Regulation of 3-hydroxy-3-methylglutaryl-CoA reductase mRNA contents in human hepatoma cell line Hep G2 by distinct classes of mevalonate-derived metabolites.

Hep G2 cells were incubated under conditions known to influence the HMG-CoA (3-hydroxy-3-methylglutaryl-CoA) reductase activity, e.g. in the presence of compactin (a competitive inhibitor of HMG-CoA reductase itself) and U18666A (a squalene-2,3-epoxide cyclase inhibitor). We studied the effects of these conditions both on the HMG-CoA reductase activity and on the reductase mRNA content. In the presence of compactin the mRNA content increased, but less than the enzyme activity, as determined after removal of the inhibitor. The increase in mRNA could be prevented by addition of mevalonate or by a combination of low-density lipoprotein (LDL) plus a low concentration of mevalonate. LDL alone prevented the compactin-induced increases in mRNA and activity only partially. The effect of U18666A on reductase mRNA content and activity was biphasic, i.e. a slight decrease at low (0.3-0.5 microM) concentrations, with a concomitant formation of polar sterols [Boogaard, Griffioen & Cohen (1987) Biochem. J. 241, 345-351], and an increase at high (20-30 microM) concentrations, with complete blockage of sterol formation. At these high concentrations of U18666A, additional compactin (2 microM) increased the reductase activity, but not the mRNA content. We conclude that non-sterol metabolites of mevalonate regulate exclusively at the enzyme level, whereas sterol metabolites regulate at the reductase mRNA level. In the latter group of regulators we distinguish mevalonate metabolites which can, and metabolites which cannot, be replaced by exogenous LDL.     

Regulation of squalene synthetase in human hepatoma cell line Hep G2 by sterols, and not by mevalonate-derived non-sterols

Incubations of Hep G2 cells for 18 h with human low-density lipoprotein (LDL) resulted in a decrease of squalene synthetase activity, whereas heavy high-density lipoprotein (hHDL) stimulated the activity. Simultaneous addition of LDL abolished the hHDL-induced stimulation, indicating that manipulating the regulatory sterol pool within the cells influenced the enzyme activity. Blocking the endogenous cholesterol synthesis either at the 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase site with compactin or at the 2,3-oxidosqualene cyclase site with the inhibitor U18666A gave rise to an elevation of the squalene synthetase activity. Simultaneous addition of mevalonate abolished the compactin-induced increase. However, at total blockade of sterol synthesis by 30 microM U18666A, added compactin and/or mevalonate did not change the enzyme activity further. It was concluded that sterols regulate the squalene synthetase activity, whereas, in contrast with the regulation of the HMG-CoA reductase activity in Hep G2 cells, mevalonate-derived non-sterols did not influence this enzyme.     

Effects of compactin, mevalonate and low-density lipoprotein on 3-hydroxy-3-methylglutaryl-coenzyme A reductase activity and low-density-lipoprotein-receptor activity in the human hepatoma cell line Hep G2.

Compactin, an inhibitor of HMG-CoA (3-hydroxy-3-methylglutaryl-CoA) reductase, decreased cholesterol synthesis in intact Hep G2 cells. However, after the inhibitor was washed away, the HMG-CoA-reductase activity determined in the cell homogenate was found to be increased. Also the high-affinity association of LDL (low-density lipoprotein) to Hep G2 cells was elevated after incubation with compactin. Lipoprotein-depleted serum, present in the incubation medium, potentiated the compactin effect compared with incubation in the presence of human serum albumin. Addition of either mevalonate or LDL prevented the compactin-induced rise in activities of both HMG-CoA reductase and LDL receptor in a comparable manner. It is concluded that in this human hepatoma cell line, as in non-transformed cells, both endogenous mevalonate or mevalonate-derived products and exogenous cholesterol are able to modulate the HMG-CoA reductase activity as well as the LDL-receptor activity.     

