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.     

Selective inhibition of cholesterol synthesis in liver versus extrahepatic tissues by HMG-CoA reductase inhibitors

Hepatic specificity of inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase may be achieved by efficient first-pass liver extraction resulting in low circulating drug levels, as with lovastatin, or by lower cellular uptake in peripheral tissues, seen with pravastatin. BMY-21950 and its lactone form BMY-22089, new synthetic inhibitors of HMG-CoA reductase, were compared with the major reference agent lovastatin and with the synthetic inhibitor fluindostatin in several in vitro and in vivo models of potency and tissue selectivity. The kinetic mechanism and the potency of BMY-21950 as a competitive inhibitor of isolated HMG-CoA reductase were comparable to the reference agents. The inhibitory potency (cholesterol synthesis assayed by 3H2O or [14C]acetate incorporation) of BMY-21950 in rat hepatocytes (IC50 = 21 nM) and dog liver slices (IC50 = 23 nM) equalled or exceeded the potencies of the reference agents. Hepatic cholesterol synthesis in vivo in rats was effectively inhibited by BMY-21950 and its lactone form BMY-22089 (ED50 = 0.1 mg/kg p.o.), but oral doses (20 mg/kg) that suppressed liver synthesis by 83-95% inhibited sterol synthesis by only 17-24% in the ileum. In contrast, equivalent doses of lovastatin markedly inhibited cholesterol synthesis in both organs. In tissue slices from rat ileum, cell dispersions from testes, adrenal, and spleen, and in bovine ocular lens epithelial cells, BMY-21950 inhibited sterol synthesis weakly in vitro with IC50 values 76- and 188-times higher than in hepatocytes; similar effects were seen for BMY-22089. However, the IC50 ratios (tissue/hepatocyte) for lovastatin and fluindostatin were near unity in these models. Thus, BMY-21950 and BMY-22089 are the first potent synthetic HMG-CoA reductase inhibitors that possess a very high degree of liver selectivity based upon differential inhibition sensitivities in tissues. This cellular uptake-based property of hepatic specificity of BMY-21950 and BMY-22089, also manifest in pravastatin, is biochemically distinct from the pharmacodynamic-based disposition of lovastatin, which along with fluindostatin exhibited potent inhibition in all tissues that were exposed to it.     

Hepatic and nonhepatic sterol synthesis and tissue distribution following administration of a liver selective HMG-CoA reductase inhibitor, CI-981: comparison with selected HMG-CoA reductase inhibitors.

Since cholesterol biosynthesis is an integral part of cellular metabolism, several HMG-CoA reductase inhibitors were systematically analyzed in in vitro, ex vivo and in vivo sterol synthesis assays using [14C]acetate incorporation into digitonin precipitable sterols as a marker of cholesterol synthesis. Tissue distribution of radiolabeled CI-981 and lovastatin was also performed. In vitro, CI-981 and PD134967-15 were equipotent in liver, spleen, testis and adrenal, lovastatin was more potent in extrahepatic tissues than liver and BMY21950, pravastatin and PD135023-15 were more potent in liver than peripheral tissues. In ex vivo assays, all inhibitors except lovastatin preferentially inhibited liver sterol synthesis; however, pravastatin and BMY22089 were strikingly less potent in the liver. CI-981 inhibited sterol synthesis in vivo in the liver, spleen and adrenal while not affecting the testis, kidney, muscle and brain. Lovastatin inhibited sterol synthesis to a greater extent than CI-981 in the spleen, adrenal and kidney while pravastatin and BMY22089 primarily affected liver and kidney. The tissue distribution of radiolabeled CI-981 and lovastatin support the changes observed in tissue sterol synthesis. Thus, we conclude that a spectrum of liver selective HMG-CoA reductase inhibitors exist and that categorizing agents as liver selective is highly dependent upon method of analysis.     

Tissue selectivity of pravastatin sodium, lovastatin and simvastatin. The relationship between inhibition of de novo sterol synthesis and active drug concentrations in the liver, spleen and testis in rat.

