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.     

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.     

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.     

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.     

Properties of 3-Hydroxy-3-methylglutaryl-coenzyme A Reductase in Villous and Crypt Cells of the Rat Small Intestine

Some properties of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase in microsomes of villous and crypt cells from the jejunal and ileal epithelia of rats fed commercial pellet were studied. The optimum pH of the microsomal reductase from villi and crypts was 7.0~7.2 and the Km for HMG-CoA was 41.7 µm. The reductase specifically required dithiothreitol for its activity. The activity was higher in ileal populations than in the jejunum. Responses of the reductase in the villous fraction to feeding cholesterol and taurocholate in combination or cholestyramine resembled those observed in crypt cells. Thus, the properties of microsomal HMG-CoA reductase in villous and crypt cells from the small intestine are similar each other, and they are possibly the same enzyme.     

Coordinate regulation of 3-hydroxy-3-methylglutaryl-coenzyme A synthase, 3-hydroxy-3-methylglutaryl-coenzyme A reductase, and prenyltransferase synthesis but not degradation in HepG2 cells

Human hepatoma HepG2 cells were used to demonstrate coordinate regulation of three enzymes of cholesterol synthesis under a variety of conditions. Addition of either delipidized serum and mevinolin or low density lipoprotein, 25-hydroxycholesterol, or mevalonic acid to HepG2 cells resulted in rapid changes both in the levels of the mRNAs and in the rates of synthesis of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) synthase, HMG-CoA reductase, and farnesyl pyrophosphate synthetase (prenyltranferase). In all cases, the changes in mRNA levels were paralleled by changes in the rates of specific protein synthesis. Pulse-chase techniques were used to determine the half-lives of all three proteins. Addition of low density lipoprotein to the media during the chase increased the rate of degradation of HMG-CoA reductase 4.6-fold but had no affect on the half-lives of HMG-CoA synthase or prenyltransferase. Therefore, we conclude that the coordinate regulation of these three enzymes under a variety of conditions occurs at the level of enzyme synthesis and not at the level of protein stability.     

Activation of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase during high fat diet feeding

The liver plays a central role in regulating cholesterol homeostasis. High fat diets have been shown to induce obesity and hyperlipidemia. Despite considerable advances in our understanding of cholesterol metabolism, the regulation of liver cholesterol biosynthesis in response to high fat diet feeding has not been fully addressed. The aim of the present study was to investigate mechanisms by which a high fat diet caused activation of liver 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA reductase) leading to increased cholesterol biosynthesis. Mice were fed a high fat diet (60% kcal fat) for 5 weeks. High fat diet feeding induced weight gain and elevated lipid levels (total cholesterol and triglyceride) in both the liver and serum. Despite cholesterol accumulation in the liver, there was a significant increase in hepatic HMG-CoA reductase mRNA and protein expression as well as enzyme activity. The DNA binding activity of sterol regulatory element binding protein (SREBP)-2 and specific protein 1 (Sp1) were also increased in the liver of mice fed a high fat diet. To validate the in vivo findings, HepG2 cells were treated with palmitic acid. Such a treatment activated SREBP-2 as well as increased the mRNA and enzyme activity of HMG-CoA reductase leading to intracellular cholesterol accumulation. Inhibition of Sp1 by siRNA transfection abolished palmitic acid-induced SREBP-2 and HMG-CoA reductase mRNA expression. These results suggest that Sp1-mediated SREBP-2 activation contributes to high fat diet induced HMG-CoA reductase activation and increased cholesterol biosynthesis. This may play a role in liver cholesterol accumulation and hypercholesterolemia.     

Altered kinetic properties of rat liver 3-hydroxy-3-methylglutaryl coenzyme A reductase following dietary manipulations

The microsomal enzyme 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase catalyzes the rate-limiting step in the cholesterogenic pathway and was proposed to be composed in situ of 2 noncovalently linked subunits (Edwards, P.A., Kempner, E.S., Lan, S.-F., and Erickson, S.K. (1985) J. Biol. Chem. 260, 10278-10282). In the present report, the activities and kinetic properties of HMG-CoA reductase in microsomes isolated from livers of rats fed on diets supplemented with either ground Amberlite XAD-2 ("X"), cholestyramine/mevinolin ("CM"), or unsupplemented, normal rat chow ("N"), were compared. The specific activities of HMG-CoA reductase in X and CM microsomes were, respectively, 5- and 83-fold higher than that of N microsomes. In NADPH-dependent kinetics of HMG-CoA reductase activated with 4.5 mM GSH, the concentration of NADPH required for half-maximal velocity (S0.5) was 209 +/- 23, 76 +/- 23, and 40 +/- 4 microM for the N, X, and CM microsomes, respectively. While reductase from X microsomes displays cooperative kinetics toward NADPH (Hill coefficient (nH) = 1.97 +/- 0.07), the enzyme from CM microsomes does not (nH = 1.04 +/- 0.07). Similarly to HMG-CoA reductase from CM microsomes, the freeze-thaw solubilized enzyme ("SOL") displays no cooperativity toward NADPH and its Km for this substrate is 34 microM. At 4.5 mM GSH, HMG-CoA reductase from X, CM, and SOL preparations has a similar Km value for [DL]-HMG-CoA, ranging between 13-16 microM, while reductase from N microsomes had a higher Km value (42 microM) for this substrate. No cooperativity towards HMG-CoA was observed in any of the tested enzyme preparations. Immunoblotting analyses of the different preparations demonstrated that the observed altered kinetics of HMG-CoA reductase in the microsomes is not due to preferential proteolytic cleavage of the native 97-100 kDa subunit of the enzyme to the noncooperative 50-55 kDa species. Moreover, it was found that the ratio enzymatic activity/immunoreactivity of the reductase increased in the order N less than X less than CM approximately equal to SOL, indicating that the activity per reductase molecule increases with the induction of the enzyme. These results are compatible with a model suggesting that dietary induction of hepatic HMG-CoA reductase may change the state of functional aggregation of its subunits.     

