Mitochondrial Oxidative Stress and Calcium-Dependent Permeability Transition are Key Players in the Mechanisms of Statins-Associated Side Effects

Statins are cholesterol-lowering medicines utilized worldwide and are associated with reduced risk of cardiovascular mortality and events. However, 0.5–10% of patients suffer from adverse effects especially on skeletal muscle. Recently, new onset of diabetes has been reported in subjects on statin therapy. Pro- and anti-oxidant effects of statins have been reported, thus fostering a debate. Previously reported data provide evidence that statins induce alterations in intracellular calcium homeostasis and mitochondrial dysfunctions that can be counteracted by antioxidants (e.g., CoQ10, creatine, and L-carnitine). Therefore, we have proposed that statin-induced inhibition of mitochondrial respiration leads to oxidative stress that opens a calcium-dependent permeability transition pore, an event that may lead to cell death. In addition, mitochondrial oxidative stress caused by statin treatment may be a signal for cellular antioxidant system responses such as catalase upregulation, possibly explaining the alleged statins’ antioxidant properties. Muscle mitochondrial dysfunction induced by statin treatment may be associated with the peripheral insulin resistance and may explain statins-induced new onset of diabetes. Together, the data presented in this review suggest that the statins’ detrimental effects can be prevented by co-administration of antioxidants

Effect of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibition by pravastatin on glucose homeostasis and beta cell function : studies in hypercholesterolemic mice and in vitro

Orientador: Helena Coutinho Franco de OliveiraTese (doutorado) - Universidade Estadual de Campinas, Instituto de BiologiaResumo: Em trabalhos anteriores publicados por nosso grupo, demonstramos que na hipercolesterolemia genética por deficiência do receptor de LDL ocorre prejuízo da secreção de insulina. Utilizando animais knockout para o receptor de LDL (LDLr-/-), modelo de hipercolesterolemia familiar, observamos que os LDLr-/- apresentaram uma secreção de insulina diminuída que pode ser revertida após remoção de colesterol das ilhotas ex vivo por metil-?-ciclodextrina. Desta maneira, levantamos a hipótese de que o tratamento in vivo com inibidores da síntese de colesterol (pravastatina) poderia normalizar a secreção de insulina pelas ilhotas pancreáticas dos camundongos LDLr-/-. Ao contrário do esperado, animais tratados por dois meses ou mais com pravastatina desenvolveram hiperglicemia e diminuição da secreção de insulina pelas ilhotas isoladas estimuladas por glicose. Estes resultados foram associados a redução na expressão de proteínas da maquinaria exocitótica (SNAREs), aumento de morte das células beta e aumento de peroxidação lipídica observado nas ilhotas dos animais LDLr-/- tratados com pravastatina. Estudos em cultura de células secretoras de insulina (INS1E) tratadas com pravastatina mostraram um aumento de morte celular e estresse oxidativo (indicado pela produção de H2O2). Como as estatinas inibem a HMG-CoA redutase, a síntese de outros importantes produtos intermediários da via da síntese de colesterol, como a coenzima Q10 (CoQ10, ou ubiquinona), são também inibidos. Assim sendo, testamos a hipótese de que os efeitos deletérios do tratamento com pravastatina são causados pela deficiência de CoQ10. Para tanto, os animais LDLr-/- receberam suplementação dietética com CoQ10 durante o tratamento com pravastatina, o que reverteu a hiperglicemia, melhorou a tolerância a glicose e corrigiu a redução da secreção de insulina estimulada por glicose. O tratamento com CoQ10 em células INS1E reduziu a morte celular e o estresse oxidativo induzidos por pravastatina. Após observar o prejuízo da função pancreática induzido por pravastatina, decidimos investigar a evolução temporal do tratamento e o possível desenvolvimento da diabetes. Camundongos tratados com pravastatina tornaram-se intolerantes a glicose e resistentes a insulina após 10 meses de tratamento. Experimentos em cultura com miotubos C2C12 tratados com pravastatina mostraram uma diminuição na sinalização de insulina, estresse oxidativo e morte celular por apoptose. Em conclusão, estes resultados mostraram que a inibição da síntese do colesterol por tratamento crônico com pravastatina causa deficiência de CoQ10, que prejudica a função e viabilidade das células beta. Os efeitos diabetogênicos das estatinas estão relacionados com o aparecimento de miotoxicidade e diminuição da sensibilidade à insulina, aumentando assim o risco de desenvolver diabetes mellitus tipo IIAbstract: We have previously demonstrated that genetic hypercholesterolemia by LDL receptor deficiency show an impairment of insulin secretion. LDL receptor knockout (LDLr-/-) mice, a model of human familial hypercholesterolemia secretes less insulin than wild type mice. By removing cholesterol from LDLr-/- islets ex vivo using methyl-?-cyclodextrin reversed this defect. Thereby, we hypothesized that in vivo treatment of LDLr-/- mice with the cholesterol inhibitor (pravastatin) would normalize glucose-stimulated insulin secretion. Contrary to our hypothesis, female LDLr-/- mice treated for two or three months with pravastatin exhibit increased glycemia and reducted glucose-stimulated insulin secretion by isolated pancreatic islets. These effects were associated with reductions in the levels of SNARE proteins, increased beta cell death and lipid peroxidation in LDLr-/- mice islets treated with pravastatin. Cell culture studies of insulin secreting cells (INS1E) treated with pravastatin showed an increase of cell death and oxidative stress (indicated by H2O2 production). By inhibiting HMG-CoA reductase, statins also inhibit the production of other intermediaries of cholesterol synthesis pathway, such as coenzyme Q10 (CoQ10). Thus, we hypothesized that the islet toxic effects of pravastatin could be caused by the decrease in CoQ10 biosynthesis. For this reason, LDLr-/- mice treated with pravastatin were supplemented with CoQ10. Diet CoQ10 supplementation reversed fasting hyperglycemia observed in pravastatin treated LDLr-/- mice, as well as, improved their glucose tolerance and restored the glucose stimulated insulin secretion. INS1E cells treated with CoQ10 were protected from cell death and oxidative stress induced by pravastatin. After that, we investigated the long term effects of pravastatin treatment and the possible development of diabetes. LDLr-/- mice became glucose intolerant and insulin resistant after 10 months of pravastatin treatment. Experiments in vitro with C2C12 myotubes treated with pravastatin showed a decrease in insulin signaling, oxidative stress and cell death by apoptosis. In conclusion, these results indicate that inhibition of cholesterol synthesis by chronic treatment with statins causes CoQ10 deficit, which impairs beta cell viability and insulin secretion. The stains diabetogenic effects are also related to myotoxicity and reduction of insulin sensitivity, thus increasing the risk of developing type II diabetes mellitusDoutoradoFisiologiaDoutora em Biologia Funcional e MolecularCAPE

