Analysis of the coordinate expression of 3-hydroxy-3-methylglutaryl coenzyme A synthase and reductase activities in Chinese hamster ovary fibroblasts

Decreased activities of both 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) synthase and HMG CoA reductase are observed in the presence of sterol in the Chinese hamster ovary (CHO) fibroblast. In three different genotypes of CHO cell mutants resistant to 25-hydroxycholesterol both enzyme activities exhibit a decreased response to 25-hydroxycholesterol compared to wild-type cells. Permanently repressed levels of both HMG CoA synthase and HMG CoA reductase activities are observed in another CHO mutant, phenotypically a mevalonate auxotroph. Mevinolin, a competitive inhibitor of HMG CoA reductase, has no effect on HMG CoA synthase activity measured in vitro. Incubation of CHO cells with sublethal concentrations of mevinolin produces an inhibition of the conversion of [14C]acetate to cholesterol and results in elevated levels of both HMG CoA synthase and HMG CoA reductase activities. Studies of CHO cells in sterol-free medium supplemented with cycloheximide indicate that continuous protein synthesis is not required for the maximal expression of HMG CoA synthase activity and provide an explanation for the lack of temporal similarity between HMG CoA synthase and reductase activities after derepression. These results support the hypothesis of a common mode of regulation for HMG CoA synthase and HMG CoA reductase activities in CHO fibroblasts.     

Location and regulation of early enzymes of sterol biosynthesis in yeast.

Acetoacetyl CoA thiolase and 3-hydroxy-3-methylglutaryl (HMG) CoA synthase were found almost entirely in the cytosol of Saccharomyces cerevisiae, whereas HMG CoA reductase was found almost entirely in mitochondria and further located in the matrix. Formation of all three enzymes was inhibited by cycloheximide, but not by chloramphenicol, indicating that they were synthesized in the cytosol. In anaerobically growing cells the levels of acetoacetyl CoA thiolase and HMG CoA synthase were decreased by ergosterol, whereas HMG CoA reductase levels were affected only slightly, suggesting that in yeast the enzymes responsible for synthesis of HMG CoA were regulated by ergosterol. Aerobically growing cells were essentially impermeable to ergosterol and cholesterol, whereas those growing anaerobically and requiring sterols were readily permeable. Mutants blocked in ergosterol formation were also permeable to sterols under aerobic conditions.     

The effect of substrates and competitive inhibitors on the phosphatase-dependent activation of hepatic hydroxymethylglutaryl CoA reductase

Previous studies have demonstrated that the in vitro activation of microsomal hepatic hydroxymethylglutaryl (HMG) CoA reductase by dephosphorylation is inhibited by HMG CoA or NADPH, the substrates of HMG CoA reductase (13). In the present study the effect of three competitive inhibitors of HMG CoA reductase on the activation of HMG CoA reductase was investigated. Adenosine-2'-monophospho-5'-diphosphoribose, a competitive inhibitor for the NADPH binding site, blocked the phosphatase-mediated activation of HMG CoA reductase. By contrast, neither compactin nor mevinolin, competitive inhibitors for the HMG CoA binding site, altered the activation of HMG CoA reductase. Moreover, the HMG CoA-mediated inhibition of the activation of HMG CoA reductase was not blocked even by very high concentrations of either compactin or mevinolin. These observations suggest that HMG CoA can bind to two sites on HMG CoA reductase. One site of HMG CoA binding serves as a catalytic site and is competitively blocked by compactin or mevinolin, and the second binding site is an allosteric site to which only HMG CoA is capable of binding. The binding of HMG CoA to this second site inhibits the activation of HMG CoA reductase by phosphatases.     

