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.     

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.     

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.     

Diurnal Rhythms of Rat Liver 3-Hydroxy-3-Methylglutaryl-Co A Reductase and D Site-Binding Protein mRNA Levels Are Not Abolished by Adrenalectomy

The effect of glucocorticoids on the diurnal rhythm of rat liver 3-hydroxy-3-methyl-glutaryl-CoA reductase (HMGR) has been controversial. Also, diurnal variation of D site-binding protein (DBP) has been suggested to be under a negative control of glucocorticoids. Here we have re-evaluated the effects of adrenal hormones on these rhythms at the level of gene expression. Sham-operated and bilaterally adrenalectomized rats were killed at 4-hr intervals and total RNA from each liver was subjected to Northern blot analysis. Diurnal variation patterns of HMGR and DBP mRNA levels in adrenalectomized rats were substantially identical to those in sham-operated rats, although DBP mRNA levels in adrenalectomized rats were slightly more abundant than in control rats. HMGR mRNA levels in adrenalectomized rats in the dark period were insensitive to a single injection of adrenal hormones, whereas the augmented levels of DBP mRNA in these animals were returned to the control levels by this treatment, indicating that glucocorticoids are prone to decrease the amplitude of variation in the DBP gene expression. The present results suggest that adrenal hormones are not critical for the generation of diurnal rhythms of these mRNAs.     

Diurnal Rhythms of Rat Liver 3-Hydroxy-3-Methylglutaryl-Co A Reductase and D Site-Binding Protein mRNA Levels Are Not Abolished by Adrenalectomy

The effect of glucocorticoids on the diurnal rhythm of rat liver 3-hydroxy-3-methyl-glutaryl-CoA reductase (HMGR) has been controversial. Also, diurnal variation of D site-binding protein (DBP) has been suggested to be under a negative control of glucocorticoids. Here we have re-evaluated the effects of adrenal hormones on these rhythms at the level of gene expression. Sham-operated and bilaterally adrenalectomized rats were killed at 4-hr intervals and total RNA from each liver was subjected to Northern blot analysis. Diurnal variation patterns of HMGR and DBP mRNA levels in adrenalectomized rats were substantially identical to those in sham-operated rats, although DBP mRNA levels in adrenalectomized rats were slightly more abundant than in control rats. HMGR mRNA levels in adrenalectomized rats in the dark period were insensitive to a single injection of adrenal hormones, whereas the augmented levels of DBP mRNA in these animals were returned to the control levels by this treatment, indicating that glucocorticoids are prone to decrease the amplitude of variation in the DBP gene expression. The present results suggest that adrenal hormones are not critical for the generation of diurnal rhythms of these mRNAs.     

Regulation of cholesterol 7 alpha-hydroxylase in the liver. Cloning, sequencing, and regulation of cholesterol 7 alpha-hydroxylase mRNA.

Monospecific antibody against purified rat liver cholesterol 7 alpha-hydroxylase cytochrome P-450 was used to screen a lambda gt11 cDNA library constructed from immuno-enriched polysomal RNA of cholestyramine-treated female rat liver. Two types of cDNA clones differing in the length of the 3'-untranslated region were identified, and DNA sequences were determined. The full length clone contains 3561 base pairs plus a long poly(A) tail. The amino acid sequence deduced from the open reading frame revealed a unique P-450 protein containing 503 amino acid residues which belonged to a new gene family designated family VII or CYP7. Southern blot hybridization experiments indicated that the minimal size of P-450 VII gene was 11 kilobase pairs (kb), and there was probably only one gene in this new family. Northern blot hybridization using specific cDNA probes revealed at least two major mRNA species of about 4.0 kb and 2.1 kb, respectively. These two mRNA species may be derived from the use of different polyadenylation signals and reverse-transcribed to two types of cDNA clones. Cholesterol 7 alpha-hydroxylase mRNAs were induced 2- to 3-fold in rat liver by cholestyramine treatment. The mRNA level was rapidly reduced upon the removal of the inducer. Similarly, cholesterol feeding induced enzyme activity, protein, and mRNA levels in the rat by 2-fold, suggesting that cholesterol is an important regulator of cholesterol 7 alpha-hydroxylase in the liver. On the other hand, dexamethasone and pregnenolone-16 alpha-carbonitrile drastically reduced the activity, protein, and mRNA levels. These experiments suggest that the induction of cholesterol 7 alpha-hydroxylase activity by cholestyramine or cholesterol and inhibition of cholesterol 7 alpha-hydroxylase activity by bile acid feedback are results of the rapid turnover of cholesterol 7 alpha-hydroxylase enzyme and mRNA levels.     

