Potent HMG-CoA Reductase Inhibitor Research Articles

In silico approach for identification of potential tetracyclic triterpenoids from mushroom as HMG-CoA reductase inhibitor

Cardiovascular disease is estimated to be responsible for one-third of all global deaths annually. It occurs mostly due to hyperlipidemia, a condition where excessive cholesterol deposits in blood vessels. A favorable target for treating hyperlipidemia involves the crucial role of inhibition of a specific enzyme known as 3-Hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA reductase). The primary goal of this present study is to identify potential HMG-CoA reductase inhibitors containing tetracyclic triterpene nucleus derived from mushrooms. A library of 86 myco-constituents bearing a tetracyclic triterpene scaffold was prepared and screened to identify potential HMG-CoA reductase inhibitors targeting proteins 1HW8 and 1HW9. For this purpose, molecular docking, ADME prediction, and molecular dynamics (MD) simulation studies were performed on this in-house prepared database. The virtual screening results exhibited M_02(c) as the best hit with promising SP Glide scores compared to standard statin drugs. In order to assess the stability and interactions, a 100 ns MD simulation was performed. Further, M_02(c) was also analysed for MMGBSA binding energy to access and validate the thermodynamic stability of the protein-ligand complex. The results of this study revealed that M_02(c) is a promising hit molecule and may emerge as a potent HMG-CoA reductase inhibitor in preventing and treating hyperlipidemia.

In vitro and in silico studies of the potential cytotoxic, antioxidant, and HMG CoA reductase inhibitory effects of chitin from Indonesia mangrove crab (Scylla serrata) shells

This study aimed to characterize chitin extracted from Indonesia mangrove crab (Scylla serrata) shells, as well as to assess its in vitro cytotoxic, antioxidant, and HMG CoA reductase inhibitory potentials. In silico molecular docking, molecular dynamic, and ADMET prediction analyses were also carried out. Chitin was extracted from mangrove crab shells using deproteination and demineralization processes, Scanning Electron Microscopy (SEM) and Fourier Transform Infrared (FTIR) characterization are then performed. The MTT method was further tested in a study of cell viability, while in vitro method was used to assess HMG CoA reductase inhibitory and antioxidant activities. The extracted chitin was found to have a moderate level of cytotoxic and antioxidant activities. In vitro studies showed that it has an IC50 of 36,65 ± 0,082 μg/mL as an HMG CoA reductase inhibitor, and decreased enzyme activity by 68.733 % at 100 μg/mL as a concentration. Furthermore, in the in silico study, chitin showed a strong affinity to several targets, including HMG CoA reductase, HMG synthase, LDL receptor, PPAR-alfa, and HCAR-2 with binding energies of −5.7; −5.8; −3.6; −5.6; −4.6 kcal/mol, respectively. Based on the ADMET properties, it had non-toxic molecules, which were absorbed and distributed across the blood-brain barrier. The molecular dynamics (MD) simulation also showed that it remained stable in the active sites of HMG CoA reductase receptor for 100 ns. These results indicated that chitin from Indonesian mangrove crab shells can be used to develop more potent HMG CoA reductase inhibitor with antioxidant and cytotoxic activities for effective dyslipidemia therapy.

Interplay of Cholesterol and Actin in Neurotransmitter GPCR Signaling: Insights from Chronic Cholesterol Depletion Using Statin.

Serotonin1A receptors are important neurotransmitter receptors in the G protein-coupled receptor (GPCR) family and modulate a variety of neurological, behavioral, and cognitive functions. We recently showed that chronic cholesterol depletion by statins, potent inhibitors of HMG-CoA reductase (the rate-limiting enzyme in cholesterol biosynthesis), leads to polymerization of the actin cytoskeleton that alters lateral diffusion of serotonin1A receptors. However, cellular signaling by the serotonin1A receptor under chronic cholesterol depletion remains unexplored. In this work, we explored signaling by the serotonin1A receptor under statin-treated condition. We show that cAMP signaling by the receptor is reduced upon lovastatin treatment due to reduction in cholesterol as well as polymerization of the actin cytoskeleton. To the best of our knowledge, these results constitute the first report describing the effect of chronic cholesterol depletion on the signaling of a G protein-coupled neuronal receptor. An important message arising from these results is that it is prudent to include the contribution of actin polymerization while analyzing changes in membrane protein function due to chronic cholesterol depletion by statins. Notably, our results show that whereas actin polymerization acts as a negative regulator of cAMP signaling, cholesterol could act as a positive modulator. These results assume significance in view of reports highlighting symptoms of anxiety and depression in humans upon statin administration and the role of serotonin1A receptors in anxiety and depression. Overall, these results reveal a novel role of actin polymerization induced by chronic cholesterol depletion in modulating GPCR signaling, which could act as a potential therapeutic target.

