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The proapoptotic BH3-only proteins Bim and Puma are downstream of endoplasmic reticulum and mitochondrial oxidative stress in pancreatic islets in response to glucotoxicity.

Wali JA, Rondas D, McKenzie MD, Zhao Y, Elkerbout L, Fynch S, Gurzov EN, Akira S, Mathieu C, Kay TW, Overbergh L, Strasser A, Thomas HE - Cell Death Dis (2014)

Bottom Line: We studied the stress pathways induced by glucotoxicity in beta cells that result in apoptosis.The antioxidant N-acetyl-cysteine also partially protected islets from glucotoxicity.Islets deficient in both Bim and Puma, but not Bim or Puma alone, were significantly protected from killing induced by the mitochondrial reactive oxygen species donor rotenone.

View Article: PubMed Central - PubMed

Affiliation: 1] St. Vincent's Institute of Medical Research, Fitzroy, VIC, Australia [2] Department of Medicine, St. Vincent's Hospital, The University of Melbourne, Fitzroy, VIC, Australia.

ABSTRACT
Apoptosis of pancreatic beta cells is a feature of type 2 diabetes and its prevention may have therapeutic benefit. High glucose concentrations induce apoptosis of islet cells, and this requires the proapoptotic Bcl-2 homology domain 3 (BH3)-only proteins Bim and Puma. We studied the stress pathways induced by glucotoxicity in beta cells that result in apoptosis. High concentrations of glucose or ribose increased expression of the transcription factor CHOP (C/EBP homologous protein) but not endoplasmic reticulum (ER) chaperones, indicating activation of proapoptotic ER stress signaling. Inhibition of ER stress prevented ribose-induced upregulation of Chop and Puma mRNA, and partially protected islets from glucotoxicity. Loss of Bim or Puma partially protected islets from the canonical ER stressor thapsigargin. The antioxidant N-acetyl-cysteine also partially protected islets from glucotoxicity. Islets deficient in both Bim and Puma, but not Bim or Puma alone, were significantly protected from killing induced by the mitochondrial reactive oxygen species donor rotenone. Our data demonstrate that high concentrations of glucose induce ER and oxidative stress, which causes cell death mediated by Bim and Puma. We observed significantly higher Bim and Puma mRNA in islets of human donors with type 2 diabetes. This indicates that inhibition of Bim and Puma, or their inducers, may prevent beta-cell destruction in type 2 diabetes.

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Inhibition of oxidative stress protects islet cells from glucotoxicity. (a and b) The frequency of cells undergoing DNA fragmentation was measured by flow cytometry after incubation of wild-type C57BL/6, Bim−/−Puma−/−, Bim−/− or Puma−/− islets with 30 μM H2O2 and 50 μM pancaspase inhibitor qVD.oph for 2 days (a), or 100 nM Rotenone for 2 days (b). Control islets were incubated for 2 days in a medium containing 5.5 mM glucose. Results represent mean±s.e.m. of n≥4 independent experiments. **P<0.01 ***P<0.001 (two-way ANOVA). (c) Mean fluorescence intensity of H2DCF staining measured by flow cytometry after incubation of MIN6 cells with 50 mM ribose with or without 1.0 mM NAC for 3 days. Results are mean±s.e.m. of n=3–4 independent experiments. *P<0.05, ***P<0.001 (one-way ANOVA). (d and e) The frequency of cells undergoing DNA fragmentation was measured by flow cytometry after incubation of wild-type C57BL/6, Puma−/− or Bim−/− islets with 50 mM ribose with or without 1.0 mM NAC for 4 days (d), or 33.3 mM glucose with our without 1.0 mM NAC for 6–7 days (e). Control islets were incubated in a medium containing 5.5 mM glucose. Results represent mean±s.e.m. of n≥5 independent experiments. **P<0.01, ***P<0.001 (two-way ANOVA)
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fig6: Inhibition of oxidative stress protects islet cells from glucotoxicity. (a and b) The frequency of cells undergoing DNA fragmentation was measured by flow cytometry after incubation of wild-type C57BL/6, Bim−/−Puma−/−, Bim−/− or Puma−/− islets with 30 μM H2O2 and 50 μM pancaspase inhibitor qVD.oph for 2 days (a), or 100 nM Rotenone for 2 days (b). Control islets were incubated for 2 days in a medium containing 5.5 mM glucose. Results represent mean±s.e.m. of n≥4 independent experiments. **P<0.01 ***P<0.001 (two-way ANOVA). (c) Mean fluorescence intensity of H2DCF staining measured by flow cytometry after incubation of MIN6 cells with 50 mM ribose with or without 1.0 mM NAC for 3 days. Results are mean±s.e.m. of n=3–4 independent experiments. *P<0.05, ***P<0.001 (one-way ANOVA). (d and e) The frequency of cells undergoing DNA fragmentation was measured by flow cytometry after incubation of wild-type C57BL/6, Puma−/− or Bim−/− islets with 50 mM ribose with or without 1.0 mM NAC for 4 days (d), or 33.3 mM glucose with our without 1.0 mM NAC for 6–7 days (e). Control islets were incubated in a medium containing 5.5 mM glucose. Results represent mean±s.e.m. of n≥5 independent experiments. **P<0.01, ***P<0.001 (two-way ANOVA)

