Effects of peroxisome proliferators gemfibrozil and clofibrate on syntheses of dolichol and cholesterol in rat liver

The effects of two peroxisome proliferators, gemfibrozil and clofibrate, on syntheses of dolichol and cholesterol in rat liver were investigated. Gemfibrozil did not affect the overall content of dolichyl phosphate, but it changed the chain-length distribution of dolichyl phosphate, increasing the levels of species with shorter isoprene units. Gemfibrozil suppressed synthesis of dolichyl phosphate from [(3)H]mevalonate and [(3)H]farnesyl pyrophosphate in rat liver. In contrast, clofibrate increased the content of dolichol (free and acyl ester forms). It remarkably enhanced dolichol synthesis from mevalonate, but did not affect dolichol synthesis from farnesyl pyrophosphate. Gemfibrozil elevated cholesterol synthesis from [(14)C]acetate, but did not affect the synthesis from mevalonate. Clofibrate suppressed cholesterol synthesis from acetate, but did not affect cholesterol synthesis from mevalonate. These results suggest that gemfibrozil suppresses synthesis of dolichyl phosphate by inhibiting, at the least, the pathway from farnesyl pyrophosphate to dolichyl phosphate. As a result, the chain-length pattern of dolichyl phosphate may show an increase in shorter isoprene units. Clofibrate may increase the content of dolichol by enhancing dolichol synthesis from mevalonate. Gemfibrozil may increase cholesterol synthesis by activating the pathway from acetate to mevalonate. Unlike gemfibrozil, clofibrate may decrease cholesterol synthesis by inhibiting the pathway from acetate to mevalonate.     

Increased hepatic dolichol and dolichyl phosphate-mediated glycosylation in rats fed cholesterol

The concentrations of dolichol and cholesterol in livers of rats maintained for 2 weeks on a diet enriched with cholesterol (1%) were significantly higher than those in animals on a normal diet. The incorporation of radioactive mevalonate into dolichol and into a dolichyl diphosphate oligosaccharide fraction by liver slices of the cholesterol-fed animals was increased over that of the control group. However, the incorporation of radioactive mevalonate into cholesterol was decreased, as was the incorporation of radioactive acetate into both dolichol and, more markedly, cholesterol. These results are consistent with cholesterol feeding causing partial inhibition of the cholesterol-biosynthetic pathway both at β-hydroxy-β-methylglutaryl coenzyme A reductase and at a step after farnesyl pyrophosphate formation, resulting in a greater flux of mevalonate to dolichol and an increase in pool sizes of precursors of β-hydroxy-β-methylglutaryl coenzyme A. Maximal activity of glycosyl transfer to dolichyl phosphate was greater in microsomal preparations from livers of cholesterol-fed animals compared with those of control animals. A corresponding higher degree of in vitro glycosylation of endogenous protein was also observed. It is concluded that the cholesterol-enriched diet caused an increase in the biosynthesis and concentration of dolichyl monophosphate which resulted in a higher level of N-glycosylation of protein. These effects were complicated by differences in the kinetics of glycosyl transfer and in its response to exogenous dolichyl monophosphate.     

Occurrence of the enzymes effecting the conversion of acetyl CoA to squalene in homogenates of hog aorta

Homogenates and subcellular fractions of the intimamedia of hog aorta have been prepared and examined for the presence of the enzymes catalyzing the conversion of acetyl CoA to squalene. Enzyme activities effecting the conversion of acetyl CoA to 3-hydroxy-3-methylglutarate (HMG); HMG CoA to mevalonic acid; mevalonic acid to 5-phosphomevalonic acid, 5-pyrophosphomevalonic acid, and isopentenyl pyrophosphate; isopentenyl pyrophosphate to farnesyl pyrophosphate; and farnesyl pyrophosphate to squalene have been demonstrated in these homogenates. The overall conversion of mevalonate to squalene has also been demonstrated with recombined fractions of hog aorta homogenates. Data are also presented that suggest that phosphatases present in the crude homogenates act to cleave farnesyl pyrophosphate to farnesol, and phospho- and pyrophosphomevalonate to mevalonate.     