Simvastatin: In Vitro Metabolic Profiling of a Potent Competitive HMG-CoA Reductase Inhibitor

Simvastatin (SV) is a semisynthetic derivative of lovastatin (LV), which is biosynthetically produced from the fungus Aspergillus terreus and has a high log p value (log p = 4.39)and thus high hepatic extraction and high efficacy in controlling cholesterol synthesis. The current study was undertaken to investigate the metabolic profile of SV using various mass spectrometry (MS) platforms. Metabolic profiling was studied in in vitro models, rat liver microsomes (RLMs), and isolated perfused rat liver hepatocytes (RLHs) using both ion trap and triple quadruple LC–MS/MS systems. A total of 29 metabolites were identified. Among them, three types of SV-related phase-I metabolites, namely exomethylene simvastatin acid (exomethylene SVA), monohydroxy SVA, and dihydrodiol SVA, were identified as new in RLMs. No phase-II metabolites were identified while incubating with RLHs.

Pharmacokinetic and pharmacodynamic interaction of Rosuvastatin calcium with guggulipid extract in rats.

Background & objectivesRosuvastatin calcium (RC) is a potent and competitive synthetic inhibitor of HMG-CoA reductase used for the treatment of dyslipidemia. Guggulipid obtained from Commiphora mukul is used in the treatment of a wide variety of diseases such as atherosclerosis, hypercholesterolemia, rheumatism, and obesity. The present study evaluates the pharmacokinetic and pharmacodynamic interactions between RC and the standardized guggulipid extract in rats.Materials and methodsThe guggulipid extract was standardized for the presence of guggulsterones. The pharmacokinetic interaction was determined after a single dose administration of RC alone or in combination with the guggulipid extract or after multiple-dose administration of RC alone or RC along with the guggulipid extract for 14 days. To determine the pharmacodynamic interaction, RC and guggulipid extract were administered to hyperlipidemic rats for 14 days. The level of significance was determined using unpaired student’s t-test, one way ANOVA, the post-ANOVA Tukey test.ResultsStandardization of guggulipid extract showed it contains 7.5%w/w of guggulsterones. Guggulipid extract increased the bioavailability of RC in both single-dose and multiple-dose studies. Guggulipid extract reduced the rate of absorption (Ka) of RC but showed an increase in maximum serum concentration (Cmax). An in-vitro study using isolated rat intestine revealed that guggulipid extract decreased the rate of absorption of RC in the intestinal lumen. The hypolipidemic activity of RC was augmented by the guggulipid extract in hyperlipidemic rats.Interpretation & conclusionTherefore it is concluded that guggulipid extract increases the bioavailability of RC by delaying its Ka and augments its hypolipidemic action. However, it is recommended that a combination of RC with guggulipid extract should be used only after an adverse effect(s) of this combination are determined.

In vitro 3-Hydroxy-3-methylglutaryl-coenzyme: A reductase inhibition assay of triphala ayurvedic formulation

