RESUMO
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
Diabetes mellitus is a product of low insulin sensibility and pancreatic ß-cell insufficiency. Rats with streptozotocin-induced diabetes during the neonatal period by the fifth day of age develop the classic diabetic picture of hyperglycemia, hypoinsulinemia, polyuria, and polydipsia aggravated by insulin resistance in adulthood. In this study, we investigated whether the effect of long-term treatment with melatonin can improve insulin resistance and other metabolic disorders in these animals. At the fourth week of age, diabetic animals started an 8-wk treatment with melatonin (1 mg/kg body weight) in the drinking water at night. Animals were then killing, and the sc, epididymal (EP), and retroperitoneal (RP) fat pads were excised, weighed, and processed for adipocyte isolation for morphometric analysis as well as for measuring glucose uptake, oxidation, and incorporation of glucose into lipids. Blood samples were collected for biochemical assays. Melatonin treatment reduced hyperglycemia, polydipsia, and polyphagia as well as improved insulin resistance as demonstrated by constant glucose disappearance rate and homeostasis model of assessment-insulin resistance. However, melatonin treatment was unable to recover body weight deficiency, fat mass, and adipocyte size of diabetic animals. Adiponectin and fructosamine levels were completely recovered by melatonin, whereas neither plasma insulin level nor insulin secretion capacity was improved in diabetic animals. Furthermore, melatonin caused a marked delay in the sexual development, leaving genital structures smaller than those of nontreated diabetic animals. Melatonin treatment improved the responsiveness of adipocytes to insulin in diabetic animals measured by tests of glucose uptake (sc, EP, and RP), glucose oxidation, and incorporation of glucose into lipids (EP and RP), an effect that seems partially related to an increased expression of insulin receptor substrate 1, acetyl-coenzyme A carboxylase and fatty acid synthase. In conclusion, melatonin treatment was capable of ameliorating the metabolic abnormalities in this particular diabetes model, including insulin resistance and promoting a better long-term glycemic control.