Obligatory Role of Early Ca²⁺ Responses in H₂O₂-Induced β-Cell Apoptosis

Abstract

Our previous study using apoptosis analysis suggested that Ca²⁺ release through inositol 1,4,5-trisphosphate (IP₃) receptors and the subsequent Ca²⁺ influx through store-operated channels (SOCs) constitute a triggering signal for H₂O₂-induced β-cell apoptosis. In the present study, we further examined the obligatory role of early Ca²⁺ responses in β-cell apoptosis induction. H₂O₂ induced elevation of the cytosolic Ca²⁺ concentration ([Ca²⁺]_c) consisting of two phases: an initial transient [Ca²⁺]_c elevation within 30 min and a slowly developing one thereafter. The first phase was almost abolished by 2-aminoethoxydiphenylborate (2-APB), which blocks IP₃ receptors and cation channels including SOCs, while the second phase was only partially inhibited by 2-APB. The inhibition by 2-APB of the second phase was not observed when 2-APB was added 30 min after the treatment with H₂O₂. 2-APB also largely inhibited elevation of the mitochondrial Ca²⁺ concentration ([Ca²⁺]_m) induced by H₂O₂ when 2-APB was applied simultaneously with H₂O₂, but not when applied 30 min after H₂O₂ application. In addition, 2-APB inhibited the release of mitochondrial cytochrome c to the cytosol induced by H₂O₂ when 2-APB was applied simultaneously with H₂O₂ but not 30 min post-treatment. H₂O₂-induced [Ca²⁺]_m elevation and cell death were not inhibited by Ru360, an inhibitor of the mitochondrial calcium uniporter (MCU). These results suggest that the H₂O₂-induced initial [Ca²⁺]_c elevation, occurring within 30 min and mediated by Ca²⁺ release through IP₃ receptors and subsequent Ca²⁺ influx through SOCs, leads to [Ca²⁺]_m elevation, possibly through a mechanism independent of MCU, thereby inducing cytochrome c release and consequent apoptosis.

Apoptosis of pancreatic β-cells is postulated to be a common feature of both type 1 and type 2 diabetes.^1,2) The ability to secrete adequate amounts of insulin depends on β-cell function and mass. Decreased β-cell mass and increased β-cell apoptosis in patients with type 2 diabetes have been demonstrated.^2,3) Chronic exposure to elevated levels of glucose and free fatty acids is thought to induce β-cell apoptosis.^1,4)

During the development of type 2 diabetes, oxidative stress due to reactive oxygen species (ROS) is likely to play a central role in β-cell dysfunction. ROS are produced not only in activated macrophages infiltrated into pancreatic islets, but also in β-cells exposed to various stimuli, including cytokines,⁵⁾ hyperglycemia,^6,7) and free fatty acids.⁸⁾ Among ROS, hydrogen peroxide (H₂O₂) is often used as an apoptosis inducer. Because the expression of catalase and glutathione peroxidase in β-cells is relatively low,^9,10) β-cells are likely to be highly sensitive to oxidative stress. H₂O₂-induced apoptosis has been reported in the β-cell lines MIN6N8a¹¹⁾ and RINm5F,¹²⁾ and the involvement of Ca²⁺ release from intracellular stores in H₂O₂-induced cell death has been suggested in MIN6N8a cells.¹¹⁾ H₂O₂ has been reported to elicit Ca²⁺ influx through store-operated channels (SOCs) in various cell types, such as HEK-293 cells,¹³⁾ endothelial cells,¹⁴⁾ and human platelets,¹⁵⁾ and to increase Ca²⁺ release from intracellular Ca²⁺ stores and Ca²⁺ permeability through the plasma membrane in isolated rat pancreatic β-cells.¹⁶⁾

