Depletion of substrates for protein prenylation increases apoptosis in human periovulatory granulosa cells

Progesterone receptor (PR) stimulation promotes survival in human and rat periovulatory granulosa cells. PR antagonists, Org 31710 and RU 486, both increase apoptosis and decrease cholesterol synthesis in these cells. The decrease in cholesterol synthesis also causes decreased synthesis of other products branching from the cholesterol synthesis pathway, including substrates for protein prenylation. In this study we focus on the link between apoptosis and prenylation in human periovulatory granulosa cells. A decreased cholesterol synthesis and increased apoptosis was verified in experiments with human periovulatory granulosa cells treated with the PR antagonists Org 31710 or RU 486 by measuring caspase-3/7 activity and incorporation of 14C-acetate into cholesterol and progesterone. Correspondingly, specific inhibition of cholesterol synthesis in periovulatory human granulosa cells using HMG-CoA reductase inhibitors (lovastatin or simvastatin) increased apoptosis, measured as caspase-3/7 activity. The increase in apoptosis caused by simvastatin or Org 31710 was partially reversed by addition of the protein prenylation precursors farnesol or geranylgeraniol. In addition, the prenylation inhibitors FTI R115777 and GGTI 2147 increased apoptosis in these cells. In conclusion our data suggest that PR antagonists increase apoptosis and reduce cholesterol synthesis in periovulatory granulosa cells and that the resulting depletion of substrates for protein prenylation may contribute to the increased apoptosis sensitivity.     

血红素氧合酶-1/一氧化碳通路参与辛伐他汀抗高血压诱发的大鼠心肌肥厚

为了探讨他汀类药物抑制心肌肥厚的作用机制,本研究应用一氧化氮合酶抑制剂左旋硝基精氨酸[N-nitro-L-arginine, L-NNA,15 mg／(kg·d)]制备大鼠高血压心肌肥厚模型,并分别给予不同剂量辛伐他汀[5或30 mg／(kg·d)进行干预。6周后测大鼠左心室功能、左心室重量指数(left ventricular mass index,LVMI)、心肌脑钠素(brain natriuretic peptide,BNP)含量、心肌羟脯氨酸含量和心肌血红素氧合酶(heme oxygenase,HO)活性。在体外培养的新生大鼠心肌细胞中,观察辛伐他汀对血管紧张素Ⅱ(angiotensin Ⅱ,Ang Ⅱ)引起的心肌细胞肥大的抑制作用与细胞血红素氧合酶-1(HO-1)表达、HO活性及CO生成间的关系。结果表明,辛伐他汀干预明显减轻L-NNA处理大鼠的心肌肥厚(LVMI值、心肌BNP和羟脯氨酸含量均显著低于单纯L-NNA处理组),改善左心室舒张功能,而且心肌HO活性显著升高。在离体培养的原代乳鼠心肌细胞,辛伐他汀浓度依赖性地抑制Ang Ⅱ引起的细胞肥大(3H-亮氨酸掺入),并相应增加HO-1 mRNA表达、HO活性和CO生成量。应用HO抑制剂锌卟啉能有效抑制辛伐他汀抗Ang Ⅱ诱导的心肌肥大作用。结果提示:辛伐他汀上调HO-1／CO通路是其抗高血压诱发的心肌肥厚的机制之一。     

Critical role for integrin-β4 in the attenuation of murine acute lung injury by simvastatin

The statins are a class of 3-hydroxy-3-methylglutaryl-coenzyme A-reductase inhibitors that are recognized to have pleiotropic properties. We previously reported the attenuation of LPS-induced murine acute lung injury (ALI) by simvastatin in vivo and identified relevant effects of simvastatin on endothelial cell (EC) signaling, activation, and barrier function in vitro. In particular, simvastatin induces the upregulation of integrin-β4, which in turn inhibits EC inflammatory responses via attenuation of MAPK signaling. The role of integrin-β4 in murine ALI protection by simvastatin, however, is unknown. We initially confirmed a time- and dose-dependent effect of simvastatin on increased integrin-β4 mRNA expression in human lung EC with peak protein expression evident at 16 h. Subsequently, reciprocal immunoprecipitation demonstrated an attenuation of LPS-induced integrin-β4 tyrosine phosphorylation by simvastatin (5 μM, 16 h). Increased expression of EC inflammatory cytokines [IL-6, IL-8, monocyte chemoattractant protein (MCP)-1, regulated on activation normal T cell expressed and secreted (RANTES)] by LPS (500 ng/ml, 4 h) was also significantly attenuated by simvastatin pretreatment (5 μM, 16 h), but this effect was reversed by cotreatment with an integrin-β4-blocking antibody. Finally, although simvastatin (20 mg/kg) conferred significant protection in murine ALI as evidenced by decreased bronchoalveolar lavage fluid cell counts, protein, inflammatory cytokines (IL-6, IL-1β, MCP-1, RANTES), decreased Evans blue dye albumin extravasation in lung tissue, and changes on lung histology, these effects were reversed by the integrin-β4-blocking antibody (IV, 1 mg/kg, 2 h before LPS). These findings support integrin-β4 as an important mediator of ALI protection by simvastatin and implicate signaling by integrin-β4 as a novel therapeutic target in patients with ALI.     

