RESUMO
Pancreatic ß-cells respond to metabolic stress by upregulating insulin secretion, however the underlying mechanisms remain unclear. Here we show, in ß-cells from overweight humans without diabetes and mice fed a high-fat diet for 2 days, insulin exocytosis and secretion are enhanced without increased Ca2+ influx. RNA-seq of sorted ß-cells suggests altered metabolic pathways early following high fat diet, where we find increased basal oxygen consumption and proton leak, but a more reduced cytosolic redox state. Increased ß-cell exocytosis after 2-day high fat diet is dependent on this reduced intracellular redox state and requires the sentrin-specific SUMO-protease-1. Mice with either pancreas- or ß-cell-specific deletion of this fail to up-regulate exocytosis and become rapidly glucose intolerant after 2-day high fat diet. Mechanistically, redox-sensing by the SUMO-protease requires a thiol group at C535 which together with Zn+-binding suppresses basal protease activity and unrestrained ß-cell exocytosis, and increases enzyme sensitivity to regulation by redox signals.
Assuntos
Dieta Hiperlipídica , Exocitose , Animais , Humanos , Camundongos , Cisteína Endopeptidases/genética , Citosol , Dieta Hiperlipídica/efeitos adversos , Glucose , Peptídeo HidrolasesRESUMO
AIMS: The exposure of isolated pancreatic islets to pro-angiogenic factors prior to their transplantation represents a promising strategy to accelerate the revascularization of the grafts. It has been shown that erythropoietin (EPO), a glycoprotein regulating erythropoiesis, also induces angiogenesis. Therefore, we hypothesized that EPO exposure of isolated islets improves their posttransplant revascularization. METHODS: Flow cytometric, immunohistochemical and quantitative real-time (qRT)-PCR analyses were performed to study the effect of EPO on the viability, cellular composition and gene expression of isolated islets. Moreover, islets expressing a mitochondrial or cytosolic H2O2 sensor were used to determine reactive oxygen species (ROS) levels. The dorsal skinfold chamber model in combination with intravital fluorescence microscopy was used to analyze the revascularization of transplanted islets. RESULTS: We found that the exposure of isolated islets to EPO (3 units/mL) for 24 h does not affect the viability and the production of ROS when compared to vehicle-treated and freshly isolated islets. However, the exposure of islets to EPO increased the number of CD31-positive cells and enhanced the gene expression of insulin and vascular endothelial growth factor (VEGF)-A. The revascularization of the EPO-cultivated islets was accelerated within the initial phase after transplantation when compared to both controls. CONCLUSION: These findings indicate that the exposure of isolated islets to EPO may be a promising approach to improve clinical islet transplantation.
Assuntos
Eritropoetina , Transplante das Ilhotas Pancreáticas , Ilhotas Pancreáticas , Eritropoetina/farmacologia , Peróxido de Hidrogênio , Neovascularização Fisiológica , Fator A de Crescimento do Endotélio Vascular/genéticaRESUMO
Dormant hematopoietic stem cells (dHSCs) are atop the hematopoietic hierarchy. The molecular identity of dHSCs and the mechanisms regulating their maintenance or exit from dormancy remain uncertain. Here, we use single-cell RNA sequencing (RNA-seq) analysis to show that the transition from dormancy toward cell-cycle entry is a continuous developmental path associated with upregulation of biosynthetic processes rather than a stepwise progression. In addition, low Myc levels and high expression of a retinoic acid program are characteristic for dHSCs. To follow the behavior of dHSCs in situ, a Gprc5c-controlled reporter mouse was established. Treatment with all-trans retinoic acid antagonizes stress-induced activation of dHSCs by restricting protein translation and levels of reactive oxygen species (ROS) and Myc. Mice maintained on a vitamin A-free diet lose HSCs and show a disrupted re-entry into dormancy after exposure to inflammatory stress stimuli. Our results highlight the impact of dietary vitamin A on the regulation of cell-cycle-mediated stem cell plasticity. VIDEO ABSTRACT.
