Mitochondrial Reactive Oxygen Species Are Obligatory Signals for Glucose-Induced Insulin Secretion
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Figures
- FIG. 1.
Glucose induces ROS production in isolated rat islets. A: Glucose challenge (16.7 mmol/l) in a 30-min static incubation triggered a threefold increase in ROS fluorescence. B: ROS production was accompanied by a classical GSIS. Quenching ROS by trolox completely blunted GSIS. A and B: Three independent experiments, n = 6 per group; ***P < 0.001, glucose 5.5 vs. 16.7 mmol/l; ###P < 0.001, glucose 16.7 vs. 16.7 mmol/l+trolox. C: Dynamic experiments using perifusion demonstrating the antioxidant-induced reduction of GSIS. Three independent experiments, P < 0.001 between vehicle and treated groups and between 16.7 mmol/l glucose vs. 16.7 mmol/l glucose + trolox. D and E: Modulation of glucose-induced ROS production and GSIS by gradual antioxidant doses. Three independent experiments, n = 6 per group; **P < 0.01 or ***P < 0.001, 16.7 mmol/l glucose + 1 nmol/l trolox vs. 16.7 mmol/l glucose + 1 μmol/l trolox; ##P < 0.01 or ### P < 0.001, 16.7 mmol/l glucose + 1 μmol/l trolox vs. 16.7 mmol/l glucose + 1 mmol/l trolox. F: In these conditions, the regression linking ROS production to insulin secretion (r = 0.899) was highly significant P < 0.001. A.U., arbitrary unit.
- FIG. 2.
ROS kinetics paralleled insulin secretion in GSIS. A: Intracellular H2O2 production measured with H2-DCFDA probe. Freshly isolated islets on 5.5 mmol/l glucose before time 0 were treated with 16.7 mmol/l glucose the lasting 30 min. ROS production appeared maximally and reached a plateau at 5 min. B: Insulin secretion immediately followed this ROS production and shared a similar profile.
- FIG. 3.
mROS mimic GSIS in β-cells islets. A: Effect of treatments on intracellular H2O2 production measured with H2-DCFDA probe. Treatments were as follows: 100 μmol/l rotenone or 20 μmol/l antimycin, inhibitors of complexes I and III, respectively, added to the 30-min static incubation in 5.5 mmol/l glucose; 1 mmol/l trolox or 1 mmol/l glutathione reduced ethyl ester (GE) cotreatment with 16.7 mmol/l glucose or rotenone (rot) or antimycin (ant); 16.7 mmol/l glucose coadministrated with the uncoupler CCCP. C: Insulin release measurement in the same conditions. A and C: Three independent experiments, n = 6 per group. ***P < 0.001, glucose 5.5 vs. 16.7 mmol/l, vs. 5.5 mmol/l glucose + rot or 5.5 mmol/l glucose + ant; ###P < 0.001, 5.5 mmol/l glucose + rot vs. 5.5 mmol/l glucose + rot + trolox or glutathione ethyl ester (GE); §§§P < 0.001, 5.5 mmol/l glucose + ant vs. 5.5 mmol/l glucose + ant + trolox or glutathione ethyl ester (GE); ‡‡‡P < 0.001, 16.7 mmol/l glucose vs. 16.7 mmol/l glucose + CCCP. E: Dynamic insulin secretion using perifusion model under rotenone alone or in presence of the antioxidant trolox. Three independent experiments, with one perifused column per case, P < 0.001 between vehicle and treated groups and between rotenone vs. rotenone + trolox. B and D: Modulation of rotenone-induced ROS production and GSIS by gradual antioxidant doses. ROS-dependent response might be established with insulin secretion in static incubation. Three independent experiments, n = 6 per group; ***P < 0.001, rotenone + 1 nmol/l trolox vs. rotenone + 1 μmol/l trolox; ###P < 0.001, rotenone + 1 μmol/l trolox vs. rotenone + 1 mmol/l trolox. F: In these conditions, the regression linking ROS production to insulin secretion (r = 0.883) was highly significant P < 0.001. ⋄, vehicle; ♦, glucose 16.7 mM; ▴, rotenone; □, rotenone + trolox.
