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cGMP-independent anti-apoptotic effect of nitric oxide on thapsigargin-induced apoptosis in the pancreatic β-cell line INS-1
Life Sciences 83 (2008) 865–870
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Life Sciences j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / l i f e s c i e
cGMP-independent anti-apoptotic effect of nitric oxide on thapsigargin-induced apoptosis in the pancreatic β-cell line INS-1 Akiko Noguchi a, Masahiro Takada a, Koichi Nakayama b, Tomohisa Ishikawa a,⁎ a b
Department of Pharmacology, Graduate School of Pharmaceutical Sciences, University of Shizuoka, 52-1 Yada, Suruga-ku, Shizuoka City, Shizuoka 422-8526, Japan Department of Molecular and Cellular Pharmacology, Faculty of Pharmaceutical Sciences, Iwate Medical University, 2-1-1 Nishitokuta, Yahaba, Iwate 028-3694, Japan
a r t i c l e
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Article history: Received 13 June 2008 Accepted 3 October 2008 Keywords: Nitric oxide Apoptosis Thapsigargin cGMP INS-1
a b s t r a c t Aims: Low concentrations of nitric oxide (NO) produced by constitutive NO synthase (cNOS) in pancreatic β-cells have been suggested to be a physiological regulator of insulin secretion. In contrast, excessive NO produced by inducible NO synthase is known to mediate β-cell apoptosis. The aim of the present study was to investigate the effect of low concentrations of NO on β-cell apoptosis. Main methods: Apoptosis of the pancreatic β-cell line INS-1 was quantitatively determined by Annexin V flow cytometry. Key findings: The 24-h incubation with 1 mM DETA/NO, a long half-life NO donor, induced β-cell apoptosis, which was insensitive to the soluble guanylate cyclase (sGC) inhibitor ODQ. In contrast, DETA/NO at lower concentrations until 300 μM concentration-dependently decreased the apoptosis induced by thapsigargin, an inhibitor of endoplasmic reticulum Ca2+-ATPase. ODQ did not affect the anti-apoptotic effect of DETA/NO. Moreover, neither the cGMP analogue 8-Br-cGMP nor the sGC activator YC-1 mimicked the anti-apoptotic effect of DETA/NO. Significance: These results suggest that low levels of NO protect β-cells from thapsigargin-induced apoptosis in a cGMP-independent manner. © 2008 Elsevier Inc. All rights reserved.
Introduction Apoptosis of pancreatic β-cells is now postulated to be a common feature of type 1 and type 2 diabetes (Donath et al., 2003; Cnop et al., 2005). In type 1 diabetes, the destruction of β-cells is due predominantly to autoimmunity through the action of pro-inflammatory cytokines such as interleukin (IL)-1β, interferon (IFN)-γ, and tumor necrosis factor (TNF)-α, which are produced by immune cells infiltrated into the islets (Eizirik and Mandrup-Poulsen, 2001). IL-1β, in combination with IFN-γ and/or TNF-α, activates the transcription factor nuclear factor (NF)-κB, which regulates a number of genes in β-cells, including that encoding inducible nitric oxide synthase (iNOS) (Cardozo et al., 2001). Although the precise role of nitric oxide (NO) in the development of type 1 diabetes remains to be fully elucidated, the excessive NO produced by iNOS is likely to account for some of the deleterious effects of IL-1β in β-cells (Kaneto et al., 1995; Darville and Eizirik, 1998). Type 2 diabetes results from both a progressive defect in β-cell insulin secretion and insulin sensitivity. The ability to secrete adequate amounts of insulin depends on β-cell function and mass. Recent studies have shown a significant reduction in β-cell mass and an increase in β-cell apoptosis in type 2 diabetic subjects (Sakuraba et al., 2002; Butler et al., 2003). These findings imply a deficiency of β-cell mass in type 2 diabetes,
⁎ Corresponding author. Tel.: +81 54 264 5694; fax: +81 54 264 5696. E-mail address: [email protected] (T. Ishikawa). 0024-3205/$ – see front matter © 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.lfs.2008.10.002
secondary to increased β-cell apoptosis. Elevated levels of glucose and free fatty acids have been assumed to induce β-cell apoptosis in type 2 diabetes (Donath et al., 2003; Cnop et al., 2005). Constitutive NO synthase (cNOS) produces a small amount of NO in response to an increase in intracellular Ca2+ concentration ([Ca2+]i). We have previously shown that NO is produced by cNOS in rat pancreatic islets in response to elevated glucose concentrations (Nakada et al., 2003), and that NO in the nM range of concentrations, which is assumed to be produced by cNOS, exerts stimulatory and inhibitory effects at low and high concentrations, respectively, on glucose-induced insulin secretion from rat pancreatic β-cells (Kaneko et al., 2003). Thus, NO produced by cNOS is suggested to function as an endogenous regulator of insulin secretion. The contribution of cNOS-produced NO to the regulation of β-cell mass, however, remains to be fully elucidated, although excessive NO produced by iNOS is well known to cause β-cell death (Kaneto et al., 1995; Darville and Eizirik, 1998; Kutlu et al., 2003). Low concentrations of NO have recently been shown to protect from apoptosis in endothelial cells (Sata et al., 2000), hepatocytes (Kim et al., 1997), and neurons (Ciani et al., 2002). The anti-apoptotic mechanisms of NO are divided into cGMP-dependent and cGMP-independent mechanisms, which are likely cell-type specific (Choi et al., 2002). In the β-cell line RINm5F, 10 μM DETA/NO, an NO donor, has been shown to inhibit apoptotic signals, such as cytochrome c release from mitochondria, caspase-3 activation, and Bcl-2 downregulation, induced by serum deprivation (Tejedo et al., 2001, 2004). However, the involvement of cGMP in the anti-apoptotic effect of NO in β-cells remains to be fully