Effect of detergents on sterol synthesis in a cell-free system of yeast

In order to obtain information about the reactivity of enzymes in sterol synthesis of yeast, the effects of some detergents were investigated. Among the detergents used, Triton X-100 was found to exert a unique action, and its effect on the incorporation of 14C-labeled acetate, mevalonate, farnesyl pyrophosphate, or S-adenosyl-L-methionine into squalene, 2,3-oxidosqualene, and sterols in a cell-free system was examined. Triton X-100 showed virtually no effect on the enzyme activities in the reactions from acetyl CoA to farnesyl pyrophosphate, but it had a marked effect on reactions from farnesyl pyrophosphate to ergosterol. Evidence was obtained suggesting that Triton X-100 apparently activated squalene synthetase (EC 2.5.1.21) but inhibited squalene epoxidase (EC 1.14.99.7) and delta 24-sterol methyltransferase (EC 2.1.1.41). The activity of epoxidase was protected from the inhibition by increasing the concentration of cell-free extracts or by the prior addition of lecithin liposomes to the reaction mixture. The inhibition of methyltransferase was partially reversed by treatment with Bio-heads SM-2, but that of epoxidase was not reversed by the treatment.     

Cholesterol Inhibition of Pentacyclic Triterpenoid Biosynthesis in Tetrahymena pyriformis*†

SYNOPSIS. Tetrahymena pyriformis synthesizes tetrahymanol and “diplopterol” from acetate, mevalonate or squalene. The formation of these pentacyclic triterpenoid alcohols is inhibited by the addition of cholesterol to the culture fluid of the ciliates. Cholesterol also inhibits the biosynthesis of squalene from acetate or mevalonic acid. The synthesis of other terpene derivatives from acetate and mevalonate continues in the presence of cholesterol, thus suggesting that a major block occurs after “isoprene” formation and before squalene formation. Further, inhibition of squalene conversion to the pentacyclic alcohols by cholesterol has been established.     

The mechanism and regulation of dolichyl phosphate biosynthesis in rat liver

Rat liver slices were pulse labeled for 6 min with [3H]mevalonolactone and then chased for 90 min with unlabeled mevalonolactone in order to study the mechanism of dolichyl phosphate biosynthesis. The cholesterol pathway was also monitored and served to verify the pulse-chase. Under conditions in which radioactivity in the methyl sterol fraction chased to cholesterol, radioactivity in alpha-unsaturated polyprenyl (pyro)-phosphate chased almost exclusively into dolichyl (pyro)phosphate. Lesser amounts of radioactivity appeared in alpha-unsaturated polyprenol and dolichol, and neither exhibited significant decline after 90 min of incubation. The relative rates of cholesterol versus dolichyl phosphate biosynthesis were studied in rat liver under four different nutritional conditions using labeled acetate, while the absolute rates of cholesterol synthesis were determined using 3H2O. From these determinations, the absolute rates of dolichyl phosphate synthesis were calculated. The absolute rates of cholesterol synthesis were found to vary 42-fold while the absolute rates of dolichyl phosphate synthesis were unchanged. To determine the basis for this effect, the rates of synthesis of cholesterol and dolichyl phosphate were quantitated as a function of [3H]mevalonolactone concentration. Plots of nanomoles incorporated into the two lipids were nearly parallel, yielding Km values on the order of 1 mM. In addition, increasing concentrations of mevinolin yielded parallel inhibition of incorporation of [3H]acetate into cholesterol and dolichyl phosphate. The specific activity of squalene synthase in liver microsomes from rats having the highest rate of cholesterol synthesis was only 2-fold greater than in microsomes from rats having the lowest rate. Taken together, the results suggest that the maintenance of constant dolichyl phosphate synthesis under conditions of enhanced cholesterogenesis is not due to saturation of the dolichyl phosphate pathway by either farnesyl pyrophosphate or isopentenyl pyrophosphate but coordinate regulation of hydroxymethylglutaryl-CoA reductase and a reaction on the pathway from farnesyl pyrophosphate to cholesterol.     

