. Virol. 1995; 69: 4600-4606Crossref PubMed Google Scholar). We removed the liver and small intestine and extracted DNA, RNA, and microsomes from these tissues 1.5–3 weeks after AdCre1 treatment. By Southern blot analysis, we found that AdCre1 treatment produced a deletion of the floxed exons in the ℓMTP gene in the liver. By EcoRV digestion, hybridization using the probe shown produced a 6.5-kb band in the intact floxed ℓMTP gene (Fig.2). Upon deletion of the floxed portion of the gene, it produced a 3.5-kb band with the removal of the DNA between the loxP sequences, which has an EcoRV site in exon 5. When we examined the blot from DNA extracted from the small intestine, it was evident that the floxed ℓMTP locus remained intact. The liver specificity is not unexpected because adenoviral vectors administered intravenously are taken up preferentially by liver cells (25Chan L. Curr. Opin. Lipidol. 1995; 6: 335-340Crossref PubMed Scopus (16) Google Scholar). For heterozygous ℓMTP knockout mice, deletion of the floxed ℓMTP allele was also detected in the liver but not the small intestine (data not shown). We next examined liver and small intestine ℓMTP mRNA levels following AdCre1 administration. We used RNase protection assay with an ℓMTP cDNA fragment and a β-actin cDNA fragment as internal control. As shown in Fig. 3 A, the ℓMTP mRNA band is essentially undetectable in the liver of Cre-treated ℓMTP−/− mice, whereas the ℓMTP mRNA level in treated ℓMTP+/− mouse liver was only slightly reduced. In comparison, the concentration of ℓMTP mRNA in the small intestine of mice of all three genotypes and in the liver of wild-type mice was unaffected by AdCre1 administration. Therefore, there was good correlation between ℓMTP mRNA expression and the presence of an undisrupted ℓMTP gene, and Cre-induced deletion of exons 5 and 6 led to the absence of detectable ℓMTP mRNA. Since the antisense probe used corresponds to a region of the ℓMTP gene 5′ to the missing exons, these results suggest that in the AdCre1-treated mouse liver, if the disrupted ℓMTP gene were transcribed at all, the RNA transcript was so unstable and its steady-state concentration so low that it was undetectable by RNase protection. To examine whether these changes at the DNA and mRNA levels are reflected at the protein level, we isolated microsomes from the liver and small intestine and performed immunoblot analysis using an ℓMTP antibody. As shown in Fig. 3 B, a ℓMTP immunoreactive band was easily detectable in wild-type mouse liver and small intestine, being slightly more intense in the latter. Following AdCre1 treatment there was a reduction in intensity of the immunoreactive band in the liver, but not small intestine, of the heterozygous floxed ℓMTP+/− mice. In the homozygous floxed ℓMTP−/− mice, the Cre gene transfer to the liver completely eliminated the ℓMTP band. Interestingly, there seemed to be a concomitant increase in the intensity of the ℓMTP band in the small intestine following AdCre1 treatment. ℓMTP has a relatively long half-life (4.4 days in HepG2 cells (26Lin M.C.M. Gordon D. Wetterau J.R. J. Lipid Res. 1995; 36: 1073-1081Abstract Full Text PDF PubMed Google Scholar)). These results indicate that in mice, within 10 days of acute interruption of ℓMTP gene transcription, there is essentially no immunoreactive ℓMTP left in the liver cells. We determined the MTP activity in the microsomes isolated from AdCre1-treated wild-type, floxed heterozygous ℓMTP+/−, and floxed homozygous ℓMTP−/− animals. The results shown in Fig. 3 C reveal that the MTP triglyceride transfer activity of microsomes isolated from the liver of heterozygous floxed ℓMTP+/− (3.7%) mice is reduced to about half that of the wild-type (7.3%), and the activity of the homozygous floxed ℓMTP−/− mice is almost down to background level (1.3%). In contrast, the intestinal MTP triglyceride transfer activity in all three groups of animals is very similar (7.8%, 8.6%, and 7.7%, respectively, for wild-type, heterozygous, and homozygous knockout animals). Therefore, in the liver and small intestine there is good correlation between ℓMTP protein expression and MTP functional activity, which suggests that ℓMTP, and not protein-disulfide isomerase, is limiting under these conditions. The plasma lipid levels of the different types of mice before and after AdCre1 treatment are shown in Table I. Before adenovirus administration, the mice were put on a high cholesterol diet, and their plasma cholesterol and triglyceride levels were similar in the three groups of animals. Following AdCre1 injection, there was a significant reduction in the plasma cholesterol concentration in the homozygous ℓMTP−/− mice, which was significantly lower than that in wild-type animals. The cholesterol level in the heterozygous animals was intermediate between those of wild-type and homozygous knockout animals. The plasma triglyceride level was not statistically different between the three groups of animals, although it tended to be lower in the homozygous mice. Because functional ℓMTP is required for apoB biogenesis (12Gordon D.A. Curr. Opin. Lipidol. 