RESUMO
Glucose-induced insulin secretion from beta-cells is often impaired in diabetic condition and by exposure to diabetogenic pharmacological agents. In pancreatic beta-cells, intracellular glucose metabolism regulates exocytosis of insulin granules, according to metabolism-secretion coupling in which glucose-induced mitochondrial ATP production plays an essential role. Impaired glucose-induced insulin secretion often results from impaired glucose-induced ATP elevation in beta-cells. Mitochondrial ATP production is driven by the proton-motive force including mitochondrial membrane potential (DeltaPsi(m)) generated by the electron transport chain. These electrons are derived from reducing equivalents, generated in the Krebs cycle and transferred from cytosol by the shuttles. Here, roles of the determinants of mitochondrial ATP production in impaired glucose-induced insulin secretion are discussed. Cytosolic alkalization, H(+) leak in the inner membrane by uncoupler (e.g. free fatty acid exposure), decrease in the supply of electron donors including NADH and FADH(2) to the respiratory chain, and endogenous mitochondrial ROS (e.g. Na(+)/K(+)-ATPase inhibition) all reduce hyperpolarlization of DeltaPsi(m) and ATP production, causing decresed glucose-induced insulin release. The decrease in the supply of NADH and FADH(2) to the respiratory chain derives from impairments in glucose metabolism including glycolysis (e.g. MODY2 and exposure to NO) and the shuttles (e.g. diabetic state and exposure to ketone body).
Assuntos
Trifosfato de Adenosina/metabolismo , Glucose/metabolismo , Células Secretoras de Insulina/metabolismo , Mitocôndrias/metabolismo , Humanos , Modelos Biológicos , Espécies Reativas de OxigênioRESUMO
We examined the effects of reduced Na(+)/K(+)-ATPase activity on mitochondrial ATP production and insulin release from rat islets. Ouabain, an inhibitor of Na(+)/K(+)-ATPase, augmented 16.7 mmol/l glucose-induced insulin release in the early period but suppressed it after a delay of 20-30 min. Unexpectedly, the ATP content in an islet decreases in the presence of 16.7 mmol/l glucose when Na(+)/K(+)-ATPase activity is diminished by ouabain, despite the reduced consumption of ATP by the enzyme. Ouabain also suppressed the increment of ATP content produced by glucose even in Ca(2+)-depleted or Na(+)-depleted conditions. That mitochondrial membrane hyperpolarization and O(2) consumption in islets exposed to 16.7 mmol/l glucose were suppressed by ouabain indicates that the glycoside inhibits mitochondrial respiration but does not produce uncoupling. Ouabain induced mitochondrial reactive oxygen species (ROS) production that was blocked by myxothiazol, an inhibitor of site III of the mitochondrial respiratory chain. An antioxidant, alpha-tocopherol, also blocked ouabain-induced ROS production as well as the suppressive effect of ouabain on ATP production and insulin release. However, ouabain did not directly affect the mitochondrial ATP production originating from succinate and ADP. These results indicate that ouabain suppresses mitochondrial ATP production by generating ROS via transduction, independently of the intracellular cationic alternation that may account in part for the suppressive effect on insulin secretion.
Assuntos
Trifosfato de Adenosina/metabolismo , Glucose/farmacologia , Insulina/metabolismo , Ilhotas Pancreáticas/fisiologia , Mitocôndrias/metabolismo , Ouabaína/farmacologia , Consumo de Oxigênio/fisiologia , Espécies Reativas de Oxigênio/metabolismo , Animais , Carbonil Cianeto p-Trifluormetoxifenil Hidrazona/farmacologia , Glucose/antagonistas & inibidores , Peróxido de Hidrogênio/farmacologia , Técnicas In Vitro , Secreção de Insulina , Ilhotas Pancreáticas/efeitos dos fármacos , Ilhotas Pancreáticas/metabolismo , Cinética , Mitocôndrias/efeitos dos fármacos , Ratos , Vitamina E/farmacologiaRESUMO
Glucose and other nutrients have been shown to stimulate insulin release from pancreatic islets under Ca2+-depleted condition when protein kinase A (PKA) and protein kinase C (PKC) are activated simultaneously. We investigated the role of metabolic nucleotide signals including ATP, ADP, and GTP in exocytosis of insulin secretory granules under Ca2+-depleted condition using electrically permeabilized rat islets. ATP under PKC activation augmented insulin release concentration-dependently by 100 nM 12-O-tetradecanoyl-phorbol-13-acetate (TPA) in Ca2+-depleted condition, while ADP could not suppress ATP-dependent insulin release in this condition. Neither GTP nor activated PKA in the absence of PKC activation increased insulin release under Ca2+-depleted condition in the presence of ATP, but both enhanced insulin secretion in the presence of ATP when PKC was activated. In conclusion, activated PKC and the presence of ATP both are required in the insulin secretory process under Ca2+-depleted condition. While PKA activation and GTP cannot substitute for PKC activation and ATP, respectively, under Ca2+-depleted condition, they enhance ATP-dependent insulin secretion when PKC is activated.
