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

Abstract

Reactive oxygen species (ROS) are important mediators of cellular signal transduction cascades such as proliferation, migration, and apoptosis. Chronic exposure of isolated β-cells to proinflammatory cytokines elevates intracellular oxidative stress leading to the demise of pancreatic β-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 β-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-1β, IFN-γ, and TNF-α; 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 β-cell.

Phagocyte-like NADPH oxidase generates ROS in INS 832/13 cells and rat islets: role of protein prenylation

“…Available evidence suggests that a protein kinase C-sensitive phosphorylation of p47 phox leads to its translocation to the membrane fraction (3). It has also been shown that functional activation of Rac1 (Rac1.GTP) is vital for the holoenzyme assembly and activation of Nox in insulin-secreting cells (38,39).…”

“…A growing body of recent evidence implicated roles for reactive oxygen species (ROS) in metabolic dysfunction of the islet ␤-cell under the duress of glucolipotoxicity, cytokines, and ceramide (26,38,39). It has been shown that increased ROS generation seen under the above experimental conditions is derived from the activation of phagocyte-like NADPH oxidase (Nox), since inhibition of this enzyme by selective inhibitors [e.g., diphenyleneiodonium chloride (DPI) or apocynin] or transfection of short interfering RNA (siRNA) against individual subunits of Nox (e.g., p47 phox ) significantly attenuated deleterious effects of aforementioned noxious stimuli (38,39).…”

Biphasic Roles of a Small G-Protein, RAC1 in Pancreatic Β-Cell

“…In addition to the above mentioned strategies for attenuating oxidative stress, inhibitors blocking Tiam1/Rac1/Nox signaling axis, 57,94,106 polyphenolic extracts supplementation, 107 stress activated kinase inhibitors, [108][109][110][111] and angiotensin receptor antagonists 112 have proven efficaciously to reduce oxidative stress and improve islet β-cell function.…”

“…91 In addition, studies have also implicated Nox in metabolic dysfunction of the islet β-cell under conditions of glucolipotoxicity and exposure to cytokines. 57,94 Generation of ROS under these conditions appears to be largely due to the activation of Nox, since inhibition of …”

“…94,96 In addition, recent studies from Sidarala and colleagues present the evidence that the Rac1-Nox2 signaling is vital in high glucose induced activation of stress activated kinases and loss in GSIS causing islet β-cell dysfunction. 97 Despite these in vitro evidences, potential roles for Nox in islet dysfunction in animal models of type 2 diabetes are minimal.…”