Regulation of cholesterol esterification and biosynthesis in monolayer cultures of normal adult rat hepatocytes.

Abstract

Adult rat parenchymal liver cells were isolated and cultured in monolayers. Cholesterol esterification in the intact cultured cells was determined by measuring incorporation of tritiated oleic acid into cell cholesterol ester. Addition of 10 microgram/ml of 25-hydroxycholesterol to the medium gave a 3- to 6-fold increase in cholesterol esterification, while the incorporation of oleic acid into phospholipids and triglycerides remained unaltered. Similar stimulation of cholesterol esterification by 25-hydroxycholesterol was also found if [14C]mevalonolactone or [3H]cholesterol (the latter presented to the cells in high density lipoproteins) were used as precursors. The stimulatory effect of 25-hydroxycholesterol was maximal after only 15-min incubation and was independent of protein synthesis. After 4 to 6 h of incubation with 25-hydroxycholesterol, its stimulatory effect was reduced significantly, and after 18 h of incubation no stimulation was observed. Thus, the liver cells in some fashion adapt to the continuing presence of 25-hydroxycholesterol. Isolated microsomes prepared from cells previously incubated with 25-hydroxycholesterol showed acyl-CoA:cholesterol acyltransferase activity 3 times that of microsomes from control cells. Incubation of isolated microsomes with 25-hydroxycholesterol increased acyl-CoA:cholesterol acyltransferase activity 2-fold. The net cellular content of ester cholesterol increased after 2 to 6 h incubation of hepatocytes with 25-hydroxycholesterol; there was a net decrease in cellular free cholesterol. Mevalonolactone (10 mM) also stimulated cholesterol esterification and inreased the cellular content of ester cholesterol (3- to 4-fold). The effectiveness of mevalonolactone did not diminish with longer periods of preincubation. Furthermore, the stimulatory effects of 25-hydroxycholesterol and mevalonolactone added together were at least 50 to 100% greater than the effects of either agent alone, suggesting that the mechanisms by which they increase cellular cholesterol esterification are different. Both 25-hydroxycholesterol and mevalonolactone rapidly inhibited microsomal 3-hydroxy-3-methylglutaryl-CoA reductase activity. Pure cholesterol had no effect on cellular cholesterol esterification or on 3-hydroxy-3-methylglutaryl-CoA reductase activity at concentrations up to 10 microgram/ml.