Interference of H2O2 with stimulus-secretion coupling in mouse pancreatic beta-cells

Abstract

1. We have reported previously that in mouse pancreatic beta-cells H2O2 hyperpolarizes the membrane and increases the ATP-sensitive K+ current recorded in the perforated patch configuration of the patch-clamp technique. The present study was undertaken to elucidate the underlying mechanisms. 2. The intracellular ATP concentration measured by chemoluminescence was reduced by H2O2. The ADP concentration increased in parallel during the first 10 min, resulting in a pronounced decrease in the ATP/ADP ratio. 3. Consistent with these results, glucose-stimulated insulin secretion from isolated islets was inhibited by H2O2. 4. Membrane hyperpolarization measured with intracellular microelectrodes in intact islets and inhibition of insulin secretion were counteracted by tolbutamide, indicating that the channels are still responsive to inhibitors and that the ATP concentration is not too low to trigger exocytosis. However, the sensitivity of the beta-cells to tolbutamide was reduced after treatment with H2O2. 5. H2O2 increased the intracellular Ca2+ activity ([Ca2+]i) in a biphasic manner. A first transient rise in [Ca2+]i due to mobilization of Ca2+ from intracellular stores was followed by a sustained increase, which was at least partly dependent on Ca2+ influx. The first phase seems to reflect Ca2+ mobilization from mitochondria. 6. Our results demonstrate that H2O2 interferes with glucose metabolism, which influences the membrane potential and ATP-sensitive K+ current via the intracellular concentration of ATP. These events finally lead to an inhibition of insulin secretion despite an increase in [Ca2+]i.

Figure 1. H₂O₂ hyperpolarizes the membrane potential by increasing the ATP-sensitive K⁺ current

A, effects of 1 mM H₂O₂ and 0.1 and 1 mM tolbutamide (tolb) on the membrane potential of mouse pancreatic β-cells in the presence of 15 mM glucose (15 G). The H₂O₂-induced hyperpolarization was irreversible but could be counteracted by inhibiting the ATP-sensitive K⁺ current with tolbutamide. The record is representative of four experiments with similar results. B, ATP-sensitive K⁺ current monitored in the perforated-patch mode in the presence of 15 mM glucose (15 G). The holding potential was -70 mV (continuous trace) and every 15 s 300 ms voltage steps to -80 and -60 mV (lower and upper dashed traces, respectively) were applied. H₂O₂ dramatically increased the current. Again the irreversible effect of H₂O₂ was at least partly counteracted by tolbutamide. The insets show the corresponding currents on an extended time scale. At the points marked by asterisks the amplifier was switched from voltage-clamp to current-clamp mode and the membrane potential was registered. The record is representative of five experiments with similar results.

Figure 2. H₂O₂ diminishes intracellular ATP content

A, mean changes in the ATP/ADP ratio monitored with isolated islets after addition of 1 mM H₂O₂in the presence of 15 mM glucose. Numbers of experiments (n) given in parentheses below the symbols in A also apply to corresponding symbols in B and C. *Values significantly different from controls. B and C, H₂O₂ induced changes in intracellular ATP (B) and ADP (C) content. Note that the H₂O₂ effect is biphasic: within the first 5 min the loss in ATP coincided with an increase in ADP whereas afterwards the concentration of both nucleotides decreased.

Figure 3. H₂O₂ decreases the insulin secretion from isolated mouse islets

Effects of 1 mM H₂O₂ and 1 mM tolbutamide (tolb) on insulin release from perifused mouse islets. Switching from 3 to 15 mM glucose (3 G, 15 G) elicited the response of the islets to glucose. H₂O₂ irreversibly inhibited glucose-stimulated insulin secretion, an effect which was blunted by tolbutamide. Values are means ±s.e.m. of four experiments. Control experiments without H₂O₂ and tolbutamide are represented by the dashed line.

Figure 4. Effect of H₂O₂ on [Ca²⁺]_i

Switching the extracellular glucose concentration from 0.5 mM (c = control) to 15 mM (15 G) led to an increase in [Ca²⁺]_i. The subsequent addition of 1 mM H₂O₂ led to a marked drop in [Ca²⁺]_i followed by a biphasic increase in [Ca²⁺]_i, a first transient phase (see arrowhead) was followed by a large sustained increase. At the time intervals marked by the hatched bars Ca²⁺ was removed from the extracellular bath solution and 1 mM EGTA was added. After a steady-state level of [Ca²⁺]_i had been reached, the Ca²⁺ ionophore ionomycin (1 μm) was applied in the absence of extracellular Ca²⁺. The experiment shown is representative of five with similar results.

Figure 5. Influence of D600, Gd³⁺ and flufenamate on H₂O₂-induced changes in [Ca²⁺]_i

In all experiments [Ca²⁺]_i was first increased by switching from 0.5 mM glucose (c = control) to 15 mM glucose (15 G) in the bath solution. Neither the L-type Ca²⁺ channel blocker D600 (100 μm; A) nor the inhibitors of non-selective cation channels and the Ca²⁺ release-activated Ca²⁺ current I_crac, Gd³⁺ (100 μm; B) or flufenamate (flufen, 100 μm; C), suppressed the second marked rise in [Ca²⁺]_i induced by 1 mM H₂O₂. Each experiment presented in this figure is representative of four experiments with similar results.

Figure 6. Effect of H₂O₂ on [Ca²⁺]_i in Ca²⁺-free solution

Raising the glucose concentration from 0.5 mM (c = control) to 15 mM (15 G) increased [Ca²⁺]_i. A subsequent switch to Ca²⁺-free solution (1 mM EGTA) restored [Ca²⁺]_i to control values. Under these conditions the addition of 1 mM H₂O₂ led to a transient increase in [Ca²⁺]_i, pointing to Ca²⁺ release from intracellular sites. The experiment shown is representative of nine with similar results.

Figure 7. Effect of the Ca²⁺ pump inhibitor thapsigargin and the mitochondria uncoupler FCCP on the first phase increase in [Ca²⁺]_i induced by H₂O₂

A, the cells were pre-incubated for 30 min with 1 μm thapsigargin. The effectiveness of the treatment is demonstrated by the complete suppression of the first initial decrease in [Ca²⁺]_i after increasing the glucose concentration from 0.5 to 15 mM (compare traces at ▾ in A and B). Ca²⁺ store depletion by thapsigargin did not prevent the first rise in [Ca²⁺]_i induced by H₂O₂ (▴). The experiment shown is representative of four with similar results. B, addition of 100 μm FCCP in the presence of 15 mM glucose suppressed the first phase of the H₂O₂-evoked rise in [Ca²⁺]_i (compare traces at ▴ in A and B). The experiment shown is representative of five with similar results.