Background: Triphala, the Ayurvedic herbal formulation composed of Terminalia chebula Retz. (Combretaceae), Terminalia bellirica Roxb. (Combretaceae), and Phyllanthus emblica L. (Euphorbiaceae) fruits. It has been reported the cholesterol-lowering effect that the reduction of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase activity was proposed as a key mechanism of action. Since, triphala formulations in equal proportion (1:1:1) and different ratios of its three fruit constituents (vata, pitta, and kapha) have been prescribed by the traditional practitioners due to the patient's body conditions. The biological activities of each formulation are needed to evaluate. Objectives: The objective of the study was to investigate phytochemicals, HMG-CoA reductase inhibitory effect, and HMG-CoA reductase molecular modeling of triphala extracts. Materials and Methods: Four triphala extracts were prepared by decoction and determined the contents of gallic acid, ellagic acid, chebulagic acid, and chebulinic acid as markers using high-pressure liquid chromatography analysis. The in vitro HMG CoA reductase assay was performed based on ultraviolet spectrophotometry, and molecular modeling was simulated using Autodock 1.5.6 to characterize the binding energy, ligand efficacy, and H-bond interaction. Results: All extracts contained gallic acid and chebulagic acid in the high contents, whereas ellagic acid and chebulinic acid were found in a small amount. The enzyme assay revealed pitta extract (at 10 μg/mL) was the most potent enzyme inhibition of 58.4' ± 0.40' (P = 0.05). Moreover, the modeling results indicated that these four markers can interact the enzyme with different configurations and binding affinities. Conclusion: Pitta extract appeared to be a potent HMG-CoA reductase inhibitor. It was a potential natural product as an alternative treatment for hypercholesterolemia.

Rosuvastatin improves myocardial and neurological outcomes after asphyxial cardiac arrest and cardiopulmonary resuscitation in rats.

Rosuvastatin, a potent HMG-CoA reductase inhibitor, is cholesterol-lowering drugs and reduce the risk of myocardial infarction and stroke. This study is to explore whether rosuvastatin improves outcomes after cardiac arrest in rats. Male Sprague-Dawley rats were subjected to 8min of cardiac arrest (CA) by asphyxia and randomly assigned to three experimental groups immediately following successful resuscitation: Sham; Control; and Rosuvastatin. The survival, hemodynamics, myocardial function, neurological outcomes and apoptosis were assessed. The 7-d survival rate was greater in the rosuvastatin treated group compared to the Control group (P=0.019 by log-rank test). Myocardial function, as measured by cardiac output and ejection fraction, was significantly impaired after CA and notably improved in the animals treated with rosuvastatin beginning at 60min after return of spontaneous circulation (ROSC) (P<0.05). Moreover, rosuvastatin treatment significantly ameliorated brain injury after ROSC, which was characterized by the increase of neurological function scores, and reduction of brain edema in cortex and hippocampus (P<0.05). Meanwhile, the levels of cardiac troponin T and neuron-specific enolase and the caspase-3 activity were significantly decreased in the Rosuvastatin group when compared with the Control group (P<0.05). In conclusion, rosuvastatin treatment substantially improves the 7-d survival rate as well as myocardial function and neurological outcomes after ROSC.

Simvastatin Treatment Upregulates Anti-Fibrotic Bone Morphogenetic Protein-7 Expression at Rat Cardiac Allograft Rejection

Background: Bone morphogenetic protein (BMP)-7 mediates ischemic tolerance and anti-fibrotic effects in various organs such as kidney and heart. Recently, reno- and podocyte-protective effects of a potent HMG-CoA reductase inhibitor, pitavastatin, were accompanied by BMP-7 upregulation. Methods: Here, we investigated the effect of simvastatin treatment on BMP-7 expression in major MHC-mismatched rat cardiac allografts subjected to ischemia-reperfusion injury and adaptive immune activation at 10 days. Results: We localized Smad2 activity and Reca-1⁺ fibroblast specific protein-1⁺ immunoreactivity, suggesting endothelial-to-mesenchymal transition, at fibrotic borderline of cardiac allografts at 10 days. Simvastatin donor and recipient combination treatment significantly upregulated cardiac allograft BMP-7 expression when compared to nontreated controls at 10 days. Conclusion: The beneficial effect of statin treatment on cardiac allograft may in part be mediated through the upregulation of BMP-7.