Assuntos
Células-Tronco Hematopoéticas/citologia , Transdução de Sinais , Tretinoína/farmacologia , Vitamina A/administração & dosagem , Animais , Vias Biossintéticas , Técnicas de Cultura de Células , Ciclo Celular/efeitos dos fármacos , Sobrevivência Celular , Dieta , Perfilação da Expressão Gênica , Células-Tronco Hematopoéticas/efeitos dos fármacos , Camundongos , Poli I-C/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Análise de Célula Única , Estresse Fisiológico , Vitamina A/farmacologia , Vitaminas/administração & dosagem , Vitaminas/farmacologiaRESUMO
High glucose-induced oxidative stress and increased NADPH oxidase-2 (NOX2) activity may contribute to the progressive decline of the functional ß-cell mass in type 2 diabetes. To test that hypothesis, we characterized, in islets from male NOX2 knockout (NOX2-KO) and wild-type (WT) C57BL/6J mice cultured for up to 3 weeks at 10 or 30 mmol/l glucose (G10 or G30), the in vitro effects of glucose on cytosolic oxidative stress using probes sensing glutathione oxidation (GRX1-roGFP2), thiol oxidation (roGFP1) or H2O2 (roGFP2-Orp1), on ß-cell stimulus-secretion coupling events and on ß-cell apoptosis. After 1-2 days of culture in G10, the glucose stimulation of insulin secretion (GSIS) was â¼1.7-fold higher in NOX2-KO vs. WT islets at 20-30 mmol/l glucose despite similar rises in NAD(P)H and intracellular calcium concentration ([Ca2+]i) and no differences in cytosolic GRX1-roGFP2 oxidation. After long-term culture at G10, roGFP1 and roGFP2-Orp1 oxidation and ß-cell apoptosis remained low, and the glucose-induced rises in NAD(P)H, [Ca2+]i and GSIS were similarly preserved in both islet types. After prolonged culture at G30, roGFP1 and roGFP2-Orp1 oxidation increased in parallel with ß-cell apoptosis, the glucose sensitivity of the NADPH, [Ca2+]i and insulin secretion responses increased, the maximal [Ca2+]i response decreased, but maximal GSIS was preserved. These responses were almost identical in both islet types. In conclusion, NOX2 is a negative regulator of maximal GSIS in C57BL/6J mouse islets, but it does not detectably contribute to the in vitro glucotoxic induction of cytosolic oxidative stress and alterations of ß-cell survival and function.
Assuntos
Glucose/toxicidade , Células Secretoras de Insulina/enzimologia , Células Secretoras de Insulina/patologia , NADPH Oxidase 2/metabolismo , Animais , Apoptose/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Citosol/metabolismo , Transportador de Glucose Tipo 2/genética , Transportador de Glucose Tipo 2/metabolismo , Glutarredoxinas/metabolismo , Glutationa/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Heme Oxigenase-1/genética , Heme Oxigenase-1/metabolismo , Insulina/metabolismo , Secreção de Insulina , Células Secretoras de Insulina/efeitos dos fármacos , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , NADPH Oxidase 2/deficiência , Oxirredução , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Compostos de Sulfidrila/metabolismo , Técnicas de Cultura de TecidosRESUMO
Mapping the in vivo distribution of endogenous oxidants in animal tissues is of substantial biomedical interest. Numerous health-related factors, including diet, physical activity, infection, aging, toxins, or pharmacological intervention, may cause redox changes. Tools are needed to pinpoint redox state changes to particular organs, tissues, cell types, and subcellular organelles. We describe a procedure that preserves the in vivo redox state of genetically encoded redox biosensors within histological tissue sections, thus providing "redox maps" for any tissue and comparison of interest. We demonstrate the utility of the technique by visualizing endogenous redox differences and changes in the context of tumor growth, inflammation, embryonic development, and nutrient starvation.
