Diphenylhydantoin Suppresses Glucose-Induced Insulin Release by Decreasing Cytoplasmic H+ Concentration in Pancreatic Islets

Abstract

Diphenylhydantoin (DPH), which is clinically used in the treatment of epilepsy, inhibits glucose-induced insulin release from pancreatic islets by a mechanism that remains unknown. In the present study, DPH is shown to suppress glucose-induced insulin release concentration-dependently. In dynamic experiments, 20 microm DPH suppressed 16.7 mm glucose-induced biphasic insulin release. DPH also suppressed insulin release in the presence of 16.7 mm glucose, 200 microm diazoxide, and 30 mm K+ without affecting the intracellular Ca2+ concentration. DPH suppressed ATP content and mitochondrial membrane hyperpolarization in the presence of 16.7 mm glucose without affecting glucose utilization, glucose oxidation, and reduced nicotinamide adenine dinucleotide phosphate fluorescence. DPH increased cytoplasmic pH in the presence of high glucose, but the increase was abolished under Na+ -deprived conditions and HCO3- -deprived conditions, suggesting that Na+ and HCO3- transport across the plasma membrane are involved in the increase in cytoplasmic pH by DPH. Alkalization by adding NH4+ to the extracellular medium also suppressed insulin release, ATP content, and mitochondrial membrane hyperpolarization. Because ATP production from the mitochondrial fraction in the presence of substrates was decreased by increased pH in the medium, DPH suppresses mitochondrial ATP production by reducing the H+ gradient across mitochondrial membrane. Using permeabilized islets, the increase in pH was shown to decrease Ca2+ efficacy at a clamped concentration of ATP in the exocytotic system. Taken together, DPH inhibits glucose-induced insulin secretion not only by inhibiting mitochondrial ATP production, but also by reducing Ca2+ efficacy in the exocytotic system through its alkalizing effect on cytoplasm.

Regulation and functional effects of ZNT8 in human pancreatic islets

“…This effect is independent of ATP levels that directly affect the exocytotic system. ATP cellular concentration decreased under metabolic stress and remained unaffected by ZNT8 overexpression (Fujimoto et al 2002, Nabe et al 2006. Further work is needed to elucidate the exact mechanism of how ZNT8 activity impacts insulin release but may be related to a more efficient release of noncrystalline insulin.…”

Metformin suppresses hepatic gluconeogenesis and lowers fasting blood glucose levels through reactive nitrogen species in mice

“…Cell lysates were then centrifuged for 3 min at 3,000×g and 4°C, and a fraction (0.4 ml) of the supernatant fraction was mixed with 0.1 ml of 2 mol/l HEPES and 0.1 ml of 1 mol/l Na 2 CO 3 . Adenine nucleotide contents were measured by a luminometric method as previously described [28,29].…”

Src activation generates reactive oxygen species and impairs metabolism–secretion coupling in diabetic Goto–Kakizaki and ouabain-treated rat pancreatic islets

“…Measurement of glucose oxidation Glucose oxidation was measured as previously described [8]. Cultured islets were preincubated in KRBB with 2.8 mmol/l glucose in the presence or absence of Src inhibitor and antioxidants at 37°C for 30 min.…”

“…Since depletion of mitochondrial DNA abolishes the glucose-induced ATP elevation, mitochondria clearly are a major source of ATP production in pancreatic beta cells [2,3]. Glucose-induced insulin secretion from beta cells is often impaired by exposure to high concentrations of fuels including glucose, NEFAs and ketone bodies, and by administration of diabetogenic pharmacological agents, all of which involve impaired glucose-induced ATP elevation in beta cells [4][5][6][7][8][9][10][11]. Thus, reduced mitochondrial ATP production plays an important role in impaired glucoseinduced insulin secretion.…”