Mentions: Previous reports showed that exposure to high concentrations of glucose or ribose lead to increased ROS production in rat and human islets.21, 22 We investigated the involvement of oxidative stress in glucose-induced islet cell killing. First, we cultured islets in hydrogen peroxide (H2O2) at low concentrations (30 μM) that have been shown to induce apoptosis but not necrosis in non-islet cells.33, 34 Absence of Bim or Puma, or both Bim and Puma, did not protect islets from H2O2-induced DNA fragmentation (Figure 6a). Addition of the pancaspase inhibitor qVD.oph also failed to inhibit H2O2 toxicity (Figure 6a). This suggests that in contrast to certain other cell types, inhibition of apoptosis does not protect islet cells from cell death caused by cytosolic oxidative stress.


The proapoptotic BH3-only proteins Bim and Puma are downstream of endoplasmic reticulum and mitochondrial oxidative stress in pancreatic islets in response to glucotoxicity.

Wali JA, Rondas D, McKenzie MD, Zhao Y, Elkerbout L, Fynch S, Gurzov EN, Akira S, Mathieu C, Kay TW, Overbergh L, Strasser A, Thomas HE - Cell Death Dis (2014)

Inhibition of oxidative stress protects islet cells from glucotoxicity. (a and b) The frequency of cells undergoing DNA fragmentation was measured by flow cytometry after incubation of wild-type C57BL/6, Bim−/−Puma−/−, Bim−/− or Puma−/− islets with 30 μM H2O2 and 50 μM pancaspase inhibitor qVD.oph for 2 days (a), or 100 nM Rotenone for 2 days (b). Control islets were incubated for 2 days in a medium containing 5.5 mM glucose. Results represent mean±s.e.m. of n≥4 independent experiments. **P<0.01 ***P<0.001 (two-way ANOVA). (c) Mean fluorescence intensity of H2DCF staining measured by flow cytometry after incubation of MIN6 cells with 50 mM ribose with or without 1.0 mM NAC for 3 days. Results are mean±s.e.m. of n=3–4 independent experiments. *P<0.05, ***P<0.001 (one-way ANOVA). (d and e) The frequency of cells undergoing DNA fragmentation was measured by flow cytometry after incubation of wild-type C57BL/6, Puma−/− or Bim−/− islets with 50 mM ribose with or without 1.0 mM NAC for 4 days (d), or 33.3 mM glucose with our without 1.0 mM NAC for 6–7 days (e). Control islets were incubated in a medium containing 5.5 mM glucose. Results represent mean±s.e.m. of n≥5 independent experiments. **P<0.01, ***P<0.001 (two-way ANOVA)
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fig6: Inhibition of oxidative stress protects islet cells from glucotoxicity. (a and b) The frequency of cells undergoing DNA fragmentation was measured by flow cytometry after incubation of wild-type C57BL/6, Bim−/−Puma−/−, Bim−/− or Puma−/− islets with 30 μM H2O2 and 50 μM pancaspase inhibitor qVD.oph for 2 days (a), or 100 nM Rotenone for 2 days (b). Control islets were incubated for 2 days in a medium containing 5.5 mM glucose. Results represent mean±s.e.m. of n≥4 independent experiments. **P<0.01 ***P<0.001 (two-way ANOVA). (c) Mean fluorescence intensity of H2DCF staining measured by flow cytometry after incubation of MIN6 cells with 50 mM ribose with or without 1.0 mM NAC for 3 days. Results are mean±s.e.m. of n=3–4 independent experiments. *P<0.05, ***P<0.001 (one-way ANOVA). (d and e) The frequency of cells undergoing DNA fragmentation was measured by flow cytometry after incubation of wild-type C57BL/6, Puma−/− or Bim−/− islets with 50 mM ribose with or without 1.0 mM NAC for 4 days (d), or 33.3 mM glucose with our without 1.0 mM NAC for 6–7 days (e). Control islets were incubated in a medium containing 5.5 mM glucose. Results represent mean±s.e.m. of n≥5 independent experiments. **P<0.01, ***P<0.001 (two-way ANOVA)