Sterol-independent regulation of 3-hydroxy-3-methylglutaryl-CoA reductase by mevalonate in Chinese hamster ovary cells. Magnitude and specificity

In this paper, we assess the relative degree of regulation of the rate-limiting enzyme of isoprenoid biosynthesis, 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, by sterol and nonsterol products of mevalonate by utilizing cultured Chinese hamster ovary cells blocked in sterol synthesis. We also examine the two other enzymes of mevalonate biosynthesis, acetoacetyl-CoA thiolase and HMG-CoA synthase, for regulation by mevalonate supplements. These studies indicate that in proliferating fibroblasts, treatment with mevalonic acid can produce a suppression of HMG-CoA reductase activity similar to magnitude to that caused by oxygenated sterols. In contrast, HMG-CoA synthase and acetoacetyl-CoA thiolase are only weakly regulated by mevalonate when compared with 25-hydroxycholesterol. Furthermore, neither HMG-CoA synthase nor acetoacetyl-CoA thiolase exhibits the multivalent control response by sterol and mevalonate supplements in the absence of endogenous mevalonate synthesis which is characteristic of nonsterol regulation of HMG-CoA reductase. These observations suggest that nonsterol regulation of HMG-CoA reductase is specific to that enzyme in contrast to the pleiotropic regulation of enzymes of sterol biosynthesis observed with oxygenated sterols. In Chinese hamster ovary cells supplemented with mevalonate at concentrations that are inhibitory to reductase activity, at least 80% of the inhibition appears to be mediated by nonsterol products of mevalonate. In addition, feed-back regulation of HMG-CoA reductase by endogenously synthesized nonsterol isoprenoids in the absence of exogenous sterol or mevalonate supplements also produces a 70% inhibition of the enzyme activity.     

A relationship between the activities of hepatic lanosterol 14 alpha-demethylase and 3-hydroxy-3-methylglutaryl-CoA reductase.

At 1-2 h after intragastric administration of ketoconazole, a cytochrome P-450 inhibitor, to rats, there was a 50-60% decrease in the activity of hepatic 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase. Inhibition reached a maximum at 6-12 h after the drug was given, but after 24 h enzyme activity was stimulated by 60%. The rates of synthesis of hepatic non-saponifiable lipids in vivo showed a similar time-dependent pattern of change. During the first few hours after drug administration, the hepatic cytochrome P-450-dependent metabolism of lanosterol was suppressed in vivo. However, 24 h after treatment, this activity was stimulated, an effect which was also observed by pre-treatment of the rats with the drug for several days. Suppression of hepatic HMG-CoA reductase and lanosterol 14 alpha-demethylase activities was accompanied by a relative increase in the accumulation of labelled polar sterols in the liver in vivo. In the intestine, ketoconazole also resulted in a rapid decline in the rate of synthesis of non-saponifiable lipids and an inhibition of lanosterol 14 alpha-demethylation in vivo. However, in contrast with the liver, there was no stimulation of non-saponifiable lipid synthesis after 24 h.     

Regulation of hepatic cholesterol biosynthesis. Effects of a cytochrome P-450 inhibitor on the formation and metabolism of oxygenated sterol products of lanosterol.

The involvement of oxygenated cholesterol precursors in the regulation of 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase activity was studied by examining the effect of ketoconazole on the metabolism of mevalonic acid, lanosterol and the lanosterol metabolites, lanost-8-ene-3 beta,32-diol,3 beta-hydroxylanost-8-en-32-al and 4,4-dimethylcholesta-8,14-dien-3 beta-ol, in liver subcellular fractions and hepatocyte cultures. Inhibition of cholesterol synthesis from mevalonate by ketoconazole at concentrations up to 30 microM was due exclusively to a suppression of cytochrome P-450LDM (LDM = lanosterol demethylase) activity, resulting in a decreased rate of lanosterol 14 alpha-demethylation. No enzyme after the 14 alpha-demethylase step was affected. When [14C]mevalonate was the cholesterol precursor, inhibition of cytochrome P450LDM was accompanied by the accumulation of several labelled oxygenated sterols, quantitatively the most important of which was the C-32 aldehyde derivative of lanosterol. There was no accumulation of the 24,25-oxide derivative of lanosterol, nor of the C-32 alcohol. Under these conditions the activity of HMG-CoA reductase declined. The C-32 aldehyde accumulated to a far greater extent when lanost-8-ene-3 beta,32-diol rather than mevalonate was used as the cholesterol precursor in the presence of ketoconazole. With both precursors, this accumulation was reversed at higher concentrations of ketoconazole in liver subcellular fractions. A similar reversal was not observed in hepatocyte cultures.     

