geranylgeranyl-pyrophosphate and squalestatin-1

Results from previous investigations have indicated that glucose-stimulated insulin secretion (GSIS) is affected by changes in cholesterol and its intermediates, but the precise link between secretion and cholesterol has not been thoroughly investigated. In this study, we show the contribution of both protein isoprenylation and cholesterol-dependent plasma membrane structural integrity to insulin secretion in INS-1E cells and mouse islets. Acute (2 h) inhibition of hydroxyl-methylglutaryl-CoA reductase by simvastatin (SIM) resulted in inhibition of GSIS without reduction in total cellular cholesterol content. This effect was prevented by cell loading with the isoprenyl molecule geranylgeranyl pyrophosphate. Chronic (24 h) inhibition of cholesterol biosynthesis resulted in inhibition of GSIS with a significant reduction in total cellular cholesterol content, which was also observed after the inhibition of cholesterol biosynthesis downstream of isoprenoid formation. Electron paramagnetic resonance analyses of INS-1E cells showed that the SIM-induced reduction in cholesterol increased plasma membrane fluidity. Thus, the blockade of cholesterol biosynthesis resulted in the reduction of availability of isoprenoids, followed by a reduction in the total cholesterol content associated with an increase in plasma membrane fluidity. Herein, we show the different contributions of cholesterol biosynthesis to GSIS, and propose that isoprenoid molecules and cholesterol-dependent signaling are dual regulators of proper β-cell function.

Topics: Animals; Anticholesteremic Agents; Bridged Bicyclo Compounds, Heterocyclic; Cells, Cultured; Cholesterol; Glucose; Insulin; Insulin Secretion; Membrane Fluidity; Membrane Microdomains; Metabolic Networks and Pathways; Mice; Polyisoprenyl Phosphates; Simvastatin; Tricarboxylic Acids

Statins, drugs commonly used to lower serum cholesterol, have been shown to stimulate osteoblast differentiation and bone formation. Statins inhibit 3-hydroxy-3-methylglutaryl (HMG)-coenzyme A reductase (HMGCR), the first step of the isoprenoid biosynthetic pathway, leading to the depletion of the isoprenoids farnesyl pyrophosphate (FPP) and geranylgeranyl pyrophosphate (GGPP). The effects of statins on bone have previously been attributed to the depletion of GGPP, because the addition of exogenous GGPP prevented statin-stimulated osteoblast differentiation in vitro. However, in a recent report, we demonstrated that the specific depletion of GGPP did not stimulate but, in fact, inhibited osteoblast differentiation. This led us to hypothesize that isoprenoids upstream of GGPP play a role in the regulation of osteoblast differentiation. We demonstrate here that the expression of HMGCR and FPP synthase decreased during primary calvarial osteoblast differentiation, correlating with decreased FPP and GGPP levels during differentiation. Zaragozic acid (ZGA) inhibits the isoprenoid biosynthetic pathway enzyme squalene synthase, leading to an accumulation of the squalene synthase substrate FPP. ZGA treatment of calvarial osteoblasts led to a significant increase in intracellular FPP and resulted in inhibition of osteoblast differentiation as measured by osteoblastic gene expression, alkaline phosphatase activity, and matrix mineralization. Simultaneous HMGCR inhibition prevented the accumulation of FPP and restored osteoblast differentiation. In contrast, specifically inhibiting GGPPS to lower the ZGA-induced increase in GGPP did not restore osteoblast differentiation. The specificity of HMGCR inhibition to restore osteoblast differentiation of ZGA-treated cultures through the reduction in isoprenoid accumulation was confirmed with the addition of exogenous mevalonate. Similar to ZGA treatment, exogenous FPP inhibited the mineralization of primary calvarial osteoblasts. Interestingly, the effects of FPP accumulation on osteoblasts were found to be independent of protein farnesylation. Our findings are the first to demonstrate that the accumulation of FPP impairs osteoblast differentiation and suggests that the depletion of this isoprenoid may be necessary for normal and statin-induced bone formation.