Tissue selectivity of pravastatin sodium (pravastatin), lovastatin and simvastatin, 3-hydroxy-3-methylglutaryl-CoA reductase inhibitors was examined by measuring inhibition of de novo sterol synthesis and active drug concentrations in the liver, spleen and testis in rats after a single oral administration (25 mg/kg) of these drugs. Regarding tissue drug concentrations, all three drugs were liver selective: concentrations of drugs in the liver were about ten-times higher than those in the spleen and testis. On the other hand, pravastatin was far more liver selective in inhibiting sterol synthesis than two other inhibitors: pravastatin inhibited de novo sterol synthesis in the liver but minimally in the spleen and testis, whereas lovastatin and simvastatin inhibited in all three tissues. Microautoradiographic and in vitro cellular-uptake studies demonstrated that pravastatin remained in the extracellular space in the spleen, whereas the other drugs entered the cell. We conclude that pravastatin exhibits a liver-selective inhibition of sterol synthesis because the agent permeates the cell membrane in the liver, but not in non-hepatic tissues.     

Different effects of 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors of sterol synthesis in various human cell types

The three vastatins examined, lovastatin, simvastatin and pravastatin, are equally strong inhibitors of the sterol synthesis in human hepatocytes in culture with IC₅₀-values of 4.1, 8.0 and 2.0 nM, respectively. However, in the human extrahepatic cells: umbilical vascular endothelial cells, retinal pigment epithelial cells, cornea fibroblasts and granulosa cells, pravastatin was much less inhibiting the sterol synthesis than lovastatin or simvastatin. It was observed as well that longer incubation with the vastatins resulted in higher IC₅₀-values. In order to show that the feedback regulation mechanism for 3-hydroxy-3-methylglutaryl-coenzyme A reductase was involved in this phenomena mRNA levels were measured in human vascular endothelial cells after incubation with the vastatins for 3.5 h and for 20 h. Indeed, lovastatin and simvastatin gave rise to higher levels of HMG-CoA reductase mRNA after 20 h than after 3.5 h of incubation. The differences observed in different human cell types can be explained by supposing that pravastatin is transported into the human hepatocyte via a liver-specific transporter. This was supported by the results of uptake experiments with ¹⁴C-labelled pravastatin and ¹⁴C-labelled simvastatin into human hepatocytes compared to that into human umbilical endothelial cells (as an example of an extrahepatic cell type). [¹⁴C]-Simvastatin was associated with both cell types, whereas [¹⁴C]-pravastatin was hardly associated with human endothelial cells, but to a similar extent as [¹⁴C]-simvastatin with human hepatocytes.     

Hep-G2 cells and primary rat hepatocytes differ in their response to inhibitors of HMG-CoA reductase

CI-981, a novel synthetic inhibitor of HMG-CoA reductase, was previously reported to be highly liver-selective using an ex vivo approach. In order to determine liver-selectivity at the cellular level, CI-981 was evaluated in cell culture and compared to lovastatin, pravastatin, fluvastatin and BMY-21950. Using human cell lines, none of the compounds tested showed liver-selectivity, i.e. strong inhibition of cholesterol synthesis in Hep-G2 cells (liver model) but weak inhibition in human fibroblasts (peripheral cell model). In contrast, all drugs tested produced equal and potent inhibition of sterol synthesis in primary cultures of rat hepatocytes, and CI-981, pravastatin and BMY-21950 were more than 100-fold more potent in rat hepatocytes compared to human fibroblasts. Since all compounds were also equally potent at inhibiting sterol synthesis in a rat subcellular system and in vivo, the data suggest that the use of Hep-G2 cells may not be the cell system of choice in which to study inhibition of hepatic cholesterogenesis or to demonstrate liver selectivity of inhibitors of HMG-CoA reductase.     

Lovastatin treatment inhibits sterol synthesis and induces HMG-CoA reductase activity in mononuclear leukocytes of normal subjects

