Increase In HMG-CoA Reductase Activity Research Articles

HMG–CoA Reductase Activation and Urinary Pellet Cholesterol Elevations in Acute Kidney Injury

Experimental acute kidney injury (AKI) activates the HMG-CoA reductase (HMGCR) gene, producing proximal tubule cholesterol loading. AKI also causes sloughing of proximal tubular cell debris into tubular lumina. This study tested whether these two processes culminate in increased urinary pellet cholesterol content, and whether the latter has potential AKI biomarker utility. Urine samples were collected from 29 critically ill patients with (n = 14) or without (n= 15) AKI, 15 patients with chronic kidney disease, and 15 healthy volunteers. Centrifuged urinary pellets underwent lipid extraction, and the extracts were assayed for cholesterol content (factored by membrane phospholipid phosphate content). In vivo HMGCR activation was sought by measuring levels of RNA polymerase II (Pol II), and of a gene activating histone mark (H3K4m3) at exon 1 of the HMGCR gene (chromatin immunoprecipitation assay of urine chromatin samples). AKI+ patients had an approximate doubling of urinary pellet cholesterol content compared with control urine samples (versus normal; P < 0.001). The values significantly correlated (r, 0.5; P < 0.01) with serum, but not urine, creatinine concentrations. Conversely, neither critical illness without AKI nor chronic kidney disease raised pellet cholesterol levels. Increased HMGCR activity in the AKI+ patients was supported by three- to fourfold increased levels of Pol II, and of H3K4m3, at the HMGCR gene (versus controls or AKI- patients). (1) Clinical AKI, like experimental AKI, induces HMGCR gene activation; (2) increased urinary pellet cholesterol levels result; and (3) urine pellet cholesterol levels may have potential AKI biomarker utility. The latter will require future testing in a large prospective trial.

Androgen‐mediated cholesterol metabolism in LNCaP and PC‐3 cell lines is regulated through two different isoforms of acyl‐coenzyme A: Cholesterol Acyltransferase (ACAT)

The objective of this work was to determine the effect of an androgen agonist, R1881, on intracellular cholesterol synthesis and esterification in androgen-sensitive (AS) prostate cancer (LNCaP) cells. We investigated the activity and expression of cholesterol metabolism enzymes, HMG-CoA-reductase and ACAT in the LNCaP and PC-3 (androgen-independent control) models. Microsomal PC-3 HMG-CoA-reductase activity was increased with R1881 despite having similar cholesterol levels while increased cholesterol levels in microsomes from LNCaPs treated with R1881 (L+) were associated with increased HMG-CoA reductase activity. Increased intracellular cholesteryl esters (CE) found in (L+) were not associated with an increased ACAT1 activity. There was no effect from androgen treatment on ACAT1 protein expression in theses cells; however, ACAT2 expression was induced upon R1881 treatment. In contrast, we found an increase in the in vitro ACAT1 activity in PC-3 cells treated with androgen (P+). Only ACAT1 expression was induced in P+. We further assessed the expression of STAT1 alpha, a transcriptional activator that modulates ACAT1 expression. STAT1 alpha expression and phosphorylation were induced in P+. To determine the role of the AR on ACAT1 expression and esterification, we treated PC-3 cells overexpressing the androgen receptor with R1881 (PAR+). AR expression was decreased in PAR+ cells; ACAT1 protein expression and cholesterol ester levels were also decreased, however, ACAT2 remained unchanged. STAT1 alpha expression was decreased in PAR+. Overall, these findings support the importance of cholesterol metabolism regulation within prostate cancer cells and unravel a novel role for STAT1 alpha in prostate cancer metabolism.

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.

