Inhibition of GSK3 enhances NRF2 stability and nuclear localisation in pancreatic beta cells

Aim: Accumulation of reactive oxygen species (i.e., oxidative stress) is a leading cause of beta cell dysfunction and apoptosis in diabetes. Nrf2 (Nuclear factor-erythroid 2 p45-related factor 2) is a master regulator of the adaptive response to oxidative stress. Its activity is tightly regulated by cytoplasmic Keap1 (Kelch-like ECH-associated protein-1). We, and others, have now demonstrated that Nrf2 is not only regulated by Keap1 but also by the insulin-regulated protein kinase Gsk3 (Glycogen synthase kinase-3). Phosphorylation of Nrf2 by Gsk3 enhances beta-TrCP binding and ubiquitylation by cullin-1, resulting in increased proteasomal degradation of Nrf2. Thus, we hypothesize that the increase in Gsk3 activity in beta cells suppresses Nrf2 levels and diminishes expression of Nrf2-target genes.

Methods: INS1 cells (beta cell line) or isolated C57BL/6J mouse islets were exposed to selective Gsk3 inhibitors (CT) alone or in combination with the Keap1 inhibitor TBE31. Upon lysis, protein and mRNA levels were measured by Western blotting and qPCR, respectively.

Results: Treating INS1 cells over 24 hours with TBE31 significantly enhanced Nrf2 protein levels. The presence of CT along with TBE31 resulted in prolonged Nrf2 stability and enhanced nuclear localization (p< 0.05). The level of mRNA for the Nrf2-target gene NAD(P)H Quinone Dehydrogenase 1 (Nqo1) was significantly enhanced by the presence of CT (p< 0.05) in both INS1 and isolated mouse islets.

Summary: We have demonstrated that Gsk3 regulates the antioxidant response of pancreatic beta cells by modifying the availability and function of Nrf2. Inhibition of Gsk3 is, therefore, a strategy to investigate for protecting beta cells from antioxidant stress.