The effects of NADPH and 3-hydroxy-3-methylglutaryl-CoA on the thiol/disulfide redox behavior of rat liver microsomal 3-hydroxy-3-methylglutaryl-CoA reductase

Microsomal 3-hydroxy-3-methylglutaryl-CoA reductase isolated from the livers of rats fed a diet containing cholestyramine (HMGR-C) is oxidized to a protein-SS-protein disulfide via a thermodynamically favorable thiol/disulfide exchange in glutathione redox buffers which approach the normal in vivo redox poise. In the presence of either substrate (NADPH or 3-hydroxy-3-methylglutaryl-CoA), the equilibrium thiol/disulfide redox behavior of HMGR-C is substantially different than that observed in the absence of substrates or in the presence of both substrates. NADPH present during redox equilibrium in a glutathione redox buffer decreases the equilibrium constant for formation of the protein-SS-protein disulfide (Kox,i) from 0.55 +/- 0.07 M to 0.18 +/- 0.02 M and increases the Kox,m for formation of an inactive protein-SS-glutathione mixed disulfide from less than 1 to 6 +/- 1. The presence of 3-hydroxy-3-methylglutaryl-CoA during redox equilibrium has a similar effect, decreasing the Kox,i for protein-SS-protein disulfide formation to 0.10 +/- 0.02 M and increasing the Kox,m for protein-SS-glutathione mixed disulfide formation to 3.8 +/- 0.9. A three-state model is developed which describes the simultaneous accumulation of protein-SS-protein and protein-SS-glutathione mixed disulfides at redox equilibrium with glutathione redox buffers. Because of the different redox behavior of the free and substrate-liganded forms of the enzyme, addition of 3-hydroxy-3-methylglutaryl-CoA or NADPH to HMGR-C at redox equilibrium results in increased reduction and activation of the enzyme.     

Localization of 3-hydroxy-3-methylglutaryl CoA reductase and 3-hydroxy-3-methylglutaryl CoA synthase in the rat liver and intestine is affected by cholestyramine and mevinolin

In the normal fed rat, both 3-hydroxy-3-methylglutaryl CoA (HMG-CoA) synthase and HMG-CoA reductase are found in high concentrations in hepatocytes that are localized periportally. The majority of the liver cells show little or no evidence of either enzyme. Addition of cholestyramine and mevinolin to the diet resulted in all liver cells showing strong positive staining for both HMG-CoA reductase and HMG-CoA synthase. These two drugs increased the hepatic HMG-CoA reductase and HMG-CoA synthase activities 92- and 6-fold, respectively, and also increased the HMG-CoA reductase activity in intestine, heart, and kidney 3- to 15-fold. We used immunofluorescence and avidin-biotin labeled antibody to localize HMG-CoA reductase in the rat intestine. In rats fed a normal diet, the most HMG-CoA reductase-positive cells were the villi of the ileum greater than jejunum greater than duodenum. Crypt cells showed no evidence of HMG-CoA reductase. Addition of cholestyramine and mevinolin to the diet led to a dramatic increase in the concentration of HMG-CoA reductase in the apical region of the villi of the ileum and jejunum and in the crypt cells of the duodenum. Hence these two drugs affected both the relative concentration and distribution of intestinal HMG-CoA reductase. Cholestyramine and mevinolin feeding induced in the liver, but not intestine, whorls of smooth endoplasmic reticulum that were proximal to the nucleus and contained high concentrations of HMG-CoA reductase. Administration of mevalonolactone led to the rapid dissolution of the hepatic whorls within 15 min, at a time when there is little or no change in the mass of HMG-CoA reductase. We conclude that the whorls are present in the livers of rats fed cholestyramine and mevinolin because the cells are deprived of a cellular product normally synthesized from mevalonate.     