The mechanism by which competitive inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase decrease serum cholesterol is incompletely understood. The few available data in humans suggest that chronic administration of the competitive inhibitor, lovastatin, decreases serum cholesterol with little or no change in total body sterol synthesis. To further define the effect of lovastatin on cholesterol synthesis in normal subjects, we investigated the effect of a single oral dose of lovastatin and a 4-week treatment period of lovastatin on mononuclear leukocyte (ML) sterol synthesis as a reflection of total body sterol synthesis. In parallel, we measured serum lipid profiles and HMG-CoA reductase activity in ML microsomes that had been washed free of lovastatin. ML sterol synthesis did not significantly decrease (23 +/- 5%, mean +/- SEM) at 3 h after a single 40-mg dose of lovastatin. With a single oral 80-mg dose, ML sterol synthesis decreased by 57 +/- 10% (P less than 0.05) and remained low for the subsequent 6 h. With both doses, total HMG-CoA reductase enzyme activity in microsomes prepared from harvested mononuclear leukocytes was induced 4.8-fold (P less than 0.01) over baseline values. Both the 20-mg bid dose and the 40-mg bid dose of lovastatin administered for a 4-week period decreased serum cholesterol by 25-34%. Lovastatin at 20 mg bid decreased ML sterol synthesis by 23 +/- 6% (P less than 0.02) and increased ML HMG-CoA reductase 3.8 times (P less than 0.001) the baseline values. Twenty four hours after stopping lovastatin, ML sterol synthesis and HMG-CoA reductase enzyme activity had returned to the baseline values. The higher dose of lovastatin (40 mg bid) decreased ML sterol synthesis by 16 +/- 3% (P less than 0.05) and induced HMG-CoA reductase to 53.7 times (P less than 0.01) the baseline value at 4 weeks. Stopping this higher dose effected a rebound in ML sterol synthesis to 140 +/- 11% of baseline (P less than 0.01), while HMG-CoA reductase remained 12.5 times baseline (P less than 0.01) over the next 3 days. No rebound in serum cholesterol was observed. From these data we conclude that in normal subjects lovastatin lowers serum cholesterol with only a modest effect on sterol synthesis. The effect of lovastatin on sterol synthesis in mononuclear leukocytes is tempered by an induction of HMG-CoA reductase enzyme quantity, balancing the enzyme inhibition by lovastatin.(ABSTRACT TRUNCATED AT 400 WORDS)     

Inhibition of rat brain prostaglandin D synthase by 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors

The effect of lovastatin, simvastatin and pravastatin, which are competitive inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, was tested on PGD synthase purified to apparent homogeneity from the rat brain. Lovastatin and simvastatin, which reportedly cause insomnia in vivo, inhibited the PGD synthase activity dose-dependently and showed an IC50 value of 100 and 75 microM, respectively. On the other hand, pravastatin, which does not cause insomnia, showed no significant effect at 100 microM and only a slight effect up to 500 microM.     

Discordant expression of the sterol pathway in lens underlies simvastatin-induced cataracts in Chbb: Thom rats

Simvastatin rapidly induced cataracts in young Chbb:Thom (CT) but not Sprague Dawley (SD) or Hilltop Wistar (HW) rats. Oral treatment for 14 but not 7 days committed CT rat lenses to cataract formation. The cholesterol to phospholipid molar ratio in lenses of treated CT rats was unchanged. Differences between strains in serum and ocular humor levels of simvastatin acid poorly correlated with susceptibility to cataracts. No significant differences were found between rat strains in the capacity of simvastatin acid to inhibit lens-basal sterol synthesis. Prolonged treatment with simvastatin comparably elevated HMG-CoA reductase protein and enzyme activity in lenses of both cataract resistant and sensitive strains. However, in contrast to SD and HW rats, where sterol synthesis was markedly increased, sterol synthesis in CT rat lenses remained at baseline. Discordant expression of sterol synthesis in CT rats may be due to inadequate upregulation of lens HMG-CoA synthase. HMG-CoA synthase protein levels, and to a much lesser extent mRNA levels, increased in lens cortex of SD but not CT rats. Because upregulation of the sterol pathway may result in increased formation of isoprene-derived anti-inflammatory substances, failure to upregulate the pathway in CT rat lenses may reflect an attenuated compensatory response to injury that resulted in cataracts.     

Hypercholesterolaemia: simvastatin and pravastatin alter cholesterol metabolism by different mechanisms

