NAD(P)H oxidase participates in the oleic acid-induced metabolic channelling during insulin secretion.

Ácidos graxos são requeridos para a manutenção da função celular e atuam como moduladores da secreção de insulina induzida. Os importantes sítios de formação de EROs são a mitocôndria e a NAD(P)H oxidase, a primeira pode alterar a produção de EROs em função da atividade metabólica e a segunda tem sua atividade regulada por diversos fatores, dentre eles a PKC .O ácido oléico, junto com o palmítico, é um dos AGs mais abundantes na circulação. O tratamento agudo (1 hora) com 100 µM de ácido oléico aumentou a secreção de insulina associado ao aumento no metabolismo do AG. A oxidação desse ácido graxo induziu aumento no conteúdo de EROs em 16,7 mM de glicose com participação da NAD(P)H oxidase. Apesar da reconhecida função da EROs como sinalizadores, a diminuição de EROs induzida pela inibição da NAD(P)H oxidase promoveu aumento relativo na oxidação da glicose. A secreção relativa de insulina aumentou após inibição da NAD(P)H oxidase, sugerindo função regulatória das EROs no metabolismo da glicose e, conseqüentemente, da secreção de insulina. Dessa forma, o ácido oléico é capaz de aumentar a secreção de insulina com participação da NAD(P)H oxidase e as EROs produzidas por essa enzima promovendo a regulação do metabolismo da glicose.Fatty acids are required to maintain cellular functioning and are able to modulate insulin secretion from pancreatic islets. The important sites of ROS production are the mitochondria and the NAD(P)H oxidase. The mitochondrial ROS release depends on cellular activity and NAD(P)H oxidase activity depends on many factors, including PKC. Acute (1 hour) exposure to oleic acid increased insulin secretion at 16.7 mM glucose. The insulin secretion induced by OA was associated to increased fatty acid oxidation and decreased glucose metabolism. Also, at 16.7 mM glucose, OA oxidation increased ROS production mediated by NAD(P)H. ROS decreased content induced by NAD(P)H oxidase inhibition induced glucose oxidation re-establishment after OA stimulus. The relative secretion was stimulated by NAD(P)H oxidase inhibition after OA stimulus. This suggests that ROS produced by NAD(P)H oxidase act as glucose metabolism regulators in the pancreatic cell. In consequence of glucose metabolism re-establishment the insulin secretion was increased. In conclusion, ROS produced by NAD(P)H oxidase are regulators of glucose metabolism. The glucose metabolism regulation may be in part responsible for the increased insulin secretion induced by ROS

Efeito da dieta suplementada com vitamina E e cobre nas respostas metabólicas e antioxidantes de matrinxã, Brycon cephalus (Gunther, 1869), frente à hipóxia.

