Cholesterol, an integral membrane component required for normal cellular function, is derived from low density lipoprotein (LDL) following endocytosis and hydrolysis in lysosomes or via de novo biosynthesis in the endoplasmic reticulum (ER). The mechanism for the ensuing movement of cholesterol from these intracellular sites to its cellular destinations is not known and defects in these transport pathways result in pathological states such as atherosclerotic lesions and Niemann Pick-C (NP-C) disease, a genetic lesion of cholesterol metabolism. A critical step towards correction of cellular cholesterol lipidoses is understanding pathways of intracellular cholesterol transport. We have used the sterol binding agent 2- hydroxypropyl-beta-cyclodextrin as a convenient and useful experimental tool to probe intracellular cholesterol transport pathways with combines biochemical and cytochemical approaches. Plasma membrane cholesterol is specifically removed by cyclodextrin which also removes cholesterol arriving at the plasma membrane from lysosomes was strongly attenuated by cyclodextrin indicating that a large portion of endocytosed cholesterol is delivered from lysosomes to the endoplasmic reticulum (ER) via the plasma membrane (PM). Several studies have provided a link between the Golgi complex and the processing of cellular cholesterol. We have previously shown that the processing of LDL-derived cholesterol in normal cells is accompanied by an enrichment of the cholesterol content of the Golgi in an anterograde fashion. Treatment of normal cells with Brefeldin A (BFA), to merge Golgi components with ER, renders esterification of lysosomal cholesterol insensitive to cyclodextrin. This finding implies that cholesterol is normally transferred from lysosomes to the PM via a PM-independent pathway. The Golgi also plays a role in shuttling cholesterol between the PM and other organelles. BFA enhances esterification of PM cholesterol suggesting a direct role for the Golgi in regulating trafficking of PM derived cholesterol to the ER. Golgi processing of cholesterol derived from LDL and the PM may play an essential role in maintaining the appropriate distribution of cholesterol within the cell. Lipoprotein lipase (LPL), is made in parenchymal cells and transported to capillaries where the enzyme is required for lipolysis of triacylglycerol in circulating lipoproteins. A genetic defect in mice, combined lipase deficiency (cld cld), results in cellular synthesis of LPL that is inactive and retained in the ER. Cultured brown adipocytes derived from cld cld newborn mice were incubated with BFA to translocate Golgi enzymes to LPL retained in the ER. BFA incubation enabled mutant cells to synthesize active lipase. Thus, production of inactive LPL in cld cld adipocytes probably results from their inability to translocate LPL from ER to Golgi for enzymatic processing to active lipase.
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