Characterization of the expression of NADPH oxidase isoforms in pancreatic beta cells and its modulation by proinflammatory cytokines

Abstract

NADPH oxidases (NOX) are reactive oxygen species (ROS)-producing isoenzymes. The isoenzymes NOX1, NOX2 and NOX4 are expressed in pancreatic beta cells and our preliminary studies indicate expression of the isoenzymes DUOX1 and DUOX2 in beta cells in vitro. However, this remains to be confirmed in vivo. In addition to oxidative stress, ROS participate in signal transduction and this function depends on its restricted production in specific intracellular microdomains: NOX intracellular location determines ROS signaling function in many tissues. The intracellular location of NOX in beta cells remains unknown. Documenting this location will indicate the role of these isoenzymes in beta cell (patho)physiology. Insulitis and beta cell destruction contribute to Type 1 Diabetes mellitus (T1DM). NADPH oxidases participate in beta cell apoptosis and cytokines released during insulitis appear to influence NOX expression, but the nature of this modulation remains uncertain. Understanding the modulation of NOX by cytokines is essential to unveil its contribution to beta cell apoptosis. In this project, we propose to characterize NADPH oxidase expression in beta cells and its modulation by pro-inflammatory cytokines. DUOX expression in rodent pancreatic islets will be confirmed by immunostaining. The intracellular location of NOX isoforms will be studied by immunocytochemistry and their relative abundance analyzed by RT-qPCR and immunostaining in INS-1E. Finally, the modulation of NOX expression by cytokines will be studied by RT-qPCR and immunostaining in INS-1E and islets grown in vitro. The knowledge obtained can advance our understanding of beta cell (patho)physiology and NOX function in this tissue. (AU)