The effect of factors released from the tumor-transformed cells on DNA synthesis, mitosis, and cellular enlargement in 3T3 fibroblasts

Quiescent serum-starved 3T3 cells can be stimulated to initiate DNA synthesis after addition of conditioned media from spontaneously tumor-transformed 3T3 cells (3T6-cells) or from SV-40-transformed 3T3 cells (SV-3T3 cells). The conditioned media were found to stimulate both the chromosome cycle (i.e., DNA synthesis and cell division) and the growth cycle (i.e., cellular enlargement). Furthermore, addition of conditioned media to quiescent 3T3 cells increased the activity of HMG CoA reductase--an enzyme previously proposed to exercise some control on cell proliferation in 3T3 cells (Larsson and Zetterberg: J. Cell. Physiol. 129:99-102, 1986. The increased activity of HMG CoA reductase after treatment with tumor cell conditioned media was correlated to the stimulatory effects on DNA synthesis. By treating 3T3 cells stimulated to resume proliferation by addition of conditioned media with mevinolin (a competitive inhibitor of HMG CoA reductase) the activity of HMG CoA reductase as well as the DNA synthesis and cell division were efficiently inhibited. In contrast, HMG CoA activity was not coupled to the cellular enlargement. Therefore, it is proposed that one set of factors present in tumor cell conditioned media preferentially stimulates the chromosome cycle by increasing the HMG-CoA reductase activity, whereas another set of factors is responsible for growth in cell size. Both types of factors are required for balanced growth.     

Cell cycle-specific growth inhibition of human breast cancer cells induced by metabolic inhibitors

Proliferation of exponentially growing breast cancer cells (lineHs578T) was blocked specifically in G₁ by 3-hydroxy-3-methylglutarylCoenzyme A (HMG CoA) reductase inhibition, as well as by inhibitionof N-linked glycosylation. As a consequence of these inhibitoryconditions, the cells were synchronized in the G₁ stage of thecell cycle. The similarities in the kinetic responses pointto the possibility that the two different types of metabolicinhibitions block cell cycle progression by common mechanisms.One possibility is that the inhibition of HMG CoA reductaseactivity also leads to a depressed rate of N-linked glycosylation,which in turn may constitute the critical event for cell cycleprogression and cell growth. In order to investigate whetherthis relationship exists in breast cancer cells, cells synchronizedin G₁ by mevinolin (an inhibitor of HMG CoA reductase) wereused. Upon addition of mevalonate, whose endogenous synthesisis catalysed by HMG CoA reductase, the cells entered S phaseafter a 4 h pre-replicative period. Mevalonate stimulation alsoled to a rapid and substantial increase in N-linked glycosylation,measured by determining the uptake of radioactive glucosamine.This metabolic event was found to be of critical importancefor the initiation of DNA synthesis. However, as soon as thecells had entered S phase, they were independent of the levelof N-linked glycosylation. breast cancer cells glycosylation HMG CoA reductase     

Control of sterol synthesis and of hydroxymethylglutaryl CoA reductase in skin fibroblasts grown from patients with homozygous type II hyperlipoproteinemia.

In skin fibroblasts grown from four children with a homozygous form of type II hyperlipoproteinemia, the feedback control of sterol synthesis and the inhibitory effect on hydroxymethylglutaryl (HMG) CoA reductase activity by serum or low density lipoprotein were present, though diminished compared with the effects in normal fibroblasts. Stimulation of HMG CoA reductase by insulin and inhibition of acetyl CoA carboxylase by serum lipids were not impaired in these type II cells, indicating a degree of specificity in the abnormal response of the reductase. A rapid and convenient method for isolation of mevalonolactone in the course of the assay of HMG CoA reductase is described.     

Characterization of HMG CoA synthase activity of rat liver and CHO-K1 cells

This report describes the characterization and partial purification of rat liver 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) synthase activity. A preliminary characterization of Chinese hamster ovary (CHO) cell HMG CoA synthase activity is also presented. Ion-exchange chromatography of ammonium sulfate precipitates of rat liver cytosol indicate the existence of two isoenzymes of HMG CoA synthase. These isoenzymes are physically, catalytically, and immunologically distinct. One of these isoenzymes, peak 1, resembles mitochondrial HMG-CoA synthase activity as evidenced by similarities in elution upon ion-exchange chromatography, inhibition by MgCl2, and cross reactivity with an antibody prepared against the mitochondrial enzyme. As peak 1 activity is unstable, further purification studies were performed on peak 2 activity. Peak 2 can be further resolved into two activities (peaks 2A and 2B) by gel filtration. In contrast, CHO-K1 cells (a permanent fibroblast line) possess only peak 2 type HMG CoA synthase activity.     

