Defective regulation of 3-hydroxy-3-methylglutaryl coenzyme A reductase in a somatic cell mutant.

A somatic cell mutant of the CHO-K1 cell selected to be resistant to the killing effects of 25-hydroxycholesterol in the absence of cholesterol is shown to be defective in the inhibition of 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase activity by 25-hydroxycholesterol, cholesterol, and lipoproteins, thus maintaining the enzyme activity found in cells in the absence of exogenous sterol constitutively. The mutants phenotype is shown to be dominant with respect to the wild type. Actinomycin D and cycloheximide prevent the increase of HMG-CoA reductase activity that occurs in the CHO-K1 cell when cholesterol is removed from medium. Degradation of the enzyme, measured during inhibition of protein synthesis by cycloheximide, occurs at the same rate in the mutant as in the wild type. Kinetic studies indicate that the Km for two substrates, the activation energy, and a break in the Arrhenius plot are the same for HMG-CoA reductase determined in wild type and mutant cells. From these studies it is concluded that the mutant is defective in the regulation of synthesis of HMG-CoA reductase. Of the four processes which determine cellular cholesterol levels: biosynthesis, esterification, efflux, and uptake, only biosynthesis is altered, demonstrating that these processes are not co-ordinately controlled as has been suggested previously.     

qPCR array检测高糖对胆固醇合成基因表达的影响

目的:高血糖易引起胆固醇在体内积聚,增加糖尿病合并动脉粥样硬化性心血管疾病的患病风险。本文通过建立稳定的实时定量PCR芯片(Real-time quantitative polymerasechain reaction array,qPCR array)检测方案,研究高糖对小鼠肝癌细胞Hepa1-6胆固醇合成基因表达的影响,探讨胆固醇合成基因在糖尿病大血管并发症发展中的作用机制。方法:以不同浓度葡萄糖(5、15、30mmo/L)和不同时间(0、6、12、18、24 h),刺激肝癌细胞Hepa1-6,利用qPCR array检测其胆固醇合成基因的表达差异。结果:与5mmol/L相比,高糖组(15、30 mmo/L)处理细胞18 h后,胆固醇合成基因CYP51、EBP、NSDHL、SQLE、FDFT1和PMVK的表达上调(P0.05),呈现剂量依赖性。与0 h相比,15 mmol/L高糖处理细胞12 h,CYP51、EBP和SQLE mRNA表达量上调(P0.01)。至24 h,CYP51、EBP降至0 h水平,而SQLE的表达量继续增加;NSDHL在12 h表达无差异,至18 h表达量发生上调(P0.05)。结论:该qPCR array检测方案能特异性检测胆固醇合成基因的表达量。高糖能够促进胆固醇合成基因的表达,使细胞内胆固醇积聚,这可能是糖尿病患者容易发生动脉粥样硬化的原因。这提示我们将胆固醇合成基因作为药物靶点可能延缓糖尿病动脉粥样硬化进展。     

Estrogen regulation of hepatic 3-hydroxy-3-methylglutaryl coenzyme A reductase and acetyl-CoA carboxylase in xenopus laevis

Administration of estradiol-17 beta to male Xenopus laevis evokes the proliferation of the endoplasmic reticulum and the Golgi apparatus and the synthesis and secretion by the liver of massive amounts of the egg yolk precursor phospholipoglycoprotein, vitellogenin. We have investigated the effects of estrogen on three key regulatory enzymes in lipid biosynthesis, 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase, the major regulatory enzyme in cholesterol and isoprenoid synthesis, and acetyl-CoA carboxylase and fatty acid synthetase, which regulate fatty acid biosynthesis. HMG-CoA reductase activity and cholesterol synthesis increase in parallel following estrogen administration. Reductase activity in estrogen stimulated Xenopus liver cells peaks at 40-100 times the activity observed in control liver cells. The increased rate of reduction of HMG-CoA to mevalonic acid is not due to activation of pre-existing HMG-CoA reductase by dephosphorylation, as the fold induction is unchanged when reductase from control and estrogen-stimulated animals is fully activated prior to assay. The estrogen-induced increase of fatty acid synthesis is paralleled by a 16- to 20-fold increase of acetyl-CoA carboxylase activity, indicating that estrogen regulates fatty acid synthesis at the level of acetyl-CoA carboxylase. Fatty acid synthetase activity was unchanged during the induction of fatty acid biosynthesis by estrogen. The induction of HMG-CoA reductase and of acetyl-CoA carboxylase by estradiol-17 beta provides a useful model for regulation of these enzymes by steroid hormones.     

