Incorporation of mevalonate into dolichol and other isoprenoids during estrogen-induced chick oviduct differentiation

Incorporation of [14C]mevalonate into dolichol and other isoprenoid compounds by chick oviduct explants has been studied. A reliable assay of dolichol biosynthesis employing several chromatographic procedures, including two-dimentional TLC, was developed. Incorporation of [14C]mevalonate into dolichol by oviduct explants was linear for at least 6 h. The effect of estrogen-induced differentiation was studied by incubation of explants obtained from chicks treated for various periods of time with diethylstilbestrol. Mevalonate incorporation into dolichol, when expressed as cpm per g of tissue, was not affected by estrogen treatment, but since the oviduct increased about 100-fold in mass during differentiation, each oviduct synthesizes about 100-fold more dolichol. In most tissues, the major product of mevalonate incorporation is cholesterol. However, although approx. 90% of the non-saponifiable 14C-labeled compounds were in the so-called 'cholesterol fraction', oviduct explants from estrogenized chicks synthesized little, if any, cholesterol. A number of cholesterol biosynthetic intermediates were observed, with compounds comigrating with squalene and lanosterol accounting for about 50% of the total. Since the estrogenized chick has serum cholesterol levels in the range of 800-900 mg/dl, these results suggest that oviduct has secondary control points which allow it to inhibit cholesterol synthesis when mevalonate is used as the precursor. In support of this hypothesis is the observation that explants from untreated chicks can incorporate mevalonate into cholesterol.     

The isolation of 2,3-oxidosqualene from the liver of rats treated with 1-dodecylimidazole, a novel hypocholesterolaemic agent

1. Non-saponifiable lipid from the livers of rats treated with 1-dodecylimidazole contained an unidentified compound that was not present in the livers from untreated animals. 2. Treated rats had lower serum cholesterol concentrations than control rats. 3. 1-Dodecylimidazole, when added to rat liver slices, inhibited the incorporation of [1-(14)C]acetate and [2-(14)C]mevalonate into digitonin-precipitable sterols and resulted in the accumulation of a labelled compound, which was chromatographically identical with the unknown compound described in 1 above. 4. Rats treated with 1-dodecylimidazole incorporated less [(14)C]mevalonate into liver digitonin-precipitable sterols than untreated animals and accumulated the unknown compound as a labelled intermediate. 5. The unknown intermediate had the same chromatographic properties, n.m.r. and mass spectra as authentic 2,3-oxidosqualene. 6. The identity of the intermediate as 2,3-oxidosqualene was further established by showing that it was incorporated into sterols by rat liver homogenates under anaerobic conditions. In addition, incubation of [(14)C]squalene with rat liver homogenates resulted in trapping of the radioactivity by the added intermediate. 7. It is suggested that the hypocholesterolaemic activity of 1-dodecylimidazole results in part from the inhibition of cholesterol biosynthesis at the level of 2,3-oxidosqualene sterol cyclase.     

Contribution of the shunt pathway of mevalonate metabolism to the regulation of cholesterol synthesis in rat liver

The modulation of the shunt pathway of mevalonate metabolism (Edmond, J., and Popják, G. (1974) J. Biol. Chem. 249, 66-71) has been studied in livers from fed, starved, and diabetic rats perfused with a physiological concentration (300 nM) of [5-14C] + [5-3H]mevalonate. Shunt activity was measured by (i) production of 14CO2 (corrected for loss of label by exchange reactions) and (ii) production of 3H2O. Contribution of exogenous mevalonate to total mevalonate production (0.06-0.11%) was assessed in parallel experiments by the incorporation of 3H2O into sterols. Inhibition of non-saponifiable lipid synthesis by starvation and diabetes is not associated with an inhibition of mevalonate production but with a major increase in shunting (7-34%) of sterol-bound mevalonate. The shunt pathway of mevalonate metabolism appears to participate in the regulation of cholesterol synthesis.     

