REVERSIBLE ACCUMULATION OF CHOLESTERYL ESTERS IN MACROPHAGES INCUBATED WITH ACETYLATED LIPOPROTEINS

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.