MODULATION OF REGULATORY OXYSTEROL FORMATION AND LOW-DENSITY-LIPOPROTEIN SUPPRESSION OF 3-HYDROXY-3-METHYLGLUTARYL COENZYME-A (HMG-COA) REDUCTASE-ACTIVITY BY KETOCONAZOLE - A ROLE FOR CYTOCHROME-P-450 IN THE REGULATION OF HMG-COA REDUCTASE IN RAT INTESTINAL EPITHELIAL-CELLS

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  • 25 June 1986
    • journal article
    • research article
    • Vol. 261  (18) , 8348-8356
Abstract
The effects of ketaconazole, a lanosterol demethylase and cytochrome P450 inhibitor, on the regulation of 3-hydroxy-3-methylglutaryl coenzyme A reductase (EC 1.1.1.34, reductase) activity and sterol biosynthesis were studied in rat intestinal epithelial cell cultures (IEC-6). Incubation of cells with 0.15-2 .mu.M ketoconazole resulted in a concentration-dependent inhibition of reductase activity. As the drug concentration approached 15 .mu.M, the reductase activity returned to control values, and at 30 .mu.M ketaconazole, a stimulation of enzyme activity was observed. The drug had no effect on reductase activity in homogenates of IEC-6 cells. Ketoconazole (0.15-30 .mu.M) caused a concentration-dependent inhibition of the incorporation of [3H]mevalonolactone into cholesterol with a concomitant accumulation of radioactivity in methyl sterols; e.g. lanosterol and 24,25-epoxylanosterol. Interestingly, the incorporation of radioactivity into polar sterols showed a biphasic response which was inversely proportional to the biphasic response of reductase activity. Thus, the incorporation of [3H]mevalonolactone into polar sterols increased at low concentrations of ketaconazole (0.15-2 .mu.M) and decreased to control values at high concentrations of the drug. Treatment of cells with ketaconazole (30 .mu.M) and [3H]mevalonolactone followed by removal of the drug and radiolabel resulted in an inhibition of reductase activity and a redistribution of radioactivity from lanosterol and 24,25-epoxylanosterol to cholesterol and polar sterols. These results suggested that the inhibition of reductase activity at low concentrations of ketoconazole (< 2 .mu.M) was due to a formation of regulatory polar sterols generated from the methyl sterols. At high concentrations of ketoconazole (30 .mu.M) where no suppression in reductase activity was observed, the conversion of exogenously added [3H]24(S),25-epoxylanosterol to polar sterols was prevented. Exogenously added 24,25-epoxylanosterol inhibited reductase activity in a dose-dependent fashion, and ketoconazole (30 .mu.M) prevented the inhibition caused by low concentrations of epxoylanosterol. The drug, however, was unable to prevent the dose-dependent suppression of reductase activity by 25-hydroxylanosterol, a reduced form of 24,25-epoxylanosterol. These results indicated that 24,25-epoxylanosterol per se was not an inhibitor of reductase activity but could be metabolized to regulatory polar sterols through a cytochrome P-450 dependent reaction which was sensitive to ketoconazole. Treatment of cells with ketoconazole totally abolished the inhibition of reductase activity by low density lipoprotein (LDL). The drug did not affect the ability of cells to take up and degrade LDL nor did it affect the intracellular movement of free cholesterol derived from LDL. Acyl-CoA-cholesterol acyltransferase activity was inhibited by ketoconazole in both control and LDL supplemented cells. However, this inhibition by the drug was not related to the prevention of LDL action on reductase. Since ketoconazole blocks formation of endogenous polar sterols, the possibility is raised that the drug prevents the LDL suppression of reductase activity by inhibiting the cytochrome P-450-dependent formation of endogenous oxysterols induced by LDL.