Effect of acetoacetyl coa thiolase amplification on sterol synthesis in the yeasts S. cerevisiae and S. uvarum

Summary The ERG10 gene was amplified inS. cerevisiae and inS. uvarum, in order to study the regulation of ergosterol biosynthesis. Transformants with 10 or 20 fold increase in acetoacetyl CoA thiolase activity show the same ergosterol (aerobiosis) and squalene (anaerobiosis) contents as the wild type strains, confirming the presence of limiting steps in the conversion of acetoacetyl CoA into sterols. Measurement of mevalonate and phosphomevalonate synthesis shows that mevalonate kinase could be repressed in transformed strains.     

Hepatic cholesterol metabolism in normo- and hyperlipidemic patients with cholesterol gallstones.

In vivo studies have shown abnormalities in cholesterol and bile acid metabolism in primary hyperlipoproteinemia (HLP). The aim of the present investigation was to determine if the increased production of cholesterol in HLP type IV can be attributed to a correspondingly high level of the hepatic 3-hydroxy-3-methylglutaryl (HMG) CoA reductase activity and if the low cholic acid: chenodeoxycholic acid synthesis ratio in HLP type II is due to some hydroxylase deficiency. Liver biopsies from 26 normolipidemic and 25 hyperlipidemic (10 type IIa, 6 type IIb, and 9 type IV) patients undergoing elective cholecystectomy were assayed for HMG CoA reductase activity, 12 alpha-hydroxylase activity, and 25-hydroxylase activity. The HMG CoA reductase activity was normal in HLP type IIa and type IIb and was increased about twice HLP type IV (P less than 0.001). The 12 alpha- and 25-hydroxylase activities were normal in all groups of patients. The results are compatible with a normal cholesterol synthesis in the liver in HLP type II. A reduced 12 alpha- or 25-hydroxylase activity cannot explain the low production of cholic acid relative to chenodeoxycholic acid in this type of HLP. The elevated HMG CoA reductase activity found in the liver of type IV patients may, however, be part of the explanation for the elevated synthesis of cholesterol often seen in these patients.     

HMG CoA reductase of intestinal mucosa and liver of the rat

Methods were developed for the determination of HMG CoA (3-hydroxy-3-methylglutaryl CoA) reductase activity in subcellular fractions of intestinal mucosa and liver of Wistar strain rats. In the liver, reductase activity was located exclusively in the microsomal fraction. In the intestinal mucosa, activity was found in both mitochondrial and microsomal fractions of crypt cells but not of villi. The microsomal HMG CoA reductases of liver and intestinal mucosa had similar kinetic characteristics and pH optima. However, the activity of the hepatic enzyme differed with age and sex of the experimental animals while that of the intestinal crypt cells did not. Cholestyramine treatment enhanced the activity of the microsomal HMG CoA reductase in both liver and intestinal mucosa. Reductase activity of the intestinal crypt cells was elevated in both jejunum and ileum. The greatest stimulation, both relatively and absolutely, was observed in the distal half of the jejunum.     

ATP-dependent degradation of 3-hydroxy-3-methylglutaryl coenzyme A reductase in permeabilized cells

A system for the assay of 3-hydroxy-3-methyglutaryl (HMG) coenzyme A (CoA) reductase in digitonin-permeabilized Chinese hamster ovary cells is described. Under these conditions, HMG-CoA reductase remained intact and associated with the endoplasmic reticulum, and values for Km (HMG-CoA), Ki (mevinolin), and active/total activity were similar to those seen in sonicated cell preparations. However, the mechanism by which this rapidly turned over (half-life approximately 2 h) enzyme is degraded was disrupted. Addition of ATP at physiological concentrations to digitonin-permeabilized cells resulted in the rapid, irreversible loss of enzyme activity. Immunoblot analysis showed that this loss of activity was followed by cleavage of the intact 97-kilodalton enzyme to a 68-kilodalton fragment which was distinct from the catalytically active fragments generated by nonspecific proteolysis in sonicated cell homogenates. Assay of a lysosomal marker enzyme confirmed that ATP-mediated inactivation and cleavage of reductase was not due to release of lysosomal proteases. The possible role of ATP in phosphorylation, inactivation, and degradation of reductase is discussed.     

