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.     

Differential regulation of the expression of 3-hydroxy-3-methylglutaryl coenzyme A reductase, synthase, and low density lipoprotein receptor genes.

The ability of mitogenic stimulation of human T lymphocytes to alter the expression of genes involved in sterol metabolism was examined. Messenger RNA levels for 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, HMG-CoA synthase, and low density lipoprotein (LDL) receptor were quantified in resting and mitogen-stimulated T lymphocytes by nuclease protection assay. Mitogenic stimulation increased HMG-CoA synthase mRNA levels by 5-fold and LDL receptor by 4-fold when cells were cultured in lipoprotein-depleted medium whereas HMG-CoA reductase gene expression was not significantly increased. When cultures were supplemented with concentrations of low density lipoprotein sufficient to saturate LDL receptors, expression of all three genes was inhibited in resting lymphocytes, as effectively as was noted with fibroblasts. Similarly, LDL down-regulated gene expression in mitogen-activated lymphocytes so that mitogenic stimulation did not increase either HMG-CoA reductase or synthase mRNA levels, although LDL receptor gene expression was enhanced. These results indicate that expression of three of the genes involved in sterol metabolism is differentially regulated by LDL and mitogenic stimulation. Moreover, the increase in rates of endogenous sterol synthesis and the activity of HMG-CoA reductase in mitogen-stimulated T lymphocytes cannot be accounted for by increases in HMG-CoA reductase mRNA levels.     

Differential translational effects of myristic acid and eicosapentaenoic acid on 3-hydroxy-3-methylglutaryl-CoA reductase from Reuber H35 hepatoma cells

The mechanisms by which saturated and polyunsaturated fatty acids may exert their effects on levels of blood cholesterol and human atherosclerosis have not been fully established. In this work, we studied the translational effects of myristic (14:0) and eicosapentaenoic (20:5) acids on 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase from Reuber H35 hepatoma cells. This enzyme is an intrinsic membrane, 96-kDa protein whose proteolysis releases an enzymatically active, 52- to 56-kDa, soluble fragment. We optimized an immunoblot procedure for quantifying small amounts of both the native and the soluble forms of HMG-CoA reductase from Reuber H35 hepatoma cells. We demonstrated that the upregulation of HMG-CoA reductase by a acid is due to an increase of the HMG-CoA reductase protein; therefore, protein synthesis would be required for the increase of HMG-CoA reductase activity caused by this fatty acid. In contrast, the downregulation of HMG-CoA reductase caused by eicosapentaenoic acid is not due to decreased protein synthesis, since similar levels of protein were found in the presence and absence of this fatty acid. Results obtained with cycloheximide as a protein-synthesis inhibitor confirm these findings.     

真菌中脱落酸生物合成代谢关键酶的初步研究*

应用逆转录PCR(RT-PCR)技术测定脱落酸产生菌Botrytis cinerea 3-羟-3-甲基戊二酰CoA(HMG-CoA)还原酶mRNA含量,结果表明经诱变筛选得到的脱落酸高产菌HMG-CoA还原酶含量显著高于野生菌,提示HMG-CoA还原酶可能为真菌ABA生物合成的关键酶。     

Phosphorylation of hepatic 3-hydroxy-3-methylglutaryl coenzyme A reductase and modulation of its enzymic activity by calcium-activated and phospholipid-dependent protein kinase

A calcium-activated and phospholipid-dependent protein kinase (protein kinase C) catalyzes the phosphorylation of both insoluble microsomal (Mr approximately 100,000) and purified soluble (Mr = 53,000) 3-hydroxy-3-methylglutaryl coenzyme A reductase. The phosphorylation and concomitant inactivation of enzymic activity of HMG-CoA reductase was absolutely dependent on Ca2+, phosphatidylserine, and diolein. Dephosphorylation of phosphorylated HMG-CoA reductase was associated with the loss of protein bound radioactivity and reactivation of enzymic activity. Maximal phosphorylation of purified HMG-CoA reductase was associated with the incorporation of 1.05 +/- 0.016 mol of phosphate/mol of native form of HMG-CoA reductase (Mr approximately 100,000). The apparent Km for purified HMG-CoA reductase and histone H1 was 0.08 mg/ml, and 0.12 mg/ml, respectively. The tumor-promoting phorbol ester, phorbol 12-myristate 13-acetate stimulated the protein kinase C-catalyzed phosphorylation of HMG-CoA reductase. Increased phosphorylation of HMG-CoA reductase by phorbol 12-myristate 13-acetate suggests a possible in vivo protein kinase C-mediated mechanism for the short-term regulation of HMG-CoA reductase activity. The identification of the protein kinase C system in addition to the reductase kinase-reductase kinase kinase bicyclic cascade systems for the modulation of the enzymic activity of HMG-CoA reductase may provide new insights into the molecular mechanisms involved in the regulation of cholesterol biosynthesis.     