In our previous study in INS-1 β-cells, we showed that H₂O₂-induced apoptosis was abolished by 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid-acetoxymethyl ester (BAPTA/AM), a chelator of intracellular Ca²⁺; by 2-aminoethoxydiphenylborate (2-APB), a blocker of inositol 1,4,5-trisphosphate (IP₃) receptors and cation channels, including SOCs; and by xestospongin D, a blocker of IP₃ receptors and was partially blocked by SKF-96365, a blocker of nonselective cation channels, including SOCs.¹⁷⁾ Thus, the H₂O₂-induced apoptosis of INS-1 β-cells is likely to be mediated by Ca²⁺ release form the endoplasmic reticulum (ER) via IP₃ receptors, and partly by Ca²⁺ influx through cation channels on the plasma membrane in INS-1 β-cells. Interestingly, the inhibitory effect of 2-APB on H₂O₂-induced apoptosis was largely attenuated when 2-APB was added 30 min after H₂O₂ application. Thus, the initial increase in the cytosolic Ca²⁺ concentration ([Ca²⁺]_c) might be obligatory for the induction of β-cell apoptosis by H₂O₂. In this paper, we present additional evidence for the obligatory role of the initial [Ca²⁺]_c elevation in H₂O₂-induced β-cell apoptosis and propose a signaling mechanism linking the initial [Ca²⁺]_c elevation and the induction of apoptosis.

MATERIALS AND METHODS

Materials

Hydrogen peroxide (H₂O₂; 30% solution), NaBH₄, and oligomycin were purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). Gadolinium(III) chloride hexahydrate (Gd³⁺), BAPTA/AM, fura-PE3-acetoxymethyl ester (fura-PE3/AM), cremophor EL, carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP), and the goat anti-mouse immunoglobulin G (IgG) horseradish peroxidase (HRP) antibody were from Sigma-Aldrich (St. Louis, MO, U.S.A.). Fetal bovine serum (FBS) was from Tissue Culture Biologicals (Tulare, CA, U.S.A.) or Life Technologies (Carlsbad, CA, U.S.A.). Penicillin G potassium and streptomycin sulfate were from Meiji Seika (Tokyo, Japan). 2-APB and Ru360 were from Merck Millipore (Darmstadt, Germany). Rhod-2/AM was from Dojindo Laboratories (Kumamoto, Japan). The anti-cytochrome c monoclonal antibody was from Abcam (Cambridge, U.K.); the anti-β-tubulin monoclonal antibody and anti-rabbit IgG HRP antibody were from Cell Signaling (Danvers, MA, U.S.A.). SuperSignal West Femto Maximum Sensitivity Substrate was from Thermo Fisher Scientific (Waltham, MA, U.S.A.). Can Get Signal Immunoreaction Enhancer Solution was from Toyobo (Osaka, Japan). Chemicals were dissolved in either purified water or dimethyl sulfoxide (DMSO) and stored at −20°C for later use. The stock solutions were dissolved in RPMI-1640 supplemented with 10 mM N-(2-hydroxyethyl)piperazine-N′-2-ethanesulfonic acid (HEPES), 1 mM sodium pyruvate, 50 µM β-mercaptoethanol, 100 µg/mL streptomycin, 100 U/mL penicillin G, 0.3 mg/mL L-glutamine, and 10% FBS before each experiment. The vehicle had little or no effect on any of the variables measured.

Cell Culture

INS-1 cells, a gift from Dr. C. Wollheim (University Medical Center, Geneva, Switzerland), were cultured in 5% CO₂/95% air at 37°C in the supplemented RPMI-1640 medium according to the method described by Asfari et al.¹⁸⁾ The medium was replaced every third day. All experiments were performed in INS-1 cells between the 18th and 48th passages.