Gonadotropin modulation of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity in desensitized luteinized rat ovary

These studies were done to examine the effect of gonadotropin on rat luteal 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase activity (the rate-limiting step in cholesterol biosynthesis) in ovaries of pregnant mare's serum gonadotropin (PMSG)-human chorionic gonadotropin (hCG) primed rats. Administration of hCG stimulated HMG CoA reductase activity in a time- and dose-dependent manner: significant increases were noted within 4 h, with maximum effects (30-40-fold increases) seen 24 h after hCG (25 IU) administration. This effect was specific in that only LH, of several hormones tested, was as effective as hCG in stimulating HMG CoA reductase activity, and no change in the activity of either liver microsomal HMG CoA reductase or luteal microsomal NADPH-cytochrome c reductase was seen after hCG. The gonadotropin-induced increase in HMG CoA reductase activity seemed to be due to a net increase in enzyme activity, not to a change in the phosphorylated/dephosphorylated state of the enzyme. Pretreatment of animals with aminoglutethimide, an inhibitor of the conversion of cholesterol to steroid (pregnenolone), prevented the hCG-induced rise in HMG CoA reductase activity, whereas treatment with 4-aminopyrazolo[3,4-d]pyrimidine (4-APP), which depletes cellular cholesterol content, led to striking increases in enzyme activity. However, the combined effects of 4-APP and hCG were additive, suggesting that the stimulating effect of hCG on HMG CoA reductase activity is not entirely due to a depletion of cellular sterol content of luteinized ovaries. Similarly, cholesteryl ester and cholesterol syntheses as measured by [14C]acetate conversion were also increased by hCG and 4-APP treatment.(ABSTRACT TRUNCATED AT 250 WORDS)     

Stimulation of the proliferation of the Madin-Darby canine kidney (MDCK) epithelial cell line by high-density lipoproteins and their induction of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity

MDCK Cells seeded on extracellular matrix- (ECM) coated dishes and exposed to medium supplemented with high-density lipoproteins (HDLs, 750 micrograms protein/ml) and transferrin (10 micrograms/ml) have a proliferative rate, final cell density, and morphological appearance similar to those of cells grown in serum-supplemented medium. The mitogenic stimulus provided by HDLs is not limited by the initial cell density at which cultures are seeded, nor is it limited in time, since cells grown in medium supplemented with transferrin and HDLs grew to at least 50 generations. The presence of HDLs in the medium is required in order for cells to survive, since cells actively proliferating in the presence of medium supplemented with HDLs and transferrin begin to die within 2 days after being transferred to medium supplemented only with transferrin. Low-density lipoprotein (LDL) is mitogenic for MDCK cells when present at low concentrations (from 2.5 to 100 micrograms protein/ml). Above 100 micrograms protein/ml, LDL is cytotoxic and therefore cannot support cell proliferation at an optimal rate. The mitogenic effect of HDLs is also observed when cells are maintained on fibronectin-coated dishes. However, the proliferative rate of the cells is suboptimal and cultures cannot be passaged on this substrate indefinitely, as they can be on ECM-coated dishes. A close association between the ability of HDLs to support cell proliferation and their ability to induce the activity of 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase is observed. HMG CoA reductase activity is 18 times higher (70 pmoles/min/10(6) cells) in proliferating cells than in confluent, nondividing cells (4 pmoles/min/10(6) cells). The HMG Coa reductase activity of sparse cells is more sensitive to induction by HDLs (eight-fold higher than control cells) than is the enzyme activity of confluent cells (two-fold higher than control levels). The dose-response relationship between the abilities of HDLs to support proliferation and to induce HMG CoA reductase activity are similar. The time course of the stimulation of proliferation and the increase in enzyme activity of sparse, quiescent cells after exposure to HDLs are parallel. The HMG CoA reductase activity of sparse MDCK cells is induced six-fold by exposure to compactin, a competitive inhibitor of HMG CoA reductase. This induction of HMG CoA reductase is prevented by mevalonic acid, not affected by LDL, and synergistically enhanced by simultaneous exposure to HDLs. HDLs effect a rescue from the cytotoxic effect of compactin, whereas LDL does not.(ABSTRACT TRUNCATED AT 400 WORDS)     

Thiolase engineering for enhanced butanol production in Clostridium acetobutylicum