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evaluated. The present study was therefore designed to investigate the effect of low concentrations of NO on β-cell apoptosis and the involvement of cGMP in it by quantitative analysis with flow cytometry. β-Cell apoptosis was induced by endoplasmic reticulum (ER) stress, which has been shown to be induced by glucotoxicity and lipotoxicity during the development of type 2 diabetes (Donath et al., 2003; Cnop et al., 2005). The present data show that low concentrations of NO protect β-cells from ER stress-induced apoptosis in a cGMP-independent manner. Materials and methods Materials 8-Bromoguanosine 3′,5′-cyclic monophosphate sodium salt monohydrate (8-Br-cGMP), diethylenetriamine nitric oxide adduct (DETA/NO), NGnitro-L-arginine (L-NNA), 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), ribonuclease A from bovine pancreas (RNase A), thapsigargin, and 3-(5′-Hydroxymethyl-2′-furyl)-1-benzyl indazole (YC-1) were obtained from Sigma (St. Louis, MO); [N-(3-aminomethyl)benzyl]acetamidine dihydrochloride (1400 W) was from Alexis Biochemicals (San Diego, CA); Annexin-V-Fluos was from Roche Diagnostics GmbH (Mannheim, Germany); fetal bovine serum (FBS) was from Tissue Culture Biologicals (Tulare, CA); penicillin G potassium, streptomycin sulfate were from Meiji Seika (Tokyo, Japan); 3,8-diamino-5-(3-diethylaminopropyl) 6phenyl-phenanthridinium iodide methiodide (propidium iodide; PI) were from Dojindo (Kumamoto, Japan); and TriDye™ 2-Log DNA Ladder (0.1– 10.0 kb) was from New England BioLabs (Ipswich, MA). We dissolved 8-Br-cGMP and 1400 W in purified water as a 100 mM solution; thapsigargin in DMSO as a 1 mM solution; ODQ and YC-1 in DMSO as a 10 mM solution; DETA/NO in 10 mM NaOH as a 1 M solution, which were stored at −20 °C for later use. The stock solutions were dissolved in RPMI 1640 medium supplemented with 10 mM HEPES, 1 mM sodium pyruvate, 50 μM 2-mercaptoethanol, 100 μg/ml streptomycin, 100 U/ml penicillin G, 0.3 mg/ml glutamine, and 10% fetal bovine serum (FBS), before each experiment. Cell culture INS-1 cells, a gift from Dr. C. Wollheim (University Medical Center, Geneva, Switzerland), were cultured at 5% CO2/95% air and 37 °C in the supplemented RPMI 1640 medium. The medium was replaced every third day (Asfari et al., 1992). All experiments were performed in INS-1 cells between the 14th and 33rd passages. Annexin V flow cytometry The harvested cells were centrifuged at 900 rpm for 5 min, and washed with phosphate-buffered saline (PBS) and then with a labeling solution composed of (in mM) 150 NaCl, 5 KCl, 1 MgCl2, 1.8 CaCl2, and 10 HEPES (pH 7.4 with NaOH). Then 100 μl of the labeling solution and 2 μl of Annexin-V-Fluos were added to each sample. After 10-min incubation at room temperature in the dark, 800 μl of the labeling solution was added, and the cells were meshed with N-No.355T nylon mesh (NBC, Tokyo, Japan). One hundred thousand cells were analyzed in triplicate for each sample with a flow cytometer (EPICS XL System II, Beckman Coulter, Fullerton, CA) within 1 h. The cells were excited with a 488-nm Argon laser and the fluorescence emission of AnnexinV-Fluos was measured in FL3 (620 nm; 605–640 nm). Although FL1 (525 nm; 505–540 nm) is generally used for the fluorescence detection of Annexin-V-Fluos, the data measured in FL3 were clearer than those in FL1 on the separation of Annexin V-negative and positive cells. We also used the characteristics that apoptotic cells shrink while necrotic cells swell, for the separation of apoptotic and necrotic cells. Cytograms of forward scatter (linear scale), which is proportional to cell size, vs. side scatter (log scale), which is
Fig.1. Thapsigargin-induced apoptosis in INS-1 cells. A: Typical flow cytometry data of INS1 cells without (Control) or with the treatment with thapsigargin (TG; 300 nM) for 24 h. Forward scatter/side scatter dot plots and histogram plots of FL3 were used to separate normal size (gate 1) and shrunk cells (gate 2) and to separate Annexin V-positive cells (lined) in gate 2, respectively. Shrunk and Annexin V-positive cells were defined to be apoptotic cells. B: Time-dependent changes in percentage of apoptotic cells in total cells examined without (Control) or with the treatment with TG (300 nM). Data are shown as mean ± SEM of four to eight experiments. ⁎⁎P b 0.01 vs. corresponding control.
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proportional to cell density, were used to separate shrunk cells (gate 2; see Fig. 1). Annexin V-positive and shrunk cells were defined to be apoptotic cells.
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Table 1 Time course for thapsigargin-induced apoptosis analyzed by flow cytometry using Annexin V labeling in INS-1 cells Time (h)
Propidium iodide flow cytometry The harvested cells were washed with PBS and then with cold PBS, and fixed with 1% formaldehyde-PBS on ice for 15 min and then with 70% ethanol-PBS at −20 °C over 12 h. The cells were permeated with 0.1% Triton X-100 (pH 7.5) in 0.1% sodium citrate at room temperature for 5 min, stained with 1 μg/ml propidium iodide (PI)-PBS and 10 μg/ ml RNaseA-PBS on ice in the dark for 30 min, and meshed with the nylon mesh. One hundred thousand cells were analyzed in triplicate for each sample with a flow cytometer, by exciting the cells with a 488-nm argon laser and by measuring the fluorescence emission of PI in FL4 (675 nm; 660–705 nm). After debris was excluded from analyses based on significantly diminished light scatter properties, data were analyzed. DNA laddering The harvested cells were washed with PBS. Approximately 5.0 × 106 cells were lysed with a lysis buffer composed of 50 mM Tris–HCl, 10 mM EDTA-4Na and 0.5% sodium-N-lauroyl sarcosinate (pH 7.8). The
Control Normal size cells (gate 1) Annexin V-positive cells in gate 1 Shrunk cells (gate 2) Annexin V-positive cells in gate 2 TG Normal size cells (gate 1) Annexin V-positive cells in gate 1 Shrunk cells (gate 2) Annexin V-positive cells in gate 2
0.5
12
76.1 ± 5.1
80.1 ± 2.6 80.2 ± 2.7
16
20
24
81.1 ± 1.5
79.2 ± 1.0
4.8 ± 1.0
1.8 ± 0.6
2.1 ± 0.3
3.3 ± 0.3
2.8 ± 0.3
7.7 ± 2.3 3.8 ± 0.9
6.0 ± 1.4 2.7 ± 0.6
7.3 ± 0.7 2.9 ± 0.4
6.1 ± 0.7 2.9 ± 0.4
7.9 ± 0.5 3.6 ± 0.4
81.1 ± 1.2
76.3 ± 3.1
50.9 ± 3.4 56.4 ± 7.1
41.2 ± 4.8
4.3 ± 0.6
1.5 ± 0.4
1.8 ± 0.2
3.6 ± 1.0
2.5 ± 0.4
6.6 ± 0.5 3.6 ± 0.4
9.3 ± 0.7 26.9 ± 3.1 3.8 ± 0.7 8.2 ± 0.6
27.8 ± 4.2 13.6 ± 2.8
41.4 ± 3.7 21.0 ± 3.6
The data show the percentage of normal size cells (gate 1), Annexin V-positive cells in gate 1, shrunk cells (gate 2), and Annexin V-positive cells in gate 2, in total cells tested (mean ± SEM of four to eight experiments). INS-1 cells were treated with thapsigargin (TG; 300 nM) for 0.5, 12, 16, 20 and 24 h.
lysates were incubated in the lysis buffer containing 0.33 mg/ml RNase A at 50 °C for 30 min and then further incubated in the lysis buffer containing 0.33 mg/ml proteinase K at 50 °C for 30 min. DNA was
Fig. 2. Pro-apoptotic effect of high-concentration DETA/NO. INS-1 cells were treated with DETA/NO (100, 300, and 1000 μM) for 24 h, and the adherent and detached cells were harvested. We determined cell death by trypan blue exclusion assay (A) and apoptotic cells by DNA fragmentation (B) and by Annexin V assay (C). D: INS-1 cells were treated with DETA/NO (1 mM) in the absence or presence of oxadiazoloquinoxalin (ODQ; 10 μM) for 24 h. Apoptotic cells were determined by Annexin V assay. Data representing the percentage of cell death (A), DNA fragmentation (B), and Annexin V-positive cells (C, D) in total cells examined or total DNA are shown as mean ± SEM of four to ten experiments. ⁎P b 0.05, ⁎⁎P b 0.01 vs. control.
