The failure of pancreatic β-cells is the key determinant for the development of type 2 diabetes (T2D). Nutrient overload caused by insulin resistance and obesity increases mitochondrial respiration and ROS generation as the byproduct in pancreatic β-cells. Due to unusually low levels of enzymes that detoxify ROS, β-cells are highly susceptible to ROS-mediated damage. To understand the role of ROS in β-cell defects and alterations of mitochondrial membrane potential, we induced ROS generation in dispersed rat and human islet cells with 10 µM menadione in the presence and absence of an ROS inhibitor, N-acetyl cysteine (1 mM), followed by CellRox Green and mitrotracker treatment, ROS and mitochondrial membrane potential detecting agents, respectively. Rat and human islet cells were trypsinized, washed, and flow cytometry analysis was performed. As anticipated, menadione induced ROS generation compared to control (no treatment), which was inhibited by N-acetyl cysteine. Measurement of the mitochondrial membrane potentials showed the similar trend as ROS generation. In order to confirm this finding and visualize individual cells, we have also obtained images using a 20X objective in a Leica inverted fluorescent microscope. Quantitation of the intensity of CellRox Green using the ImageJ Image processing program corroborated the results obtained from the flow cytometry analysis. We have also determined the effects of menadione on ROS generation in insulin-secreting alpha-cells versus glucagon-secreting β-cells, which showed the similar patterns. Next, we determined the effects of SR-135, a peroxynitrite decomposing catalyst, on ROS generation induced by excess nutrients (25 mM glucose and 500 µM free fatty acids). Cells treated with both excess nutrients and SR-135 (10µM) for 2 days, unexpectedly, showed higher intensity of CellRox Green than those treated with excess nutrients alone. Furthermore, a control drug SRB (the same chemical structure as SR-135 but no peroxynitrite decomposing catalyst activity) brought down the intensity. Interestingly, the levels of nitrotyrosine, considered as the footprint of peroxynitrite, were higher in cells treated with excess nutrients alone than those treated with both excess nutrients and SR-135. We postulate that CellRox Green and nitrotyrosine detect two distinct species of ROS, superoxide and peroxynitrite, respectively. The ability to measure distinct ROS generation in β-cells would be valuable in elucidating the mechanisms by which excess nutrients cause β-cell defects, leading to type 2 diabetes.
|Commitee:||Neumann, William L., Schober, Joseph|
|School:||Southern Illinois University at Edwardsville|
|School Location:||United States -- Illinois|
|Source:||MAI 58/01M(E), Masters Abstracts International|
|Keywords:||Beta cell, ROS, Type 2 diabetes|
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