Diabetogenic effect of pravastatin is associated with insulin resistance and myotoxicity in hypercholesterolemic mice

HMG-CoA reductase inhibitors (statins) are cholesterol-lowering drugs widely used to treat hypercholesterolemia and prevent cardiovascular disease. Statins are generally well tolerated, but adverse reactions may occur, particularly myopathy and new onset of diabetes. The exact mechanism of statin-induced myopathy and diabetes has not been fully elucidated. We have previously shown that treatment of hypercholesterolemic (LDLr-/-) mice with pravastatin for 2months decreased pancreatic islet insulin secretion and increased oxidative stress and cell death, but no glucose intolerance was observed. The purpose of the current work was to study long-term pravastatin effects on glucose homeostasis, insulin sensitivity, muscle protein turnover and cell viability.MethodsLDLr(-/-) mice were treated with pravastatin for 3, 6 and 10months. Glucose tolerance, insulin resistance and glucose-stimulated insulin secretion were evaluated. The rates of protein synthesis and degradation were determined in gastrocnemius muscle after 10months of treatment. Insulin signalling, oxidative stress and cell death were analysed in vitro using C2C12 myotubes.ResultsAfter 6 and 10months of treatment, these mice became glucose intolerant, and after 10months, they exhibited marked insulin resistance. Reduced islet glucose-stimulated insulin secretion was observed after the 3rdmonth of treatment. Mice treated for 10months showed significantly decreased body weight and increased muscle protein degradation. In addition, muscle chymotrypsin-like proteasomal activity and lysosomal cathepsin were markedly elevated. C2C12 myotubes exposed to increasing concentrations of pravastatin presented dose-dependent impairment of insulin-induced Akt phosphorylation, increased apoptotic markers (Bax protein and cleaved caspase-3) and augmented superoxide anion production.ConclusionsIn addition to reduced insulin secretion, long-term pravastatin treatment induces insulin resistance and muscle wasting. These results suggest that the diabetogenic effect of statins is linked to the appearance of myotoxicity induced by oxidative stress, impaired insulin signalling, proteolysis and apoptosis171CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICO - CNPQCOORDENAÇÃO DE APERFEIÇOAMENTO DE PESSOAL DE NÍVEL SUPERIOR - CAPESFUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO - FAPESP300937/2018-0sem informação2017/17728-