3-Hydroxy-3-methylglutaryl-coenzyme A reductase in the lobster mandibular organ: regulation by the eyestalk

The mandibular organ (MO) of the lobster, Homarus americanus, produces the isoprenoid methyl farnesoate (MF), a compound related to insect juvenile hormone (JH). To better understand the synthesis and regulation of MF, we studied 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase (HMGR), the rate-limiting enzyme in isoprenoid biosynthesis. Lobster HMGR had a K m of 11.4 μM for HMG-CoA, a K m of 14.8 μM for NADPH, and was at least 2000-fold more selective for this cofactor than for NADH. Lovastatin and mevalonic acid inhibited HMGR, with K I values of 1.3 nM and 25.3 μM, respectively, whereas MF, farnesoic acid, cholesterol, 20-hydroxyecdysone, and progesterone had no effect. Approximately 75% of the HMGR activity in lobster MO was soluble. Similar levels of HMGR activity were observed in all regions of the MO. Eyestalk removal increased MF synthesis and the activity of farnesoic acid O-methyltransferase (FAOMeT, the final step in MF synthesis) in the MO by 10.7- and 5.7-fold, respectively, and caused a 3.1-fold increase of HMGR activity. Injection of the eyestalk ablated lobsters with an extract of two sinus glands (SG), a neuroendocrine organ in the eyestalk, decreased MF synthesis, FAOMeT activity and HMGR activity to 3, 8, and 20%, respectively, of the levels observed in saline-treated animals. The regulation of crustacean HMGR by the SG suggests that the lobster MO is a useful model system for investigating the cellular regulation of HMGR activity.

HYPOTHALAMIC DIGOXIN, CEREBRAL DOMINANCE, AND LIPID METABOLISM

The present study assessed the biochemical differences in lipid metabolism between right hemispheric dominant and left hemispheric dominant individuals. The HMG CoA reductase activity and the serum isoprenoidal metabolites--digoxin, dolichol, and ubiquinone--were studied. The results showed that right hemispheric dominant individuals had (i) increased HMG CoA reductase activity, (ii) elevated serum digoxin levels, (iii) reduced serum ubiquinone levels, (iv) increased serum tryptophan and reduced tyrosine, (v) increased serum dolichol levels, and (vi) decreased RBC membrane Na+-K+ ATPase activity and serum magnesium levels. Left hemispheric dominant individuals had the opposite patterns. Right hemispheric dominance represents a hyperdigoxinemic state with membrane sodium-potassium ATPase inhibition and an upregulated isoprenoid pathway. Left hemispheric dominance represents the reverse pattern with hypodigoxinemia/membrane sodium-potassium ATPase stimulation and a downregulated isoprenoid pathway. Cerebral dominance can regulate lipid metabolism.

THE CONCEPT OF CEREBRAL CHEMICAL DOMINANCE

The study assessed the biochemical differences between right hemispheric dominant and left hemispheric dominant individuals detected by handedness and the dichotic listening test. The isoprenoid metabolites--digoxin, dolichol, and ubiquinone, glycoconjugate metabolism, free radical metabolism, and the RBC membrane composition were studied in individuals with differing hemispheric dominance. The results showed that all right hemispheric dominant individuals and 50% of left hemispheric dominant individuals had increased HMG CoA reductase activity, elevated digoxin and dolichol levels. The serum magnesium, RBC membrane Na+-K+ ATPase activity and serum ubiquinone levels were reduced in all right hemispheric dominant individuals and 50% of left hemispheric dominant individuals. The tryptophan-derived neurotrans­mittors--serotonin, quinolinic acid, strychnine, and nicotine--were increased while the tyrosine derived neurotransmittors--dopamine, noradrena1ine, and morphine--were reduced in all right hemispheric dominant individuals­ and 50% of left hemispheric dominant individuals. The other 50% of left hemispheric dominant individuals had decreased HMG CoA reductase activity, reduced digoxin, and dolichol levels. The serum magnesium, RBC membrane Na+-K+ ATPase activity, and serum ubiquinone levels were increased in this group. The tryptophan derived neurotrans­mittors--serotonin, quinolinic acid, strychnine, and nicotine were reduced,­ while the tyrosine-derived neurotransmittors--dopamine, noradrenaline, and morphine--­were increased in the rest (50% of left hemispheric dominant individuals). Hemispheric dominance detected by the dichotic listening test and handedness has no correlation with cerebral chemical dominance.­

