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Impaired Insulin Secretion by Diphenyleneiodium Associated with Perturbation of Cytosolic Ca²⁺ Dynamics in Pancreatic β-Cells

Department of Medicine and Clinical Science, Graduate School of Medical Sciences, Kyushu University, Fukuoka 812-8582, Japan

Address all correspondence and requests for reprints to: Masanori Iwase, M.D., Ph.D., Department of Medicine and Clinical Science, Graduate School of Medical Sciences, Kyushu University, Maidashi 3-1-1, Higashi-ku, Fukuoka 812-8582, Japan. E-mail: iwase{at}intmed2.med.kyushu-u.ac.jp.

	Abstract

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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 ([Ca²⁺]_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 Ca²⁺-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 [Ca²⁺]_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.

	Introduction

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DIPHENYLENEIODIUM (DPI), A COMPOUND consisting of two benzene rings attached to a positively charged iodine molecule, was initially identified as a potent hypoglycemic agent (1). This activity was ascribed to the inhibition of mitochondrial oxidation (2), i.e. nicotinamide dinucleotide (NADH)-ubiquinone reductase (complex I) (3), which results in suppression of hepatic gluconeogenesis. However, the toxic effect on mitochondria prevented the use of DPI clinically. Later, DPI was also reported to inhibit neutrophil oxidase, i.e. NADH phosphate (NADPH) oxidase, in a manner similar to the inhibition of complex I (4). The inhibitory actions of DPI are explained by its removal of an electron from flavoproteins such as flavin adenine dinucleotide and the formation of phenyl radicals, which inhibit the catalytic sites of the enzymes (5). DPI is widely used as an uncompetitive inhibitor of flavoenzymes, especially NADPH oxidase, to inhibit the production of reactive oxygen species (ROS) both in vitro and in vivo. In addition to professional phagocytes, NADPH oxidase in nonphagocytic cells has recently attracted significant interest in many aspects of cellular functions and is increasingly reported to be involved in various conditions including tumorigenesis, cardiovascular diseases, and metabolic diseases through increased production of ROS (6). In this context, the beneficial effects of DPI have been experimentally shown in metabolic syndrome (7), hepatic fibrosis (8), cerebral ischemia (9), pancreatitis (10), and the enhancement of glucose uptake in skeletal muscle cells (11).

Oliveira et al. (12) demonstrated the expression of phagocyte-type NADPH oxidase components in rat pancreatic islets, i.e. Nox2 (gp91^phox) and p22^phox as membrane-associated components, as well as the expression of p47^phox, p67^phox, and p40^phox in the cytosol. Furthermore, we identified the expression of multiple Nox isoforms, Nox1, Nox2, and Nox4, as well as homologs of cytosolic subunits, Noxo1 (homolog of p47^phox) and Noxa1 (homolog of p67^phox) in rat islets and β-cell line RINm5F (RIN) cells (13). Although the exact role of NADPH oxidase in β-cells remains obscure, we previously reported that DPI suppressed glucose-stimulated insulin secretion from rat islets in a dose-dependent manner (13). To elucidate the mechanisms of DPI-induced impairment of insulin secretion, we investigated in the present study the effects of DPI on glucose- and nonfuel-stimulated insulin secretion, islet glucose metabolism, and intracellular Ca²⁺ concentration ([Ca²⁺]_i) in β-cells. The results showed that DPI suppressed islet glucose metabolism and ATP content through its known inhibition of complex I in the mitochondrial respiratory chain. Furthermore, DPI altered [Ca²⁺]_i dynamics in response to secretagogues, and DPI per se induced an increase in [Ca²⁺]_i at least in part via the L-type voltage-dependent Ca²⁺ channel (L-VDCC). This increase in [Ca²⁺]_i was associated with a transient increase in insulin secretion and was attenuated by mitochondrial inhibitors or the exogenous addition of low concentrations of H₂O₂.