Mevalonolactone inhibits the rate of synthesis and enhances the rate of degradation of 3-hydroxy-3-methylglutaryl coenzyme A reductase in rat hepatocytes

3-Hydroxy-3-methylglutaryl(HMG)-coenzyme A reductase purified from rat liver in the absence of protease inhibitors is composed of two distinct polypeptides of Mr = 51,000 and 52,500. Antibody raised to enzyme purified from rats fed a diet supplemented with cholestyramine and mevinolin inactivated HMG-CoA reductase. The antibody specifically precipitated a polypeptide of Mr = 94,000 from rat liver cells that had been previously incubated with [35S]methionine. The immunoprecipitation of the 35S-labeled polypeptide of Mr = 94,000 was prevented by addition of unlabeled pure HMG-CoA reductase (Mr = 51,000 and 52,500). Incubation of rat liver cells with mevalonolactone resulted in a decreased activity of HMG-CoA reductase and in a 40% decrease in the rate of incorporation of [35S]methionine into the immunoprecipitable reductase polypeptide of Mr = 94,000. In pulse-chase experiments, mevalonolactone enhanced the rate of degradation of the Mr = 94,000 polypeptide 3-fold. We propose that endogenous microsomal HMG-CoA reductase has a subunit of Mr = 94,000 and that the synthesis and degradation of this polypeptide are regulated by either mevalonolactone or, more likely, a product of mevalonolactone metabolism.     

Roles of nuclear receptors in the up-regulation of hepatic cholesterol 7alpha-hydroxylase by cholestyramine in rats

Nuclear receptors are involved in regulating the expression of cholesterol 7alpha-hydroxylase (CYP7A1), however, their roles in the up-regulation of CYP7A1 by cholestyramine (CSR) are still unclear. In the present study, male Wistar rats were divided into four groups and fed [high sucrose + 10% lard diet] (H), [H + 3% CSR diet] (H + CSR), [H + 0.5% cholesterol + 0.25% sodium cholate diet] (C), or [C + 3% CSR diet] (C + CSR) for 2 weeks. Cholestyramine decreased serum and liver cholesterol levels significantly in rats fed C-based diets, but had no effect on these parameters in rats fed H-based diets. Cholestyramine raised hepatic levels of CYP7A1 mRNA and activity in both groups. The gene expression of hepatic ATP-binding cassettes A1 and G5, regulated by liver X receptor (LXR), were unchanged and down-regulated by cholestyramine, respectively. The mRNA levels of the hepatic ATP-binding cassette B11 and short heterodimer partner (SHP), regulated by farnesoid X receptor (FXR), were not changed by cholestyramine. C-based diets, which contained cholesterol and cholic acid, increased SHP mRNA levels compared to H-based diets. Consequently, in rats fed the C+CSR diet, hepatic FXR was activated by dietary bile acids, but the hepatic CYP7A1 mRNA level was increased 16-fold compared to that in rats fed an H diet. These results suggest that cholestyramine up-regulates the expression of CYP7A1 independently via LXR- or FXR-mediated pathways in rats.     

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.     

Hepatic P-450 cholesterol 7 alpha-hydroxylase. Regulation in vivo at the protein and mRNA level in response to mevalonate, diurnal rhythm, and bile acid feedback

Cholesterol 7 alpha-hydroxylase (P-450 Ch7 alpha) catalyzes the first and rate-limiting step in the hepatic conversion of cholesterol to bile acids. P-450 Ch7 alpha activity in rat liver is regulated at three independent levels: (a) feedback inhibition by bile acids (long term regulation); (b) midterm regulation through the diurnal cycle; (c) short term modulation by hormones and dietary factors. P-450 Ch7 alpha was purified to apparent homogeneity and in active form (turnover number = 10-15 min-1 P-450(-1)) from cholestyramine-fed female rats, and rabbit anti-P-450 Ch7 alpha polyclonal antibodies were then prepared. Liver microsomes were isolated from rats fed normal diet or diet containing the bile acid sequestrant cholestyramine and were then killed at either the apex (midnight) or nadir (noon) of the diurnal rhythm of P-450 Ch7 alpha activity. Direct comparison of microsomal P-450 Ch7 alpha enzyme activity levels with P-450 Ch7 alpha protein (Western blotting) and mRNA levels (Northern and slot blots) revealed that the 2.5-3-fold induction of P-450 Ch7 alpha activity with cholestyramine feeding can be fully accounted for by an increase in P-450 Ch7 alpha protein and mRNA. Turnover numbers of 7-9 nmol of 7 alpha-hydroxycholesterol/min/nmol of microsomal P-450 Ch7 alpha were observed for both induced and uninduced animals. Similarly, the postmidnight decrease in enzyme activity could be generally accounted for by a decrease in P-450 Ch7 alpha protein and mRNA, suggesting that these species have relatively short half-lives. The short term regulation of P-450 Ch7 alpha was examined following treatment with the cholesterol precursor mevalonic acid. A 2.5-fold increase in hepatic microsomal P-450 Ch7 alpha activity occurred within 150 min and was accompanied by a significant elevation of P-450 Ch7 alpha mRNA (up to 3-6-fold increase). These findings establish that hepatic cholesterol 7 alpha-hydroxylase activity is regulated in response to long term, midterm, and short term control factors primarily at a pretranslational level and that this regulation is of greater importance than proposed mechanisms based on allosteric effects of bile acids on P-450 Ch7 alpha protein, changes in cholesterol availability, or reversible phosphorylation of a putative P-450 Ch7 alpha phosphoprotein.     