Measurement of [Ca²⁺]_c

[Ca²⁺]_c was measured in fura-PE3-loaded cells using dual-wavelength fluorometry. INS-1 cells were incubated with 4 µM fura-PE3/AM for 2.5 h at 37°C, placed in a chamber mounted on the stage of an inverted microscope (ECLIPSE Ti; Nikon, Tokyo), and continuously superfused with HEPES-Krebs buffer solution (HK solution: in mM, 129 sodium chloride (NaCl), 4.7 potassium chloride (KCl), 1.2 magnesium sulfate (MgSO₄), 1 calcium chloride (CaCl₂), 1.2 potassium dihydrogen phosphate (KH₂PO₄), 10 HEPES, 5 sodium bicarbonate (NaHCO₃), 0.1% bovine serum albumin (BSA), 2.8 glucose; pH 7.4 with sodium hydroxide (NaOH); 37°C) at a flow rate of 1 mL/min. The fura-PE3 fluorescence was measured with an AQUACOSMOS system (Hamamatsu Photonics, Shizuoka, Japan), with alternating excitation of cells at 340 and 380 nm and monitoring of the resultant emission at 510 nm. Pairs of fluorescence images at 340 and 380 nm were captured every 20 s and were converted to the 340/380 ratio images. The 340/380 ratio was used to determine the relative changes in [Ca²⁺]_c. Drugs were applied in the HK solution used for superfusion.

Measurement of the Mitochondrial Ca²⁺ Concentration ([Ca²⁺]_m)

[Ca²⁺]_m was measured in rhod-2-loaded cells. In order to assess mitochondrial Ca²⁺ specifically, rhod-2/AM was reduced to dihiydro-rhod-2/AM by adding NaBH₄ just before loading, in accordance with the manufacturer’s protocol. The cells were loaded with 2 µM dihydro-rhod-2/AM and 0.03% cremophor EL for 1–2 h at 37°C, placed in a chamber mounted on the stage of an inverted microscope (ECLIPSE Ti; Nikon, Tokyo), and continuously superfused with HK solution at a flow rate of 1 mL/min. Fluorescence images were acquired with an AQUACOSMOS system (Hamamatsu Photonics), with excitation at 540 nm and emission at 580 nm every 8 s. The relative changes in the fluorescence intensity were used as a measure of [Ca²⁺]_m.

Measurement of Cytochrome c Release

Cytochrome c release was assessed by Western blot analysis of the cytosolic fraction. INS-1 cells were collected after treatment with H₂O₂ for 18 h. The cytosolic fraction was prepared using a MitoSciences Cell Fractionation Kit (Abcam) according to the manufacturer's instructions, and protein levels were assessed using a BCA protein assay kit (Thermo Fisher Scientific). For Western blot analyses, the same amount of each protein extract was applied for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The protein extract was fractionated by SDS-PAGE and electroblotted onto a polyvinylidene difluoride (PVDF) membrane using a SNAP id system (Merck Millipore). Membranes were blocked for 20 s with Blocking One (Nacalai Tesque, Kyoto, Japan) in Tris-buffered saline containing Tween 20 (TBS-T) and then incubated with a 1 : 1000 dilution of anti-cytochrome c or anti-β-tubulin antibodies for 10 min at room temperature. The secondary antibody was HRP-conjugated anti-mouse IgG or goat anti-rabbit IgG used at a 1 : 800 or 1 : 1000 dilution, respectively. All washes were performed in TBS-T. Blots were visualized using the SuperSignal West Femto Maximum Sensitivity Substrate and recorded with an ImageQuant LAS 4000 (GE Healthcare, Uppsala, Sweden). The signal intensity was quantified using MultiGauge software (FUJIFILM, Tokyo, Japan).

Cell Viability Assay

Cell viability was assessed with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) viability assays using Cell Counting Kit-8 (Dojindo Laboratories). A suspension of INS-1 cells (100 µL/well) was seeded in a 96-well plate, and the cells were pre-incubated in a humidified 5% CO₂ incubator at 37°C. Cell Counting Kit-8 solution (10 µL) was added to each well of the plate 18 h after H₂O₂ application. The absorbance at 450 nm was measured at time 0. The cells were incubated for 2 h in the incubator, and the absorbance at 450 nm was measured again. The difference between the absorbance at 2 h and the absorbance at time 0 was used as a measure of cell viability.

Statistical Analysis

Data were expressed as the mean±standard error of the mean (S.E.M.) Results were compared using Dunnett’s multiple comparison test. A p-value of less than 0.05 was considered statistically significant.