Molecular rationale delineating the role of lycopene as a potent HMG-CoA reductase inhibitor: in vitro and in silico study

This study initially aimed to depict the molecular rationale evolving the role of lycopene in inhibiting the enzymatic activity of β-hydroxy-β-methylglutaryl-CoA (HMG-CoA) reductase via in vitro and in silico analysis. Our results illustrated that lycopene exhibited strong HMG-CoA reductase inhibitory activity (IC50 value of 36 ng/ml) quite better than pravastatin (IC50 = 42 ng/ml) and strong DPPH free radical scavenging activity (IC50 value = 4.57 ± 0.23 μg/ml) as compared to ascorbic acid (IC50 value = 9.82 ± 0.42 μg/ml). Moreover, the Ki value of lycopene (36 ng/ml) depicted via Dixon plot was well concurred with an IC50 value of 36 ± 1.8 ng/ml. Moreover, molecular informatics study showed that lycopene exhibited binding energy of −5.62 kcal/mol indicating high affinity for HMG-CoA reductase than HMG-CoA (ΔG: −5.34 kcal/mol). Thus, in silico data clearly demonstrate and support the in vitro results that lycopene competitively inhibit HMG-CoA reductase activity by binding at the hydrophobic portion of HMG-CoA reductase.

Bioactivity guided fractionation and hypolipidemic property of a novel HMG-CoA reductase inhibitor from Ficus virens Ait.

BackgroundThe current perspective for the search of 3-hydroxy-3-methyl-glutaryl-coenzyme A (HMG-CoA) reductase inhibitor has been shifted towards a natural agent also having antioxidant property. Thus, this study was intended to isolate and identify the bioactive compounds from methanolic extract of Ficus virens bark (FVBM) and to evaluate their antioxidant, HMG-CoA reductase inhibitory and hypolipidemic activity.MethodsBioactivity guided fractionation and isolation of bioactive compound from FVBM extract has been done to isolate and characterize the potent HMG-CoA reductase (HMGR) inhibitor with antioxidant activity by using repeated extensive column chromatography followed by spectroscopic methods, including Infrared (IR), 1H & 13C nuclear magnetic resonance (NMR) and Mass spectrum analysis. The in vitro HMGR inhibition and enzyme kinetic assay was determined using HMG-CoA as substrate. In addition, antioxidant activity of the new isolated compound, was measured using 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assay and FRAP value. In-silico molecular informatics of HMGR enzyme type inhibition and pharmacokinetics data of the new compound was further evaluated through molecular docking and ADME-T studies. Further, in-vivo hypolipidemic property of FVBM extract and newly isolated compound was also analyzed in triton-WR 1339 induced rats.ResultsThereby, we report the discovery of n-Octadecanyl-O-α-D-glucopyranosyl(6′ → 1″)-O-α-D-glucopyranoside (F18) as a novel HMG-CoA reductase inhibitor with strong antioxidant property. This inhibitor exhibited not only higher free radical scavenging activity but also marked HMG-CoA reductase inhibitory activity with an IC50 value of 84 ± 2.8 ng/ml. This inhibitory activity concurred with kinetic study that showed inhibition constant (Ki) of 84 ng/ml via an uncompetitive mode of inhibition. The inhibition was also corroborated by molecular docking analysis and in silico pharmacokinetics data. The in vivo study revealed that administration of FVBM extract (at higher dose, 100 mg/rat) and the inhibitor (1 mg/rat) to Triton WR-1339-induced hyperlipidemic rats significantly ameliorated the altered levels of plasma lipids and lipoproteins including hepatic HMG-CoA reductase activity; this effect was comparable to the effect of standard drug atorvastatin.ConclusionsThe in vitro, in silico and in vivo results clearly demonstrated the antioxidant potential and therapeutic efficacy of the inhibitor as an alternate drug against hyperlipidemia.Electronic supplementary materialThe online version of this article (doi:10.1186/s12944-015-0013-6) contains supplementary material, which is available to authorized users.