Propionate inhibits hepatocyte lipid synthesis

Oat bran lowers serum cholesterol in animals and humans. Propionate, a short-chain fatty acid produced by colonic bacterial fermentation of soluble fiber, is a potential mediator of this action. We tested the effect of propionate on hepatocyte lipid synthesis in rats using [1-14C]acetate, 3H2O, and [2-14C]mevalonate as precursors. Propionate produced a statistically significant inhibition of cholesterol biosynthesis from [1-14C]acetate at a concentration of 1.0 mM and from 3H2O and [2-14C]mevalonate at concentrations of 2.5 mM. Propionate also produced a significant inhibition of fatty acid biosynthesis at concentrations of 2.5 mM using [1-14C]acetate as a precursor. The demonstration of propionate-mediated inhibition of cholesterol and fatty acid biosynthesis at these concentrations suggests that propionate may inhibit cholesterol and fatty acid biosynthesis in vivo and may mediate in part the hypolipidemic effects of soluble dietary fiber. Further studies are needed to clarify this action of propionate and to establish the exact mechanisms by which the inhibition occurs.     

The shunt pathway of mevalonate metabolism in the isolated perfused rat kidney

The shunt pathway of mevalonate metabolism (Edmond, J., and Popják, G. (1974) J. Biol. Chem. 249, 66-71) has been studied in isolated kidneys from rats perfused with physiological concentrations of variously labeled [14C]- and [3H]mevalonates. The rate of operation of the shunt pathway was quantified by the production of either 14CO2 or 3H2O from the tracers. The measured rates of 14CO2 production from [14C] mevalonate were converted to rates of mitochondrial acetyl-CoA production by methods which take into account underestimations of metabolic rates derived from 14CO2 production. We have shown that the sex difference in renal shunting of mevalonate (Wiley, M. H., Howton, M. M., and Siperstein, M. D. (1979) J. Biol. Chem. 254, 837-842) occurs at physiological levels of substrate. The shunt pathway diverts up to 17% of the flux of mevalonate entering the cholesterol synthesis pathway in the kidney. It may, therefore, play a role in the long term regulation of cholesterol synthesis in this organ, as had been hypothesized by Edmond and Popják.     

The preparation and purification of isolated rat corpus-luteum cells and their use in studying the relationship between cholesterol biosynthesis and the lutropin-stimulated formation of progesterone.

Isolated luteal cells, prepared from superovulated rat ovaries by digestion with collagenase, were subjected to density-gradient centrifugation on Percoll to give a more highly purified preparation of luteal cells than has been reported previously. The cells formed progesterone when incubated in vitro; lutropin stimulated this steroidogenesis. Progesterone formation was linear for at least 2 h; a minimal lutropin concentration of 1.0 ng/ml was needed for stimulation and concentrations of 3.0 and 100 ng/ml gave half-maximal and maximal responses respectively. The cells were unresponsive towards hormones other than lutropin. Exposure to lutropin raised the cellular cyclic AMP concentration, and dibutyryl cyclic AMP, but not dibutyryl cyclic GMP, was as effective in stimulating steroidogenesis as was lutropin. Aminoglutethimide, an inhibitor of cholesterol side-chain cleavage, completely blocked progesterone formation by the cells, showing cholesterol side-chain cleavage to be an obligatory step in steroidogenesis by these cells. Neither the activity of 3-hydroxy-3-methylglutaryl-CoA reductase nor the incorporation of radioactively labelled acetate or mevalonate into cholesterol by cells incubated in vitro were detectable unless the rats had been treated previously with 4-aminopyrazolo[3,4-d]pyrimidine. In cells from rats so treated, compactin was found to block almost completely the incorporation of radioactively labelled acetate, but not of mevalonate, into cholesterol, indicating that this inhibitor acts in corpus luteum in the same way as it does in other tissues. In cells from rats not treated with 4-aminopyrazolo[3,4-d]pyrimidine compactin had no effect on progesterone formation in vitro, showing cholesterol biosynthesis to be unnecessary for the rapid steroidogenic response by luteal cells to lutropin.     

Effect of geraniol on fatty-acid and mevalonate metabolism in the human hepatoma cell line Hep G2.