Sterol regulatory element-binding proteins induce an entire pathway of cholesterol synthesis

To evaluate the effects of sterol regulatory element-binding proteins (SREBPs) on the expression of the individual enzymes in the cholesterol synthetic pathway, we examined expression of these genes in the livers from wild-type and transgenic mice overexpressing nuclear SREBP-1a or -2. As estimated by a Northern blot analysis, overexpression of nuclear SREBP-1a or -2 caused marked increases in mRNA levels of the whole battery of cholesterogenic genes. This SREBP activation covers not only rate-limiting enzymes such as HMG CoA synthase and reductase that have been well established as SREBP targets, but also all the enzyme genes in the cholesterol synthetic pathway tested here. The activated genes include mevalonate kinase, mevalonate pyrophosphate decarboxylase, isopentenyl phosphate isomerase, geranylgeranyl pyrophosphate synthase, farnesyl pyrophosphate synthase, squalene synthase, squalene epoxidase, lanosterol synthase, lanosterol demethylase, and 7-dehydro-cholesterol reductase. These results demonstrate that SREBPs activate every step of cholesterol synthetic pathway, contributing to an efficient cholesterol synthesis.     

Prenylation of mammalian Ras protein in Xenopus oocytes.

Ras protein requires an intermediate of the cholesterol biosynthetic pathway for posttranslational modification and membrane anchorage. This step is necessary for biological activity. Maturation of Xenopus laevis oocytes induced by an oncogenic human Ras protein can be inhibited by lovastatin or compactin, inhibitors of the synthesis of mevalonate, an intermediate of cholesterol biosynthesis. This inhibition can be overcome by mevalonic acid or farnesyl diphosphate, a cholesterol biosynthetic intermediate downstream of mevalonate, but not by squalene, an intermediate after farnesyl pyrophosphate in the pathway. This study supports the idea that in Xenopus oocytes, the Ras protein is modified by a farnesyl moiety or its derivative. Furthermore, an octapeptide with the sequence similar to the C-terminus of the c-H-ras protein inhibits the biological activity of Ras proteins in vivo, suggesting that it competes for the enzyme or enzymes responsible for transferring the isoprenoid moiety (prenylation) in the oocytes. This inhibition of Ras prenylation by the peptide was also observed in vitro, using both Saccharomyces cerevisiae and Xenopus oocyte extracts. These observations show that Xenopus oocytes provide a convenient in vivo system for studies of inhibitors of the posttranslational modification of the Ras protein, especially for inhibitors such as peptides that do not penetrate cell membranes.     

Posthatching evolution of in vivo mevalonate metabolism in chick liver

The in vivo mevalonate incorporation into total nonsaponifiable lipids by chick liver was minimal after hatching and drastically increased between 1-5 days. The hepatic synthesis of different cholesterol precursors emerged sequentially after hatching. Between 1-5 days increased strongly the conversion of mevalonate into squalene and also the formation of oxygenated lanosterol derivatives from squalene. The conversion of squalene became completely active at day 8. Cholesterol formation from lanosterol derivatives was completely activated between 8-11 days. Results in this paper demonstrate for the first time the accumulation of a fraction of nonsaponifiable lipids identified as lanosterol derivatives and cholesterol precursors formed from [5-14C]mevalonate in experiments carried out in vivo. Postnatal evolution of these oxysterols may explain the great increase of 3-hydroxy-3-methylglutaryl-CoA reductase activity found in chick liver between 5-11 days, simultaneous or posterior to the diminution of the oxygenated cholesterol precursors.     

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.     

Mechanism of squalene biosynthesis: evidence against the involvement of free nerolidyl pyrophosphate

Several mechanisms that utilize farnesyl pyrophosphate and nerolidyl pyrophosphate as condensing substrates have been postulated for the asymmetric condensation reaction in squalene biosynthesis. Although there is ample evidence that farnesyl pyrophosphate is a substrate for this reaction, there has been no information concerning the role of nerolidyl pyrophosphate. We have made the following observations that demonstrate that nerolidyl pyrophosphate cannot be a free intermediate in squalene biosynthesis. (a) There is no significant interconversion of farnesyl pyrophosphate and nerolidyl pyrophosphate in a squalene-synthesizing system from yeast. (b) Nerolidyl-1-(3)H(2) pyrophosphate is not converted to squalene in the presence or absence of farnesyl pyrophosphate. (c) The addition of unlabeled nerolidyl pyrophosphate to incubation mixtures does not alter the relative loss of alpha-hydrogens from farnesyl pyrophosphate during its conversion to squalene. The synthesis of nerolidyl-1-(3)H(2) pyrophosphate is described. Chromatographic methods for the separation of pyrophosphate esters of triprenols and terpenols are included.     

