P043 Ciliary neurotrophic factor (CNTF) downregulates CAMKII-AMPK pathway protecting MIN6 cells against apoptosis induced by IL-1beta

Abstract

Introduction Background: Diabetes Mellitus is a metabolic syndrome of multiple etiologies, resulting from a lack of insulin and sometimes associated with an increase in resistance to the hormone by insulin-target tissues. DM1 is characterized by the infiltration of macrophages and T-type CD4+ and T-CD8+ cells in the pancreas, due to the failure of the autoimmune system, causing inflammation and leading to the release of nitric oxide, free radicals, and cytokines such as IL1β and IFNγ. These pro-inflammatory cytokines activate pro-apoptotic mechanisms leading to a loss in functional beta-cell mass. These mechanisms can be reproduced in vitro by the exposure of beta cells to pro-inflammatory cytokines, such as IL-1β. In contrast, CNTF is a cytokine that promotes pancreatic islet cell survival. CaMKII is Calmodulin-Ca2+ activated enzyme that acts in memory formation and release process. This enzyme is important in the insulin secretion and in, together with LKB1, the masters AMPK activators. AMPK is a kinase that acts on pancreatic beta cells as a sensor of the cellular energy state. It is well known that in pancreatic islets AMPK plays an important role in regulating insulin secretion and inhibition of AMPK protects beta cells from apoptosis mediated either by cytokines and/or induced T cell CD8+ and CD4+. Methods MIN6 cells, Wildtype or AMPKkd, were pre-treated with (N Group) ou Without (C Group) 1nM of CNTF before the exposure to IL-1β (10 ng/ml). After the treatment, the proteins and the total mRNA were extract and the protein and mRNA of the AMPK pathway were analyzed. Furthermore, importants apoptosis markers, such as a caspase-3 cleavage, iNOS expression and DNA fragmentation were measured. Results Our results indicate that apoptosis induced by IL-1β in MIN6 cells requires activation of the CaMKII-AMPK pathway; furthermore, CNTF inhibits MIN6 cell apoptosis through downregulation of AMPK decreasing pCaMKII. Moreover, IL1β activate AMPK mainly through CaMKII phosphorylation. Finally, IL1β-induced MIN6 cell apoptosis via an increase in iNOS that was reversed by CNTF. Conclusion IL1β upregulates AMPK-INOS pathway, ultimately leading to beta cell death. In this context, CNTF protects beta cells against apoptosis, induced by either IL1β or Alloxan through the reduction of the activity of the CaMKII-AMPK-INOS pathway.