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

Llanos P, Contreras-Ferrat A, Barrientos G, Valencia M, Mears D, Hidalgo C - PLoS ONE (2015)

Bottom Line: Inhibitory ryanodine or NAC did not affect insulin secretion induced by glucose plus carbachol, which engages inositol 1,4,5-trisphosphate receptors.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 S-glutathionylation to similar levels, measured by a proximity ligation assay; in contrast, NAC significantly reduced the RyR2 S-glutathionylation increase produced by stimulatory glucose.

View Article: PubMed Central - PubMed

Affiliation: Institute for Research in Dental Sciences, Facultad de Odontología, Universidad de Chile, Santiago, Chile; Center of Molecular Studies of the Cell, Facultad de Medicina, Universidad de Chile, Santiago, Chile.

ABSTRACT
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 S-glutathionylation to similar levels, measured by a proximity ligation assay; in contrast, NAC significantly reduced the RyR2 S-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.

No MeSH data available.


Related in: MedlinePlus

Incubation with exogenous H2O2 increases [Ca2+]i in pancreatic β-cells via activation of RyR-mediated Ca2+ release.(A) Records of [Ca2+]i vs time obtained from rat pancreatic β-cells pre-incubated for 1 h with 2 μM fura-2-AM in Hanks basal solution (2.8 mM glucose). Control: cells were kept in basal Hanks solution. H2O2: cells were pre-incubated for 1 h with 100 μM H2O2 in basal Hanks solution. H2O2 + Rya ON: cells were pre-incubated with 200 μM ryanodine (Rya) for 12 h and were then incubated for 1 h with 100 μM H2O2 (in ryanodine-free solution) prior to recording in basal Hanks solution (H2O2 free). Rya ON: cells were pre-incubated with 200 μM ryanodine for 12 h. At right, quantification of these results, given as Mean ± SEM, N = 3–7. Statistical significance was determined with one-way ANOVA followed by Tukey multiple comparison test. ***: p <0.001. (B) Average record (N = 3) of Ca2+ signals elicited by 100 μM H2O2 in the absence of ryanodine. (C) Average record (N = 3) of Ca2+ signals registered in cells pre-incubated with 200 μM ryanodine for 12 h (Rya ON); 100 μM H2O2 or 90 mM KCl were added in succession, as indicated by the arrows.
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pone.0129238.g007: Incubation with exogenous H2O2 increases [Ca2+]i in pancreatic β-cells via activation of RyR-mediated Ca2+ release.(A) Records of [Ca2+]i vs time obtained from rat pancreatic β-cells pre-incubated for 1 h with 2 μM fura-2-AM in Hanks basal solution (2.8 mM glucose). Control: cells were kept in basal Hanks solution. H2O2: cells were pre-incubated for 1 h with 100 μM H2O2 in basal Hanks solution. H2O2 + Rya ON: cells were pre-incubated with 200 μM ryanodine (Rya) for 12 h and were then incubated for 1 h with 100 μM H2O2 (in ryanodine-free solution) prior to recording in basal Hanks solution (H2O2 free). Rya ON: cells were pre-incubated with 200 μM ryanodine for 12 h. At right, quantification of these results, given as Mean ± SEM, N = 3–7. Statistical significance was determined with one-way ANOVA followed by Tukey multiple comparison test. ***: p <0.001. (B) Average record (N = 3) of Ca2+ signals elicited by 100 μM H2O2 in the absence of ryanodine. (C) Average record (N = 3) of Ca2+ signals registered in cells pre-incubated with 200 μM ryanodine for 12 h (Rya ON); 100 μM H2O2 or 90 mM KCl were added in succession, as indicated by the arrows.

Mentions: Addition of H2O2 stimulates RyR-mediated CICR in other cell types [30]. The results illustrated in Fig 6 led us to hypothesize that addition of H2O2 activates RyR-mediated Ca2+ release in pancreatic β-cells; the resulting increase in [Ca2+]i would cause the increase in insulin secretion induced by H2O2. To test this hypothesis, we measured [Ca2+]i with the fluorescent probe fura-2 (Fig 7A). Incubation for 1 h of disaggregated β-cells with H2O2 increased [Ca2+]i from a basal level of 99.7 ± 21 nM to 455.2 ± 69.6 nM. Cells pre-incubated with inhibitory ryanodine for 12 h displayed an average value of [Ca2+]i = 142.6 ± 21.5 nM, which did not change after addition of H2O2 (Fig 7A). As illustrated in Fig 7B, H2O2 addition to control cells increased [Ca2+]i rapidly (within 10 s) to a value of 324 ± 5.4 nM (mean value, first minute after H2O2 addition, N = 3). This increase occurred as a consequence of RyR-mediated Ca2+ release since overnight incubation with inhibitory ryanodine prevented the fast [Ca2+]i increase produced by H2O2 (Fig 7C). Yet, these same cells did respond to subsequent addition of 90 mM KCl with a marked increase in [Ca2+]i (Fig 7C). The observations that disaggregated β-cells incubated overnight with inhibitory ryanodine maintained [Ca2+]i at resting levels, and responded to KCl, show that Ca2+ homeostasis and depolarization-induced Ca2+ influx through voltage-gated Ca2+ channels remained largely unaffected by this treatment.


