Existing evidence in multiple cell types, including the pancreatic β-cell, implicates post-translational phosphorylation and prenylation of individual components as a requisite for the optimal activation of Nox (9
). The main objective of the current study was to determine the functional status of Nox in islets derived from the ZDF rat, a well-studied model for obesity and type 2 diabetes, and to determine potential regulation of Nox components in human islets under the duress of glucolipotoxicity and diabetes. Our data suggested a significant activation of Nox and associated ROS generation in the ZDF islets compared with those derived from the ZLC islets. Lastly, our data in diabetic human islets corroborated our findings in the ZDF islets.
Several recent studies have demonstrated activation of Nox after exposure to physiologic concentrations of glucose in a variety of insulin-secreting cells (10
). Data from studies of pharmacologic and molecular biologic inhibition of Nox revealed that a tonic increase in Nox-derived ROS is necessary for GSIS (10
) and that prenylation of Rac1 appears to be necessary for glucose-induced Nox activation and ROS generation in isolated β-cells (9
). Emerging evidence also implicates Nox in metabolic dysfunction of the islet β-cell under conditions of glucolipotoxicity and exposure to cytokines (16
). These studies demonstrated an increase in the expression and phosphorylation of Nox subunits (i.e., p47phox
), together with significant activation of Rac1. In addition, the activation status of Rac1 was under the precise control of Tiam1, a guanine nucleotide exchange factor for Rac1 in β-cells (27
). In further support of this, we reported a marked reduction in high glucose-, high palmitate-, and cytokine-induced Rac1 and Nox activation and ROS generation in isolated β-cells after exposure to NSC23766, a selective inhibitor of the Tiam1/Rac1 signaling axis (16
). Taken together, previous in vitro findings implicated participatory roles of Nox in exerting effects at the mitochondrial level, including loss in membrane potential, cytochrome C release, and activation of caspase-3 culminating in islet β-cell dysfunction (16
In addition to an increase in p47phox
expression, Rac1 activation, and ROS generation, we observed a significant increase in the phosphorylation of JNK1/2 in the ZDF islets compared with the control islets. Similar changes in the activation of JNK1/2 were demonstrable in INS 832/13 cells after incubation with high glucose or palmitate. Selective inhibition of JNK1/2 using SP600125 markedly attenuated caspase-3 activation under glucotoxic conditions, suggesting that JNK1/2 activation lies upstream to mitochondrial dysfunction and caspase-3 activation. These data are in accord with findings of Cunha et al. (28
) demonstrating significant inhibition of palmitate-induced JNK activation and cell apoptosis in INS-1E cells by SP600125 and L-TAT-JNKi, a small peptide inhibitor of JNK. Along these lines, several recent studies have also demonstrated inhibition of caspase-3 activation after inhibition of JNK1/2 activation in models of cellular apoptosis (21
The observed reduction of ERK1/2 activation under glucolipotoxic conditions in the ZDF rat islets in vivo and in the INS 832/13 cells in vitro are indicative of impaired metabolic function and β-cell proliferation. Our current findings on reduction in ERK1/2 phosphorylation in INS 832/13 cells are in accord with studies of Costes et al. (32
), who demonstrated a significant reduction in ERK1/2 phosphorylation in MIN6 cells after exposure to 25 mmol/L glucose for 24 h. From the results of further studies, these investigators concluded that glucotoxic conditions downregulate the ERK1/2-cAMP-responsive element–binding protein-signaling pathway, leading to the apoptotic demise of the β-cell.
Recent studies by Zhang et al. (33
) demonstrated a significant increase in JNK1/2 phosphorylation and reduction in ERK1/2 phosphorylation during mevastatin-induced apoptosis of salivary adenoid carcinoma cells, suggesting a potential inverse relationship between JNK1/2 and ERK1/2 phosphorylation in the induction of cellular apoptosis.
Together, our observations in INS 832/13 cells, ZDF islets, and diabetic human islets support involvement of the Nox–ROS stress-activated signaling axis in the metabolic dysfunction; however, additional studies are needed to substantiate this formulation. Recent studies by Nakayama et al. (34
) demonstrated the functional activation of Nox in islets of db/db
mice and in Otsuka Long-Evans Tokushima Fatty rats. Treatment of these animals with angiotensin II type-1 receptor antagonists reduced Nox activation and oxidative stress. It may be germane to point out that Valle et al. (35
) recently examined potential changes in Nox in islets derived from obese animals fed a high-fat diet. In contrast to islets from db/db
mice, Otsuka Long-Evans Tokushima Fatty rats (34
), and ZDF rat (current study), islets from animals fed a high-fat diet exhibited markedly lower expression levels of p47phox
subunits and ROS production compared with control rat islets. These investigators attributed this toward increased glucose oxidation and GSIS seen in islets from animals fed a high-fat diet in response to glucose (35
On the basis of the existing information and our current findings, we propose the following model for Nox-mediated induction of β-cell dysfunction in diabetes (): Exposure of isolated β-cells to glucolipotoxic conditions or islets derived from the diabetic condition in ZDF rats or humans results in increased activation of Rac1 and Nox. Consequential generation of ROS and the associated oxidative stress, in turn, promote activation of JNK1/2 and mitochondrial dysregulation. Alternatively, activation of the cytosolic Nox–ROS–JNK1/2 signaling pathway increases superoxide generation that impairs the functional efficiency of mitochondria. This proposal is supported by findings of Bindokas et al. (36
) that demonstrated excessive superoxide levels in islet mitochondria from the ZDF rat.
FIG. 8. Proposed model for Nox-induced ROS-mediated mitochondrial dysregulation in diabetes. Based on the data accrued from the current studies, we propose a model for the Nox–ROS–JNK signaling in the metabolic dysfunction of the pancreatic β-cell (more ...)
In summary, our current findings implicate Nox as one of the sources of oxidative stress in the diabetic islet. It will be interesting to determine if pharmacologic intervention of Nox activation seen in islets under diabetic conditions can be restored to its normal function. Such intervention modalities include NSC23766, a selective inhibitor of Tiam1/Rac1, which we have used in in vitro experiments to restore mitochondrial function in β-cells exposed to elevated glucose, lipids, and cytokines (16
). In this context, recent investigations have successfully used NSC23766, a selective inhibitor of the Tiam1–Rac1 signaling axis, to correct Nox-mediated effects on cellular function in vitro and in vivo (15
). Using the streptozotocin diabetic mouse model, Shen et al. (37
) demonstrated a regulatory role for Rac1 in hyperglycemia-induced apoptosis in cardiomyocytes. They demonstrated that upregulation of Rac1, Nox activity, and increased ROS generation led to apoptosis of cardiomyocytes under the duress of hyperglycemia. Treatment of diabetic db
mice with NSC23766 significantly inhibited Nox activity and cell apoptosis (37
). Additional studies are needed to pinpoint the regulatory roles of Tiam1–Rac1–Nox–ROS signaling in the metabolic dysfunction in the diabetic islet.