Monoterpenes have multiple pharmacological effects on the metabolism of mevalonate. Geraniol, a dietary monoterpene, has in vitro and in vivo anti-tumor activity against several cell lines. We have studied the effects of geraniol on growth, fatty-acid metabolism, and mevalonate metabolism in the human hepatocarcinoma cell line Hep G2. Up to 100 micromol geraniol/L inhibited the growth rate and 3-hydroxymethylglutaryl coenzyme A reductase (HMG-CoA) reductase activity of these cells. At the same concentrations, it increased the incorporation of cholesterol from the medium in a dose-dependent manner. Geraniol-treated cells incorporated less 14C-acetate into nonsaponifiable lipids, inhibiting its incorporation into cholesterol but not into squalene and lanosterol. This is indicative of an inhibition in cholesterol synthesis at a step between lanosterol and cholesterol, a fact confirmed when cells were incubated with 3H-mevalonate. The incorporation of 3H-mevalonate into protein was also inhibited, whereas its incorporation into fatty acid increased. An inhibition of delta5 desaturase activity was demonstrated by the inhibition of the conversion of 14C-dihomo-gamma-linolenic acid into arachidonic acid. Geraniol has multiple effects on mevalonate and lipid metabolism in Hep G2 cells, affecting cell proliferation. Although mevalonate depletion is not responsible for cellular growth, it affects cholesterogenesis, protein prenylation, and fatty-acid metabolism.     

Cell-cycle-dependent, differential prenylation of proteins

Isoprenylated proteins related to cell growth have been detected during proliferation. Since cholesterogenesis (isoprenoid synthesis) is mandatory for cell proliferation, the observation of a temporally coordinated protein prenylation during the cell division cycle might constitute obligatory processes in the signalling pathway for initiating DNA replication and/or in maintaining the growing state. We have found such a definitive cell-cycle-phase-dependent pattern of prenylation for various classes of cytosolic and nuclear matrix proteins in synchronized HepG2 cells. Characteristic [3H]mevalonate incorporation began to increase during mid-to-late G1, just after cholesterol synthesis reached its apex, and peaked just prior to or coincident with mid S. Incorporation then declined subsequent to S (during G2) as cells approached mitosis. Prior to the rise in mevalonate incorporation into proteins, during early-to-mid G1, steady-state [14C]acetate incorporation into chromatographically resolved cholesterogenic lipid intermediates displayed a maximum only into cholesterol. However, during the late-G1/S interval, a singular peak of 14C incorporation was found for the farnesyl moiety (farnesol/nerolidol plus farnesyl diphosphate). Except for the farnesyl moiety, none of the other polyisoprenoids detected by our procedures showed any fluctuation in 14C incorporation subsequent to mid G1. These results support the proposal that subsequent to peak cholesterol synthesis in early-to-mid G1, the generation of a cholesterol-pathway-dependent set of post-translationally modified, polyisoprenylated proteins could constitute an obligatory step leading to the duplication of the cellular genome, thereby impelling transit through the cell cycle. The well known high flux through cholesterogenesis in tumors, which manifests an intrinsic lack of sensitivity to feedback inhibition and operates continuously, is consonant with this proposal.     

Inhibition of the biosynthesis of sterols by phenyl and phenolic compounds in rat liver

The presence of mitochondria increased the incorporation of [2-(14)C]mevalonate into sterols in a cell-free system from rat liver. Various phenyl and phenolic compounds inhibited the incorporation of mevalonate when added in vitro. p-Hydroxycinnamate, a metabolite of tyrosine, was the most powerful inhibitor among the compounds tested. Catechol, resorcinol and quinol were inhibitory at high concentrations. Organic acids lacking an aromatic ring were not inhibitory. Two hypocholesterolaemic drugs, Clofibrate (alpha-p-chlorophenoxyisobutyrate) and Clofenapate [alpha,4-(p-chlorophenyl)phenoxyisobutyrate], which are known to affect some step before the formation of mevalonate in the biosynthesis of cholesterol in vivo, showed inhibition at a step beyond the formation of mevalonate in vitro. The presence of the aromatic ring and the carboxyl group in a molecule appears to be necessary for the inhibition.     