	Materials and Methods

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Chemicals
DPI, diazoxide, mastoparan, carbamoylcholine chloride (carbachol), thapsigargin, nifedipine, verapamil, SKF96365, rotenone, carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP), antimycin A, ruthenium red, and H₂O₂ were purchased from Sigma-Aldrich (St. Louis, MO). EGTA and Fura 2/AM were obtained from Dojindo (Kumamoto, Japan). 12-O-tetradecanoylphorbol-13-acetate (TPA) and dimethylsulfoxide were purchased from Wako Biochemicals (Osaka, Japan).

Islet isolation and cell culture
Pancreatic tissue was harvested from male Sprague Dawley rats (body weight 250–350 g; Kyudo, Kumamoto, Japan) and islets were isolated by collagenase digestion (14). The islets were handpicked under a stereomicroscope (Leica MZ8; Leica, Heerbrugg, Switzerland) and transferred to RPMI 1640 medium (Sigma-Aldrich) containing 10% fetal bovine serum (Life Technologies, Grand Island, NY), 100 U/ml penicillin, 100 µg/ml streptomycin, and 11.0 mM glucose in 60- x 15-mm Petri dishes (Sumilon, Akita, Japan). All experiments were performed according to the guidelines of the animal experimentation ethics committee of Kyushu University. Radiation-induced rat insulinoma RIN cells were purchased from the American Type Culture Collection (Rockville, MD) and grown at 37 C under a humidified, 5% CO₂ atmosphere in RPMI 1640 medium, supplemented with 10% fetal bovine serum, 25.0 mM glucose, 2.0 mM L-glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin as recommended by the manufacturer. Cells were plated at a density of 2 x 10⁵ per 60- x 15-mm Petri dish and grown to confluence within 7 d. All experiments were performed with cells between passages 38 and 47.

Static insulin secretion
Experiments were carried out in Krebs-Ringer bicarbonate buffer (KRB) containing (in mM) 118.4 NaCl, 4.7 KCl, 1.2 MgSO₄, 1.2 KH₂PO₄, 2.4 CaCl₂, and 20 NaHCO₃ (equilibrated with 95% O₂ and 5% CO₂, pH 7.4) and supplemented with 0.2% BSA (Sigma-Aldrich). Islets (150–200 µm in diameter) cultured for 3 d were picked from the culture dish at random. In glucose-stimulation experiments (n = 4), triplicate batches of five islets were preincubated in KRB containing 3.3 mM glucose at 37 C for 30 min and incubated in KRB in the presence of 3.3 mM glucose with and without 1 or 10 µM DPI for 1 h under continuous shaking and then in the presence of 16.7 mM glucose with and without 1 or 10 µM DPI for another hour. In membrane depolarization experiments (n = 4), triplicate batches of five islets were preincubated for 1 h in KRB containing 3.3 mM glucose with and without 10 µM DPI and then incubated with 250 µM diazoxide with and without high K⁺ plus KRB (in mM: 94.8 NaCl, 28.8 KCl, 1.2 MgSO₄, 1.2 KH₂PO₄, 2.4 CaCl₂, and 20 NaHCO₃) containing 3.3 mM glucose and/or 10 µM DPI for 1 h under continuous shaking. In mastoparan experiments (n = 4), triplicate batches of five islets were washed in Ca²⁺-free KRB containing 1 mM EGTA (Ca²⁺-free KRB/EGTA) and preincubated for 30 min in Ca²⁺-free KRB/EGTA containing 3.3 mM glucose. Islets were incubated in Ca²⁺-free KRB/EGTA in the presence of 3.3 mM glucose with and without 10 µM mastoparan and/or 10 µM DPI for 1 h and then in the presence of 16.7 mM glucose with and without 10 µM mastoparan and/or 10 µM DPI for another hour. In TPA experiments (n = 4), triplicate batches of five islets were preincubated for 1 h in KRB or Ca²⁺-free KRB/EGTA containing 3.3 mM glucose with and without 10 µM DPI and then incubated with and without 500 nM TPA and/or 10 µM DPI in KRB or Ca²⁺-free KRB/EGTA containing 3.3 mM glucose for 1 h. The effect of the acute DPI application (10 min) on insulin secretion was investigated by two successive static incubations with 3.3 or 16.7 mM glucose, with and without 10 µM DPI for a total of 20 min (n = 5). After triplicate batches of five islets were preincubated in KRB containing 3.3 mM glucose at 37 C for 30 min, islets were transferred into another tube and incubated in KRB in the presence of 3.3 or 16.7 mM glucose with and without 10 µM DPI for 10 min under continuous shaking. A batch of five islets was transferred into another tube within approximately 40 sec, and thus islets transfer per a static incubation required less than 10 min. After the first static incubation, islets were transferred into another tube of the same experimental group and incubated for another 10 min until islets were removed. Experiments were always performed in a parallel fashion in islets incubated with and without DPI dissolved in dimethylsulfoxide. After incubation, the medium was removed and analyzed for insulin by RIA (Eiken, Tokyo, Japan).

