H₂O₂ has dual effects on glucose-induced insulin secretion.

<p>(A) Insulin secretion in basal glucose (2.8 mM) was determined in islets incubated for 1 h with different concentrations of H₂O₂. An increase in insulin secretion in basal glucose was observed at 100 μM H₂O₂. (B) Insulin secretion in stimulatory glucose (16.7 mM) in islets incubated for 1 h with different concentrations of H₂O₂. Mean ± SEM, N = 3–5. Statistical significance was determined with one-way ANOVA followed by Tukey multiple comparison test. *: p <0.05; **: p <0.01.</p

Incubation with exogenous H₂O₂ or glucose increases ROS generation.

<p>ROS accumulation was detected by confocal microscopy in islets or cells loaded with CM-H₂DCFDA (10 μM), which cytoplasmic esterase enzymes convert to the redox-sensitive fluorescence reporter H₂DCFDA. Representative images correspond to isolated islets or pancreatic β-cells incubated for 1 h in Krebs bicarbonate buffer containing: (A) 2.8 mM glucose; (B) 16.7 mM glucose; (C) 2.8 mM glucose plus 100 μM H₂O₂ and (D) Quantification of ROS production with the redox-sensitive fluorescence reporter H₂DCFDA. Similar results were obtained in three independent experiments. Scale bars: 50 μm, islet; 10 μm, β-cell. Statistical significance was determined with one-way ANOVA followed by Tukey multiple comparison test. *: p <0.05; **: p <0.01; ***p <0.001.</p

Schematic model of GSIS.

<p>Previous studies (thick arrows/lines) have established that an increase in extracellular glucose, the principal physiological insulin secretagogue, stimulates glucose uptake into β-cells via the GLUT-2 transporter. The ensuing accelerated metabolism of intracellular glucose stimulates ROS production and increases the cytoplasmic ATP/ADP ratio. The increase in cytoplasmic ATP promotes closure of ATP-sensitive K⁺ channels (K_ATP) leading to membrane depolarization and activation of Ca²⁺ influx through voltage-gated Ca²⁺ channels (VGCC). Based on our results, we propose (gray arrows) that the ROS increase induced by glucose promotes RyR2 oxidation (<i>S</i>-glutathionylation), which makes possible RyR2-mediated calcium-induced calcium release (CICR) in response to the small and localized [Ca²⁺]_i increase produced by Ca²⁺ influx. The glucose-induced ATP increase may also contribute to stimulate CICR mediated by oxidized RyR2 channels (broken arrow). The subsequent [Ca²⁺]_i increase promotes insulin exocytosis.</p

Glucose-Dependent Insulin Secretion in Pancreatic β-Cell Islets from Male Rats Requires Ca²⁺ Release via ROS-Stimulated Ryanodine Receptors

<div><p>Glucose-stimulated insulin secretion (GSIS) from pancreatic β-cells requires an increase in intracellular free Ca²⁺ concentration ([Ca²⁺]). Glucose uptake into β-cells promotes Ca²⁺ influx and reactive oxygen species (ROS) generation. In other cell types, Ca²⁺ and ROS jointly induce Ca²⁺ release mediated by ryanodine receptor (RyR) channels. Therefore, we explored here if RyR-mediated Ca²⁺ release contributes to GSIS in β-cell islets isolated from male rats. Stimulatory glucose increased islet insulin secretion, and promoted ROS generation in islets and dissociated β-cells. Conventional PCR assays and immunostaining confirmed that β-cells express RyR2, the cardiac RyR isoform. Extended incubation of β-cell islets with inhibitory ryanodine suppressed GSIS; so did the antioxidant N-acetyl cysteine (NAC), which also decreased insulin secretion induced by glucose plus caffeine. Inhibitory ryanodine or NAC did not affect insulin secretion induced by glucose plus carbachol, which engages inositol 1,4,5-trisphosphate receptors. Incubation of islets with H₂O₂ in basal glucose increased insulin secretion 2-fold. Inhibitory ryanodine significantly decreased H₂O₂-stimulated insulin secretion and prevented the 4.5-fold increase of cytoplasmic [Ca²⁺] produced by incubation of dissociated β-cells with H₂O₂. Addition of stimulatory glucose or H₂O₂ (in basal glucose) to β-cells disaggregated from islets increased RyR2 <i>S</i>-glutathionylation to similar levels, measured by a proximity ligation assay; in contrast, NAC significantly reduced the RyR2 <i>S</i>-glutathionylation increase produced by stimulatory glucose. We propose that RyR2-mediated Ca²⁺ release, induced by the concomitant increases in [Ca²⁺] and ROS produced by stimulatory glucose, is an essential step in GSIS.</p></div

RyR inhibition prevents H₂O₂-dependent insulin secretion.