Assuntos
Imagem Molecular/métodos , Sondas Moleculares/metabolismo , Transgenes , Animais , Células HEK293 , Humanos , Camundongos , Camundongos Nus , Sondas Moleculares/genética , OxirreduçãoRESUMO
OBJECTIVES: The aim of the study was to evaluate the potential changes induced by fish oil (FO) supplementation on the redox status of pancreatic islets from healthy rats. To test whether these effects were due to eicosapentaenoic acid and docosahexaenoic acid (ω-3), in vitro experiments were performed. METHODS: Rats were supplemented with FO, and pancreatic islets were obtained. Islets were also treated in vitro with palmitate (P) or eicosapentaenoic acid + docosahexaenoic acid (ω-3). Insulin secretion (GSIS), glucose oxidation, protein expression, and superoxide content were analyzed. RESULTS: The FO group showed a reduction in superoxide content. Moreover, FO reduced the expression of NAD(P)H oxidase subunits and increased superoxide dismutase, without altering ß-cell function. Palmitate increased ß-cell reactive oxygen species (ROS) production, apoptosis, and impaired GSIS. Under these conditions, ω-3 triggered a parallel reduction in ROS production and ß-cell apoptosis induced by P and protected against the impairment in GSIS. There was no difference in mitochondrial ROS production. CONCLUSIONS: Our results show that ω-3 protect pancreatic islets from alterations induced by P. In vivo FO supplementation modulates the redox state of pancreatic ß-cell. Considering that in vitro effects do not involve mitochondrial superoxide production, we can speculate that this protection might involve NAD(P)H oxidase activity.
Assuntos
Antioxidantes/administração & dosagem , Suplementos Nutricionais , Ácidos Docosa-Hexaenoicos/administração & dosagem , Ácido Eicosapentaenoico/administração & dosagem , Ilhotas Pancreáticas/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Administração Oral , Animais , Apoptose/efeitos dos fármacos , Biomarcadores/sangue , Glicemia/efeitos dos fármacos , Glicemia/metabolismo , Glutationa/metabolismo , Insulina/sangue , Células Secretoras de Insulina/efeitos dos fármacos , Células Secretoras de Insulina/metabolismo , Células Secretoras de Insulina/patologia , Ilhotas Pancreáticas/metabolismo , Ilhotas Pancreáticas/patologia , Masculino , NADPH Oxidases/metabolismo , Oxirredução , Ácido Palmítico/toxicidade , Ratos Wistar , Espécies Reativas de Oxigênio/metabolismo , Superóxido Dismutase/metabolismo , Superóxidos/metabolismo , Fatores de Tempo , Técnicas de Cultura de TecidosRESUMO
The glucose stimulation of insulin secretion by pancreatic ß-cells depends on increased production of metabolic coupling factors, among which changes in NADPH and ROS (reactive oxygen species) may alter the glutathione redox state (EGSH) and signal through changes in thiol oxidation. However, whether nutrients affect EGSH in ß-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 ß-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 Ca(2+) concentration and in mitochondrial pH. They were unaffected by overexpression of SOD2 (superoxide dismutase 2) 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, the present study demonstrates that glucose and other nutrients acutely reduce mitochondrial, but not cytosolic/nuclear, EGSH in pancreatic ß-cells under control conditions.
