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PubMed1991

Stereospecificity of the inhibition by etomoxir of fatty acid and cholesterol synthesis in isolated rat hepatocytes.

The racemates of substituted 2-oxiranecarboxylates are potent inhibitors of fatty acid oxidation and fatty acid and cholesterol synthesis. We show in the accompanying paper [Agius L, Peak M and Sherratt HSA, Biochem Pharmacol 42: 1711-1715, 1991] that only the R-enantiomer of etomoxir, a potent hypoglycaemic compound, inhibits fatty acid oxidation in hepatocytes. We demonstrate in this paper that although the R-enantiomer of etomoxir is esterified to its CoA-ester more readily than the S-enantiomer, both the R- and S-enantiomers are equally potent inhibitors of fatty acid and cholesterol synthesis from acetate in rat hepatocytes. The inhibition of fatty acid synthesis is not due to direct inhibition of fatty acid synthetase and the inhibition of cholesterol synthesis occurs at a site proximal to formation of mevalonate. Since the S-enantiomer inhibits fatty acid and cholesterol synthesis but not fatty acid oxidation the inhibition of the biosynthetic pathways is not coupled to inhibition of fatty acid oxidation.

Animals↗
PubMed1997

HMG-CoA reductase inhibitors induce apoptosis in mouse proximal tubular cells in primary culture.

Renal cyst formation in polycystic diseases or after nephron reduction is attributed to enhanced tubular cell proliferation with unbalanced cell death. The induction of tubular cell death could be effective to reduce renal cyst formation. In this study, we examined the effects of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors on apoptosis in mouse proximal tubular (MPT) cells in primary culture. After treatment with HMG-CoA reductase inhibitors, the extracted DNA was analyzed by gel-electrophoresis under ultraviolet light. Apoptosis was evaluated quantitatively by estimating the ratio of fragmented DNA over intact DNA. For morphologic studies, cells were stained with Hoechst 33,258. DNA ladder pattern of 200 kDa typical of apoptosis and significant increase in DNA fragmentation were seen after 24 hours of treatment with lovastatin, a HMG-CoA reductase inhibitor. Staining with the Hoechst dye revealed cleavage of nucleus into pieces under the same condition. Geranylgeranylpyrophosphate (20 microM) and mevalonate (500 microM) completely reversed the effect of lovastatin, while farnesylpyrophosphate (20 microM) partially reversed it. Other products of HMG-CoA pathway such as cholesterol, ubiquinone, dolichol, and isopentenyladenine had no effect. Perillic acid and alpha-hydoxyfarnesylphosphonic acid, isoprenylation inhibitors, induced apoptosis of the cells. A treatment with lovastatin caused actin filament disruption. Cytochalasin D, an inhibitor of actin polymerization, induced apoptosis. Interleukin-1 beta-converting enzyme inhibitor II, a protease inhibitor, had no effect on the apoptosis induced by either HRI or cytochalasin D. The present study suggests that in mouse proximal tubules, HMG-CoA reductase inhibitors induce apoptosis via inhibition of isoprenoid production, and disruption of actin filaments may play a role in the apoptosis induction.

Actins↗
PubMed1985

Sites of gibberellin biosynthesis in pea seedlings.

Potential sites of gibberellin biosynthesis in 10-day-old ;Alaska' pea (Pisum sativum L.) seedlings were investigated using a cell-free ezyme system capable of incorporating [(14)C]-mevalonic acid into ent-kaurene. In peas, ent-kaurene is assumed to be a committed intermediate in the gibberellin biosynthetic pathway. Comparative results from enzyme assays using extracts from shoot tips, leaf blades, internodes, and root tips indicate that the highest capacity for ent-kaurene (and presumably gibberellin) synthesis is in those tissues with the greatest potential for growth. The highest rates were obtained with extracts prepared from the fifth (youngest) internode, the fourth (youngest) expanded leaf, and the shoot tip itself. This report represents the first direct evidence that the enzymes responsible for early stages in gibberellin biosynthesis occur in internode tissues with potential for rapid elongation.