Drosophila longevity is not affected by heterochromatin-mediated gene silencing

Two highly conserved histone deacetylases, Sir2 and Rpd3, have been linked to caloric restriction and the extension of longevity. Because the Drosophila forms of each protein can silence genes in either euchromatin or heterochromatin, we determined whether longevity extension is mediated by silencing in the latter domain. When silencing was increased and decreased using mutations that affect heterochromatin protein 1 (HP1), but have no direct effect upon Sir2 or Rpd3, lifespan was unaffected. Heterochromatin-mediated gene silencing was then modulated without directly influencing HP1 as well as the deacetylases, again yielding no effect on lifespan. Mortality rates were unchanged by all manipulations, indicating that euchromatic targets are likely to be the effectors of deacetylase-mediated longevity extension in Drosophila [corrected]     

External control of the Drosophila melanogaster lifespan by combination of 3D oscillating low-frequency electric and magnetic fields

We demonstrate that the lifespan of Drosophila melanogaster population is controllable by a combination of external three-dimensional oscillating low-frequency electric and magnetic fields (3D OLFEMFs). The lifespan was decreased or increased in dependence of the parameters of the external 3D OLFEMFs. We propose that metabolic processes in D. melanogaster’s body are either accelerated (in the case of reduced lifespan) or slowed down (in the case of increased lifespan) in function of 3D OLFEMFs that induce vibrational motions on sub-cellular and larger scales.     

Mevalonate cascade regulation of airway mesenchymal cell autophagy and apoptosis: a dual role for p53

Statins inhibit the proximal steps of cholesterol biosynthesis, and are linked to health benefits in various conditions, including cancer and lung disease. We have previously investigated apoptotic pathways triggered by statins in airway mesenchymal cells, and identified reduced prenylation of small GTPases as a primary effector mechanism leading to p53-mediated cell death. Here, we extend our studies of statin-induced cell death by assessing endpoints of both apoptosis and autophagy, and investigating their interplay and coincident regulation. Using primary cultured human airway smooth muscle (HASM) and human airway fibroblasts (HAF), autophagy, and autophagosome formation and flux were assessed by transmission electron microscopy, cytochemistry (lysosome number and co-localization with LC3) and immunoblotting (LC3 lipidation and Atg12-5 complex formation). Chemical inhibition of autophagy increased simvastatin-induced caspase activation and cell death. Similarly, Atg5 silencing with shRNA, thus preventing Atg5-12 complex formation, increased pro-apoptotic effects of simvastatin. Simvastatin concomitantly increased p53-dependent expression of p53 up-regulated modulator of apoptosis (PUMA), NOXA, and damage-regulated autophagy modulator (DRAM). Notably both mevalonate cascade inhibition-induced autophagy and apoptosis were p53 dependent: simvastatin increased nuclear p53 accumulation, and both cyclic pifithrin-α and p53 shRNAi partially inhibited NOXA, PUMA expression and caspase-3/7 cleavage (apoptosis) and DRAM expression, Atg5-12 complex formation, LC3 lipidation, and autophagosome formation (autophagy). Furthermore, the autophagy response is induced rapidly, significantly delaying apoptosis, suggesting the existence of a temporally coordinated p53 regulation network. These findings are relevant for the development of statin-based therapeutic approaches in obstructive airway disease.     