The 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor simvastatin, reduced low-density-lipoprotein (LDL) cholesterol in hypercholesterolaemic patients by 40% (P less than 0.001). The reduction in LDL cholesterol was accompanied by a significant decrease in the esterified/free cholesterol ratio of the patients' LDL from 2.51 +/- 0.13 to 2.06 +/- 0.14 (P less than 0.01). This change led to a significant increase (P less than 0.05) in the capacity of the LDL to suppress [14C]acetate incorporation into cholesterol in mononuclear leucocytes. Furthermore, [14C]acetate incorporation into the patients mononuclear leucocytes was significantly lower (P less than 0.02) following drug treatment (117 +/- 22 vs. 162 +/- 29 nmol/mg cell protein). Comparison of simvastatin with another HMG-CoA reductase inhibitor pravastatin, showed similar reduction in LDL cholesterol. Pravastatin treatment however, did not result in a reduction in the LDL esterified/free cholesterol ratio or in the changes in cellular cholesterol synthesis and its regulation by LDL which accompanied simvastatin treatment. The activity of the enzyme acyl-coenzyme A: cholesterol acyltransferase (ACAT) in patients' mononuclear cells remained unchanged after treatment with either drug. Results of the study show that while the drugs are equally effective in lowering LDL cholesterol, simvastatin has additional compositional effects on LDL which increase its capacity to regulate mononuclear leucocyte cholesterologenesis.     

Tissue selectivity of the cholesterol-lowering agents lovastatin, simvastatin and pravastatin in rats in vivo

Tissue selectivity of lovastatin, simvastatin and pravastatin was determined in male rats. Peak levels of active drug were found in all tissues examined between 0.5 and 2 hours after oral administration. The area under the curve describing 24 hour exposure of the tissues to drug indicated that the drugs were preferentially concentrated in the liver. However, the concentration of pravastatin was approximately 50% that of either lovastatin or simvastatin in the liver and 3-6 times higher in peripheral tissues. These studies demonstrate that the hydrophobic prodrugs, lovastatin and simvastatin show greater selectivity than the hydrophilic agent pravastatin towards the liver which is the target organ for inhibitors of 3-hydroxy-3-methylglutaryl-coenzyme A reductase.     

Depletion of substrates for protein prenylation increases apoptosis in human periovulatory granulosa cells

Progesterone receptor (PR) stimulation promotes survival in human and rat periovulatory granulosa cells. PR antagonists, Org 31710 and RU 486, both increase apoptosis and decrease cholesterol synthesis in these cells. The decrease in cholesterol synthesis also causes decreased synthesis of other products branching from the cholesterol synthesis pathway, including substrates for protein prenylation. In this study we focus on the link between apoptosis and prenylation in human periovulatory granulosa cells. A decreased cholesterol synthesis and increased apoptosis was verified in experiments with human periovulatory granulosa cells treated with the PR antagonists Org 31710 or RU 486 by measuring caspase-3/7 activity and incorporation of 14C-acetate into cholesterol and progesterone. Correspondingly, specific inhibition of cholesterol synthesis in periovulatory human granulosa cells using HMG-CoA reductase inhibitors (lovastatin or simvastatin) increased apoptosis, measured as caspase-3/7 activity. The increase in apoptosis caused by simvastatin or Org 31710 was partially reversed by addition of the protein prenylation precursors farnesol or geranylgeraniol. In addition, the prenylation inhibitors FTI R115777 and GGTI 2147 increased apoptosis in these cells. In conclusion our data suggest that PR antagonists increase apoptosis and reduce cholesterol synthesis in periovulatory granulosa cells and that the resulting depletion of substrates for protein prenylation may contribute to the increased apoptosis sensitivity.     

Effect of lovastatin on acyl-CoA: cholesterol O-acyltransferase (ACAT) activity and the basolateral-membrane secretion of newly synthesized lipids by CaCo-2 cells.

Lovastatin, a potent competitive inhibitor of 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase activity, was used to study the regulation of cholesterol metabolism and the basolateral-membrane secretion of triacylglycerol and cholesterol in the human intestinal cell line CaCo-2. At 0.1 microgram/ml, lovastatin decreased 3H2O incorporation into cholesterol by 71%. In membranes prepared from cells incubated with lovastatin for 18 h, HMG-CoA reductase activity was induced 4-8-fold. Mevalonolactone prevented this induction. In intact cells, lovastatin (10 micrograms/ml) decreased cholesterol esterification by 50%. The reductase inhibitor decreased membrane acyl-CoA:cholesterol O-acyltransferase (ACAT) activity by 50% at 5 micrograms/ml. ACAT inhibition by lavastatin was not reversed by adding excess of cholesterol or fatty acyl-CoA to the assay. Lovastatin, in the presence or absence of mevalonolactone, decreased the basolateral secretion of newly synthesized cholesteryl esters and triacylglycerols. Lovastatin also inhibited the esterification of absorbed cholesterol and the secretion of this newly synthesized cholesteryl ester. Lovastatin is a potent inhibitor of cholesterol synthesis in CaCo-2 cells. Moreover, it is a direct inhibitor of ACAT activity, independently of its effect on HMG-CoA reductase and cholesterol synthesis.     