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electrophoresed on 2.0% agarose gel. Gels were stained with 0.5 μg/ml ethidium bromide for 15 min and visualized under UV light. Statistical analysis Data are shown as the mean ± SEM. Comparisons were made using unpaired t-test or one-way analysis of variance (ANOVA) followed by Tukey–Kramer test or Williams' test, as appropriate. Results Thapsigargin-induced β-cell apoptosis The incubation of INS-1 cells with thapsigargin (300 nM), an inhibitor of ER Ca2+-ATPase, increased shrunk and Annexin V-positive cells in a time-dependent manner (Fig. 1 and Table 1). The β-cell apoptosis induced by the treatment with thapsigargin for 24 h was not significantly affected by the iNOS inhibitor 1400 W (30 μM) or the nonselective NOS inhibitor L-NNA (1.0 mM; data not shown, n = 7). Pro-apoptotic effect of NO The effect of exogenously applied NO on apoptosis of INS-1 cells was investigated with DETA/NO, an NO donor with a long half-life (20 h) (Mooradian et al., 1995). DETA/NO at 1 mM markedly induced apoptosis of INS-1 cells, although DETA/NO at lower concentrations until 300 μM did not induce it (Fig. 2A, B, and C). The pro-apoptotic effect of 1 mM DETA/NO was not affected by ODQ (10 μM), a sGC inhibitor (Fig. 2D). We have previously shown that the same concentration of ODQ completely abolishes the [Ca2+]i elevation induced by NO in rat pancreatic β-cells (Ishikawa et al., 2003). Anti-apoptotic effect of low concentrations of NO The effect of low concentrations of NO on thapsigargin-induced apoptosis was next investigated in INS-1 cells. DETA/NO at low concentrations until 300 μM concentration-dependently decreased the apoptosis induced by the treatment with thapsigargin (300 nM), an inhibitor of endoplasmic reticulum Ca2+-ATPase, for 24 h (Fig. 3A). We also confirmed that 300 μM DETA/NO decreased the DNA laddering induced by the thapsigargin treatment (Fig. 3B). The anti-apoptotic effect of 300 μM DETA/NO was not affected by ODQ (Fig. 4A). In addition, either the cell-permeable cGMP analogue 8-Br-cGMP (3, 10, 30 or 100 μM; Fig. 4B) or sGC activator YC-1 (10 μM; Fig. 4C) had no effect on the thapsigargin-induced apoptosis. Discussion In the present study, apoptosis of the pancreatic β-cell line INS-1 was quantitatively determined by Annexin V flow cytometry. In accordance with previous studies showing apoptotic effect of NO in β-cells (Kaneto et al., 1995; Kutlu et al., 2003), the NO donor DETA/NO at 1 mM induced apoptosis of INS-1 cells. In contrast, DETA/NO at lower concentrations was shown to inhibit ER stress-induced apoptosis triggered by thapsigargin in the same cell line. The anti-apoptotic action of NO in β-cells is also suggested by recent studies by Tejedo et al. (2001), which has shown that 10 μM DETA/NO inhibits serum deprivation-induced apoptotic signals, i.e., caspase-3 activation, cytochrome c release, and Bcl-2 down-regulation, in the β-cell line RINm5F, and by Kitiphongspattana et al. (2007), which has shown that an NO synthase inhibitor represses ER stress-induced expression of genes involved in antioxidant defense and ER oxidative protein folding in the β-cell line MIN6. Thus, NO seems to function as an anti-apoptotic and pro-apoptotic modulator at low and high concentrations, respectively, in β-cells. The involvement of cGMP in the anti-apoptotic effects of NO appears to be dependent on cell type. The effects are suggested to be
Fig. 3. Anti-apoptotic effects of low-concentration DETA/NO. A: INS-1 cells were incubated for 24 h with thapsigargin (TG; 300 nM) and DETA/NO (30, 100 or 300 μM). Apoptotic cells were determined by Annexin V assay. Data representing the percentage of apoptotic cells in total cells examined are shown as mean ± SEM of four to eight experiments. ⁎⁎P b 0.01 vs. control. ††P b 0.01 vs. TG alone. B: Gel electrophoresis of DNA extracts from INS-1 cells. INS-1 cells were incubated for 24 h without (control; 2nd and 3rd lanes) or with TG (300 nM; 4th and 5th lanes) or TG (300 nM) plus DETA/NO (300 μM; 6th and 7th lanes). Apoptotic cells were determined by qualitative DNA laddering.
mediated via NO/cGMP pathway in PC12 cells (Kim et al., 1999), hepatocytes (Kim et al., 1997), and B lymphocytes (Genaro et al., 1995), but seem to be due to direct action by NO in hepatocytes (Kim et al., 1997) and endothelial cells (Ceneviva et al., 1998). In the β-cell line RINm5F, Tejedo et al. (2001) have shown that ODQ and KT-5823, a protein kinase G (PKG) inhibitor, abrogate the inhibitory effects of 10 μM DETA/NO on serum deprivation-induced apoptotic signals, i.e., caspase-3 activation, cytochrome c release, and Bcl-2 down-regulation. They further showed that these actions were fully reverted by the Src inhibitor PP1, suggesting the involvement of c-Src in the cGMPdependent, anti-apoptotic actions of NO (Tejedo et al., 2001). However, in their subsequent report using the same β-cell line, KT5823 failed to inhibit the c-Src activation by NO, although the PKG inhibitor partially decreased NO-induced phosphorylation of Akt and Bad (Tejedo et al., 2004). They proposed, therefore, that NO antiapoptotic action is mediated through at least two pathways; the cGMPdependent pathway and the cSrc-dependent one. The latter was suggested to lead to the activation of phosphatidylinositol 3-kinase
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Fig. 4. Anti-apoptotic effect of low-concentration DETA/NO is independent of NO/sGC/cGMP pathway. A: INS-1 cells were treated for 24 h with thapsigargin (TG; 300 nM) or TG (300 nM) plus DETA/NO (300 μM) in the absence or presence of oxadiazoloquinoxalin (ODQ; 10 μM). B, C: INS-1 cells were treated for 24 h with thapsigargin (TG; 300 nM) plus 8-BrcGMP (3, 10, 30 or 100 μM; B) or the soluble guanylate cyclase activator YC-1 (10 μM; C). Apoptotic cells were determined by Annexin V assay. The data representing the percentage of apoptotic cells in total cells examined are shown as mean ± SEM of four to seven experiments. ⁎P b 0.05, ⁎⁎P b 0.01 vs. control. †P b 0.05 vs. TG; NS: not significant.