Experimental model of escape phenomenon in hamsters and the effectiveness of YM‐53601 in the model

1. The aim of this study was to establish an experimental model of the escape phenomenon, in which plasma cholesterol, initially reduced by a 3-hydroxy-3-methylglutaryl CoA (HMG-CoA) reductase inhibitor such as pravastatin, increases again on long-term administration. We also evaluated the efficacy of YM-53601 ((E)-2-[2-fluoro-2- (quinuclidin-3-ylidene) ethoxy]-9H-carbazole monohydrochloride), a squalene synthase inhibitor, in this model. 2. Pravastatin inhibited cholesterol biosynthesis in hamster primary hepatocytes (IC(50), 14 nM). After pre-treatment with pravastatin, in contrast, almost no effect on cholesterol biosynthesis was seen. 3. In hamsters fed a high fat diet, 3 mg kg(-1) pravastatin for 9 days decreased plasma non-HDL cholesterol (total cholesterol - high density lipoprotein cholesterol) (P<0.01), but this effect was lost between 17 and 27 days of treatment, accompanied by an increase in HMG-CoA reductase activity. No such increase in plasma non-HDL cholesterol was seen with YM-53601 at 30 mg kg(-1) after 9 (P<0.001), 17 (P<0.01) or 27 (P<0.001) days of treatment. Replacement of pravastatin with YM-53601 caused a decrease in plasma non-HDL cholesterol by 53% (P<0.001) and in HMG-CoA reductase activity. 4. This animal model thus satisfactorily replicates the escape phenomenon observed in humans and may therefore be useful in evaluation of lipid-lowering agents, specifically comparison of HMG-CoA reductase inhibitors. Further, YM-53601 may be useful in the treatment of hypercholesterolemia without induction of the escape phenomenon.

Age-related changes of cholesterol and dolichol biosynthesis in rat liver

Ageing has been defined as a gradually decreased ability to maintain homeostatic potential and increased risk to die, associated with a tissue accumulation of altered proteins and lipids. Among other, increased concentration of an isoprenoid compound, dolichol (Dol), in mammalian tissues during ageing has been reported and it has been considered as a new biomarker of ageing. However, the mechanism and the role of this accumulation is still unknown. Aim of this work was to study the mechanism of age-dependent Dol accumulation in the liver analysing the activity of the hepatic rate-limiting enzyme of isoprenoid biosynthesis, the 3-hydroxy 3-methylglutaryl CoA reductase (HMGCoA reductase), the Dol synthesis by mevalonate (MVA), the Dol level in the plasma, and the cholesterol (Chol) synthesis and content of ageing rat fed ad libitum (AL) or subjected to the effect of food restriction. Since the caloric restrictions are the most reproducible way to slow ageing and to extend life span, animals on these nutritional regimens were used to study ageing related mechanisms. The data show that during ageing the hepatic Dol accumulation is associated with an increase of HMGCoA reductase activity, which is affected by diet restriction, and with an increase of MVA incorporation in Dol and Chol, which is not. In addition, the liver of aged rats maintains the capability to regulate its Chol content and to modify Chol delivery into the blood.

HYPOTHALAMIC DIGOXIN, CEREBRAL DOMINANCE, AND MEMBRANE BIOCHEMISTRY

The present study assessed the biochemical differences in membrane composition/function between right hemispheric dominant and left hemispheric dominant individuals. The HMG CoA reductase activity, serum isoprenoid metabolite--digoxin--serum magnesium, and the RBC membrane composition/Na+-K+ ATPase activity were studied. The results showed that right hemispheric dominant individuals had (i) increased HMG CoA reductase activity and elevated digoxin levels, (ii) decreased RBC membrane Na+-K+ ATPase activity and serum magnesium levels, and (iii) increased cholesterol:phospholipid ratio of RBC membranes with reduced membrane glycoconjugates. Left hemispheric dominant individuals had the opposite patterns. Right hemispheric dominance represents a hyperdigoxinemic state with membrane sodium-potassium ATPase inhibition. Left hemispheric dominance represents the reverse pattern with hypodigoxinemia and membrane sodium-potassium ATPase stimulation. Cerebral dominance can regulate membrane structure/function.