Measurement of [Ca²⁺]_i
Measurements were carried out in KRB containing (in mM) 128.8 NaCl, 4.8 KCl, 1.2 MgSO₄, 1.2 KH₂PO₄, 2.5 CaCl₂, 5 NaHCO₃, and 10 HEPES (KRBH; equilibrated with 95% O₂, 5% CO₂, and NaOH, pH 7.4), supplemented with 0.1% BSA, as described previously (14). Ca²⁺-free KRBH containing 1 mM EGTA was used to investigate the effect of the absence of extracellular Ca²⁺. Islets were attached to a glass-bottom culture dish (MatTek, Ashland, MA) precoated with 100 µg/ml poly-L-lysine (Sigma-Aldrich) in RPMI 1640-based medium containing 2.8 mM glucose with and without 10 µM DPI for 30 min at 37 C in a 95% O₂-5% CO₂ incubator. The dissociated RIN cells attached to a glass-bottom culture dish in RPMI 1640-based medium containing 25.0 mM glucose for 2 d at 37 C in a 95% O₂-5% CO₂ incubator. The islets and RIN cells were washed gently twice in KRBH containing 2.8 mM glucose, loaded with 3 µM fura 2/AM in KRBH containing 2.8 mM glucose with and without 10 µM DPI and then incubated at 37 C for 30 min in a 5% CO₂ incubator. The loading solution was then removed, and the islets or RIN cells were washed once in KRBH, before being placed on the stage of an inverted microscope (Leica DM IRB) and superfused at 1 ml/min at 37 C with KRBH containing the drugs used in each experiment. The fura 2-loaded single whole cell was illuminated by excitation at 340 nm (F340) and 380 nm (F380) alternately every 8–9 sec in 30 min through a x10 fluorite objective, and the emission signals at 510 nm were detected by a CCD camera (C4742-95-12ER; Hamamatsu Photonics, Hamamatsu, Japan). The F340/F380 ratio image was produced using a dual-excitation microfluorescence system (Aquacosmos version 1.3; Hamamatsu Photonics). β-Cells per islet were confirmed by applying 100 µM tolbutamide (Aventis Pharmaceuticals, Frankfurt, Germany), and RIN cells were confirmed by applying 500 µM carbachol in each experiment. The F340/F380 ratio was used as described for [Ca²⁺]_i by adjusting the baseline ratio as 1.0. In each experiment (n = 3–5), measurements were made in control and DPI-treated islets or RIN cells in a parallel fashion.

Islet glucose oxidation
After preincubation in KRB with 3.3 mM glucose with and without 10 µM DPI for 60 min at 37 C, triplicate batches of 10 islets, 150–200 µm in diameter, were placed into a 1-ml glass cup with 16.7 mM glucose and 37 kBq D-[U-¹⁴C]glucose (Amersham Pharmacia Biotech, Piscataway, NJ) with and without 10 µM DPI in 100 µl KRB (pH 7.4) (15). For mastoparan experiments, Ca²⁺-free KRB/EGTA and 10 µM mastoparan were used. Each cup with its contents was placed in a 20-ml glass scintillation vial, bubbled with 95% O₂ and 5% CO₂, and then sealed airtight with a rubber stopper. The vials were shaken for 90 min at 37 C. Islet glucose metabolism was stopped with 100 µl 0.05 mM antimycin A dissolved in 70% (vol/vol) ethanol, which was injected through the stopper into the cup. In the next step, 250 µl hyamine hydroxide (Packard, Meriden, CT) was injected into the vial. CO₂ was released from the incubation medium by injecting 100 µl 0.4 M Na₂HPO₄ (Wako Biochemicals) (pH 6.0) into the cup. To allow CO₂ trapping by hyamine hydroxide, the vials were shaken for another 120 min at 37 C. In each experiment (n = 3–4), parallel incubations were performed without islets. The cup was removed, and 5 ml scintillation fluid was added to the vial. After vortexing, radioactivity was counted in a liquid scintillation counter (LSC 1000; Aloka, Tokyo, Japan).

