Studies on the rate of efflux of cholesterol from cultured human skin fibroblasts

The cholesterol content of normal human skin fibroblasts was increased (approximately doubled) by incubating cells in the presence of a high concentration of low density lipoprotein. Cholesterol efflux from these cells was then studied as a function of time and as a function of acceptor concentration. High density lipoprotein from which essentially all of the cholesterol had been removed by heptane extraction was used as a model acceptor (cholesterol-depleted high density lipoprotein). Using a sensitive enzymatic assay, it was possible to measure the increase in medium cholesterol and the decrease in cell cholesterol content simultaneously. Release was approximately a linear function of time for at least 6-12 h. A maximal rate of release was obtained at 20 micrograms of protein/ml (50% of excess stored sterol released in about 12 h); increasing the acceptor concentration 10-fold (to 200 micrograms/ml) failed to increase efflux rate. Comparison of the rates of fall of free and ester cholesterol levels suggested that hydrolysis of the ester may be rate-limiting when cholesterol-depleted high density lipoprotein is used as the acceptor. The results imply that above saturating concentrations of acceptor, acceptor-cell interaction is no longer limiting and that the rate of efflux of cholesterol under such conditions depends on intracellular processes necessary to make cholesterol available to the acceptor (e.g. hydrolysis of cholesterol esters and transfer of cholesterol from intracellular sites to the plasma membrane). Whether or not the concentrations of acceptor bathing cells in vivo is normally rate-limiting remains to be determined.