1997; 8: 131-137Crossref PubMed Scopus (81) Google Scholar, 14Wetterau J.R. Lin M.C.M. Jamil H. Biochim. Biophys. Acta. 1997; 1345: 136-150Crossref PubMed Scopus (286) Google Scholar), we analyzed the plasma for apoB-100 and apoB-48 expression by immunoblot analysis. We took plasma from these mice 2 weeks following AdCre1 treatment. In animals that were on regular chow (Fig. 3 D,left panel), there was no difference in wild-type and heterozygous knockout animals; both had clearly detectable apoB-48, but barely detectable apoB-100. In homozygous knockout animals, apoB-100 was undetectable and apoB-48 was markedly reduced and barely detectable. In wild-type animals that were fed a high cholesterol diet (Fig. 3 D, right panel), plasma apoB-100 and apoB-48 were clearly detected on the blot, with apoB-48 being a much more intense band than the apoB-100 band. In comparison, the AdCre1-treated heterozygous floxed ℓMTP+/− mice had mildly reduced apoB-48 and markedly reduced apoB-100 bands. When we analyzed the plasma from AdCre1-treated homozygous floxed ℓMTP−/− mice, we found a marked reduction in the intensity of the apoB-48 band and an almost complete disappearance of the apoB-100 band. Therefore, specific inactivation of the ℓMTP locus in the liver leads to an essentially complete absence of circulating apoB-100 and a marked decrease in plasma apoB-48. These changes would be consistent with the annulment of apoB-100 and apoB-48 production by the liver with the preservation of apoB-48 production in the small intestine in the AdCre1-treated homozygous floxed ℓMTP−/− mice. We next analyzed the plasma lipoproteins in the different groups of mice by FPLC analysis (Fig. 4). Plasma was obtained from wild-type, heterozygous floxed ℓMTP+/−, and homozygous floxed ℓMTP−/−mice 10 to 21 days following AdCre1 treatment. In chow-fed animals, in comparison with wild-type, the ℓMTP−/− mice displayed a complete absence of the VLDL and IDL/LDL peaks (Fig. 4 A,top panel) and a markedly reduced HDL peak. In heterozygous animals, only minor reductions were observed in the VLDL and IDL/LDL regions, with no change in the HDL region compared with wild-type animals. Among animals that were fed a high cholesterol diet, wild-type mice developed a prominent VLDL peak, a substantial IDL/LDL, and a prominent HDL peak. In the Cre-treated homozygous floxed ℓMTP−/− mice, there was essentially a complete absence of detectable lipoproteins in the VLDL and IDL/LDL fractions. There was a slight reduction in the HDL peak. Therefore, the markedly reduced apoB-100 production resulting from the almost complete inhibition of MTP activity in the ℓMTP−/− mice was sufficient to abrogate a VLDL/IDL/LDL response to high cholesterol diet feeding. The heterozygous ℓMTP+/− mice, which had intermediate ℓMTP mass and MTP activity (Fig. 3), displayed an intermediate lipoprotein phenotype (Fig. 4 A, middle panel). The plasma lipoprotein triglyceride was entirely in the VLDL region (Fig.4 A, bottom panel) in wild-type animals. It was essentially abolished in ℓMTP−/−mice. To explore the mechanism of the marked VLDL deficiency in the ℓMTP−/− mice, we measured the rate of triglyceride production in these animals. The intravenous administration of Triton WR1339 inhibits the catabolism of triglyceride-rich lipoproteins. The accumulation of plasma triglyceride following Triton treatment reflects the triglyceride secretion rate from the liver and intestine. Because the animals were on a fat-free diet during this experiment, the secretion came exclusively from the liver. As shown in Fig.4 B, the triglyceride secretion rate in ℓMTP−/− mice (0.09 ± 0.04 mg/min/100 g) was reduced to less than one-tenth that in the wild-type controls (1.02 ± 0.19 mg/min/100 g). The rate in heterozygous knockout mice was intermediate (0.52 ± 0.07 mg/min/100 g). Thus, the absence of VLDL/IDL/LDL in the knockout animals was a result of failure of production, not increased catabolism. Pulse-chase experiments on cultured hepatocytes isolated from wild-type and homozygous knockout animals revealed that there was complete failure of secretion of apoB in ℓMTP−/− animals, which would account for the absence of VLDL production in these animals. Albumin production was normal (Fig. 4 C). In conclusion, we have produced a viable abetalipoproteinemia gene knockout mouse model using a Cre/loxP strategy. We found that the liver-specific disruption of the ℓMTP gene was sufficient to completely abrogate the plasma VLDL/LDL response to a high cholesterol diet. Because conventional knockout of the ℓMTP gene is embryonic lethal in the homozygous state, the conditional knockout mice will be a valuable model for studying the metabolic defect and pathophysiology of abetalipoproteinemia as well as the role of MTP in apoB biosynthesis and the biogenesis of apoB-containing lipoproteins in vivo. We thank Dr. Frank Graham (McMaster University, Hamilton, Canada) for providing AdCre1, Drs. David Gordon and Haris Jamil (Bristol-Myers Squibb Pharmaceutical Research Institute) for providing antibody to MTP and helpful discussions, Dr. Kazuhiro Oka and Maria Merched-Sauvage for assistance with AdCre1 production, Dr. Hye-Jeong Lee for primary hepatocyte culture, and Sylvia Ledesma for expert secretarial assistance." @default.
• W2016286787 created "2016-06-24" @default.
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• W2016286787 date "1999-03-01" @default.
• W2016286787 modified "2023-10-16" @default.
• W2016286787 title "Liver-specific Inactivation of the Abetalipoproteinemia Gene Completely Abrogates Very Low Density Lipoprotein/Low Density Lipoprotein Production in a Viable Conditional Knockout Mouse" @default.
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• W2016286787 doi "https://doi.org/10.1074/jbc.274.10.6051" @default.
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