Journal Article↗
PubMed2006

A role for geranylgeranylation in interleukin-1beta secretion.

OBJECTIVE: Mevalonate kinase deficiency (MKD) is an autosomal-recessive disorder characterized by recurring episodes of inflammation. MK catalyzes the phosphorylation of mevalonic acid, which is an early step in isoprenoid biosynthesis. The goal of our study was to determine whether a temporary shortage of certain isoprenoid end products and/or the accumulation of mevalonic acid is the cause of interleukin-1beta (IL-1beta) secretion in MKD. METHODS: We studied the effect of the addition of intermediate metabolites and inhibitors of the isoprenoid biosynthesis pathway on IL-1beta secretion by peripheral blood mononuclear cells (PBMCs) of patients with MKD and healthy controls. RESULTS: Inhibition of enzymes involved in geranylgeranyl pyrophosphate (GGPP) synthesis or geranylgeranylation of proteins led to a marked increase of lipopolysaccharide-stimulated IL-1beta secretion in PBMCs of control subjects. Furthermore, the increased IL-1beta secretion by PBMCs of patients with MKD was reversed by supplementation with GGPP as well as with mevalonic acid. IL-1beta secretion was increased only when control PBMCs were incubated with excessive amounts of mevalonic acid. Finally, a reduction in IL-1beta secretion by MKD PBMCs was also observed when sterol biosynthesis was inhibited, favoring nonsterol isoprenoid biosynthesis. CONCLUSION: Our results indicate that a shortage of geranylgeranylated proteins, rather than an excess of mevalonate, is likely to cause increased IL-1beta secretion by PBMCs of patients with MKD.

Anti-Inflammatory Agents↗
PubMed2001

Labeling patterns of chloroplastidic isoprenoids in cultured cells of liverwort Ptychanthus striatus.

Incorporation studies administering 2H- and 13C-labeled mevalonate (MVA) and 13C-labeled glucose to suspension cultured cells of the liverwort, Ptychanthus striatus, were carried out in order to examine the biosynthesis of the phytyl side-chain of chlorophyll a. Administration of 13C- and 2H-labeled MVA provided evidence for the involvement of the MVA pathway in the phytyl side-chain biosynthesis and preferential labeling of the farnesyl diphosphate (FPP)-derived portion. An alternate labeling pattern in the phytyl side-chain was observed which was slightly different to the non-equivalent labeling in other liverworts, such as Heteroscyphus planus and Lophocolea heterophylla and in the hornwort, Anthoceros punctatus. The labeling pattern observed after the administration of 13C-labeled glucose revealed the simultaneous involvement of the non-MVA pathway in the phytol biosynthesis of P. striatus cells.

Acetylation↗
PubMed2006

Microbial sensors for small molecules: development of a mevalonate biosensor.

We describe a novel biosensor strain for detection and quantification of a small molecule, mevalonate. The biosensor strain is an Escherichia coli mevalonate auxotroph that expresses the green fluorescent protein and reports on the mevalonate concentration in the growth medium through a change in growth rate. A model describing the growth rate dependence on mevalonate was developed in order to use the biosensor strain for high-throughput screening (HTS) and quantitative measurement of mevalonate in the extracellular environment. In general, this method should be applicable to the quantification of any small molecule for which an auxotroph can be developed and will be useful for HTS of evolved metabolic pathways for which there is no readily available screen or selection.

Aldose-Ketose Isomerases↗
PubMed2001

Inhibition of isoprenoid biosynthesis causes insulin resistance in 3T3-L1 adipocytes.

Lovastatin treatment caused down-regulation of the insulin-responsive glucose transporter 4 (Glut4) and up-regulation of Glut1 in 3T3-L1 adipocytes. These changes in protein expression were associated with a marked inhibition of insulin-stimulated glucose transport. Lovastatin had no effect on cell cholesterol levels, but its effects were reversed by mevalonate, demonstrating that inhibition of isoprenoid biosynthesis causes insulin resistance in 3T3-L1 adipocytes. These findings support the notion that whole body insulin resistance may arise as a result of perturbations in general biochemical pathways, rather than primary defects in insulin signalling.