<p>Islets were pre-incubated for 1 h at 37°C in Krebs bicarbonate buffer supplemented with 2.8 mM glucose. Groups of 15 islets were then incubated for 1 h at 37°C in the presence or absence of 100 μM H₂O₂ in basal glucose (2.8 mM) to measure insulin secretion. Rya ON: islets were pre-incubated with 200 μM ryanodine for 12 h before the 1 h incubation period used to measure insulin secretion. Values represent Mean ± SEM; N = 3. Statistical significance was determined with one-way ANOVA followed by Tukey multiple comparison test. *: p <0.05.</p

Overnight incubation of pancreatic islets with 200 μM ryanodine inhibits insulin secretion stimulated by glucose but not by glucose plus carbachol.

<p>Insulin secretion was determined in groups of 15 islets after incubation for 1 h at 37°C in basal (2.8 mM) or stimulatory glucose (16.7 mM). (A, left) Rya ON: islets were pre-incubated with 200 μM ryanodine for 12 h before determination of insulin secretion after 1 h incubation in ryanodine-free solutions. (A, right) Rya 1 h: islets were pre-incubated with 100 μM ryanodine for 1 h before determination of insulin secretion after 1 h incubation in ryanodine-free solutions; G: glucose. (B) CCh: 30 μM carbachol was added during the 1 h incubation period used to measure insulin secretion. All data represent Mean ± SEM; N = 3 experiments (each condition in triplicate). Statistical significance was determined with one-way ANOVA followed by Tukey multiple comparison test. *: p <0.05; **: p <0.01; ***p <0.001.</p

Video_1_Mitochondrial Calcium Increase Induced by RyR1 and IP3R Channel Activation After Membrane Depolarization Regulates Skeletal Muscle Metabolism.AVI

<p>Aim: We hypothesize that both type-1 ryanodine receptor (RyR1) and IP₃-receptor (IP₃R) calcium channels are necessary for the mitochondrial Ca²⁺ increase caused by membrane depolarization induced by potassium (or by electrical stimulation) of single skeletal muscle fibers; this calcium increase would couple muscle fiber excitation to an increase in metabolic output from mitochondria (excitation-metabolism coupling).</p><p>Methods: Mitochondria matrix and cytoplasmic Ca²⁺ levels were evaluated in fibers isolated from flexor digitorium brevis muscle using plasmids for the expression of a mitochondrial Ca²⁺ sensor (CEPIA3mt) or a cytoplasmic Ca²⁺ sensor (RCaMP). The role of intracellular Ca²⁺ channels was evaluated using both specific pharmacological inhibitors (xestospongin B for IP₃R and Dantrolene for RyR1) and a genetic approach (shIP₃R1-RFP). O₂ consumption was detected using Seahorse Extracellular Flux Analyzer.</p><p>Results: In isolated muscle fibers cell membrane depolarization increased both cytoplasmic and mitochondrial Ca²⁺ levels. Mitochondrial Ca²⁺ uptake required functional inositol IP₃R and RyR1 channels. Inhibition of either channel decreased basal O₂ consumption rate but only RyR1 inhibition decreased ATP-linked O₂ consumption. Cell membrane depolarization-induced Ca²⁺ signals in sub-sarcolemmal mitochondria were accompanied by a reduction in mitochondrial membrane potential; Ca²⁺ signals propagated toward intermyofibrillar mitochondria, which displayed increased membrane potential. These results are compatible with slow, Ca²⁺-dependent propagation of mitochondrial membrane potential from the surface toward the center of the fiber.</p><p>Conclusion: Ca²⁺-dependent changes in mitochondrial membrane potential have different kinetics in the surface vs. the center of the fiber; these differences are likely to play a critical role in the control of mitochondrial metabolism, both at rest and after membrane depolarization as part of an “excitation-metabolism” coupling process in skeletal muscle fibers.</p

Prolonged Activation of the Htr2b Serotonin Receptor Impairs Glucose Stimulated Insulin Secretion and Mitochondrial Function in MIN6 Cells

<div><p>Aims</p><p>Pancreatic β-cells synthesize and release serotonin (5 hydroxytryptamine, 5HT); however, the role of 5HT receptors on glucose stimulated insulin secretion (GSIS) and the mechanisms mediating this function is not fully understood. The aims of this study were to determine the expression profile of 5HT receptors in murine MIN6 β-cells and to examine the effects of pharmacological activation of 5HT receptor Htr2b on GSIS and mitochondrial function.</p><p>Materials and Methods</p><p>mRNA levels of 5HT receptors in MIN6 cells were quantified by RT qPCR. GSIS was assessed in MIN6 cells in response to global serotonergic activation with 5HT and pharmacological Htr2b activation or inhibition with BW723C86 or SB204741, respectively. In response to Htr2b activation also was evaluated the mRNA and protein levels of PGC1α and PPARy by RT-qPCR and western blotting and mitochondrial function by oxygen consumption rate (OCR) and ATP cellular content.</p><p>Results</p><p>We found that mRNA levels of most 5HT receptors were either very low or undetectable in MIN6 cells. By contrast, Htr2b mRNA was present at moderate levels in these cells. Preincubation (6 h) of MIN6 cells with 5HT or BW723C86 reduced GSIS and the effect of 5HT was prevented by SB204741. Preincubation with BW723C86 increased PGC1α and PPARy mRNA and protein levels and decreased mitochondrial respiration and ATP content in MIN6 cells.</p><p>Conclusions</p><p>Our results indicate that prolonged Htr2b activation in murine β-cells decreases glucose-stimulated insulin secretion and mitochondrial activity by mechanisms likely dependent on enhanced PGC1α/PPARy expression.</p></div

