A possible translational control of 3-hydroxy-3-methylglutaryl coenzyme A reductase induction by ML-236B(Compactin) in isolated rat hepatocytes

ML-236B (“Compactin”), a competitive inhibitor of 3-hydroxy-3-methylglutaryl(HMG)-CoA reductase, increased the cholesterol synthesis and the HMG-CoA reductase activity in isolated rat hepatocytes. These increases were prevented by 0.2 mM puromycin, but not by 10 μg/ml actinomycin D and 40 μg/ml α-amanitin. These results indicated that the increases in cholesterol synthesis and HMG-CoA reductase activity by ML-236B required the enzyme synthesis but not newly synthesized mRNA. The regulatory site of feed-back inhibition by cholesterol for the HMG-CoA reductase synthesis in liver may be at the translational level.     

Tissue-selective inhibition of cholesterol synthesis in vivo by pravastatin sodium, a 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor

Tissue selectivity of pravastatin sodium (pravastatin) in inhibition of cholesterol synthesis was investigated and its effect was compared with other 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors, such as lovastatin, simvastatin and ML-236B. Inhibition of cholesterol synthesis in vivo was measured by incorporation of radioactivity into the sterol fraction 1 h after intraperitoneal injection of [14C]acetate to mice. The drugs were orally administered to mice 2 h before the acetate injection. When pravastatin at a dose of 20 mg/kg was administered to mice, about 90% inhibition of cholesterol synthesis was observed in liver and ileum, but the inhibition was less than 14% in kidney, spleen, adrenal, testis, prostate and brain. This tissue selectivity of pravastatin was also demonstrated even in varying doses (5-100 mg/kg) and time (75-180 min) after drug administration. Other 3-hydroxyl-3-methylglutaryl coenzyme A reductase inhibitors did not show such a tissue-selective inhibition of sterol synthesis under the same conditions. These results obtained with the in vivo study were confirmed in vitro by the inhibition of sterol synthesis in various cultured cells and rats lenses, as well as by cellular uptake of 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors.     

Effect of 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor pravastatin on urinary 6 beta-hydroxycortisol excretion: a preliminary study.

In this preliminary study, the levels of urinary 6 beta-hydroxycortisol and urinary free cortisol and the 6 beta-hydroxycortisol/free cortisol ratio were determined in normal volunteers and in patients with heterozygous familial hypercholesterolemia before and after Pravastatin administration (10 mg/d for 2 weeks). Urinary 6 beta-hydroxycortisol and 6 beta-hydroxycortisol/free cortisol ratio increased significantly in both groups after Pravastatin administration (P less than 0.05). The percent increase of 6 beta-hydroxycortisol/free cortisol did not differ significantly when the two groups were compared. Our preliminary results suggest that Pravastatin induces hepatic microsomal 6 beta-hydroxylase both in normal volunteers and in patients with heterozygous familial hypercholesterolemia.     

Influence of 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor, pravastatin, on corticosteroid metabolism in patients with heterozygous familial hypercholesterolemia.

The present study examined whether hypolipidemic therapy with a potent 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor, pravastatin, influences corticosteroid metabolism in patients with heterozygous familial hypercholesterolemia (FH). Urinary excretion of tetrahydrocortisone, tetrahydrocortisol, 6 beta-hydroxycortisol and free cortisol were determined in 22 patients with heterozygous FH before and after pravastatin administration (10 mg/day for 2 months). Pravastatin induced a statistically significant decrease in serum total cholesterol in patients with heterozygous FH from 6.9 +/- 0.1 to 5.9 +/- 0.1 mmol/l (p less than 0.05). No significant changes were seen in the urinary tetrahydrocortisone, tetrahydrocortisol and free cortisol levels before and after pravastatin therapy. Urinary excretion of 6 beta-hydroxycortisol was significantly (p less than 0.05) increased after pravastatin administration. These results suggest that the hypolipidemic effect of pravastatin in patients with heterozygous FH does not influence the corticosteroid metabolism. The increase in urinary 6 beta-hydroxycortisol may be caused by pravastatin-induced hepatic microsomal 6 beta-hydroxylase induction.     

