Mechanisms of inhibition by apolipoprotein C of apolipoprotein E-dependent cellular metabolism of human triglyceride-rich lipoproteins through the low density lipoprotein receptor pathway.

E Sehayek,S Eisenberg

doi:10.1016/s0021-9258(18)55263-7

Abstract

The mechanism of inhibition by apolipoprotein C of the uptake and degradation of triglyceride-rich lipoproteins from human plasma via the low density lipoprotein (LDL) receptor pathway was investigated in cultured human skin fibroblasts. Very low density lipoprotein (VLDL) density subfractions and intermediate density lipoprotein (IDL) with or without added exogenous recombinant apolipoprotein E-3 were used. Total and individual (C-I, C-II, C-III-1, and C-III-2) apoC molecules effectively inhibited apoE-3-mediated cell metabolism of the lipoproteins through the LDL receptor, with apoC-I being most effective. When the incubation was carried out with different amounts of exogenous apoE-3 and exogenous apoC, it was shown that the ratio of apoE-3 to apoC determined the uptake and degradation of VLDL. Excess apoE-3 overcame, at least in part, the inhibition by apoC. ApoC, in contrast, did not affect LDL metabolism. Neither apoA-I nor apoA-II, two apoproteins that do not readily associate with VLDL, had any effect on VLDL cell metabolism. The inhibition of VLDL and IDL metabolism cannot be fully explained by interference of association of exogenous apoE-3 with or displacement of endogenous apoE from the lipoproteins. IDL is a lipoprotein that contains both apoB-100 and apoE. By using monoclonal antibodies 4G3 and 1D7, which specifically block cell interaction by apoB-100 and apoE, respectively, it was possible to assess the effects of apoC on either apoprotein. ApoC dramatically depressed the interaction of IDL with the fibroblast receptor through apoE, but had only a moderate effect on apoB-100. The study thus demonstrates that apoC inhibits predominantly the apoE-3-dependent interaction of triglyceride-rich lipoproteins with the LDL receptor in cultured fibroblasts and that the mechanism of inhibition reflects association of apoC with the lipoproteins and specific concentration-dependent effects on apoE-3 at the lipoprotein surface.

Highlights

That the ratioof apoE-3 toapoC determined the uptake Some lipoproteins that are naturally rich in apoE, e.g. pand degradationof VLDL
Total and individual apoC, apoC-I, apoC-11, previously described [9].The lipoprotein-containing mediumwas apoC-111-1,and apoC-111-2 wereprepared from human plasma VLDL removed and the cells were washed with PBS, 0.2% albumin and apoproteins by gel filtration on Sephadex G-150 followed by reincubated for 2 h with medium containing 10 pCi of [2-14C]acetate chromatography on DEAE-cellulose (apoC-I, apoC-11,apoC-111-1, (55 mCi/mmol)
The Studies with ExogenousupoE-3"The chemical composition mixture (1ml) was applied to a 0.9 X 25-cm column packed with 8% and apoprotein profile of VLDL-I, -11, and -111 and of IDL

Summary

EXPERIMENTAL PROCEDURES

Preparation of Lipoproteins, “‘I-Lipoproteins, and “‘I-Apoproteins-VLDL, VLDL density subfractions (I-III), IDL, and LwDeLre prepared from the plasma of healthy normal volunteers as recently. Regulation by ApoE-3 and ApoC of VLDL Cellular Metabolism a similar procedure. Total and individual apoC, apoC-I, apoC-11, previously described [9].The lipoprotein-containing mediumwas apoC-111-1,and apoC-111-2 wereprepared from human plasma VLDL removed and the cells were washed with PBS, 0.2% albumin and apoproteins by gel filtration on Sephadex G-150 (apoC) followed by reincubated for 2 h with medium containing 10 pCi of [2-14C]acetate chromatography on DEAE-cellulose (apoC-I, apoC-11,apoC-111-1, (55 mCi/mmol). I1were prepared bygel filtration from human plasma high density sterols was determined [26].Cultures incubated without lipoproteins lipoprotein [24]. The purity of apoproteins was checked by SDS- and served to determine the capacity of the cells to synthesize 14C-labeled urea-PAGE. ApoC-I was not identified in any of the other apoC AnalyticalMethods-Lipoprotein and cell protein were determined preparations. Lipoproteins (300 pg of protein) were incubated with different "c amounts of apolipoproteins for 60min at 37 in 0.9% NaCl, 20 mM

RESULTS

L D L - 111

ApoC-ApoE-3 r

DISCUSSION