Cholesterol Biosynthesis and Its Regulation in Dissociated Cell Cultures of Fetal Rat Brain: Developmental Changes and the Role of 3-Hydroxy-3-Methylglutaryl Coenzyme A Reductase

Primary cultures of cells dissociated from fetal rat brain were utilized to define the developmental changes in cholesterol biosynthesis and the role of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase in the regulation of these changes. Cerebral hemispheres of fetal rats of 15-16 days of gestation were dissociated mechanically into single cells and grown in the surface-adhering system. Cholesterol biosynthesis, studied as the rate of incorporation of [14C]acetate into digitonin-precipitable sterols, was shown to exhibit two distinct increases in synthetic rates, a prominent increase after 6 days in culture and a smaller one after 14 days in culture. Parallel measurements of HMG-CoA reductase activity also demonstrated two discrete increases in enzymatic activity, and the quantitative and temporal aspects of these increases were virtually identical to those for cholesterol synthesis. These data indicate that cholesterol biosynthesis undergoes prominent alterations with maturation and suggest that these alterations are mediated by changes in HMG-CoA reductase activity. The timing of the initial prominent peak in both cholesterol biosynthesis and HMG-CoA reductase activity at 6 days was found to be the same as the timing of the peak in DNA synthesis, determined as the rate of incorporation of [3H]thymidine into DNA. The second, smaller peak in reductase activity and sterol biosynthesis at 14 days occurred at the time of the most rapid rise in activity of the oligodendroglial enzyme, 2':3'-cyclic nucleotide 3'-phosphohydrolase (CNP). These latter observations suggest an intimate relationship of the sterol biosynthetic pathway with cellular proliferation and with oligodendroglial differentiation in developing mammalian brain.     

ACTH induction of 3-hydroxy-3-methylglutaryl coenzyme A reductase, cholesterol biosynthesis, and steroidogenesis in primary cultures of bovine adrenocortical cells

The current studies demonstrate that corticosteroidogenesis can be maintained by primary cultures of bovine adrenocortical cells under lipoprotein-depleted conditions. The cholesterol necessary as substrate for steroid synthesis was found to arise from de novo synthesis within these cells. Adrenocorticotropin (ACTH) increased 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase activity 5-fold within 12 h after addition to the medium. The increase in activity apparently represented accumulation of enzyme as determined by protein blotting and immunodetection. The predominant immunodetectable species of HMG-CoA reductase from bovine adrenal cells was 97,000 daltons; no higher molecular mass species was detectable. The ACTH induction of HMG-CoA reductase activity could be prevented after inhibition of cholesterol conversion to pregnenolone with clotrimazole. These results are suggestive that ACTH increases adrenocortical cholesterol biosynthesis and HMG-CoA reductase activity after conversion of a cellular pool of cholesterol and/or oxysterol into steroid. The increased rate of cholesterol biosynthesis is then capable of maintaining ACTH-promoted steroid production. This is the first study, in vitro, to demonstrate an ACTH-promoted accumulation of HMG-CoA reductase of adrenocortical cells.     

Topogenesis and Homeostasis of Fatty Acyl-CoA Reductase 1

Peroxisomal fatty acyl-CoA reductase 1 (Far1) is essential for supplying fatty alcohols required for ether bond formation in ether glycerophospholipid synthesis. The stability of Far1 is regulated by a mechanism that is dependent on cellular plasmalogen levels. However, the membrane topology of Far1 and how Far1 is targeted to membranes remain largely unknown. Here, Far1 is shown to be a peroxisomal tail-anchored protein. The hydrophobic C terminus of Far1 binds to Pex19p, a cytosolic receptor harboring a C-terminal CAAX motif, which is responsible for the targeting of Far1 to peroxisomes. Far1, but not Far2, was preferentially degraded in response to the cellular level of plasmalogens. Experiments in which regions of Far1 or Far2 were replaced with the corresponding region of the other protein showed that the region flanking the transmembrane domain of Far1 is required for plasmalogen-dependent modulation of Far1 stability. Expression of Far1 increased plasmalogen synthesis in wild-type Chinese hamster ovary cells, strongly suggesting that Far1 is a rate-limiting enzyme for plasmalogen synthesis.     