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

Llanos P, Contreras-Ferrat A, Barrientos G, Valencia M, Mears D, Hidalgo C - PLoS ONE (2015)

Incubation with exogenous H2O2 increases [Ca2+]i in pancreatic β-cells via activation of RyR-mediated Ca2+ release.(A) Records of [Ca2+]i vs time obtained from rat pancreatic β-cells pre-incubated for 1 h with 2 μM fura-2-AM in Hanks basal solution (2.8 mM glucose). Control: cells were kept in basal Hanks solution. H2O2: cells were pre-incubated for 1 h with 100 μM H2O2 in basal Hanks solution. H2O2 + Rya ON: cells were pre-incubated with 200 μM ryanodine (Rya) for 12 h and were then incubated for 1 h with 100 μM H2O2 (in ryanodine-free solution) prior to recording in basal Hanks solution (H2O2 free). Rya ON: cells were pre-incubated with 200 μM ryanodine for 12 h. At right, quantification of these results, given as Mean ± SEM, N = 3–7. Statistical significance was determined with one-way ANOVA followed by Tukey multiple comparison test. ***: p <0.001. (B) Average record (N = 3) of Ca2+ signals elicited by 100 μM H2O2 in the absence of ryanodine. (C) Average record (N = 3) of Ca2+ signals registered in cells pre-incubated with 200 μM ryanodine for 12 h (Rya ON); 100 μM H2O2 or 90 mM KCl were added in succession, as indicated by the arrows.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4457734&req=5

pone.0129238.g007: Incubation with exogenous H2O2 increases [Ca2+]i in pancreatic β-cells via activation of RyR-mediated Ca2+ release.(A) Records of [Ca2+]i vs time obtained from rat pancreatic β-cells pre-incubated for 1 h with 2 μM fura-2-AM in Hanks basal solution (2.8 mM glucose). Control: cells were kept in basal Hanks solution. H2O2: cells were pre-incubated for 1 h with 100 μM H2O2 in basal Hanks solution. H2O2 + Rya ON: cells were pre-incubated with 200 μM ryanodine (Rya) for 12 h and were then incubated for 1 h with 100 μM H2O2 (in ryanodine-free solution) prior to recording in basal Hanks solution (H2O2 free). Rya ON: cells were pre-incubated with 200 μM ryanodine for 12 h. At right, quantification of these results, given as Mean ± SEM, N = 3–7. Statistical significance was determined with one-way ANOVA followed by Tukey multiple comparison test. ***: p <0.001. (B) Average record (N = 3) of Ca2+ signals elicited by 100 μM H2O2 in the absence of ryanodine. (C) Average record (N = 3) of Ca2+ signals registered in cells pre-incubated with 200 μM ryanodine for 12 h (Rya ON); 100 μM H2O2 or 90 mM KCl were added in succession, as indicated by the arrows.
Mentions: Addition of H2O2 stimulates RyR-mediated CICR in other cell types [30]. The results illustrated in Fig 6 led us to hypothesize that addition of H2O2 activates RyR-mediated Ca2+ release in pancreatic β-cells; the resulting increase in [Ca2+]i would cause the increase in insulin secretion induced by H2O2. To test this hypothesis, we measured [Ca2+]i with the fluorescent probe fura-2 (Fig 7A). Incubation for 1 h of disaggregated β-cells with H2O2 increased [Ca2+]i from a basal level of 99.7 ± 21 nM to 455.2 ± 69.6 nM. Cells pre-incubated with inhibitory ryanodine for 12 h displayed an average value of [Ca2+]i = 142.6 ± 21.5 nM, which did not change after addition of H2O2 (Fig 7A). As illustrated in Fig 7B, H2O2 addition to control cells increased [Ca2+]i rapidly (within 10 s) to a value of 324 ± 5.4 nM (mean value, first minute after H2O2 addition, N = 3). This increase occurred as a consequence of RyR-mediated Ca2+ release since overnight incubation with inhibitory ryanodine prevented the fast [Ca2+]i increase produced by H2O2 (Fig 7C). Yet, these same cells did respond to subsequent addition of 90 mM KCl with a marked increase in [Ca2+]i (Fig 7C). The observations that disaggregated β-cells incubated overnight with inhibitory ryanodine maintained [Ca2+]i at resting levels, and responded to KCl, show that Ca2+ homeostasis and depolarization-induced Ca2+ influx through voltage-gated Ca2+ channels remained largely unaffected by this treatment.

Bottom Line: Inhibitory ryanodine or NAC did not affect insulin secretion induced by glucose plus carbachol, which engages inositol 1,4,5-trisphosphate receptors.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 S-glutathionylation to similar levels, measured by a proximity ligation assay; in contrast, NAC significantly reduced the RyR2 S-glutathionylation increase produced by stimulatory glucose.

View Article: PubMed Central - PubMed

Affiliation: Institute for Research in Dental Sciences, Facultad de Odontología, Universidad de Chile, Santiago, Chile; Center of Molecular Studies of the Cell, Facultad de Medicina, Universidad de Chile, Santiago, Chile.

ABSTRACT
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 S-glutathionylation to similar levels, measured by a proximity ligation assay; in contrast, NAC significantly reduced the RyR2 S-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.

No MeSH data available.


Related in: MedlinePlus