3T3 Cells↗
PubMed2002

Lovastatin is a potent inhibitor of meningioma cell proliferation: evidence for inhibition of a mitogen associated protein kinase.

Lovastatin inhibits 3-hydroxy 3-methylglutaryl coenzyme A (HMG-CoA) reductase the rate limiting enzyme for synthesis of mevalonic acid, a precursor for cholesterol, farnesyl and geranylgeranyl pyrophosphate isoprenoids. Recent studies suggest it also has growth inhibitory properties. Posttranslational farnesyl or geranylgeranylation of low molecular weight GTP-binding proteins such as RAS and RHO are thought to be an essential step in activation of phosphorylation cascades such as the RAS-RAF-1-MEK-1-MAPK/ERK pathway which stimulate cell proliferation. In this study, we evaluated lovastatin effects on meningioma cell proliferation and activation of the MEK-1-MAPK/ERK pathway. The effect of lovastatin on cell proliferation was assessed in eight human meningioma cell cultures stimulated by platelet derived growth factor (PDGF)-BB, cerebrospinal fluid (CSF), and fetal bovine serum (FBS). Concomitant lovastatin effects on phosphorylation/activation of mitogen-activated protein kinase/extracellular signal regulated kinase (MAPK/ERK) kinase (MEK-1) and MAPK/ERK were assessed by Western blot. Whether lovastatin acts via a mevalonate-dependent mechanism was also evaluated. Coadministration of lovastatin completely blocked PDGF-BB, CSF, and FBS stimulation of [3H]-thymidine incorporation and cell proliferation. Lovastatin inhibited PDGF-BB's stimulatory effect in a dose dependent manner. Concomitant with its growth inhibitory effects, lovastatin reduced phosphorylation/activation of MEK-1/2 in five meningiomas and MAPK/ERK in seven. Coadministration of mevalonate with lovastatin partially restored PDGF's mitogenic effect. Lovastatin is a potent inhibitor of meningioma cell proliferation which may act in part by reducing activation of MEK-1-MAPK/ERK pathway. Additional studies are warranted to assess whether lovastatin and similar HMG-CoA reductase inhibitors represent a new adjunctive chemotherapy for recurrent meningiomas.

Aged↗
PubMed1991

Nucleotide sequence of the ERG12 gene of Saccharomyces cerevisiae encoding mevalonate kinase.

The nucleotide sequence of the ERG12 gene, encoding mevalonate kinase, from Saccharomyces cerevisiae is presented. The longest open reading frame may code for a protein containing 443 amino acids with a deduced relative molecular mass of 48,500. The analysis of the nucleotide sequence reveals a complete identity with the yeast gene RAR1, isolated elsewhere by complementation of a rar1 mutation involved in the stability of plasmids with weak ARS. In addition, we show that mevalonate kinase is not a rate-limiting enzyme; however its sensitivity to FFP could be a key regulatory mechanism in the sterol pathway of yeast.

Amino Acid Sequence↗
PubMed2003

Atorvastatin inhibits expression of minichromosome maintenance proteins in vascular smooth muscle cells.

3-Hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors have been reported to inhibit vascular smooth muscle cell growth, a key event in the pathogenesis of proliferative vascular diseases. The mechanism by which HMG-CoA reductase inhibitors exert their antiproliferative activity is not fully understood, especially their effect on DNA replication. We therefore investigated the effects of atorvastatin on minichromosome maintenance (MCM) protein 6 and 7 expression in vascular smooth muscle cells, two proteins essential for initiation of DNA replication. Stimulation of quiescent rat aortic vascular smooth muscle cells with fetal bovine serum induced MCM6 and MCM7 protein and mRNA expression, which was potently attenuated by atorvastatin in a dose-dependent fashion. Mevalonate completely abrogated the inhibitory effect on serum-induced MCM6 and MCM7 expression, demonstrating that biosynthesis of isoprenoids was likely the specific pathway blocked by atorvastatin. Transient transfection experiments revealed that atorvastatin inhibited MCM6 and MCM7 promoter activity, implicating a transcriptional mechanism. The MCM6 and MCM7 promoters contain several E2F sites critical for their transcriptional activation. Activity of a luciferase reporter plasmid containing four E2F elements was also strongly inhibited by atorvastatin. The inhibitory effect of atorvastatin on MCM6 and MCM7 was reversed by adenoviral-mediated overexpression of E2F, indicating that their downregulation by atorvastatin involves an E2F-dependent mechanism. These findings demonstrate that MCM proteins play an essential role during the proliferation of vascular smooth muscle cells and may provide a novel therapeutic target for proliferative vascular diseases. Inhibition of MCM6 and MCM7 expression by blocking E2F function may contribute importantly to the inhibition of vascular smooth muscle cell DNA synthesis by atorvastatin.

