Abstract
Oxidative stress is thought to promote pancreatic β-cell dysfunction and contribute to both type 1 and type 2 diabetes. Reactive oxygen species (ROS), such as superoxide and hydrogen peroxide, are mediators of oxidative stress that arise largely from electron leakage during oxidative phosphorylation. Reports that β-cells express low levels of antioxidant enzymes, including catalase and GSH peroxidases, have supported a model in which β-cells are ill-equipped to detoxify ROS. This hypothesis seems at odds with the essential role of β-cells in the control of metabolic homeostasis and organismal survival through exquisite coupling of oxidative phosphorylation, a prominent ROS-producing pathway, to insulin secretion. Using glucose oxidase to deliver H2O2 continuously over time and Amplex Red to measure extracellular H2O2 concentration, we found here that β-cells can remove micromolar levels of this oxidant. This detoxification pathway utilizes the peroxiredoxin/thioredoxin antioxidant system, as selective chemical inhibition or siRNA-mediated depletion of thioredoxin reductase sensitized β-cells to continuously generated H2O2 In contrast, when delivered as a bolus, H2O2 induced the DNA damage response, depleted cellular energy stores, and decreased β-cell viability independently of thioredoxin reductase inhibition. These findings show that β-cells have the capacity to detoxify micromolar levels of H2O2 through a thioredoxin reductase-dependent mechanism and are not as sensitive to oxidative damage as previously thought.
Highlights
Oxidative stress is thought to promote pancreatic -cell dysfunction and contribute to both type 1 and type 2 diabetes
When 100 M H2O2 is added as a bolus to our medium conditions, more than 50% disappears after 5 min, and nearly the entire dose is gone within 30 min, regardless of the presence of cells (Fig. 1A)
When thioredoxin reductase is either inhibited or depleted, INS 832/13 cells become significantly more sensitized to increasing concentrations of menadione (Fig. 6, H and I). These results suggest that thioredoxin reductase, which maintains the reduced, active pool of peroxiredoxins, is necessary for -cell detoxification of continuously generated H2O2
Summary
When examining the responses of -cells to H2O2, it is common to use a single or repeated dose of diluted H2O2 added to the cell culture medium [32]. Exposure to H2O2 delivered continuously over 30 min at glucose oxidase concentrations of 30 milliunits/ml or lower fails to activate the DNA damage response and energy-sensing pathways and does not modify the peroxiredoxin active site (Fig. 4). High concentrations of glucose oxidase (40 milliunits/ml or greater) that continuous H2O2 delivery elicits similar responses to bolus addition (Fig. 4C), suggesting the existence of a threshold effect for H2O2 removal This threshold level, -cells can detoxify H2O2. When thioredoxin reductase is either inhibited or depleted, INS 832/13 cells become significantly more sensitized to increasing concentrations of menadione (Fig. 6, H and I) These results suggest that thioredoxin reductase, which maintains the reduced, active pool of peroxiredoxins, is necessary for -cell detoxification of continuously generated H2O2. These results suggest that the peroxiredoxin/thioredoxin system, supported by thioredoxin reductase, protects -cells from H2O2-mediated DNA damage
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