Effects of 3 beta-[2-(diethylamino)ethoxy]androst-5-en-17-one on the synthesis of cholesterol and ubiquinone in rat intestinal epithelial cell cultures

The relationship between cholesterol and ubiquinone synthesis in rat intestinal epithelial cell cultures was examined by using 3 beta-[2-(diethylamino)ethoxy]androst-5-en-17-one hydrochloride (U18666A). Addition of U18666A to cells caused a greater than 90% inhibition of incorporation of [3H]acetate into cholesterol and an apparent large increase in the incorporation of [3H]acetate and [3H]mevalonate into ubiquinone. However, the incorporation of 4-hydroxy[U-14C]benzoate, a ring precursor of ubiquinone, was unchanged. The apparent increase of 3H incorporation into ubiquinone was found to be due to the formation of a contaminant that has been identified as squalene 2,3:22,23-dioxide. Following incubation of cells with U18666A, its removal from the medium resulted in a decrease in squalene 2,3:22,23-dioxide labeling and a corresponding increase in the polar sterol fraction. These results demonstrate that U18666A inhibits the reaction catalyzed by 2,3-oxidosqualene cyclase (EC 5.4.99.7). As a result, the isoprenoid precursors are diverted not to ubiquinone as has been suggested but to squalene 2,3:22,23-dioxide, a metabolite not on the cholesterol biosynthetic pathway. Removal of the drug allows cyclization of squalene 2,3:22,23-dioxide, leading to formation of compounds with chromatographic properties of polar sterols.     

Modulation of regulatory oxysterol formation and low density lipoprotein suppression of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase activity by ketoconazole. A role for cytochrome P-450 in the regulation of HMG-CoA reductase in rat intestinal epithelial cells

The effects of ketoconazole, a lanosterol demethylase and cytochrome P450 inhibitor, on the regulation of 3-hydroxy-3-methylglutaryl coenzyme A reductase (EC 1.1.1.34, reductase) activity and sterol biosynthesis were studied in rat intestinal epithelial cell cultures (IEC-6). Incubation of cells with 0.15-2 microM ketoconazole resulted in a concentration-dependent inhibition of reductase activity. As the drug concentration approached 15 microM, the reductase activity returned to control values, and at 30 microM ketoconazole, a stimulation of enzyme activity was observed. The drug had no effect on reductase activity in homogenates of IEC-6 cells. Ketoconazole (0.15-30 microM) caused a concentration-dependent inhibition of the incorporation of [3H] mevalonolactone into cholesterol with a concomitant accumulation of radioactivity in methyl sterols; e.g. lanosterol and 24,25-epoxylanosterol. Interestingly, the incorporation of radioactivity into polar sterols showed a biphasic response which was inversely proportional to the biphasic response of reductase activity. Thus, incorporation of [3H]mevalonolactone into polar sterols increased at low concentrations of ketoconazole (0.15-2 microM) and decreased to control values at high concentrations of the drug. Treatment of cells with ketoconazole (30 microM) and [3H]mevalonolactone followed by removal of the drug and radiolabel resulted in an inhibition of reductase activity and a redistribution of radioactivity from lanosterol and 24,25-epoxylanosterol to cholesterol and polar sterols. These results suggested that the inhibition of reductase activity at low concentrations of ketoconazole (less than 2 microM) was due to a formation of regulatory polar sterols generated from the methyl sterols. At high concentrations of ketoconazole (30 microM) where no suppression in reductase activity was observed, the conversion of exogenously added [3H]24(S),25-epoxylanosterol to polar sterols was prevented. Exogenously added 24,25-epoxylanosterol inhibited reductase activity in a dose-dependent fashion, and ketoconazole (30 microM) prevented the inhibition caused by low concentrations of epoxylanosterol. The drug, however, was unable to prevent the dose-dependent suppression of reductase activity by 25-hydroxylanosterol, a reduced form of 24,25-epoxylanosterol. These results indicated that 24,25-epoxylanosterol per se was not an inhibitor of reductase activity but could be metabolized to regulatory polar sterols through a cytochrome P-450 dependent reaction which was sensitive to ketoconazole. Treatment of cells with ketoconazole totally abolished the inhibition of reductase activity by low density lipoprotein (LDL).(ABSTRACT TRUNCATED AT 400 WORDS)     

