Reduced hepatic LDL-receptor, 3-hydroxy-3-methylglutaryl coenzyme A reductase and sterol carrier protein-2 expression is associated with pregnancy loss in the diabetic rat

Hepatic low density lipoprotein receptor (LDLR), 3-hydroxy-3-methylglutaryl coenyzme A reductase (HMGR), cholesterol 7α-hydroxylase, and sterol carrier protein-2 are important proteins associated with the uptake, synthesis, degradation and transport of cellular cholesterol. Since cholesterol is critically important for steroid hormone synthesis and is an essential component in membrane biosynthesis, this study investigated whether these proteins are altered in the normal pregnant and streptozotocin (STZ)-induced diabetic pregnant rat. The goal of these experiments was to determine whether diabetic reproductive dysfunction is associated with a significant change in maternal cholesterol homeostasis. Diabetic animals were grouped based on their ability or inability to maintain pregnancy up to day 15 post-conception. LDLR and HMGR mRNA levels were significantly reduced in animals which did not maintain pregnancy whereas diabetic animals with fetuses had normal LDLR and HMGR mRNA levels. Hepatic LDLR, HMGR, and SCP2 protein levels were examined in normal pregnant and diabetic pregnant animals by Western blot analysis. SCP2 levels were reduced in all diabetic animals, particularly in the diabetic animals which lost their fetuses. The decline in SCP2 was associated with an increase in sterol carrier protein-X (SCPx), a protein related to SCP2. SCPx has been shown to have thiolytic activity and is thought to have a role in β-oxidation of fatty acids. HMGR was also significantly reduced in diabetic animals which lost their fetuses. Cholesterol 7α-hydroxylase mRNA was slightly, but not significantly, reduced in all diabetic animals. Serum very low density lipoprotein (VLDL) +LDL cholesterol increased significantly in the STZ-treated diabetic rats while the HDL cholesterol levels declined in these animals. Reduced hepatic LDLR and HMGR mRNA levels were consistently associated with reduced serum progesterone and an inability to maintain pregnancy. The results of this study suggest that the maintenance of maternal cholesterol metabolism is a critical factor directly associated with successful pregnancy outcome in the diabetic rat.     

Use of 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors in various forms of dyslipidemia

The inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase are highly effective in treating severe elevations of serum cholesterol, and are being widely used for this purpose. In our laboratory, these drugs have been used for the treatment of other forms of dyslipidemia including primary moderate hypercholesterolemia, primary mixed hyperlipidemia, diabetic dyslipidemia, hyperlipidemia of the nephrotic syndrome, and primary hypoalphalipoproteinemia. In these conditions, the HMG CoA reductase inhibitors proved effective in substantially decreasing levels of both low-density lipoproteins and very low density lipoproteins, as well as apolipoprotein B. In some patients, they may even increase levels of high-density lipoproteins. The primary mode of action of HMG CoA reductase inhibitors appears to be to increase the synthesis of hepatic receptors for lipoproteins containing apolipoprotein B, although a reduction in synthesis of these lipoproteins has not been ruled out with certainty. Regardless of mechanisms, drugs of this type appear to have the potential for effective therapy of various forms of dyslipidemia beyond primary severe hypercholesterolemia.     

Monotherapy with hMG-CoA reductase inhibitors and secondary prevention in coronary artery disease