Topics: Animals; Bridged Bicyclo Compounds, Heterocyclic; Cell Differentiation; Cell Proliferation; Cells, Cultured; Farnesyltranstransferase; Hydroxymethylglutaryl-CoA Reductase Inhibitors; Lovastatin; Osteoblasts; Polyisoprenyl Phosphates; Rats; Rats, Sprague-Dawley; Sesquiterpenes; Terpenes; Tricarboxylic Acids

We performed a focused siRNA screen in an A549 dengue type 2 New Guinea C subgenomic replicon cell line (Rluc-replicon) that contains a Renilla luciferase cassette. We found that siRNA mediated knock down of mevalonate diphospho decarboxylase (MVD) inhibited viral replication of the Rluc-replicon and DEN-2 NGC live virus replication in A549 cells. When the Rluc-replicon A459 cells were grown in delipidated media the replicon expression was suppressed and MVD knock down could further sensitize Renilla expression. Hymeglusin and zaragozic acid A could inhibit DEN-2 NGC live virus replication in K562 cells, while lovastatin could inhibit DEN-2 NGC live virus replication in human peripheral blood mononuclear cells. Renilla expression could be rescued in fluvastatin treated A549 Rluc-replicon cells after the addition of mevalonate, and partially restored with geranylgeranyl pyrophosphate, or farnesyl pyrophosphate. Our data suggest genetic and pharmacological modulation of cholesterol biosynthesis can regulate dengue virus replication.

Topics: Bridged Bicyclo Compounds, Heterocyclic; Carboxy-Lyases; Cholesterol; Dengue Virus; Fatty Acids, Monounsaturated; Fluvastatin; Gene Knockdown Techniques; Humans; Hydroxymethylglutaryl-CoA Reductase Inhibitors; Indoles; K562 Cells; Mevalonic Acid; Polyisoprenyl Phosphates; Replicon; RNA, Small Interfering; Sesquiterpenes; Tricarboxylic Acids; Virus Replication

A sensitive, nonradioactive analytical method has been developed to simultaneously determine the concentrations of farnesyl pyrophosphate (FPP) and geranylgeranyl pyrophosphate (GGPP) in cultured cells. Following extraction, enzyme assays involving recombinant farnesyl protein transferase or geranylgeranyl protein transferase I are performed to conjugate FPP or GGPP to dansylated peptides. The reaction products are then separated and quantified by high-performance liquid chromatography coupled to a fluorescence detector at the excitation wavelength 335 nm and the emission wavelength 528 nm. The retention times for farnesyl-peptide and geranylgeranyl-peptide are 8.4 and 16.9 min, respectively. The lower limit of detection is 5 pg of FPP or GGPP ( approximately 0.01 pmol). A linear response has been established over a range of 5-1000 pg ( approximately 0.01-2 pmol) with good reproducibility. The method has been used to determine the levels of FPP (0.125+/-0.010 pmol/10(6)cells) and GGPP (0.145+/-0.008 pmol/10(6)cells) in NIH3T3 cells. Furthermore, changes in FPP and GGPP levels following treatment of cells with isoprenoid biosynthetic pathway inhibitors were measured. This method is suitable for the determination of the concentrations of FPP and GGPP in any cell type or tissue.

Topics: 3T3 Cells; Alkyl and Aryl Transferases; Animals; Bridged Bicyclo Compounds, Heterocyclic; Chromatography, High Pressure Liquid; Evaluation Studies as Topic; Hydroxymethylglutaryl-CoA Reductase Inhibitors; Lovastatin; Mass Spectrometry; Mice; Polyisoprenyl Phosphates; Sesquiterpenes; Tricarboxylic Acids

We have used the potent squalene synthase inhibitor squalestatin I to investigate the regulation of isoprenoid metabolism in rat liver Fresh-frozen liver pieces from normal rats and rats infused with squalestatin I at 16 micrograms h-1 for 16 h were assayed for farnesyl pyrophosphate (FPP) and geranylgeranyl pyrophosphate (GGPP) by HPLC after dephosphorylation. Levels of FPP and GGPP were 5.4 +/- 1.6 nmol g-1 and 1.6 +/- 0.7 nmol g-1 (n = 13) wet wt., respectively, in control livers and 110 + 41 nmol g-1 and 3.0 +/- 2.2 nmol g-1 (n = 13) in livers from squalestatin I infused rats. In order to determine the relative level of isopentenyl pyrophosphate, liver slices from normal and squalestatin I infused rats were labeled to steady-state with [3H]acetate. Analysis of isoprenoid pyrophosphate intermediates by radio-HPLC after dephosphorylation indicated that squalestatin I brought about a 20-fold increase in the relative level of FPP (confirming direct analysis) and a 5-fold increase in the relative level of IPP. No change in either of these compounds was observed in livers from cholesterol-fed rats. To determine if squalestatin I altered the synthesis of nonsterol products, rats were subjected to long term subcutaneous infusion. After 14 days of infusion of 15 micrograms h-1, the median chain length of hepatic dolichol and dolichyl phosphate increased from C95 to C115 and the levels of these lipids increased approximately 3-fold. In addition, dolichyl phosphate mannose synthase activity in microsomes from squalestatin I treated rats was increased relative to controls when assayed in the absence of dolichyl phosphate. Squalestatin I affected ubiquinone metabolism to a lesser extent: chain lengths shifted from a Q10/Q9 ratio of 0.118 +/- 0.021 in the normal rat to 0.185 +/- 0.016 in the squalestatin I treated animals, and levels rose by approximately 90%. These results suggest that the isoprenoid pyrophosphate intermediates are shared by the cholesterol, dolichol and ubiquinone pathways and further show that the dolichol and ubiquinone pathways are not saturated. Apparently, under normal conditions, the levels of these intermediates are maintained relatively constant by coordinate enzyme regulation, thereby ensuring a constant rate of synthesis of nonsterols.

