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.     

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.     

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.     

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.     

Regulation of 3-hydroxy-3-methylglutaryl-coenzyme A reductase in human hepatoma cell line Hep G2. Effects of inhibitors of cholesterol synthesis on enzyme activity.

Incubating Hep G2 cells for 18 h with triparanol, buthiobate and low concentrations (less than 0.5 microM) of U18666A, inhibitors of desmosterol delta 24-reductase, of lanosterol 14 alpha-demethylase and of squalene-2,3-epoxide cyclase (EC 5.4.99.7) respectively, resulted in a decrease of the HMG-CoA (3-hydroxy-3-methylglutaryl-coenzyme A) reductase activity. However, U18666A at concentrations higher than 3 microM increased the HMG-CoA reductase activity in a concentration-dependent manner. None of these inhibitors influenced directly the reductase activity in Hep G2 cell homogenates. Analysis by t.l.c. of 14C-labelled non-saponifiable lipids formed from either [14C]acetate or [14C]mevalonate during the cell incubations confirmed the sites of action of the drugs used. Beside the 14C-labelled substrates of the blocked enzymes and 14C-labelled cholesterol, another non-saponifiable lipid fraction was observed, which behaves as polar sterols on t.l.c. This was the case with triparanol and at those concentrations of U18666A that decreased the reductase activity, suggesting that polar sterols may play a role in suppressing the reductase activity. In the presence of 30 microM-U18666A (sterol formation blocked) the increase produced by simultaneously added compactin could be prevented by addition of mevalonate. This indicates the existence of a non-sterol mevalonate-derived effector in addition to a sterol-dependent regulation. LDL (low-density lipoprotein), which was shown to be able to decrease the compactin-induced increase in reductase activity, could not prevent the U18666A-induced increase. On the contrary, LDL enhanced the U18666A effect, showing that the LDL regulation is not merely the result of introducing cholesterol to the cells.     

Effect of vitamin D3 derivatives on cholesterol synthesis and HMG-CoA reductase activity in cultured cells

Treatment of logarithmically growing rat intestinal epithelial cells (IEC-6) in culture with vitamin D3 (cholecalciferol), 25-hydroxy vitamin D3 (25-hydroxy cholecalciferol), 1,25-dihydroxy vitamin D3 (1,25-dihydroxycholecalciferol), and 24,25 dihydroxy vitamin D3 (24(R),25-dihydroxycholecalciferol), caused an inhibition of the cholesterol biosynthetic pathway at two separate sites. At concentrations greater than 2 micrograms/ml, the hydroxylated forms of vitamin D3 caused an accumulation of methyl sterols indicating an inhibition of lanosterol demethylation. Vitamin D3, however, had little effect on lanosterol demethylation. A second site of inhibition occurs at 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA reductase), the rate limiting enzyme in cholesterol biosynthesis at concentrations less than 2 micrograms/ml. All vitamin D3 compounds, except 1,25-dihydroxy vitamin D3, inhibited HMG-CoA reductase activity in a concentration-dependent manner. The lack of inhibition of HMG-CoA reductase activity by 1,25-dihydroxy vitamin D3 in IEC-6 cells was not due to impaired uptake, since 1,25-dihydroxy vitamin D3 caused an accumulation of methyl sterols under similar conditions. The inhibition of HMG-CoA reductase activity and cholesterol synthesis by vitamin D3 and 25-hydroxy vitamin D3 was also observed in other cell culture lines such as human skin fibroblasts (GM-43), transformed human liver cells (Hep G2), and mouse peritoneal macrophages (J-774). On the other hand, 1,25-hydroxy vitamin D3 showed effects on HMG-CoA reductase activity that varied with the cell line. In J-774 and human skin fibroblasts, 1,25-dihydroxy vitamin D3 showed a biphasic effect on reductase activity such that at low concentrations reductase activity was inhibited but was restored to control values at high concentrations.(ABSTRACT TRUNCATED AT 250 WORDS)     

Lipid requirement for cell cycling : The effect of selective inhibition of lipid synthesis

Tissue culture cells require lipid which must be provided exogenously or synthesized via endogenous pathways. The exogenous supplies can be largely removed by growing cells in medium containing delipidized serum. Pathways for synthesis of lipid can then be blocked at three steps: (1) fatty acids by removal of biotin, an essential coenzyme; (2) phosphatidylcholine and sphingomyelin by deleting choline from the growth medium; and (3) cholesterol by inhibiting HMG-CoA reductase with 25-hydroxycholesterol. Sustained proliferation is prevented when lipid synthesis is blocked at any one of these steps. Cell proliferation resumes upon restoring synthesis with biotin, choline, or mevalonate (the product of the HMG-CoA reductase reaction) or by providing the lipid end products oleic acid or cholesterol. Using a combined cytophotometric-autoradiographic analysis to determine cell cycle distributions we have demonstrated that prereplicative (G1) cell cycle arrests develop in parallel with the proliferative inhibition. Each of the G1 arrests can be reversed by restoring the synthetic pathways or their lipid products. These observations suggest a causal relationship between the supply of lipids and passage through G1.     