Although thrombolytic drugs, percutaneous transluminal coronary angioplasty, and coronary artery bypass grafting have provided major advances in the treatment of coronary artery disease, the use of lipid-lowering drugs for secondary prevention has significantly reduced cardiovascular events in the population with coronary artery disease. Secondary prevention trials using HMG-CoA reductase inhibitors include the Familial Atherosclerosis Treatment Study (FATS), the Monitored Atherosclerosis Regression Study (MARS), the Canadian Coronary Atherosclerosis Intervention Trial (CCAIT), the Asymptomatic Carotid Artery Progression Study (ACAPS), the Multi Anti-Atheroma Study (MAAS), the Scandinavian Simvastatin Survival Study (4S), the Pravastatin Limitation of Atherosclerosis in Coronary Arteries (PLAC I), the Regression Growth Evaluation Statin Study (REGRESS), the Pravastatin Multinational Study, and the Pravastatin, Lipids, and Atherosclerosis in Carotids (PLAC II). Mean changes from baseline of lipid fractions in these trials included: total cholesterol 18 to 35% reduction low-density lipoprotein (LDL) cholesterol 26 to 46% reduction high-density lipoprotein (HDL) cholesterol 5 to 15% increase and triglyceride 7 to 22% reduction. Angiographic regression or lack of progression was statistically demonstrated in the FATS, MARS, CCAIT, MAAS, PLAC I, and REGRESS trials. Cardiovascular events decreased 25 to 92% in all trials, and there was a significant reduction in both cardiovascular and total mortality in the 4S. The greater reduction in cardiovascular events than in anatomic changes suggests that the HMG-CoA reductase inhibitors stabilized the surface of plaques. Monotherapy with HMG-CoA reductase inhibitors provides the clinical opportunity to modify the natural history of coronary artery disease.     

Regulation of hamster adrenal 3-hydroxy-3-methylglutaryl coenzyme A reductase activity

In the adrenal gland of the hamster, the activity of 3-hydroxy-3-methyl-glutaryl coenzyme A reductase [mevalonate:NADP+ oxidoreductase (acylating CoA); EC 1.1.1.34] is mainly located in the postmitochondria fraction. This enzyme exhibits a diurnal rhythm, with a maximum at 1900 h. Peak activity levels are about 1667 pmol/mg protein . min, roughly 8 times the minimum levels. Plasma corticosteroid concentrations closely parallel reductase activity levels. The administration of ACTH or metyrapone (a drug which increases in vivo ACTH secretion) to hamsters enhanced the adrenal reductase activity, whereas aminoglutethimide, an inhibitor of the side chain cleavage of cholesterol, diminished it. The feeding of a 5% cholesterol diet and treatment with 4-aminopyrazolopyrimidine resulted in an increase and a decrease in plasma cholesterol, respectively. Neither treatment significantly altered adrenal 3-hydroxy-3-methylglutaryl coenzyme A reductase activity or the cholesterol content of the gland. Hamster adrenals contain very little free cholesterol (7-11 microgram/mg protein) and even less esterified cholesterol (0.5-2.0 microgram/mg protein). These findings suggest that in the hamster the adrenal gland may be largely autonomous in cholesterol production.     

β-羟甲基戊二酰辅酶A还原酶基因多态性与冠心病相关性的研究

目的研究汉族人群β-羟甲基戊二酰辅酶A还原酶(HMGCR)基因型和等位基因频率分布的遗传背景,探讨HMGCR多态性与冠心病的相关性。方法采用聚合酶链反应-单链构象多态性分析及测序技术,对湖北地区汉族123例健康人以及117例冠心病病人HMGCR多态性基因型和等位基因频率分布进行分析,研究HMGCR多态性对血脂、脂蛋白和载脂蛋白水平的影响。结果冠心病组内TT/CC(TT+CC)基因型与TT基因型相比,血清TC和LDL-C水平均有显著性差异(P<0·05)。冠心病组与对照组相比,2725A/G位点基因型间血脂、脂蛋白及载脂蛋白水平均无显著性差异(P>0·05)。结论HMGCR基因3089T/C位点基因多态性可能与冠心病存在相关性,而2725A/G位点多态性与血脂水平的相关性不明显。     

3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors interfere with angiogenesis by inhibiting the geranylgeranylation of RhoA

