IDs are novel oxidative stress-responsive genes in beta-cells that regulate redox status and survival through effects on mitochondria and the NFE2L2 pathway

Bensellam, Mohammed;Montgomery, Magdalene;Luzuriaga, Jude;Chan, Jeng Yie;Laybutt, D. Ross
(2014) Islet Study Group meeting 2014: Pancreatic Islet Cell Plasticity in Health and Diabetes — Location: Lausanne, Switzerland (12.September.2014)

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  • Bensellam, MohammedUCLouvain
    Author
  • Montgomery, Magdalene
    Author
  • Luzuriaga, Jude
    Author
  • Chan, Jeng Yie
    Author
  • Laybutt, D. Ross
    Author
Abstract
Background: Oxidative stress is a central mechanism of beta-cell glucotoxicity in type 2 diabetes. Inhibitor of Differentiation (ID) proteins are transcriptional regulators induced by hyperglycemia in islets, but the mechanisms involved and their role in beta-cells are not clear. Here we investigated: 1) whether oxidative stress regulates ID expression in beta-cells, and 2) the role of ID expression in beta-cell pathophysiology under conditions of oxidative stress. Methods: Fixed pancreata and isolated islets from db/+ and db/db mice were used to verify the expression of IDs and antioxidant genes. MIN6 beta-cells and isolated islets from Id1 and Id3-KO mice were cultured for 0-48h in the presence or absence of H2O2 (100-300 µM) or ribose (5-50 mM) to induce oxidative stress. RNA interference was used to silence the expression of Id1, Id3 and Maf(F/G/K) in MIN6 cells. mRNA and protein levels were measured by real-time RT-PCR, western blot and immunocytochemistry. H2O2 levels were assessed by DCFDA probe, mitochondrial morphology by Mitotracker probe, oxygen consumption by Clark electrode and apoptosis by DNA fragmentation ELISA. Results: ID1-4 expression was upregulated in the islets of diabetic db/db mice with parallel changes in the expression of multiple antioxidant genes. In MIN6 cells, ribose and H2O2 treatment increased the mRNA levels of Id1-4 in a time- and concentration-dependent manner with parallel changes in the expression of antioxidant genes. Furthermore, ribose treatment increased ID1 and ID3 nuclear localisation. In ribose-treated cells, inhibition of Id1 and/or Id3 reduced the expression of multiple antioxidant genes, including heme oxygenase. Additive effects were observed when both isoforms were inhibited. These effects were accompanied by ~2-fold increase in H2O2 levels (p<0.01), 18% reduction in oxygen consumption (p<0.01) and ~2-fold increase in beta-cell apoptosis (p<0.001). Furthermore, Id1/3 inhibition induced mitochondrial fragmentation similar to that observed in the presence of ribose. Similarly, ribose-induced apoptosis in islets was potentiated in Id1-KO islets and to a stronger extent in Id3-KO islets (1.6-fold; p<0.01). Finally, under oxidative stress, Id1/3 inhibition further increased NFE2L2 nuclear localization but represses the expression of its interacting partners MafK, MafF, and to a lesser extent MafG. Interestingly, inhibition of Maf(F/G/K) mimicked the effects of Id1/3 inhibition on antioxidant gene expression and apoptosis. Conclusion: We have identified IDs as a novel family of oxidative stress-responsive genes in beta-cells. IDs modulate the redox status through a link to the NFE2L2-MAF pathway and mitochondria and thereby, may promote beta-cell survival under conditions of oxidative stress.
Affiliations
  • Garvan Institute of Medical ResearchDiabetes Division

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Bensellam, M., Montgomery, M., Luzuriaga, J., Chan, J. Y., & Laybutt, D. R. (2014). IDs are novel oxidative stress-responsive genes in beta-cells that regulate redox status and survival through effects on mitochondria and the NFE2L2 pathway. Islet Study Group meeting 2014: Pancreatic Islet Cell Plasticity in Health and Diabetes, Lausanne, Switzerland. https://hdl.handle.net/2078.5/91978