Negative regulation of cell proliferation by mevalonate or one of the mevalonate phosphates

The role of mevalonate and its products in the regulation of cellular proliferation was examined using 6-fluoromevalonate (Fmev), a compound that blocks the conversion of mevalonate pyrophosphate to isopentenyl pyrophosphate. Fmev suppressed DNA synthesis by a variety of transformed and malignant T cell, B cell, and myeloid cell lines. In contrast to results previously reported with mitogen-stimulated human peripheral blood T cell DNA synthesis, low concentrations of low density lipoprotein (LDL) alone could not restore proliferation to these cell lines. The same concentrations of LDL were able to provide sufficient cholesterol and support the growth of all cell lines when mevalonate synthesis was blocked with a specific inhibitor of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase, lovastatin. Fmev-mediated inhibition was totally prevented in some but not all cell lines when the concentration of exogenous LDL was increased 5-10-fold above that required to permit proliferation of lovastatin-blocked cells. Residual HMG-CoA reductase activity of cells cultured with LDL inversely correlated with the restoration of growth to Fmev-blocked cultures. Confirmation of the critical role of HMG-CoA reductase activity and mevalonate synthesis in the inhibition of cellular proliferation by Fmev was obtained by demonstrating that the specific inhibitor of this enzyme, lovastatin, restored proliferation of Fmev-blocked cells. Furthermore, supplementation of cultures with mevalonate, the product of HMG-CoA reductase activity, markedly inhibited proliferation of Fmev-blocked cells. These findings indicate that mevalonate or one of the mevalonate phosphates, which accumulates in Fmev-blocked cells, is a critical negative regulator of cellular proliferation.     

Inhibition of cholesterol synthesis and cell growth by 24(R,S),25-iminolanosterol and triparanol in cultured rat hepatoma cells

24(R,S),25-Iminolanosterol (IL) and triparanol added to cultures of rat hepatoma cells, H4-II-C3 (H4), interrupt the conversion of lanosterol to cholesterol and, depending on their concentrations, cause the accumulation in the cells of intermediates in the lanosterol to cholesterol conversion. At 45 microM, both substances cause the accumulation of 5 alpha-cholesta-8(9),24-dien-3 beta-ol (zymosterol), and at the low concentration of 4.5 microM, they cause the accumulation of cholesta-5.24-dien-3 beta-ol (desmosterol). The effect of intermediate concentrations of 9 or 22.5 microM of either substance is to cause the accumulation in the cells of three sterols: cholesta-5,7,24-trien-3 beta-ol, zymosterol, and desmosterol. The synthesis of these intermediary sterols, not found normally in H4 cells, is particularly pronounced in cultures kept in lipid-depleted media that contain the inhibitors and proceeds by the use of endogenous substrates at the expense of cholesterol. The synthesis of cholesterol from [14C]acetate or [2-14C]mevalonate is completely blocked by either inhibitor even at 4.5 microM. IL or triparanol inhibits the growth of H4 cells. Cells seeded into either full growth or lipid-depleted medium containing 22.5 microM IL will not grow unless the media are supplemented with low density lipoproteins (60 micrograms/ml). Supplementation of the media with 4.6 mM mevalonate does not counteract the inhibitory effect of IL on cell growth.     

The absolute rate of cholesterol biosynthesis in monocyte-macrophages from normal and familial hypercholesterolaemic subjects.