RESULTS

Effects of 2-APB and Gd³⁺ on H₂O₂-Induced [Ca²⁺]_c Elevation

H₂O₂ (100 µM) increased [Ca²⁺]_c in INS-1 cells. The increase occurred in two phases: an initial transient [Ca²⁺]_c elevation within around 30 min and a slowly developing elevation thereafter (Fig. 1A). 2-APB (100 µM), which blocks IP₃ receptors and non-selective cation channels including SOCs, significantly inhibited both the first phase and second phase of the [Ca²⁺]_c elevation; however, the effect was somewhat different, i.e., 2-APB almost abolished the first phase, while it only partially inhibited the second phase (Figs. 1A, B). The contribution of Ca²⁺ influx through non-selective cation channels to the [Ca²⁺]_c elevation was further confirmed by using Gd³⁺, which blocks cation channels but not IP₃ receptors. Gd³⁺ (10 µM), also significantly inhibited the first and second phases, although the inhibition by Gd³⁺ was less than that by 2-APB (Figs. 1A, B). These results suggest that Ca²⁺ release via IP₃ receptors and Ca²⁺ influx via non-selective cation channels, most probably SOCs, are involved in H₂O₂-induced apoptosis of INS-1 cells.

Fig. 1. Effects of 2-APB and Gd³⁺ on H₂O₂-Induced [Ca²⁺]_c Elevation

Typical traces of relative changes in [Ca²⁺]_c after the addition of H₂O₂ (100 µM; time 0) in the absence and presence of 2-APB (100 µM) or Gd³⁺ (10 µM) in INS-1 cells loaded with fura-PE-3 (A). 2-APB or Gd³⁺ was added at time 0. Relative changes in [Ca²⁺]_c were measured at 10 and 50 min after the addition of H₂O₂ (B). The bar graphs show the mean±S.E.M. of 77–99 cells from two experiments. ** p<0.01, * p<0.05.

Time Dependent Effect of 2-APB on H₂O₂-Induced [Ca²⁺]_c Elevation

Our previous study showed that the inhibitory effect of 2-APB on H₂O₂-induced apoptosis was largely attenuated when 2-APB was added 30 min after treatment with H₂O₂.¹⁷⁾ We therefore performed similar experiments on [Ca²⁺]_c measurements. 2-APB or BAPTA/AM, a chelator of intracellular Ca²⁺, was applied 30 min after the exposure to H₂O₂ (100 µM). With this protocol, 2-APB did not inhibit the H₂O₂-induced [Ca²⁺]_c elevation, while BAPTA/AM completely inhibited it (Figs. 2A, B).

Fig. 2. Time Dependent Effect of 2-APB on H₂O₂-Induced [Ca²⁺]_c Elevation

Typical traces of relative changes in [Ca²⁺]_c after the addition of H₂O₂ (100 µM; time 0). 2-APB (100 µM) or BAPTA/AM (10 µM) was applied 30 min after the addition of H₂O₂ (A). Relative changes in [Ca²⁺]_c were measured at 10 and 50 min after the addition of H₂O₂ (B). Bar graphs show the mean±S.E.M. of 100–182 cells from two experiments. ** p<0.01.

Time-Dependent Effect of 2-APB on H₂O₂-Induced [Ca²⁺]_m Elevation

H₂O₂ (100 µM) induced a gradual increase in [Ca²⁺]_m (Fig. 3A). We confirmed that subsequent exposure to FCCP plus oligomycin, which diminishes the membrane potential of mitochondria, decreased [Ca²⁺]_m. The simultaneous application of 2-APB (100 µM) with H₂O₂ significantly inhibited the [Ca²⁺]_m elevation (Figs. 3B, D). However, when 2-APB (100 µM) was added 30 min after H₂O₂ application, it had no apparent inhibitory effect on the [Ca²⁺]_m elevation (Figs. 3C, D). These results suggest that the H₂O₂-induced, 2-APB-senstive transient [Ca²⁺]_c elevation leads to a slow increase in [Ca²⁺]_m.