Adenovirus E2 Proteins↗
PubMed2003

Fluvastatin induces apoptosis of vascular endothelial cells: blockade by glucocorticoids.

Statins block de novo synthesis of cholesterol by inhibiting the enzyme, HMG CoA reductase. The product of this reaction, mevalonic acid, is also a precursor of isoprenoids, molecules required for the activation of signaling G-proteins, such as Ras. Signal transduction pathways involving Ras are important for cell survival and this may be why statins induce apoptotic death of several cell types. Given that statins are used to treat vascular disease, surprisingly no studies have been conducted on vascular endothelial cells. Here we show that fluvastatin (FS), at concentrations from 1-2 microM, blocks growth and induces apoptosis of the endothelial cell line, EA.hy 926. Considerable redundancy is known to exist in cell signaling and in vivo toxicity of FS might be prevented by other signaling pathways, like those activated by adrenal or sex steroids. RT-PCR analysis revealed the expression of the androgen and glucocorticoid receptor in EA.hy 926 cells. Although the androgen, dihydrotestesterone (DHT) had no effect, the glucocorticoid, dexamethasone (Dex), blocked FS-induced apoptosis. Cell cycle analysis revealed that 24 h exposure to FS prevented cells from leaving G(1) and 24-48 h later a marked sub-G(1) peak was observed. Dex was able to reduce the sub-G(1) peak, but it failed to block accumulation of cells in G(1), indicating that it's effect was specific for blockade of apoptosis, and not specific to an effect on FS alone. This study strongly suggests that glucocorticoids have a role to play in preventing vascular injury and they may provide the reason why statins are not inherently toxic to vascular endothelial cells, in vivo.

Anticholesteremic Agents↗
PubMed2002

A second gene for peroxisomal HMG-CoA reductase? A genomic reassessment.

HMG-CoA reductase (HMGCR) catalyzes the conversion of HMG-CoA to mevalonate, the rate-limiting step of eukaryotic isoprenoid biosynthesis, and is the main target of cholesterol-lowering drugs. The classical form of the enzyme is a transmembrane-protein anchored to the endoplasmic reticulum. However, during the last years several lines of evidence pointed to the existence of a second isoform of HMGCR localized in peroxisomes, where mevalonate is converted further to farnesyl diphosphate. This finding is relevant for our understanding of the complex regulation and compartmentalization of the cholesterogenic pathway. Here we review experimental evidence suggesting that the peroxisomal activity might be due to a second HMGCR gene in mammals. We then present a comprehensive analysis of completely sequenced eukaryotic genomes, as well as the human and mouse genome drafts. Our results provide evidence for a large number of independent duplications of HMGCR in all eukaryotic kingdoms, but not for a second gene in mammals. We conclude that the peroxisomal HMGCR activity in mammals is due to alternative targeting of the ER enzyme to peroxisomes by an as yet uncharacterized mechanism.

Animals↗
PubMed1992

Cholesterol and mevalonic acid modulation in cell metabolism and multiplication.