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.     

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.     

Potential role of nonstatin cholesterol lowering agents

Although statins, 3β-hydroxy-3β-methylglutaryl coenzyme A reductase (HMGR) inhibitors, have revolutionized the management of cardiovascular diseases by lowering serum low density lipoproteins, many patients suffer from their side effects. Whether the statin side effects are related to their intrinsic toxicity or to the decrease of HMGR main isoprenoid end products, which are essential compounds for cell viability, is still debated. In addition to HMGR, the key and rate limiting step of cholesterol synthesis, many enzymes are involved in this multi-step pathway whose inhibition could be taken into account for a "nonstatin approach" in the management of hypercholesterolemia. In particular, due to their unique position downstream from HMGR, the inhibition of squalene synthase, farnesyl diphosphate farnesyltransferase (FDFT1), squalene epoxidase (SQLE), and oxidosqualene cyclase:lanosterol synthase (OSC) should decrease plasma levels of cholesterol without affecting ubiquinone, dolichol, and isoprenoid metabolism. Thus, although FDFT1, SQLE and OSC are little studied, they should be considered as perspective targets for the development of novel drugs against hypercholesterolemia. Here, structure-function relationships of FDFT1, SQLE, and OSC are reviewed highlighting the advantages that the downstream inhibition of HMGR could provide when compared to the statin-based therapy.     

Posttranslational Regulation of Fatty Acyl-CoA Reductase 1, Far1, Controls Ether Glycerophospholipid Synthesis

Plasmalogens are a major subclass of ethanolamine and choline glycerophospholipids in which a long chain fatty alcohol is attached at the sn-1 position through a vinyl ether bond. This ether-linked alkyl bond is formed in peroxisomes by replacement of a fatty acyl chain in the intermediate 1-acyl-dihydroxyacetone phosphate with a fatty alcohol in a reaction catalyzed by alkyl dihydroxyacetone phosphate synthase. Here, we demonstrate that the enzyme fatty acyl-CoA reductase 1 (Far1) supplies the fatty alcohols used in the formation of ether-linked alkyl bonds. Far1 activity is elevated in plasmalogen-deficient cells, and conversely, the levels of this enzyme are restored to normal upon plasmalogen supplementation. Down-regulation of Far1 activity in response to plasmalogens is achieved by increasing the rate of degradation of peroxisomal Far1 protein. Supplementation of normal cells with ethanolamine and 1-O-hexadecylglycerol, which are intermediates in plasmalogen biosynthesis, accelerates degradation of Far1. Taken together, our results indicate that ether lipid biosynthesis in mammalian cells is regulated by a negative feedback mechanism that senses cellular plasmalogen levels and appropriately increases or decreases Far1.     

Scavenger receptor class B type I affects cholesterol homeostasis by magnifying cholesterol flux between cells and HDL.

Results from several laboratories clearly indicate that expression of scavenger receptor class B type I (SR-BI) enhances the bidirectional flux of cholesterol between cells and lipoproteins. Because the activity of HMG-CoA reductase, the key enzyme in cholesterol biosynthesis, is regulated by cell cholesterol content, we designed experiments to investigate the effect of SR-BI expression on the activity of this enzyme and on net cellular cholesterol mass. In addition, we compared the function of SR-BI with its human homolog, CD36 and LIMPII analogous 1. Our experiments demonstrate that both receptors enhance the flux of unesterified or free cholesterol bidirectionally, down a concentration gradient. Receptor-mediated cholesterol flux can effectively modulate multiple aspects of cellular cholesterol metabolism, including the pool that regulates the activity of HMG-CoA reductase. We also found that constitutive expression of SR-BI alters the steady state level of cellular cholesterol and phospholipid when SR-BI-expressing cells are maintained in medium containing serum lipoproteins. All of these effects are proportional to the level of receptor on the cell surface. These data indicate that the level of SR-BI expression determines both the rate of free cholesterol flux and the steady state level of cellular cholesterol.     

