Abstract

In vitro studies have shown that the binding site for microsomal triglyceride transfer protein (MTP) is within the first 17% of apoB (apoB-17). Expression of apoB-48 in McArdle cells decreases endogenous lipoprotein production; however, overexpression of human apoB in transgenic mice does not decrease endogenous mouse apoB expression. To assess this inconsistency, adenoviruses expressing human apoB-17 (AdB17) or apoB-17-beta (which contains apoB-17 plus a small lipid-binding beta-sheet region of apoB, AdB-17beta) were produced. Hepatoma cells were infected with AdB17 or AdB17-beta with AdLacZ, an adenovirus expressing beta-galactosidase, as a control. Overexpression of apoB-17 and apoB-17-beta in hepatoma cells to levels 2- to 3-fold greater than that of endogenous apoB did not alter endogenous apoB production. This was also true in the presence of oleic acid and N-acetyl-leucyl-leucyl-norleucinal. High levels of apoB-17 or beta-galactosidase expression reduced apoB-100 production; however, control protein production was also reduced. To assess the effects of apoB-17 expression in vivo, mice of three different strains were injected with AdB17. Two days after injection, plasma apoB-17 was approximately 24 times the amount of endogenous apoB in the C57BL/6 mice, 2 times the apoB-100 in human apoB transgenic mice, and 4 times the apoB-48 in apoE knockout mice. Overexpression of apoB-17 did not decrease apoB-100 or apoB-48 concentrations in mouse plasma as assessed by Western blot analysis. These results demonstrate that although the apoB-17 binds to MTP in vitro, it does not alter endogenous apoB expression in mice or in hepatoma cells.

Highlights

  • In vitro studies have shown that the binding site for microsomal triglyceride transfer protein (MTP) is within the first 17% of Apolipoprotein B (apoB)

  • HepG2 cells were grown in minimum essential medium (MEM); containing 10% fetal bovine serum (FBS). 293 cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% FBS, and McArdle-RH7777 cells were grown in DMEM containing 10% FBS and 10% horse serum

  • The standard infection protocol involved using about 107 virus particles for each 35-mm well, which is a multiplicity of infection (MOI) of approximately 10

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Summary

Introduction

In vitro studies have shown that the binding site for microsomal triglyceride transfer protein (MTP) is within the first 17% of apoB (apoB-17). Expression of apoB-48 in McArdle cells decreases endogenous lipoprotein production; overexpression of human apoB in transgenic mice does not decrease endogenous mouse apoB expression. To assess this inconsistency, adenoviruses expressing human apoB-17 (AdB17) or apoB-17-␤ (which contains apoB-17 plus a small lipid-binding ␤-sheet region of apoB, AdB-17␤) were produced. Overexpression of apoB-17 did not decrease apoB-100 or apoB-48 concentrations in mouse plasma as assessed by Western blot analysis These results demonstrate that the apoB-17 binds to MTP in vitro, it does not alter endogenous apoB expression in mice or in hepatoma cells. ApoB-100 is primarily expressed in the liver, and is secreted into the plasma It is the major protein in very low density lipoproteins and LDL.

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