Phagocyte-like NADPH oxidase promotes cytokine-induced mitochondrial dysfunction in pancreatic beta-cells: evidence for regulation by Rac1

Subasinghe W, Syed I, Kowluru A. Phagocyte-like NADPH oxidase promotes cytokine-induced mitochondrial dysfunction in pancreatic beta-cells: evidence for regulation by Rac1. Am J Physiol Regul Integr Comp Physiol 300: R12-R20, 2011. First published October 13, 2010; doi:10.1152/ajpregu.00421.2010.-Reactive oxygen species (ROS) are important mediators of cellular signal transduction cascades such as proliferation, migration, and apoptosis. Chronic exposure of isolated beta-cells to proinflammatory cytokines elevates intracellular oxidative stress leading to the demise of pancreatic beta-cells culminating in the onset of diabetes. Although the mitochondrial electron transport chain is felt to be the primary source of ROS, several lines of recent evidence suggest that phagocyte-like NADPH oxidase plays a central role in cytokine-mediated ROS generation and apoptosis of beta-cells. However, the precise mechanisms underlying the regulation of NADPH oxidase remain unknown. To address this, insulin-secreting INS 832/13 cells were treated with cytomix (IL-1 beta, IFN-gamma, and TNF-alpha; 10 ng/ml each) for different time intervals (0-24 h). A significant, time-dependent increase in NADPH oxidase activation/intracellular ROS production, p47(phox) subunit, but not p67(phox) subunit, expression of the phagocyte-like NADPH oxidase were demonstrable under these conditions. Furthermore, siRNA-p47(phox) transfection or exposure of INS 832/13 cells to apocynin, a selective inhibitor of NADPH oxidase, markedly attenuated cytomix-induced ROS generation in these cells. Cytomix-mediated mitochondrial dysfunction in INS 832/13 cells was evident by a significant loss of mitochondrial membrane potential (MMP) and upregulated caspase 3 activity. Cytomix treatment also caused a transient (within 15 min) activation of Rac1, a component of the NADPH oxidase holoenzyme. Furthermore, GGTI-2147 and NSC23766, known Rac1 inhibitors, not only attenuated the cytomix-induced Rac1 activation but also significantly prevented loss of MMP (NSC23766 > GGTI-2147). However, NSC23766 had no effect on cytomix-induced NO generation or caspase 3 activation, suggesting additional regulatory mechanisms might underlie these signaling steps. Together, these findings suggested that Rac1-mediated regulation of phagocyte-like NADPH oxidase contributes to cytokine-mediated mitochondrial dysfunction in the beta-cell.