The diet is an essential factor for the growth and maintenance of vital functions as well as to ability fish to reply the stressors. The reduction of oxygen concentration in the water may occur mainly in fish culture ponds, and may reduce the growth rate of the animals due to metabolism changes. Therefore the supplementation of the diet with copper and vitamin E can improve the animals defenses. Matrinxã specimens, Brycon cephalus, promising specie for fisheries, were fed with commercial ration (Co) and rations supplemented with vitamin E (E) and vitamin E+copper (E+Cu) during 30 days following by exposure to hypoxia (Hpx) during 24h (DO = 2,87 mgO2/L). The hematological variables [hematocrit (Hct), hemoglobin concentration ([Hb]), red blood cells (RBC), mean corpuscular volume (MCV), mean hemoglobin concentration (MHC) and mean corpuscular hemoglobin concentratrion (MCHC)]; metabolic changes such as concentrations of glucose, lactate, piruvate, ammonia, glycogen and protein in liver, red and white muscles and plasma (exception of glycogen) and, the oxidative stress parameters as concentration of peroxide lipids (HP), activity of superoxide dismutase (SOD), glutathione peroxidase (GHS-Px) and catalase (CAT) (exception in plasma) in the same tissues, including the gills and heart. The diets E and E+Cu may increase the aerobic capability of matrinxã when in hypoxic condition as there were increases of [Hb] and MCHC in group E+Cu and an increase in MCHC in E accompanied by an increase in the pyruvate and no change in lactate in most of tissues of fish fed wit supplemented diets. These results support the view that the both supplemented diets, and mainly the E+Cu, seem to increased the aerobic capacity of matrinxã during hypoxia. Hypoxia increased HP concentration only in the plasma and the supplemented diet change the oxidative stress, hematological and metabolical parameters. The diet E reduced HP concentration in the heart and red muscles during hypoxia and the diet E+Cu increased the enzyme concentration and activity without generated HP increase, even in conditions of higher aerobic metabolism. The obtained results suggest that the supplemented diets increased the aerobic capacity of matrinxã during hypoxia without increasing oxidative stress, while the metabolic adjustments front to the reduced concentration of O2 had been efficient to maintain the O2 delivery to the tissues since the hypoxia only modified the parameters analyzed when in association with the differentiated diets.Financiadora de Estudos e ProjetosA dieta é fator essencial para o crescimento e manutenção das funções vitais principalmente frente a situações adversas. Em pisciculturas ocorrem situações de redução na concentração de oxigênio na água que podem reduzir o crescimento dos animais uma vez que interferem no metabolismo. A suplementação da dieta com vitaminas e metais essenciais, dentre eles o cobre e vitamina E, que tem função antioxidante, podem melhorar as defesas dos animais frente a estressores. Exemplares de matrinxã, Brycon cephalus, espécie promissora para piscicultura, foram alimentados durante 30 dias com ração comercial (Co) e ração suplementada com vitamina E (E) e vitamina E+cobre (E+Cu) e, posteriormente, foram expostos a hipóxia (Hpx) durante 24h (OD = 2,87 mgO2/L). As alterações hematológicas [hematócrio (Hct), concentração de hemoglobina ([Hb]), número de eritrócitos (RBC) volume corpuscular médio (VCM), concentração de hemoglobina média (HCM) e concentração média de hemoglobina corpuscular (CHCM)]; alterações metabólicas [concentrações de glicose, lactato, piruvato, amônia, glicogênio e proteína no plasma (exceção do glicogênio), fígado, músculos vermelho e branco] e os parâmetros de estresse oxidativo [(concentração de hidroperóxidos de lipídios (HP), atividade das enzimas superóxido dismutase (SOD), glutationa peroxidase (GHS-Px) e catalase (CAT)] nos mesmos tecidos com exceção do plasma, além de brânquias e coração foram analisadas em normóxia e hipóxia. A dieta E e, principalmente, a E+Cu parecem ter aumentado a capacidade aeróbica de matrinxã quando em hipóxia, pois houve aumento na [Hb] e CHCM no grupo E+Cu e aumento no CHCM em E, além de aumento no piruvato e ausência de aumento no lactato na maioria dos tecidos dos animais tratados com dieta suplementada. A hipóxia gerou aumento na concentração de HP somente no plasma e a dieta suplementada alterou parâmetros de estresse oxidativo, assim como hematológicos e metabólicos. A vitamina E reduziu HP em coração e músculo vermelho quando em hipóxia e a vitamina E + cobre aumentou atividade enzimática sem gerar aumento na concentração de HP, mesmo em condições de maior metabolismo aeróbico. Os resultados obtidos sugerem que as dietas suplementadas aumentaram a capacidade aeróbica de matrinxã em hipóxia sem aumentar o estresse oxidativo nesses animais, enquanto os ajustes metabólicos frente à reduzida concentração de O2 foram eficientes em manter o aporte de O2 para os tecidos, já que a hipóxia só alterou os parâmetros analisados quando em associação com as dietas diferenciadas

Acute nutrient regulation of mitochondrial glutathione redox state in pancreatic β-cells