The true rate of cholesterogenesis in cultured monocyte-macrophages was determined from the incorporation of [2-14C]acetate into cholesterol, using the desmosterol (cholesta-5,24-dien-3 beta-ol) that accumulated in the presence of the drug triparanol to estimate the specific radioactivity of the newly formed sterols. It was shown that this procedure could be successfully adapted for use with cultured monocytes despite the accumulation of other unidentified biosynthetic intermediates. In cells maintained in 20% (v/v) whole serum approx. 25% of the sterol carbon was derived from exogenous acetate. Cholesterol synthesis was as high in normal cells as in cells from homozygous familial hypercholesterolaemic (FH) subjects and accounted for 50% of the increase in cellular cholesterol. The addition of extra low-density lipoprotein (LDL) reduced cholesterol synthesis, apparently through a decrease in the activity of 3-hydroxy-3-methylglutaryl-CoA reductase (HMG-CoA reductase). When incubated in lipoprotein-deficient serum some cells did not survive, but those that remained showed a normal increase in protein content; the amount of cellular protein and cholesterol in each well did not increase and cholesterol synthesis was reduced by over 80%. HMG-CoA reductase activity fell less dramatically and the proportion of sterol carbon derived from exogenous acetate increased, suggesting that the low rate of cholesterogenesis with lipoprotein-deficient serum was due to a shortage of substrate. The results indicate that under normal conditions monocyte-macrophages obtain cholesterol from endogenous synthesis rather than through receptor-mediated uptake of LDL, and that synthesis together with non-saturable uptake of LDL provides the majority of the cholesterol required to support growth.     

The regulation of 3-hydroxy-3-methylglutaryl-CoA reductase activity, cholesterol esterification and the expression of low-density lipoprotein receptors in cultured monocyte-derived macrophages.

Human blood monocytes cultured in medium containing 20% whole serum showed the greatest activity of 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase and [14C]acetate incorporation into non-saponifiable lipids around the 7th day after seeding, the period of greatest growth. Although there was enough low-density lipoprotein (LDL) in the medium to saturate the LDL receptors that were expressed by normal cells at that time, HMG-CoA reductase activity and acetate incorporation were as high in normal cells as in cells from familial-hypercholesterolaemic (FH) patients. Both the addition of extra LDL, which interacted with the cells by non-saturable processes, and receptor-mediated uptake of acetylated LDL significantly reduced reductase activity and increased incorporation of [14C]oleate into cholesteryl esters in normal cells and cells from FH patients ('FH cells'), and reduced the expression of LDL receptors in normal cells. Pre-incubation for 20h in lipoprotein-deficient medium apparently increased the number of LDL receptors expressed by normal cells but reduced the activity of HMG-CoA reductase in both normal and FH cells. During subsequent incubations the same rate of degradation of acetylated LDL and of non-saturable degradation of LDL by FH cells was associated with the same reduction in HMG-CoA reductase activity, although LDL produced a much smaller stimulation of oleate incorporation into cholesteryl esters. In normal cells pre-incubated without lipoproteins, receptor-mediated uptake of LDL could abolish reductase activity and the expression of LDL receptors. The results suggested that in these cells, receptor-mediated uptake of LDL might have a greater effect on reductase activity and LDL receptors than the equivalent uptake of acetylated LDL. It is proposed that endogenous synthesis is an important source of cholesterol for growth of normal cells, and that the site at which cholesterol is deposited in the cells may determine the nature and extent of the metabolic events that follow.     

The effect of (-)-hydroxycitrate on the activity of the low-density-lipoprotein receptor and 3-hydroxy-3-methylglutaryl-CoA reductase levels in the human hepatoma cell line Hep G2.

(-)-Hydroxycitrate, a potent inhibitor of ATP citrate-lyase, was tested in Hep G2 cells for effects on cholesterol homoeostasis. After 2.5 h and 18 h incubations with (-)-hydroxycitrate at concentrations of 0.5 mM or higher, incorporation of [1,5-14C]citrate into fatty acids and cholesterol was strongly inhibited. This most likely reflects an effective inhibition of ATP citrate-lyase. Cholesterol biosynthesis was decreased to 27% of the control value as measured by incorporations from 3H2O, indicating a decreased flux of carbon units through the cholesterol-synthetic pathway. After 18 h preincubation with 2 mM-(-)-hydroxycitrate, the cellular low-density-lipoprotein (LDL) receptor activity was increased by 50%, as determined by the receptor-mediated association and degradation. Measurements of receptor-mediated binding versus LDL concentration suggests that this increase was due to an increase in the numbers of LDL receptors. Simultaneously, enzyme levels of 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase as determined by activity measurements increased 30-fold. Our results suggest that the increases in HMG-CoA reductase and the LDL receptor are initiated by the decreased flux of carbon units in the cholesterol-synthetic pathway, owing to inhibition of ATP citratelyase. A similar induction of HMG-CoA reductase and LDL receptor was also found after preincubations of cells with 0.3 microM-mevinolin, suggesting that the underlying mechanism for this induction is identical for both drugs.     