Cholesterol in animals is a major structural component of cell membranes. It may therefore play a functional role in the modulation of cell osmolarity, the process of pinocytosis and the activities of membrane-associated proteins such as ionic pumps, immune responses, etc. A major relationship exists between the cell-growth processes and the cholesterol biosynthetic pathway. The cholesterol needed for new membranes may be derived either from endogenous synthesis or from exogenous sources, principally plasma low-density-lipoproteins (LDL) which enter the cells by receptor-mediated endocytosis. Both these pathways are enhanced in rapidly growing cells. Conversely, if synthesis is inhibited and no exogenous cholesterol is available, cell growth is blocked. The 3-hydroxy-3-methylglutaryl CoA (HMGCoA) reductase (the rate-limiting reaction in cholesterol biosynthesis) is the enzyme which catalyzes the conversion of HMGCoA to mevalonic acid. It has been suggested that mevalonate may play an important role in cell proliferation. All cells need at least two products synthesized from mevalonate in order to proliferate, and the only one yet identified is cholesterol. Other melavonate-derived potential candidates as cell-cycle and cell-survival products include the dolichols ubiquinone side chains, isopentenyladenosine derivatives, etc. Furthermore, it has recently been shown that membrane association appears to be an important function in mevalonate-derive modifications of several important proteins such as cellular membrane G proteins, those coded for by oncogenes (ras proteins) and lamins (nuclear proteins). In recent years the development of cholesterol-synthesis-inhibiting drugs, for lowering plasma cholesterol levels has mainly been centred on the control of HMGCoA reductase activity (vastatins). However, because mevalonic acid is the precursor of numerous metabolites, any reduction of such activity may potentiate pleiotropic effects. Vastatins are now, therefore, receiving increased attention as potential pharmacological tools for the control of abnormal cell growth in pathological situations, i.e. tumours and vascular smooth muscle cell proliferation under atherogenic conditions. In our laboratories, we have demonstrated that simvastatin can prevent arterial myocyte proliferation both in vivo and in vitro. Simvastatin can also inhibit in vitro the rate of human glioma cell growth, since it shows a strong synergistic inhibitory effect on cell proliferation when used in association with anticancer agents such as Carmustine or beta-interferon. Both simvastatin-induced cell growth inhibition and the synergy observed with these drugs can be completely reversed by incubating cells with mevalonate. This shows that the effect of simvastatin of cell proliferation is due to its specific inhibitory activity on intracellular mevalonate synthesis.

Animals↗
PubMed1980

Stereochemistry of the side chain oxidation of 5 beta-cholestane-3 alpha,7 alpha,12 alpha-triol in man.

Previous in vitro studies have shown that the oxidation of the side chain of bile acid precursors can start with either microsomal or mitochondrial enzyme systems. The microsomal system oxidizes the terminal methyl group (C-26) of the side chain that originates from C-2 of mevalonic acid, and the mitochondrial system oxidizes the terminal methyl group (C-27) derived from C'-3 of mevalonic acid. We administered [2-14C]-mevalonic acid to a patient with a complete bile fistula and isolated from the bile 3 alpha,7 alpha,12 alpha-trihydroxy[1,7,15,22,26-14C]5 beta-cholestanoic acid, a precursor in the synthesis of cholic acid which has undergone partial oxidation of the side chain. This compound was chemically decarboxylated and the liberated CO2 was trapped and counted. The recovery from control experiments using [24-14C]cholic acid was 82.6 +/- 2.3% (+/- 1 S.D.). However, following decarboxylation of 3 alpha,7 alpha,12 alpha-trihydroxy[14C5]5 beta-cholestanoic acid, only 7.5 +/- 0.58% (+/- S.D.) of the C-26 radioactivity was recovered. This study suggests that the major pathway of side chain oxidation of 5 beta-cholestane-3 alpha,7 alpha,12 alpha-triol starts with hydroxylation of the methyl group derived from C'-3 of mevalonate (C-27) by mitochondrial enzymes. It is also concluded that the 3 alpha,7 alpha,12 alpha-trihydroxy-5 beta-cholestan-27-oic acid formed in man has the R configuration at carbon 25.

Bile↗
PubMed2006

Manipulation of isoprenoid biosynthesis as a possible therapeutic option in mevalonate kinase deficiency.