The glucose stimulation of insulin secretion by pancreatic β-cells depends on increased production of metabolic coupling factors, among which changes in NADPH and reactive oxygen species (ROS) may alter the glutathione redox state (EGSH) and signal through changes in thiol oxidation. However, whether nutrients affect EGSH in β-cell subcellular compartments is unknown. Using redox-sensitive GFP2 fused to glutaredoxin 1 and its mitochondria-targeted form, we studied the acute nutrient regulation of EGSH in the cytosol/nucleus or the mitochondrial matrix of rat islet cells. These probes were mainly expressed in β-cells and reacted to low concentrations of exogenous H2O2 and menadione. Under control conditions, cytosolic/nuclear EGSH was close to -300 mV and unaffected by glucose (from 0 to 30 mM). In comparison, mitochondrial EGSH was less negative and rapidly regulated by glucose and other nutrients, ranging from -280 mV in the absence of glucose to -299 mV in 30 mM glucose. These changes were largely independent from changes in intracellular Ca2+ concentration and in mitochondrial pH. They were unaffected by overexpression of SOD2 and mitochondria-targeted catalase, but were inversely correlated with changes in NAD(P)H autofluorescence, suggesting that they indirectly resulted from increased NADPH availability rather than from changes in ROS concentration. Interestingly, the opposite regulation of mitochondrial EGSH and NAD(P)H autofluorescence by glucose was also observed in human islets isolated from two donors. In conclusion, this study demonstrates that glucose and other nutrients acutely reduce mitochondrial but not cytosolic/nuclear EGSH in pancreatic β-cells under control conditions

Potential contribution of translational factors to triiodo-l-hyronine-induced Insulin synthesis by pancreatic beta cells

Thyroid hormones (THs) are known to regulate protein synthesis by acting at the transcriptional level and inducing the expression of many genes. However, little is known about their role in protein expression at the post-transcriptional level, even though studies have shown enhancement of protein synthesis associated with mTOR/p70S6K activation after triiodo-l-thyronine (T3) administration. On the other hand, the effects of TH on translation initiation and polypeptidic chain elongation factors, being essential for activating protein synthesis, have been poorly explored. Therefore, considering that preliminary studies from our laboratory have demonstrated an increase in insulin content in INS-1E cells in response to T3 treatment, the aim of the present study was to investigate if proteins of translational nature might be involved in this effect. INS-1E cells were maintained in the presence or absence of T3 (10(-6) or 10(-8) M) for 12 hours. Thereafter, insulin concentration in the culture medium was determined by radioimmunoassay, and the cells were processed for Western blot detection of insulin, eukaryotic initiation factor 2 (eIF2), p-eIF2, eIF5A, EF1A, eIF4E binding protein (4E-BP), p-4E-BP, p70S6K, and p-p70S6K. It was found that, in parallel with increased insulin generation, T3 induced p70S6K phosphorylation and the expression of the translational factors eIF2, eIF5A, and eukaryotic elongation factor 1 alpha (eEF1A). In contrast, total and phosphorylated 4E-BP, as well as total p70S6K and p-eIF2 content, remained unchanged after T3 treatment. Considering that (i) p70S6K induces S6 phosphorylation of the 40S ribosomal subunit, an essential condition for protein synthesis; (ii) eIF2 is essential for the initiation of messenger RNA translation process; and (iii) eIF5A and eEF1A play a central role in the elongation of the polypeptidic chain during the transcripts decoding, the data presented here lead us to suppose that a part of T3-induced insulin expression in INS-1E cells depends on the protein synthesis activation at the post-transcriptional level, as these proteins of the translational machinery were shown to be regulated by T3226637642FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO - FAPESP08/56446-

Antioxidant defenses and biochemical changes in the neotropical fish pacu, Piaractus mesopotamicus: Responses to single and combined copper and hypercarbia exposure

This study investigated the potentially detrimental effects of copper and elevated aquatic CO2 (hypercarbia), alone or in combination, on pacu, Piaractus mesopotamicus. Fish were exposed for 48 h to control (no copper addition in normocarbia), to 400 mu g Cu2+L-1, to hypercarbic (1% CO2; PCO2=6.9 mm Hg) water and to 400 mu g Cu2+L-1+ hypercarbia. In liver the single factors caused an increase in lipid hydroperoxide concentration that was not observed when the factors were combined. Copper exposure elicited increased hepatic superoxide dismutase activity, irrespective of aquatic CO2 level. On the other hand, the effects of copper on hepatic glutathione peroxidase activity were dependent on water CO2 levels. The two stressors combined did not affect hepatic catalase activity. Hypercarbic water caused a decline in plasma glucose concentration, but this was not observed when hypercarbia was combined with copper exposure. Copper caused a decrease in branchial Na+/K+-ATPase activity that was independent of water CO2 level. Copper caused an increase in branchial metallothionein concentration that was independent of water CO2 level. Thus, branchial metallothionein and Na+/K+-ATPase were effective biomarkers of copper exposure that were not affected by water CO2 level. (C) 2012 Elsevier Inc. All rights reserved.CNPqCNPqAquapeixe Aquaculture (Conchal, SP, Brazil)Aquapeixe Aquaculture (Conchal, SP, Brazil)Sao Paulo State Research Foundation (FAPESP)Sao Paulo State Research Foundation - FAPESP [Proc. 03/06105-7