Simvastatin stimulates VEGF release via p44/p42 MAP kinase in vascular smooth muscle cells

It has been shown that 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors (statins) modulate vascular smooth muscle cell functions. In the present study, we investigated the effect of simvastatin on vascular endothelial growth factor (VEGF) release, and the underlying mechanism, in a rat aortic smooth muscle cell line, A10 cells. Administration of simvastatin increased the VEGF level in rat plasma in vivo. In cultured cells, simvastatin significantly stimulated VEGF release in a dose-dependent manner. Simvastatin induced the phosphorylation of p44/p42 MAP kinase but not p38 MAP kinase or SAPK (stress-activated protein kinase)/JNK (c-Jun N-terminal kinase). PD98059 and U-0126, inhibitors of the upstream kinase that activates p44/p42 MAP kinase, significantly reduced the simvastatin-induced VEGF release in a dose-dependent manner. The phosphorylation of p44/p42 MAP kinase induced by simvastatin was reduced by PD98059 or U-0126. Moreover, a bolus injection of PD98059 truly suppressed the simvastatin-increased VEGF level in rat plasma in vivo. These results strongly suggest that p44/p42 MAP kinase plays a role at least partly in the simvastatin-stimulated VEGF release in vascular smooth muscle cells.     

Inhibition of geranylgeranylation mediates sensitivity to CHOP-induced cell death of DLBCL cell lines

Prenylation is a post-translational hydrophobic modification of proteins, important for their membrane localization and biological function. The use of inhibitors of prenylation has proven to be a useful tool in the activation of apoptotic pathways in tumor cell lines. Rab geranylgeranyl transferase (Rab GGT) is responsible for the prenylation of the Rab family. Overexpression of Rab GGTbeta has been identified in CHOP refractory diffuse large B cell lymphoma (DLBCL). Using a cell line-based model for CHOP resistant DLBCL, we show that treatment with simvastatin, which inhibits protein farnesylation and geranylgeranylation, sensitizes DLBCL cells to cytotoxic treatment. Treatment with the farnesyl transferase inhibitor FTI-277 or the geranylgeranyl transferase I inhibitor GGTI-298 indicates that the reduction in cell viability was restricted to inhibition of geranylgeranylation. In addition, treatment with BMS1, a combined inhibitor of farnesyl transferase and Rab GGT, resulted in a high cytostatic effect in WSU-NHL cells, demonstrated by reduced cell viability and decreased proliferation. Co-treatment of BMS1 or GGTI-298 with CHOP showed synergistic effects with regard to markers of apoptosis. We propose that inhibition of protein geranylgeranylation together with conventional cytostatic therapy is a potential novel strategy for treating patients with CHOP refractory DLBCL.     

Sphingosylphosphorylcholine-induced contraction of feline ileal smooth muscle cells is mediated by Galphai3 protein and MAPK

We studied the mechanism of sphingosylphosphorylcholine (SPC)-induced contraction in feline ileal smooth muscle cells. Western blotting revealed that G protein subtypes of Galpha(i1), Galpha(i3) and Galpha(o) existed in feline ileum. Galpha(i3) antibody penetration into permeabilized cells decreased SPC-induced contraction. In addition, incubation of [35S]guanosine 5'-O-(3-thiotriphosphate) ([35S]GTPgammaS) with membrane fraction increased its binding to Galpha(i3) subtype after SPC treatment, suggesting that the signalling pathways invoked by SPC were mediated by Galpha(i3) protein. MAPK kinase (MEK) inhibitor PD98059 blocked the contraction significantly, but p38 mitogen-activated protein kinase (MAPK) inhibitor SB202190 did not. Chelerythrine and neomycin also inhibited the contraction. However, cotreatment of PD98059 and chelerythrine showed no significant difference. Phosphorylation of p44/42 MAPK was increased by SPC treatment, which was reversed by pretreatment of inhibitors of signalling molecules that decreased SPC-induced contraction previously. The same result was obtained in the assay of MAPK activity.     