Measurement of islet ATP content
ATP content was determined as described previously (14). Briefly, islets were preincubated in KRB containing 3.3 mM glucose at 37 C for 30 min, then triplicate batches of 10 islets, 150–200 µm in diameter, were placed in 0.5 ml KRB with 3.3 or 16.7 mM glucose under continuous shaking for 60 min at 37 C (n = 4). The reaction was stopped by the addition of 0.5 ml trichloroacetic acid at a final concentration of 5%. The tubes were immediately mixed with vortex and then sonicated in ice-cold water for 3 min. They were centrifuged (2000 x g for 3 min), and a fraction (0.7 ml) of the supernatant was mixed with 1 ml water-saturated diethyl ether. The ether phase containing trichloroacetic acid was discarded. This step was repeated four times. The ATP concentration was measured by adding 100 µl luciferin-luciferase solution (Enliten ATP Assay System; Promega, Madison, WI) to a fraction sample (20 µl) in a bioluminometer (MiniLumat LB 9506; Berthold, Bad Wildbad, Germany). To construct a standard curve, blanks and ATP standards were run through the entire procedure, including the extraction steps.

Data analysis
Values are expressed as mean ± SEM. Statistical significance was evaluated by unpaired or paired t test or ANOVA with Scheffé’s F test as a post hoc test. P < 0.05 was considered significant.

	Results

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We investigated the effects of DPI on glucose- or nonfuel-stimulated insulin secretion from rat islets (Figs. 1 and 2). DPI at 1 or 10 µM had no effects on insulin secretion in the presence of 3.3 mM glucose, whereas DPI dose-dependently inhibited insulin secretion stimulated by 16.7 mM glucose as we reported previously (13) (Fig. 1A). DPI at 10 µM totally inhibited glucose-stimulated insulin secretion (percentage of control, 9.2 ± 1.2%; P < 0.001). Moreover, 10 µM DPI significantly suppressed insulin secretion induced by membrane depolarization using high-K⁺ buffer containing diazoxide (Fig. 1B, percentage of control, 36.0 ± 5.3%; P < 0.01). Furthermore, DPI significantly suppressed insulin secretion induced through the activation of protein kinase C (PKC) with 500 nM TPA in both the presence and absence of extracellular Ca²⁺ (Fig. 2A; percentage of control, 30.2 ± 10.6% with extracellular Ca²⁺, P < 0.01; percentage of control, 42.0 ± 4.7% without extracellular Ca²⁺, P < 0.01). As shown in Fig. 2B, however, DPI did not affect insulin release elicited by 10 µM mastoparan, wasp venom, which is known to activate directly the distal exocytotic machinery (16), at 3.3 and 16.7 mM glucose under stringent Ca²⁺-free conditions.

View larger version (10K): [in this window] [in a new window]   FIG. 1. A, DPI at 1 or 10 µM had no effects on insulin secretion from isolated rat islets in the presence of 3.3 mM glucose, whereas 10 µM DPI totally inhibited glucose-stimulated insulin secretion. B, DPI (10 µM) significantly suppressed insulin secretion induced by membrane depolarization using a high-K+ buffer containing diazoxide. Data are mean ± SEM values of four experiments. *, P < 0.01; **, P < 0.001 vs. without DPI by Student’s t test.

