Craig W Y, Nutik R, Cooper A D
Department of Medicine, Stanford University, California 94305.
J Biol Chem. 1988 Sep 25;263(27):13880-90.
The regulation of lipoprotein assembly and secretion at a molecular level is incompletely understood. To begin to identify the determinants of apoprotein synthesis and distribution among lipoprotein classes, we have examined the effects of chylomicron remnants which deliver triglyceride and cholesterol, and beta very low density lipoprotein (beta VLDL), which deliver primarily cholesterol, on apolipoprotein synthesis and secretion by the human hepatoma Hep G2. Hep G2 cells were incubated with remnants or beta VLDL for 24 h, the medium was changed and the cells then incubated with [35S]methionine. The secreted lipoproteins were separated by gradient ultracentrifugation and the radiolabeled apoproteins were isolated by immunoprecipitation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis and counted. Remnants caused a 14-fold, and beta VLDL a 7-fold, increase in VLDL apoprotein (apo) secretion; the apoB/apoE ratio in this class was unchanged. Preincubation with either of the lipoproteins also stimulated low density lipoprotein apoB secretion. Preincubation with beta VLDL, but not with remnants, significantly increased apoE and apoA-I secreted in high density lipoprotein (HDL). In addition, the apoE/apoA-I ratio precipitated from the HDL of beta VLDL-treated cells by anti-apoE was 2.2-fold higher than that precipitated by anti-apoA-I. There was no difference in the ratios precipitated from control HDL. This was due to the secretion of a lipoprotein, subsequently isolated by immunoaffinity chromatography, that contained predominantly apoE. When Hep G2 cells were preincubated with oleic acid alone, total apoprotein secretion was not altered. However, cholesterol-rich liposomes stimulated secretion of newly synthesized apoE, but not apoB, while apoA-I secretion was variably affected. Cholesterol-poor liposomes had no effect. Thus, lipid supply is a determinant of apoprotein synthesis and secretion, and cholesterol may be of particular importance in initiating apoprotein synthesis.
脂蛋白组装和分泌在分子水平上的调控尚未完全明了。为了开始确定载脂蛋白合成及在脂蛋白类别中分布的决定因素，我们研究了携带甘油三酯和胆固醇的乳糜微粒残粒以及主要携带胆固醇的β极低密度脂蛋白（β-VLDL）对人肝癌Hep G2细胞载脂蛋白合成和分泌的影响。将Hep G2细胞与残粒或β-VLDL一起孵育24小时，更换培养基，然后将细胞与[35S]甲硫氨酸一起孵育。分泌的脂蛋白通过梯度超速离心分离，放射性标记的载脂蛋白通过免疫沉淀、十二烷基硫酸钠-聚丙烯酰胺凝胶电泳分离并计数。残粒使极低密度脂蛋白（VLDL）载脂蛋白（apo）分泌增加14倍，β-VLDL使其增加7倍；此类中的apoB/apoE比值未改变。用任何一种脂蛋白预孵育也刺激低密度脂蛋白apoB的分泌。用β-VLDL而非残粒预孵育显著增加了高密度脂蛋白（HDL）中分泌的apoE和apoA-I。此外，抗apoE从β-VLDL处理细胞的HDL中沉淀出的apoE/apoA-I比值比抗apoA-I沉淀出的高2.2倍。从对照HDL中沉淀出的比值没有差异。这是由于分泌了一种脂蛋白，随后通过免疫亲和层析分离，该脂蛋白主要含有apoE。当Hep G2细胞仅用油酸预孵育时，总载脂蛋白分泌未改变。然而，富含胆固醇的脂质体刺激新合成的apoE分泌，但不刺激apoB分泌，而apoA-I分泌受到不同程度的影响。贫胆固醇脂质体没有作用。因此，脂质供应是载脂蛋白合成和分泌的一个决定因素，胆固醇在启动载脂蛋白合成中可能特别重要。
