Identification and localization of reactive oxygen species during nutrient-induced insulin secretion: role of NADPH oxidase.

Abstract

In vivo and in vitro data provide evidences of oxygen reactive species (ROS) as one of the common factors between insulin resistance, decreased insulin secretion and diabetes. However, ROS are known to be important during cell signaling and other events such as proliferation, apoptosis and insulin secretion. Free Fatty acids (FFA) have a direct effect on pancreatic beta cell, controlling insulin secretion and cell viability. One of the important effects of FFA on beta cell is the activation of NADPH oxidase. The aim of this study is to investigate the role of ROS and NADPH oxidase on insulin secretion and beta cell dysfunction after exposure to nutrients. To test this, the following strategies will be used: 1) islets from control and knockout mice for gp91phox subunit (gp91 phox-/-) and 2) interference RNA to delete the p22 phox subunit in cell lines. Pancreatic islets and beta cells will be incubated with different glucose concentrations and FFA (palmitate and oleate) and infected with adenovirus/lentivirus expressing genetically encoded probes target to different compartments (mitochondria, cytoplasm, peroxissomes, endoplasmic reticulum) to measure oxidative stress in dynamic and real time experiments. Cell viability, changes in calcium , metabolism and signalling pathways will also be addressed. In addition, we will use an insulin resistance animal model (ob/ob) to analyze the importance of NADPH oxidase and ROS production on beta cell dysfunction. A better understanding of the role of NADPH oxidase e and FFA on ROS production, insulin secretion and signaling is of fundamental importance to elucidate the mechanisms of diabetes development.