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.     

Inhibition of cholesterol synthesis and esterification regulates high density lipoprotein interaction with isolated epithelial cells of human small intestine

The effect of two inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, lovastatin and monacolin L, and an inhibitor of acyl coenzyme A:cholesterol acyltransferase (ACAT), Sandoz compound 58-035, on the interaction of 125I-labeled high density lipoprotein-3 (HDL3) with isolated human enterocytes was studied. Both HMG-CoA reductase inhibitors inhibited cholesterol synthesis and 125I-labeled HDL3 binding and degradation by enterocytes; a strong correlation between changes in cholesterol synthesis and interaction of 125I-labeled HDL3 with cells was observed. Lovastatin caused reduction of the apparent number of 125I-labeled HDL3 binding sites without affecting the binding affinity. No changes of cell cholesterol content were observed after incubation of cells with lovastatin. Mevalonic acid reversed the effect of lovastatin on 125I-labeled HDL3 binding. Lovastatin blocked up-regulation of the HDL receptor in response to loading of cells with nonlipoprotein cholesterol and modified cholesterol-induced changes of 125I-labeled HDL3 degradation. Lovastatin also reduced HDL-mediated efflux of endogenously synthesized cholesterol from enterocytes. The ACAT inhibitor caused a modest increase of 125I-labeled HDL3 binding to enterocytes and significantly decreased its degradation; both effects correlated with inhibition of cholesteryl ester synthesis. The results allow us to assume that the intracellular free cholesterol pool may play a key role in regulation of the HDL receptor.     

Inhibition of farnesylation blocks growth but not differentiation in FRTL-5 thyroid cells.

The cholesterol lowering drug lovastatin, a competitive inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, blocks DNA synthesis and proliferation of thyrotropin (TSH) primed FRTL-5 rat thyroid cells. The blockade can be completely prevented and/or reversed by mevalonate and largely prevented and/or reversed by farnesol whereas cholesterol and/or other non-sterol mevalonate derivatives such as ubiquinone, dolichol or isopentenyladenosine are ineffective. TSH-dependent augmentation of cyclic-AMP and cAMP dependent differentiated functions, such as iodide uptake, are unaffected by lovastatin. 3H-Thymidine incorporation into DNA is also decreased by alpha-hydroxyfarnesyl-phosphonic acid, an inhibitor of protein farnesylation which mimicks the effect of lovastatin since it also leaves unaffected TSH stimulated iodide uptake. It is suggested that the HMG-CoA reductase inhibitor lovastatin affects cell proliferation mainly through inhibition of protein farnesylation which results in altered function proteins relevant for proliferation control, notably p21ras and/or other small GTPases.     

Progesterone-receptor antagonists and statins decrease de novo cholesterol synthesis and increase apoptosis in rat and human periovulatory granulosa cells in vitro

Progesterone-receptor (PR) stimulation promotes survival in rat and human periovulatory granulosa cells. To investigate the mechanisms involved, periovulatory rat granulosa cells were incubated in vitro with or without the PR-antagonist Org 31710. Org 31710 caused the expected increase in apoptosis, and expression profiling using cDNA microarray analysis revealed regulation of several groups of genes with functional and/or metabolic connections. This regulation included decreased expression of genes involved in follicular rupture, increased stress responses, decreased angiogenesis, and decreased cholesterol synthesis. A decreased cholesterol synthesis was verified in experiments with both rat and human periovulatory granulosa cells treated with the PR-antagonists Org 31710 or RU 486 by measuring incorporation of [14C]acetate into cholesterol, cholesterol ester, and progesterone. Correspondingly, specific inhibition of cholesterol synthesis in periovulatory rat granulosa cells using 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors (lovastatin, mevastatin, or simvastatin) increased apoptosis, measured as DNA fragmentation and caspase-3/7 activity. The increase in apoptosis caused by simvastatin was reversed by addition of the cholesterol synthesis-intermediary mevalonic acid. These results show that PR antagonists reduce cholesterol synthesis in periovulatory granulosa cells and that cholesterol synthesis is important for granulosa cell survival.     