(PI3K)/Akt system. The present quantitative analysis by flow cytometry demonstrated that ODQ, a sGC inhibitor, failed to abrogate the antiapoptotic effect of NO, and that 8-Br-cGMP and YC-1, a sGC activator, failed to inhibit the apoptosis induced by thapsigargin. These results suggest that the protection by NO from ER stress-induced apoptosis is independent of cGMP in β-cells. The absence of cGMP-dependent antiapoptotic effect of NO may be due to the differences in induction of apoptosis and cell lines. The protective effect of NO on β-cell apoptosis may result from activation of the PI3K/Akt survival pathway. Tejedo et al. have recently shown that 10 μM DETA/NO induces phosphorylation of Akt and Bad and that the PI3K inhibitor LY 294002 blocks the protective action of NO on DNA fragmentation in serum-depleted RINm5F cells (Tejedo et al., 2004). They also showed that the PKG inhibitor KT-5823 partially inhibited the phosphorylation of Akt and Bad (Tejedo et al., 2004). However, the present study showed that the protective effect of NO in the thapsigargin-treated INS-1 cells is independent of cGMP.
Thus, the anti-apoptotic NO signaling in the serum-depleted RINm5F cells may be somewhat different from that in the thapsigargin-treated INS-1 cells. NO has also been shown to directly inhibit the activity of caspases by S-nitrosylation of the enzyme in hepatocytes (Kim et al., 1997) and endothelial cells (Ceneviva et al., 1998). In addition, Kitiphongspattana et al. (2007) have recently shown that inhibition of NO synthase represses the ER stress-induced gene expression (Gclc, Prx1, Gpx-1, and Grp78/BiP) in the β-cell line MIN6, suggesting that NO exerts a protective action via the upregulation of these genes involved in antioxidant defense and ER protein folding during the ER stress response. Further studies are required to elucidate the anti-apoptotic NO signaling in ER stress-induced β-cell apoptosis. As an alternative possibility, we hypothesized that NO exhibits anti-apoptotic activity by inhibiting an elevation of intracellular Ca2+ concentration ([Ca2+]i) in thapsigargin-induced apoptosis, because we have previously shown that NO at 160–200 nM inhibits intracellular Ca2+ oscillation induced by 11.1 mM glucose through a mechanism
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independent of cGMP (Kaneko et al., 2003). However, we found that the thapsigargin-induced apoptosis was not affected by 10 μM BAPTA/ AM, a chelator of intracellular Ca2+ (Noguchi, unpublished data). It is therefore unlikely that the anti-apoptotic effect of NO is due to its inhibitory effect on [Ca2+]i. Several studies have shown that NO-induced apoptosis in pancreatic β-cells is independent of activation of NO/cGMP pathway. cGMP-independent pro-apoptotic effect of NO has also been shown in β-cell lines, INS-1 (Størling et al., 2005), RINm5F (Tejedo et al., 1999; Sjhölm et al., 2000), and MIN6 (Oyadomari et al., 2001). In contrast, NO induces apoptosis of the β-cell line HIT-T15 in a cGMP-dependent manner (Loweth et al., 1997). In the present study, the sGC inhibitor ODQ had no effect on the pro-apoptotic effect of 1 mM DETA/NO, suggesting the cGMP-independent pro-apoptotic effect of NO in INS-1 cells. The underlying cause of the discrepancy between the mechanism of pro-apoptotic effects of NO is not clear, but it might be due to the specific nature of the NO donor used, cell clone differences and/or differences in experimental conditions. Since NO at pathological levels has been shown to induce DNA damage or mitochondrial damage, high concentrations of NO could induce apoptosis via inhibition of ribonucrease reductase, mitochondrial respiratory chain, and/or aconitase (Cnop et al., 2005; Choi et al., 2002). Moreover, NO has recently been suggested to induce β-cell apoptosis through ER stress (Oyadomari et al., 2001). In summary, exogenously applied NO at high concentrations induces apoptosis, whereas low concentrations of NO inhibit the apoptosis induced by ER stress. Moreover, both the pro- and anti-apoptotic effects of NO are suggested to be independent of the sGC/cGMP pathway. Thus, NO is likely to protect from apoptosis at physiological levels and to induce apoptosis at pathological levels in pancreatic β-cells. Acknowledgments The authors thank Prof. Hiromitsu Nakauchi (University of Tokyo) for his advice on flow cytometry and Prof. Ichiro Niki (Oita University) for his advice on INS-1 cells. This study was supported by a Grant-in-Aid for Scientific Research from Japan Society for the Promotion of Science, and by Suzuken Memorial Foundation. References Asfari, M., Janjic, D., Meda, P., Li, G., Halban, P.A., Wollheim, C.B., 1992. Establishment of 2-mercaptoethanol-dependent differentiated insulin-secreting cell lines. Endocrinology 130, 167–178. Butler, A.E., Janson, J., Bonner-Weir, S., Ritzel, R., Rizza, R.A., Butler, P.C., 2003. β-cell deficit and increased β-cell apoptosis in humans with type 2 diabetes. Diabetes 52, 102–110. Cardozo, A.K., Kruhøffer, M., Leeman, R., Ørntoft, T., Eizirik, D.L., 2001. Identification of novel cytokine-induced genes in pancreatic β-cells by high-density oligonucleotide arrays. Diabetes 50, 909–920. Ceneviva, G.D., Tzeng, E., Hoyt, D.G., Yee, E., Gallagher, A., Engelhardt, J.F., Kim, Y.M., Billiar, T.R., Watkins, S.A., Pitt, B.R., 1998. Nitric oxide inhibits lipopolysaccharideinduced apoptosis in pulmonary artery endothelial cells. American Journal of Physiology 275, L717–728. Choi, B.M., Pae, H.O., Jang, S.I., Kim, Y.M., Chung, H.T., 2002. Nitric oxide as a proapoptotic as well as anti-apoptotic modulator. Journal of Biochemistry and Molecular Biology 35, 116–126. Ciani, E., Virgili, M., Contestabile, A., 2002. Akt pathway mediates a cGMP-dependent survival role of nitric oxide in cerebellar granule neurones. Journal of Neurochemistry 81, 218–228.