Time-Dependent Effect of 2-APB on H₂O₂-Induced Cytochrome c Release

We next examined the effect of 2-APB on cytochrome c release from mitochondria, a biochemical marker of apoptosis. The mass of living cells was apparently decreased by treatment with H₂O₂ (100 µM) for 18 h (Fig. 4A). 2-APB (100 µM) inhibited the effect of H₂O₂ on cell mass when simultaneously applied with H₂O₂, but had little effect when applied 30 min after H₂O₂ application (Fig. 4A). In the cells in each condition, the release of cytochrome c into the cytosol was measured. Cytochrome c release was nearly abolished by 2-APB (100 µM) when 2-APB was added simultaneously with H₂O₂. However, when 2-APB was applied 30 min after H₂O₂ application, cytochrome c release was not inhibited, but rather tended to increase (Fig. 4B).

Fig. 3. Time-Dependent Effect of 2-APB on H₂O₂-Induced [Ca²⁺]_m Elevation

Changes in [Ca²⁺]_m after the addition of H₂O₂ (100 µM) in INS-1 cells loaded with dihydro-rhod-2 (A–C). 2-APB (10 µM) was added simultaneously with H₂O₂ (B) or 30 min after the addition of H₂O₂ (C). Vertical bars represent the S.E.M. The effect of 2-APB on the H₂O₂-induced [Ca²⁺]_m elevation was assessed by measuring the relative changes in [Ca²⁺]_m at 80 min (D). The values were normalized to the averaged value of [Ca²⁺]_m at 80 min in panel A. The bar graphs show the mean±S.E.M. of 12–29 cells from three experiments. *** p<0.001.

Effects of Ru360 on [Ca²⁺]_m and Cell Death

Finally, the contribution of the mitochondrial Ca²⁺ uniporter (MCU) to the H₂O₂-induced [Ca²⁺]_m elevation was investigated using Ru360, an MCU inhibitor. Surprisingly, Ru360 (10 µM) had little effect on the [Ca²⁺]_m elevation induced by H₂O₂ (100 µM; Fig. 5B), suggesting little involvement of MCU in H₂O₂-induced β-cell apoptosis. Therefore, we further investigated the effect of Ru360 on H₂O₂-induced cell death in INS-1 cells using the MTT assay. Cell death induced by exposure to H₂O₂ (100 µM) for 18 h was significantly reduced by 2-APB (100 µM), but not by Ru360 (10 µM; Fig. 5C).

Fig. 4. Time-Dependent Effect of 2-APB on H₂O₂-Induced Cytochrome c Release

Typical images of INS-1 cells after exposure without (a) and with H₂O₂ (100 µM; b–d) for 18 h (A). 2-APB (100 µM) was added simultaneously with H₂O₂ (c) or 30 min after the addition of H₂O₂ (d). Cytochrome c release in the cells shown in panel A was analyzed by Western blotting with an anti-cytochrome c antibody (B). Similar results were obtained in three independent experiments.

Fig. 5. Effects of the MCU Inhibitor Ru360 on H₂O₂-Induced [Ca²⁺]_m Elevation and Cell Death

Typical traces of the changes in [Ca²⁺]_m after the addition of H₂O₂ (100 µM) in INS-1 cells loaded with dihydro-rhod-2 in the absence (A) and presence of Ru360 (10 µM; B). Ru360 was added 30 min before the addition of H₂O₂. Vertical bars represent the S.E.M. H₂O₂-induced cell death was assessed with the MTT assay (C). The values were normalized to control values from cells without H₂O₂. The bar graphs show the mean±S.E.M. of 13–26 cells from two experiments. *** p<0.001.