HMG-CoA reductase inhibition causes neurite loss by interfering with geranylgeranylpyrophosphate synthesis

To determine whether neurite outgrowth depends upon the mevalonate pathway, we blocked mevalonate synthesis in nerve growth factor-treated PC12 cells or primary cortical neurones with atorvastatin, a 3-hydroxymethylglutaryl coenzyme A (HMG-CoA) reductase inhibitor, and substituted different intermediates of the mevalonate pathway. We show that HMG-CoA reductase inhibition causes a profound reduction of neurite length, neurite loss and ultimatively cell death in undifferentiated and pre-differentiated PC12 cells and also in rat primary cortical neurones. Geranylgeranylpyrophosphate, but not farnesylpyrophosphate, squalene or cholesterol, completely compensated for the lack of mevalonate. Our data indicate that, under HMG-CoA reductase inhibition, geranylgeranylpyrophosphate rather than farnesylpyrophosphate or cholesterol is critical for neurite outgrowth and/or maintenance. Loss of neurites is an early manifestation of various neurodegenerative disorders, and dysfunction of isoprenylation might play a role in their pathogenesis.     

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.     

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.     

ACTH induction of 3-hydroxy-3-methylglutaryl coenzyme A reductase, cholesterol biosynthesis, and steroidogenesis in primary cultures of bovine adrenocortical cells

The current studies demonstrate that corticosteroidogenesis can be maintained by primary cultures of bovine adrenocortical cells under lipoprotein-depleted conditions. The cholesterol necessary as substrate for steroid synthesis was found to arise from de novo synthesis within these cells. Adrenocorticotropin (ACTH) increased 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase activity 5-fold within 12 h after addition to the medium. The increase in activity apparently represented accumulation of enzyme as determined by protein blotting and immunodetection. The predominant immunodetectable species of HMG-CoA reductase from bovine adrenal cells was 97,000 daltons; no higher molecular mass species was detectable. The ACTH induction of HMG-CoA reductase activity could be prevented after inhibition of cholesterol conversion to pregnenolone with clotrimazole. These results are suggestive that ACTH increases adrenocortical cholesterol biosynthesis and HMG-CoA reductase activity after conversion of a cellular pool of cholesterol and/or oxysterol into steroid. The increased rate of cholesterol biosynthesis is then capable of maintaining ACTH-promoted steroid production. This is the first study, in vitro, to demonstrate an ACTH-promoted accumulation of HMG-CoA reductase of adrenocortical cells.     

Characterization of peroxisomal 3-hydroxy-3-methylglutaryl coenzyme A reductase in UT2 cells: sterol biosynthesis, phosphorylation, degradation, and statin inhibition

We have previously identified a CHO cell line (UT2 cells) that expresses only one 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase protein which is localized exclusively in peroxisomes [Engfelt, H.W., Shackelford, J.E., Aboushadi, N., Jessani, N., Masuda, K., Paton, V.G., Keller, G.A., and Krisans, S.K. (1997) J. Biol. Chem. 272, 24579-24587]. In this study, we utilized the UT2 cells to determine the properties of the peroxisomal reductase independent of the endoplasmic reticulum (ER) HMG-CoA reductase. We demonstrated major differences between the two proteins. The peroxisomal reductase is not the rate-limiting enzyme for cholesterol biosynthesis in UT2 cells. The peroxisomal reductase protein is not phosphorylated, and its activity is not altered in the presence of inhibitors of cellular phosphatases. Its rate of degradation is not accelerated in response to mevalonate. Finally, the degradation process is not blocked by N-acetyl-Leu-Leu-norleucinal (ALLN). Furthermore, the peroxisomal HMG-CoA reductase is significantly more resistant to inhibition by statins. Taken together, the data support the conclusion that the peroxisomal reductase is functionally and structurally different from the ER HMG-CoA reductase.     

Cytotoxicity of cholesterol oxides and their effects on cholesterol metabolism in cultured human aortic smooth muscle cells

Incubation of monolayers of cultured human aortic smooth muscle cells with oxygenated sterols (25-hydroxycholesterol, 7-ketocholesterol, or cholesterol 5,6-epoxide) markedly inhibited growth though the viability of the culture was not affected. The effects on growth was concentration dependent, and 25-hydroxycholesterol was the most potent inhibitor of cellular growth as measured by decreased incorporation of thymidine into DNA and suppression of HMG-CoA reductase activity. The inhibitory effect of 25-hydroxycholesterol on cellular growth was not reversible if the cultures were grown in medium with normal fetal calf serum. However, in medium with delipidated serum, addition of purified cholesterol partially prevented growth inhibition induced by 25-hydroxycholesterol. Purified cholesterol, independently or in combination with tocopherol had no toxic effect on cellular growth. Addition of cholesterol oxides to the incubation medium stimulated lysosomal activation and release of acid phosphatase into the culture medium. The effect was concentration dependent and inversely related to cellular growth.     

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.     

