Impaired insulin secretion by diphenyleneiodium associated with perturbation of cytosolic Ca²⁺ dynamics in pancreatic β-cells

Pancreatic islets express the superoxide-producing nicotinamide adenine dinucleotide phosphate (NADPH) oxidase system, but its role remains unknown. To address this, we studied the mechanisms of impaired insulin secretion induced by diphenyleneiodium (DPI), an NADPH oxidase inhibitor. We investigated the effects of DPI on glucose- and nonfuel-stimulated insulin secretion, islet glucose metabolism, and intracellular Ca²⁺ concentration ([Ca ²⁺]_i) dynamics in rat islets and β-cell line RINm5F cells. DPI did not affect insulin secretion at 3.3 mM glucose but totally suppressed insulin secretion stimulated by 16.7 mM glucose (percentage of control, 9.2 ± 1.2%; P <0.001). DPI also inhibited insulin release by high K⁺-induced membrane depolarization (percentage of control, 36.0 ± 5.3%; P <0.01) and protein kinase C activation (percentage of control, 30.2 ± 10.6% in the presence of extracellular Ca²⁺, P <0.01; percentage of control, 42.0 ± 4.7% in the absence of extracellular Ca²⁺, P <0.01). However, DPI had no effect on mastoparan-induced insulin secretion at 3.3 and 16.7 mM glucose under Ca ²⁺-free conditions. DPI significantly suppressed islet glucose oxidation and ATP content through its known inhibitory action on complex I in the mitochondrial respiratory chain. On the other hand, DPI altered [Ca ²⁺]_i dynamics in response to high glucose and membrane depolarization, and DPI per se dose-dependently increased [Ca²⁺] _i. The DPI-induced [Ca²⁺]_i rise was associated with a transient increase in insulin secretion and was attenuated by removal of extracellular Ca²⁺, by L-type voltage-dependent Ca²⁺ channel blockers, by mitochondrial inhibitors, or by addition of 0.1 or 1.0 μM H₂O₂ exogenously. Our results showed that DPI impairment of insulin secretion involved altered Ca²⁺ signaling, suggesting that NADPH oxidase may modulate Ca²⁺ signaling in β-cells.