- FIG. 4.
NADH and ATP increases are not necessary for mROS-induced insulin secretion. Ratio of either ROS production as measured with H2-DCFDA (A), NADH (B), insulin secretion (C), or ATP (D) compared with basal condition (5.5 mmol/l glucose) in three independent experiments, n = 4 per group. A and C: Static islet incubation for 30 min in 16.7 mmol/l glucose cotreated with trolox abolished ROS production and blunted insulin secretion; ***P < 0.001, glucose 16.7 vs. 5.5 mmol/l; ###P < 0.001, 16.7 mmol/l glucose + trolox vs. 16.7 mmol/l glucose alone. B and D: Both NADH and ATP were increased, although insulin secretion was abolished when ROS were blunted; *P < 0.05 or **P < 0.001, 16.7 mmol/l glucose + trolox vs. 16.7 mmol/l glucose alone. Conversely, 100 μmol/l rotenone (rot) stimulated ROS production and insulin secretion (A and C), independently of NADH or ATP (B and D), which were unchanged compared with controls. ***P < 0.001, 5.5 mmol/l glucose + rotenone vs. 5.5 mmol/l glucose; §§§P < 0.001 or §P < 0.05, 5.5 mmol/l glucose + rotenone vs. 5.5 mmol/l glucose + rotenone + trolox.
- FIG. 5.
Intracellular calcium profiles after glucose, mitochondrial blocker, or KCl treatment. A: Intracellular calcium expressed as the ratio of fura-2 probe fluorescence (340 nm/380 nm) from basal conditions (5 mmol/l glucose) to 16.7 mmol/l glucose. Addition of the antioxidant trolox significantly reverses the [Ca2+]i mobilization classically observed with glucose concentration increase. C: The experiment was repeated except that instead of raising glucose, 100 μmol/l rotenone was added to islets. Addition of rotenone first led to a transient fall in [Ca2+]i the first 8 min, followed by a large and sustained increase. B: Investigation of calcium origin using 1 mmol/l EGTA after removal of Ca2+ from the extracellular bath solution. Increasing the glucose concentration or adding rotenone first triggered a typical biphasic response, and the presence of EGTA resulted in a profound decrease of fluorescence fura-2 ratio 340:380 for both glucose and rotenone. D: Investigation of calcium origin using 1 μmol/l thapsigargin in islets treated with either 16.7 mmol/l glucose or 100 μmol/l rotenone did not suppress the massive increase of [Ca2+]i. The experiment shown is representative of three (glucose and mitochondrial blockers) or two (KCl) with similar results.
- FIG. 6.
Islet preservation under the different mitochondrial treatments. A: To exclude a damaging and nonspecific oxidative stress, lipid peroxidation was evaluated. Hydroperoxides were evaluated on islets treated in static incubation for 30 min with 100 μmol/l rotenone, 1 μmol/l CCCP, or 20 μmol/l antimycine compared with basal conditions, 5.5 mmol/l glucose. No difference in these groups compared with glucose was observed. B: Insulin resecretion after the 100 μmol/l rotenone or 20 μmol/l antimycin treatment consisted to expose islets to GSIS after they were stimulated with the mitochondrial blockers for 30 min and insulin was measured in the milieu. They were replaced in basal glucose (5.5 mmol/l) for 30 min further, and insulin was reevaluated before a GSIS was done. In these conditions, insulin release was absent in the milieu in basal conditions, and the ability of glucose to restimulate insulin secretion was totally maintained after the pharmacological treatments. Three independent experiments, n = 6; ***P < 0.001, glucose 16.7 vs. 5.5 mmol/l. Bars represent SE in all figures. They were not represented for perifused-islet experiments. Ant, antimycin; rot, rotenone.
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