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, Ross
(2014) 50th Annual Meeting of the European Association for the Study of Diabetes — Location: Vienna, Austria (15.September.2014)

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  • Bensellam, Mohammedorcid-logoUCLouvain
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  • Montgomery, Magdalene
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  • Luzuriaga, Jude
    Author
  • Chan, Jeng-Yie
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  • Laybutt, Ross
    Author
Abstract
Background: Oxidative stress is a central mechanism of beta-cell glucotoxicity, but the underlying signaling pathways are only partially understood. 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 diabetic db/db mice and their normoglycemic db/+ littermates were used to verify the expression of IDs and antioxidant genes. Insulin-secreting 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 and/or Id3 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, immunostaining showed that ribose treatment increased ID1 and ID3 nuclear localisation. In ribose-treated cells, siRNA-mediated 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. Glutathione peroxidase activity was also reduced after Id1/3 knockdown. 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 and MafF. Conclusion: We have identified IDs as a novel family of oxidative stress-responsive genes in beta-cells as well as an unexpected role for Id1 and Id3 in the modulation of redox status. Such modulation is likely to stem from the maintenance of an adequate mitochondrial adaptation and upholding of the expression of NFE2L2 interacting partners. The maintenance of an adequate mitochondrial-antioxidant response by Ids may promote beta-cell survival under conditions of oxidative stress.
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  • Garvan Institute of Medical Research

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Bensellam, M., Montgomery, M., Luzuriaga, J., Chan, J.-Y., & Laybutt, 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. Diabetologia : clinical and experimental diabetes and metabolism, 57, S49. https://doi.org/10.1007/s00125-014-3355-0 (Original work published 2014)