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Aims/hypothesis: Nicotinamide nucleotide transhydrogenase (NNT) is involved in mitochondrial NADPH production and its spontaneous inactivating mutation (Nnt Tr [Tr, truncated]) is usually considered to be the main cause of the lower glucose tolerance of C57BL/6J vs C57BL/6N mice. However, the impact of this mutation on glucose tolerance remains disputed. Here, we singled out the impact of Nnt Tr from that of other genetic variants between C57BL/6J and C57BL/6N mice on mitochondrial glutathione redox state (E GSH ), glucose-stimulated insulin secretion (GSIS) and glucose tolerance., Methods: Male and female N5BL/6J mice that express wild-type Nnt (Nnt WT ) or Nnt Tr (N5-WT and N5-Tr mice) on the C57BL/6J genetic background were obtained by crossing N5BL/6J Nnt WT/Tr heterozygous mice. C57BL/6J and C57BL/6N mice were from Janvier Labs. The Nnt genotype was confirmed by PCR and the genetic background by whole genome sequencing of one mouse of each type. Glucose tolerance was assessed by IPGTT, ITT and fasting/refeeding tests. Stimulus-secretion coupling events and GSIS were measured in isolated pancreatic islets. Cytosolic and mitochondrial E GSH were measured using the fluorescent redox probe GRX1-roGFP2 (glutaredoxin 1 fused to redox-sensitive enhanced GFP)., Results: The Nnt genotype and genetic background of each type of mouse were confirmed. As reported previously in C57BL/6N vs C57BL/6J islets, the glucose regulation of mitochondrial (but not cytosolic) E GSH and of NAD(P)H autofluorescence was markedly improved in N5-WT vs N5-Tr islets, confirming the role of NNT in mitochondrial redox regulation. However, ex vivo GSIS was only 1.2-1.4-times higher in N5-WT vs N5-Tr islets, while it was 2.4-times larger in C57BL/6N vs N5-WT islets, questioning the role of NNT in GSIS. In vivo, the ITT results did not differ between N5-WT and N5-Tr or C57BL/6N mice. However, the glucose excursion during an IPGTT was only 15-20% lower in female N5-WT mice than in N5-Tr and C57BL/6J mice and remained 3.5-times larger than in female C57BL/6N mice. Similar observations were made during a fasting/refeeding test. A slightly larger (~30%) impact of NNT on glucose tolerance was found in males., Conclusions/interpretation: Although our results confirm the importance of NNT in the regulation of mitochondrial redox state by glucose, they markedly downsize the role of NNT in the alteration of GSIS and glucose tolerance in C57BL/6J vs C57BL/6N mice. Therefore, documenting an Nnt WT genotype in C57BL/6 mice does not provide proof that their glucose tolerance is as good as in C57BL/6N mice., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Aims/hypothesis: The mechanisms responsible for beta cell compensation in obesity and for beta cell failure in type 2 diabetes are poorly defined. The mRNA levels of several metallothionein (MT) genes are upregulated in islets from individuals with type 2 diabetes, but their role in beta cells is not clear. Here we examined: (1) the temporal changes of islet Mt1 and Mt2 gene expression in mouse models of beta cell compensation and failure; and (2) the role of Mt1 and Mt2 in beta cell function and glucose homeostasis in mice., Methods: Mt1 and Mt2 expression was assessed in islets from: (1) control lean (chow diet-fed) and diet-induced obese (high-fat diet-fed for 6 weeks) mice; (2) mouse models of diabetes (db/db mice) at 6 weeks old (prediabetes) and 16 weeks old (after diabetes onset) and age-matched db/+ (control) mice; and (3) obese non-diabetic ob/ob mice (16-week-old) and age-matched ob/+ (control) mice. MT1E, MT1X and MT2A expression was assessed in islets from humans with and without type 2 diabetes. Mt1-Mt2 double-knockout (KO) mice, transgenic mice overexpressing Mt1 under the control of its natural promoter (Tg-Mt1) and corresponding control mice were also studied. In MIN6 cells, MT1 and MT2 were inhibited by small interfering RNAs. mRNA levels were assessed by real-time RT-PCR, plasma insulin and islet MT levels by ELISA, glucose tolerance by i.p. glucose tolerance tests and overnight fasting-1 h refeeding tests, insulin tolerance by i.p. insulin tolerance tests, insulin secretion by RIA, cytosolic free Ca 2+ concentration with Fura-2 leakage resistant (Fura-2 LR), cytosolic free Zn 2+ concentration with Fluozin-3, and NAD(P)H by autofluorescence., Results: Mt1 and Mt2 mRNA levels were reduced in islets of murine models of beta cell compensation, whereas they were increased in diabetic db/db mice. In humans, MT1X mRNA levels were significantly upregulated in islets from individuals with type 2 diabetes in comparison with non-diabetic donors, while MT1E and MT2A mRNA levels were unchanged. Ex vivo, islet Mt1 and Mt2 mRNA and MT1 and MT2 protein levels were downregulated after culture with glucose at 10-30 mmol/l vs 2-5 mmol/l, in association with increased insulin secretion. In human islets, mRNA levels of MT1E, MT1X and MT2A were downregulated by stimulation with physiological and supraphysiological levels of glucose. In comparison with wild-type (WT) mice, Mt1-Mt2 double-KO mice displayed improved glucose tolerance in association with increased insulin levels and enhanced insulin release from isolated islets. In contrast, isolated islets from Tg-Mt1 mice displayed impaired glucose-stimulated insulin secretion (GSIS). In both Mt1-Mt2 double-KO and Tg-Mt1 models, the changes in GSIS occurred despite similar islet insulin content, rises in cytosolic free Ca 2+ concentration and NAD(P)H levels, or intracellular Zn 2+ concentration vs WT mice. In MIN6 cells, knockdown of MT1 but not MT2 potentiated GSIS, suggesting that Mt1 rather than Mt2 affects beta cell function., Conclusions/interpretation: These findings implicate Mt1 as a negative regulator of insulin secretion. The downregulation of Mt1 is associated with beta cell compensation in obesity, whereas increased Mt1 accompanies beta cell failure and type 2 diabetes.

