The role of RNA oxidation in islet dysfunction in Type 2 diabetes

Oxidative stress and reactive oxygen species (ROS) play a key role in the development of insulin resistance, β-cell dysfunction, and impaired glucose tolerance in type 2 diabetes (T2D). ROS initiate reactions that promote the modification of biological molecules and aberrant signalling to result in cellular dysfunction and death. RNA could be a key target for ROS due to its localization within the cells and lack of RNA repair mechanisms. The nucleobase guanosine, is highly sensitive to oxidation, which forms 8-oxoguanosine (8-oxoGuo), a product strongly associated with T2D morbidity. However, the pathway by which 8-oxoGuo is formed in patients with T2D and whether 8-oxoGuo has a causal role in disease progression and islet dysfunction remains unknown. In this study, we examined the reactivity of 8-oxoGuo and related modified nucleoside with INS-1 cells, as β-cell model, and assessed the pathways responsible for ROS formation on exposure of β-cells to pro-inflammatory cytokines or glucolipotoxicity (GLT). Initial studies focused on the effect of the treatments on intracellular thiol concentration and the formation of hydrogen peroxide (H2O2). Exposure of INS-1 cells to TNFα, IL-1β or GLT conditions (25 mM glucose / 100 mM palmitic acid) resulted in a significant time-dependent loss in thiols. Exposure to TNFα and/or IL-1β, but not GLT, resulted in an increase in H2O2 formation. Experiments were also performed with INS-1E cells treated with 8-oxoGuo, 8-oxodeoxyguanosine (8-oxodG), 8-oxoGTP, 8-chloroguanosine (8ClG), and 8-chlorodeoxyguanosine (8CldG). This resulted in a non-significant loss in thiols, but an elevation in the production of H2O2, particularly with 8ClG after 4 and 24 h exposure times. In both sets of experiments, evidence was obtained for the alteration of 8-oxoGuo and 8-oxodG within the cellular RNA and DNA respectively. Overall, this project provides new data regarding oxidative pathways in different T2D models of β-cell dysfunction