View larger version (13K): [in this window] [in a new window]   FIG. 2. A, DPI (10 µM) significantly suppressed insulin secretion induced by PKC activation with 500 nM TPA in both the presence and absence of extracellular Ca2+. B, DPI (10 µM) did not affect insulin release elicited by 10 µM mastoparan at 3.3 and 16.7 mM glucose under stringent Ca2+-free conditions. Data are mean ± SEM values of four experiments. *, P < 0.01 vs. without DPI by Student’s t test.

View larger version (29K): [in this window] [in a new window]   FIG. 3. A, Preincubation for 60 min followed by perifusion with 1 µM DPI did not affect the changes in [Ca2+]i of islet β-cells during stimulation with 16.7 mM glucose. B, Preincubation for 60 min with 10 µM DPI had no effects on [Ca2+]i of islet β-cells stimulated by 16.7 mM glucose. C, Coperifusion of 10 µM DPI with 16.7 mM glucose induced a rapid rise in [Ca2+]i but suppressed its rise during the following period. D, Preincubation for 60 min followed by perifusion with 10 µM DPI consistently increased [Ca2+]i of islet β-cells in the presence of 2.8 and 16.7 mM glucose. E, The 60-min preincubation and perifusion with 10 µM DPI dampened the abrupt increase in [Ca2+]i of islet β-cells by membrane depolarization using high-K+ buffer with diazoxide. Solid line, with DPI; broken line, without DPI. Data are mean values of three to four experiments in each group.

View larger version (11K): [in this window] [in a new window]   FIG. 4. A, DPI (10 µM) markedly suppressed glucose oxidation rate at 16.7 mM glucose. *, P < 0.01 vs. without DPI by Student’s t test. B, ATP content was significantly reduced in islets incubated with 10 µM DPI in the presence of 3.3 or 16.7 mM glucose. *, P < 0.01 vs. without DPI; #, P < 0.01 vs. 3.3 mM glucose without DPI by ANOVA with Scheffé’s F test as a post hoc test. C, Under stringent Ca2+-free conditions, 10 µM DPI also reduced the glucose oxidation rate at 16.7 mM glucose. Mastoparan (10 µM) significantly suppressed the glucose oxidation rate, and DPI had mild additional effects. *, P < 0.01 vs. without DPI and mastoparan; , P < 0.05 vs. mastoparan alone by ANOVA with Scheffé’s F test as a post hoc test. Data are mean ± SEM values of three to four experiments.

View larger version (18K): [in this window] [in a new window]   FIG. 5. A, DPI at 10 or 100 µM significantly increased [Ca2+]i in β-cells of rat islets. B, Ca2+-free buffer significantly suppressed the [Ca2+]i elevation induced by 10 µM DPI in β-cells of rat islets. C, DPI increased [Ca2+]i in a dose-dependent manner in β-cell line RIN cells. D, Ca2+-free buffer also suppressed the DPI-induced elevation in [Ca2+]i in RIN cells. Data are mean values of four to five experiments in each group.

View larger version (26K): [in this window] [in a new window]   FIG. 6. A, Dihydropyridine type L-VDCC Ca2+ channel blocker, 5 µM nifedipine, significantly reduced the increase in [Ca2+]i, as did another phenylalkylamine type L-VDCC blocker, 40 µM verapamil in RIN cells (B). C, Carbachol (500 µM), an acetylcholine analog, significantly increased [Ca2+]i in RIN cells, whereas combined stimulation with DPI and carbachol induced a 4-fold increase compared with carbachol alone. D, Thapsigargin (2 µM), a potent inhibitor of sarcoendoplasmic reticulum Ca2+-type ATPases, completely inhibited the increase in [Ca2+]i induced by carbachol but not that induced by DPI in RIN cells. Data are mean values of four experiments.

View larger version (18K): [in this window] [in a new window]   FIG. 7. Effects of mitochondrial inhibitors on DPI-induced rise in [Ca2+]i in RIN cells. A, Rotenone (10 µM), a selective NADH-dehydrogenase inhibitor in complex I, significantly suppressed the DPI-induced rise in [Ca2+]i. B, Antimycin A (1.0 µM), a complex III inhibitor, also suppressed the rise in [Ca2+]i. C, FCCP (5 µM), a mitochondrial uncoupler, increased [Ca2+]i and significantly prevented DPI-induced rise in [Ca2+]i. D, Ruthenium red (10 µM), an inhibitor of mitochondrial Ca2+ uniporter, tended to lower the peak of [Ca2+]i elevation and delayed recovery from the elevated [Ca2+]i level. Data are mean values of three to four experiments.

