Transient oxidative stress damages mitochondrial machinery inducing persistent beta-cell dysfunction

Abstract

Transient exposure of beta-cells to oxidative stress interrupts the transduction of signals normally coupling glucose metabolism to insulin secretion. We investigated putative persistence of effects induced by one transient oxidative stress (200 microm H(2)O(2), 10 min) on insulin secreting cells following recovery periods of days and weeks. Three days after oxidative stress INS-1E cells and rat islets exhibited persistent dysfunction. In particular, the secretory response to 15 mm glucose was reduced by 40% in INS-1E cells stressed 3 days before compared with naïve cells. Compared with non-stressed INS-1E cells, we observed reduced oxygen consumption (-43%) and impaired glucose-induced ATP generation (-46%). These parameters correlated with increased mitochondrial reactive oxygen species formation (+60%) accompanied with down-regulation of subunits of the respiratory chain and decreased expression of genes responsible for mitochondrial biogenesis (TFAM, -24%; PGC-1alpha, -67%). Three weeks after single oxidative stress, both mitochondrial respiration and secretory responses were recovered. Moreover, such recovered INS-1E cells exhibited partial resistance to a second transient oxidative stress and up-regulation of UCP2 (+78%) compared with naïve cells. In conclusion, one acute oxidative stress induces beta-cell dysfunction lasting over days, explained by persistent damages in mitochondrial components.

Figures

FIGURE 1.

Apoptosis/proliferation, secretory responses, and mitochondrial morphology of INS-1E cells during days following oxidative stress. A and B, INS-1E cells were cultured for the indicated time points without stress (Control) or following transient oxidative stress (200 μm H2O2 for 10 min at time 0, Stressed). A, cell apoptosis and (B) proliferation were measured by the TUNEL assay and BrdUrd incorporation, respectively. TUNEL (A)- or BrdUrd (B)-positive cells were counted under fluorescent microscope in at least three separate fields (each containing ∼70 cells) of four independent experiments, and graphical results are depicted as a percentage of positive cells over the total amount of insulin positive cells. C and D, insulin secretion tested in INS-1E cells cultured for 3 (C) and 5 (D) days following transient exposure to 200 μm H2O2 for 10 min (Stressed + 3d and Stressed + 5d) or without oxidative stress (Control). Insulin release was measured following a 30-min incubation at basal 2.5 mm glucose (2.5 Glc), stimulatory 15 mm glucose (15 Glc), and 2.5 Glc with 30 mm KCl (2.5 Glc + KCl). Values are means ± S.D. of 1 representative out of 5 different experiments performed in quintuplicate. E and F, visualization under laserscan confocal microscope of INS-1E cells stained for mitochondria using MitoTracker. 3 (E) and 5 (F) days after transient exposure to 200 μm H2O2 for 10 min (Stressed + 3d and Stressed + 5d) were compared with non-stressed cells (Control). Pictures shown are representative of three independent experiments. *, p < 0.05; ***, p < 0.005 versus basal secretion (2.5 Glc); #, p < 0.05; ##, p < 0.01; ###, p < 0.005 versus corresponding controls.

FIGURE 2.

Mitochondrial activation in INS-1E cells 3 days post-stress. INS-1E cells were cultured for 3 days following transient oxidative stress (200 μm H2O2 for 10 min, Stressed + 3d) before analyses. A, ΔΨm was monitored as Rhodamine-123 fluorescence and hyperpolarization was induced by raising glucose from basal 2.5 mm to stimulatory 15 mm (see arrow, Glucose). Complete depolarization of the mitochondrial membrane was evoked by addition of 1 μm of the uncoupler FCCP (see arrow, FCCP). B, real-time cytosolic ATP changes were monitored in luciferase expressing INS-1E cells as bioluminescence. ATP generation was induced by raising glucose from 2.5 to 15 mm (see arrow, Glucose) and collapsed by addition of the mitochondrial poison 2 mm NaN3 (see arrow, NaN3). C, total cellular ATP concentrations were determined following a 10-min incubation period at 2.5 (2.5 Glc) and 15 mm (15 Glc) glucose. Results are means ± S.D. of 1 representative out of 3 independent experiments, each performed in triplicate. D, O2 consumption in INS-1E cells was induced by raising glucose from 0 to 15 mm (see arrow, Glucose). Tangents corresponding to glucose-induced respiration in control and stressed cells are shown as dashed and dotted lines, respectively. Traces are representative of four independent experiments. E, isolated mitochondria were permeabilized, placed into an oxymeter chamber, and stabilized for at least 10 min without substrates (basal). Then, complex I-dependent O2 consumption was stimulated by the addition of 5 mm NADH (NADH). F, O2 consumption was induced in intact mitochondria by adding 5 mm succinate (succinate), followed by further 150 μm ADP addition (succinate + ADP). Columns represent means ± S.E. of four independent experiments. *, p < 0.01; ***, p < 0.001 versus basal non-stressed controls; #, p < 0.05; ##, p < 0.01; and ###, p < 0.005 versus corresponding controls.

FIGURE 3.