Stimulation of liver cholesterol synthesis by actinomycin D

1. An eightfold increase in the incorporation of [2-(14)C]acetate into liver cholesterol in vivo was observed 24hr. after starved rats had been given actinomycin D (0.5mg./kg. of body wt.). Liver cholesterol radioactivity declined faster in the treated animals, suggesting a greater rate of cholesterol turnover. 2. Liver slices from treated animals showed a tenfold increase in the incorporation of [2-(14)C]acetate into cholesterol; conversion into CO(2) and into fatty acids was less markedly increased, and conversion into ketone bodies was not significantly affected. 3. The patterns of conversion into liver cholesterol in vivo of the lactone and the sodium salt of mevalonic acid differed markedly. The former was converted at a faster rate and to a greater extent than the latter. Treatment with actinomycin D increased the conversion of both forms of mevalonic acid into liver cholesterol, but only to a small extent. 4. Stimulation of the incorporation of acetate into cholesterol occurred at 4hr. after the administration of actinomycin D but not at 2hr. The response was abolished by the simultaneous administration of dl-ethionine or puromycin. 5. Pre-feeding with a cholesterol-rich diet greatly diminished the stimulation of conversion of acetate into cholesterol caused by actinomycin D, though it did not completely suppress it. Adrenalectomized animals responded to the drug, but much less markedly. 6. It is concluded that actinomycin D stimulates the synthesis of cholesterol in the liver at a stage in the pathway before mevalonic acid, by a mechanism that probably requires protein synthesis. A likely site would be the beta-hydroxy-beta-methylglutaryl-CoA reductase, the rate-limiting enzyme in cholesterol biosynthesis. Some possible mechanisms by which the drug may lead to increased activity of this enzyme are considered.     

Effect of 26-oxygenosterols from Ganoderma lucidum and their activity as cholesterol synthesis inhibitors

Ganoderma lucidum is a medicinal fungus belonging to the Polyporaceae family which has long been known in Japan as Reishi and has been used extensively in traditional Chinese medicine. We report the isolation and identification of the 26-oxygenosterols ganoderol A, ganoderol B, ganoderal A, and ganoderic acid Y and their biological effects on cholesterol synthesis in a human hepatic cell line in vitro. We also investigated the site of inhibition in the cholesterol synthesis pathway. We found that these oxygenated sterols from G. lucidum inhibited cholesterol biosynthesis via conversion of acetate or mevalonate as a precursor of cholesterol. By incorporation of 24,25-dihydro-[24,25-3H2]lanosterol and [3-3H]lathosterol in the presence of ganoderol A, we determined that the point of inhibition of cholesterol synthesis is between lanosterol and lathosterol. These results demonstrate that the lanosterol 14alpha-demethylase, which converts 24,25-dihydrolanosterol to cholesterol, can be inhibited by the 26-oxygenosterols from G. lucidum. These 26-oxygenosterols could lead to novel therapeutic agents that lower blood cholesterol.     

Screening of microbial secondary metabolites inhibiting cholesterol biosynthesis with the use of hepatoblastoma G2]

The culture of hepatoblastoma G2 (Hep G2) cells is proposed as an effective model for screening of microbial metabolites--inhibitors of sterol biosynthesis. This model can be applied at early stages of screening procedures and is quite effective for testing of crude extracts of producers' culture broth. The test is based on measurement inhibition of the radiolabelled precursors incorporation in cholesterol and separate fractions of lipids by microbial metabolites in Hep G2 cells. That allows not only to reveal inhibitors of cholesterol biosynthesis, but also to evaluate mechanism of action, including ability to inhibit the synthesis of cholesterol ethers. The cholesterol biosynthesis inhibition was tested at 150 microbial cultures (actinomycetes and imperfect fungi), isolated from soil. The ability to inhibit 14C-acetate incorporation into cholesterol was found in 15-20% of microbial cultures possessing antifungal activity of extracts (culture broth and mycelium).     

The regulation of the biosynthesis of ubiquinone in the rat.