3-Hydroxy-3-methylglutaryl-coenzyme A reductase and T cell receptor alpha subunit are differentially degraded in the endoplasmic reticulum.

3-Hydroxy-3-methylglutaryl-coenzyme A reductase (HMG-CoA reductase) is located in the endoplasmic reticulum (ER) and responds to rapid degradation which is regulated by mevalonate or sterols. T cell antigen receptor alpha chain (TCR alpha) is also known to be rapidly degraded within the ER. In both cases, the membrane domains of the proteins have a crucial role in their rapid degradation. In order to investigate protein degradation in the ER, we compared the degradation of HMG-CoA reductase and TCR alpha in the same Chinese hamster ovary cells. Among the protease inhibitors tested, N-acetyl-leucyl-leucyl-methioninal blocks the degradation of HMG-CoA reductase and also inhibits the degradation of TCR alpha. On the other hand, N-tosyl-L-phenylalanine chloromethyl ketone and N-carbobenzoxy-L-phenylalanine chloromethyl ketone inhibit the degradation of TCR alpha but have no effect on the degradation of HMG-CoA reductase. Diamide, a thiol-oxidizing agent, blocks the degradation of both HMG-CoA reductase and TCR alpha. Perturbation of cellular Ca2+ attenuates the rapid degradation of HMG-CoA reductase but does not affect the degradation of TCR alpha. Furthermore, thapsigargin, a selective ER Ca(2+)-ATPase inhibitor, and Co2+, a potent Ca2+ antagonist, increase the half-life of HMG-CoA reductase but not that of TCR alpha. Energy inhibitors diminish the rapid degradation of HMG-CoA reductase but not that of TCR alpha. These results suggest that although HMG-CoA reductase and TCR alpha appear to be degraded in the same subcellular compartment, the mechanisms responsible for degradation differ.     

Allosteric activation of rat liver cytosolic 3-hydroxy-3-methylglutaryl coenzyme A reductase kinase by nucleoside diphosphates

Extensively purified rat liver cytosolic 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase kinase was used to examine the role of ADP in inactivation of HMG-CoA reductase (EC 1.1.1.34). Solubilized HMG-CoA reductase was a suitable substrate for HMG-CoA reductase kinase. At sufficiently high concentrations of solubilized HMG-CoA reductase, reductase kinase activity approached that measured using microsomal HMG-CoA reductase as substrate. Inactivation of solubilized HMG-CoA reductase by HMG-CoA reductase kinase required both MgATP and ADP. Other nucleoside diphosphates, including alpha, beta-methylene-ADP, could replace ADP. HMG-CoA reductase kinase catalyzed phosphorylation of bovine serum albumin fraction V by [gamma-32P]ATP. This process also required a nucleoside diphosphate (e.g. alpha, beta-methylene-ADP). Nucleoside diphosphates thus act on HMG-CoA reductase kinase, not on HMG-CoA reductase. For inactivation of HMG-CoA reductase, the ability of nucleoside triphosphates to replace ATP decreased in the order ATP greater than dATP greater than GTP greater than ITP, UTP. TTP and CTP did not replace ATP. Both for inactivation of HMG-CoA reductase and for phosphorylation of bovine serum albumin protein, the ability of nucleoside diphosphates to replace ADP decreased in the order ADP greater than CDP, dADP greater than UDP. GDP did not replace ADP. Nucleoside di- and triphosphates thus appear to bind to different sites on HMG-CoA reductase kinase. Nucleoside diphosphates act as allosteric activators of HMG-CoA reductase kinase. For inactivation of HMG-CoA reductase by HMG-CoA reductase kinase, Km for ATP was 140 microM and the activation constant, Ka, for ADP was 1.4 mM. The concentration of ADP required to modulate reductase kinase activity in vitro falls within the physiological range. Modulation of HMG-CoA reductase kinase activity, and hence of HMG-CoA reductase activity, by changes in intracellular ADP concentrations thus may represent a control mechanism of potential physiological significance.     