Chronic Use Of Pravastatin Reduces Insulin Exocytosis And Increases Beta-cell Death In Hypercholesterolemic Mice

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)We have previously demonstrated that hypercholesterolemic LDL receptor knockout (LDLr-/-) mice secrete less insulin than wild-type mice. Removing cholesterol from isolated islets using methyl-beta-cyclodextrin reversed this defect. In this study, we hypothesized that in vivo treatment of LDLr-/- mice with the HMGCoA reductase inhibitor pravastatin would improve glucose-stimulated insulin secretion. Female LDLr-/- mice were treated with pravastatin (400 mg/L) for 1-3 months. Isolated pancreatic islets were assayed for insulin secretion rates, intracellular calcium oscillations, cholesterol levels, NAD(P)H and SNARE protein levels, apoptosis indicators and lipidomic profile. Two months pravastatin treatment reduced cholesterol levels in plasma, liver and islets by 35%, 25% and 50%, respectively. Contrary to our hypothesis, pravastatin treatment increased fasting and fed plasma levels of glucose and decreased markedly (40%) fed plasma levels of insulin. In addition, ex vivo glucose stimulated insulin secretion was significantly reduced after two and three months (36-48%, p<0.05) of pravastatin treatment. Although reducing insulin secretion and insulinemia, two months pravastatin treatment did not affect glucose tolerance because it improved global insulin sensitivity. Pravastatin induced islet dysfunction was associated with marked reductions of exocytosis-related SNARE proteins (SNAP25, Syntaxin 1A, VAMP2) and increased apoptosis markers (Bax/Bcl2 protein ratio, cleaved caspase-3 and lower NAD(P)H production rates) observed in pancreatic islets from treated mice. In addition, several oxidized phospholipids, tri- and diacylglycerols and the proapoptotic lipid molecule ceramide were identified as markers of pravastatin-treated islets. Cell death and oxidative stress (H2O2 production) were confirmed in insulin secreting INS-1E cells treated with pravastatin. These results indicate that chronic treatment with pravastatin impairs the insulin exocytosis machinery and increases beta-cell death. These findings suggest that prolonged use of statins may have a diabetogenic effect. (C) 2016 Elsevier Ireland Ltd. All rights reserved.3444252Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP), Brazil [2011/50400-0, 2011/51349-8]Conselho Nacional de Desenvolvimento Cientifico e Tecnologico (CNPq), BrazilCNPqFAPESPCAPESFundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES

Palmitate and insulin counteract glucose-induced thioredoxin interacting protein (TXNIP) expression in insulin secreting cells via distinct mechanisms.

Glucose and palmitate synergistically stimulate insulin secretion, but chronically elevated they induce apoptotic β-cell death. The glucotoxic effect has been attributed, at least partly, to the upregulation of the oxidative stress marker thioredoxin interacting protein (TXNIP). Palmitate downregulates TXNIP expression, the functional significance of which is still under debate. This study examines the mechanism and consequence of palmitate-mediated TXNIP regulation in insulin secreting cells. Palmitate (600 μM) reduced TXNIP mRNA levels in isolated human and mouse islets independently of FFAR1/GPR40. Similar effects of palmitate were observed in INS-1E cells and mimicked by other long chain fatty acids. The lowering of TXNIP mRNA was significant already 1 h after addition of palmitate, persisted for 24 h and was directly translated to changes in TXNIP protein. The pharmacological inhibition of palmitate-induced phosphorylation of AMPK, ERK1/2, JNK and PKCα/β by BML-275, PD98059, SP600125 and Gö6976, respectively, did not abolish palmitate-mediated TXNIP downregulation. The effect of palmitate was superimposed by a time-dependent (8 h and 24 h) decline of TXNIP mRNA and protein. This decline correlated with accumulation of secreted insulin into the medium. Accordingly, exogenously added insulin reduced TXNIP mRNA and protein levels, an effect counteracted by the insulin/IGF-1 receptor antagonist linsitinib. The inhibition of PI3K and Akt/PKB increased TXNIP mRNA levels. The histone deacetylase (HDAC1/2/3) inhibitor MS-275 completely abrogated the time-dependent, insulin-mediated reduction of TXNIP, leaving the effect of palmitate unaltered. Acute stimulation of insulin secretion and chronic accentuation of cell death by palmitate occurred independently of TXNIP regulation. On the contrary, palmitate antagonized glucose-augmented ROS production. In conclusion, glucose-induced TXNIP expression is efficiently antagonized by two independent mechanisms, namely via an autocrine activation of insulin/IGF-1 receptors involving HDAC and by palmitate attenuating oxidative stress of β-cells