Two major regulatory steps in cholesterol synthesis by human renal cancer cells.

Two major mechanisms regulating cholesterol biosynthesis exist in a human renal cancer cell line, Caki-1. Caki-1 is a newly established cell line whose characteristics of rapid growth and active cholesterol synthesis qualify it as a potentially valuable tool for elucidation of regulatory mechanism of cholesterol synthesis and transport. In the absence of exogenous cholesterol, cholesterol is the dominant sterol arising from labeled acetate and mevalonate. As expected, in the presence of exogenous cholesterol, the conversion of acetate to cholesterol and the activity of 3-hydroxy-3-methylglutaryl coenzyme A reductase (EC 1.1.1.34) is markedly reduced and this inhibition is released when cholesterol is removed from the medium. An unexpected and possibly unique finding is the inhibition of the conversion of mevalonate to cholesterol in the presence of exogenous cholesterol. This second major control process results in the accumulation of squalene and may involve additional late steps in cholesterol biosynthesis or metabolism. The occurrence of two major mechanisms regulating cholesterol synthesis may be a unique property of renal cancer cells or a previously unrecognized characteristic of a variety of cultured cells.     

Bisphosphonates used for the treatment of bone disorders inhibit squalene synthase and cholesterol biosynthesis.

Some bisphosphonates used for the treatment of bone disorders are also potent inhibitors of squalene synthase, a critical enzyme for sterol biosynthesis. Among seven drugs tested, YM 175 (cycloheptylaminomethylene-1,1-bisphosphonic acid) was the most potent inhibitor of rat liver microsomal squalene synthase (Ki = 57 nM) and sterol biosynthesis from [14C]mevalonate in rat liver homogenate (IC50 = 17 nM). EB 1053 (3-(1-pyrolidino)-1-hydroxypropylidene-1,1-bisphosphonic acid) and PHPBP (3-(1-piperidino)-1-hydroxypropylidene-1,1-bisphosphonic acid) were less potent inhibitors in both these assays. Pamidronate and alendronate were poor inhibitors of squalene synthase (IC50 > 10 microM) but were potent inhibitors of sterol biosynthesis from mevalonate (IC50 = 420 and 168 nM, respectively), suggesting that the latter two agents may have inhibited other enzymes involved in the synthesis of farnesyl pyrophosphate from mevalonate. Etidronate and clodronate were inactive in both these assays. YM 175 also inhibited sterol biosynthesis in mouse macrophage J774 cells (IC50 = 64 microM) and in rats, when administered acutely, it inhibited cholesterol biosynthesis in the liver (ED50 = 30 mg/kg, s.c.). Structural modifications on YM 175 to enhance cell permeability may result in a new class of cholesterol-lowering agents.     

Gorgosterol biosynthesis: localization of squalene formation in the zooxanthellar component of various gorgonians

Four species of gorgonians: three related pseudoplexaurids Pseudoplexaura porosa, P. flagellosa and P. wagenaari; and the unrelated Pseudopterogorgia americana, are sources of zooxanthellae capable, in purified broken cell preparations, of converting [14C]labeled farnesyl pyrophosphate into squalene. More extensive studies with P. porosa and P. americana zooxanthellae preparations characterize the conversion as enzymatic and demonstrate farnesyl pyrophosphate and reduced pyridine nucleotide as substrates. NADPH and NADH are essentially equivalent. Anaerobic conditions are not required. Despite numerous attempts zooxanthellae formation of sterols, including gorgosterol, from radioactive substrates was unsuccessful. By enzyme studies, we can show only the conversion of mevalonate into squalene as a zooxanthellae capability.     