Angiogenesis is implicated in the pathogenesis of cancer, rheumatoid arthritis, and atherosclerosis and in the treatment of coronary artery and peripheral vascular disease. Here, cholesterol-lowering agents, 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors, are shown to interfere with angiogenesis. In vivo, the HMG-CoA reductase inhibitor simvastatin dose-dependently inhibited capillary growth in both vascular endothelial growth factor-stimulated chick chorioallantoic membranes and basic fibroblast growth factor-stimulated mouse corneas. In vitro, the development of tubelike structures by human microvascular endothelial cells cultured on 3D collagen gels was inhibited at simvastatin concentrations similar to those found in the serum of patients on therapeutic doses of this agent. HMG-CoA reductase inhibitors interfered with angiogenesis via inhibition of the geranylgeranylation and membrane localization of RhoA. Simvastatin inhibited membrane localization of RhoA with a concentration dependence similar to that for the inhibition of tube formation, whereas geranylgeranyl pyrophosphate, the substrate for the geranylgeranylation of Rho, reversed the effect of simvastatin on tube formation and on the membrane localization of RhoA. Furthermore, tube formation was inhibited by GGTI, a specific inhibitor of the geranylgeranylation of Rho; by C3 exotoxin, which inactivates Rho; and by the adenoviral expression of a dominant-negative RhoA mutant. The expression of a dominant-activating RhoA mutant reversed the effect of simvastatin on tube formation. Finally, HMG-CoA reductase inhibitors inhibited signaling by vascular endothelial growth factor, Akt, and focal adhesion kinase, three RhoA-dependent pathways known to be involved in angiogenesis. This study demonstrates a new relationship between lipid metabolism and angiogenesis and an antiangiogenic effect of HMG-CoA reductase inhibitors with possible important therapeutic implications.     

Rapid effect of 3-hydroxy-3-methylglutaryl coenzyme a reductase inhibition on coronary endothelial function

Treatment with 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors (statins) decreases cardiovascular event rates in hypercholesterolemic patients. Whether statins exert effects within 24 hours on the coronary vasculature in patients with endothelial dysfunction has not been elucidated. Twenty-seven patients with stable angina pectoris and average low-density lipoprotein cholesterol concentrations of 138+/-9 mg/dL at baseline were allocated to treatment with placebo (14 patients) or 40 mg/d pravastatin (13 patients) in a randomized, double-blind, prospective trial. Coronary endothelial function was assessed before and 24 hours after single treatment by quantitative coronary angiography during intracoronary infusion of nitroglycerin or increasing concentrations of acetylcholine (0.01, 0.1, and 1 micromol/L). Coronary blood flow reserve was measured by Doppler velocimetry during adenosine infusion. Intracoronary acetylcholine infusion induced abnormal vasoconstriction in both groups before treatment, indicating coronary endothelial dysfunction. Treatment with a single oral 40-mg dose of pravastatin significantly attenuated acetylcholine-mediated vasoconstriction after 24 hours (mean+/-SE decrease in luminal diameter before and after treatment: 0.01 micromol/L, 6.1+/-2.2% versus 3.0+/-1.2%; 0.1 micromol/L, 15.6+/-2.6% versus 7.4+/-1.8%; P<0.05; 1 micromol/L, 22.9+/-2.9% versus 13.2+/-2.6%; P<0.05). There was no significant difference in the response to acetylcholine in the placebo group (8.1+/-2.4% versus 9.7+/-2.4%, 16.1+/-2.9% versus 16.8+/-3.2%, and 21.4+/-3.9% versus 23.3+/-4.2%). The response to nitroglycerin infusion was not altered in both groups. Increase in coronary blood flow in response to adenosine and coronary flow reserve remained unchanged during placebo and statin treatment. Serum concentrations of blood lipids and high-sensitive C-reactive protein were not significantly altered after 24 hours in response to placebo or pravastatin therapy. Statin treatment improves endothelium-dependent coronary vasomotion within 24 hours in the absence of significant cholesterol reduction. The full text of this article is available online at http://www.circresaha.org.     