Effect of estrogens on β-hydroxy-β-methylglutaryl coenzyme a reductase activity and cholesterol levels

The effect of estrogens on hepatic β-hydroxy-β-methylglutaryl coenzyme A reductase activity and cholesterol in serum and liver of ovarietcomized rats on normal diet, 2% cholestyramine diet or 2% cholesterol diet was investigated. Estrogen administration to ovariectomized rats on normal diet resulted in increased reductase activity and was correlated with decreased serum cholesterol and increased liver cholesterol levels wlth mestranol (ME), ethinyl estradiol (EE) and estradiol benzoate (EB, 250 μg) but increased serum and liver cholesterol levels with 25 μg and 100 μg EB administration. The increased stimulation of reductase activity by estrogen administration was absolished when rats were fed a 2% cholesterol diet. Cholestyramine feeding markedly increased reductase activity in livers of ovariectomized rats. These studies show that estrogens are not absolutely required for the stimulation of reductase activity and therefore is consistent with the model in which cholesterol functions as a feedback repressor of reductase activity.     

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.     

Properties of purified rat hepatic 3-hydroxy-3-methylglutaryl coenzyme A reductase and regulation of enzyme activity

3-Hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase from rat liver microsomes has been purified to apparent homogeneity with recoveries of approximately 50%. The enzyme obtained from rats fed a diet supplemented with cholestyramine had specific activities of approximately 21,500 nmol of NADPH oxidized/min/mg of protein. After amino acid analysis a specific activity of 31,000 nmol of NADPH oxidized/min/mg of amino acyl mass was obtained. The s20,w for HMG-CoA reductase was 6.14 S and the Stokes radius was .39 nm. The molecular weight of the enzyme was 104,000 and the enzyme subunit after sodium dodecyl sulfate-polyacrylamide gel electrophoresis was 52,000. Antibodies prepared against the homogeneous enzyme specifically precipitated HMG-CoA reductase from crude and pure fractions of the enzyme. Incubation of rat hepatocytes for 3 h in the presence of lecithin dispersions, compactin, or rat serum resulted in significant increases in the specific activity of the microsomal bound reductase. Immunotitrations indicated that in all cases these increases were associated with an activated form of the reductase. However activation of the enzyme accounted for only a small percentage of the total increase in enzyme activity; the vast majority of the increase was apparently due to an increase in the number of enzyme molecules. In contrast, when hepatocytes were incubated with mevalonolactone the lower enzyme activity which resulted was primarily due to inactivation of the enzyme with little change in the number of enzyme molecules. Immunotitrations of microsomes obtained from rats killed at the nadir or peak of the diurnal rhythm of 3-hydroxy-3-methylglutaryl-CoA reductase indicated that the rhythm results both from enzyme activation and an increased number of reductase molecules.     

Tissue-specific expression of genes encoding apolipoprotein E and apolipoprotein A-I in rabbits

We determined the site of synthesis of apolipoprotein (apo) E and apo-A-I in rabbit by measuring in vitro translational activity of their mRNAs from the liver and from the intestine. Poly(A+) RNA isolated from liver and intestinal epithelium of rabbits fed either a chow diet or a cholesterol-rich diet was translated in vitro in the rabbit reticulocyte lysate system using [35S] methionine as the labeled precursor. Newly synthesized apolipoproteins were immunoprecipitated with specific antisera and quantitated after electrophoresed on 10% polyacrylamide slab gels in the presence of 0.2% sodium dodecyl sulfate. The levels of liver apo-E and apo-A-I mRNAs from chow-fed rabbits are 0.41 and 0.002% of total translatable mRNA, respectively. The level of liver apo-A-I mRNA in the rabbit is approximately 500-fold lower than the reported level of apo-A-I mRNA in rat and human livers. Rabbit intestinal apo-E and apo-A-I mRNAs levels are 0.0036 and 0.67%, respectively. Our results indicate that in rabbits apo-E is synthesized primarily in the liver and that apo-A-I is synthesized primarily in the intestine. When rabbits are fed a cholesterol-rich diet, liver and intestinal apo-E in mRNA levels and intestinal apo-A-I mRNA levels are not changed. In contrast, the liver apo-A-I mRNA level increases 5-fold in response to the cholesterol-rich diet. However, because the intestinal liver apo-A-I mRNA level is so low, the 5-fold induction only increases liver mRNA levels to 2.7% of the corresponding intestinal apo-A-I mRNA level.     

In situ determination of the functional size of hepatic 3-hydroxy-3-methylglutaryl-CoA reductase by radiation inactivation analysis

Radiation inactivation analysis of liver pieces yielded a target size of 210 kDa for hepatic 3-hydroxy-3-methylglutaryl CoA (HMG-CoA) reductase [S)-mevalonate:NADP+ oxidoreductase (CoA-acylating), EC 1.1.1.34) from rats fed a normal diet. Feeding a diet containing mevinolin and colestipol, which causes a marked increase in enzyme activity, resulted in a reduction of the target size to 120 kDa. These results are consistent with those obtained by radiation inactivation and immunoblotting analysis of isolated microsomes and suggest that the increase in HMG-CoA reductase activity caused by these dietary agents is accompanied by a change from a dimer to a monomer form of the enzyme.