Biosynthetic thiolases catalyze the condensation of two molecules acetyl‐CoA to acetoacetyl‐CoA and represent key enzymes for carbon–carbon bond forming metabolic pathways. An important biotechnological example of such a pathway is the clostridial n‐butanol production, comprising various natural constraints that limit titer, yield, and productivity. In this study, the thiolase of Clostridium acetobutylicum, the model organism for solventogenic clostridia, was specifically engineered for reduced sensitivity towards its physiological inhibitor coenzyme A (CoA‐SH). A high‐throughput screening assay in 96‐well microtiter plates was developed employing Escherichia coli as host cells for expression of a mutant thiolase gene library. Screening of this library resulted in the identification of a thiolase derivative with significantly increased activity in the presence of free CoA‐SH. This optimized thiolase comprised three amino acid substitutions (R133G, H156N, G222V) and its gene was expressed in C. acetobutylicum ATCC 824 to assess the effect of reduced CoA‐SH sensitivity on solvent production. In addition to a clearly delayed ethanol and acetone formation, the ethanol and butanol titers were increased by 46% and 18%, respectively, while the final acetone concentrations were similar to the vector control strain. These results demonstrate that thiolase engineering constitutes a suitable methodology applicable to improve clostridial butanol production, but other biosynthetic pathways involving thiolase‐mediated carbon flux limitations might also be subjected to this new metabolic engineering approach. Biotechnol. Bioeng. 2013; 110: 887–897. © 2012 Wiley Periodicals, Inc.     

Effects of 25-hydroxycholesterol, cholesterol, and isoprenoid derivatives on the G1 progression in Swiss 3T3 cells

The effect of inhibition of 3-Hydroxy-3-methylglutaryl Coenzyme A reductase (HMG CoA reductase) on cell cycle progression in proliferating 3T3 cells was studied. It was found that short transient exposures to the HMG CoA reductase inhibitor 25-hydroxycholesterol temporarily blocked the cell cycle traverse in the postmitotic half of G1 (G1pm), whereas cells in the subsequent cell cycle phases were unaffected. The kinetics of the cell cycle delay, induced by 25-hydroxycholesterol, resembled the kinetics of the delay induced by serum depletion, which also inhibited the activity of HMG CoA reductase. In contrast to the case of serum depletion, platelet derived growth factor (PDGF), which efficiently prevented the decrease of HMG CoA reductase in serum-free medium, was not capable of preventing the growth inhibitory effect following treatment by 25-hydroxycholesterol. However, cholesterol and two isoprenoids, dolichol and coenzyme Q, were effective in this respect. In addition, dolichol counteracted the cell cycle delay following short periods of serum starvation.     

Effect of Serum Lipoproteins on Growth and Sterol Synthesis in Cultured Rat Brain Glial Cells

Cells dissociated from brains of 1-day-old rats were cultured in medium containing either lipoprotein-deficient serum (LPDS) or LPDS plus various lipoprotein fractions. Increases in number of cells and in DNA content served as a measure of cell growth. Cholesterol synthesis was measured from the incorporation of [14C]acetate into total nonsaponifiable lipids and digitonin-precipitable sterols, and from the activity of the enzyme 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase. The data indicated that cholesterol biosynthesis from acetate was reduced in cells cultured in medium containing either LPDS plus low-density lipoproteins (LDL), high-density lipoproteins (HDL), or total lipoproteins (LP) and that this reduction was accompanied by a reduction in the activity of the HMG CoA reductase and an increase in the esterified sterol content. The reduction in cholesterol synthesis from acetate was maximal in cells cultured in the presence of HDL, whereas the maximal reduction in the activity of HMG CoA reductase occurred in cells cultured in the presence of LP. The presence of LDL or LP in the culture medium enhanced the cell growth but the presence of HDL did not. Esterified sterol content was highest in cells cultured in the medium containing LPDS plus LP and was not detected in cells cultured in LPDS medium. It is inferred from these data that rat brain glial cells in culture are able to utilize cholesterol in lipoproteins, that the presence of LDL in the medium enhances cell growth, and that reduced cholesterol synthesis in the presence of lipoproteins may occur at the HMG CoA reductase step as well as at some other step(s).     