Biological disulfides: the third messenger? Modulation of phosphofructokinase activity by thiol/disulfide exchange

Rabbit muscle phosphofructokinase is rapidly inactivated at pH 8.0 by incubation with low concentrations of oxidized glutathione, Coenzyme A glutathione mixed disulfide, and oxidized Coenzyme A. The inactivation is first order in disulfide concentration over the concentration ranges examined (50-200 microM), and is approximately 8-fold slower at pH 7.0 than at pH 8.0. The substrates ATP and fructose 6-phosphate protect against inactivation while effector molecules such as AMP, cAMP, and citrate do not. The oxidation of the enzyme by disulfides is fully reversible. The equilibrium constant for the reaction Ered + GSSG in equilibrium Eox + GSH at pH 8.0 is 7.1 in the absence of substrates and 2.5 in the presence of 0.1 mM ATP. For comparison, the equilibrium constant for the reaction CoASH + GSSG in equilibrium CoASSG + GSH was found to be 3.1 at pH 8.0. These equilibrium constants for thiol/disulfide exchange are such that modulation of phosphofructokinase activity by thiol/disulfide exchange in vivo is feasible. The ability of the thiol/disulfide ratio in vivo to modulate the activity of the fructose 6-phosphate/fructose 1,6-diphosphate futile cycle is discussed. The possibility is considered that modulation of the thiol/disulfide ratio in vivo may serve as a "third messenger" in response to cAMP levels, and that the activity of key enzymes of glycolysis/gluconeogenesis may be regulated in response to changing thiol/disulfide ratios.     

Functional size of rat hepatic 3-hydroxy-3-methylglutaryl coenzyme A reductase as determined by radiation inactivation

The functional molecular weight of rat liver 3-hydroxy-3-methylglutaryl-CoA reductase was determined by radiation inactivation. Both isolated hepatic microsomes and primary hepatocytes were irradiated with high energy electrons at -135 degrees C, and the residual microsomal enzyme activity was subsequently determined. The loss of enzyme activity in both irradiated microsomes and microsomes isolated from irradiated hepatocytes followed a single exponential decay which corresponded to a molecular mass of 200 kDa. This minimal molecular size of the functional enzyme was unaffected by either addition of cholestyramine to the rat diet or addition of 25-hydroxycholesterol plus mevalonate to the isolated rat hepatocytes. In addition, surviving enzyme protein was determined by immunoprecipitation of radiolabeled enzyme from hepatocytes that had been incubated with [35S]methionine before irradiation. The target size for loss of the monomer subunits was 98 kDa. The simplest interpretation of these results is that rat liver 3-hydroxy-3-methylglutaryl-CoA reductase in situ is a noncovalently linked dimer of the Mr = 97,200 enzyme subunit.     

Diurnal rhythm of rat liver mRNAs encoding 3-hydroxy-3-methylglutaryl coenzyme A reductase. Correlation of functional and total mRNA levels with enzyme activity and protein

Rat liver 3-hydroxy-3-methylglutaryl coenzyme A reductase exhibits a diurnal rhythm of activity which coincides with a diurnal rhythm of reductase protein and reductase mRNA levels. This diurnal rhythm of reductase activity, polypeptide mass, and mRNA exists in rats fed a normal diet (unsupplemented rat chow) and in rats fed a diet supplemented with cholestyramine plus or minus mevinolin. Levels of reductase protein were determined by 8 M urea/sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting. Reductase mRNA was measured by in vitro translation or blot hybridization of liver RNA. Functional reductase mRNA levels in rats fed a normal diet were approximately 10-fold higher during the middle of the dark cycle than during the middle of the light cycle. Maximum induction of functional reductase mRNA was observed in rats fed cholestyramine and mevinolin. This latter level was 157-fold higher than the level measured at the diurnal low point in rats fed a normal diet. Blot hybridization of liver RNA showed two predominant mRNAs of 4.6 and 4.2 kilobase pairs and a minor species at 6.9 kilobase pairs. These mRNAs exhibited a diurnal rhythm for rats on all three diets and reached peak levels during the 12-h dark period. These data indicate that the diurnal rhythm of reductase mass and activity is closely paralleled by the level of its mRNA.     