Video_4_Mitochondrial Calcium Increase Induced by RyR1 and IP3R Channel Activation After Membrane Depolarization Regulates Skeletal Muscle Metabolism.AVI

<p>Aim: We hypothesize that both type-1 ryanodine receptor (RyR1) and IP₃-receptor (IP₃R) calcium channels are necessary for the mitochondrial Ca²⁺ increase caused by membrane depolarization induced by potassium (or by electrical stimulation) of single skeletal muscle fibers; this calcium increase would couple muscle fiber excitation to an increase in metabolic output from mitochondria (excitation-metabolism coupling).</p><p>Methods: Mitochondria matrix and cytoplasmic Ca²⁺ levels were evaluated in fibers isolated from flexor digitorium brevis muscle using plasmids for the expression of a mitochondrial Ca²⁺ sensor (CEPIA3mt) or a cytoplasmic Ca²⁺ sensor (RCaMP). The role of intracellular Ca²⁺ channels was evaluated using both specific pharmacological inhibitors (xestospongin B for IP₃R and Dantrolene for RyR1) and a genetic approach (shIP₃R1-RFP). O₂ consumption was detected using Seahorse Extracellular Flux Analyzer.</p><p>Results: In isolated muscle fibers cell membrane depolarization increased both cytoplasmic and mitochondrial Ca²⁺ levels. Mitochondrial Ca²⁺ uptake required functional inositol IP₃R and RyR1 channels. Inhibition of either channel decreased basal O₂ consumption rate but only RyR1 inhibition decreased ATP-linked O₂ consumption. Cell membrane depolarization-induced Ca²⁺ signals in sub-sarcolemmal mitochondria were accompanied by a reduction in mitochondrial membrane potential; Ca²⁺ signals propagated toward intermyofibrillar mitochondria, which displayed increased membrane potential. These results are compatible with slow, Ca²⁺-dependent propagation of mitochondrial membrane potential from the surface toward the center of the fiber.</p><p>Conclusion: Ca²⁺-dependent changes in mitochondrial membrane potential have different kinetics in the surface vs. the center of the fiber; these differences are likely to play a critical role in the control of mitochondrial metabolism, both at rest and after membrane depolarization as part of an “excitation-metabolism” coupling process in skeletal muscle fibers.</p

Video_8_Mitochondrial Calcium Increase Induced by RyR1 and IP3R Channel Activation After Membrane Depolarization Regulates Skeletal Muscle Metabolism.AVI

<p>Aim: We hypothesize that both type-1 ryanodine receptor (RyR1) and IP₃-receptor (IP₃R) calcium channels are necessary for the mitochondrial Ca²⁺ increase caused by membrane depolarization induced by potassium (or by electrical stimulation) of single skeletal muscle fibers; this calcium increase would couple muscle fiber excitation to an increase in metabolic output from mitochondria (excitation-metabolism coupling).</p><p>Methods: Mitochondria matrix and cytoplasmic Ca²⁺ levels were evaluated in fibers isolated from flexor digitorium brevis muscle using plasmids for the expression of a mitochondrial Ca²⁺ sensor (CEPIA3mt) or a cytoplasmic Ca²⁺ sensor (RCaMP). The role of intracellular Ca²⁺ channels was evaluated using both specific pharmacological inhibitors (xestospongin B for IP₃R and Dantrolene for RyR1) and a genetic approach (shIP₃R1-RFP). O₂ consumption was detected using Seahorse Extracellular Flux Analyzer.</p><p>Results: In isolated muscle fibers cell membrane depolarization increased both cytoplasmic and mitochondrial Ca²⁺ levels. Mitochondrial Ca²⁺ uptake required functional inositol IP₃R and RyR1 channels. Inhibition of either channel decreased basal O₂ consumption rate but only RyR1 inhibition decreased ATP-linked O₂ consumption. Cell membrane depolarization-induced Ca²⁺ signals in sub-sarcolemmal mitochondria were accompanied by a reduction in mitochondrial membrane potential; Ca²⁺ signals propagated toward intermyofibrillar mitochondria, which displayed increased membrane potential. These results are compatible with slow, Ca²⁺-dependent propagation of mitochondrial membrane potential from the surface toward the center of the fiber.</p><p>Conclusion: Ca²⁺-dependent changes in mitochondrial membrane potential have different kinetics in the surface vs. the center of the fiber; these differences are likely to play a critical role in the control of mitochondrial metabolism, both at rest and after membrane depolarization as part of an “excitation-metabolism” coupling process in skeletal muscle fibers.</p