Regulation of bile acid synthesis. IV. Interrelationship between cholesterol and bile acid biosynthesis pathways

Under most experimental conditions, the activities of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA reductase) and cholesterol 7 alpha-hydroxylase, change together in parallel directions. It has been suggested that newly synthesized cholesterol may be the preferred substrate for cholesterol 7 alpha-hydroxylase, which may account for the observed synchronous behavior of the two enzymes. To test this hypothesis, mevinolinic acid, a potent competitive inhibitor of HMG-CoA reductase, was administered as a single intravenous bolus (10 mg/kg) to rats with a chronic bile fistula. Bile acid synthesis was determined following inhibition of HMG-CoA reductase by mevinolinic acid over a 27-h time course and specific activities of HMG-CoA reductase and cholesterol 7 alpha-hydroxylase were determined in liver microsomes. At 3, 6, and 27 h after a bolus dose of mevinolinic acid, bile acid synthesis was reduced by 54 +/- 5%, 42 +/- 8%, and 23 +/- 13%, respectively, from preinfusion baseline. Within 30 min after administration of mevinolinic acid, HMG-CoA reductase activity was inhibited by at least 87%. At 0.5, 1.5, 3, 6, and 27 h after mevinolinic acid injection, cholesterol 7 alpha-hydroxylase activity was decreased by 6%, 25%, 54%, 41%, and 17%, respectively. By 27 h, the activities of both enzymes had returned to baseline levels. The reduction of bile acid synthesis correlated closely with the observed changes in the activities of cholesterol 7 alpha-hydroxylase. In vitro addition of mevinolinic acid (up to 20 microM) to rat liver microsomes failed to inhibit cholesterol 7 alpha-hydroxylase activity, suggesting no direct effect of mevinolinic acid on enzyme activity. When a bolus dose of mevinolinic acid was coupled with a continuous infusion of mevalonate, the product of the reaction catalyzed by HMG-CoA reductase, the mevinolinic acid-induced decrease in cholesterol 7 alpha-hydroxylase activity and bile acid synthesis was prevented. The results of this study provide evidence that, under the experimental conditions described, there is a linkage between the rates of cholesterol synthesis and the activities of cholesterol 7 alpha-hydroxylase. The data also emphasize the importance of the newly synthesized cholesterol in the regulation of cholesterol 7 alpha-hydroxylase activity.     

Formation of mevalonic acid, sterols and bile acids from [1-14C]acetyl-CoA and [2-14C]malonyl-CoA in the liver of rabbits with experimental hypercholesterolemia

The effect of cholesterol diet on the rate of mevalonic acid biosynthesis from 1-14C acetyl-CoA, 2-14C malonyl-CoA and the incorporation of these substrates into sterols and bile acids in rabbit liver were studied. Simultaneously, the activities of 3-hydroxy-3-methylglutaryl-CoA reductase (HMG-CoA reductase) and acetyl-CoA carboxylase and the biosynthesis of fatty acids from acetyl-CoA and malonyl-CoA were measured. Hypercholesterolemia was found to be concomitant with the inhibition of acetyl-CoA carboxylase activity only in cell-free (700 g) and mitochondrial fractions and slightly decreased the incorporation of acetyl-CoA and malonyl-CoA into fatty acids in the postmitochondrial fraction. The HMG-CoA reductase activity in all subcellular fractions except for the postmicrosomal one was inhibited under these conditions. A significant decrease of acetyl-CoA incorporation and an increase in malonyl-CoA incorporation into mevalonic acid in all liver fractions except for microsomal one were observed in rabbits with hypercholesterolemia. These data provide evidence for the existence of two pathways of mevalonic acid synthesis from the above-said substrates that are differently sensitive to cholesterol. Cholesterol feeding resulted in a decreased synthesis of the total unsaponified fraction including cholesterol from acetyl-CoA, malonyl-CoA and mevalonic acid. The rate of incorporation of these substrates into lanosterol was unchanged. All the indicated substrates (acetyl-CoA, malonyl-CoA, mevalonic acid) are precursors of bile acid synthesis in rabbit liver. Cholesterol feeding and the subsequent development of hypercholesterolemia resulted in bile acid synthesis stimulation, preferentially in the formation of the cholic + deoxycholic acids from these precursors.