Studies on the biosynthesis of tetrahymanol in Tetrahymena pyriformis. The mechanism of inhibition by cholesterol

Tetrahymanol biosynthesis by the protozoan Tetrahymena pyriformis was progressively inhibited by the inclusion of cholesterol in the growth medium. Studies with labelled precursors of tetrahymanol have established that there are two major sites of inhibition in whole cells. The inhibition at the first site, between acetate and mevalonate, occurred rapidly after addition of cholesterol. The activity of 3-hydroxy-3-methylglutaryl-CoA reductase (EC 1.1.1.34), a predominantly cytosolic enzyme in this organism, was not inhibited in cholesterol-grown cells nor by addition of cholesterol directly to the assay medium. The second major site of inhibition in whole cells is between mevalonate and squalene and this is accompanied by inhibition of the enzyme that converts farnesyl-pyrophosphate into squalene (squalene synthetase). Squalene cyclase is partially inhibited. The conversion of mevalonate into tetrahymanol in vitro was not inhibited by the addition of cholesterol to the assay medium. Tetrahymanol added to the culture medium is taken up by the cells but does not inhibit endogenous biosynthesis. It is suggested that cholesterol inhibits the later stages of tetrahymanol biosynthesis by causing a change in membrane structure and function which alters the activity of membrane-bound enzymes.     

The activity and kinetic properties of mevalonate kinase in superovulated rat ovary

Methods are described for the assay and partial purification of mevalonate kinase from superovulated rat ovary. The total activity of mevalonate kinase in superovulated rat ovary was 1.6+/-0.14units/g wet wt.; it was unchanged by the administration of luteinizing hormone in vivo. The K(m) of a partially purified preparation of mevalonate kinase for dl-Mevalonate was 3.6+/-0.5mum; its K(m) for MgATP(2-) was 120+/-7.7mum. The enzyme was inhibited by geranyl pyrophosphate and farnesyl pyrophosphate, but not by isopentenyl pyrophosphate or 3,3'-dimethylallyl pyrophosphate. dl-mevalonate 5-phosphate inhibited at high concentrations. With both geranyl pyrophosphate and farnesyl pyrophosphate the inhibition was competitive with respect to MgATP(2-). The K(i) for inhibition by geranyl pyrophosphate was 1.3+/-0.2mum; the K(i) for inhibition by farnesyl pyrophosphate was 1.0+/-0.3mum. These findings are discussed with reference to the control by luteinizing hormone of steroidogenesis from acetate.     

Polyisoprenoid amphiphilic compounds as inhibitors of squalene synthesis and other microsomal enzymes.

Analogues of farnesyl pyrophosphate containing a farnesyl moiety and a variety of amine residues replacing the pyrophosphate have been synthesized. Most of these compounds were effective inhibitors of the synthesis of squalene and presqualene pyrophosphate from farnesyl pyrophosphate. 50% inhibition was obtained at concentrations between 50 and 100 micron. These analogues also inhibited other microsomal enzymes so they apparently function as general inhibitors of microsomal enzymes.     

Regulation of macrophage cholesterol efflux through hydroxymethylglutaryl-CoA reductase inhibition: a role for RhoA in ABCA1-mediated cholesterol efflux

The cholesterol biosynthetic pathway produces numerous signaling molecules. Oxysterols through liver X receptor (LXR) activation regulate cholesterol efflux, whereas the non-sterol mevalonate metabolite, geranylgeranyl pyrophosphate (GGPP), was recently demonstrated to inhibit ABCA1 expression directly, through antagonism of LXR and indirectly through enhanced RhoA geranylgeranylation. We used HMG-CoA reductase inhibitors (statins) to test the hypothesis that reduced synthesis of mevalonate metabolites would enhance cholesterol efflux and attenuate foam cell formation. Preincubation of THP-1 macrophages with atorvastatin, dose dependently (1-10 microm) stimulated cholesterol efflux to apolipoprotein AI (apoAI, 10-60%, p < 0.05) and high density lipoprotein (HDL(3)) (2-50%, p < 0.05), despite a significant decrease in cholesterol synthesis (2-90%). Atorvastatin also increased ABCA1 and ABCG1 mRNA abundance (30 and 35%, p < 0.05). Addition of mevalonate, GGPP or farnesyl pyrophosphate completely blocked the statin-induced increase in ABCA1 expression and apoAI-mediated cholesterol efflux. A role for RhoA was established, because two inhibitors of Rho protein activity, a geranylgeranyl transferase inhibitor and C3 exoenzyme, increased cholesterol efflux to apoAI (20-35%, p < 0.05), and macrophage expression of dominant-negative RhoA enhanced cholesterol efflux to apoAI (20%, p < 0.05). In addition, atorvastatin increased the RhoA levels in the cytosol fraction and decreased the membrane localization of RhoA. Atorvastatin treatment activated peroxisome proliferator activated receptor gamma and increased LXR-mediated gene expression suggesting that atorvastatin induces cholesterol efflux through a molecular cascade involving inhibition of RhoA signaling, leading to increased peroxisome proliferator activated receptor gamma activity, enhanced LXR activation, increased ABCA1 expression, and cholesterol efflux. Finally, statin treatment inhibited cholesteryl ester accumulation in macrophages challenged with atherogenic hypertriglyceridemic very low density lipoproteins indicating that statins can regulate foam cell formation.     