Characterization of 3-hydroxy-3-methylglutaryl coenzyme A reductase in human adrenal cortex

Our study compares the properties of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase from a human metastatic virilizing carcinoma and that from normal adrenal glands obtained from kidney donors. Optimal conditions for enzyme assay were obtained when a 50-mM imidazole buffer (pH 7.2) containing 5 mM EDTA, 250 mM NaCl, 1 mM phenyl-methylsulfonylfluoride, 0.1 mM leupeptin, and 5.5 mM dithiothreitol (DTT) was used. A 30-min preincubation period preceding addition of substrates enhanced reductase activity by 1.75-fold. In crude microsomal preparations, Km values were similar for both tumor and normal tissues and varied between 4 and 5 microM (S)HMG-CoA. The presence of NaF in homogenization and incubation media decreased the maximum velocity, but not the Km. A partially purified rat liver phosphorylase phosphatase preparation or a similar preparation from the carcinoma restored to maximal levels the reductase activity of microsomes prepared in the presence of NaF. A Km of 96 microM NADP was found for the carcinoma microsomal preparation. Preincubation of microsomes in the presence of monothioglycerol or DTT resulted in an increased reductase activity, suggesting a possible inactive enzyme precursor(s) consisting of disulfide-linked units. Reactions of the DTT-activated enzyme incubated in the presence of increasing amounts of NADPH showed sigmoidal kinetics. Under reducing conditions, sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting revealed the presence of a 92.5K mol wt protein band that reacted with a rat antireductase antibody. Reductase activity in different regions of the carcinoma varied from 679-1763 pmol/mg protein, with an average of 1146 pmol. In three normal adrenal glands we found values of 23.4, 48.1, and 36 pmol. We concluded that the expression of HMG-CoA reductase activity was elevated in human adrenal carcinoma.     

Multitasking of the 3-hydroxy-3-methylglutaryl coenzyme a reductase inhibitor: Beyond cardiovascular diseases

Statins can profoundly affect cellular metabolism by inhibiting 3-hydroxy-3-methylglutaryl coenzyme A reductase, which is the rate-limiting enzyme responsible for cholesterol synthesis. Many physicians prescribe statins to lower plasma cholesterol levels, which has beneficial effects in both the primary and secondary prevention of coronary artery disease. However, in vitro, in vivo, animal, and clinical studies have all shown that statins may also have important pleiotropic properties. In fact, a number of clinical studies have suggested that statins are involved in modulating diseases such as cancer, osteoporosis, and dementia (including Alzheimer’s disease). However, because these studies have been only preliminary and observational in nature, large randomized, placebo-controlled studies are needed to confirm the modulatory role of statins in these important diseases.     

Activation and inactivation of 3-hydroxy-3-methylglutaryl coenzyme a reductase in aged rat liver

Activation and inactivation of hepatic 3-hydroxy-3-methylglutaryl coenzyme A reductase, which is a key enzyme of cholesterol synthesis, were examined in young and aged rats in vitro.The reductase activity in the liver microsomes of aged rats was always lower than that of young rats at various experimental conditions in vitro. But activation and inactivation of the reductase could operate on liver microsomes in aged rats as well as in young rats. Function of hepatic cytosol activator in aged rats seemed to be identical to that in young rats.     

Withdrawal of 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors elicits oxidative stress and induces endothelial dysfunction in mice

3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors (statins) improve endothelial function. We determined whether withdrawal of statin therapy affects endothelium-dependent relaxation in mice and studied the underlying mechanism. Mice were treated with daily injections of cerivastatin (2 mg/kg per day SC), atorvastatin (1 and 10 mg/kg per day SC), or placebo. Vascular reactivity was studied in aortic rings from these mice after 10 days of treatment and after cessation of therapy for several days. Both statins improved endothelium-dependent relaxation to acetylcholine. Compared with control, withdrawal of statin treatment transiently (from day 4 to 7) attenuated endothelium-dependent relaxation. In vessels from animals subjected to atorvastatin withdrawal, the antioxidant tiron restored relaxations. Vascular superoxide anion generation was unaffected by statin therapy but was increased during withdrawal. In mice lacking the gp91phox subunit of the NADPH oxidase, no attenuation of acetylcholine-induced relaxation and no increase in superoxide generation were observed after withdrawal of atorvastatin. In human umbilical vein endothelial cells, statins, which decrease the membrane association of NADPH oxidase-activating Rac-1, increased the activity of this GTPase in whole-cell lysates. Withdrawal of statins induced a translocation of Rac-1 from the cytosol to the membrane and transiently increased NADPH-induced lucigenin chemiluminescence in membrane preparations. Rac-1 inactivation by Clostridium difficile toxin B inhibited the cerivastatin-induced oxygen radical production in human umbilical vein endothelial cells. These observations indicate that the withdrawal of statins induces endothelial dysfunction. The underlying mechanism involves activation of a gp91phox-containing NADPH oxidase by Rac-1 and the subsequent scavenging of endothelium-derived NO by superoxide anions generated from this enzyme.     

