Intracellular location and transport of cholesterol was studied in normal cells and those with genetic defects in lipid metabolism by fluorescence and electron microscopy. Golgi was identified as regulatory organelle for cholesterol transport. In normal fibroblasts both trans Golgi vacuoles and cis/medial Golgi cisternae accumulate cholesterol in response to LDL uptake suggesting a route of transport for cholesterol from trans Golgi to plasma membrane and cis/medial Golgi to endoplasmic reticulum (ER). In NP-C fibroblasts defective in cholesterol metabolism, cholesterol accumulates in trans Golgi cisternae suggesting impaired cholesterol tranport through Golgi. Lipid metabolites of ceramide formed in Golgi, are also affected by cholesterol enrichment of Golgi membranes. Ability of cells to process lipids and lipoproteins could depend on modulation of cholesterol enriched membrane traffic through Golgi. Studies on human adrenal cells indicate a direct lysosomal transfer pathway may transport LDL cholesterol to ER for esterification. Perilipin is present at the surface of intracellular lipid droplets. ER, vimentin and peroxisomes have a close spatial relationship to developing lipid droplets in 3T3-L1 adipocytes. Electron microscopy showed that particles associated with surface layer of lipid droplets are identical to IMP of membrane bilayers, indicating that the lipid droplet surface is derived from a membrane leaflet, probably ER where triacylglycerol is synthesized. Perilipin is located on this monolayered surface of the lipid droplet core. Perilipin was also located on surface of cholesterol ester enriched lipid droplets in Y1 mouse adrenal cells which synthesize steroids upon hormonal stimulation. Thus, perilipin is present in cells which have capacity to hydrolyze triacylglycerol or cholesterol ester via hormone sensitive lipase. We located lipoprotein lipase (LPL) and hepatic lipase (HL) in livers and cultured hepatocytes from newborn mice. In contrast to hepatocytes from normal mice those from cld/cld mice retain LPL intracellularly in ER and lysosomes. Liver and hepatocyte cultures from cld/cld mice contain HL in the extracellular environment. Studies with monensin indicate that HL is transported to Golgi in both normal and cld/cld hepatocytes. Thus cld/cld hepatocytes are capable of transporting HL but not LPL from ER to Golgi for processing and secretion. Cultured cld/cld) brown adipocytes retain LPL in ER although studies indicate Golgi enzymes necessary for processing of LPL are present and active in these cells. Findings in brown adipocytes and hepatocytes indicate a primary transport defect specific for LPL in cld/cld cells.