Low-dose simvastatin improves survival and ventricular function via eNOS in congestive heart failure

3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors increase endothelial nitric oxide synthase (eNOS) activity by multiple mechanisms. We previously reported that genetic overexpression of eNOS improves survival and cardiac function in congestive heart failure (CHF). In the present study, we tested the hypothesis that low-dose treatment with an 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor exerts beneficial effects on survival and/or cardiac function in a murine model of CHF. Mice were subjected to permanent ligation of the left coronary artery and randomized to receive either saline vehicle or simvastatin (0.25 mg/kg) 2 h after myocardial infarction and daily (0.25 mg/kg) for 7 days, followed by 21 days of administration every other day for a total duration of 28 days. Myocardial infarct size was not reduced by simvastatin therapy (P = not significant between groups). Simvastatin treatment did significantly (P < 0.05) improve survival (45%) compared with vehicle treatment (25%). In addition, simvastatin treatment significantly improved (P < 0.01) left ventricular function and significantly (P < 0.01) abrogated cardiac hypertrophy and pulmonary edema compared with vehicle treatment. The protective effects of simvastatin were abrogated by delayed initiation of treatment or genetic ablation of eNOS. In conclusion, low-dose simvastatin therapy significantly improves survival and cardiac function and reduces both cardiac hypertrophy and pulmonary edema via an eNOS-dependent mechanism in a murine model of CHF.     

A mild impairment of mitochondrial electron transport has sex-specific effects on lifespan and aging in mice

Impairments of various aspects of mitochondrial function have been associated with increased lifespan in various model organisms ranging from Caenorhabditis elegans to mice. For example, disruption of the function of the 'Rieske' iron-sulfur protein (RISP) of complex III of the mitochondrial electron transport chain can result in increased lifespan in the nematode worm C. elegans. However, the mechanisms by which impaired mitochondrial function affects aging remain under investigation, including whether or not they require decreased electron transport. We have generated knock-in mice with a loss-of-function Risp mutation that is homozygous lethal. However, heterozygotes (Risp(+/P224S)) were viable and had decreased levels of RISP protein and complex III enzymatic activity. This decrease was sufficient to impair mitochondrial respiration and to decrease overall metabolic rate in males, but not females. These defects did not appear to exert an overtly deleterious effect on the health of the mutants, since young Risp(+/P224S) mice are outwardly normal, with unaffected performance and fertility. Furthermore, biomarkers of oxidative stress were unaffected in both young and aged animals. Despite this, the average lifespan of male Risp(+/P224S) mice was shortened and aged Risp(+/P224S) males showed signs of more rapidly deteriorating health. In spite of these differences, analysis of Gompertz mortality parameters showed that Risp heterozygosity decreased the rate of increase of mortality with age and increased the intrinsic vulnerability to death in both sexes. However, the intrinsic vulnerability was increased more dramatically in males, which resulted in their shortened lifespan. For females, the slower acceleration of age-dependent mortality results in significantly increased survival of Risp(+/P224S) mice in the second half of lifespan. These results demonstrate that even relatively small perturbations of the mitochondrial electron transport chain can have significant physiological effects in mammals, and that the severity of those effects can be sex-dependent.     

Abrogation of insulin-like growth factor-I (IGF-I) and insulin action by mevalonic acid depletion: synergy between protein prenylation and receptor glycosylation pathways

The vasculoprotective effects of hydroxy-3-methylglutaryl (HMG)-CoA reductase inhibitors (statins) correlate with cholesterol lowering. HMG-CoA reductase inhibitors also disrupt cellular processes by the depletion of isoprenoids and dolichol. Insulin and insulin-like growth factor (IGF) signaling appear particularly prone to such disruption as intracellular receptor processing requires dolichol for correct N-glycosylation, whereas downstream signaling through Ras requires the appropriate prenylation (farnesol). We determined how HMG-CoA reductase inhibition affected the mitogenic effects of IGF-I and metabolic actions of insulin in 3T3-L1 cells and examined the respective roles of receptor glycosylation and Ras prenylation. IGF-I- and insulin-induced proliferation was significantly reduced by all statins tested, although cerivastatin (10 nm) had the greatest effect (p < 0.005). Although inhibitors of Ras prenylation induced similar results (10 microm FTI-277 89% +/- 7.4%, p < 0.01), the effect of HMG-CoA reductase inhibition could only be partially reversed by farnesyl pyrophosphate refeeding. Treatment with statins resulted in decreased membrane expression of receptors and accumulation of proreceptors, suggesting disruption of glycosylation-dependent cleavage. Glycosylation inhibitors inhibited IGF-I-induced proliferation (tunicamycin p < 0.005, castanospermine p < 0.01, deoxymannojirimycin p < 0.01). High concentrations of statin were necessary to impair insulin-mediated glucose uptake (300 nm = 33% +/- 12% p < 0.05), and this process was not effected by farnesyl transferase inhibition. Gycosylation inhibitors mimicked the effect of statin treatment (tunicamycin p < 0.001, castanospermine p < 0.05, deoxymannojirimycin p < 0.05), and there was insulin proreceptor accumulation. These data imply that HMG-CoA reductase inhibitors disrupt IGF-I signaling by combined effects on Ras prenylation and IGF receptor glycosylation, whereas insulin signaling is only affected by disrupted receptor glycosylation.     