Isolation of a monocot 3-hydroxy-3-methylglutaryl coenzyme A reductase gene that is elicitor-inducible

The rice (Oryza sativa) phytoalexins, momilactones and oryzalexins, are synthesized by the isoprenoid pathway. An early step in this pathway, one that is rate-limiting in mammalian systems, is catalyzed by the enzyme 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR). A gene that encodes this enzyme has been isolated from rice, and found to contain an open reading frame of 1527 bases. The encoded protein sequence of the rice HMGR appears to be conserved with respect to other HMGR proteins, and 1 or 2 membrane-spanning domains characteristic of plant HMGRs are predicted by a hydropathy plot of the amino acid sequence. The protein is truncated at its 5 end, and shows reduced sequence conservation in this region as compared to other plant sequences. The rice genome contains a small family of HMGR genes. The isolated gene, HMGR I, is expressed at low levels in both vegetative and floral organs of rice plants. It is not induced in plants by wounding, but is strongly and rapidly induced in suspension cells by a fungal cell wall elicitor from the pathogenMagnaporthe grisea, causal agent of rice blast disease. This suggests that HMGR I may be important in the induction of rice phytoalexin biosynthesis in response to pathogen attack, and therefore may play a key role as a component of the inducible defense mechanism in rice.     

Purification of 3-hydroxy-3-methylglutaryl coenzyme A reductase.

This paper describes a rapid purification procedure for 3-hydroxy-3-methylglutaryl coenzyme A reductase, the major regulatory enzyme in hepatic cholesterol biosynthesis. A freeze-thaw technique is used for solubilizing the enzyme from rat liver microsomal membranes. No detergents or other stringent conditions are required. The purification procedure employs Blue Dextran-Sepharose-4B affinity chromatography, and purification can be carried out from microsomal membranes to purified enzyme in 8 to 10 hours. The purified enzyme has a specific activity of 517 nmoles/min/mg protein, and it is 975-fold purified with respect to the original microsomal membrane suspension. SDS polyacrylamide gel electrophoresis of the purified enzyme shows only trace impurities; the subunit molecular weight for the enzyme measured by this technique is 47,000.     

Preventive effect of pravastatin sodium, a potent inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A reductase, on coronary atherosclerosis and xanthoma in WHHL rabbits

In this paper, we examined whether the development of atherosclerosis in the Watanabe heritable hyperlipidemic (WHHL) rabbit, an animal model of familial hypercholesterolemia in man, could be prevented by the reduction of serum cholesterol levels. Pravastatin sodium (the generic name of CS-514), a potent inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, was used as a cholesterol-lowering drug. The drug was administered orally to 12 WHHL rabbits (2-3 months old) at a dose of 50 mg/kg per day for 24 weeks, and 13 animals were given water as control. In the treated group, serum cholesterol, phospholipid and triacylglycerol levels were significantly reduced by 28%, 32% and 16%, respectively, as compared with those of the control group. Although the prevention of development of the aortic atherosclerosis was not significant, the progression of coronary atherosclerosis was significantly prevented. The incidence of atherosclerosis in four main coronary arteries was reduced from 42% (control group) to 19% (treated group, P less than 0.01), and the development of lesion of coronary arteries evaluated by area of lesion was reduced from 19.7% (control group) to 9.1% (treated group, P less than 0.05). Histopathological findings supported the above observations. In addition, development of xanthoma in digital joints was also reduced from 90.4% (control group) to 58.3% (treated group, P less than 0.005). These results suggest that the development of coronary atherosclerosis and xanthoma in WHHL rabbit was reduced by continuous reduction of serum cholesterol levels treated with pravastatin sodium.     

