Abstract
The coordinated control of cholesterol biosynthesis and esterification by 25-hydroxycholesterol was studied in the macrophage-like cell line P388D1. Since 25-hydroxycholesterol rapidly stimulated incorporation of [3H]oleate into the cholesteryl ester fraction of these cells, we have tested the possibility that the well-known inhibition of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA reductase) by 25-hydroxycholesterol might be the indirect consequence of an increased cholesterol esterification rather than a direct effect on HMG-CoA reductase. The experimental results show that progesterone, an inhibitor of acyl-CoA:cholesterol acyltransferase (ACAT), when added together with 25-hydroxycholesterol, abolished the increased cholesterol esterification without affecting the inhibition of HMG-CoA reductase by 25-hydroxycholesterol. Thus, uncoupling cholesterol esterification had no effect on 25-hydroxycholesterol's ability to inhibit HMG-CoA reductase. Unexpectedly, pretreatment of P388D1 cells with 25-hydroxycholesterol resulted in no elevation of ACAT activity as measured in broken cell preparations. Therefore, the possibility that 25-hydroxycholesterol stimulated cholesteryl ester formation by increasing the amount of cholesterol available for esterification, rather than by acting directly on ACAT activity, was considered. Labeling experiments using [14C]-cholesterol have provided evidence for this assumption.
Highlights
The coordinated control of cholesterol biosynthesis and esterification by 25-hydroxycholesterol was studied in the macrophage-likecell line P388D1.Since 25-hydroxycholesterol rapidly stimulated incorporation of ['Hloleate into the cholesteryl ester fraction of these cells, we have tested the possibility that the well-known inhibition of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCoA reductase) by 25-hydroxycholesterol might be the indirect consequence of an increased cholesterol esterification rather than a direct effect on HMGCoA reductase
It is of interest that in studies in microsomes where both 3-hydroxy-3-methylglutarylcoenzyme A (HMG-CoA) reductase and acyl-CoA:cholesterol acyltransferase (ACAT) activities were measured, an increase in the free cholesterol concentration of microsomes resulted in a decreased HMG-CoA reductase activity and an increase in ACAT activity [8]
The present experiments using [''C]acetate and ['HIoleate have extended this work, confirming the results obtained in hepatocytes by Drevon, Weinstein, and Abbreviations: HMG-CoA, 3-hydroxy-3-methylglutarylcoenzyme A; ACAT, acyl-CoA:cholesterol acyltransferase; MEM, Eagle's minimum essential medium; NCS, neonatal calf serum; BSA, bovine
Summary
The coordinated control of cholesterol biosynthesis and esterification by 25-hydroxycholesterol was studied in the macrophage-likecell line P388D1.Since 25-hydroxycholesterol rapidly stimulated incorporation of ['Hloleate into the cholesteryl ester fraction of these cells, we have tested the possibility that the well-known inhibition of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCoA reductase) by 25-hydroxycholesterol might be the indirect consequence of an increased cholesterol esterification rather than a direct effect on HMGCoA reductase. The data suggestthat 25-hydroxycholestero1may stimulate the formation of cholesteryl esters in P388D1 macrophage-like cells by increasing the rate of entry of free cholesterol into the ACAT substrate pool. Effect of time of incubation with 45-hydroxycholesterl on incorporation of ["C]aCet.te and rH)oleate into free cholesterol and cholesteryl esters
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