The urinary excretion of p-hydroxybenzoate was not altered by ubiquinone feeding, but, although decreased considerably, was not eliminated in protein deficiency. The incorporation of p-hydroxy[U-14C]benzaldehyde into ubiquinone in vivo increased in cold-exposed and p-chlorophenoxyisobutyrate (clofibrate)-fed rats, and these changes were parallel with the changes in the incorporation of [2-14C]mevalonate under these conditions. Starvation, cholesterol feeding and cholic acid feeding resulted in the decreased incorporation of p-hydroxy[U-14C]benzaldehyde into ubiquinone, confirming the decreased ubiquinone synthesis. Feeding exogenous ubiquinone increased the hepatic ubiquinone concentration, but did not cause any decrease in the incorporation of p-hydroxy[U-14C]benzaldehyde into ubiquinone, indicating the absence of a feedback control.     

Lack of adaptation in lipogenesis by hepatoma 9121

The "minimal deviation" hepatoma 9121, implanted in rats, was shown to biosynthesize fatty acids from acetate-1-(14)C at the same rate as normal rat liver but faster than host liver. Feeding the host animals a fat-deficient diet caused fatty acid biosynthesis to be increased 3- to 13-fold in liver, but the dietary regimen did not influence fatty acid biosynthesis in the tumor tissue. Oxygen consumption and the oxidation of acetate and mevalonate to CO(2) were all affected by the dietary manipulation in liver but not in hepatoma. The fat-deficient diet decreased incorporation of acetate and mevalonate into cholesterol by the liver of control animals, increased it in the liver of host animals, and had no effect on this process in hepatoma. Thus, the transplantable tumor has lost the adaptive power of its parent tissue to respond to the dietary stimulus. The changes in fatty acid composition in total lipids in response to the fasting and refeeding were also markedly different in hepatoma from those in liver of the host animals. These results support the concept that this tumor is characterized by a loss of some metabolic controls.     

Alterations in cholesterol and fatty acid biosynthesis in rat liver homogenates by aryloxy acids (Short Communication)

2,4-Dichlorophenoxyacetic acid and 2,4,5-trichlorophenoxyacetic acid inhibited the incorporation of [2-(14)C]mevalonate into cholesterol and non-saponifiable lipids. Both compounds inhibited the conversion of [1-(14)C]isopentenyl pyrophosphate into cholesterol and the synthesis of cholesterol and fatty acids from [2-(14)C]acetate. There was no inhibition of the conversion of [1-(14)C]mevalonate into CO(2). At low concentrations (0.5mm) of the compounds there was a stimulation of acetate incorporation into fatty acids.     

Effect of p-chlorophenoxyisobutyrate (CPIB) fed to rats on hepatic biosynthesis and catabolism of ubiquinone

The effect of p-chlorophenoxyisobutyrate (CPIB) feeding on cholesterol and ubiquinone metabolism in rats was investigated. The results obtained from acetate-1-(14)C and mevalonate-2-(14)C incorporation studies both in vivo and in vitro confirm the results of other workers that CPIB feeding caused a metabolic block in the conversion of acetate to mevalonate, thereby inhibiting over-all steroidogenesis. Liver ubiquinone synthesis was inhibited in CPIB-fed rats, but a block in the catabolism of the ubiquinone resulted in accumulation of ubiquinone in CPIB-fed animals.     

Rat liver microsomal cholesterol biosynthesis and drug oxidase activity are affected by chromium ions (Cr3+) in vitro

Chromium ions (Cr3+)evoked a biphasic curve of changes of rat liver microsomal cholesterol biosynthesis using [14C]acetate and/or [14C]mevalonate as precursors. While for the lower range of Cr3+ concentrations the rate of cholesterol biosynthesis rises, at concentrations above 8 X 10(-6) M they evoke a decrease in the cholesterol biosynthesis, up to 50% down on its control value at a concentration of 8 X 10(-4) M. Differences were more pronounced when using [14C]mevalonate instead of [14C]acetate as precursor. The activity of the microsomal enzyme biphenyl-4-hydroxylase showed an equally intense rise to that of cholesterol biosynthesis up to a 8 X 10(-6) M Cr3+ concentration. Above this concentration, however, the activity of the enzyme starts to drop. NADPH-cytochrome c reductase and NADPH-oxidase were decreased at all Cr3+ concentrations used, which cover a 100-fold range. Lineweaver-Burk plots of the cytoplasmic glucose-6-phosphate dehydrogenase demonstrated an uncompetitive mechanism of inhibition by Cr3+ ions. The results are discussed in terms of the possible significance of the Cr3+ concentration-dependent effects on cholesterol biosynthesis, with the observed atherosclerosis in Cr-deficient humans.     

