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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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Glucose toxicity activates the PERK and IRE1α arms of the UPR pathway. (a) Quantitative RT-PCR of wild-type C57BL/6 islets cultured for 1 to 3 days either in a control medium or in a medium containing 50 mM ribose. Relative mRNA expression levels for Chop, Atf4, Bip, Pdia4, P58 and XBP1-unspliced/spliced were calculated by normalizing to the signal for β-Actin mRNA in each sample and comparison with islets cultured in a control medium. Results represent mean±s.e.m. of n=3–6 independent experiments. *P<0.05, ***P<0.001 when comparing islets cultured in ribose with those cultured in a control medium (one-way ANOVA). (b) Eight hundred wild-type C57BL/6 islets were cultured in a control medium or in a medium containing 50 mM ribose. Positive control islets were cultured in 5 μm thapsigargin. Western blotting was performed with antibodies to CHOP (31 kDa), BiP (78 kDa) and β-Actin (45 kDa) (loading control). Results are representative of three independent experiments
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fig2: Glucose toxicity activates the PERK and IRE1α arms of the UPR pathway. (a) Quantitative RT-PCR of wild-type C57BL/6 islets cultured for 1 to 3 days either in a control medium or in a medium containing 50 mM ribose. Relative mRNA expression levels for Chop, Atf4, Bip, Pdia4, P58 and XBP1-unspliced/spliced were calculated by normalizing to the signal for β-Actin mRNA in each sample and comparison with islets cultured in a control medium. Results represent mean±s.e.m. of n=3–6 independent experiments. *P<0.05, ***P<0.001 when comparing islets cultured in ribose with those cultured in a control medium (one-way ANOVA). (b) Eight hundred wild-type C57BL/6 islets were cultured in a control medium or in a medium containing 50 mM ribose. Positive control islets were cultured in 5 μm thapsigargin. Western blotting was performed with antibodies to CHOP (31 kDa), BiP (78 kDa) and β-Actin (45 kDa) (loading control). Results are representative of three independent experiments

Mentions: We next determined whether ER stress-mediated UPR can be induced by glucose. For in vitro treatment, we used the reducing sugar ribose (50 mM), which can enter the cell through glucose transporters and be metabolized through the pentose phosphate pathway and glycolysis. Like other reducing sugars, including glucose or fructose, ribose induces islet cell death through glycation and formation of ROS,22 but over a shorter time frame. The levels of mRNAs for UPR pathway components were examined in wild-type islets by qPCR. Expression of Chop but not ATF4 increased in islets cultured with ribose, and this increase was statistically significant after 2 days (Figure 2a). Moreover, IRE1α-mediated splicing of XBP1 gradually increased with ribose treatment and was significantly increased after 3 days, whereas unspliced XBP1 mRNA expression did not change (Figure 2a). There was a marginal decrease in expression of the ATF6-regulated chaperones BiP and Pdia4 after the first day of ribose incubation, but expression of the cochaperone P58 was not affected by the exposure of islets to ribose (Figure 2a). Treatment of cultured islets with 33.3 mM glucose for 7 days produced the same results (data not shown).


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)

Glucose toxicity activates the PERK and IRE1α arms of the UPR pathway. (a) Quantitative RT-PCR of wild-type C57BL/6 islets cultured for 1 to 3 days either in a control medium or in a medium containing 50 mM ribose. Relative mRNA expression levels for Chop, Atf4, Bip, Pdia4, P58 and XBP1-unspliced/spliced were calculated by normalizing to the signal for β-Actin mRNA in each sample and comparison with islets cultured in a control medium. Results represent mean±s.e.m. of n=3–6 independent experiments. *P<0.05, ***P<0.001 when comparing islets cultured in ribose with those cultured in a control medium (one-way ANOVA). (b) Eight hundred wild-type C57BL/6 islets were cultured in a control medium or in a medium containing 50 mM ribose. Positive control islets were cultured in 5 μm thapsigargin. Western blotting was performed with antibodies to CHOP (31 kDa), BiP (78 kDa) and β-Actin (45 kDa) (loading control). Results are representative of three independent experiments
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3973197&req=5

fig2: Glucose toxicity activates the PERK and IRE1α arms of the UPR pathway. (a) Quantitative RT-PCR of wild-type C57BL/6 islets cultured for 1 to 3 days either in a control medium or in a medium containing 50 mM ribose. Relative mRNA expression levels for Chop, Atf4, Bip, Pdia4, P58 and XBP1-unspliced/spliced were calculated by normalizing to the signal for β-Actin mRNA in each sample and comparison with islets cultured in a control medium. Results represent mean±s.e.m. of n=3–6 independent experiments. *P<0.05, ***P<0.001 when comparing islets cultured in ribose with those cultured in a control medium (one-way ANOVA). (b) Eight hundred wild-type C57BL/6 islets were cultured in a control medium or in a medium containing 50 mM ribose. Positive control islets were cultured in 5 μm thapsigargin. Western blotting was performed with antibodies to CHOP (31 kDa), BiP (78 kDa) and β-Actin (45 kDa) (loading control). Results are representative of three independent experiments
Mentions: We next determined whether ER stress-mediated UPR can be induced by glucose. For in vitro treatment, we used the reducing sugar ribose (50 mM), which can enter the cell through glucose transporters and be metabolized through the pentose phosphate pathway and glycolysis. Like other reducing sugars, including glucose or fructose, ribose induces islet cell death through glycation and formation of ROS,22 but over a shorter time frame. The levels of mRNAs for UPR pathway components were examined in wild-type islets by qPCR. Expression of Chop but not ATF4 increased in islets cultured with ribose, and this increase was statistically significant after 2 days (Figure 2a). Moreover, IRE1α-mediated splicing of XBP1 gradually increased with ribose treatment and was significantly increased after 3 days, whereas unspliced XBP1 mRNA expression did not change (Figure 2a). There was a marginal decrease in expression of the ATF6-regulated chaperones BiP and Pdia4 after the first day of ribose incubation, but expression of the cochaperone P58 was not affected by the exposure of islets to ribose (Figure 2a). Treatment of cultured islets with 33.3 mM glucose for 7 days produced the same results (data not shown).

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