Assuntos
Trifosfato de Adenosina/farmacologia , Cálcio/fisiologia , Exocitose/fisiologia , Insulina/metabolismo , Ilhotas Pancreáticas/metabolismo , Trifosfato de Adenosina/análogos & derivados , Adenilil Imidodifosfato/farmacologia , Animais , Exocitose/efeitos dos fármacos , Secreção de Insulina , Ilhotas Pancreáticas/efeitos dos fármacos , Cinética , Masculino , Ratos , Ratos Wistar , Acetato de Tetradecanoilforbol/farmacologiaRESUMO
A brief exposure to elevated glucose augments the insulin secretory response of islets to subsequent stimulation. The site of this priming effect of glucose in the mechanism of the regulation of insulin secretion is not completely known, however. Insulin release triggered by a depolarizing concentration of K+ in the presence of basal glucose is markedly enhanced in primed rat islets. To clarify the role of priming on Ca(2+) and ATP efficacy in the exocytotic apparatus, islets were electrically permeabilized to vary the intracellular Ca(2+) and ATP concentrations according to the extracellular medium, and insulin release was evaluated. Ca(2+) and ATP efficacy in Ca(2+)- and ATP-dependent insulin secretion was not affected by priming, and alteration of the intracellular Ca(2+) concentration after depolarization cannot account for the phenomenon. There was no difference in ATP content before depolarization between nonprimed and primed islets. Moreover, the decline in ATP level after depolarization with basal glucose was observed in both primed and nonprimed islets. However, a reduced decline in ATP level in the early phase was observed in primed islets. In addition, oligomycin, a mitochondrial metabolism inhibitor, abolished the difference in ATP level between primed and nonprimed islets, suggesting that mitochondrial ATP production may be linked to the phenomenon.
Assuntos
Trifosfato de Adenosina/metabolismo , Glucose/farmacologia , Insulina/metabolismo , Ilhotas Pancreáticas/efeitos dos fármacos , Ilhotas Pancreáticas/fisiologia , Potássio/farmacologia , Animais , Cálcio/metabolismo , Relação Dose-Resposta a Droga , Eletrofisiologia , Técnicas In Vitro , Secreção de Insulina , Membranas Intracelulares/efeitos dos fármacos , Membranas Intracelulares/metabolismo , Ilhotas Pancreáticas/metabolismo , Masculino , Manoeptulose/farmacologia , Oligomicinas/farmacologia , Concentração Osmolar , Permeabilidade , Ratos , Ratos Wistar , Acetato de Tetradecanoilforbol/farmacologia , Fatores de TempoRESUMO
To investigate the effects of chronic exposure to ketone bodies on glucose-induced insulin secretion, we evaluated insulin release, intracellular Ca2+ and metabolism, and Ca2+ efficacy of the exocytotic system in rat pancreatic islets. Fifteen-hour exposure to 5 mM d-beta-hydroxybutyrate (HB) reduced high glucose-induced insulin secretion and augmented basal insulin secretion. Augmentation of basal release was derived from promoting the Ca2+-independent and ATP-independent component of insulin release, which was suppressed by the GDP analog. Chronic exposure to HB affected mostly the second phase of glucose-induced biphasic secretion. Dynamic experiments showed that insulin release and NAD(P)H fluorescence were lower, although the intracellular Ca2+ concentration ([Ca2+](i)) was not affected 10 min after exposure to high glucose. Additionally, [Ca2+](i) efficacy in exocytotic system at clamped concentrations of ATP was not affected. NADH content, ATP content, and ATP-to-ADP ratio in the HB-cultured islets in the presence of high glucose were lower, whereas glucose utilization and oxidation were not affected. Mitochondrial ATP production shows that the respiratory chain downstream of complex II is not affected by chronic exposure to HB, and that the decrease in ATP production is due to decreased NADH content in the mitochondrial matrix. Chronic exposure to HB suppresses glucose-induced insulin secretion by lowering the ATP level, at least partly by inhibiting ATP production by reducing the supply of NADH to the respiratory chain. Glucose-induced insulin release in the presence of aminooxyacetate was not reduced, which implies that chronic exposure to HB affects the malate/aspartate shuttle and thus reduces NADH supply to mitochondria.