Subcellular localization of squalene synthase in human hepatoma cell line Hep G2.

Using the Hep G2 cell line as a model for the human hepatocyte the question was studied whether Hep G2-peroxisomes could be able to synthesize cholesterol. Hep G2 cell homogenates were applied to density gradient centrifugation on Nycodenz, resulting in good separation between the organelles. The different organelle fractions were characterized by assaying the following marker enzymes: catalase for peroxisomes, glutamate dehydrogenase for mitochondria and esterase for endoplasmic reticulum. Squalene synthase activity was not detectable in the peroxisomal fraction. Incubation of Hep G2 cells with U18666A, an inhibitor of the cholesterol synthesis at the site of oxidosqualene cyclase, together with heavy high density lipoprotein, which stimulates the efflux of cholesterol, led to a marked increase in the activity of squalene synthase as well as HMG-CoA reductase, whereas no significant effect on the marker enzymes was observed. Neither enzyme activity was detectable in the peroxisomal density gradient fraction, suggesting that in Hep G2-peroxisomes cholesterol synthesis from the water-soluble early intermediates of the pathway cannot take place. Both stimulated and non-stimulated cells gave rise to preparations where squalene synthase activity was comigrating with the reductase activity at the lower density side of the microsomal fraction; however, it was also present at the high density side of the microsomal peak, where reductase activity was not detected.     

Isoprenoid synthesis in isolated embryonic Drosophila cells. Sterol-independent regulatory signal molecule is distal to isopentenyl 1-pyrophosphates

Embryonic Drosophila cells (Kc cells) were used to further characterize sterol-independent modulation of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase activity. 3-Methyl-3-5-dihydroxyvalerate (mevalonate), 3-fluoromethyl-3,5-dihydroxyvalerate (fluoromevalonate), and 3-ethyl-3,5-dihydroxyvalerate (homomevalonate) were tested as modulators. Although mevalonate caused a rapid, reversible suppression of reductase activity, fluoro- and homomevalonate increased activity; fluoromevalonate was more effective than homomevalonate. Mevalonate, added simultaneously with fluoromevalonate, blocked the analogue's effect on Kc cell reductase activity. However, mevalonate did not suppress an established fluoromevalonate increase in HMG-CoA reductase activity. Fluoromevalonate blocked [1-14C, 5-3H]mevalonate conversion to 14CO2- and 3H-labeled lipids and [3H] mevalonate 5-pyrophosphate accumulated. Neither protein nor RNA synthesis were required for mevalonate-mediated suppression of reductase activity. However, fluoromevalonate's effect on reductase activity required protein synthesis. Furthermore, in the absence of protein synthesis, fluoromevalonate-stabilized Kc cell HMG-CoA reductase activity. We have concluded that mevalonate, fluoromevalonate, homomevalonate, and compactin (mevinolin) modulated HMG-CoA reductase activity because they altered isoprenoid carbon flow to a post-isopentenyl 1-pyrophosphate regulatory, signal molecule.     

Down-regulation of mammalian 3-hydroxy-3-methylglutaryl coenzyme A reductase activity with highly purified liposomal cholesterol.