Treatment of CHO-K1 cells with 25-hydroxycholesterol produces a more rapid loss of 3-hydroxy-3-methylglutaryl-coenzyme A reductase activity than can be accounted for by enzyme turnover

A key enzyme in the regulation of mammalian cellular cholesterol biosynthesis is 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMG-CoA reductase). It is well established that treatment with the compound 25-hydroxycholesterol lowers HMG-CoA reductase activity in cultured Chinese hamster ovary (CHO-K1) cells. After brief incubation (0-4 h) with 25-hydroxycholesterol (0.5 microgram/ml), cellular HMG-CoA reductase activity is decreased to 40% of its original level. This also occurs in the presence of exogenous mevinolin, a competitive inhibitor of HMG-CoA reductase which has previously been shown to inhibit its degradation. The inhibition of HMG-CoA reductase activity by 25-hydroxycholesterol is complete after 2 h. Radio-immune precipitation analysis of the native enzyme under these conditions shows a degradation half-life which is considerably longer than that of the observed inhibition. Studies with sodium fluoride, phosphatase 2A, bacterial alkaline phosphatase and calf alkaline phosphatase indicate that the observed loss of activity is not due to phosphorylation. These data are not consistent with described mechanisms of HMG-CoA reductase activity regulation by phosphorylation or degradation but are consistent with a novel mechanism that regulates the catalytic efficiency of this enzyme.     

3-Hydroxy-3-methylglutaryl-coenzyme A reductase from Haloferax volcanii: purification, characterization, and expression in Escherichia coli.

Prior work from this laboratory characterized eukaryotic (hamster) and eubacterial (Pseudomonas mevalonii) 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductases. We report here the characterization of an HMG-CoA reductase from the third domain, the archaea. HMG-CoA reductase of the halobacterium Haloferax volcanii was initially partially purified from extracts of H. volcanii. Subsequently, a portion of the H. volcanii lovastatin (formerly called mevinolin) resistance marker mev was subcloned into the Escherichia coli expression vector pT7-7. While no HMG-CoA reductase activity was detectable following expression in E. coli, activity could be recovered after extracts were exposed to 3 M KCl. Following purification to electrophoretic homogeneity, the specific activity of the expressed enzyme, 24 microU/mg, equaled that of homogeneous hamster or P. mevalonii HMG-CoA reductase. Activity was optimal at pH 7.3. Kms were 66 microM (NADPH) and 60 microM [(S)-HMG-CoA]. (R)-HMG-CoA and lovastatin inhibited competitively with (S)-HMG-CoA. H. volcanii HMG-CoA reductase also catalyzed the reduction of mevaldehyde [optimal activity at pH 6.0; Vmax 11 microU/mg; Kms 32 microM (NADPH), 550 microM [(R,S)-mevaldehyde]] and the oxidative acylation of mevaldehyde [optimal activity at pH 8.0; Vmax 2.1 microU/mg; Kms 350 microM (NADP+), 300 microM (CoA), 470 microM [(R,S)-mevaldehyde]]. These properties are comparable to those of hamster and P. mevalonii HMG-CoA reductases, suggesting a similar catalytic mechanism.     

8-bromoadenosine cyclic 3',5'-phosphate rapidly increases 3-hydroxy-3-methylglutaryl coenzyme A reductase mRNA in human granulosa cells: role of cellular sterol balance in controlling the response to tropic stimulation

Exposure of cultured human granulosa cells to 8-bromoadenosine cyclic 3',5'-phosphate (8-bromo-cAMP) resulted in a rapid increase in the content of the mRNA for 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, a rate-limiting enzyme in the de novo synthesis of cholesterol. HMG-CoA reductase mRNA levels increased within 2 h of stimulation and remained elevated for at least 6 h. Treatment of granulosa cells with 25-hydroxycholesterol, a soluble cholesterol analogue, in combination with aminoglutethimide to block conversion of cellular sterols to pregnenolone, resulted in suppression of HMG-CoA reductase mRNA. When cells were stimulated with 8-bromo-cAMP in the presence of 25-hydroxycholesterol and aminoglutethimide, the increase in HMG-CoA reductase mRNA provoked by the tropic agent was markedly attenuated. This indicates that 8-bromo-cAMP raises HMG-CoA reductase mRNA levels indirectly by accelerating steroidogenesis and depleting cellular sterol pools, thus relieving sterol-mediated negative feedback of HMG-CoA reductase gene expression. 25-Hydroxycholesterol in the presence of aminoglutethimide suppressed low-density lipoprotein (LDL) receptor mRNA, but 8-bromo-cAMP effected a significant stimulation of LDL receptor mRNA levels when added with hydroxysterol and aminoglutethimide. These findings reveal differential regulation of HMG-CoA reductase and LDL receptor mRNAs in the presence of sterol negative feedback.     