Assuntos
Ácido 3-Hidroxibutírico/farmacologia , Cálcio/metabolismo , Glucose/farmacologia , Insulina/metabolismo , Ilhotas Pancreáticas/enzimologia , Ilhotas Pancreáticas/metabolismo , NAD/metabolismo , Adaptação Fisiológica/efeitos dos fármacos , Animais , Células Cultivadas , Relação Dose-Resposta a Droga , Interações Medicamentosas , Ativação Enzimática/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Exocitose/efeitos dos fármacos , Exocitose/fisiologia , Secreção de Insulina , Ilhotas Pancreáticas/efeitos dos fármacos , Masculino , Taxa de Depuração Metabólica , NAD/antagonistas & inibidores , Ratos , Ratos Wistar , Fatores de TempoRESUMO
Tacrolimus is widely used for immunosuppressant therapy, including various organ transplantations. One of its main side effects is hyperglycemia due to reduced insulin secretion, but the mechanism remains unknown. We have investigated the metabolic effects of tacrolimus on insulin secretion at a concentration that does not influence insulin content. Twenty-four-hour exposure to 3 nM tacrolimus reduced high glucose (16.7 mM)-induced insulin secretion (control 2.14 +/- 0.08 vs. tacrolimus 1.75 +/- 0.02 ng.islet(-1).30 min(-1), P < 0.01) without affecting insulin content. In dynamic experiments, insulin secretion and NAD(P)H fluorescence during a 20-min period after 10 min of high-glucose exposure were reduced in tacrolimus-treated islets. ATP content and glucose utilization of tacrolimus-treated islets in the presence of 16.7 mM glucose were less than in control (ATP content: control 9.69 +/- 0.99 vs. tacrolimus 6.52 +/- 0.40 pmol/islet, P < 0.01; glucose utilization: control 103.8 +/- 6.9 vs. tacrolimus 74.4 +/- 5.1 pmol.islet(-1).90 min(-1), P < 0.01). However, insulin release from tacrolimus-treated islets was similar to that from control islets in the presence of 16.7 mM alpha-ketoisocaproate, a mitochondrial fuel. Glucokinase activity, which determines glycolytic velocity, was reduced by tacrolimus treatment (control 65.3 +/- 3.4 vs. tacrolimus 49.9 +/- 2.8 pmol.islet(-1).60 min(-1), P < 0.01), whereas hexokinase activity was not affected. These results indicate that glucose-stimulated insulin release is decreased by chronic exposure to tacrolimus due to reduced ATP production and glycolysis derived from reduced glucokinase activity.
Assuntos
Glucoquinase/antagonistas & inibidores , Glucoquinase/metabolismo , Glucose/farmacologia , Insulina/metabolismo , Ilhotas Pancreáticas/enzimologia , Ilhotas Pancreáticas/metabolismo , Animais , Células Cultivadas , Relação Dose-Resposta a Droga , Interações Medicamentosas , Ativação Enzimática/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Secreção de Insulina , Ilhotas Pancreáticas/efeitos dos fármacos , Masculino , Taxa de Depuração Metabólica , Ratos , Ratos WistarRESUMO
INTRODUCTION AND AIM: To clarify the effects of a high fat-diet on insulin secretion from genetically diabetic beta cells, Goto-Kakizaki rats and Wistar rats were subjected to oral glucose tolerance test (OGTT) after 12-week high-fat feeding. METHODOLOGY: We compared Wistar and Goto-Kakizaki (GK) rats fed a high-fat diet (45% fat content) for 12 weeks, measuring insulin secretion and insulin release. RESULTS: Insulin secretion during oral glucose tolerance test (OGTT) was enhanced in high-fat diet-fed Wistar rats (WF) with normal glucose tolerance. Insulin secretion in high-fat diet-fed GK rats (GF) during OGTT also was enhanced together with deteriorated glucose tolerance. Basal insulin release from the isolated perfused pancreas at 3.3 m glucose in WF was comparable to that in normal chow-fed Wistar rats (WN), but basal insulin release in GF was remarkably higher than in normal chow-fed GK rats (GN). Stimulated insulin release induced by 16.7 m glucose was remarkably increased in WF compared with WN. Total insulin release at 16.7 m glucose in both GK rat groups was similar and minimal. CONCLUSION: These results indicate that normal pancreatic beta-cells have the ability to secrete sufficient insulin to compensate for the insulin resistance induced by a high-fat diet. In contrast, glucose metabolism in diabetic rats after high-fat diet deteriorated partly because of insufficient insulin secretion caused by genetic defects and lipotoxicity due to chronically high FFA levels.