Is it Simvastatin harmful in children? A case report

Non-alcoholic fatty liver disease (NAFLD) is the most common liver disease in children living in Western countries. Hyperlipidemia, obesity and insulin resistance are common components of the metabolic syndrome, which is frequently associated with NAFLD. Since patients with NAFLD are at high risk to develop cardiovascular disease (CVD), statins are frequently prescribed to patients with NAFLD and hyperlipidemia. The 3-Hydroxy-3-methyl-coenzyme A reductase (HMG-CoA reductase) is the rate limiting enzyme in cholesterol biosynthesis. Simvastatin is a lactone that is readily hydrolyzed in vivo to the corresponding ?-hydroxyacid, a potent inhibitor of HMG-CoA reductase. Under treatment with simvastatin, an improvement of enzymatic antioxidant parameters has been described in subjects with hypercholesterolemia. The safety and effectivity of statins in pediatric patients with NAFLD or non-alcoholic steatohepatitis (NASH), and their effect on hepatic fat infiltration or the extent of hepatic fibrosis are not known. Also, no evidences of the effects of a non therapeutic ingestion of this drug on the glutathione homeostasis and in children have been reported. We describe the case of a obese 4-year-old girl in whom an accidental overdose of simvastatin led to decrease levels of glutathione in blood with increase of the GSSG/GSH ratio. No adverse reactions were registered. All laboratory test were normal during the follow up. Only a 35% decrease of Glutathione was observed such as a possible mechanism of mithocondrial toxicity and depletion of the glutathione pool after the intake of excessive dose of HMG-CoA reductase inhibitors. Further RCTs are needed in order to establish the safety and efficacy to use of statin for pediatric NAFLD or NASH.

Erythrocyte membrane cholesterol and lipid core growth in a rabbit model of atherosclerosis: modulatory effects of rosuvastatin

Abstract Background Lipid core expansion is partly responsible for the conversion of a stable atherosclerotic lesion to a rupture-prone plaque. Intraplaque hemorrhage contributes to the accumulation of cholesterol within unstable plaques. In the present study, we investigated, using a rabbit model of atherosclerosis, the extent to which diet-induced increases in cholesterol content of erythrocyte membranes (CEM) contribute to lipid core expansion and the modulatory effect of rosuvastatin use. Methods and results Rabbits fed with atherogenic diet (0.75% cholesterol) for 5months exhibited advanced atherosclerotic lesions (mean plaque area, 0.39±0.03mm 2 ), and lipid core size was associated with the concentration–time integral (CTI) of CEM levels ( r =0.567, P =0.004) independent of other established predictors of lipid core size. Further experiments were performed by feeding rabbits atherogenic diet (1% cholesterol) for 3months, followed by either normal diet or normal diet plus rosuvastatin for the next 3months. Although no differences were observed in total plaque area between both groups, administration of rosuvastatin was associated with significantly smaller lipid cores, fewer macrophages within the lipid core, less microvessels as well as with lower CTI of CEM levels compared to normal diet alone. Moreover, intraplaque erythrocyte membranes covered a smaller lipid core area in rabbits under rosuvastatin plus normal diet as opposed to rabbits under diet alone. Conclusions Increased CEM levels, induced by high-cholesterol diet, are associated with lipid core growth. Ingestion of a potent HMG-CoA reductase inhibitor (rosuvastatin) may decrease CEM levels, and this effect may contribute to regression of the lipid core.

Erythrocyte membrane cholesterol and lipid core growth in a rabbit model of atherosclerosis: Modulatory effects of rosuvastatin

BackgroundLipid core expansion is partly responsible for the conversion of a stable atherosclerotic lesion to a rupture-prone plaque. Intraplaque hemorrhage contributes to the accumulation of cholesterol within unstable plaques. In the present study, we investigated, using a rabbit model of atherosclerosis, the extent to which diet-induced increases in cholesterol content of erythrocyte membranes (CEM) contribute to lipid core expansion and the modulatory effect of rosuvastatin use. Methods and resultsRabbits fed with atherogenic diet (0.75% cholesterol) for 5months exhibited advanced atherosclerotic lesions (mean plaque area, 0.39±0.03mm2), and lipid core size was associated with the concentration–time integral (CTI) of CEM levels (r=0.567, P=0.004) independent of other established predictors of lipid core size. Further experiments were performed by feeding rabbits atherogenic diet (1% cholesterol) for 3months, followed by either normal diet or normal diet plus rosuvastatin for the next 3months. Although no differences were observed in total plaque area between both groups, administration of rosuvastatin was associated with significantly smaller lipid cores, fewer macrophages within the lipid core, less microvessels as well as with lower CTI of CEM levels compared to normal diet alone. Moreover, intraplaque erythrocyte membranes covered a smaller lipid core area in rabbits under rosuvastatin plus normal diet as opposed to rabbits under diet alone. ConclusionsIncreased CEM levels, induced by high-cholesterol diet, are associated with lipid core growth. Ingestion of a potent HMG-CoA reductase inhibitor (rosuvastatin) may decrease CEM levels, and this effect may contribute to regression of the lipid core.