Phosphorylation and modulation of the enzymic activity of native and protease-cleaved purified hepatic 3-hydroxy-3-methylglutaryl-coenzyme A reductase by a calcium/calmodulin-dependent protein kinase

A Ca2+/calmodulin-dependent kinase has been purified which catalyzed the phosphorylation and concomitant inactivation of both the microsomal native (100,000 Da) and protease-cleaved purified 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMG-CoA reductase) (53,000 Da) fragments. This low molecular weight brain cytosolic Ca2+/calmodulin-dependent kinase phosphorylates histone H1, synapsin I, and purified HMG-CoA reductase as major substrates. The kinase, purified by sequential chromatography on DEAE-cellulose, calmodulin affinity resin, and high performance liquid chromatography (TSKG 3000 SW) is an electrophoretically homogeneous protein of approximately 110,000 Da. The molecular weight of the holoenzyme, substrate specificity, subunit protein composition, subunit autophosphorylation, subunit isoelectric points, and subunit phosphopeptide analysis suggest that this kinase of Mr 110,000 may be different from other previously reported Ca2+/calmodulin-dependent kinases. Maximal phosphorylation by the low molecular form of Ca2+/calmodulin-dependent kinase of purified HMG-CoA reductase revealed a stoichiometry of approximately 0.5 mol of phosphate/mol of 53,000-Da enzyme. Dephosphorylation of phosphorylated and inactivated native and purified HMG-CoA reductase revealed a time-dependent loss of 32P-bound radioactivity and reactivation of enzyme activity. Based on the results reported here, we propose that HMG-CoA reductase activity may be modulated by yet another kinase system involving covalent phosphorylation. The elucidation of a Ca2+/calmodulin-dependent HMG-CoA reductase kinase-mediated modulation of HMG-CoA reductase activity involving reversible phosphorylation may provide new insights into the molecular mechanisms involved in the regulation of cholesterol biosynthesis.     

Characterization of active and inactive forms of rat hepatic 3-hydroxy-3-methylglutaryl coenzyme A reductase

Rat hepatic 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase was purified to homogeneity using agarose-HMG-CoA affinity chromatography. Additional protein was isolated from the affinity column with 0.5 M KCl that demonstrated no HMG-CoA reductase activity, yet comigrated with purified HMG-CoA reductase on sodium dodecyl sulfate-polyacrylamide gels. This protein was determined to be an inactive form of HMG-CoA reductase by tryptic peptide mapping, reaction with anti-HMG-CoA reductase antibody, and coelution with purified HMG-CoA reductase from a molecular-sieving high-performance liquid chromatography column. This inactive protein was present in at least fourfold greater concentration than active HMG-CoA reductase, and could not be activated by rat liver cytosolic phosphoprotein phosphatases. Immunotitration studies with microsomal and solubilized HMG-CoA reductase isolated in the presence and absence of proteinase inhibitors suggested that the inactive protein was not generated from active enzyme during isolation of microsomes or freeze-thaw solubilization of HMG CoA reductase.     

Regulation of rat liver cell 3-hydroxy-3-methylglutaryl coenzyme A reductase by methoxypolyoxyethylated cholesterol

A water-soluble derivative of cholesterol, methoxypolyoxyethylated (MPOE) cholesterol, has been synthesized and used to study the regulation of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, the key regulatory enzyme in cholesterol biosynthesis. MPOE cholesterol causes a specific, rapid and linear decline in HMG-CoA reductase in cultured rat liver cells. MPOE cholesterol is not a direct allosteric inhibitor of HMG-CoA reductase, does not appear to regulate HMG-CoA reductase through changes in membrane environment, and does not change the phosphorylation state and level of activation of rat liver cell HMG-CoA reductase. In order to confirm our data, which were consistent with a model in which MPOE cholesterol regulates the amount of HMG-CoA reductase and not its activity, we made direct measurements of reductase mRNA levels. The decline in HMG-CoA reductase in MPOE cholesterol-treated rat liver cells is preceded by the rapid disappearance of HMG-CoA reductase mRNA. As a water-soluble cholesterol derivative, MPOE cholesterol represents a useful model compound for studies on the regulation of the level of HMG-CoA reductase and its cognate mRNA.     

Effects of hypocholesterolaemic agents on the expression and activity of 3-hydroxy-3-methylglutaryl-CoA reductase in the fat body of the German cockroach

In the fat body of adult Blattella germanica females, the expression of 3-hydroxy-3-methylglutaryl-CoA reductase (HMG-CoA reductase) during the first reproductive cycle is parallel to that of vitellogenin, suggesting a functional link between the mevalonate pathway, and vitellogenesis and reproduction. We have studied the effects of compactin and fluvastatin, two inhibitors of HMG-CoA reductase, on the expression and activity of the enzyme in the fat body, and on the ootheca formation, ootheca viability, and number of larvae per viable ootheca. Short-term assays showed that both compounds reduce the protein levels and enzymatic activity of HMG-CoA reductase, and long-term experiments revealed that fluvastatin impairs embryo development.