Identification and regulation of a rat liver cDNA encoding farnesyl pyrophosphate synthetase

CR39 is a cholesterol-repressible rat liver cDNA previously isolated by differential hybridization (Clarke, C.F., Tanaka, R.D., Svenson, K., Wamsley, M., Fogelman, A.M., and Edwards, P.A. (1987) Mol. Cell. Biol. 7, 3138-3146). To precisely identify the function of CR39 a fusion protein was constructed that contained the amino-terminal region of the bacterial protein anthranilate synthetase fused to the full length CR39 polypeptide. Affinity purified antisera directed against the fusion protein inactivated rat liver cytosolic prenyltransferase activity in vitro. In addition, affinity purified antisera made to purified chicken prenyltransferase cross-reacted with the fusion protein containing CR39. Rat hepatic prenyltransferase activity and enzyme mass were quantitated in animals fed diets or drugs known to alter endogenous cholesterol biosynthesis. Rats fed a diet supplemented with cholestyramine and mevinolin showed a 3.5-fold increase in activity and a 5.0-fold increase in mass of cytosolic prenyltransferase. A diet supplemented with cholesterol resulted in approximately a 4.0-fold decrease in hepatic enzyme activity and a 10-fold decrease in enzyme mass. Under these same dietary regimens the mass of prenyltransferase in the testes remained unchanged. We conclude that CR39 encodes the prenyltransferase of cholesterol biosynthesis, farnesyl pyrophosphate synthetase. Furthermore, in the liver this enzyme shows coordinate regulation with two other enzymes, 3-hydroxy-3-methylglutaryl-CoA reductase and 3-hydroxy-3-methylglutaryl-CoA synthase, in response to cholesterol feeding and hypocholesterolemic drugs.     

Regulation of rat liver 3-hydroxy-3-methylglutaryl coenzyme A synthase and the chromosomal localization of the human gene

3-Hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) synthase was purified to homogeneity from rat liver cytoplasm. The active enzyme is a dimer composed of identical subunits of Mr = 53,000. The amino acid composition and the NH2-terminal sequence are presented. Partial cDNA clones for the enzyme were isolated by screening of a rat liver lambda gt11 expression library with antibodies raised against the purified protein. The identity of the clones was confirmed by hybrid selection and translation. When rats were fed diets supplemented with cholesterol, cholestyramine, or cholestyramine plus mevinolin, the hepatic protein mass of cytoplasmic synthase, as determined by immunoblotting, was 25, 160, and 1100%, respectively, of the mass observed in rats fed normal chow. Comparable changes in enzyme activity were observed. Approximately 9-fold increases in both HMG-CoA synthase mRNA mass and synthase mRNA activity were observed when control diets were supplemented with cholestyramine and mevinolin. When rats were fed these two drugs and then given mevalonolactone by stomach intubation, there was a 5-fold decrease of synthase mRNA within 3 h. These results indicate that cytoplasmic synthase regulation occurs primarily at the level of mRNA. This regulation is rapid and coordinate with that observed for HMG-CoA reductase. The chromosomal localization of human HMG-CoA synthase was determined by examining a panel of human-mouse somatic cell hybrids with the rat cDNA probe. Interestingly, the synthase gene resides on human chromosome 5, which has previously been shown to contain the gene for HMG-CoA reductase. Regional mapping, performed by examination of a series of chromosome 5 deletion mutants and by in situ hybridization to human chromosomes indicates that the two genes are not tightly clustered.     

Activation of rat liver microsomal 3-hydroxy-3-methylglutaryl coenzyme A reductase by NADPH. Effects of dietary treatments

The sigmoidal curves observed for rat liver microsomal 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase with NADPH as the varied substrate were markedly affected by feeding the animals diets containing colestipol, mevinolin and colestipol or cholesterol. Feeding of mevinolin and colestipol decreased the S0.5 for NADPH from 270 to 40 microM, while cholesterol feeding increased the value to 1.3 mM. Immuno-blotting analysis revealed that the Mr 100,000 form of HMG-CoA reductase predominated in cases where the S0.5 value was lowest, and the Mr 200,000 species was the major form where the S0.5 values were highest. Activation of HMG-CoA reductase by NADPH was not due to conversion of the Mr 200,000 form to the 100,000 form.     

Succinylation and inactivation of 3-hydroxy-3-methylglutaryl-CoA synthase by succinyl-CoA and its possible relevance to the control of ketogenesis.