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Sakuraba, H., Mizukami, H., Yagihashi, N., Wada, R., Hanyu, C., Yagihashi, S., 2002. Reduced beta-cell mass and expression of oxidative stress-related DNA damage in the islet of Japanese Type II diabetic patients. Diabetologia 45, 85–96. Sata, M., Kakoki, M., Nagata, D., Nishimatsu, H., Suzuki, E., Aoyagi, T., Sugiura, S., Kojima, H., Nagano, T., Kangawa, K., Matsuo, H., Omata, M., Nagai, R., Hirata, Y., 2000. Adrenomedullin and nitric oxide inhibit human endothelial cell apoptosis via a cyclic GMP-independent mechanism. Hypertension 36, 83–88. Sjhölm, Å., Berggren, P.O., Cooney, R.V., 2000. γ-tocopherol partially protects insulinsecreting cells against functional inhibition by nitric oxide. Biochemical and Biophysical Research Communications 277, 334–340. Størling, J., Binzer, J., Andersson, A.K., Züllig, R.A., Tonnesen, M., Lehmann, R., Spinas, G.A., Sandler, S., Billestrup, N., Mandrup-Poulsen, T., 2005. Nitric oxide contributes to cytokine-induced apoptosis in pancreatic beta cells via potentiation of JNK activity and inhibition of Akt. Diabetologia 48, 2039–2050. Tejedo, J., Bernabé, J.C., Ramírez, R., Sobrino, F., Bedoya, F.J., 1999. NO induces a cGMPindependent release of cytochrome c from mitochondria which precedes caspase 3 activation in insulin producing RINm5F cells. FEBS Letters 459, 238–243. Tejedo, J.R., Ramírez, R., Cahuana, G.M., Rincón, P., Sobrino, F., Bedoya, F.J., 2001. Evidence for involvement of c-Src in the anti-apoptotic action of nitric oxide in serum-deprived RINm5F cells. Cellular Signalling 13, 809–817. Tejedo, J.R., Cahuana, G.M., Ramírez, R., Esbert, M., Jiménez, J., Sobrino, F., Bedoya, F.J., 2004. Nitric oxide triggers the phosphatidylinositol 3-kinase/Akt survival pathway in insulin-producing RINm5F cells by arousing Src to activate insulin receptor substrate-1. Endocrinology 145, 2319–2327.
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Life Sciences j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / l i f e s c i e
cGMP-independent anti-apoptotic effect of nitric oxide on thapsigargin-induced apoptosis in the pancreatic β-cell line INS-1 Akiko Noguchi a, Masahiro Takada a, Koichi Nakayama b, Tomohisa Ishikawa a,⁎ a b
Department of Pharmacology, Graduate School of Pharmaceutical Sciences, University of Shizuoka, 52-1 Yada, Suruga-ku, Shizuoka City, Shizuoka 422-8526, Japan Department of Molecular and Cellular Pharmacology, Faculty of Pharmaceutical Sciences, Iwate Medical University, 2-1-1 Nishitokuta, Yahaba, Iwate 028-3694, Japan
a r t i c l e
i n f o
Article history: Received 13 June 2008 Accepted 3 October 2008 Keywords: Nitric oxide Apoptosis Thapsigargin cGMP INS-1
a b s t r a c t Aims: Low concentrations of nitric oxide (NO) produced by constitutive NO synthase (cNOS) in pancreatic β-cells have been suggested to be a physiological regulator of insulin secretion. In contrast, excessive NO produced by inducible NO synthase is known to mediate β-cell apoptosis. The aim of the present study was to investigate the effect of low concentrations of NO on β-cell apoptosis. Main methods: Apoptosis of the pancreatic β-cell line INS-1 was quantitatively determined by Annexin V flow cytometry. Key findings: The 24-h incubation with 1 mM DETA/NO, a long half-life NO donor, induced β-cell apoptosis, which was insensitive to the soluble guanylate cyclase (sGC) inhibitor ODQ. In contrast, DETA/NO at lower concentrations until 300 μM concentration-dependently decreased the apoptosis induced by thapsigargin, an inhibitor of endoplasmic reticulum Ca2+-ATPase. ODQ did not affect the anti-apoptotic effect of DETA/NO. Moreover, neither the cGMP analogue 8-Br-cGMP nor the sGC activator YC-1 mimicked the anti-apoptotic effect of DETA/NO. Significance: These results suggest that low levels of NO protect β-cells from thapsigargin-induced apoptosis in a cGMP-independent manner. © 2008 Elsevier Inc. All rights reserved.
Introduction Apoptosis of pancreatic β-cells is now postulated to be a common feature of type 1 and type 2 diabetes (Donath et al., 2003; Cnop et al., 2005). In type 1 diabetes, the destruction of β-cells is due predominantly to autoimmunity through the action of pro-inflammatory cytokines such as interleukin (IL)-1β, interferon (IFN)-γ, and tumor necrosis factor (TNF)-α, which are produced by immune cells infiltrated into the islets (Eizirik and Mandrup-Poulsen, 2001). IL-1β, in combination with IFN-γ and/or TNF-α, activates the transcription factor nuclear factor (NF)-κB, which regulates a number of genes in β-cells, including that encoding inducible nitric oxide synthase (iNOS) (Cardozo et al., 2001). Although the precise role of nitric oxide (NO) in the development of type 1 diabetes remains to be fully elucidated, the excessive NO produced by iNOS is likely to account for some of the deleterious effects of IL-1β in β-cells (Kaneto et al., 1995; Darville and Eizirik, 1998). Type 2 diabetes results from both a progressive defect in β-cell insulin secretion and insulin sensitivity. The ability to secrete adequate amounts of insulin depends on β-cell function and mass. Recent studies have shown a significant reduction in β-cell mass and an increase in β-cell apoptosis in type 2 diabetic subjects (Sakuraba et al., 2002; Butler et al., 2003). These findings imply a deficiency of β-cell mass in type 2 diabetes,
⁎ Corresponding author. Tel.: +81 54 264 5694; fax: +81 54 264 5696. E-mail address: [email protected] (T. Ishikawa). 0024-3205/$ – see front matter © 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.lfs.2008.10.002
secondary to increased β-cell apoptosis. Elevated levels of glucose and free fatty acids have been assumed to induce β-cell apoptosis in type 2 diabetes (Donath et al., 2003; Cnop et al., 2005). Constitutive NO synthase (cNOS) produces a small amount of NO in response to an increase in intracellular Ca2+ concentration ([Ca2+]i). We have previously shown that NO is produced by cNOS in rat pancreatic islets in response to elevated glucose concentrations (Nakada et al., 2003), and that NO in the nM range of concentrations, which is assumed to be produced by cNOS, exerts stimulatory and inhibitory effects at low and high concentrations, respectively, on glucose-induced insulin secretion from rat pancreatic β-cells (Kaneko et al., 2003). Thus, NO produced by cNOS is suggested to function as an endogenous regulator of insulin secretion. The contribution of cNOS-produced NO to the regulation of β-cell mass, however, remains to be fully elucidated, although excessive NO produced by iNOS is well known to cause β-cell death (Kaneto et al., 1995; Darville and Eizirik, 1998; Kutlu et al., 2003). Low concentrations of NO have recently been shown to protect from apoptosis in endothelial cells (Sata et al., 2000), hepatocytes (Kim et al., 1997), and neurons (Ciani et al., 2002). The anti-apoptotic mechanisms of NO are divided into cGMP-dependent and cGMP-independent mechanisms, which are likely cell-type specific (Choi et al., 2002). In the β-cell line RINm5F, 10 μM DETA/NO, an NO donor, has been shown to inhibit apoptotic signals, such as cytochrome c release from mitochondria, caspase-3 activation, and Bcl-2 downregulation, induced by serum deprivation (Tejedo et al., 2001, 2004). However, the involvement of cGMP in the anti-apoptotic effect of NO in β-cells remains to be fully