DISCUSSION

Our previous study using apoptosis analysis suggested that an H₂O₂-induced, IP₃ receptor-mediated initial [Ca²⁺]_c elevation, occurring within 30 min, functions as a trigger for β-cell apoptosis. The conclusion is supported by the following findings: The inhibitory effect of 2-APB or BAPTA/AM, a chelator of intracellular Ca²⁺, on H₂O₂-induced β-cell apoptosis was largely attenuated when the compounds were added 30 min after start of the H₂O₂ treatment. In addition, in β-cells treated with H₂O₂ for 30 min, apoptosis observed after 18 h was comparable to that in cells treated with H₂O₂ for 18 h.¹⁷⁾ The present study provides further evidence for the obligatory role of an initial [Ca²⁺]_c elevation in H₂O₂-induced β-cell apoptosis. The initial transient [Ca²⁺]_c elevation, gradually developing [Ca²⁺]_m elevation, and cytochrome c release induced by H₂O₂ were all inhibited by 2-APB when the inhibitor was applied simultaneously with H₂O₂. In contrast, the inhibitory effects were not observed when 2-APB was applied 30 min after H₂O₂ application. Moreover, a larger, gradually developing [Ca²⁺]_c elevation, observed from around 30 min after H₂O₂ application, was less sensitive to 2-APB. The findings that BAPTA/AM abolished the [Ca²⁺]_c elevation (the present study) but did not inhibit the apoptosis (the previous study¹⁷⁾) when applied 30 min after H₂O₂ exposure also support the less possible involvement of the second phase [Ca²⁺]_c elevation in H₂O₂-induced β-cell apoptosis. These results suggest that the H₂O₂-induced initial transient elevation of [Ca²⁺]_c tightly couples to [Ca²⁺]_m elevation, thereby inducing cytochrome c release and consequent apoptosis. However, H₂O₂-induced [Ca²⁺]_m elevation and cell death were not affected by the MCU inhibitor Ru360, implying little involvement of MCU in the Ca²⁺ coupling between the ER and mitochondria.

The H₂O₂-induced [Ca²⁺]_c elevation occurred in two phases. The first transient phase, observed within 30 min after H₂O₂ application, was almost abolished by 2-APB and was partly inhibited by Gd³⁺. These results are consistent with our previous study, which showed that H₂O₂-induced β-cell apoptosis was abolished by 2-APB or xestospongin D, an IP₃ receptor inhibitor, and partially inhibited by SKF-96365, an inhibitor of cation channels, including SOCs. Thus, IP₃ receptor-mediated Ca²⁺ release from the ER and subsequent Ca²⁺ influx through SOCs are likely to induce the transient [Ca²⁺]_c elevation. The present results, showing that both the H₂O₂-induced [Ca²⁺]_m elevation and cytochrome c release were inhibited by 2-APB, are further evidence for the obligatory role of the initial [Ca²⁺]_c elevation in H₂O₂-induced β-cell apoptosis.

The cytochrome c release observed after H₂O₂ application was inhibited by 2-APB, suggesting that Ca²⁺ release through IP₃ receptors on the ER occurs upstream of cytochrome c release from mitochondria in H₂O₂-induced β-cell apoptosis. In many apoptosis models, cytochrome c, a component of the electron transfer system in mitochondria, is released from mitochondria during the induction of apoptosis. Released cytochrome c binds to apoptotic protease activating factor-1(APAF-1) and forms an apoptosome complex that triggers apoptosis.¹⁹⁾ The elevation of [Ca²⁺]_m appears to induce the formation of the mitochondrial permeability transition pore (PTP), a large proteinaceous complex localized to contact sites between the inner (IMM) and outer mitochondrial membranes (OMM) that mediates OMM permeabilization. Cytochrome c can be released from mitochondria through the PTP.²⁰⁾ In addition, the elevation of [Ca²⁺]_m appears to promote the dissociation of cytochrome c from cardiolipin, an anionic phospholipid n the IMM, through which cytochrome c binds to the IMM. The dissociation is a crucial step in cytochrome c release from mitochondria.²⁰⁾ Cytochrome c also appears to be released through a pathway involving pro-apoptotic proteins such as Bax, Bak, and tBid, in which pore formation is triggered by the tBid-induced oligomerization of Bak or Bax.²⁰⁾ Because H₂O₂-induced β-cell apoptosis is accompanied by [Ca²⁺]_m elevation, the former mechanism involving PTP is likely involved.

