Glucose-stimulated insulin secretion by pancreatic β-cells results in a high level of metabolic activity and ROS production. Excess ROS can cause cellular damage leading to apoptosis. We recently reported that the cytokine interleukin 6 (IL-6) stimulates autophagy and protects β-cells from ROS-induced damage and apoptosis via the autophagic degradation of an antioxidant response repressor, KEAP1. This results in increased protein levels of the master antioxidant factor, NRF2. IL-6 also stimulates early markers of mitophagy in a NRF2-dependent manner. Interestingly, these effects are associated with noncanonical translocation of NRF2 to the mitochondria. Here, we set out to validate these findings and determine how NRF2 mitochondrial translocation is regulated. Using fluorescence imaging of mt-keima, we demonstrate that IL-6 stimulates mitophagy. This occurs without effects on mitochondrial function as measured by the Seahorse MitoStress Test. We also find that IL-6 rapidly stimulates NRF2 mitochondrial translocation, an effect that does not occur in response to treatment with hydrogen peroxide or a chemical inhibitor of KEAP1. These data indicate that liberating NRF2 from KEAP1 alone does not lead to NRF2 mitochondrial translocation, suggesting that IL-6 regulates NRF2 localization by additional mechanisms. NRF2 has extensive post-translational modifications, some of which are associated with nuclear localization. Therefore, we hypothesized that IL-6 induces specific NRF2 post-translational modifications that promote mitochondrial translocation. Indeed, we found that IL-6 stimulates serine phosphorylation on NRF2. Based on our findings described here, we propose that selective modification of NRF2 regulates its distinct subcellular localization and that selective translocation determines the mechanism of NRF2-mediated antioxidant response that functions to reduce ROS and restore cellular homeostasis in pancreatic β-cells.
M. Marasco: Employee; Spouse/Partner; Eli Lilly and Company. A.M. Conteh: None. C. Muralidharan: None. E. Pfeifer: None. A.K. Linnemann: None.
National Institutes of Health (K01DK102492), (R03DK115990 to A.K.L.)