Succinyl-CoA (3-carboxypropionyl-CoA) inactivates ox liver mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase (EC 4.1.3.5) in a time-dependent manner, which is partially prevented by the presence of substrates of the enzyme. The inactivation is due to the enzyme catalysing its own succinylation. Complete inactivation corresponds to about 0.5 mol of succinyl group bound/mol of enzyme dimer. The succinyl-enzyme linkage appears to be a thioester bond and is probably formed with the active-site cysteine residue that is normally acetylated by acetyl-CoA. Succinyl-CoA binds to 3-hydroxy-3-methylglutaryl-CoA synthase with a binding constant of 340 microM and succinylation occurs with a rate constant of 0.57 min-1. Succinyl-enzyme breaks down with a half-life of about 40 min (k = 0.017 min-1) at 30 degrees C and pH 7 and is destabilized by the presence of acetyl-CoA and succinyl-CoA. A control mechanism is postulated in which flux through the 3-hydroxy-3-methylglutaryl-CoA cycle of ketogenesis is regulated according to the extent of succinylation of 3-hydroxy-3-methylglutaryl-CoA synthase.     

Control of 3-hydroxy-3-methylglutaryl coenzyme A reductase by endogenously synthesized sterols in vitro and in vivo.

Isolated rat hepatocytes converted mevalonolactone into sterol intermediates and fatty acids 6- to 8-fold faster than mevalonate salt at concentrations less than 6 X 10(-4) M. Incubation of hepatocytes for 3 h normally results in induction of 3-hydroxy-3-methylglutaryl-CoA reductase. This increase in enzyme activity was inhibited by mevalonolactone and by mevalonate salt; at each concentration between 6 X 10(-4) M and 6 X 10(-8) M the lactone was a more effective inhibitor than the salt. The increase in enzyme activity was completely prevented by 6 X 10(-4) M lactone, and at this concentration the cells synthesized from the lactone an amount of sterol per hour which approximated that leavingthe cells in the same period. Administration of mevalonolactone to intact rats resulted in a dose-dependent inhibition of hepatic 3-hydroxy-3-methylglutaryl-CoA reductase activity. At the highest dose (400 mg of (RS)-mevalonolactone/200 g of rat) enzyme activities declined 85% within 45 min and were still suppressed below normals after 28 h. Mevalonolactone treatment resulted in increases in liver cholesterol content and in the cholesterol ester concentration of liver microsomes. The results demonstrate that the activity of hepatic 3-hydroxy-3-methylglutaryl-CoA reductase can be controlled by the rate of endogenous sterol synthesis both in vitro and in vivo.     

Independent regulation of 3-hydroxy-3-methylglutaryl-coenzyme A reductase and chylomicron remnant receptor activities in rat liver.

Treatment of rats with pharmacological doses of oestrogen resulted in a 3-fold decrease in the activity of hepatic 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMG-CoA reductase) and a 4-fold increase in saturable binding of 125I-labelled chylomicron remnants to liver membranes in vitro. Intragastric administration of mevalonolactone to rats did not affect the capacity of the liver membranes to bind to labelled chylomicron remnants even though there was a substantial decrease in the activity of HMG-CoA reductase. Similar results were obtained after cholesterol feeding. Simultaneous treatment of rats with cholestyramine and compactin increased hepatic HMG-CoA reductase activity 6-fold. However, liver membranes derived from these animals showed no change in their capacity to bind to labelled chylomicron remnants in vitro. Administration of mevalonolactone to the cholestyramine/compactin-treated animals also failed to produce a change in remnant-binding capacity. Although administration of mevalonolactone alone produced a significant 3-fold decrease in the activity of hepatic HMG-CoA reductase it was unable to suppress significantly the increase in enzyme activity caused by treatment with cholestyramine and compactin.     

3-Hydroxy-3-methylglutaryl coenzyme A synthase. Evidence for an acetyl-S-enzyme intermediate and identification of a cysteinyl sulfhydryl as the site of acetylation.

