Mahlberg F H, Glick J M, Lund-Katz S, Rothblat G H
Department of Physiology and Biochemistry, Medical College of Pennsylvania, Philadelphia 19129.
J Biol Chem. 1991 Oct 25;266(30):19930-7.
We have demonstrated previously that HDL-mediated efflux of plasma membrane cholesterol is independent of specific binding of apolipoproteins to the high density lipoprotein (HDL) receptor in either control or cholesterol-enriched cells (Karlin, J. B., Johnson, W. J., Benedict, C. R., Chacko, G. K., Phillips, M. C., and Rothblat, G. H. (1987) J. Biol. Chem. 262, 12557-12564 and Johnson, W. J., Mahlberg, F. H., Chacko, G. K., Phillips, M. C., and Rothblat, G. H. (1988) J. Biol. Chem. 263, 14099-14106). The present studies were conducted to determine if the process for removal of intracellular (lysosomal) cholesterol is similar to that of membrane cholesterol or if, in contrast, it is selectively regulated by specific apolipoproteins of HDL. For these reasons, we examined the influence of each of the major apolipoproteins of human HDL, apoAI, apoAII, and apoCs on the metabolism of membrane and lysosomal cholesterol in a macrophage foam cell model. We developed an experimental system which allows, for the first time, the simultaneous determination of lysosomal hydrolysis of cholesteryl ester and efflux and esterification of both lysosomal and membrane cholesterol. J774 and elicited mouse peritoneal macrophages were loaded with cholesteryl ester within lysosomes through phagocytosis of sonicated lipid droplets. Membrane and lysosomal pools of cholesterol were differentially radiolabeled. Discoidal complexes of egg phosphatidylcholine and purified apolipoproteins having a similar size and composition were used as cholesterol acceptors. Our results demonstrate that lysosomal hydrolysis of cholesteryl ester is independent of the presence of extracellular acceptors. Lysosomal production of cholesterol stimulates the esterification by acyl-CoA:cholesterol acyltransferase of membrane and lysosomal cholesterol. All the particles tested induce the efflux of both pools of cholesterol at a similar ratio. As efflux is stimulated, esterification by acyl-CoA:cholesterol acyltransferase is reduced. We conclude that none of these apolipoproteins selectively influences the efflux or the esterification of membrane of lysosomal cholesterol. In addition, we observe that particles containing apoAI are the most efficient acceptors, but this effect is not linked to specific binding to the HDL receptor.
我们之前已经证明，在对照细胞或富含胆固醇的细胞中，高密度脂蛋白（HDL）介导的质膜胆固醇流出独立于载脂蛋白与高密度脂蛋白（HDL）受体的特异性结合（卡林，J.B.，约翰逊，W.J.，本尼迪克特，C.R.，查科，G.K.，菲利普斯，M.C.，和罗斯布拉特，G.H.（1987）《生物化学杂志》262，12557 - 12564以及约翰逊，W.J.，马尔伯格，F.H.，查科，G.K.，菲利普斯，M.C.，和罗斯布拉特，G.H.（1988）《生物化学杂志》263，14099 - 14106）。进行本研究以确定细胞内（溶酶体）胆固醇的去除过程是否与膜胆固醇的去除过程相似，或者相反，它是否由HDL的特定载脂蛋白选择性调节。出于这些原因，我们在巨噬细胞泡沫细胞模型中研究了人类HDL的每种主要载脂蛋白，即载脂蛋白AI、载脂蛋白AII和载脂蛋白C对膜胆固醇和溶酶体胆固醇代谢的影响。我们开发了一种实验系统，首次能够同时测定胆固醇酯的溶酶体水解以及溶酶体胆固醇和膜胆固醇的流出与酯化。通过吞噬超声处理的脂质滴，使J774细胞和诱导的小鼠腹腔巨噬细胞在溶酶体内负载胆固醇酯。膜胆固醇池和溶酶体胆固醇池用不同的放射性标记。将具有相似大小和组成的卵磷脂酰胆碱和纯化载脂蛋白的盘状复合物用作胆固醇受体。我们的结果表明，胆固醇酯的溶酶体水解与细胞外受体的存在无关。溶酶体胆固醇的产生刺激膜胆固醇和溶酶体胆固醇通过酰基辅酶A：胆固醇酰基转移酶进行酯化。所有测试的颗粒以相似的比例诱导两个胆固醇池的流出。随着流出受到刺激，酰基辅酶A：胆固醇酰基转移酶的酯化作用降低。我们得出结论，这些载脂蛋白均未选择性地影响溶酶体胆固醇膜的流出或酯化。此外，我们观察到含有载脂蛋白AI的颗粒是最有效的受体，但这种作用与与HDL受体的特异性结合无关。