The mechanism of lack of hypocholesterolemic effects of pravastatin sodium,a 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor,in rats

In order to clarify the reason why pravastatin, a 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase inhibitor, did not show hypocholesterolemic effects in rats, the changes of various parameters affecting the serum cholesterol levels by pravastatin were determined in rats and rabbits, as a comparison. In rabbits, pravastatin administration at 50 mg/kg for 14 days decreased serum and liver cholesterol by 40% and 8%, respectively. The hepatic LDL receptor activity was increased 1.7-fold, and VLDL cholesterol secretion was decreased. Cholesterol 7α-hydroxylase activity was not changed. In contrast, in rats, serum cholesterol was increased by 14% at 50 mg/kg and 27% at 250 mg/kg for 7 days, respectively. At 250 mg/kg, liver cholesterol was significantly increased by 11%. Under these conditions, neither the hepatic LDL receptor activity nor cholesterol 7α-hydroxylase was changed, and VLDL cholesterol secretion was increased. At 250 mg/kg, net cholesterol synthesis in rat liver was increased after 7 days of consecutive administration. These results imply that in rats, stimulated net cholesterol synthesis caused the increase of liver cholesterol followed by the increase of VLDL cholesterol secretion, and resulted in the raise of plasma cholesterol. Although hepatic HMG-CoA reductase was induced almost the same fold in both animals at 50 mg/kg, the induced HMG-CoA reductase activity in rats might overcome the inhibitory capability of pravastatin, resulting in an increase of net cholesterol synthesis, but not in rabbits. This overresponse to pravastatin in rats might cause the lack of hypocholesterolemic effects of this drug.     

The 3-hydroxy-3-methylglutaryl co-enzyme A reductase inhibitor pravastatin enhances neurite outgrowth in hippocampal neurons

Epidemiological studies demonstrate a relationship between statin [3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor] usage and reduced risk of developing Alzheimer's disease. To determine whether statins affect neuronal development, we treated cultured rat hippocampal neurons with pravastatin. After 4-48 h of treatment, pravastatin significantly increased the number of neurites produced by each cell and caused a corresponding increase in levels of the membrane phospholipid phosphatidylcholine. Pravastatin treatment also significantly increased neurite length and branching but did not affect cellular cholesterol levels. Co-incubation with mevalonate, but not cholesterol, abolished the stimulatory effect of pravastatin on neurite outgrowth. Treatment of neurons with isoprenoids also abolished the effect of pravastatin on neurite growth, suggesting that pravastatin may stimulate neuritogenesis by preventing isoprenylation of signaling molecules such as the Rho family of small GTPases. A specific inhibitor of geranylgeranylation, but not farnesylation, mimicked the stimulatory effect of pravastatin on neuritogenesis. Pravastatin treatment significantly decreased levels of membrane-associated RhoA. These data suggest that pravastatin treatment increases neurite outgrowth and may do so via inhibiting the activity of geranylgeranylated proteins such as RhoA.     

CS-514, a competitive inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A reductase: tissue-selective inhibition of sterol synthesis and hypolipidemic effect on various animal species

CS-514 is a tissue-selective inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A reductase, a key enzyme in cholesterol synthesis. For the microsomal enzyme from rat liver, the mode of inhibition is competitive with respect to hydroxymethylglutaryl-CoA, and the Ki value is 2.3 X 10(-9) M. CS-514 also strongly inhibited the sterol synthesis from [14C]acetate in cell-free enzyme systems from rat liver and in freshly isolated rat hepatocytes; the concentrations required for 50% inhibition were 0.8 ng/ml and 2.2 ng/ml, respectively. On the other hand, the inhibition by CS-514 was much less in the cells from nonhepatic tissues such as freshly isolated rat spleen cells, and cultured mouse L cells and human skin fibroblasts. In addition, the cellular uptake of 14C-labeled CS-514 by isolated rat spleen cells or mouse L cells was less than one-tenth of that by isolated hepatocytes. These differences between hepatic and nonhepatic cells were further confirmed by the fact that CS-514 orally administered to rats inhibited sterol synthesis selectively in liver and intestine, the major sites of cholesterogenesis. CS-514 markedly reduced serum cholesterol levels in dogs, monkeys and rabbits, including Watanabe heritable hyperlipidemic (WHHL) rabbits, an animal model for familial hypercholesterolemia in man, but did not reduce those in rats and mice. In the former case, preferential lowering of atherogenic lipoproteins was observed in all of the animals tested. The biliary neutral sterols significantly decreased, whereas the amount of biliary bile acids was not affected by administration of the drug to dogs.     