Drosophila sbo regulates lifespan through its function in the synthesis of coenzyme Q in vivo

CoQ is an essential electron carrier in the mitochondrial respiratory chain of both eukaryotes and prokaryotes. It consists of a benzoquinone head group and a hydrophobic polyisoprenoid tail. The genes (COQ1-9) involved in CoQ biosynthesis have been characterized in yeast. In this study, we generated and molecularly characterized a mutant allele of a novel Drosophila gene, sbo, which encodes a protein that is predicted to catalyze the prenylation of p-hydroxybenzoate with the isoprenoid chain during the process of CoQ synthesis. Expression of sbo in yeast rescues the lethality of ?COQ2 mutant cells, indicating that sbo is a functional homolog of COQ2. HPLC results show that the levels of CoQ(9) and CoQ(10) were significantly reduced in sbo heterozygous adult flies. Furthermore, the mean lifespans of males and females heterozygous for sbo are extended by 12.5% and 30.8%, respectively. Homozygous sbo animals exhibit reduced activities of the insulin/insulin-like growth factor signaling (IIS) pathway. Taken together, we conclude that sbo is an essential gene for Drosophila development, mutation of which leads to an extension of lifespan most likely by altering endogenous CoQ biosynthesis.     

Simvastatin enhances induction of inducible nitric oxide synthase in 3T3-L1 adipocytes

The present study was designed to determine whether hydroxymethylglutaryl-CoA reductase inhibitors (statins) modulate the NO production via iNOS in adipocytes stimulated by lipopolysaccharide (L) and tumour necrosis factor-alpha (T). Well-differentiated 3T3-L1 adipocytes significantly produced NO by LT-treatment. Pre-incubation with simvastatin, a lipophilic statin, pravastatin, a hydrophilic one, or Y27632, an inhibitor of Rho kinase, further enhanced the production of NO. The effect of simvastatin was offset by mevalonate and geranylgeranyl pyrophosphate (GGPP) but not by squalene. The mRNA level for iNOS parallelled the NO production. The NF-kappaB was activated by the LT-treatment and was further enhanced by simvastatin, pravastatin or Y27632 addition. Mevalonate and GGPP completely offset the effect of simvastatin. Statins and Y27632 also further increased the interleukin-6 secretion in the LT-treated 3T3-L1 adipocytes. These results suggest that statins, especially lipophilic type, enhance induction of iNOS by inhibiting the small GTP-binding protein signal in adipocytes.     

Isoprenoids and Related Pharmacological Interventions: Potential Application in Alzheimer’s Disease

Two major isoprenoids, farnesyl pyrophosphate and geranylgeranyl pyrophosphate, serve as lipid donors for the posttranslational modification (known as prenylation) of proteins that possess a characteristic C-terminal motif. The prenylation reaction is catalyzed by prenyltransferases. The lipid prenyl group facilitates to anchor the proteins in cell membranes and mediates protein-protein interactions. A variety of important intracellular proteins undergo prenylation, including almost all members of small GTPase superfamilies as well as heterotrimeric G protein subunits and nuclear lamins. These prenylated proteins are involved in regulating a wide range of cellular processes and functions, such as cell growth, differentiation, cytoskeletal organization, and vesicle trafficking. Prenylated proteins are also implicated in the pathogenesis of different types of diseases. Consequently, isoprenoids and/or prenyltransferases have emerged as attractive therapeutic targets for combating various disorders. This review attempts to summarize the pharmacological agents currently available or under development that control isoprenoid availability and/or the process of prenylation, mainly focusing on statins, bisphosphonates, and prenyltransferase inhibitors. Whereas statins and bisphosphonates deplete the production of isoprenoids by inhibiting the activity of upstream enzymes, prenyltransferase inhibitors directly block the prenylation of proteins. As the importance of isoprenoids and prenylated proteins in health and disease continues to emerge, the therapeutic potential of these pharmacological agents has expanded across multiple disciplines. This review mainly discusses their potential application in Alzheimer's disease.     