Potential Contribution of Translational Factors to Triiodo-L-Thyronine-Induced Insulin Synthesis by Pancreatic Beta Cells

Background: Thyroid hormones (THs) are known to regulate protein synthesis by acting at the transcriptional level and inducing the expression of many genes. However, little is known about their role in protein expression at the post-transcriptional level, even though studies have shown enhancement of protein synthesis associated with mTOR/p70S6K activation after triiodo-l-thyronine (T3) administration. On the other hand, the effects of TH on translation initiation and polypeptidic chain elongation factors, being essential for activating protein synthesis, have been poorly explored. Therefore, considering that preliminary studies from our laboratory have demonstrated an increase in insulin content in INS-1E cells in response to T3 treatment, the aim of the present study was to investigate if proteins of translational nature might be involved in this effect. Methods: INS-1E cells were maintained in the presence or absence of T3 (10(-6) or 10(-8) M) for 12 hours. Thereafter, insulin concentration in the culture medium was determined by radioimmunoassay, and the cells were processed for Western blot detection of insulin, eukaryotic initiation factor 2 (eIF2), p-eIF2, eIF5A, EF1A, eIF4E binding protein (4E-BP), p-4E-BP, p70S6K, and p-p70S6K. Results: It was found that, in parallel with increased insulin generation, T3 induced p70S6K phosphorylation and the expression of the translational factors eIF2, eIF5A, and eukaryotic elongation factor 1 alpha (eEF1A). In contrast, total and phosphorylated 4E-BP, as well as total p70S6K and p-eIF2 content, remained unchanged after T3 treatment. Conclusions: Considering that (i) p70S6K induces S6 phosphorylation of the 40S ribosomal subunit, an essential condition for protein synthesis; (ii) eIF2 is essential for the initiation of messenger RNA translation process; and (iii) eIF5A and eEF1A play a central role in the elongation of the polypeptidic chain during the transcripts decoding, the data presented here lead us to suppose that a part of T3-induced insulin expression in INS-1E cells depends on the protein synthesis activation at the post-transcriptional level, as these proteins of the translational machinery were shown to be regulated by T3.Fundacao de Amparo a Pesquisa do Estado de Sao Paulo [FAPESP: 08/56446-9]Fundacao de Amparo a Pesquisa do Estado de Sao Paul

Unveiling a common mechanism of apoptosis in β-cells and neurons in Friedreich's ataxia.

Friedreich's ataxia (FRDA) is a neurodegenerative disorder associated with cardiomyopathy and diabetes. Effective therapies for FRDA are an urgent unmet need; there are currently no options to prevent or treat this orphan disease. FRDA is caused by reduced expression of the mitochondrial protein frataxin. We have previously demonstrated that pancreatic β-cell dysfunction and death cause diabetes in FRDA. This is secondary to mitochondrial dysfunction and apoptosis but the underlying molecular mechanisms are not known. Here we show that β-cell demise in frataxin deficiency is the consequence of oxidative stress-mediated activation of the intrinsic pathway of apoptosis. The pro-apoptotic Bcl-2 family members Bad, DP5 and Bim are the key mediators of frataxin deficiency-induced β-cell death. Importantly, the intrinsic pathway of apoptosis is also activated in FRDA patients' induced pluripotent stem cell-derived neurons. Interestingly, cAMP induction normalizes mitochondrial oxidative status and fully prevents activation of the intrinsic pathway of apoptosis in frataxin-deficient β-cells and neurons. This preclinical study suggests that incretin analogs hold potential to prevent/delay both diabetes and neurodegeneration in FRDA