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evaluated. The present study was therefore designed to investigate the effect of low concentrations of NO on β-cell apoptosis and the involvement of cGMP in it by quantitative analysis with flow cytometry. β-Cell apoptosis was induced by endoplasmic reticulum (ER) stress, which has been shown to be induced by glucotoxicity and lipotoxicity during the development of type 2 diabetes (Donath et al., 2003; Cnop et al., 2005). The present data show that low concentrations of NO protect β-cells from ER stress-induced apoptosis in a cGMP-independent manner. Materials and methods Materials 8-Bromoguanosine 3′,5′-cyclic monophosphate sodium salt monohydrate (8-Br-cGMP), diethylenetriamine nitric oxide adduct (DETA/NO), NGnitro-L-arginine (L-NNA), 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), ribonuclease A from bovine pancreas (RNase A), thapsigargin, and 3-(5′-Hydroxymethyl-2′-furyl)-1-benzyl indazole (YC-1) were obtained from Sigma (St. Louis, MO); [N-(3-aminomethyl)benzyl]acetamidine dihydrochloride (1400 W) was from Alexis Biochemicals (San Diego, CA); Annexin-V-Fluos was from Roche Diagnostics GmbH (Mannheim, Germany); fetal bovine serum (FBS) was from Tissue Culture Biologicals (Tulare, CA); penicillin G potassium, streptomycin sulfate were from Meiji Seika (Tokyo, Japan); 3,8-diamino-5-(3-diethylaminopropyl) 6phenyl-phenanthridinium iodide methiodide (propidium iodide; PI) were from Dojindo (Kumamoto, Japan); and TriDye™ 2-Log DNA Ladder (0.1– 10.0 kb) was from New England BioLabs (Ipswich, MA). We dissolved 8-Br-cGMP and 1400 W in purified water as a 100 mM solution; thapsigargin in DMSO as a 1 mM solution; ODQ and YC-1 in DMSO as a 10 mM solution; DETA/NO in 10 mM NaOH as a 1 M solution, which were stored at −20 °C for later use. The stock solutions were dissolved in RPMI 1640 medium supplemented with 10 mM HEPES, 1 mM sodium pyruvate, 50 μM 2-mercaptoethanol, 100 μg/ml streptomycin, 100 U/ml penicillin G, 0.3 mg/ml glutamine, and 10% fetal bovine serum (FBS), before each experiment. Cell culture INS-1 cells, a gift from Dr. C. Wollheim (University Medical Center, Geneva, Switzerland), were cultured at 5% CO2/95% air and 37 °C in the supplemented RPMI 1640 medium. The medium was replaced every third day (Asfari et al., 1992). All experiments were performed in INS-1 cells between the 14th and 33rd passages. Annexin V flow cytometry The harvested cells were centrifuged at 900 rpm for 5 min, and washed with phosphate-buffered saline (PBS) and then with a labeling solution composed of (in mM) 150 NaCl, 5 KCl, 1 MgCl2, 1.8 CaCl2, and 10 HEPES (pH 7.4 with NaOH). Then 100 μl of the labeling solution and 2 μl of Annexin-V-Fluos were added to each sample. After 10-min incubation at room temperature in the dark, 800 μl of the labeling solution was added, and the cells were meshed with N-No.355T nylon mesh (NBC, Tokyo, Japan). One hundred thousand cells were analyzed in triplicate for each sample with a flow cytometer (EPICS XL System II, Beckman Coulter, Fullerton, CA) within 1 h. The cells were excited with a 488-nm Argon laser and the fluorescence emission of AnnexinV-Fluos was measured in FL3 (620 nm; 605–640 nm). Although FL1 (525 nm; 505–540 nm) is generally used for the fluorescence detection of Annexin-V-Fluos, the data measured in FL3 were clearer than those in FL1 on the separation of Annexin V-negative and positive cells. We also used the characteristics that apoptotic cells shrink while necrotic cells swell, for the separation of apoptotic and necrotic cells. Cytograms of forward scatter (linear scale), which is proportional to cell size, vs. side scatter (log scale), which is
Fig.1. Thapsigargin-induced apoptosis in INS-1 cells. A: Typical flow cytometry data of INS1 cells without (Control) or with the treatment with thapsigargin (TG; 300 nM) for 24 h. Forward scatter/side scatter dot plots and histogram plots of FL3 were used to separate normal size (gate 1) and shrunk cells (gate 2) and to separate Annexin V-positive cells (lined) in gate 2, respectively. Shrunk and Annexin V-positive cells were defined to be apoptotic cells. B: Time-dependent changes in percentage of apoptotic cells in total cells examined without (Control) or with the treatment with TG (300 nM). Data are shown as mean ± SEM of four to eight experiments. ⁎⁎P b 0.01 vs. corresponding control.
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proportional to cell density, were used to separate shrunk cells (gate 2; see Fig. 1). Annexin V-positive and shrunk cells were defined to be apoptotic cells.
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Table 1 Time course for thapsigargin-induced apoptosis analyzed by flow cytometry using Annexin V labeling in INS-1 cells Time (h)
Propidium iodide flow cytometry The harvested cells were washed with PBS and then with cold PBS, and fixed with 1% formaldehyde-PBS on ice for 15 min and then with 70% ethanol-PBS at −20 °C over 12 h. The cells were permeated with 0.1% Triton X-100 (pH 7.5) in 0.1% sodium citrate at room temperature for 5 min, stained with 1 μg/ml propidium iodide (PI)-PBS and 10 μg/ ml RNaseA-PBS on ice in the dark for 30 min, and meshed with the nylon mesh. One hundred thousand cells were analyzed in triplicate for each sample with a flow cytometer, by exciting the cells with a 488-nm argon laser and by measuring the fluorescence emission of PI in FL4 (675 nm; 660–705 nm). After debris was excluded from analyses based on significantly diminished light scatter properties, data were analyzed. DNA laddering The harvested cells were washed with PBS. Approximately 5.0 × 106 cells were lysed with a lysis buffer composed of 50 mM Tris–HCl, 10 mM EDTA-4Na and 0.5% sodium-N-lauroyl sarcosinate (pH 7.8). The
Control Normal size cells (gate 1) Annexin V-positive cells in gate 1 Shrunk cells (gate 2) Annexin V-positive cells in gate 2 TG Normal size cells (gate 1) Annexin V-positive cells in gate 1 Shrunk cells (gate 2) Annexin V-positive cells in gate 2
0.5
12
76.1 ± 5.1
80.1 ± 2.6 80.2 ± 2.7
16
20
24
81.1 ± 1.5
79.2 ± 1.0
4.8 ± 1.0
1.8 ± 0.6
2.1 ± 0.3
3.3 ± 0.3
2.8 ± 0.3
7.7 ± 2.3 3.8 ± 0.9
6.0 ± 1.4 2.7 ± 0.6
7.3 ± 0.7 2.9 ± 0.4
6.1 ± 0.7 2.9 ± 0.4
7.9 ± 0.5 3.6 ± 0.4
81.1 ± 1.2
76.3 ± 3.1
50.9 ± 3.4 56.4 ± 7.1
41.2 ± 4.8
4.3 ± 0.6
1.5 ± 0.4
1.8 ± 0.2
3.6 ± 1.0
2.5 ± 0.4
6.6 ± 0.5 3.6 ± 0.4
9.3 ± 0.7 26.9 ± 3.1 3.8 ± 0.7 8.2 ± 0.6
27.8 ± 4.2 13.6 ± 2.8
41.4 ± 3.7 21.0 ± 3.6
The data show the percentage of normal size cells (gate 1), Annexin V-positive cells in gate 1, shrunk cells (gate 2), and Annexin V-positive cells in gate 2, in total cells tested (mean ± SEM of four to eight experiments). INS-1 cells were treated with thapsigargin (TG; 300 nM) for 0.5, 12, 16, 20 and 24 h.