Aims: Whether H 2 O 2 contributes to the glucose-dependent stimulation of insulin secretion (GSIS) by pancreatic β cells is highly controversial. We used two H 2 O 2 -sensitive probes, roGFP2-Orp1 (reduction/oxidation-sensitive enhanced green fluorescent protein fused to oxidant receptor peroxidase 1) and HyPer (hydrogen peroxide sensor) with its pH-control SypHer, to test the acute effects of glucose, monomethyl succinate, leucine with glutamine, and α-ketoisocaproate on β cell cytosolic and mitochondrial H 2 O 2 concentrations. We then tested the effects of low H 2 O 2 and menadione concentrations on insulin secretion. Results: RoGFP2-Orp1 was more sensitive than HyPer to H 2 O 2 (response at 2-5 vs. 10 μ M ) and less pH-sensitive. Under control conditions, stimulation with glucose reduced mitochondrial roGFP2-Orp1 oxidation without affecting cytosolic roGFP2-Orp1 and HyPer fluorescence ratios, except for the pH-dependent effects on HyPer. However, stimulation with glucose decreased the oxidation of both cytosolic probes by 15 μ M exogenous H 2 O 2 . The glucose effects were not affected by overexpression of catalase, mitochondrial catalase, or superoxide dismutase 1 and 2. They followed the increase in NAD(P)H autofluorescence, were maximal at 5 m M glucose in the cytosol and 10 m M glucose in the mitochondria, and were partly mimicked by the other nutrients. Exogenous H 2 O 2 (1-15 μ M ) did not affect insulin secretion. By contrast, menadione (1-5 μ M ) did not increase basal insulin secretion but reduced the stimulation of insulin secretion by 20 m M glucose. Innovation: Subcellular changes in β cell H 2 O 2 levels are better monitored with roGFP2-Orp1 than HyPer/SypHer. Nutrients acutely lower mitochondrial H 2 O 2 levels in β cells and promote degradation of exogenously supplied H 2 O 2 in both cytosolic and mitochondrial compartments. Conclusion: The GSIS occurs independently of a detectable increase in β cell cytosolic or mitochondrial H 2 O 2 levels.