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.

Effect of FR194738, a potent inhibitor of squalene epoxidase, on cholesterol metabolism in HepG2 cells

( E)- N-ethyl- N-(6,6-dimethyl-2-hepten-4-ynyl)-3-[2-methyl-2-(3-thienylmethoxy)propyloxy]benzylamine hydrochloride (FR194738) inhibited squalene epoxidase activity in HepG2 cell homogenates with an IC 50 value of 9.8 nM. In the study using intact HepG2 cells, FR194738 inhibited cholesterol synthesis from [ 14C]acetate with an IC 50 value of 4.9 nM, and induced intracellular [ 14C]squalene accumulation. On the other hand, the 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase inhibitor simvastatin reduced both cholesterol and squalene synthesis from [ 14C]acetate. Incubation with simvastatin for 18 h produced increases in HMG-CoA reductase activity in HepG2 cells, which was related to the degree of reduction in cholesterol synthesis. The HMG-CoA reductase activity increased by 13- and 19-fold at the concentrations of simvastatin that inhibited cholesterol synthesis by 65% and 82%, respectively. In contrast, FR194738 did not increase HMG-CoA reductase activity at the concentrations that inhibited cholesterol synthesis by 24% and 69%, and moderate increase (4.6-fold) was observed at the concentration that inhibited cholesterol synthesis by 90%. These results suggest that non-sterol metabolite(s) derived from mevalonate prior to the squalene epoxidation step in the cholesterol synthetic cascade have a regulatory role in the suppression of HMG-CoA reductase activity. We speculate that FR194738 inhibits cholesterol synthesis with a minimal change of the regulator(s) and would be highly effective in the treatment of hypercholesterolemia.

Essential fatty acids as possible mediators of the actions of statins

Statins and polyunsaturated fatty acids have similar actions: both enhance endothelial nitric oxide synthesis, inhibit the production of pro-inflammatory cytokines, lower cholesterol levels, prevent atherosclerosis and are of benefit in coronary heart disease, stroke and osteoporosis. Statins enhance the conversion of linoleic acid and eicosapentaenoic acid to their long chain derivatives. Animals with essential fatty acid deficiency show an increase in HMG-CoA reductase activity, which reverts to normalcy following topical application of linoleic acid. Similarly to statins, polyunsaturated fatty acids also inhibit HMG-CoA reductase activity. In view of the similarity in their actions and as statins influence essential fatty acid metabolism, it is suggested that essential fatty acids and their metabolites may serve as second messengers of the actions of statins.

The Regulatory Effects Of Thyroid Hormone On The Activity Of 3-Hydroxy-3-Methylglutaryl Coenzyme A Reductase

The effects of 3, 3', 5-triiodothyronine (T3) on 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase activity were evaluated in the C100 baby hamster kidney cell line. Cells cultured in Minimal Essential Medium (MEM) were supplemented with 10% thyroid hormone-depleted fetal bovine serum (THDS-MEM) and had a 70.1% lower level of HMG-CoA reductase activity than the cells grown in a medium supplemented with fetal bovine serum (FBS). When T3 was added to THDS-MEM, the reduction of the reductase activity was blocked in a dose-dependent manner. In the cells grown in THDS-MEM for 48 hours, T3 (10-6 M) treatment rapidly increased HMG-CoA reductase activity, achieving the control level six hours after treatment. Such effects of T3 were blocked by actinomycin D (5 μg/ml) or cycloheximide (10 μg/ml). The transcriptional rate of the HMG-CoA reductase gene did not change significantly regardless of the presence of T3, while T3 inhibited the 25-hydroxycholesterol-mediated decay of the reductase mRNA significantly. Our results show that T3-dependent regulation of HMG-CoA reductase activity, via the de novo synthesis of the reductase enzyme, seems to be mediated at least partially by the stabilization of HMG-CoA reductase mRNA.

