Real time imaging of intracellular hydrogen peroxide in pancreatic islets

A real-time method to measure intracellular hydrogen peroxide (H 2 O 2 ) would be very impactful in characterizing rapid changes that occur in physiologic and pathophysiologic states. Current methods do not provide the sensitivity, specificity and spatiotemporal resolution needed for such experiments on intact cells. We developed the use of HyPer, a genetic indicator for H 2 O 2 that can be expressed in the cytosol (cyto-HyPer) or the mitochondria (mito-HyPer) of live cells. INS-1 cells or islets were permeabilized and the cytosolic HyPer signal was a linear function of extracellular H 2 O 2 , allowing fluorescent cyto-HyPer signals to be converted into H 2 O 2 concentrations. Glucose increased cytosolic H 2 O 2 , an effect that was suppressed by overexpression of catalase. Large perturbations in pH can influence the HyPer signal, but inclusion of HEPES [4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid] in the perfusate prevented pH changes, but did not affect glucose-induced cyto-HyPer signals, suggesting that this effect is largely pH-independent. Using the assay, two fundamental questions were addressed. Knockdown of superoxide dismutase 2 (SOD2), the mitochondrial form of SOD, completely suppressed glucose-induced H 2 O 2 . Furthermore, glucose also induced mitochondrial superoxide and H 2 O 2 production, which preceded the appearance of cytosolic H 2 O 2 . Therefore, glucose-induced H 2 O 2 largely originated from mitochondria. Finally, the glucose-induced HyPer signal was less than 1/20th of that induced by toxic levels of H 2 O 2 . Overall, the use of HyPer for real-time imaging allowed resolution of acute changes in intracellular levels of H 2 O 2 and will have great utility for islet studies involving mechanisms of H 2 O 2 -mediated signaling and oxidative stress.