Phagocyte-like NADPH oxidase (Nox2) promotes activation of p38MAPK in pancreatic β-cells under glucotoxic conditions: Evidence for a requisite role of Ras-related C3 botulinum toxin substrate 1 (Rac1)

Abstract

It is well established that glucotoxicity (caused by high glucose concentrations; HG) underlies pathogenesis of islet dysfunction in diabetes. We have recently demonstrated that Nox2 plays a requisite role in the generation of reactive oxygen species (ROS) under HG conditions, resulting in mitochondrial dysregulation and loss of islet β-cell function. Herein, we investigated roles of Nox2 in the regulation of downstream stress kinase (p38MAPK) activation under HG conditions (20mM; 24h) in normal rodent islets and INS-1 832/13 cells. We observed that gp91-ds-tat, a specific inhibitor of Nox2, but not its inactive analog, significantly attenuated HG-induced Nox2 activation, ROS generation and p38MAPK activation, thus suggesting that Nox2 activation couples with p38MAPK activation. Since Rac1, is an integral member of the Nox2 holoenzyme, we also assessed the effects of Rac1 inhibitors (EHT 1864, NSC23766 and Ehop-016) on HG-induced p38MAPK activation in isolated β-cells. We report a significant inhibition of p38MAPK phosphorylation by Rac1 inhibitors, implying a regulatory role for Rac1 in promoting the Nox2-p38MAPK signaling axis in the β-cell under the duress of HG. 2-Bromopalmitate, a known inhibitor of protein (Rac1) palmitoylation, significantly reduced HG-induced p38MAPK phosphorylation. However, GGTI-2147, a specific inhibitor of geranylgeranylation of Rac1, failed to exert any significant effects on HG-induced p38MAPK activation. In conclusion, we present the first evidence that the Rac1-Nox2 signaling module plays novel regulatory roles in HG-induced p38MAPK activation and loss in glucose-stimulated insulin secretion (GSIS) culminating in metabolic dysfunction and the onset of diabetes.

Inhibition of Prenylation Promotes Caspase 3 Activation, Lamin B Degradation and Loss in Metabolic Cell Viability in Pancreatic β-Cells

“…We also conclude that inhibition of farnesylation increases susceptibility of non-farnesylated proteins, such as lamin B, for degradation by pro-apoptotic caspases, such as caspase 3 leading to loss in metabolic cell viability. Together, these findings affirm our working model that functional inactivation of prenylation pathway in islet β-cells under the duress of metabolic stress and diabetes leads to increased stress kinase activation [21, 22, 31] dysregulation of metabolic events that are essential for islet β-cell function, including proliferation and survival [13]. …”

“…Pharmacological inhibition of Rac1, observed under glucotoxic conditions, markedly attenuated p38MAPK activation [22]. Furthermore, we have demonstrated requirement protein farnesylation in glucose-induced activation of ERK1/2, a signaling kinase involved in β-cell proliferation [17].…”

“…Previous observations from our laboratory in INS-1 832/13 cells, rodent and human islets have demonstrated regulatory roles for Rac1, a geranylgeranylated protein, in the activation of stress kinases, such as p38MAPK under glucotoxic conditions [22]. Pharmacological inhibition of Rac1, observed under glucotoxic conditions, markedly attenuated p38MAPK activation [22].…”

“…In these studies, INS-1 832/13 cells were incubated in the absence (diluent]) or presence of simvastatin (0-30 µM) and FTI-277 (15 µM) for different time intervals as described in the text. Earlier studies have demonstrated that both of these agents, in the stated concentration range, significantly inhibited MVA and protein prenylation pathways in a variety of cells, including the islet β-cell [16, 17, 19-22]. …”

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

“…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. However, a recent study systematically examined the functional status of Nox in islets from Zucker Diabetic Fatty [ZDF] rat, which develops obesity, hyperinsulinemia, hyperglycemia and a decline in β-cell function.…”