A Reappraisal of the Risks and Benefits of Treating to Target with Cholesterol Lowering Drugs

Atherosclerotic cardiovascular disease (CVD) is the number one cause of death globally, and lipid modification, particularly lowering of low density lipoprotein cholesterol (LDLc), is one of the cornerstones of prevention and treatment. However, even after lowering of LDLc to conventional goals, a sizeable number of patients continue to suffer cardiovascular events. More aggressive lowering of LDLc and optimization of other lipid parameters like triglycerides (TG) and high density lipoprotein cholesterol (HDLc) have been proposed as two potential strategies to address this residual risk. These strategies entail use of maximal doses of highly potent HMG CoA reductase inhibitors (statins) and combination therapy with other lipid modifying agents. Though statins in general are fairly well tolerated, adverse events like myopathy are dose related. There are further risks with combination therapy. In this article, we review the adverse effects of lipid modifying agents used alone and in combination and weigh these effects against the evidence demonstrating their efficacy in reducing cardiovascular events, cardiovascular mortality, and all cause mortality. For patients with established CVD, statins are the only group of drugs that have shown consistent reductions in hard outcomes. Though more aggressive lipid lowering with high dose potent statins can reduce rates of non fatal events and need for interventions, the incremental mortality benefits remain unclear, and their use is associated with a higher rate of drug related adverse effects. Myopathy and renal events have been a significant concern with the use of high potency statin drugs, in particular simvastatin and rosuvastatin. For patients who have not reached target LDL levels or have residual lipid abnormalities on maximal doses of statins, the addition of other agents has not been shown to improve clinical outcomes and carries an increased risk of adverse events. The clinical benefits of drugs to raise HDLc remain unproven. In patients without known cardiovascular disease, there is conflicting evidence as to the benefits of aggressive pursuit of numerical lipid targets, particularly with respect to all cause mortality. Certainly, in statin intolerant patients, alternative agents with a low side effect profile are desirable. Bile acid sequestrants are an effective and safe choice for decreasing LDLc, and omega-3 fatty acids are safe agents to decrease TG. There remains an obvious need to design and carry out large scale studies to help determine which agents, when combined with statins, have the greatest benefit on cardiovascular disease with the least added risk. These studies should be designed to assess the impact on clinical outcomes rather than surrogate endpoints, and require a comprehensive assessment and reporting of safety outcomes.

Mechanism of AMPK suppression of LXR‐dependent Srebp‐1c transcription

Activation of AMP‐activated protein kinase (AMPK) inhibits hepatic fatty acid synthesis by suppressing sterol regulatory element‐binding protein (SREBP)‐1c via inhibition of endogenous liver X receptor (LXR) ligand production and SREBP‐1c processing. However, whether AMPK directly inhibits ligand‐induced LXR activity remained undetermined. In this study we used a series of mutant Srebp‐1c promoter linked to a luciferase reporter to determine the inhibitory mechanism in rat hepatoma McA‐RH7777 cells. AMPK activation by either AICAR or metformin decreases Srebp‐1c promoter activity by about 75%. When endogenous LXR ligand production was blocked by the potent HMG CoA reductase inhibitor compactin, T0901317‐induced Srebp‐1c promoter activity was decreased by AICAR or metformin treatment. In the mutant Srebp‐1c promoter in which two LXR elements are intact but the sterol regulatory element (SRE) is disrupted, the fold inductions of the promoter activity by T0901317 without AMPK activators are significantly higher than those with AMPK activators. Furthermore, AMPK activation attenuates induction of endogenous SREBP‐1c mRNA by T0901317. These results indicate that AMPK directly inhibits ligand‐induced LXR activity in addition to blocking production of endogenous LXR ligands.

