Analysis of the role of microsomal triglyceride transfer protein in the liver of tissue-specific knockout mice
Martin Raabe, Murielle M. Véniant, Meghan A. Sullivan, Constance H. Zlot, Johan Björkegren, Lars Bo Nielsen, Jinny S. Wong, Robert L. Hamilton, Stephen G. Young
Martin Raabe, Murielle M. Véniant, Meghan A. Sullivan, Constance H. Zlot, Johan Björkegren, Lars Bo Nielsen, Jinny S. Wong, Robert L. Hamilton, Stephen G. Young
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Analysis of the role of microsomal triglyceride transfer protein in the liver of tissue-specific knockout mice

Abstract

A deficiency in microsomal triglyceride transfer protein (MTP) causes the human lipoprotein deficiency syndrome abetalipoproteinemia. However, the role of MTP in the assembly and secretion of VLDL in the liver is not precisely understood. It is not clear, for instance, whether MTP is required to move the bulk of triglycerides into the lumen of the endoplasmic reticulum (ER) during the assembly of VLDL particles. To define MTP’s role in hepatic lipoprotein assembly, we recently knocked out the mouse MTP gene (Mttp). Unfortunately, achieving our objective was thwarted by a lethal embryonic phenotype. In this study, we produced mice harboring a “floxed” Mttp allele and then used Cre-mediated recombination to generate liver-specific Mttp knockout mice. Inactivating the Mttp gene in the liver caused a striking reduction in VLDL triglycerides and large reductions in both VLDL/LDL and HDL cholesterol levels. The Mttp inactivation lowered apo B-100 levels in the plasma by >95% but reduced plasma apo B-48 levels by only ∼20%. Histologic studies in liver-specific knockout mice revealed moderate hepatic steatosis. Ultrastructural studies of wild-type mouse livers revealed numerous VLDL-sized lipid-staining particles within membrane-bound compartments of the secretory pathway (ER and Golgi apparatus) and few cytosolic lipid droplets. In contrast, VLDL-sized lipid-staining particles were not observed in MTP-deficient hepatocytes, either in the ER or in the Golgi apparatus, and there were numerous cytosolic fat droplets. We conclude that MTP is essential for transferring the bulk of triglycerides into the lumen of the ER for VLDL assembly and is required for the secretion of apo B-100 from the liver.

Authors

Martin Raabe, Murielle M. Véniant, Meghan A. Sullivan, Constance H. Zlot, Johan Björkegren, Lars Bo Nielsen, Jinny S. Wong, Robert L. Hamilton, Stephen G. Young

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Figure 1

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Generation and characterization of a floxed Mttp allele. (a) Schematic o...
Generation and characterization of a floxed Mttp allele. (a) Schematic of the sequence-replacement gene-targeting strategy. The map of the wild-type Mttp allele (Mttpwt) spans the Mttp promoter (the sequences upstream from exon 1) and exons 1–3. The location of the 1.3-kb 3′ flanking probe (5) is indicated. Recombination of the gene-targeting vector with the cognate sequences in the chromosomal DNA produces a mutant Mttp allele, Mttpflox, in which the promoter and exon 1 of the Mttp allele are flanked by loxP sites (filled triangles). An additional loxP site is located downstream from the neo. Cre-mediated excision of both the neo and the promoter/exon 1 fragment produces a null Mttp allele (designated MttpΔ). (b) Illustration of the PvuII fragments produced by different Cre-mediated recombination events: excision of both the neo and the promoter/exon 1 fragment (in an MttpΔ allele), excision of the promoter/exon 1 fragment alone (in an MttpΔex1 allele), and excision of the neo alone (in an MttpΔneo allele). (c) A Southern blot of PvuII-digested genomic DNA from offspring of Mttpwt/flox/deleter-Cre intercrosses. The blot was hybridized with the 3′ flanking probe. Each of the 3 possible Cre-mediated recombination events was observed (MttpΔex1, MttpΔneo, and MttpΔ). Southern blots of PvuII-cleaved genomic DNA did not distinguish between the Mttpflox and MttpΔex1 alleles. The mouse Mttp gene contains SacI sites located 2 kb 5′ and 2.5 kb 3′ to the gene fragment illustrated in a. The gene-targeting event introduced a new SacI site. The recombination events could also be analyzed, therefore, with SacI-cleaved genomic DNA (Mttpwt, 16.5 kb; Mttpflox, 8 kb; MttpΔneo, 7 kb; MttpΔex1, 12.5 kb; and MttpΔ, 11.5 kb). (d) Southern blot illustrating Cre-mediated recombination in different tissues of an Mttpflox/flox/Mx1-Cre mouse in which Cre expression had been induced with pIpC. DNA samples from various tissues were digested with PvuII; the blot was hybridized with the 3′ flanking probe.