Abstract
The effects of a transient exposure to hydrogen peroxide (10 min at 200 microM H(2)O(2)) on pancreatic beta cell signal transduction and insulin secretion have been evaluated. In rat islets, insulin secretion evoked by glucose (16.7 mM) or by the mitochondrial substrate methyl succinate (5 mM) was markedly blunted following exposure to H(2)O(2). In contrast, the secretory response induced by plasma membrane depolarization (20 mM KCl) was not significantly affected. Similar results were obtained in insulinoma INS-1 cells using glucose (12.8 mM) as secretagogue. After H(2)O(2) treatment, glucose no longer depolarized the membrane potential (DeltaPsi) of INS-1 cells or increased cytosolic Ca(2+). Both DeltaPsi and Ca(2+) responses were still observed with 30 mM KCl despite an elevated baseline of cytosolic Ca(2+) appearing approximately 10 min after exposure to H(2)O(2). The mitochondrial DeltaPsi of INS-1 cells was depolarized by H(2)O(2) abolishing the hyperpolarizing action of glucose. These DeltaPsi changes correlated with altered mitochondrial morphology; the latter was not preserved by the overexpression of the antiapoptotic protein Bcl-2. Mitochondrial Ca(2+) was increased following exposure to H(2)O(2) up to the micromolar range. No further augmentation occurred after glucose addition, which normally raises this parameter. Nevertheless, KCl was still efficient in enhancing mitochondrial Ca(2+). Cytosolic ATP was markedly reduced by H(2)O(2) treatment, probably explaining the decreased endoplasmic reticulum Ca(2+). Taken together, these data point to the mitochondria as primary targets for H(2)O(2) damage, which will eventually interrupt the transduction of signals normally coupling glucose metabolism to insulin secretion.
Highlights
The control of insulin secretion in the pancreatic beta cell depends on the precise tuning of glucose metabolism leading to signal transduction [1, 2]
Our results show that the defective glucose-induced insulin secretion observed after H2O2 treatment correlates with altered mitochondrial activation seen as a loss of mitochondrial membrane potential (⌬⌿m), decreased ATP generation, and impaired responses of mitochondrial Ca2ϩ concentration ([Ca2ϩ]m)
Effect of H2O2 on Insulin Secretion in Rat Islets and INS-1 Cells—Rat pancreatic islets were maintained in culture (11.1 mM glucose) for 2– 4 days prior to the experiments
Summary
M H2O2 followed by a 5-min recovery period in basal buffer supplemented with 100 units/ml catalase. The rat beta cell line INS-1, which was used in the present study, exhibits similar low baseline catalase levels (1.0 units/mg protein) as primary rat islets [22]. Taken together, these bindings call for a better understanding of the cellular mechanisms linking oxidative stress to impaired insulin secretion. Our results show that the defective glucose-induced insulin secretion observed after H2O2 treatment correlates with altered mitochondrial activation seen as a loss of mitochondrial membrane potential (⌬⌿m), decreased ATP generation (measured on-line in living cells), and impaired responses of mitochondrial Ca2ϩ concentration ([Ca2ϩ]m). We examined whether overexpression of Bcl-2 could prevent the alteration of mitochondrial morphology during H2O2 treatment
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