View larger version (18K): [in this window] [in a new window]   FIG. 8. H2O2 at 0.1 µM (A) or 1.0 µM (B) together with DPI prevented the rapid rise in [Ca2+]i induced by DPI. H2O2 alone transiently increased [Ca2+]i followed by a sustained elevation. Broken line, DPI alone; dotted line, H2O2 alone; solid line, H2O2 and DPI. Data are mean values of three experiments.

View larger version (12K): [in this window] [in a new window]   FIG. 9. The effect of the acute DPI application (10 min) on insulin secretion was investigated by two successive static incubations with 3.3 or 16.7 mM glucose, with and without 10 µM DPI. DPI significantly enhanced insulin secretion at 3.3 mM glucose for the initial 10 min but did not affect glucose-stimulated insulin secretion. However, in the second static incubation (10 min) after transferring islets into another tube of the same experimental group immediately, the enhancing effect at 3.3 mM glucose disappeared, whereas DPI significantly suppressed glucose-stimulated insulin secretion. Data are mean ± SEM values of five experiments. *, P < 0.05; **, P < 0.01 vs. without DPI by Student’s t test.

	Discussion

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ROS have cytotoxic effects under oxidative stress, whereas at low concentrations, ROS act as secondary messengers and thus might be important for normal cellular function (17). We and others have shown that pancreatic islets express multiple NADPH oxidase components, and their combined expression patterns seem to allow constitutive superoxide production and further stimulation by agonists (12, 13). In fact, islet ROS production was enhanced by high glucose, palmitate, and proinflammatory cytokines, which was completely inhibited by DPI (18). In the present study, low concentrations of H₂O₂ attenuated the increased [Ca²⁺]_i evoked by DPI, suggesting that reduced endogenous H₂O₂ production may contribute to the [Ca²⁺]_i elevation in β-cells. This is theoretically compatible with DPI acting to potently inhibit membrane-bound NADPH oxidase. NADPH oxidase catalyzes the transfer of electrons from NADPH to O₂; NADPH + 2O₂ 2O₂^– + NADP⁺ + H⁺, and H₂O₂ is synthesized with dismutation of O₂^– by superoxide dismutase. In contrast, exogenous H₂O₂ treatment increases [Ca²⁺]_i in a variety of cell types including β-cells (19, 20). As shown in Fig. 8, H₂O₂ transiently increased [Ca²⁺]_i, followed by a sustained elevation (20). H₂O₂ readily permeates through the plasma membrane and inhibits glycolytic and mitochondrial enzymes, leading to a reduction in cytosolic ATP levels and subsequent failure of Ca²⁺ pumping into the endoplasmic reticulum. H₂O₂ releases Ca²⁺ from intracellular stores and then increases Ca²⁺ permeability of the plasma membrane (20). The pattern of [Ca²⁺]_i rise was different between DPI and H₂O₂, with a more rapid onset of [Ca²⁺]_i elevation evoked by DPI, suggesting that the main site of DPI action may be the extracellular compartment. The combined application of DPI and H₂O₂ showed only a slow rise in [Ca²⁺]_i and no fast peak. This may be explained by the addition of H₂O₂, which could compensate for the suppressed production of H₂O₂ by DPI-induced inhibition of NADPH oxidase. However, the reduced ATP contents (Fig. 4B) may cause a slower and sustained increase in [Ca²⁺]_i. Although DPI impairs mitochondrial metabolism, as revealed by reduced glucose oxidation, the contribution of nonmitochondrial metabolism, which is more vulnerable to ROS (20), remains to be determined. In addition, metabolic secretion-coupling factors other than ATP, e.g. citrate and NADPH (21), may be involved in DPI-induced impairment of insulin secretion.