Prenylation of Rho G-Proteins: a Novel Mechanism Regulating Gene Expression and Protein Stability in Human Trabecular Meshwork Cells

Endogenous prenylation with sesquiterpene or diterpene isoprenoids facilitates membrane localization and functional activation of small monomeric GTP-binding proteins. A direct effect of isoprenoids on regulation of gene expression and protein stability has also been proposed. In this study, we determined the role of sesquiterpene or diterpene isoprenoids on the regulation of Rho G-protein expression, activation, and stability in human trabecular meshwork (TM) cells. In both primary and transformed human TM cells, limiting endogenous isoprenoid synthesis with lovastatin, a potent HMG-CoA reductase inhibitor, elicited marked increases in RhoA and RhoB mRNA and protein content. The effect of lovastatin was dose-dependent with newly synthesized inactive protein accumulating in the cytosol. Supplementation with geranylgeranyl pyrophosphate (GGPP) prevented, while inhibition of geranylgeranyl transferase-I mimicked, the effects of lovastatin on RhoA and RhoB protein content. Similarly, lovastatin-dependent increases in RhoA and RhoB mRNA expression were mimicked by geranylgeranyl transferase-I inhibition. Interestingly, GGPP supplementation selectively promoted the degradation of newly synthesized Rho proteins which was mediated, in part, through the 20S proteasome. Functionally, GGPP supplementation prevented lovastatin-dependent decreases in actin stress fiber organization while selectively facilitating the subcellular redistribution of accumulated Rho proteins from the cytosol to the membrane and increasing RhoA activation. Post-translational prenylation with geranylgeranyl diterpenes selectively facilitates the expression, membrane translocation, functional activation, and turnover of newly synthesized Rho proteins. Geranylgeranyl prenylation represents a novel mechanism by which active Rho proteins are targeted to the 20S proteasome for degradation in human TM cells.

Study of the Effects of Monacolin K and Other Constituents of Red Yeast Rice on Obesity, Insulin-Resistance, Hyperlipidemia, and Nonalcoholic Steatohepatitis Using a Mouse Model of Metabolic Syndrome

Purpose. Nonalcoholic fatty liver disease (NAFLD) is a progressive and intractable disease associated with metabolic syndrome. Red yeast rice (RYR) contains monacolin K, a potent inhibitor of HMG-CoA reductase, and its consumption decreases cholesterol and triglyceride levels. We examined the efficacy of RYR constituents using a novel metabolic syndrome-NAFLD mouse model (MSG mice). Methods. Two types of RYR grown under different culture conditions were used. 1P-DU contained only 0.002 g/100 g of monacolin K, whereas 3P-D1 contained 0.131 g/100 g. MSG mice were divided into three groups: control (C) group fed standard food, RYR-C group fed standard food with 1% 1P-DU, and RYR-M group fed standard food with 1% 3P-D1. Mice were examined from 12 to 24 weeks of age. Results. Serum insulin, leptin, and liver damage as well as macrophage aggregation in visceral fat in RYR-C and RYR-M groups were lower than those in C group. The serum adiponectin levels in RYR-C group were significantly higher than those in RYR-M and C groups. Conclusions. RYR was effective against obesity-related inflammation, insulin resistance, and NAFLD in MSG mice irrespective of monacolin K levels. GABA and various peptides produced during fermentation were determined as the active constituents of RYR.

12/15-Lipoxygenase gene knockout severely impairs ischemia-induced angiogenesis due to lack of Rac1 farnesylation