Simvastatin enhances aquaporin-2 surface expression and urinary concentration in vasopressin-deficient Brattleboro rats through modulation of Rho GTPase

Statins are 3-hydroxyl-3-methyglutaryl-CoA reductase inhibitors that are commonly used to inhibit cholesterol biosynthesis. Emerging data have suggested that they also have "pleotropic effects," including modulating actin cytoskeleton reorganization. Here, we report an effect of simvastatin on the trafficking of aquaporin-2 (AQP2). Specifically, simvastatin induced the membrane accumulation of AQP2 in cell cultures and kidneys in situ. The effect of simvastatin was independent of protein kinase A activation and phosphorylation at AQP2-Ser(256), a critical event involved in vasopressin (VP)-regulated AQP2 trafficking. Further investigation showed that simvastatin inhibited endocytosis in parallel with downregulation of RhoA activity. Overexpression of active RhoA attenuated simvastatin's effect, suggesting the involvement of this small GTPase in simvastatin-mediated AQP2 trafficking. Finally, the effect of simvastatin on urinary concentration was investigated in VP-deficient Brattleboro rats. Simvastatin acutely (3-6 h) increased urinary concentration and decreased urine output in these animals. In summary, simvastatin regulates AQP2 trafficking in vitro and urinary concentration in vivo via events involving downregulation of Rho GTPase activity and inhibition of endocytosis. Our study provides an alternative mechanism to regulate AQP2 trafficking, bypassing the VP-vasopressin receptor signaling pathway.     

Unprenylated RhoA Contributes to IL-1β Hypersecretion in Mevalonate Kinase Deficiency Model through Stimulation of Rac1 Activity

Protein prenylation is a post-translational modification whereby non-sterol isoprenoid lipid chains are added, thereby modifying the molecular partners with which proteins interact. The autoinflammatory disease mevalonate kinase deficiency (MKD) is characterized by a severe reduction in protein prenylation. A major class of proteins that are affected are small GTPases, including Rac1 and RhoA. It is not clear how protein prenylation of small GTPases relates to GTP hydrolysis activity and downstream signaling. Here, we investigated the contribution of RhoA prenylation to the biochemical pathways that underlie MKD-associated IL-1β hypersecretion using human cell cultures, Rac1 and RhoA protein variants, and pharmacological inhibitors. We found that when unprenylated, the GTP-bound levels of RhoA decrease, causing a reduction in GTPase activity and increased protein kinase B (PKB) phosphorylation. Cells expressing unprenylated RhoA produce increased levels of interleukin 1β mRNA. Of other phenotypic cellular changes seen in MKD, increased mitochondrial potential and mitochondrial elongation, only mitochondrial elongation was observed. Finally, we show that pharmacological inactivation of RhoA boosts Rac1 activity, a small GTPase whose activity was earlier implied in MKD pathogenesis. Together, our data show that RhoA plays a pivotal role in MKD pathogenesis through Rac1/PKB signaling toward interleukin 1β production and elucidate the effects of protein prenylation in monocytes.     

Simvastatin enhances induction of inducible nitric oxide synthase in 3T3-L1 adipocytes

The present study was designed to determine whether hydroxymethylglutaryl-CoA reductase inhibitors (statins) modulate the NO production via iNOS in adipocytes stimulated by lipopolysaccharide (L) and tumour necrosis factor-α (T). Well-differentiated 3T3-L1 adipocytes significantly produced NO by LT-treatment. Pre-incubation with simvastatin, a lipophilic statin, pravastatin, a hydrophilic one, or Y27632, an inhibitor of Rho kinase, further enhanced the production of NO. The effect of simvastatin was offset by mevalonate and geranylgeranyl pyrophosphate (GGPP) but not by squalene. The mRNA level for iNOS parallelled the NO production. The NF-κB was activated by the LT-treatment and was further enhanced by simvastatin, pravastatin or Y27632 addition. Mevalonate and GGPP completely offset the effect of simvastatin. Statins and Y27632 also further increased the interleukin-6 secretion in the LT-treated 3T3-L1 adipocytes. These results suggest that statins, especially lipophilic type, enhance induction of iNOS by inhibiting the small GTP-binding protein signal in adipocytes.