H₂O₂ modulates the catalytic activity of enzymes by redox modification of cysteine residues (22), including the translocation and activation of serine/threonine kinases such as PKC (23). Regarding ion channel modulation by ROS, H₂O₂ modulates L-VDCC activity by altering the reduction state of cysteinyl sulfydryls of the 1c subunit of L-VDCC (24). Recently, Pi et al. (25) reported that 1–4 µM H₂O₂ stimulated insulin secretion in β-cells and that H₂O₂ was endogenously generated in response to glucose, thus contributing to glucose-stimulated insulin secretion. The same group also demonstrated that mitochondrial respiratory chain inhibitors, such as rotenone and antimycin A, increased cellular H₂O₂ levels, which may explain the suppression of DPI-induced [Ca²⁺]_i elevation by mitochondrial inhibitors in the present study. In addition, DPI was reported to completely suppress superoxide generation induced by a PKC activator in islets (12). This might be consistent with DPI suppressing insulin secretion stimulated with TPA under stringent Ca²⁺-free conditions (Fig. 2A). To our knowledge, the effect of DPI on PKC activity has not been reported previously. However, DPI has no effects on the cytosolic components, including p47^phox (4), which is activated by PKC phosphorylation (6). Therefore, DPI seems to influence Ca²⁺ handling via alteration of H₂O₂ rather than interfering with PKC activity.

O’Donnell et al. (5) have reported that the targets of iodonium compounds are flavin-containing enzymes that function in one-electron transfer, and DPI readily reacts with flavin adenine dinucleotide by electron abstraction and leads to the formation of ortho-substituted phenyl radical, which can then form covalent adducts to the flavin or heme moieties directly or to adjacent amino acids near the active site, resulting in inhibition of oxidase activities. DPI inhibits the activity of NADPH oxidase (4), nitric oxidase synthase (26), xanthine oxidase (27), and NADPH cytochrome P450 oxidoreductase (28). DPI is widely used as an uncompetitive inhibitor of flavoenzymes, especially NADPH oxidase, to inhibit the production of ROS, because NADPH oxidase is highly sensitive to DPI (5). Using specific NADPH oxidase inhibitors will be required to study the precise roles of NADPH oxidase in β-cell function, although presently it seems difficult in our hand to specifically inhibit NADPH oxidase activity using molecular techniques partly due to the complex isoforms of its components known to be expressed in β-cells, e.g. Nox isoforms 1, 2, and 4; p47^phox and its homolog Noxo1; and p67^phox and its homolog Noxa1. We reported previously that another NADPH oxidase inhibitor, apocynin, which inhibits p47^phox translocation to the plasma membrane, did not inhibit insulin secretion (13). We speculated that Noxo1 (homolog of p47^phox) might be used instead of p47^phox in ROS production. Nonspecific effects of DPI in our results may be excluded, because mastoparan-induced insulin secretion was not impaired by DPI (Fig. 2B) and the removal of DPI allowed the islets to restore their responses to a subsequent stimulation with high glucose (Fig. 3B). Mastoparan has been used to prime G protein activation in a receptor-independent manner with various cell types. Mastoparan also induced the mitochondrial permeability transition (29) and thus inhibited the glucose oxidation rate (Fig. 4B). Mastoparan-induced insulin secretion remained intact in DPI-treated islets, suggesting that glucose- and Ca²⁺-independent, but mastoparan-sensitive, mechanisms might be operative in these islets.