Assuntos
Glucose/farmacologia , Glutationa/metabolismo , Células Secretoras de Insulina/metabolismo , Mitocôndrias/efeitos dos fármacos , Animais , Cálcio/metabolismo , Catalase/metabolismo , Núcleo Celular/metabolismo , Citosol/metabolismo , Células HEK293 , Humanos , Peróxido de Hidrogênio/metabolismo , Concentração de Íons de Hidrogênio , Insulina/metabolismo , Secreção de Insulina , Células Secretoras de Insulina/efeitos dos fármacos , Mitocôndrias/fisiologia , NADP/metabolismo , Oxirredução , Ratos , Espécies Reativas de Oxigênio/metabolismo , Vitamina K 3/metabolismoRESUMO
Using the ROS (reactive oxygen species)-sensitive fluorescent dyes dichlorodihydrofluorescein and dihydroethidine, previous studies yielded opposite results about the glucose regulation of oxidative stress in insulin-secreting pancreatic ß-cells. In the present paper, we used the ratiometric fluorescent proteins HyPer and roGFP1 (redox-sensitive green fluorescent protein 1) targeted to mitochondria [mt-HyPer (mitochondrial HyPer)/mt-roGFP1 (mitochondrial roGFP1)] to monitor glucose-induced changes in mitochondrial hydrogen peroxide concentration and glutathione redox state in adenovirus-infected rat islet cell clusters. Because of the reported pH sensitivity of HyPer, the results were compared with those obtained with the mitochondrial pH sensors mt-AlpHi and mt-SypHer. The fluorescence ratio of the mitochondrial probes slowly decreased (mt-HyPer) or increased (mt-roGFP1) in the presence of 10 mmol/l glucose. Besides its expected sensitivity to H2O2, mt-HyPer was also highly pH sensitive. In agreement, changes in mitochondrial metabolism similarly affected mt-HyPer, mt-AlpHi and mt-SypHer fluorescence signals. In contrast, the mt-roGFP1 fluorescence ratio was only slightly affected by pH and reversibly increased when glucose was lowered from 10 to 2 mmol/l. This increase was abrogated by the catalytic antioxidant Mn(III) tetrakis (4-benzoic acid) porphyrin but not by N-acetyl-L-cysteine. In conclusion, due to its pH sensitivity, mt-HyPer is not a reliable indicator of mitochondrial H2O2 in ß-cells. In contrast, the mt-roGFP1 fluorescence ratio monitors changes in ß-cell mitochondrial glutathione redox state with little interference from pH changes. Our results also show that glucose acutely decreases rather than increases mitochondrial thiol oxidation in rat ß-cells.
Assuntos
Glutationa/análise , Proteínas de Fluorescência Verde/análise , Peróxido de Hidrogênio/análise , Células Secretoras de Insulina/química , Medições Luminescentes/métodos , Mitocôndrias/química , Animais , Glutationa/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Humanos , Peróxido de Hidrogênio/metabolismo , Concentração de Íons de Hidrogênio , Células Secretoras de Insulina/metabolismo , Células Secretoras de Insulina/ultraestrutura , Cinética , Masculino , Mitocôndrias/metabolismo , Concentração Osmolar , Oxirredução , Ratos , Ratos Wistar , Espécies Reativas de Oxigênio/metabolismo , Sensibilidade e EspecificidadeRESUMO
OBJECTIVES: Reactive oxygen species (ROS) are involved in many physiological and pathological processes. In the present study, we analysed whether the synthetic glucocorticoid dexamethasone induces oxidative stress in cultured pancreatic islets and whether the effects of dexamethasone on insulin secretion, gene expression, and viability can be counteracted by concomitant incubation with N-acetylcysteine (NAC). METHODS: ROS production was measured by dichlorofluorescein (DCFH-DA) assay, insulin secretion by radioimmunoassay, intracellular calcium dynamics by fura-2-based fluorescence, gene expression by real-time polymerase chain reaction analyses and cell viability by the MTS assay. RESULTS: Dexamethasone (Dexa) increased ROS production and decreased glucose-stimulated insulin secretion after 72 hours incubation. Intracellular ROS levels were decreased and the insulin secretion capacity was recovered by concomitant treatment with Dexa+NAC. The total insulin content and intracellular Ca2+ levels were not modulated in either Dexa or Dexa+NAC groups. There was a decrease in the NAD(P)H production, used as an indicator of viability, after dexamethasone treatment. Concomitant incubation with NAC returned viability to control levels. Dexa also decreased synaptotagmin VII (SYT VII) gene expression. In contrast, the Dexa+NAC group demonstrated an increased expression of SYT VII compared to controls. Surprisingly, treatment with NAC decreased the gene expression of the antioxidant enzyme copper zinc superoxide dismutase soluble. DISCUSSION: Our results indicate that dexamethasone increases ROS production, decreases viability, and impairs insulin secretion in pancreatic rat islets. These effects can be counteracted by NAC, which not only decreases ROS levels but also modulates the expression of genes involved in the secretory pathway and those coding for antioxidant enzymes.