AbstractTo understand the mechanisms by which 15(S)-hydroxyeicosatetraenoic acid (15(S)-HETE) activates Rac1 in the induction of angiogenesis, we studied the role of 3-hydroxy-3-methylglutaryl–coenzyme A (HMG-CoA) reductase and αPix. 15(S)-HETE stimulated Rac1 in a sustained manner in human dermal microvascular endothelial cells (HDMVECs). Simvastatin, a potent inhibitor of HMG-CoA reductase, suppressed 15(S)-HETE–induced Rac1 activation in HDMVECs affecting their migration and tube formation. 15(S)-HETE by inducing HMG-CoA reductase expression caused increased farnesylation and membrane translocation of Rac1 where it became activated by Src-dependent αPix stimulation. Mevalonate rescued 15(S)-HETE–induced Rac1 farnesylation and membrane translocation in HDMVECs and the migration and tube formation of these cells from inhibition by simvastatin. Down-regulation of αPix inhibited 15(S)-HETE–induced HDMVEC migration and tube formation. Hind-limb ischemia induced Rac1 farnesylation and activation leading to increased angiogenesis and these effects were blocked by simvastatin and rescued by mevalonate in WT mice. In contrast, hind-limb ischemia failed to induce Rac1 farnesylation and activation as well as angiogenic response in 12/15-Lox−/− mice. Activation of Src and αPix were also compromised at least to some extent in 12/15-Lox−/− mice compared with WT mice in response to hind-limb ischemia. Together, these findings demonstrate for the first time that HMG-CoA reductase plays a determinant role in 12/15-Lox–induced angiogenesis.

Drug-Drug Interaction Study to Assess the Effects of Multiple-Dose Pitavastatin on Steady-State Warfarin in Healthy Adult Volunteers

Warfarin, an antagonist of vitamin K, which inhibits clotting factor synthesis, is prescribed for thrombosis prophylaxis and treatment and is known to have a narrow therapeutic range. Pitavastatin is a potent HMG-CoA reductase inhibitor. In this study, the influence of multiple-dose pitavastatin (4 mg once daily) on steady-state warfarin pharmacodynamic and pharmacokinetic profiles was investigated in 24 healthy male participants whose international normalized ratio (INR) was maintained by individualized doses of warfarin. The ratio of the least squares mean of prothrombin time and INR was 0.989 (90% confidence interval [CI], 0.955-1.023) and 0.993 (0.956-1.209), respectively (test: warfarin + pitavastatin; reference: warfarin only). The geometric mean ratios of C(max) and AUC were 1.034 (90% CI, 0.994-1.075) and 1.066 (1.035-1.099), respectively, for R-warfarin and 1.033 (0.995-1.073) and 1.058 (1.026-1.092), respectively, for S-warfarin. Warfarin pharmacodynamic profiles and pharmacokinetic profiles did not differ between the warfarin monotherapy and the coadministration of pitavastatin and warfarin. No drug-drug interaction between pitavastatin and warfarin was demonstrated.

Mechanism of AMPK Suppression of LXR-dependent Srebp-1c Transcription

Activation of AMP-activated protein kinase (AMPK) inhibits hepatic fatty acid synthesis by suppressing sterol regulatory element-binding protein (SREBP)-1c, a master regulator of hepatic lipogenic gene expression. Using a model cell line rat hepatoma McA-RH7777 (CRL-1601) that mimics the behavior of the intact liver by producing high levels of SREPB-1c mRNA and protein, we previously showed that AMPK suppresses hepatic Srebp-1c transcription by inhibiting endogenous liver X receptor (LXR) ligand production and SREBP-1c processing. However, whether AMPK directly inhibits ligand-induced LXR activity remained undetermined. In this study we used a series of mutant Srebp-1c promoter linked to a luciferase reporter to determine the inhibitory mechanism in rat hepatoma McA-RH7777 cells. AMPK activation by either AICAR or metformin decreases Srebp-1c promoter activity by about 75%. Normally, the synthetic LXR ligand T0901317 compound increases the wild-type Srebp-1c promoter activity by about 3-fold, which is similar to that observed in the presence of AICAR or metformin. When endogenous LXR ligand production was blocked by the potent HMG CoA reductase inhibitor compactin, T0901317-induced Srebp-1c promoter activity was decreased by AICAR or metformin treatment. In the mutant Srebp-1c promoter in which two LXR elements are intact but the sterol regulatory element (SRE) is disrupted, the fold inductions of the promoter activity by T0901317 without AMPK activators are significantly higher than those with AMPK activators. Furthermore, AMPK activation attenuates induction of endogenous SREBP-1c mRNA by T0901317. These results indicate that AMPK directly inhibits ligand-induced LXR activity in addition to blocking production of endogenous LXR ligands.