Supernatant protein factor stimulates HMG-CoA reductase in cell culture and in vitro

Supernatant protein factor (SPF) is a 46-kDa cytosolic protein that stimulates squalene monooxygenase in vitro and, unexpectedly, cholesterol synthesis in cell culture. Because squalene monooxygenase is not thought to be rate-limiting with regard to cholesterol synthesis, we investigated the possibility that SPF might stimulate other enzymes in the cholesterol biosynthetic pathway. Substitution of [(14)C]mevalonate for [(14)C]acetate in McARH7777 hepatoma cells expressing SPF reduced the 1.8-fold increase in cholesterol synthesis by half, suggesting that SPF acted on or prior to mevalonate synthesis. This conclusion was supported by the finding that substitution with [(14)C]mevalonate completely blocked an SPF-induced increase in squalene synthesis. Evaluation of 2,3-oxidosqualene synthesis from [(14)C]mevalonate demonstrated that SPF also stimulated squalene monooxygenase (1.3-fold) in hepatoma cells. Immunoblot analysis showed that SPF did not increase HMG-CoA reductase or squalene monooxygenase enzyme levels, indicating a direct effect on enzyme activity. Addition of purified recombinant SPF to rat liver microsomes stimulated HMG-CoA reductase by about 1.5-fold, and the SPF-concentration/activation curve paralleled that for the SPF-mediated stimulation of squalene monooxygenase. These results reveal that SPF directly stimulates HMG-CoA reductase, the rate-limiting step of the cholesterol biosynthetic pathway, as well as squalene monooxygenase, and suggest a new means by which cholesterol synthesis can be rapidly modulated in response to hormonal and environmental signals.     

Studies on the regulation of glycoprotein biosynthesis. An investigation of the rate-limiting steps of dolichyl phosphate biosynthesis.

The possible role of HMG-CoA (3-hydroxy-3-methylglutaryl coenzyme A) reductase (the rate-controlling enzyme of cholesterol biosynthesis) in regulating the rate of dolichyl phosphate biosynthesis in rat liver was investigated. Rats were either fasted 48 h or fed diets supplemented with the drug cholestyramine. The activity of HMG-CoA reductase was 5000-fold greater in liver from cholestyramine-fed rats as compared to fasted rats. The activity of dolichyl phosphate synthetase, the prenyl transferase responsible for the biosynthesis of dolichyl phosphate from farnesyl pyrophosphate and isopentenyl pyrophosphate, was similar in both nutritional conditions and was markedly less active than HMG-CoA reductase even in the fasted state. Acetate incorporation into cholesterol was 2200-fold greater in liver slices from cholestyramine-fed rats as compared to fasted rats. By contrast, acetate incorporation into dolichyl phosphate was only 6-fold higher. Further studies suggested that the levels of farnesyl pyrophosphate and isopentenyl pyrophosphate are several hundred-fold greater in liver from cholestyramine-treated rats. From these results, it is concluded that the rate of dolichyl phosphate biosynthesis in rat liver is not regulated by the activity of HMG-CoA reductase but is probably regulated at the level of dolichyl phosphate synthetase.