Chinese hamster ovary-215 cells (CHO-215) cannot synthesize C27 and C28 sterols because of a defect in the reaction that decarboxylates 4-carboxysterols [Plemenitas, A., Havel, C.M. & Watson, J.A. (1990) J. Biol. Chem. 265, 17012-17017]. Thus, CHO-215 cell growth is dependent on an exogenous metabolically functional source of cholesterol. We used CHO-215 cells to (a) determine whether highly purified (> 99.5%) cholesterol, in egg lecithin liposomes, could down-regulate derepressed 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase activity and if so (b) determine whether the loss in reductase catalytic activity correlated kinetically with the synthesis and accumulation of detectable oxycholesterol derivatives. Liposomal cholesterol (26-39 microM) supported maximum CHO-215 growth and initiated suppression of HMG-CoA reductase activity at concentrations greater than 50 microM. Maximum suppression (50-60%) of reductase activity was achieved with 181.3 microM liposomal cholesterol in 6 h. Also, regulatory concentrations of highly purified liposomal [3H]cholesterol were not converted (biologically or chemically) to detectable levels of oxy[3H]cholesterol derivatives during 3-6 h incubations. Lastly, a broad-spectrum cytochrome P450 inhibitor (miconazole) had no effect on liposomal cholesterol-mediated suppression of HMG-CoA reductase activity. These observations established that (a) highly purified cholesterol, incorporated into egg lecithin liposomes, can signal the down-regulation of derepressed mammalian cell HMG-CoA reductase activity and (b) if oxycholesterol synthesis was required for liposomal cholesterol-mediated down-regulation, the products had to be more potent than 24-, 25-, or 26-/27-hydroxycholesterol.     

Isoprenoid synthesis in Halobacterium halobium. Modulation of 3-hydroxy-3-methylglutaryl coenzyme a concentration in response to mevalonate availability

Halobacterium halobium was evaluated as a potentially simpler biological model to study the regulation of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase activity (content) in response to mevalonate availability. H. halobium's HMG-CoA reductase was soluble and required NADPH as its reduced coenzyme. Maximum HMG-CoA reductase activity (4-10 nmol/min/mg of soluble protein) was obtained in buffers which contained 3.5 M KCl. Mevinolin (a) blocked growth of H. halobium, (b) was a competitive inhibitor of HMG-CoA reductase (Ki = 20 nM), (c) did not cause the paradoxical increase in assayable reductase activity, as reported for eukaryotic cells, and (d) caused a rapid (within 30 min) 8-12-fold accumulation of intracellular HMG-CoA. Mevalonate blocked and reversed mevinolin-mediated HMG-CoA accumulation. Although mevinolin-treated cell's growth was restored by mevalonate, HMG-CoA reductase's activity was not. Thus, H. halobium is a unique biological model which allows one to study the regulation of intracellular HMG-CoA concentration and not HMG-CoA reductase activity (content) in response to mevalonate availability.     

Regulation of cholesterol synthesis in primary rat hepatocyte culture cells. Possible regulatory site at sterol demethylation.

Primary rat hepatocyte culture cells were used to study the acute regulation of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase activity in response to 25-hydroxycholesterol, 3 beta,5 alpha,6 beta-cholestantriol, and mevalonolactone. All three effectors caused a rapid suppression of HMG-CoA reductase activity. 25-Hydroxycholesterol also caused an increase in the ratio of newly synthesized methyl sterols to newly synthesized C27-sterols. Furthermore, in 25-hydroxycholesterol-treated cells, the relative contribution of delta 24-sterol precursors to the nonsaponifiable lipid fraction increased. Di- and trimethyl-diene sterols were the dominant methyl sterols synthesized in the presence of 25-hydroxycholesterol. 3 beta,5 alpha,6 beta-Cholestrantriol (50 microM) also caused a very strong (97%) suppression of sterol demethylation; 4,4-dimethylmonoene sterols were more prominent (23%) in cells treated with 3 beta,5 alpha,6 beta-cholestrantriol, than in cells treated with 25-hydroxycholesterol (2%). The rates of both unesterified and esterified sterol synthesis increased as a function of exogenous mevalonolactone concentration. C27-sterol synthesis was saturated at a concentration of (R)-mevalonolactone which produced only a 33% suppression of HMG-CoA reductase activity. However, there was a direct relationship between the accumulation of methyl sterols and the decrease in HMG-CoA reductase activity. With the aid of triparanol, it was demonstrated that the suppression of HMG-CoA reductase activity by mevalonolactone was linked with the ability of the cells to convert squalene-2,3-epoxide into sterols. The results described in the present article support an important and perhaps necessary relationship between the rate of methyl sterol conversion of C27-sterols and the suppression or inhibition of HMG-Coa reductase in primary hepatocyte culture cells.