Some studies have investigated the effect of DPI on ion channels using patch-clamp techniques, although the results are inconsistent. First, carotid body type 1 (glomus) cells (30) and pulmonary smooth muscle cells (31) were studied to investigate whether NADPH oxidase functions as an O₂ sensor. Carotid body cells transmit changes in arterial oxygen pressure to the central nervous system, and pulmonary arteries constrict in response to hypoxia. DPI inhibited inward K⁺ and Ca²⁺ currents in both cell types, and also transiently inhibited voltage-dependent K⁺ channels, but not the ATP-dependent variety. The reduction in K⁺ current led to membrane depolarization and opening of voltage-dependent Ca²⁺ channels, leading to a transient increase in [Ca²⁺]_i. These findings are consistent with our results in β-cells, although the effect of DPI on L-VDCC remains to be verified using patch-clamp techniques. Second, the functional role of NADPH oxidase is extensively studied in neutrophils because of chronic granulomatous disease with genetically defective NADPH oxidase. Stimulation of neutrophils by various receptor agonists increases [Ca²⁺]_i, predominantly via store-operated calcium channels. DPI had no effect on basal [Ca²⁺]_i, but enhanced the increase in [Ca²⁺]_i induced by receptor agonists to 149% of the control levels (32). Third, the effect of DPI on sarcolemmal L-VDCC function in cardiac myocytes was tested to determine whether NADPH oxidase modulates L-VDCC during hypoxia (33). DPI did not alter basal L-VDCC function but enhanced the sensitivity of L-VDCC to isoproterenol. In both neutrophils and cardiac myocytes, DPI enhances Ca²⁺ entry in response to agonists. However, it was recently reported that DPI did not affect the [Ca²⁺]_i rise induced by a cholecystokinin analog in pancreatic acinar cells (10). These differences may be explained by differences in the types of cells used, the experimental conditions, and the expressed isoforms of NADPH oxidase components.

Among mitochondrial inhibitors studied, only FCCP protonophore increased [Ca²⁺]_i, because uncoupling by FCCP collapsed the mitochondrial membrane-potential driving Ca²⁺ uptake and led to the release of Ca²⁺ from mitochondrial stores (34). Furthermore, mitochondrial Ca²⁺ uniporter inhibitor ruthenium red delayed recovery from the elevated [Ca²⁺]_i. This suggests that the DPI-induced increased [Ca²⁺]_i may be taken up by mitochondria, because the latter acts as rapid and reversible Ca²⁺ buffers during cell stimulation (35). Mitochondrial Ca²⁺ uptake induces some functional sequences such as the open probability of the permeability transition pore, which mediates the release of cytochrome c, an apoptosis-inducing factor (35). In fact, DPI was reported to induce apoptosis of several cell types including leukemia cell line (36), endothelial cells (37), and retinal pigment epithelial cells (38). In our preliminary study, the activity of execution protease caspase-3, measured using the caspase-3/CPP32 colorimetric protease assay (39), was significantly enhanced in RIN cells 12 h after incubation with DPI compared with untreated cells (percentage of control, 181.3 ± 18.1%; P < 0.01). This suggests that DPI-induced apoptosis may be associated with enhanced mitochondrial Ca²⁺ uptake.

In conclusion, we demonstrated that DPI, an NADPH oxidase inhibitor, suppressed glucose- and nonfuel-stimulated insulin secretion. DPI suppressed islet glucose metabolism and ATP content and altered [Ca²⁺]_i dynamics in response to high glucose and membrane depolarization. Furthermore, DPI induced an increase in basal [Ca²⁺]_i probably via activated L-VDCC, and this was attenuated by mitochondrial inhibitors as well as by exogenous H₂O₂ at low concentrations. DPI-induced increase in [Ca²⁺]_i was associated with a transient augmentation in basal insulin secretion. Our study suggests that NADPH oxidase may modulate Ca²⁺ signaling in β-cells, potentially reinforcing the importance of H₂O₂ as a signaling molecule in insulin secretion.

	Acknowledgments

 
We are grateful to Prof. H. Sumimoto (Medical Institute of Bioregulation, Kyushu University) for the critical reading our manuscript.

	Footnotes

 
Disclosure Statement: The authors have nothing to disclose.

First Published Online July 10, 2008

¹ H.I. and N.S. contributed equally to this work.

Abbreviations: [Ca²⁺]_i, Intracellular Ca²⁺ concentration; DPI, diphenyleneiodium; FCCP, carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone; KRB, Krebs-Ringer bicarbonate buffer; L-VDCC, L-type voltage-dependent Ca²⁺ channel; NADH, nicotinamide dinucleotide; NADPH, NADH phosphate; PKC, protein kinase C; ROS, reactive oxygen species; TPA, 12-O-tetradecanoylphorbol-13-acetate.

Received February 7, 2008.

Accepted for publication June 30, 2008.

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