A somatic cell mutant with a kinetically altered 3-hydroxy-3-methylglutaryl coenzyme A reductase

Deinococcus radiophilus strain ATCC 27603 contains, apart from endonuclease DraI [1], two additional sequence-specific endonucleases. These enzymes, designated DraII and DraIII, recognize nucleotide sequences with novel specificities, PuG↓GNCCPy and CACNN↓GTG, respectively.     

Coordinate regulation of cholesterol synthesis and 3-hydroxy-3-methylglutaryl coenzyme A synthase but not 3-hydroxy-3-methylglutaryl coenzyme A reductase in C-6 glia

The effects on cholesterol biosynthesis of growth of cultured C-6 glial cells in serumfree medium ± supplementation with linoleic or linolenic acid were studied. Markedly higher activities of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA reductase, EC 1.1.1.34) were observed in cells grown in linoleate- or linolenate-supplemented versus nonsupplemented medium. After 48 h HMG-CoA reductase activities were two-and four-fold higher in cells supplemented with 20 and 100 μm linoleate, respectively. The increase in activity became apparent after 24 h and was marked after 48 h. Rates of incorporation of [¹⁴C]acetate or ³H₂O into sterols did not reflect the changes in reductase activity. Thus, in cells supplemented with 50 μm linoleate for 24 and 48 h rates of incorporation of [¹⁴C]acetate were 75–80% lower than rates in nonsupplemented cells. This difference resulted because over the first 24 h of the experiment a fivefold increase in the rate of sterol synthesis occurred in the nonsupplemented cells, whereas essentially no change occurred in the linoleate-supplemented cells; little further change occurred between 24 and 48 h in the nonsupplemented and the linoleate-supplemented cells. That the difference in sterol synthesis under these experimental conditions could be mediated at the level of HMG-CoA synthase (EC 4.1.3.5) was suggested by two series of findings, i.e., first, similar quantitative and temporal changes in the activity of this enzyme, and, second, no change in the activity of acetoacetyl-CoA thiolase (EC 2.3.1.9) or the incorporation of [¹⁴C]mevalonate into sterols. Thus, the data suggest that HMG-CoA synthase, and not HMG-CoA reductase, may direct the rate of cholesterol biosynthesis under these conditions of serum-free growth ± supplementation with polyunsaturated fatty acid.     

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.     

ATP-dependent degradation of 3-hydroxy-3-methylglutaryl coenzyme A reductase in permeabilized cells

A system for the assay of 3-hydroxy-3-methyglutaryl (HMG) coenzyme A (CoA) reductase in digitonin-permeabilized Chinese hamster ovary cells is described. Under these conditions, HMG-CoA reductase remained intact and associated with the endoplasmic reticulum, and values for Km (HMG-CoA), Ki (mevinolin), and active/total activity were similar to those seen in sonicated cell preparations. However, the mechanism by which this rapidly turned over (half-life approximately 2 h) enzyme is degraded was disrupted. Addition of ATP at physiological concentrations to digitonin-permeabilized cells resulted in the rapid, irreversible loss of enzyme activity. Immunoblot analysis showed that this loss of activity was followed by cleavage of the intact 97-kilodalton enzyme to a 68-kilodalton fragment which was distinct from the catalytically active fragments generated by nonspecific proteolysis in sonicated cell homogenates. Assay of a lysosomal marker enzyme confirmed that ATP-mediated inactivation and cleavage of reductase was not due to release of lysosomal proteases. The possible role of ATP in phosphorylation, inactivation, and degradation of reductase is discussed.