lysates were incubated in the lysis buffer containing 0.33 mg/ml RNase A at 50 °C for 30 min and then further incubated in the lysis buffer containing 0.33 mg/ml proteinase K at 50 °C for 30 min. DNA was
Fig. 2. Pro-apoptotic effect of high-concentration DETA/NO. INS-1 cells were treated with DETA/NO (100, 300, and 1000 μM) for 24 h, and the adherent and detached cells were harvested. We determined cell death by trypan blue exclusion assay (A) and apoptotic cells by DNA fragmentation (B) and by Annexin V assay (C). D: INS-1 cells were treated with DETA/NO (1 mM) in the absence or presence of oxadiazoloquinoxalin (ODQ; 10 μM) for 24 h. Apoptotic cells were determined by Annexin V assay. Data representing the percentage of cell death (A), DNA fragmentation (B), and Annexin V-positive cells (C, D) in total cells examined or total DNA are shown as mean ± SEM of four to ten experiments. ⁎P b 0.05, ⁎⁎P b 0.01 vs. control.
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electrophoresed on 2.0% agarose gel. Gels were stained with 0.5 μg/ml ethidium bromide for 15 min and visualized under UV light. Statistical analysis Data are shown as the mean ± SEM. Comparisons were made using unpaired t-test or one-way analysis of variance (ANOVA) followed by Tukey–Kramer test or Williams' test, as appropriate. Results Thapsigargin-induced β-cell apoptosis The incubation of INS-1 cells with thapsigargin (300 nM), an inhibitor of ER Ca2+-ATPase, increased shrunk and Annexin V-positive cells in a time-dependent manner (Fig. 1 and Table 1). The β-cell apoptosis induced by the treatment with thapsigargin for 24 h was not significantly affected by the iNOS inhibitor 1400 W (30 μM) or the nonselective NOS inhibitor L-NNA (1.0 mM; data not shown, n = 7). Pro-apoptotic effect of NO The effect of exogenously applied NO on apoptosis of INS-1 cells was investigated with DETA/NO, an NO donor with a long half-life (20 h) (Mooradian et al., 1995). DETA/NO at 1 mM markedly induced apoptosis of INS-1 cells, although DETA/NO at lower concentrations until 300 μM did not induce it (Fig. 2A, B, and C). The pro-apoptotic effect of 1 mM DETA/NO was not affected by ODQ (10 μM), a sGC inhibitor (Fig. 2D). We have previously shown that the same concentration of ODQ completely abolishes the [Ca2+]i elevation induced by NO in rat pancreatic β-cells (Ishikawa et al., 2003). Anti-apoptotic effect of low concentrations of NO The effect of low concentrations of NO on thapsigargin-induced apoptosis was next investigated in INS-1 cells. DETA/NO at low concentrations until 300 μM concentration-dependently decreased the apoptosis induced by the treatment with thapsigargin (300 nM), an inhibitor of endoplasmic reticulum Ca2+-ATPase, for 24 h (Fig. 3A). We also confirmed that 300 μM DETA/NO decreased the DNA laddering induced by the thapsigargin treatment (Fig. 3B). The anti-apoptotic effect of 300 μM DETA/NO was not affected by ODQ (Fig. 4A). In addition, either the cell-permeable cGMP analogue 8-Br-cGMP (3, 10, 30 or 100 μM; Fig. 4B) or sGC activator YC-1 (10 μM; Fig. 4C) had no effect on the thapsigargin-induced apoptosis. Discussion In the present study, apoptosis of the pancreatic β-cell line INS-1 was quantitatively determined by Annexin V flow cytometry. In accordance with previous studies showing apoptotic effect of NO in β-cells (Kaneto et al., 1995; Kutlu et al., 2003), the NO donor DETA/NO at 1 mM induced apoptosis of INS-1 cells. In contrast, DETA/NO at lower concentrations was shown to inhibit ER stress-induced apoptosis triggered by thapsigargin in the same cell line. The anti-apoptotic action of NO in β-cells is also suggested by recent studies by Tejedo et al. (2001), which has shown that 10 μM DETA/NO inhibits serum deprivation-induced apoptotic signals, i.e., caspase-3 activation, cytochrome c release, and Bcl-2 down-regulation, in the β-cell line RINm5F, and by Kitiphongspattana et al. (2007), which has shown that an NO synthase inhibitor represses ER stress-induced expression of genes involved in antioxidant defense and ER oxidative protein folding in the β-cell line MIN6. Thus, NO seems to function as an anti-apoptotic and pro-apoptotic modulator at low and high concentrations, respectively, in β-cells. The involvement of cGMP in the anti-apoptotic effects of NO appears to be dependent on cell type. The effects are suggested to be
Fig. 3. Anti-apoptotic effects of low-concentration DETA/NO. A: INS-1 cells were incubated for 24 h with thapsigargin (TG; 300 nM) and DETA/NO (30, 100 or 300 μM). Apoptotic cells were determined by Annexin V assay. Data representing the percentage of apoptotic cells in total cells examined are shown as mean ± SEM of four to eight experiments. ⁎⁎P b 0.01 vs. control. ††P b 0.01 vs. TG alone. B: Gel electrophoresis of DNA extracts from INS-1 cells. INS-1 cells were incubated for 24 h without (control; 2nd and 3rd lanes) or with TG (300 nM; 4th and 5th lanes) or TG (300 nM) plus DETA/NO (300 μM; 6th and 7th lanes). Apoptotic cells were determined by qualitative DNA laddering.
mediated via NO/cGMP pathway in PC12 cells (Kim et al., 1999), hepatocytes (Kim et al., 1997), and B lymphocytes (Genaro et al., 1995), but seem to be due to direct action by NO in hepatocytes (Kim et al., 1997) and endothelial cells (Ceneviva et al., 1998). In the β-cell line RINm5F, Tejedo et al. (2001) have shown that ODQ and KT-5823, a protein kinase G (PKG) inhibitor, abrogate the inhibitory effects of 10 μM DETA/NO on serum deprivation-induced apoptotic signals, i.e., caspase-3 activation, cytochrome c release, and Bcl-2 down-regulation. They further showed that these actions were fully reverted by the Src inhibitor PP1, suggesting the involvement of c-Src in the cGMPdependent, anti-apoptotic actions of NO (Tejedo et al., 2001). However, in their subsequent report using the same β-cell line, KT5823 failed to inhibit the c-Src activation by NO, although the PKG inhibitor partially decreased NO-induced phosphorylation of Akt and Bad (Tejedo et al., 2004). They proposed, therefore, that NO antiapoptotic action is mediated through at least two pathways; the cGMPdependent pathway and the cSrc-dependent one. The latter was suggested to lead to the activation of phosphatidylinositol 3-kinase
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Fig. 4. Anti-apoptotic effect of low-concentration DETA/NO is independent of NO/sGC/cGMP pathway. A: INS-1 cells were treated for 24 h with thapsigargin (TG; 300 nM) or TG (300 nM) plus DETA/NO (300 μM) in the absence or presence of oxadiazoloquinoxalin (ODQ; 10 μM). B, C: INS-1 cells were treated for 24 h with thapsigargin (TG; 300 nM) plus 8-BrcGMP (3, 10, 30 or 100 μM; B) or the soluble guanylate cyclase activator YC-1 (10 μM; C). Apoptotic cells were determined by Annexin V assay. The data representing the percentage of apoptotic cells in total cells examined are shown as mean ± SEM of four to seven experiments. ⁎P b 0.05, ⁎⁎P b 0.01 vs. control. †P b 0.05 vs. TG; NS: not significant.
