Cycloheximide sensitivity in regulation of acyl coenzyme A:cholesterol acyltransferase activity in Chinese hamster ovary cells. 2. Effect of sterol endogenously synthesized

Abstract

We reported in another paper [Chang, C. C. Y., Doolittle, G. M., & Chang, T. Y. (1986) Biochemistry (preceding paper in this issue)] that in Chinese hamster ovary (CHO) cells activation of acyl coenzyme A:cholesterol acyltransferase (ACAT) activity by treating cells with cycloheximide was abolished by providing exogenous sterol in the medium. We now report that providing 20 mM DL-mevalonate to cells grown in sterol-free medium increases the ACAT activity by approximately 6-fold and diminishes the cycloheximide activation effect. The mevalonate supplement has no significant effect on the rate of triglyceride or polar lipid synthesis, [3H]cholesterol efflux, or bulk protein degradation in cells. The activation of ACAT by mevalonate is prevented by adding a specific squalene oxide cyclase inhibitor to cells, indicating the requirement for endogenous sterol synthesis to mediate the mevalonate effect. In sterol-free medium, if sterol synthesis is blocked by specific enzyme inhibitors or through mutation, the ACAT activation by cycloheximide is again abolished. These results support the hypothesis that there may exist a short-lived factor(s) serving directly or indirectly as an endogenous ACAT inhibitor(s), the inhibitory action of this (these) factor(s) is (are) abolished, and its (their) turnover rate(s) is (are) increased by either exogenous sterol or by sterol endogenously synthesized. Conversely, removing exogenous sterol coupled with blocking endogenous sterol synthesis decreases the turnover rate(s) of the inhibitor(s), rendering its (their) action insensitive to intracellular degradation over the time period studied.

Isolation and characterization of Chinese hamster ovary cell mutants defective in intracellular low density lipoprotein-cholesterol trafficking.

“…There was no difference found between reconstituted ACAT activities in the cell extracts obtained from each cell line grown with or without LDL (Table I). This is consistent with previous work from this laboratory (10,15,16,23) that suggested that changes in the ACAT protein content are not responsible for the regulation of enzyme activity by LDL. The reconstituted ACAT activities in CT60 and 25-RA cell extracts were found to be similar but the reconstituted ACAT activities in CT52 cell extracts were always '~50% greater than values obtained with 25-RA cell extracts.…”

Effects of prolonged ethanol intake and malnutrition on rat pancreas.

“…The mechanism of cholesterol ester accumulation in acinar cells of alcoholic or malnourished rats is unknown. Wilson et al suggested that longterm ethanol feeding would cause pancreatic cholesterol ester accumulation by affecting exchange of cholesterol between serum and pancreatic tissue,38 and it is well known that cholesterol uptake by the cell stimulates acyl-CoA cholesterol-acyltransferase (ACAT) activity producing cholesterol ester.39 40 This proposed mechanism is in agreement with our data, because serum cholesterol concentrations of rats with pancreatic steatosis (groups C, D, and E) were higher than those in rats with normal pancreatic morphology (groups A and B) (Table IV). (Table III, Table IV) and steatosis in alcoholic rats is also present without hypercholesterolaemia as has been reported by Tsukamoto et al 30 One such factor could be the metabolism of ethanol inside acinar cells inducing cholesterol ester formation.…”

Acyl-coenzyme A:cholesterol acyltransferase 1 blockage enhances autophagy in the neurons of triple transgenic Alzheimer's disease mouse and reduces human P301L-tau content at the presymptomatic stage

“…ACAT activity assay in intact cells was performed as previously described (Chang and Chang, 1986). The incubation time with [ 3 H]-oleate-BSA was 2 h.…”