Homogeneous liver 3-hydroxy-3-methylglutaryl coenzyme A synthase, which catalyzes the condensation of acetyl-CoA with acetoacetyl-CoA to form 3-hydroxy-3-methylglutaryl-CoA, also carries out: (a) a rapid transacetylation from acetyl-CoA to 31-dephospho-CoA and (b) a slow hydrolysis of acetyl-CoA to acetate and CoA. Transacetylation and hydrolysis occur at 50 and 1 percent, respectively, the rate of the synthasecatalyzed condensation reaction. It appears that an acetyl-enzyme intermediate is involved in the transacetylase and hydrolase reactions of 3-hydroxy-3-methylglutaryl-CoA synthase, as well as in the over-all condensation process. Covalent binding to the enzyme of a [14C]acetyl group contributed by [1(-14)C]acetyl-CoA is indicated by migration of the [14C]acetyl group with the dissociated synthase upon electrophoresis in dodecyl sulfate-urea and by precipitation of [14C]acetyl-enzyme with trichloroacetic acid. At 0 degrees and a saturating level of acetyl-CoA, the synthase is rapidly (less than 20 s) acetylated yielding 0.6 acetyl group/enzyme dimer. Performic acid oxidation completely deacetylates the enzyme, suggesting the site of acetylation to be a cysteinyl sulfhydryl group. Proteolytic digestion of [14C]acetyl-S-enzyme under conditions favorable for intramolecular S to N acetyl group transfer quantitatively liberates a labeled derivative with a [14C]acetyl group stable to performic acid oxidation. The labeled oxidation product is identified as N-[14C]acetylcysteic acid, thus demonstrating a cysteinyl sulfhydryl group as the original site of acetylation. The ability of the acetylated enzyme, upon addition of acetoacetyl-CoA, to form 3-hydroxy-3-methylglutaryl-CoA indicates that the acetylated cysteine residue is at the catalytic site.     

Effects of mevinolin in rat liver: evidence for a lack of coupling between synthesis of hydroxymethylglutaryl-CoA reductase and cholesterol 7 alpha-hydroxylase activity

The effect of treatment of rats with the hydroxymethylglutaryl-CoA reductase inhibitor, mevinolin, on 7 alpha-hydroxylation of cholesterol was studied. Treatment with 0.1% mevinolin in diet for 3 days was found to have an inhibitory effect on 7 alpha-hydroxylation of cholesterol (about 35%). Treatment with cholestyramine increased 7 alpha-hydroxylation of both exogenously added and endogenous microsomal cholesterol 3-4-fold. Combined treatment with both cholestyramine and mevinolin decreased this stimulation to 2-2.5-fold. Treatment with 2% cholesterol in diet increased 7 alpha-hydroxylation of exogenous cholesterol about 2-fold and 7 alpha-hydroxylation of endogenous cholesterol about 3.5-fold. The stimulatory effect of cholesterol was reduced or abolished when 0.1% mevinolin was added to the cholesterol-containing diet. With the exception of the experiments with cholesterol in the diet, all experiments including mevinolin gave a marked stimulation (up to 60-fold) of the hydroxymethylglutaryl-CoA reductase activity under the in vitro conditions employed. The concentration of free cholesterol in the liver microsomes was not significantly changed in any of these experiments. It is concluded that there is no coupling between induction of synthesis of hydroxymethylglutaryl-CoA reductase protein and cholesterol 7 alpha-hydroxylase activity. The inhibitory effect of mevinolin on cholesterol 7 alpha-hydroxylase activity under experimental conditions where most of the effect of mevinolin on hydroxymethylglutaryl-CoA reductase was abolished by treatment with cholesterol suggest that the effect of mevinolin on the cholesterol 7 alpha-hydroxylase may be independent of its effect on cholesterol synthesis. The over-all results do not favour the hypothesis that cholesterol synthesis and cholesterol availability are the most important determinants for the regulation of the cholesterol 7 alpha-hydroxylase.     

Specificity of the effect of dietary cholesterol on rat liver microsomal 3-hydroxy-3-methylglutaryl-coenzyme a reductase activity.

Dietary cholesterol lowers the activity of rat liver microsomal 3-hydroxy-3-methylglutaryl-CoA reductase without affecting various other liver microsomal enzymes. This is consistent with a specific regulatory mechanism and distinguishes the action of cholesterol on 3-hydroxy-3-methylglutaryl-CoA reductase from that of at least one other stimulus known to affect this enzyme.     

Stimulatory effect of mevinolin on rat liver phosphatidic acid phosphatase

The activity of the soluble form of phosphatidic acid phosphatase in rat liver was stimulated about 2.5-fold by inclusion of mevinolin, a competitive hydroxymethylglutaryl-CoA reductase inhibitor, in the diet (0.1%). The stimulatory effect of mevinolin was present also after dietary addition of cholestyramine (5%) or intraperitoneal administration of ethanol. Addition of cholesterol (2%) to the diet totally abolished the stimulation by mevinolin on phosphatidic acid phosphatase. The results support a correlation between the synthesis of the rate-limiting enzyme in cholesterol biosynthesis and the activity of the apparent rate-limiting enzyme in triacylglycerol biosynthesis.     