Interestingly, the ER and mitochondria interact structurally and functionally. Specific subdomains, known as mitochondria-associated membranes (MAM), function in lipid synthesis and in Ca²⁺ transmission between the ER and mitochondria.²¹⁾ The ER-mitochondrial contacts have been visualized, and localized Ca²⁺ changes have been monitored using drug-inducible fluorescent interorganelle linkers in living cells, providing direct evidence for the existence of high-Ca²⁺ microdomains between the ER and mitochondria.²²⁾ It is thus likely that ER-mitochondria coupling is important for Ca²⁺ signaling in apoptosis. The present data, showing that H₂O₂-induced [Ca²⁺]_m elevation and cytochrome c release were inhibited by 2-APB, might support this notion.

For elevations in [Ca²⁺]_m, Ca²⁺ must cross two mitochondrial membranes, the OMM and IMM. Voltage-dependent anion channels are responsible for Ca²⁺ permeability in the OMM.²³⁾ In the IMM, the MCU complex has a critical role in Ca²⁺ permeability. The MCU complex consists of the channel-forming subunit MCU and its regulators, which include mitochondrial Ca²⁺ uptake 1 (MICU1) and mitochondrial Ca²⁺ uniporter regulator 1 (MCUR1), among others.²⁴⁾ A crucial role for MCU in glucose-induced [Ca²⁺]_m elevation in β-cells has been reported.²⁵⁾ The involvement of MCU was investigated using the MCU selective inhibitor Ru360, which has greater membrane permeability than another MCU inhibitor, Ruthenium red.^26,27) Surprisingly, Ru360 did not inhibit H₂O₂-induced [Ca²⁺]_m elevation and cell death in INS-1 cells. The same concentration of Ru360 has been shown to inhibit IL-1β-induced increases in [Ca²⁺]_m in RINm5F β-cells.²⁸⁾ The [Ca²⁺]_m increase was observed at 12 and 24 h of incubation with IL-1β, but was not induced until 8 h, indicating delayed entry of Ca²⁺ into mitochondria. The spatio-temporal mechanism for [Ca²⁺]_m elevation might be different in IL-1β- and H₂O₂-induced apoptosis. The mechanism underlying the H₂O₂-induced elevation of [Ca²⁺]_m in β-cells remains unclear, and further investigation is needed.

When 2-APB was added 30 min after the addition of H₂O₂, the release of cytochrome c tended to increase. In addition to inhibiting IP₃ receptors and cation channels, 2-APB acts as a potent uncoupling agent for gap junction channels formed by certain connexins, in particular connexin36 (Cx36) and Cx50.²⁹⁾ Cx36, which forms gap junctions between β-cells in pancreatic islets, has been shown to protect mouse β-cells against cytotoxic drugs and cytokines that induce apoptosis.³⁰⁾ Thus, 2-APB might enhance apoptosis by inhibiting gap junctions under some conditions.

In summary, the results of this study support the hypothesis proposed in our previous study: IP₃ receptor-mediated Ca²⁺ release from the ER and subsequent store-operated Ca²⁺ entry play an obligatory role in H₂O₂-induced β-cell apoptosis. An initial transient [Ca²⁺]_c elevation leads to prolonged [Ca²⁺]_m elevation, probably through a mechanism independent of MCU, thereby inducing cytochrome c release and consequent apoptosis.

Acknowledgments

This study was supported by a Grant-in-Aid for Scientific Research (C) from Japan Society for the Promotion of Science. The authors thank Mr. M. Takada and Mrs. Y. Sayama for their technical assistance. Some materials were supplied by Chugai Pharmaceutical Co., Ltd.

Conflict of Interest

Taiji Sato is an employee of Chugai Pharmaceutical Co., Ltd. The other authors declare no conflict of interest.

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