Regulatory effects of sterols and bile acids on hepatic 3-hydroxy-3-methylglutaryl CoA reductase and cholesterol 7alpha-hydroxylase in the rat

Specific activities of the hepatic microsomal enzymes 3-hydroxy-3-methylglutaryl CoA (HMG CoA) reductase and cholesterol 7alpha-hydroxylase were studied in rats fed sterols and bile acids. The administration of bile acids (taurocholate, taurodeoxycholate, taurochenodeoxycholate) at a level of 1% of the diet for 1 wk reduced the activity of HMG CoA reductase. Taurocholate and taurodeoxycholate, but not taurochenodeoxycholate, inhibited cholesterol 7alpha-hydroxylase. Dietary sitosterol produced increases in the specific activity of HMG CoA reductase (3.6-fold) and cholesterol 7alpha-hydroxylase (1.4-fold), and biliary cholesterol concentrations in this group more than doubled. Compared with controls fed the stock diet, the simultaneous administration of sitosterol and taurochenodeoxycholate resulted in a 60% decrease of HMG CoA reductase activity and no change in cholesterol 7alpha-hydroxylase activity or biliary cholesterol concentration. Rats fed sitosterol plus taurocholate had nearly normal HMG CoA reductase activity, but cholesterol 7alpha-hydroxylase was inhibited and biliary cholesterol remained high. Bile acid secretion rates and biliary bile acid composition were similar in controls and sterol-fed animals. In all groups receiving bile acids, biliary secretion of bile acids was nearly doubled and bile acid composition was shifted in the direction of the administered bile acid. It is concluded that the composition of the bile acid pool influences the hepatic concentrations of the rate-controlling enzymes of bile acid synthesis.     

Regulatory effects of dietary sterols and bile acids on rat intestinal HMG CoA reductase

The specific activity (concentration) of microsomal HMG CoA reductase of intestinal crypt cells was studied in rats fed sterols and bile acids, either singly or in combination. It was found that the basal activity of the reductase was not suppressed by the administration of relatively large amounts of bile acid (taurocholate or taurochenodeoxycholate). Bile acids reduced the specific activity of the reductase only in rats in which the activity of the enzyme had first been enhanced by biliary diversion or by sitosterol feeding. In addition, bile acid feeding abolished the diurnal elevation of reductase activity that normally occurs between midnight and 2 a.m. In no case did bile acids reduce enzyme activity below basal levels. A pronounced (60%) reduction of intestinal HMG CoA reductase activity was observed in rats fed cholesterol and bile acid in combination. This reduction in activity could not be ascribed to an increase in intestinal bile acid flux but was associated with an increase in sterol concentration within the intestinal crypt cells. These results indicate that dietary sterols and bile acids both play a role in the regulation of intestinal HMG CoA reductase.     

Cyclic AMP-sensitive activation of hepatic sterol synthesis and 3-hydroxy-3-methylglutaryl coenzyme A reductase.

We previously showed that preincubation of a 10,000 g supernatant (S(10)) from rat liver for 20 min at 37 degrees C dramatically increased the subsequent incorporation of [(14)C]acetate into sterols. No activation was seen with [(14)C]mevalonate as substrate. In the present studies we have examined the effect of preincubation on HMG CoA reductase. When microsomes were isolated from S(10) by calcium precipitation, preincubation of S(10) increased the specific activity of HMG CoA reductase threefold. No activation of HMG CoA reductase was observed in microsomes isolated by ultracentrifugation. Activation was cyclic AMP-sensitive. When cyclic AMP (0.001-1.0 mM) and MgATP (1 mM) were present during the preincubation period, there was little or no activation of HMG CoA reductase activity or of sterol synthesis from acetate. MgATP alone did not prevent activation. Neither cyclic AMP nor MgATP was inhibitory when present only during the assay of sterol synthesis. We propose that the in vitro activation represents the reversal of a physiologic cyclic AMP-mediated mechanism for the control of hepatic HMG CoA reductase. That a phosphoprotein phosphatase may catalyze the activation was supported by the observation that sodium fluoride, an inhibitor of phosphoprotein phosphatases, inhibited the activation. These results suggest that hormone-induced changes in the cellular level of cyclic AMP may regulate the activity of HMG CoA reductase and the rate of hepatic cholesterol synthesis.