(PI3K)/Akt system. The present quantitative analysis by flow cytometry demonstrated that ODQ, a sGC inhibitor, failed to abrogate the antiapoptotic effect of NO, and that 8-Br-cGMP and YC-1, a sGC activator, failed to inhibit the apoptosis induced by thapsigargin. These results suggest that the protection by NO from ER stress-induced apoptosis is independent of cGMP in β-cells. The absence of cGMP-dependent antiapoptotic effect of NO may be due to the differences in induction of apoptosis and cell lines. The protective effect of NO on β-cell apoptosis may result from activation of the PI3K/Akt survival pathway. Tejedo et al. have recently shown that 10 μM DETA/NO induces phosphorylation of Akt and Bad and that the PI3K inhibitor LY 294002 blocks the protective action of NO on DNA fragmentation in serum-depleted RINm5F cells (Tejedo et al., 2004). They also showed that the PKG inhibitor KT-5823 partially inhibited the phosphorylation of Akt and Bad (Tejedo et al., 2004). However, the present study showed that the protective effect of NO in the thapsigargin-treated INS-1 cells is independent of cGMP.
Thus, the anti-apoptotic NO signaling in the serum-depleted RINm5F cells may be somewhat different from that in the thapsigargin-treated INS-1 cells. NO has also been shown to directly inhibit the activity of caspases by S-nitrosylation of the enzyme in hepatocytes (Kim et al., 1997) and endothelial cells (Ceneviva et al., 1998). In addition, Kitiphongspattana et al. (2007) have recently shown that inhibition of NO synthase represses the ER stress-induced gene expression (Gclc, Prx1, Gpx-1, and Grp78/BiP) in the β-cell line MIN6, suggesting that NO exerts a protective action via the upregulation of these genes involved in antioxidant defense and ER protein folding during the ER stress response. Further studies are required to elucidate the anti-apoptotic NO signaling in ER stress-induced β-cell apoptosis. As an alternative possibility, we hypothesized that NO exhibits anti-apoptotic activity by inhibiting an elevation of intracellular Ca2+ concentration ([Ca2+]i) in thapsigargin-induced apoptosis, because we have previously shown that NO at 160–200 nM inhibits intracellular Ca2+ oscillation induced by 11.1 mM glucose through a mechanism
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independent of cGMP (Kaneko et al., 2003). However, we found that the thapsigargin-induced apoptosis was not affected by 10 μM BAPTA/ AM, a chelator of intracellular Ca2+ (Noguchi, unpublished data). It is therefore unlikely that the anti-apoptotic effect of NO is due to its inhibitory effect on [Ca2+]i. Several studies have shown that NO-induced apoptosis in pancreatic β-cells is independent of activation of NO/cGMP pathway. cGMP-independent pro-apoptotic effect of NO has also been shown in β-cell lines, INS-1 (Størling et al., 2005), RINm5F (Tejedo et al., 1999; Sjhölm et al., 2000), and MIN6 (Oyadomari et al., 2001). In contrast, NO induces apoptosis of the β-cell line HIT-T15 in a cGMP-dependent manner (Loweth et al., 1997). In the present study, the sGC inhibitor ODQ had no effect on the pro-apoptotic effect of 1 mM DETA/NO, suggesting the cGMP-independent pro-apoptotic effect of NO in INS-1 cells. The underlying cause of the discrepancy between the mechanism of pro-apoptotic effects of NO is not clear, but it might be due to the specific nature of the NO donor used, cell clone differences and/or differences in experimental conditions. Since NO at pathological levels has been shown to induce DNA damage or mitochondrial damage, high concentrations of NO could induce apoptosis via inhibition of ribonucrease reductase, mitochondrial respiratory chain, and/or aconitase (Cnop et al., 2005; Choi et al., 2002). Moreover, NO has recently been suggested to induce β-cell apoptosis through ER stress (Oyadomari et al., 2001). In summary, exogenously applied NO at high concentrations induces apoptosis, whereas low concentrations of NO inhibit the apoptosis induced by ER stress. Moreover, both the pro- and anti-apoptotic effects of NO are suggested to be independent of the sGC/cGMP pathway. Thus, NO is likely to protect from apoptosis at physiological levels and to induce apoptosis at pathological levels in pancreatic β-cells. Acknowledgments The authors thank Prof. Hiromitsu Nakauchi (University of Tokyo) for his advice on flow cytometry and Prof. Ichiro Niki (Oita University) for his advice on INS-1 cells. This study was supported by a Grant-in-Aid for Scientific Research from Japan Society for the Promotion of Science, and by Suzuken Memorial Foundation. References Asfari, M., Janjic, D., Meda, P., Li, G., Halban, P.A., Wollheim, C.B., 1992. Establishment of 2-mercaptoethanol-dependent differentiated insulin-secreting cell lines. Endocrinology 130, 167–178. Butler, A.E., Janson, J., Bonner-Weir, S., Ritzel, R., Rizza, R.A., Butler, P.C., 2003. β-cell deficit and increased β-cell apoptosis in humans with type 2 diabetes. Diabetes 52, 102–110. Cardozo, A.K., Kruhøffer, M., Leeman, R., Ørntoft, T., Eizirik, D.L., 2001. Identification of novel cytokine-induced genes in pancreatic β-cells by high-density oligonucleotide arrays. Diabetes 50, 909–920. Ceneviva, G.D., Tzeng, E., Hoyt, D.G., Yee, E., Gallagher, A., Engelhardt, J.F., Kim, Y.M., Billiar, T.R., Watkins, S.A., Pitt, B.R., 1998. Nitric oxide inhibits lipopolysaccharideinduced apoptosis in pulmonary artery endothelial cells. American Journal of Physiology 275, L717–728. Choi, B.M., Pae, H.O., Jang, S.I., Kim, Y.M., Chung, H.T., 2002. Nitric oxide as a proapoptotic as well as anti-apoptotic modulator. Journal of Biochemistry and Molecular Biology 35, 116–126. Ciani, E., Virgili, M., Contestabile, A., 2002. Akt pathway mediates a cGMP-dependent survival role of nitric oxide in cerebellar granule neurones. Journal of Neurochemistry 81, 218–228.
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