The mechanisms of control of glucagon secretion are largely debated. In particular, the paracrine role of somatostatin (SST) is unclear. We studied its role in the control of glucagon secretion by glucose and K ATP channel blockers, using perifused islets and the in situ perfused pancreas. The involvement of SST was evaluated by comparing glucagon release of control tissue or tissue without paracrine influence of SST (pertussis toxin-treated islets, or islets or pancreas from Sst -/- mice). We show that removal of the paracrine influence of SST suppresses the ability of K ATP channel blockers or K ATP channel ablation to inhibit glucagon release, suggesting that in control islets, the glucagonostatic effect of K ATP channel blockers/ablation is fully mediated by SST. By contrast, the glucagonostatic effect of glucose in control islets is mainly independent of SST for low glucose concentrations (0-7 mmol/L) but starts to involve SST for high concentrations of the sugar (15-30 mmol/L). This demonstrates that the glucagonostatic effect of glucose only partially depends on SST. Real-time quantitative PCR and pharmacological experiments indicate that the glucagonostatic effect of SST is mediated by two types of SST receptors, SSTR2 and SSTR3. These results suggest that alterations of the paracrine influence of SST will affect glucagon release., (© 2018 by the American Diabetes Association.)

Objective: The glucose stimulation of insulin secretion (GSIS) by pancreatic β-cells critically depends on increased production of metabolic coupling factors, including NADPH. Nicotinamide nucleotide transhydrogenase (NNT) typically produces NADPH at the expense of NADH and ΔpH in energized mitochondria. Its spontaneous inactivation in C57BL/6J mice was previously shown to alter ATP production, Ca 2+ influx, and GSIS, thereby leading to glucose intolerance. Here, we tested the role of NNT in the glucose regulation of mitochondrial NADPH and glutathione redox state and reinvestigated its role in GSIS coupling events in mouse pancreatic islets., Methods: Islets were isolated from female C57BL/6J mice (J-islets), which lack functional NNT, and genetically close C57BL/6N mice (N-islets). Wild-type mouse NNT was expressed in J-islets by adenoviral infection. Mitochondrial and cytosolic glutathione oxidation was measured with glutaredoxin 1-fused roGFP2 probes targeted or not to the mitochondrial matrix. NADPH and NADH redox state was measured biochemically. Insulin secretion and upstream coupling events were measured under dynamic or static conditions by standard procedures., Results: NNT is largely responsible for the acute glucose-induced rise in islet NADPH/NADP + ratio and decrease in mitochondrial glutathione oxidation, with a small impact on cytosolic glutathione. However, contrary to current views on NNT in β-cells, these effects resulted from a glucose-dependent reduction in NADPH consumption by NNT reverse mode of operation, rather than from a stimulation of its forward mode of operation. Accordingly, the lack of NNT in J-islets decreased their sensitivity to exogenous H 2 O 2 at non-stimulating glucose. Surprisingly, the lack of NNT did not alter the glucose-stimulation of Ca 2+ influx and upstream mitochondrial events, but it markedly reduced both phases of GSIS by altering Ca 2+ -induced exocytosis and its metabolic amplification., Conclusion: These results drastically modify current views on NNT operation and mitochondrial function in pancreatic β-cells.