Involvement of calcium in the mevalonate-accelerated degradation of 3-hydroxy-3-methylglutaryl-CoA reductase

3-Hydroxy-3-methylglutaryl-coenzyme A reductase (HMG-CoA reductase), the rate-limiting enzyme in the biosynthesis of cholesterol and isoprenoids, is subject to rapid degradation which is regulated by mevalonate (MVA)-derived metabolic products. HMG-CoA reductase is an integral membrane protein of the endoplasmic reticulum, the largest nonmitochondrial pool of cellular Ca2+. To assess the possible role of Ca2+ in the regulated degradation of HMG-CoA reductase, we perturbed cellular Ca2+ concentration and followed the fate of HMG-CoA reductase and of HMGal, a fusion protein consisting of the membrane domain of HMG-CoA reductase and the soluble bacterial enzyme beta-galactosidase. The degradation of HMGal mirrors that of HMG-CoA reductase, demonstrating that the membrane domain of HMG-CoA reductase is sufficient to confer regulated degradation (Skalnik, D.G., Narita, H., Kent, C., and Simoni, R.D. (1988) J. Biol. Chem. 263, 6836-6841; Chun, K.T., Bar-Nun, S., and Simoni, R.D. (1990) J. Biol. Chem. 265, 22004-22010). In this study we show that the MVA-dependent accelerated rates of degradation of HMG-CoA reductase and HMGal in cells maintained in Ca(2+)-free medium are 2-3-fold slower than the rate of degradation in cells grown in high (1.8-2 mM) Ca2+ concentration. This effect is reversed upon addition of Ca2+ to the medium. Furthermore, when cells maintained in high Ca2+ are treated with 1 microM ionomycin, the MVA-dependent accelerated degradation of HMG-CoA reductase and HMGal is also reduced about 2-3-fold. This inhibition is not due to a Ca(2+)-dependent uptake or incorporation of MVA into sterols, since these processes are not affected in the absence of external Ca2+. In addition, cobalt, a known antagonist of Ca(2+)-dependent cellular functions, totally abolishes (IC50 = 520 microM in the presence of 1.8 mM extracellular Ca2+) the MVA-accelerated degradation of HMGal. These results suggest that Ca2+ plays a major role in the regulated degradation of HMG-CoA reductase.     

Purification and regulation of mevalonate kinase from rat liver

Mevalonate kinase may play a key role in regulating cholesterol biosynthesis because its activity may be regulated via feedback inhibition by intermediates in the cholesterol biosynthetic pathway. To study the regulation of mevalonate kinase, the enzyme was purified to homogeneity from rat liver, and monospecific antibody against mevalonate kinase was prepared. The purified mevalonate kinase had a dimeric structure composed of identical subunits, and the Mr of the enzyme determined by gel chromatography was 86,000. Based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the subunit Mr was 39,900. The pI for mevalonate kinate was 6.2. The levels of mevalonate kinase protein and enzyme activity were determined in the livers of rats treated with either cholesterol-lowering agents (cholestyramine, pravastatin, and lovastatin) or with dietary modifications. Diets containing cholestyramine alone or cholestyramine and either pravastatin or lovastatin increased mevalonate kinase activity 3-6-fold. Mevalonate kinase activity decreased approximately 50% in rats treated with diets containing either 5% cholesterol or 5% cholesterol and 0.5% cholic acid. Fasting did not significantly change mevalonate kinase activity. The amount of mevalonate kinase protein in the liver was quantitated using immunoblots, and the changes in the levels of kinase activity induced by either drug treatment or by cholesterol feeding were correlated with similar changes in the levels of mevalonate kinase protein. Therefore, under these experimental conditions, mevalonate kinase activity in the liver was regulated principally by changes in the rates of enzyme synthesis and degradation.