OBJECTIVE: In cells from patients with the autoinflammatory disorder mevalonate kinase (MK) deficiency, which includes the hyperimmunoglobulin D with periodic fever syndrome, MK becomes the rate-limiting enzyme in the isoprenoid biosynthesis pathway. This suggests that up-regulation of residual MK activity in these patients could be a way in which to prevent or alleviate the associated symptoms. We studied the effect of 2 specific inhibitors of isoprenoid biosynthetic enzymes on the residual activity of MK in cells from patients with MK deficiency. METHODS: Skin fibroblasts from MK-deficient patients and from controls were cultured for 7 days with either simvastatin, an inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A reductase, or zaragozic acid A, an inhibitor of squalene synthase. Following culture, MK activity, MK protein levels, MVK messenger RNA levels, and the effect on the pathway flux toward non-sterol isoprenoid biosynthesis were determined. RESULTS: Treatment of the fibroblasts with either of the inhibitors led to a marked increase in residual MK enzyme activity, which was largely attributable to increased MVK gene transcription. This effect was even more pronounced when the cells were cultured in lipoprotein-depleted medium. The flux toward nonsterol isoprenoid end-product synthesis was reduced when cells were treated with simvastatin but was partly restored by concomitant treatment with zaragozic acid A. CONCLUSION: Our results indicate that manipulations of the isoprenoid biosynthesis pathway that promote the synthesis of nonsterol isoprenoids may provide an interesting therapeutic option for the treatment of MK deficiency.

Bridged Bicyclo Compounds, Heterocyclic↗
PubMed2005

Simvastatin induces caspase-independent apoptosis in LPS-activated RAW264.7 macrophage cells.

Macrophages participate in several inflammatory pathologies such as sepsis and arthritis. We examined the effect of simvastatin on the LPS-induced proinflammatory macrophage RAW264.7 cells. Co-treatment of LPS and a non-toxic dose of simvastatin induced cell death in RAW264.7 cells. The cell death was accompanied by disruption of mitochondrial membrane potential (MMP), genomic DNA fragmentation, and caspase-3 activation. Surprisingly, despite caspase-dependent apoptotic cascade being completely blocked by Z-VAD-fmk, a pan-caspase inhibitor, the cell death was only partially repressed. In the presence of Z-VAD-fmk, DNA fragmentation was blocked, but DNA condensation, disruption of MMP, and nuclear translocation of apoptosis inducing factor were obvious. The cell death by simvastatin and LPS was effectively decreased by both the FPP and GGPP treatments as well as mevalonate. Our findings indicate that simvastatin triggers the cell death of LPS-treated RAW264.7 cells through both caspase-dependent and -independent apoptotic pathways, suggesting a novel mechanism of statins for the severe inflammatory disease therapy.

Animals↗
PubMed2004

Defects in the N-linked oligosaccharide biosynthetic pathway in a Trypanosoma brucei glycosylation mutant.

Concanavalin A (ConA) kills the procyclic (insect) form of Trypanosoma brucei by binding to its major surface glycoprotein, procyclin. We previously isolated a mutant cell line, ConA 1-1, that is less agglutinated and more resistant to ConA killing than are wild-type (WT) cells. Subsequently we found that the ConA resistance phenotype in this mutant is due to the fact that the procyclin either has no N-glycan or has an N-glycan with an altered structure. Here we demonstrate that the alteration in procyclin N-glycosylation correlates with two defects in the N-linked oligosaccharide biosynthetic pathway. First, ConA 1-1 has a defect in activity of polyprenol reductase, an enzyme involved in synthesis of dolichol. Metabolic incorporation of [3H]mevalonate showed that ConA 1-1 synthesizes equal amounts of dolichol and polyprenol, whereas WT cells make predominantly dolichol. Second, we found that ConA 1-1 synthesizes and accumulates an oligosaccharide lipid (OSL) precursor that is smaller in size than that from WT cells. The glycan of OSL in WT cells is apparently Man9GlcNAc2, whereas that from ConA 1-1 is Man7GlcNAc2. The smaller OSL glycan in the ConA 1-1 explains how some procyclin polypeptides bear a Man4GlcNAc2 modified with a terminal N-acetyllactosamine group, which is poorly recognized by ConA.

Animals↗