Minor L K, Mahlberg F H, Jerome W G, Lewis J C, Rothblat G H, Glick J M
Ortho Pharmaceutical Corporation, Department of Biological Research, Raritan, New Jersey 08869.
Exp Mol Pathol. 1991 Apr;54(2):159-71. doi: 10.1016/0014-4800(91)90028-v.
Rabbit aortic smooth muscle cells take up lipid droplets when they are presented using an inverted culture technique. These droplets were localized in secondary lysosomes as demonstrated by staining for acid phosphatase. Initially, 69% of the cell volume was occupied by lipid, and 94% of the lipid was in lysosomes. After a 24-hr clearance period, the cell volume occupied by lipid decreased to 53%, although there was no change in the fraction of cell lipid that was in lysosomes. To confirm that hydrolysis of droplet lipid was occurring in lysosomes, cultures were exposed to medium containing Sandoz 58-035, an inhibitor of acyl CoA:cholesterol acyl transferase, for 24 hr in the presence and absence of chloroquine, ammonium chloride, or methylamine. Although the hydrolysis of cholesteryl oleate was sensitive to these lysosomotropic agents, the hydrolysis of triolein was not. Using reconstituted LDL containing cholesteryl oleate and triolein, we demonstrated that the hydrolyses of cholesteryl oleate and triolein were equally sensitive to the lysosomotropic agents when the cells were not loaded with droplet lipid. However, in cells loaded with lipid, hydrolysis of LDL cholesteryl ester was sensitive to the lysosomotropic agents but hydrolysis of triolein was not. We therefore conclude that both droplet lipids were hydrolyzed in lysosomes, and we attribute the failure of the lysosomotropic agents to inhibit fully the hydrolysis of droplet triolein to the presence of a large mass of free fatty acids in the lysosome that maintains a sufficiently low pH to sustain the triglyceridase activity, but not the cholesteryl esterase activity, of the lysosomal acid lipase.
当采用倒置培养技术呈现脂质滴时，兔主动脉平滑肌细胞会摄取它们。如通过酸性磷酸酶染色所示，这些脂质滴定位于次级溶酶体中。最初，细胞体积的69%被脂质占据，且94%的脂质存在于溶酶体中。经过24小时的清除期后，脂质占据的细胞体积降至53%，尽管溶酶体中细胞脂质的比例没有变化。为了证实脂质滴中的脂质水解发生在溶酶体中，在有和没有氯喹、氯化铵或甲胺的情况下，将培养物暴露于含有酰基辅酶A：胆固醇酰基转移酶抑制剂桑多兹58 - 035的培养基中24小时。虽然油酸胆固醇酯的水解对这些溶酶体促渗剂敏感，但三油酸甘油酯的水解则不然。使用含有油酸胆固醇酯和三油酸甘油酯的重组低密度脂蛋白，我们证明当细胞未加载脂质滴时，油酸胆固醇酯和三油酸甘油酯的水解对溶酶体促渗剂同样敏感。然而，在加载了脂质的细胞中，低密度脂蛋白胆固醇酯的水解对溶酶体促渗剂敏感，但三油酸甘油酯的水解则不敏感。因此，我们得出结论，两种脂质滴中的脂质都在溶酶体中被水解，并且我们将溶酶体促渗剂未能完全抑制脂质滴三油酸甘油酯水解归因于溶酶体中大量游离脂肪酸的存在，这些游离脂肪酸维持了足够低的pH值以维持溶酶体酸性脂肪酶的甘油三酯酶活性，但不能维持胆固醇酯酶活性。