Mitochondrial respiratory chain subunits in INS-1E cells over days post-stress. INS-1E cells were subjected to transient oxidative stress (200 μm H2O2 for 10 min) and further cultured for recovery periods. Then, mitochondria were isolated for immunoblot analysis. A, representative immunoblotting revealing subunit proteins of the 5 complexes 3 days after transient oxidative stress. Lanes from left to right show standards (Std) for the five subunits (ND6 for complex I, FeS for complex II, COX I for complex IV, core2 for complex III, and α subunit for complex V), non-stressed INS-1E mitochondria (Control), and stressed mitochondria (Stressed + 3d). B, time course presenting subunit protein changes at times 0, 10 min, 6 h, 1 day, and 3 days after transient (10 min) exposure to 200 μm H2O2. Quantitative analysis of relative band densities (relative changes to time 0), as shown in A, is presented as means ± S.E. of four independent experiments; *, p < 0.05; **, p < 0.01 versus corresponding time 0.

FIGURE 4.

ROS generation in INS-1E cells over days post-stress. INS-1E cells were subjected to transient oxidative stress (200 μm H2O2 for 10 min) and further cultured for recovery periods. Then, mitochondria were isolated for endogenous ROS generation. A and B, 3 days after transient exposure to exogenous ROS, mitochondrion-derived ROS generation was measured in the absence of electron donors (basal) or induced by treatments with (A) 5 mm NADH (NADH) and (B) 5 mm succinate (succinate). CAT (200 units/ml) was added in the assay to obtain values corresponding to negative controls (NADH + CAT in A or succinate + CAT in B). Columns represent relative increases over non-stimulated groups as means ± S.E. of five independent determinations; *, p < 0.05; ***, p < 0.005 versus basal ROS generation in non-stressed control mitochondria; #, p < 0.05; ##, p < 0.01 versus corresponding controls.

FIGURE 5.

Functionality of rat islets 3 days post-stress. Isolated rat islets were transiently stressed with 200 μm H2O2 for 10 min and further maintained in culture for 3 days before analyses. A, insulin secretion was assayed by perifusion (10 islets per chamber) in islets stimulated with 16.7 mm glucose for 30 min (horizontal dotted trace) after 15 min at basal 2.8 mm glucose and then back to basal glucose for another 15 min. Traces are means ± S.D. of three chambers of one out of four individual experiments, each performed in triplicate. B, representative immunoblotting showing levels of the mitochondrial complex subunits (I, II, III, and V) of rat islets stressed 3 days before analysis.

FIGURE 6.

Recovery of islet function, INS-1E secretory responses, and mitochondrial integrity. Following transient oxidative stress (200 μm H2O2 for 10 min), rat islets and INS-1E cells were cultured for recovery periods of 2, 3, and 4 weeks before analyses. Stressed cells (Stressed + 2w, 3w, and 4w, respectively) were compared with cells stressed 3 days before (Stressed + 3d) and to non-stressed cells (Control). A, insulin secretion was measured over a 60-min incubation period at basal 2.8 mm glucose (2.8 Glc) and stimulatory 16.7 mm glucose (16.7 Glc) in rat islets cultured in normal media for 3 weeks following transient oxidative stress. B, mitochondria were isolated from INS-1E cells for respiratory rate measurements. O2 consumption was induced by 5 mm succinate (succinate) and succinate plus 150 μm ADP (succinate + ADP). O2 consumption is expressed as stimulated rates relative to corresponding non-stressed controls. Bars represent means ± S.E. of five independent measurements; *, p < 0.05; **, p < 0.01; and ***, p < 0.005 versus corresponding 3 days post-stress; #, p < 0.05; ##, p < 0.01; and ###, p < 0.005 versus corresponding controls. 2 (C) and 3 (E) weeks following transient oxidative stress or without oxidative stress, secretory function was tested in INS-1E cells by measuring glucose-stimulated insulin secretion. Basal insulin release at 2.5 mm basal glucose (2.5 Glc) was compared with stimulated secretion at 15 mm glucose (15 Glc) Values are means ± S.D. of one representative out of three different experiments performed in triplicate. D and F, visualization of mitochondria by confocal microscopy of INS-1E cells stained with MitoTracker. 2 (D) and 3 (F) weeks after transient exposure to 200 μm H2O2 for 10 min. ***, p < 0.005 versus basal secretion (A, 2.8 Glc; C and E, 2.5 Glc); #, p < 0.05 versus corresponding controls.

FIGURE 7.

Sensitivity of recovered INS-1E cells to a second oxidative stress. After 3 weeks of recovery post-stress, cells were exposed for a second time to the same oxidative attack of 200 μm H2O2 for 10 min. Stressed cells allowed recovery period (Stressed + 3w) were compared with naïve cells (Control) for responses to a new oxidative stress (Control + 2nd stress and Stressed + 2nd stress). Cell apoptosis (A) and proliferation (B) were measured by the TUNEL assay and BrdUrd incorporation, respectively, as described in the legend of Fig. 1. C, insulin secretion tested in INS-1E cells cultured for 3 weeks following transient exposure to 200 μm H2O2 for 10 min or without oxidative stress. Directly after the second oxidative stress, insulin release was measured at basal 2.5 mm (2.5 Glc) and stimulatory 15 mm glucose (15 Glc). Values are means ± S.D. of one representative out of four different experiments performed in triplicate. D, mitochondrial activation was measured as changes in ΔΨm induced by raising glucose from basal 2.5 mm to stimulatory 15 mm (see arrow, Glucose), and complete depolarization of the mitochondrial membrane was evoked by addition of 1 μm of the uncoupler FCCP (see arrow, FCCP). The 10-min 200 μm H2O2 stress was performed at basal 2.5 mm glucose (H2O2), and then 15 mm glucose stimulation was done after neutralization of H2O2 by adding 100 units/ml CAT. Bars are means ± S.D. of one out of four independent experiments, each performed in triplicate.