Specificity of the effect of dietary cholesterol on rat liver microsomal 3-hydroxy-3-methylglutaryl-coenzyme A reductase activity (Short Communication)

Dietary cholesterol lowers the activity of rat liver microsomal 3-hydroxy-3-methylglutaryl-CoA reductase without affecting various other liver microsomal enzymes. This is consistent with a specific regulatory mechanism and distinguishes the action of cholesterol on 3-hydroxy-3-methylglutaryl-CoA reductase from that of at least one other stimulus known to affect this enzyme.     

A new method for assaying rat liver microsomal 3-hydroxy-3-methylglutaryl-coenzyme A reductase activity and its application in a study of the effect of dietary cholesterol on this effect of dietary cholesterol on this enzyme.

A new method suitable for measuring rat liver 3-hydroxy-3-methylglutaryl-CoA reductase activity is described and its advantages over methods previously available are discussed. An accurate time course was measured for the inhibition of liver microsomal 3-hydroxy-3-methylglutaryl-CoA reductase activity by dietary cholesterol; this enzyme was affected 1 1/4 h after the rats began to consume a cholesterol-rich diet. In this experiment there was no correlation between concentrations of microsomal cholesterol ester and the activity of 3-hydroxy-3-methylglutary-CoA reductase.     

New substrates and inhibitors of 3-hydroxy-3-methylglutaryl-CoA reductase

Methyl (RS)-5-bromo-3-hydroxy-3-methyl-pentanoate was prepared by bromination of methyl mevalonate and used for the formation of 4-carboxy-3-hydroxy-3-methylbutyl thioether derivatives by reaction with N-octanoyl-cysteamine, pantetheine, phosphopantetheine and coenzyme A. These thiols were also converted to the (RS)-3-hydroxy-3-methylglutaryl thioester derivatives. The thioesters formed with pantetheine and phosphopantetheine are substrates of 3-hydroxy-3-methylglutaryl-CoA reductase; Km and V values are similar to those of the superior CoA-derivative. The corresponding thioether derivatives in which the oxygen next to sulfur of the substrates is replaced by hydrogen, are inhibitors of the reductase. The inhibition is competitive with 3-hydroxy-3-methylglutaryl-CoA varied, and noncompetitive with NADPH varied. For each of the corresponding pairs of thioester and thioether derivatives Km (substrate) is nearly identical with Ki (inhibitor). The specificity and stereospecificity of the inhibitor action are also shown.     

Oxysterol regulators of 3-hydroxy-3-methylglutaryl-CoA reductase in liver. Effect of dietary cholesterol

Hepatic regulatory oxysterols were analyzed to determine which oxysterols were present in livers of mice fed a cholesterol-free diet and whether repression of 3-hydroxy-3-methylglutaryl-CoA reductase following cholesterol feeding was accompanied by an increase in one or more oxysterols. Analysis of free and esterified sterols from mice fed a cholesterol-free diet resulted in the identification and quantitation of six regulatory oxysterols: 24-hydroxycholesterol, 25-hydroxycholesterol, 26-hydroxycholesterol, 7 alpha-hydroxycholesterol, 7 beta-hydroxycholesterol, and 7-ketocholesterol. Following the addition of cholesterol to the diet for 1 or 2 nights, hepatic 3-hydroxy-3-methylglutaryl-CoA reductase activity declined and the levels of oxysterols, especially those of the side-chain-hydroxylated sterols, increased. Total 3-hydroxy-3-methylglutaryl-CoA reductase repressor units attributable to identified free oxysterols increased 2.5- and 6-fold after 1 and 2 nights, respectively, of cholesterol feeding. The amounts of esterified 24-, 25-, and 26-hydroxycholesterol also increased, with the increase in esterified 24-hydroxycholesterol being the greatest. The 24-hydroxycholesterol was predominantly the 24S epimer and the 26-hydroxycholesterol was predominantly the 25R epimer, indicating enzymatic catalysis of their formation. The observed correlation between increased levels of regulatory oxysterols and repression of 3-hydroxy-3-methylglutaryl-CoA reductase in cholesterol-fed mice is consistent with a hypothesis that intracellular oxysterol metabolites regulate the level of the reductase.