Mouse peritoneal macrophages accumulate large amounts of cholesteryl ester when incubated with human low-density lipoprotein that has been modified by chemical acetylation (acetyl-LDL). This accumulation is related to a high-affinity cell surface binding site that mediates the uptake of acetyl-LDL by adsorptive endocytosis and its delivery to lysosomes. The current studies demonstrate that the cholesteryl ester accumulation can be considered in terms of a two-compartment model: the incoming cholesteryl esters of acetyl-LDL are hydrolyzed in lysosomes, and the resultant free cholesterol is re-esterified in the cytosol where the newly formed esters are stored as lipid droplets. The following biochemical and morphologic evidence supports the hydrolysis-re-esterification mechanism. Incubation of macrophages with acetyl-LDL markedly increased the rate of cholesteryl ester synthesis from [ 14C]oleate, and this was accompanied by an increase in the acyl-CoA:cholesteryl cholesteryl acyltransferase activity of cell-free extracts. When macrophages were incubated with reconstituted acetyl-LDL in which the endogenous cholesterol was replaced with [ 3H]cholesteryl linoleate, the [ 3H]cholesteryl linoleate was hydrolyzed, and at least one-half of the resultant [ 3H]cholesterol was re-esterified to form [ 3H]cholesteryl oleate, which accumulated within the cell. The lysosomal enzyme inhibitor chloroquine inhibited the hydrolysis of the [ 3H]cholesteryl linoleate, thus preventing the formation of [ 3H]cholesteryl oleate and leading to the accumulation of unhydrolyzed [ 3H]cholesteryl linoleate within the cells. In the electron microscope, macrophages incubated with acetyl-LDL had numerous cytoplasmic lipid droplets that were not surrounded by a limiting membrane. The time course of droplet accumulation was similar to the time course of cholesteryl ester accumulation as measured biochemically. When acetyl-LDL was removed from the incubation medium, biochemical and morphological studies showed that cytoplasmic cholesteryl esters were rapidly hydrolyzed and that the resultant free cholesterol was excreted from the cell.
ASJC Scopus subject areas
- Cell Biology