Mentions: Previous reports showed that exposure to high concentrations of glucose or ribose lead to increased ROS production in rat and human islets.21, 22 We investigated the involvement of oxidative stress in glucose-induced islet cell killing. First, we cultured islets in hydrogen peroxide (H2O2) at low concentrations (30 μM) that have been shown to induce apoptosis but not necrosis in non-islet cells.33, 34 Absence of Bim or Puma, or both Bim and Puma, did not protect islets from H2O2-induced DNA fragmentation (Figure 6a). Addition of the pancaspase inhibitor qVD.oph also failed to inhibit H2O2 toxicity (Figure 6a). This suggests that in contrast to certain other cell types, inhibition of apoptosis does not protect islet cells from cell death caused by cytosolic oxidative stress.

Bottom Line: We studied the stress pathways induced by glucotoxicity in beta cells that result in apoptosis.The antioxidant N-acetyl-cysteine also partially protected islets from glucotoxicity.Islets deficient in both Bim and Puma, but not Bim or Puma alone, were significantly protected from killing induced by the mitochondrial reactive oxygen species donor rotenone.

View Article: PubMed Central - PubMed

Affiliation: 1] St. Vincent's Institute of Medical Research, Fitzroy, VIC, Australia [2] Department of Medicine, St. Vincent's Hospital, The University of Melbourne, Fitzroy, VIC, Australia.

ABSTRACT
Apoptosis of pancreatic beta cells is a feature of type 2 diabetes and its prevention may have therapeutic benefit. High glucose concentrations induce apoptosis of islet cells, and this requires the proapoptotic Bcl-2 homology domain 3 (BH3)-only proteins Bim and Puma. We studied the stress pathways induced by glucotoxicity in beta cells that result in apoptosis. High concentrations of glucose or ribose increased expression of the transcription factor CHOP (C/EBP homologous protein) but not endoplasmic reticulum (ER) chaperones, indicating activation of proapoptotic ER stress signaling. Inhibition of ER stress prevented ribose-induced upregulation of Chop and Puma mRNA, and partially protected islets from glucotoxicity. Loss of Bim or Puma partially protected islets from the canonical ER stressor thapsigargin. The antioxidant N-acetyl-cysteine also partially protected islets from glucotoxicity. Islets deficient in both Bim and Puma, but not Bim or Puma alone, were significantly protected from killing induced by the mitochondrial reactive oxygen species donor rotenone. Our data demonstrate that high concentrations of glucose induce ER and oxidative stress, which causes cell death mediated by Bim and Puma. We observed significantly higher Bim and Puma mRNA in islets of human donors with type 2 diabetes. This indicates that inhibition of Bim and Puma, or their inducers, may prevent beta-cell destruction in type 2 diabetes.

Show MeSH
Related in: MedlinePlus