High glucose-induced oxidative stress and increased NADPH oxidase-2 (NOX2) activity may contribute to the progressive decline of the functional β-cell mass in type 2 diabetes. To test that hypothesis, we characterized, in islets from male NOX2 knockout (NOX2-KO) and wild-type (WT) C57BL/6J mice cultured for up to 3 weeks at 10 or 30 mmol/l glucose (G10 or G30), the in vitro effects of glucose on cytosolic oxidative stress using probes sensing glutathione oxidation (GRX1-roGFP2), thiol oxidation (roGFP1) or H 2 O 2 (roGFP2-Orp1), on β-cell stimulus-secretion coupling events and on β-cell apoptosis. After 1-2 days of culture in G10, the glucose stimulation of insulin secretion (GSIS) was ∼1.7-fold higher in NOX2-KO vs. WT islets at 20-30 mmol/l glucose despite similar rises in NAD(P)H and intracellular calcium concentration ([Ca 2+ ] i ) and no differences in cytosolic GRX1-roGFP2 oxidation. After long-term culture at G10, roGFP1 and roGFP2-Orp1 oxidation and β-cell apoptosis remained low, and the glucose-induced rises in NAD(P)H, [Ca 2+ ] i and GSIS were similarly preserved in both islet types. After prolonged culture at G30, roGFP1 and roGFP2-Orp1 oxidation increased in parallel with β-cell apoptosis, the glucose sensitivity of the NADPH, [Ca 2+ ] i and insulin secretion responses increased, the maximal [Ca 2+ ] i response decreased, but maximal GSIS was preserved. These responses were almost identical in both islet types. In conclusion, NOX2 is a negative regulator of maximal GSIS in C57BL/6J mouse islets, but it does not detectably contribute to the in vitro glucotoxic induction of cytosolic oxidative stress and alterations of β-cell survival and function., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

In rat pancreatic islets, β-cell gene expression, survival, and subsequent acute glucose stimulation of insulin secretion (GSIS) are optimally preserved by prolonged culture at 10 mM glucose (G10) and markedly altered by culture at G5 or G30. Here, we tested whether pharmacological glucokinase (GK) activation prevents these alterations during culture or improves GSIS after culture. Rat pancreatic islets were cultured 1-7 days at G5, G10, or G30 with or without 3 μM of the GK activator Ro 28-0450 (Ro). After culture, β-cell apoptosis and islet gene mRNA levels were measured, and the acute glucose-induced increase in NAD(P)H autofluorescence, intracellular calcium concentration, and insulin secretion were tested in the absence or presence of Ro. Prolonged culture of rat islets at G5 or G30 instead of G10 triggered β-cell apoptosis and reduced their glucose responsiveness. Addition of Ro during culture differently affected β-cell survival and glucose responsiveness depending on the glucose concentration during culture: it was beneficial to β-cell survival and function at G5, detrimental at G10, and ineffective at G30. In contrast, acute GK activation with Ro increased the glucose sensitivity of islets cultured at G10 but failed at restoring β-cell glucose responsiveness after culture at G5 or G30. We conclude that pharmacological GK activation prevents the alteration of β-cell survival and function by long-term culture at G5 but mimics glucotoxicity when added to G10. The complex effects of glucose on the β-cell phenotype result from changes in glucose metabolism and not from an effect of glucose per se., (Copyright © 2015 the American Physiological Society.)

The glucose stimulation of insulin secretion by pancreatic β-cells depends on increased production of metabolic coupling factors, among which changes in NADPH and ROS (reactive oxygen species) may alter the glutathione redox state (EGSH) and signal through changes in thiol oxidation. However, whether nutrients affect EGSH in β-cell subcellular compartments is unknown. Using redox-sensitive GFP2 fused to glutaredoxin 1 and its mitochondria-targeted form, we studied the acute nutrient regulation of EGSH in the cytosol/nucleus or the mitochondrial matrix of rat islet cells. These probes were mainly expressed in β-cells and reacted to low concentrations of exogenous H2O2 and menadione. Under control conditions, cytosolic/nuclear EGSH was close to -300 mV and unaffected by glucose (from 0 to 30 mM). In comparison, mitochondrial EGSH was less negative and rapidly regulated by glucose and other nutrients, ranging from -280 mV in the absence of glucose to -299 mV in 30 mM glucose. These changes were largely independent from changes in intracellular Ca(2+) concentration and in mitochondrial pH. They were unaffected by overexpression of SOD2 (superoxide dismutase 2) and mitochondria-targeted catalase, but were inversely correlated with changes in NAD(P)H autofluorescence, suggesting that they indirectly resulted from increased NADPH availability rather than from changes in ROS concentration. Interestingly, the opposite regulation of mitochondrial EGSH and NAD(P)H autofluorescence by glucose was also observed in human islets isolated from two donors. In conclusion, the present study demonstrates that glucose and other nutrients acutely reduce mitochondrial, but not cytosolic/nuclear, EGSH in pancreatic β-cells under control conditions.

