title: Real-time imaging of intracellular hydrogen peroxide in pancreatic islets.
abstract: A real-time method to measure intracellular hydrogen peroxide (H2O2) 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 H2O2that 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 H2O2, allowing fluorescent cyto-HyPer signals to be converted into H2O2concentrations. Glucose increased cytosolic H2O2, 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 H2O2Furthermore, glucose also induced mitochondrial superoxide and H2O2production, which preceded the appearance of cytosolic H2O2Therefore, glucose-induced H2O2largely originated from mitochondria. Finally, the glucose-induced HyPer signal was less than 1/20th of that induced by toxic levels of H2O2Overall, the use of HyPer for real-time imaging allowed resolution of acute changes in intracellular levels of H2O2and will have great utility for islet studies involving mechanisms of H2O2-mediated signaling and oxidative stress.
authors.affiliation: Diabetes Research Institute, University of Washington, Seattle, WA 98195-8055, U.S.A.
authors.forename: Adam
authors.initials: A
authors.lastname: Neal
authors.affiliation: Diabetes Research Institute, University of Washington, Seattle, WA 98195-8055, U.S.A.
authors.forename: Austin
authors.initials: A
authors.lastname: Rountree
authors.affiliation: Diabetes Research Institute, University of Washington, Seattle, WA 98195-8055, U.S.A.
authors.forename: Kelly
authors.initials: K
authors.lastname: Kernan
authors.affiliation: Diabetes Research Institute, University of Washington, Seattle, WA 98195-8055, U.S.A.
authors.forename: Brian
authors.initials: B
authors.lastname: Van Yserloo
authors.affiliation: Department of Pain and Anesthesiology, University of Washington, Seattle, WA 98195, U.S.A.
authors.forename: Huiliang
authors.initials: H
authors.lastname: Zhang
authors.affiliation: Diabetes Research Institute, University of Washington, Seattle, WA 98195-8055, U.S.A.
authors.forename: Benjamin J
authors.initials: BJ
authors.lastname: Reed
authors.affiliation: Diabetes Research Institute, University of Washington, Seattle, WA 98195-8055, U.S.A.
authors.forename: William
authors.initials: W
authors.lastname: Osborne
authors.affiliation: Department of Pain and Anesthesiology, University of Washington, Seattle, WA 98195, U.S.A.
authors.forename: Wang
authors.initials: W
authors.lastname: Wang
authors.affiliation: Diabetes Research Institute, University of Washington, Seattle, WA 98195-8055, U.S.A.
authors.forename: Ian R
authors.initials: IR
authors.lastname: Sweet
mesh_terms: D000818:Animals; D002374:Catalase / Q000378:metabolism; D006801:Humans; D006861:Hydrogen Peroxide / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007328:Insulin / Q000378:metabolism; D007515:Islets of Langerhans / Q000378:metabolism*; D008297:Male; D008928:Mitochondria / Q000378:metabolism; D018384:Oxidative Stress; D010101:Oxygen Consumption; D011419:Propidium / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D013482:Superoxide Dismutase / Q000378:metabolism; D013481:Superoxides / Q000378:metabolism; D000099156:Superoxide Dismutase 2
chemical_list: D002374:Catalase; D006861:Hydrogen Peroxide; D007328:Insulin; D011419:Propidium; D013482:Superoxide Dismutase; D013481:Superoxides; D000099156:Superoxide Dismutase 2
keywords: glucose uptake; hydrogen peroxide; mitochondria; oxidative stress