Hypercholesterolemia and 3-hydroxy-3-methylglutaryl coenzyme A reductase regulation in aged female rats

Coronary heart disease is less prevalent in pre-menopausal women than in men, but increases at the onset of menopause. This delay is due to estrogen protective effects. The rise of cholesterolemia is one of the main risk factors for coronary disease. Since 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) is the rate-limiting enzyme of the cholesterol biosynthetic pathway, it plays a pivotal role in cholesterol homeostasis maintenance. Aim of this study is to investigate whether HMGR is involved in the cholesterolemia increase that occurs during aging, and to consider its potential role as a target for estrogen protective effects. “In vivo” studies have been performed using the livers of 12-month-old female rats (whose estrogen level decrease is comparable to the one detected at the occurrence of human menopause), 12-month-old female rats treated with 17-β-estradiol, and 3-month-old untreated male and female rats. The results indicated hypercholesterolemic status and a significant increase of HMGR activity according to a reduced activation of AMPK detected in treated rats compared to controls. Furthermore, 17-β estradiol treatment reduced HMGR activity restoring AMPK activation. These findings highlight the correlation between estrogen and HMGR short-term regulation, and suggest the presence of another mechanism underlying the protective role of estrogen in age-related diseases.     

Molecular cloning of 3-hydroxy-3-methylglutaryl coenzyme a reductase and evidence for regulation of its mRNA.

A recombinant plasmid containing a 1.2-kilobase cDNA for 3-hydroxy-3-methylglutaryl coenzyme A reductase was isolated from a cDNA library prepared from UT-1 cells, a clone of Chinese hamster ovary cells that has markedly elevated reductase activity. This plasmid, designated pRed-10, was identified by differential colony hybridization and hybrid-selected mRNA translation. The mRNA that hybridized to pRed-10 directed the synthesis in vitro of a 90,000-dalton protein that was immunoprecipitated by an antireductase antibody. The same 90,000-dalton protein was immunoprecipitated when UT-1 cells were pulse labeled with [35S]methionine in vivo and rapidly solubilized with boiling NaDodSO4. By blot hybridization, pRed-10 hybridized to mRNAs of 4.2 and 4.7 kilobases in UT-1 cells. Both mRNAs were reduced to undetectable levels when low density lipoprotein, a suppressor of the reductase, was present in the culture medium. These data indicate that the primary translation product of reductase mRNA is a 90,000-dalton protein and that LDL suppresses the reductase in UT-1 cells by drastically reducing the level of its mRNA.     

Mevalonate-mediated suppression of 3-hydroxy-3-methylglutaryl coenzyme A reductase function in alpha-toxin-perforated cells.

The regulation of mevalonic acid synthesis requires both nonsterol isopentenoid and sterol regulatory signal molecules. A primary target of this multivalent control process is the enzyme which catalyzes mevalonate synthesis: 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase (EC 1.1.1.34). In this report Staphylococcus aureus alpha-toxin perforated Chinese hamster ovary cells were used to facilitate the identification of isopentenoidogenic reactions and metabolites required for mevalonate-mediated loss of HMG-CoA reductase activity. alpha-Toxin-perforated cells retained the capacity to decrease, upon demand, HMG-CoA reductase activity and protein in response to mevalonate or isopentenoid pyrophosphate esters. Also, it was deduced with highly specific metabolic inhibitors, that conversion of farnesyl 1-diphosphate to squalene was required for mevalonate-mediated suppression of reductase activity. Since squalene (2 microM) did not downregulate reductase activity, pre-squalene pyrophosphate or a derivative, or polyprenyl-1-pyrophosphate-generated inorganic pyrophosphate, or a combination of these metabolites are proposed as candidate regulatory nonsterol isopentenoid signal molecules.