HMG-CoA reductase regulation: use of structurally diverse first half-reaction squalene synthetase inhibitors to characterize the site of mevalonate-derived nonsterol regulator production in cultured IM-9 cells

The activity of HMG-CoA reductase (HMGR) is tightly regulated, in part through post-transcriptional mechanisms that are mediated by nonsterol products of mevalonate metabolism. Previous reports have suggested that these mediators are derived from farnesyl pyrophosphate (FPP). Recent studies have implicated FPP hydrolysis products (e.g., farnesol), the squalene synthetase (SQS) reaction products presqualene pyrophosphate (PSQPP) and squalene, or their metabolites. To distinguish among these possible mediators, we evaluated the ability of HMGR and SQS inhibitors to induce compensatory increases in HMGR activity in cultured IM-9 cells. Mevinolin (HMGR inhibitor) produced predicted increases in HMGR activity that were related to the degree of cholesterolgenesis inhibition (e.g., 4-fold, 9-fold, and 17-fold increases relative to 50%, 76%, and 90% inhibition, respectively). By contrast, a variety of structurally distinct reversible, competitive, first half-reaction SQS inhibitors all reduced cholesterolgenesis by up to 90% with no appreciable increases in HMGR activity. These observations strongly suggest that nonsterol-mediated post-transcriptional mechanisms regulating HMGR activity remain intact after SQS first half-reaction inhibition, indicating that nonsterol regulator production is independent of SQS action and ruling out PSQPP, squalene and their metabolites as possible mediators. Unexpectedly, the SQS mechanism-based irreversible inactivator, zaragozic acid A (ZGA) exhibited the greatest degree of HMGR modulation, producing 5-fold, 11-fold, and 40-fold increases in HMGR activity at concentrations that produced 25%, 50%, and 75% cholesterolgenesis inhibition, respectively. The markedly greater magnitude of HMGR stimulation by ZGA versus mevinolin at similar levels of cholesterolgenesis inhibition suggests that ZGA may directly interfere with the production or action of the nonsterol regulator.

Lysophosphatidylcholine increases 3-Hydroxy-3-methylglutaryl-coenzyme A reductase gene expression in CaCo-2 cells

BACKGROUND & AIMS: The small intestine plays an important role in cholesterol homeostasis. The aim of this study was to examine the regulation of cholesterol synthesis by lysophosphatidylcholine in intestinal cells. METHODS: CaCo-2 cells cultured on semipermeable supports were incubated with taurocholate and lysophosphatidylcholine, and cholesterol synthesis rate, 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase activity, mass, and messenger RNA abundance were estimated. RESULTS: Lysophosphatidylcholine increased the rate of cholesterol synthesis as estimated by HMG-CoA reductase activity and acetate or water incorporation into sterols. Reductase was also increased by lysophosphatidylinositol or lysophosphatidylethanolamine but not by lysophosphatidylserine. Lysophosphatidylcholine increased HMG-CoA reductase messenger RNA and mass, suggesting that lysophosphatidylcholine regulated reductase at the level of gene expression. The various lysophospholipids caused the efflux of cellular cholesterol into the apical medium, and the amount effluxed correlated with the observed increase in reductase activity. Adding cholesterol to micelles containing lysophosphatidylcholine prevented the increase in HMG-CoA reductase activity and mass. CONCLUSIONS: Lysophosphatidylcholine increased cholesterol synthesis by increasing the expression of HMG-CoA reductase at the level of the gene and protein. Efflux of cellular cholesterol and the need to replace this lost cholesterol account for the observed changes in cholesterol metabolism. (Gastroenterology 1996 Apr;110(4):1068-76)

Effects of beta-blockers on HMG CoA reductase and LDL receptor activity in cultured human skin fibroblasts

