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 H₂O₂ 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 H₂O₂, allowing fluorescent cyto-HyPer signals to be converted into H₂O₂ concentrations. 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 H₂O₂ Furthermore, glucose also induced mitochondrial superoxide and H₂O₂ production, which preceded the appearance of cytosolic H₂O₂ Therefore, glucose-induced H₂O₂ largely originated from mitochondria. Finally, the glucose-induced HyPer signal was less than 1/20th of that induced by toxic levels of H₂O₂ Overall, the use of HyPer for real-time imaging allowed resolution of acute changes in intracellular levels of H₂O₂ and will have great utility for islet studies involving mechanisms of H₂O₂-mediated signaling and oxidative stress.

Keywords: glucose uptake; hydrogen peroxide; mitochondria; oxidative stress.

Figure. 1.. Schematic of the dual channel fluidics/imaging chamber.

A Bioptechs FCS2, a closed system, parallel plate flow cell, was modified in order to accommodate two inflow and outflow ports. The gaskets were then cut so that the ports supplied and drained flow to and from two separated cell channels. For cells one Parafilm gasket was used, and for islets, two were stacked prior to cutting the rectangular holes. This system allowed for two perifusions to be carried out in parallel such as when comparing genetically altered islets or cells to control cells.

Figure 2.. Real time (A) and steady state (B) responses of cyto-HyPer in permeabilized INS-1 cells.

(A) INS-1 cells were imaged during perifusion with stepwise increasing amounts of H₂O₂ in the buffer as indicated (typical calibration curve). (B) Steady state values were calculated as the average of the final five minutes of each H₂O₂ concentration. A trendline was drawn through the linear region from 65 to 600 nM, where 30 nM was indistinguishable from 0 nM fluorescence

Figure 3.. Effect of catalase overexpression on cyto-HyPer.

(A) Western analysis of cytosolic catalase in islets and mitochondrial catalase in INS-1 cells Glucose-induced increase in cyto-HyPer signal is suppressed in response to (B) overexpression of cytosolic catalase in rat islets (n = 4) and (C) overexpression of mitochondrial catalase in INS-1 cells (n = 4), demonstrating specificity of HyPer for H₂O₂.

Figure 4.. Lack of effect of glucose-induced changes in pH on cytosolic H₂O₂.

(A) Glucose-stimulated pH in the presence and absence of 20 mM HEPES in the buffer (n = 3). (B) Glucose-induced changes in cytosolic H₂O₂ by islets in the presence or absence of 20 mM HEPES (n = 3).

Figure. 5.. Production and conversion of H₂O₂.

H₂O₂ is produced in the cytosol and mitochondria by conversion of superoxide and the action of SOD. H₂O₂ in the cytosol and mitochondria are detected by cyto-HyPer and mito-HyPer respectively.

Figure 6.. Effect of SOD2 (mitochondrial SOD) knockdown on glucose-induced increase in H₂O₂.

Response in H₂O₂ to glucose by INS-1 cells cultured for 3 days in the presence (n= 2) or absence of shRNA (n = 4) for SOD2.

Figure 7.. Kinetics of superoxide, mitochondrial and cytosolic H₂O₂ in response to glucose.

(A) Confocal imaging of islets and islet cells infected with or without mito-HyPer and cyto-HyPer. Top row of panels: images of whole islets loaded with or without MitoTracker Red. Middle row of panels: single islet cell expressing mito-HyPer and loaded with MitoTracker Red. Bottom panel: image of single islet cell expressing cyto-HyPer. White scale bar is 10 microns in length. (B) Kinetic responses of islets loaded with MitoSOX Red (a sensor for mitochondrial superoxide), or expressing mito-HyPer or cyto-HyPer (n = 4). (C) Lack of response of mito-HyPer to low levels of extracellular H₂O₂ in permeabilized islets (typical response).

Figure 8.. Comparison of glucose-induced H₂O₂ levels vs. H₂O₂ observed in the presence of toxic concentrations of extracellular H₂O₂.

(A) Islets were incubated for 15 minutes in the presence of the indicated concentrations of H₂O₂ and then assessed for viability as reflected by membrane patency at the times shown. (Red cells no longer have membrane integrity.) (B) Cytosolic H₂O₂ in response to glucose and extracellular H₂O₂ (typical response). Toxic concentrations of extracellular H₂O₂ were accompanied by supraphysiologic cytosolic levels of H₂O₂. (C) Oxygen consumption rate and insulin secretion rate as a function of exposure to various concentrations of glucose in the culture media (n = 3). Prior and subsequent to the culture period, acute islet response to an increase in glucose concentration from 3 to 20 mM was assessed.