Mechanisms Of Pancreatic Beta Cell Dysfunction In Diabetes

Diabetes is a serious medical condition characterized by decreased insulin secretion from pancreatic β-cells and decreased insulin sensitivity in the peripheral tissues, resulting in elevated levels of blood glucose. According to the International Diabetes Federation, about 387 million cases have been reported worldwide in the year 2013 and it is estimated that about 500 million people would be affected by 2050. Type 2 diabetes, which accounts for about 90% of the total number of cases, is caused by decreased insulin sensitivity in the peripheral tissues and decreased glucose-stimulated insulin secretion from the pancreatic β-cells. The underlying mechanisms involved in β-cell dysfunction under hyperglycemic conditions are currently under investigation. Previous studies in our laboratory have implicated the role of Rac1-Nox2-induced oxidative stress in pancreatic β-cell dysfunction in models of impaired insulin secretion and T2DM. Studies in pancreatic islets derived from the ZDF rat and human diabetic donors have revealed increased activation of Rac1 and Nox2 subunits in these models. Further investigations have suggested the involvement of stress kinases in the activation of downstream apoptotic pathways, leading to β-cell death. Therefore, the primary objective of my dissertation project is to determine the role of Rac1-Nox2-derived oxidative stress in the activation of p38MAPK and p53 tumor suppressor, culminating in β-cell death under glucotoxic conditions. Our studies have revealed that exposure of clonal β-cells (INS-1 832/13 cells) and rodent islets to glucotoxic conditions, results in the activation of p38MAPK and p53. We first, examined the regulatory role of Rac1-Nox2 holoenzyme in the activation of p38MAPK. We utilized pharmacological agents which target Rac1 and Nox2 function by various mechanisms and observed that inhibition of Rac1-Nox2 holoenzyme prevented HG-induced activation of p38MAPK. Since, it is well established that p38MAPK induces apoptosis via p53-dependent mechanisms, we next examined the regulation of p53 under these conditions. We observed that HG-induced p53 phosphorylation was significantly blocked in the presence of inhibitors of Rac1 [EHT1864] and p38MAPK [SB203580]. Additionally, co-provision of Simvastatin, a global inhibitor of protein prenylation, and GGTI-2147, an inhibitor of geranylgeranylation, blocked HG-induced p53 phosphorylation, indicating that Rac1 prenylation is requisite for these signaling events. Furthermore, using cell death detection assay, we observed that inhibition of Rac1 [EHT1864] prevented HG-induced β-cell death. In our next set of studies, we verified our in vitro findings in INS-1 832/13 cells and rodent islets using human islets and islets derived from the ZDF rat. We observed increased activation of p38MAPK-p53 signaling axis in these models, thereby demonstrating the role of Rac1-p38MAPK-p53 signaling pathway in β-cell apoptosis under glucotoxic stress. In conclusion, we were able to demonstrate that sustained activation of Rac1-Nox2 enzyme complex leads to excess ROS generation, and the resulting oxidative stress activates downstream p38MAPK-p53 signaling axis, which in turn, promotes activation of apoptotic genes, ultimately resulting in β-cell death. Our studies provide evidence that therapeutic intervention of this signaling pathway could be used as a tool for the prevention of β-cell dysfunction and the onset of type 2 diabetes