Previous reports, based on clinical trials and animal experiments, suggest that beta-blockers may be useful in the prevention of atherosclerosis. Betaxolol, a new beta1-selective blocker, was shown to decrease plasma total and LDL cholesterol levels or to have no adverse effect on those [1-4]. While many reports deal with metabolism of triglyceride and high density lipoprotein, fewer publications about cholesterol metabolism are currently available. To clarify the mechanism by which beta-blockers affect lipid metabolism, we examined the effects of beta-blockers on HMG CoA reductase and LDL receptor activity in cultured human skin fibroblasts. L-propranolol, a nonselective beta-blocker, increased HMG CoA reductase activity and decreased LDL receptor activity. However, d-propranolol had no major effects on HMG CoA reductase activity. These results suggest that beta-blockers act on HMG CoA reductase through the beta receptors. Beta1-blocking action should decrease HMG CoA reductase activity and increase LDL receptor activity. In fact, betaxolol, a beta1-selective blocker, decreased HMG CoA reductase activity and increased LDL receptor activity, but metoprolol had no major effect. We speculate that the discrepancy between betaxolol and metoprolol in the effect on HMG CoA reductase and the LDL receptor might be due to the difference of the extent of beta1-selectivity. We conclude that beta1-selective blockers are antihypertensive agents potentially valuable in the prevention of atherosclerosis.

Effects of a novel 2,3-oxidosqualene cyclase inhibitor on the regulation of cholesterol biosynthesis in HepG2 cells.

Within the cholesterol biosynthesis cascade, the enzyme 2,3-oxidosqualene cyclase [EC 5.4.99.7] is of special interest due to its dual function: cyclization of 2,3-monoepoxysqualene to lanosterol and 2,3;22,23-diepoxysqualene to oxylanosterol. Further determination of the significance of this enzyme for the intracellular cholesterol homeostasis was done with BIBX 79, a new potent, specific inhibitor of this enzyme. In HepG2 cells the effects of BIBX 79 on cholesterol biosynthesis, 2,3-oxidosqualene cyclase as well as HMG-CoA reductase activities were studied. BIBX 79 is a potent inhibitor of sterol biosynthesis in HepG2 cells (IC50 4 x 10(-9) M). No other enzyme within the cholesterol biosynthesis cascade was significantly inhibited as was evidenced by a radio HPLC detection system. In contrast to simvastatin, no direct interaction with HMG-CoA reductase was observed. When incubating HepG2 cells for 16 h with the HMG-CoA reductase inhibitor simvastatin (10(-6)-10(-10) M) HMG-CoA reductase activity was increased up to 180%. BIBX 79 did also affect HMG-CoA reductase activity under these conditions: in concentrations of BIBX 79 "> or =" 10(-9) "< or =" 10(-7) M, where a partial inhibition of 2,3-oxidosqualene cyclase is observed, HMG-CoA reductase activity was decreased. However, higher concentrations of BIBX 79 that totally blocked 2,3-oxidosqualene cyclase led to an increase in HMG-CoA reductase activity. This effect of BIBX 79 on HMG-CoA reductase is thought to be mainly mediated by oxysterols that are formed by the cyclization of 2,3;22,23-diepoxysqualene. 2,3;22,23-Diepoxysqualene is preferentially cyclized by the 2,3-oxidosqualene cyclase and, consequently, only high inhibitor concentrations will also block 2,3;22,23-diepoxysqualene cyclization. Thus, by partial blockade of this enzyme, both an inhibition of lanosterol and subsequently cholesterol formation as well as a concomitant effect on HMG-CoA reductase can be achieved. Both effects complement each other and lead to an effective control of cholesterol biosynthesis. It is therefore concluded that 2,3-oxidosqualene cyclase plays a crucial role in the regulation of intracellular cholesterol homeostasis. 2,3-Oxidosqualene cyclase inhibitors offer an attractive approach for novel lipid-lowering agents.

