Studies on the link between HMG-CoA reductase and cholesterol 7~hydroxylase in rat liver

Under most experimental conditions, there is a covariation between the rate-limiting enzyme in cholesterol biosynthesis, HMG-CoA reductase, and the rate-limiting enzyme in bile acid biosynthesis, cholesterol 7a-hydroxylase. The most simple explanation for the coupling between the two enzymes is that newly synthesized cholesterol is a substrate for an unsaturated cholesterol 7a-hydroxylase and that substrate availability is of major regdatory importance for this enzyme. The following results seem, however, to rule out that such a simple regulatory mechanism is of major importance and that HMG-CoA reductase activity per se is of importance in the regulation of cholesterol 7a-hydroxylase. I ) The apparent degree of saturation of cholesterol 7a-hydroxylase, as measured in vitro in rat liver microsomes, was found to be relatively high (70-90%) under most experimental conditions, including starvation, cholestyramine treatment, and cholesterol treatment. A significant decrease in the degree of saturation was obtained first after a drastic reduction of total concentration of cholesterol in the microsomes by treatment with high doses of triparanol, an inhibitor of cholesterol biosynthesis. 2) The stimulatory effect of cholesterol feeding on cholesterol 7a-hydroxylase activity in rats seems to be an effect on the enzyme activity (enzyme induction?) rather than an effect on substrate availability. Thus, the stimulatory effect of cholesterol feeding was retained also after almost complete removal of the endogenous cholesterol by extraction with acetone. 3) Biliary drainage leads to a several-fold increase in the activity of both HMG-CoA reductase and cholesterol 7a-hydroxylase. The latter increase, however, cannot be due to the increased HMG-CoA reductase activity per se since infusion of cholesterol-enriched Intralipid to bile fistula rats led to a depressed HMG-CoA reductase activity with little or no effect on cholesterol 7a-hydroxylase. Similarly, depression of HMG-C& reductase by use of mevalonate in the drinking water had little or no effect on cholesterol 7a-hydroxy1ase.M It is concluded that microsomal cholesterol concentration, degree of substrate saturation, and levels of HMG-CoA reductase are not major direct regulators for cholesterol 07a-hydroxylase activity in rat liver. Bjorkhem, I., and J-E. Akerlund. Studies on the link between HMG-CoA reductase and cholesterol 7a-hydroxylase in rat liver. J. Lipid Res. 1988. 29: 136-143. Supplementary key words bile acids cholesterol synthesis cholestyramine mevalonate Intralipid Under most experimental conditions, there is a covariation between the rate-limiting enzyme in cholesterol biosynthesis, HMG-CoA reductase, and the rate-limiting enzyme in bile acid biosynthesis, cholesterol 7a-hydroxylase (for a review, see ref. 1). The reason for this very important covariation is poorly understood. Both enzymes may be regulated by the same factor(s), or the changes in the activity of cholesterol 7a-hydroxylase may be secondary to the changes in the activity of HMG-CoA reductase. In one particular experimental situation, however, the activities of the two enzymes change in opposite directions. Thus, treatment of rats with dietary cholesterol leads to a depression of HMG-CoA reductase activity and a stimulation of cholesterol 7a-hydroxylase activity. The simplest explanation for the latter finding is that the cholesterol load leads to increased availability of substrate for cholesterol 7a-hydroxylase. If so, the coupling between HMG-CoA reductase activity and cholesterol 7a-hydroxylase activity observed under most conditions may be due to the fact that increased HMG-CoA reductase activity leads to increased supply of substrate for cholesterol 7a-hydroxylase. There are some reports supporting this simple model for regulation of cholesterol 7a-hydroxylase. Mitropoulos et al. (2) reported that administration of mevalonic acid to rats led to an increased rate of 7a-hydroxylation of cholesterol. It was suggested that this increase could have been due to an increased intracellular pool of cholesterol in the environment of the HMG-CoA reductase that may have acted as a substrate for cholesterol 7a-hydroxylase (2). Shefer et al. ( 3 ) reported that cholesterol-free acetone powder prepared from rat liver microsomes obtained from rats fed a diet containing 2% cholesterol had about the same cholesterol 7ahydroxylase activity as corresponding preparations from control rats ( 3 ) . Results from some other experiments are, however, difficult to explain. In a recent work from this laboratory, it was shown that treatment of rats with cholestanol, a substrate analogue to cholesterol, also led to an increased 7ahydroxylation of cholesterol (4). This finding is better explained by an effect of the feeding on the enzyme activity than by an effect on the substrate availability. Results Abbreviations: HMG-CoA, 3-hydroxy-3-methylglutaryl CoA; DTT, dithiothreitol. 136 Journal of Lipid Research Volume 29, 1988 by gest, on N ovem er 6, 2017 w w w .j.org D ow nladed fom of experiments in which the relative rates of 7a-hydroxylation of exogenous and endogenous cholesterol were measured by isotope dilution-mass spectrometry are also difficult to fit with a regulatory model where the substrate pool is of major importance (4, 5). In addition, there seems to be little or no correlation between the concentration of free cholesterol in the microsomes and cholesterol 7a-hydroxylase activity under several different conditions (5, 6). A prerequisite for a regulatory model, in which the substrate pool of cholesterol is of major importance, is that the cholesterol 7a-hydroxylase is unsaturated under the conditions employed. If the enzyme is fully saturated with substrate, changes in the substrate pool should be of little or no regulatory importance. In a study by Bostrom (7), a purified cytochrome P-450 LM4 fraction with a high specific cholesterol 7a-hydroxylase activity was found to have a KM towards cholesterol of 36 FM. Since the concentration of free cholesterol in the microsomes is in the millimolar range, it is difficult to believe that an enzyme with such a low KM can be unsaturated to a degree allowing a threefold increase in activity as a consequence of an increased pool of substrate. On the other hand, the purified cholesterol-free enzyme may have properties different from those of the membrane-bound enzyme. In addition, it is always difficult to determine the degree of substrate saturation when the substrate is lipophilic and insoluble in aqueous medium. In the present study, attempts were made to measure the degree of saturation of cholesterol 7a-hydroxylase in crude liver microsomes from untreated rats and rats subjected to different treatments. In addition, we have repeated the experiments by Shefer et al. (3), and assayed cholesterol 7a-hydroxylase activity in acetone powder prepared from untreated and cholesterol-treated rats. A highly accurate technique has been used for the assay, based on isotope dilution-mass spectrometry. We have also investigated the possibility that HMG-CoA reductase activity per se is of importance for cholesterol 7a-hydroxylase activity by selective depression of HMG-CoA reductase activity in bilefistula rats and cholestyramine-treated rats in which both HMG-CoA reductase activity and cholesterol 7a-hydroxylase activity are stimulated. The results do not favor the hypothesis that substrate availability or HMG-CoA reductase activity are of major direct regulatory importance for cholesterol 7a-hydroxylase activity. MATERIALS AND METHODS