Early embryonic lethality caused by targeted disruption of the 3-hydroxy-3-methylglutaryl-CoA reductase gene

The endoplasmic reticulum (ER) enzyme 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, which converts HMG-CoA to mevalonate, catalyzes the ratelimiting step in cholesterol biosynthesis. Because this mevalonate pathway also produces several non-sterol isoprenoid compounds, the level of HMG-CoA reductase activity may coordinate many cellular processes and functions. We used gene targeting to knock out the mouse HMG-CoA reductase gene. The heterozygous mutant mice (Hmgcr+/-) appeared normal in their development and gross anatomy and were fertile. Although HMG-CoA reductase activities were reduced in Hmgcr+/- embryonic fibroblasts, the enzyme activities and cholesterol biosynthesis remained unaffected in the liver from Hmgcr+/- mice, suggesting that the haploid amount of Hmgcr gene is not rate-limiting in the hepatic cholesterol homeostasis. Consistently, plasma lipoprotein profiles were similar between Hmgcr+/- and Hmgcr+/+ mice. In contrast, the embryos homozygous for the Hmgcr mutant allele were recovered at the blastocyst stage, but not at E8.5, indicating that HMG-CoA reductase is crucial for early development of the mouse embryos. The lethal phenotype was not completely rescued by supplementing the dams with mevalonate. Although it has been postulated that a second, peroxisome-specific HMG-CoA reductase could substitute for the ER reductase in vitro, we speculate that the putative peroxisomal reductase gene, if existed, does not fully compensate for the lack of the ER enzyme at least in embryogenesis.     

The cytotoxicity of mevalonate, lovastatin and carminomycin with respect to MOLT-4 human malignant T-lymphoblasts in vitro]

It was shown in vitro that high concentrations of lovastatin, a competitive inhibitor of hydroxymethyl glutaryl coenzyme A (HMG-CoA) reductase inhibited human malignant cells MOLT-4. The activity of lovastatin in doses of 50-250 microM was dose-dependent. Addition of mevalonate in a concentration of 3 mM to the growth medium completely prevented the cytotoxic effect of 100 microM of lovastatin. At the same time, exogenous mevalonate did not decrease the cytotoxicity of the anthracycline antibiotic carminomycin. Moreover, in a high concentration (7 mM) mevalonate slowly but significantly inhibited the growth of the malignant target cells and the effect was added to the cytotoxic effect of carminomycin low concentrations (0.08 to 0.175 microgram/ml). The results and the literature data suggested that combination of mevalonate, HMG-CoA reductase inhibitors and anthracyclines could be useful in tumor chemotherapy. The suggestion needs further investigation.     

Feedback inhibition of the cholesterol biosynthetic pathway in patients with Smith-Lemli-Opitz syndrome as demonstrated by urinary mevalonate excretion

Smith-Lemli-Opitz syndrome (SLOS) is a genetic disorder characterized by low plasma cholesterol and high 7-dehydrocholesterol (7-DHC). Synthesis of cholesterol and 7-DHC and its metabolites is regulated by HMG-CoA reductase, whose activity can be measured by 24-h excretion of its product mevalonate. We devised a simple, non-invasive method for collecting 24-h urine in our subjects. With a background of a very low cholesterol diet, mean mevalonate excretion did not differ between controls and SLOS children, indicating that SLOS subjects have normal HMG-CoA reductase activity. In a short term feeding study, the effects of a high cholesterol diet in SLOS subjects include a significant 55% increase in plasma cholesterol levels and 39% decrease in mevalonate excretion and no change in plasma 7-DHC levels. However, in four SLOS subjects, fed a high cholesterol diet for 2-3 years, plasma cholesterol levels continued to increase, urinary mevalonate excretion remained low and total 7-DHC decreased significantly, likely from decreased total sterol synthesis. Thus, in SLOS subjects, HMG-CoA reductase activity was normal and was subject to normal cholesterol induced feedback inhibition. However, total sterol synthesis in SLOS may still be decreased because of increased diversion of mevalonate into the shunt pathway away from sterol synthesis.     

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.     

Linkage of the isoprenoid biosynthetic pathway with induction of DNA synthesis in mouse lymphocytes. Effects of compactin on mitogen-induced lymphocytes in serum-free medium

Concanavalin A induction of DNA synthesis in mouse spleen lymphocytes cultured in serum-free medium was shown to be very sensitive to inhibition by compactin (ML-236B), a specific competitive inhibitor of hydroxymethylglutaryl-CoA reductase. As low as 0.1 microM compactin could give 98% inhibition of mitogen induction of a 5.10(6) cells/ml culture. This inhibition could be reversed completely by addition of exogenous mevalonate, but could not be reversed by either exogenous cholesterol or isopentenyladenine. Oxygenated sterol inhibition of mitogen-induced DNA synthesis could be reversed by cholesterol or by mevalonate, whereas cyclic AMP inhibition could not be reversed by either compound. These results suggest that endogenous cholesterol production is a necessary but not sufficient factor co-ordinated with mitogen-induced DNA synthesis, and that the presence of some additional product of mevalonate metabolism is involved also. Isopentenyladenine, though, did not have as significant effect of alleviating any of the above inhibitions. Since mevalonate could not relieve cyclic AMP inhibition, but could overcome compactin inhibition, cyclic AMP inhibition cannot be explained as due only to blockage of mevalonate production.