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.     

Reversible inactivation-reactivation of 3-hydroxy-3-methylglutaryl coenzyme A reductase of rat intestine

3-Hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase in the ileum of rats was inactivated by Mg2+-ATP and reversibly reactivated by cytoplasmic activator from the liver. The mevalonate kinase reaction was presumably not involved in this inactivation. Studies of nucleotide specificity for the inactivation revealed that ATP was most effective in the reaction among the nucleotides tested. In contrast to the hepatic microsomal HMG-CoA reductase, more than one-half of intestinal reductase existed in an active form. These observations indicated the presence of phosphorylation-dephosphorylation mechanism for modulation of intestinal HMG-CoA reductase.     

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.     

Regulation of 3-hydroxy-3-methylglutaryl coenzyme A reductase mRNA levels by L-triiodothyronine

In hypophysectomized rats, hepatic 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase activity, immunoreactive 97-kilodalton (97-kDa) protein, and mRNA were all reduced to undetectable levels. Administration of triiodothyronine (T3) resulted in large increases in all three after a 36-h lag period. HMG-CoA reductase activity, immunoreactive 97-kDa protein levels, and reductase mRNA levels were tightly correlated. Feeding hypophysectomized rats diets containing the bile acid sequestrant colestipol, together with the potent reductase inhibitor mevinolin, resulted in an increase in HMG-CoA reductase activity similar to that seen with T3 but a lesser stimulation of reductase mRNA levels. These results suggest that agents which cause depletion of mevalonate-derived products may share in part with T3 a common mechanism for increasing levels of HMG-CoA reductase activity in order to satisfy cellular needs for these products. Dexamethasone treatment, which is known to prevent the T3-mediated stimulation of reductase activity, caused a marked decrease in 97-kDa immunoreactive material but had little effect on reductase mRNA levels.     

Cytoplasmic 3-hydroxy-3-methylglutaryl coenzyme A synthase from the hamster. I. Isolation and sequencing of a full-length cDNA

We here report the isolation and nucleotide sequencing of a full-length 3.3-kilobase cDNA for the cytoplasmic form of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) synthase, a regulated enzyme in the cholesterol biosynthetic pathway. The cDNA was isolated from UT-1 cells, a compactin-resistant line of Chinese hamster ovary cells. UT-1 cells produce large amounts of mRNA for HMG-CoA synthase and the next enzyme in the pathway, HMG-CoA reductase, as a result of growth in the presence of compactin, a competitive inhibitor of the reductase. The identity of the cDNA for HMG-CoA synthase was confirmed through comparison of the NH2-terminal amino acid sequence predicted from the cDNA with that determined chemically from the purified enzyme. Anti-peptide antibodies directed against the amino acid sequence predicted from the cDNA precipitated HMG-CoA synthase activity from liver cytoplasm. The feeding of cholesterol to hamsters led to a decrease of more than 85% in the amount of mRNA for HMG-CoA synthase and HMG-CoA reductase in hamster liver. These data indicate that the mRNAs for cytoplasmic HMG-CoA synthase and for HMG-CoA reductase, two sequential enzymes in the cholesterol biosynthetic pathway, are coordinately regulated by cholesterol.     

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.     

Regulation of mevalonate 5-pyrophosphate decarboxylase in isolated cells from chick intestinal epithelium.

The present studies were undertaken to determine whether mevalonate 5-pyrophosphate decarboxylase (EC 4.1.1.33) is subject to physiological regulation in the intestinal mucosa. Activity was determined in epithelial cells isolated in a villus-to-crypt gradient from chicks fed on different diets in order to vary the sterol flux across the intestinal epithelium. When animals were fed on cholesterol, decarboxylase activity was decreased in all the cell fractions studied, although percentages of inhibition were maximum in crypts of jejunum and ileum. In contrast, decreased sterol flux as a consequence of cholestyramine feeding stimulated decarboxylase activity, especially in villi of the duodenum, where values increased 3-fold with respect to controls. On the other hand, the total cellular sterol content was significantly increased by the cholesterol diet. In duodenum and jejunum, 20-30% of the total cholesterol was in the esterified form under these conditions. However, dietary cholestyramine did not significantly affect amounts of total cellular cholesterol in any of the cell fractions. These results demonstrate that mevalonate 5-pyrophosphate decarboxylase activity changes considerably under different dietary situations and that the existence of secondary sites in the physiological regulation of sterol synthesis in the intestinal mucosa should be considered.     

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.