Regulation of 3-hydroxy-3-methylglutaryl-coenzyme A reductase activity in the rat ileum: Effects of bile acids and lovastatin

We investigated the effects of intestinal bile acid flux, orientation of the 7-hydroxy group, and administration of lovastatin on the regulation of intestinal 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase activity in the rat ileum. HMG-CoA reductase activities in villous and crypt cells from the ileal mucosa were similar, and the study was performed on whole mucosa that contained both cell types. Taurocholate feeding decreased ileal reductase activity 48%, whereas tauroursocholate, the 7β-hydroxy epimer of taurocholate, had no effect. Feeding lovastatin (inhibitor of HMG-CoA reductase) stimulated total ileal HMG-CoA reductase activity threefold in washed microsomes, which were dissociated from the inhibitor. However, the proportion of active enzyme in the ileum of lovastatin-fed rats was 50% lower than in controls, whereas there was no change in the percentage of expressed enzyme with bile acid treatments. Interruption of the enterohepatic circulation (bile fistula) increased HMG-CoA reductase activity in the ileum 73%. Duodenal infusion of taurocholate to bile-fistula rats significantly decreased microsomal HMG-CoA reductase activity in the ileal mucosa. In contrast, infusion of the 7β-hydroxy epimer tauroursocholate failed to inhibit the derepressed HMG-CoA reductase activity in the ileum of bile-fistula rats. The inhibition of intestinal HMG-CoA reductase activity by taurocholate occurred without accumulation of mucosal cholesterol. Furthermore, the stimulation of total ileal HMG-CoA reductase activity by lovastatin treatment was observed without a decrease in mucosal cholesterol. In summary, the regulation of ileal HMG-CoA reductase activity by the intestinal luminal flux of bile acids is dependent on the orientation of the hydroxyl groups. Taurocholate is a potent inhibitor, whereas the 7β-hydroxylated bile acid tauroursocholate, which is more hydrophilic, fails to repress intestinal HMG-CoA reductase activity. Lovastatin, a competitive inhibitor of HMG-CoA reductase, stimulates the formation of inactive reductase in the ileum without a change in enterocyte cholesterol concentration.

Studies on rat liver phosphatidate phosphohydrolase and plasma lipids: Effect of HMG-CoA reductase inhibitors

Normolipidemic rats were treated with HMG-CoA reductase inhibitors (lovastatin or pravastatin) for periods of 1–3 days. Administration of these drugs reduced plasma triacylglycerol levels. Lovastatin-treated rats displayed a 30% lower hepatic triacylglycerol secretion rate compared to controls as estimated using Triton WR-1339. Lovastatin in a dose of 0.1% in the diet for 3 days increased hepatic phosphatidate phosphohydrolase (PAP) activity 2- to 3-fold, both in the cytosolic and microsomal fractions. Similar effects were seen in the presence of 0.2% pravastatin. PAP in both fractions was stimulated to a lesser extent by treatment with 0.1% pravastatin. These differences in PAP activity obtained by different treatment regimens were preserved during purification of cytosolic PAP on hydroxylapatite. The increase in PAP activity upon treatment with lovastatin or pravastatin was gradual and occurred simultaneously with the apparent increase in HMG-CoA reductase activity. In rats treated with the reductase inhibitors, the activity of microsomal and cytosolic PAP was inversely correlated to plasma triacylglycerol levels. The results indicate that hepatic triacylglycerol and cholesterol synthesis might be co-ordinately regulated, and also suggest that the activity of PAP is rapidly modulated in concert with changes in plasma triacylglycerol levels.

Presence of Cholesterol 7α-Hydroxylase Enzyme Protein in COS Cells Leads to Increased HMG CoA Reductase Activity

Transfection of COS-cells with a cDNA coding for human cholesterol 7α-hydroxylase resulted in significant production of cholesterol 7α-hydroxylase enzyme and intracellular accumulation of the product, 7α-hydroxycholesterol. Presence of this enzyme activity was always associated with increased HMG CoA reductase activity. In five different independent transfection experiments resulting in a cholesterol 7α-hydroxylase activity of 0.26 ± 0.05 pmol/min/mg in the transfected cells, the HMG CoA reductase activity increased to 158 ± 14 % of that of the control cells (p<0.01). This change was not associated with significant changes in the cholesterol content or LDL-receptor expression of the COS-cells. It is evident that the two key enzymes in cholesterol synthesis and degradation interact with each other also in extra-hepatic cells that are unable to degrade cholesterol into bile acids. Possible mechanisms for the finding is discussed.