Topics: Animals; Bridged Bicyclo Compounds, Heterocyclic; Chromatography, High Pressure Liquid; Enzyme Inhibitors; Farnesyl-Diphosphate Farnesyltransferase; Hemiterpenes; Liver; Male; Organophosphorus Compounds; Polyisoprenyl Phosphates; Rats; Rats, Sprague-Dawley; Sesquiterpenes; Tricarboxylic Acids

The effects of squalestatin 1 on rat brain and liver homogenates and on Chinese hamster ovary tissue culture cells have been investigated. This compound effectively inhibits squalene biosynthesis in a highly selective manner. Cytoplasmic farnesyl pyrophosphate and geranylgeranyl pyrophosphate synthases are not affected, which is also the case for microsomal cis-prenyltransferase. In tissue culture cells, squalestatin 1 inhibits cholesterol biosynthesis completely, but does not alter dolichol synthesis or protein isoprenylation to a great extent. Incorporation of [3H]mevalonate into ubiquinone-9 and -10 increases 3-4-fold, probably as a result of increased synthesis of this lipid. Squalestatin 1 appears not only to be an effective inhibitor of cholesterol biosynthesis, but also to be more specific than other inhibitors used earlier in various in vitro and in vivo systems.

Topics: Animals; Bridged Bicyclo Compounds; Bridged Bicyclo Compounds, Heterocyclic; CHO Cells; Cricetinae; Lipids; Male; Mevalonic Acid; Polyisoprenyl Phosphates; Rats; Rats, Sprague-Dawley; Sesquiterpenes; Tricarboxylic Acids; Ubiquinone

We have identified and characterized two novel allyl pyrophosphatase activities from rat liver microsomes. One specifically hydrolyzes farnesyl pyrophosphate (FPP) to farnesol and the other converts geranylgeranyl pyrophosphate (GGPP) to geranylgeranol. Hence, we named them farnesyl pyrophosphatase (FPPase) and geranylgeranyl pyrophosphatase (GGPPase) activities, respectively. Other allyl pyrophosphates, i.e., isopentenyl pyrophosphate, dimethyl allyl pyrophosphate, and geranyl pyrophosphate, did not act as substrates for these activities. Both activities are metal ion independent and exhibit acidic pH optima (5.5 and 6.0). Microsomal FPPase has a Km for FPP of 7 microM and a specific activity of 6.8 nmol/min/mg protein at pH 5.5. GGPP is a potent noncompetitive inhibitor of FPPase. FPP has no inhibitory effect on GGPPase activity. Microsomal GGPPase has a Km for GGPP of 12 microM and a specific activity of 14 nmol/min/mg protein. The Km of FPPase activity for FPP increases with an increase in pH. The GGPPase activity remains unaffected with an increase in pH. Metal ions Zn2+ and Mn2+ are potent inhibitors of GGPPase activity. Zaragozic acid B is a weak inhibitor of FPPase/GGPPase activities as compared to squalene synthase. GGPPase activity is inhibited with a fourfold higher IC50 (20 microM) as compared to FPPase (5 microM). Hence, the FPPase and GGPPase activities can be differentiated by zaragozic acid B inhibition. Kinetic analysis of inhibition of FPPase by zaragozic acid B further indicates that it is a mixed type noncompetitive inhibitor.

Topics: Animals; Bridged Bicyclo Compounds; Bridged Bicyclo Compounds, Heterocyclic; Cations, Divalent; Farnesyl-Diphosphate Farnesyltransferase; Hydrogen-Ion Concentration; In Vitro Techniques; Kinetics; Microsomes, Liver; Polyisoprenyl Phosphates; Pyrophosphatases; Rats; Sesquiterpenes; Substrate Specificity; Tricarboxylic Acids