Regulation of hepatic cholesterol biosynthesis by berberine during hyperhomocysteinemia

Hyperhomocysteinemia, an elevation of blood homocysteine levels, is a metabolic disorder associated with dysfunction of multiple organs. We previously demonstrated that hyperhomocysteinemia stimulated hepatic 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase leading to hepatic lipid accumulation and liver injury. The liver plays an important role in cholesterol biosynthesis and overall homeostasis. HMG-CoA reductase catalyzes the rate-limiting step in cholesterol biosynthesis. Hepatic HMG-CoA reductase is a major target for lowering cholesterol levels in patients with hypercholesterolemia. The aim of the present study was to examine the effect of berberine, a plant-derived alkaloid, on hepatic cholesterol biosynthesis in hyperhomocysteinemic rats and to identify the underlying mechanism. Hyperhomocysteinemia was induced in Sprague-Dawley rats by feeding a high-methionine diet for 4 wk. HMG-CoA reductase activity was markedly elevated in the liver of hyperhomocysteinemic rats, which was accompanied by hepatic lipid accumulation. Activation of HMG-CoA reductase was caused by an increase in its gene expression and a reduction in its phosphorylation (an inactive form of the enzyme). Treatment of hyperhomocysteinemic rats with berberine for 5 days inhibited HMG-CoA reductase activity and reduced hepatic cholesterol content. Such an inhibitory effect was mediated by increased phosphorylation of HMG-CoA reductase. Berberine treatment also improved liver function. These results suggest that berberine regulates hepatic cholesterol biosynthesis via increased phosphorylation of HMG-CoA reductase. Berberine may be therapeutically useful for the management of cholesterol homeostasis.     

Induction of macrophage elastase (MMP-12) gene expression by statins

The statins (including mevastatin and lovastatin) are a widely prescribed class of serum-cholesterol lowering drugs that function by inhibiting 3-hydroxymethylglutaryl coenzyme A (HMG CoA) reductase activity and cellular sterol synthesis. Statins are also widely being appreciated for their inhibitory effects upon inflammation, primarily mediated through direct regulation of inflammatory gene expression. Here we report that statins are also capable of increasing the expression of macrophage elastase (MMP-12). The induction of MMP-12 in mouse macrophages by statins is specific for HMG CoA reductase inhibition, rescued by mevalonate and not observed after inhibition of subsequent steps in the cholesterol biosynthetic pathway. Modulation of cholesterol metabolism may lead to changes in MMP-12 expression and subsequent impacts during physiological and pathophysiological states. We conclude that statins, in addition to their previously described anti-inflammatory properties, may promote the production of some proteinases from activated macrophages.     

Feedback regulation of 3-hydroxy-3-methylglutaryl coenzyme A reductase in Saccharomyces cerevisiae.

In eukaryotic cells all isoprenoids are synthesized from a common precursor, mevalonate. The formation of mevalonate from 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) is catalyzed by HMG-CoA reductase and is the first committed step in isoprenoid biosynthesis. In mammalian cells, synthesis of HMG-CoA reductase is subject to feedback regulation at multiple molecular levels. We examined the state of feedback regulation of the synthesis of the HMG-CoA reductase isozyme encoded by the yeast gene HMG1 to examine the generality of this regulatory pattern. In yeast, synthesis of Hmg1p was subject to feedback regulation. This regulation of HMG-CoA reductase synthesis was independent of any change in the level of HMG1 mRNA. Furthermore, regulation of Hmg1p synthesis was keyed to the level of a nonsterol product of the mevalonate pathway. Manipulations of endogenous levels of several isoprenoid intermediates, either pharmacologically or genetically, suggested that mevalonate levels may control the synthesis of Hmg1p through effects on translation.     

