NADPH Oxidase NOX2 Defines a New Antagonistic Role for Reactive Oxygen Species and cAMP/PKA in the Regulation of Insulin Secretion

In insulin-secreting cells, expression of NADPH oxidase (NOX), a potent source of ROS, has been reported, along with controversial findings regarding its function. Here, the role of NOXs was investigated: first by expression and cellular localization in mouse and human pancreatic islets, and then by functional studies in islets isolated from Nox isoform–specific knockout mice. Both human and mouse β-cells express NOX, in particular NOX2. With use of Nox isoform–specific knockout mice, functional analysis revealed Nox2 as the predominant isoform. In human islets, NOX2 colocalized with both insulin granules and endosome/lysosome membranes. Nox2-deficient islets stimulated with 22.8 mmol/L glucose exhibited potentiation of insulin release compared with controls, an effect confirmed with in vitro knockdown of Nox2. The enhanced secretory function in Nox2-deficient islets was associated with both lower superoxide levels and elevated cAMP concentrations. In control islets, GLP-1 and other cAMP inducers suppressed glucose-induced ROS production similarly to Nox2 deficiency. Inhibiting cAMP-dependent protein kinase reduced the secretory response in Nox2-null islets, although not in control islets. This study ascribes a new role for NOX2 in pancreatic β-cells as negative modulator of the secretory response, reducing cAMP/PKA signaling secondary to ROS generation. Results also show reciprocal inhibition between the cAMP/PKA pathway and ROS.NOX enzymes generate superoxide by transferring one electron from NADPH to oxygen (1). The best known NOX isoform is the phagocyte NADPH oxidase (NOX), a multicomponent complex comprising a membrane catalytic heterodimer, the flavocytochrome b_558, formed by gp91^phox (also referred to as NOX2) and p22^phox (where phox is phagocyte oxidase). The cytosolic regulatory subunits are composed of p40^phox, p47^phox, p67^phox, and GTPases Rac1 or Rac2 (1). Assembly of cytosolic elements to membrane catalytic core initiates the activation of NOX. To date, seven isoforms of NOX (NOX1–5 and dual oxidases DUOX1–2) have been identified with different activation mechanisms and heterogeneous tissue distribution (1). In addition to microbial attack by professional phagocytes, physiopathological roles of NOX have recently attracted attention in nonphagocytic cells, including pancreatic β-cells (2–7). Reactive oxygen species (ROS), such as superoxide and hydrogen peroxide, might participate in β-cell dysfunction (8). The redox imbalance favored by high metabolic rate and a relatively low detoxifying system has contributed to the general concept that β-cells are sensitive to ROS, although they can handle rather high concentrations of H₂O₂ (9).NOX family represents one of the potential sources of ROS in insulin-secreting cells (4). Both rat islets and insulinoma express membrane-associated catalytic components Nox1, Nox2, Nox4, and p22^phox, as well as cytosolic regulators p40^phox, p47^phox, and p67^phox and their homologs Noxo1 and Noxa1 (3,5,6). Regarding their putative function in the β-cell, NOXs have been implicated in glucose-induced ROS production in MIN-6 cells (10). Knockdown of p47^phox results in total inhibition of glucose-stimulated insulin secretion and lowers ROS (11). In animal models of type 2 diabetes, islets exhibit increased NOX components Nox2 and p22^phox, correlating with increased oxidative stress (12). Activation of Nox and accompanying ROS generation were demonstrated in Zucker diabetic fatty (ZDF) rat and diabetic human islets (13). However, inhibition of islet NOX using diphenyleneiodonium (DPI) impairs glucose-stimulated insulin secretion (6) along with blunted glucose-induced superoxide production (5,10). These conflicting findings regarding NOX activity and β-cell function might be attributed to poor specificity of old-generation NOX inhibitors, such as apocynin and DPI (14). The former has been shown to function as a general ROS scavenger, and the latter is a nonspecific inhibitor of electron transporters (1,15).In the current study, we first investigated relative expression levels of the different catalytic subunits of NOXs in both human and mouse pancreatic islets. Then, subcellular distribution of the identified predominant NOX isoform NOX2 was assessed in human islet cells. For avoidance of poor specificity of NOX inhibitors, islets isolated from Nox isoform–specific–deficient mice were used to investigate the contribution of NOXs in insulin secretory function.