Coenzyme Q(10) protects against -cell toxicity induced by pravastatin treatment of hypercholesterolemia

New onset of diabetes is associated with the use of statins.We have recently demonstrated that pravastatin-treated hypercholesterolemic LDL receptor knockout (LDLr-/-) mice exhibit reductions in insulin secretion and increased islet cell death and oxidative stress. Here, we hypothesized that these diabetogenic effects of pravastatin could be counteracted by treatment with the antioxidant coenzyme Q (10) (CoQ (10)), an intermediate generated in the cholesterol synthesis pathway. LDLr (-/-) mice were treated with pravastatin and/or CoQ (10) for 2 months. Pravastatin treatment resulted in a 75% decrease of liver CoQ (10) content. Dietary CoQ (10) supplementation of pravastatin-treated mice reversed fasting hyperglycemia, improved glucose tolerance (20%) and insulin sensitivity (>2-fold), and fully restored islet glucose-stimulated insulin secretion impaired by pravastatin (40%). Pravastatin had no effect on insulin secretion of wild-type mice. In vitro, insulin-secreting INS1E cells cotreated with CoQ (10) were protected from cell death and oxidative stress induced by pravastatin. Simvastatin and atorvastatin were more potent in inducing dose-dependent INS1E cell death (10-15-fold), which were also attenuated by CoQ (10) cotreatment. Together, these results demonstrate that statins impair -cell redox balance, function and viability. However, CoQ (10) supplementation can protect the statins detrimental effects on the endocrine pancreas23471104711059CAPES - Coordenação de Aperfeiçoamento de Pessoal e Nível SuperiorFAPESP – Fundação de Amparo à Pesquisa Do Estado De São Paulosem informação310546/2014-12011-17708-0; 2011/50400-0; 2013/07607-

Chronic Use Of Pravastatin Reduces Insulin Exocytosis And Increases β-cell Death In Hypercholesterolemic Mice.

We have previously demonstrated that hypercholesterolemic LDL receptor knockout (LDLr(-/-)) mice secrete less insulin than wild-type mice. Removing cholesterol from isolated islets using methyl-beta-cyclodextrin reversed this defect. In this study, we hypothesized that in vivo treatment of LDLr(-/-) mice with the HMGCoA reductase inhibitor pravastatin would improve glucose-stimulated insulin secretion. Female LDLr(-/-) mice were treated with pravastatin (400mg/L) for 1-3 months. Isolated pancreatic islets were assayed for insulin secretion rates, intracellular calcium oscillations, cholesterol levels, NAD(P)H and SNARE protein levels, apoptosis indicators and lipidomic profile. Two months pravastatin treatment reduced cholesterol levels in plasma, liver and islets by 35%, 25% and 50%, respectively. Contrary to our hypothesis, pravastatin treatment increased fasting and fed plasma levels of glucose and decreased markedly (40%) fed plasma levels of insulin. In addition, ex vivo glucose stimulated insulin secretion was significantly reduced after two and three months (36-48%, p<0.05) of pravastatin treatment. Although reducing insulin secretion and insulinemia, two months pravastatin treatment did not affect glucose tolerance because it improved global insulin sensitivity. Pravastatin induced islet dysfunction was associated with marked reductions of exocytosis-related SNARE proteins (SNAP25, Syntaxin 1A, VAMP2) and increased apoptosis markers (Bax/Bcl2 protein ratio, cleaved caspase-3 and lower NAD(P)H production rates) observed in pancreatic islets from treated mice. In addition, several oxidized phospholipids, tri- and diacylglycerols and the proapoptotic lipid molecule ceramide were identified as markers of pravastatin-treated islets. Cell death and oxidative stress (H2O2 production) were confirmed in insulin secreting INS-1E cells treated with pravastatin. These results indicate that chronic treatment with pravastatin impairs the insulin exocytosis machinery and increases β-cell death. These findings suggest that prolonged use of statins may have a diabetogenic effect.344-34