Instructions for authors

The aim of the present study was to examine hypothesis that the enhanced cholesterologenesis, found in rats with experimental chronic renal failure (CRF) resulted from the increased gene expression of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase – the rate limiting enzyme in the cholesterologenesis pathway, responsible for mevalonate synthesis. Wistar rats were used and experimental CRF was achieved by 5/6 nephrectomy model. We examined: (a) the changes in the rat liver microsomal HMG-CoA reductase activity, (b) the rat liver HMG-CoA reductase mRNA abundance in various times of day. Obtained data indicates that the increased activity of HMG-CoA reductase in the liver of rats with experimental CRF parallel enhanced mRNA level and suggests that enhanced cholesterol biosynthesis, observed in experimental CRF is at least in part due to the increased HMG-CoA reductase gene expression. The results also indicate that the physiological diurnal rhythm of HMG-CoA reductase activity is preserved in the course of experimental CRF.     

Defective elongation of fatty acids in a recessive 25-hydroxycholesterol-resistant mutant cell line

The Chinese hamster ovary recessive mutant, crB, has been selected for its resistance to the cytotoxic effects of 25-hydroxycholesterol in sterol-free media (Sinensky, M., Logel, J., and Torget, R. (1982) J. Cell. Physiol. 113, 314-319). Growth of crB in a chemically defined lipid-poor medium is very slow and is enhanced by a mixture of saturated and unsaturated fatty acids. Incorporation of [3H]acetate into total fatty acids is 4-fold lower in crB compared to that in parental Chinese hamster ovary K1 and in contrast to the wild-type cells, crB cells are unable to synthesize either stearate or oleate. In addition, crB cells can not elongate exogenous palmitate, while they are capable of desaturating exogenous stearate. The mutant cells are also pleiotropically defective in the regulation of mRNA levels for the enzymes of cholesterol biosynthesis. 25-Hydroxycholesterol is a poor regulator of the synthesis and degradation of the rate-limiting enzyme, 3-hydroxy-3-methylglutaryl-coenzyme A reductase in crB in comparison to the wild-type Chinese hamster ovary K1 cells. The defect in the elongation of fatty acids is reversed in revertants of crB selected for their ability to grow in lipid-poor medium. Such revertants exhibit normal regulation of 3-hydroxy-3-methylglutaryl-CoA reductase activity by 25-hydroxycholesterol. Regulation of reductase activity in crB cells can also be restored by supplementing the culture medium with a mixture of fatty acids that restores normal growth rate. The defective regulation of reductase in crB does not appear to be due to nonspecific adverse effects of fatty acid starvation nor is it due to any gross change in the fatty acid composition of cellular phospholipids. These results strongly suggest a direct relationship between the fatty acid auxotrophy of crB and defective regulation of the enzymes of cholesterol biosynthesis.     

A potential role for phospholipids in the regulation of 3-hydroxy-3-methylglutaryl-coenzyme A reductase in cultured C-6 glial cells. Effects of N,N-dimethylethanolamine.

The relation of the activity of the microsomal enzyme, 3-hydroxy-3-methylglutaryl coenzyme A reductase, to cellular phospholipid composition was studied in C-6 glial cells. Phospholipid composition was perturbed by growth of cells in the naturally occurring amino alcohol, N,N-dimethylethanolamine. After growth of C-6 glia in 5 mM N,N-dimethylethanolamine for 24 h, reductase activity was diminished by 50%. A similar diminution in cholesterol synthesis was observed. This effect was not accompanied by any parallel change in cell growth, DNA synthesis, protein synthesis, fatty acid synthetase activity, or microsomal NADPH-cytochrome c reductase activity. The inhibition of reductase activity by N,N-dimethylethanolamine was prevented by the addition of equimolar concentrations of choline to the culture medium and, also, could be reversed completely by removal of N,N-dimethylethanolamine from the culture medium. The effect of N,N-dimethylethanolamine on reductase was associated with the formation of phosphatidyl-N,N-dimethylethanolamine which accumulated primarily at the expense of phosphatidylcholine and, after 24 h, accounted for 27% of total phospholipid phosphorus. The data demonstrate that incorporation of N,N-dimethylethanolamine into the polar head group of cellular phospholipids has a major impact on the regulation of the reductase. These observations may have particular relevance for the mechanisms of regulation of this enzyme, the cellular adaptation to alterations in membrane lipid composition, and the regulation of cholesterol synthesis in the developing nervous system.