It is well established that regular physiological stimulation by glucose plays a crucial role in the maintenance of the β-cell differentiated phenotype. In contrast, prolonged or repeated exposure to elevated glucose concentrations both in vitro and in vivo exerts deleterious or toxic effects on the β-cell phenotype, a concept termed as glucotoxicity. Evidence indicates that the latter may greatly contribute to the pathogenesis of type 2 diabetes. Through the activation of several mechanisms and signaling pathways, high glucose levels exert deleterious effects on β-cell function and survival and thereby, lead to the worsening of the disease over time. While the role of high glucose-induced β-cell overstimulation, oxidative stress, excessive Unfolded Protein Response (UPR) activation, and loss of differentiation in the alteration of the β-cell phenotype is well ascertained, at least in vitro and in animal models of type 2 diabetes, the role of other mechanisms such as inflammation, O-GlcNacylation, PKC activation, and amyloidogenesis requires further confirmation. On the other hand, protein glycation is an emerging mechanism that may play an important role in the glucotoxic deterioration of the β-cell phenotype. Finally, our recent evidence suggests that hypoxia may also be a new mechanism of β-cell glucotoxicity. Deciphering these molecular mechanisms of β-cell glucotoxicity is a mandatory first step toward the development of therapeutic strategies to protect β-cells and improve the functional β-cell mass in type 2 diabetes., (Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.)

Background: Glucose increases the expression of glycolytic enzymes and other hypoxia-response genes in pancreatic beta-cells. Here, we tested whether this effect results from the activation of Hypoxia-Inducible-factors (HIF) 1 and 2 in a hypoxia-dependent manner., Methodology/principal Findings: Isolated rat islets and insulin-secreting INS-1E cells were stimulated with nutrients at various pO₂ values or treated with the HIF activator CoCl₂. HIF-target gene mRNA levels and HIF subunit protein levels were measured by real-time RT-PCR, Western Blot and immunohistochemistry. The formation of pimonidazole-protein adducts was used as an indicator of hypoxia. In INS-1E and islet beta-cells, glucose concentration-dependently stimulated formation of pimonidazole-protein adducts, HIF1 and HIF2 nuclear expression and HIF-target gene mRNA levels to a lesser extent than CoCl₂ or a four-fold reduction in pO₂. Islets also showed signs of HIF activation in diabetic Lepr(db/db) but not non-diabetic Lepr(db/+) mice. In vitro, these glucose effects were reproduced by nutrient secretagogues that bypass glycolysis, and were inhibited by a three-fold increase in pO₂ or by inhibitors of Ca²⁺ influx and insulin secretion. In INS-1E cells, small interfering RNA-mediated knockdown of Hif1α and Hif2α, alone or in combination, indicated that the stimulation of glycolytic enzyme mRNA levels depended on both HIF isoforms while the vasodilating peptide adrenomedullin was a HIF2-specific target gene., Conclusions/significance: Glucose-induced O₂ consumption creates an intracellular hypoxia that activates HIF1 and HIF2 in rat beta-cells, and this glucose effect contributes, together with the activation of other transcription factors, to the glucose stimulation of expression of some glycolytic enzymes and other hypoxia response genes.

Aim/hypothesis: Rat pancreatic islet cell apoptosis is minimal after prolonged culture in 10 mmol/l glucose (G10), largely increased in 5 mmol/l glucose (G5) and moderately increased in 30 mmol/l glucose (G30). This glucose-dependent asymmetric V-shaped profile is preceded by parallel changes in the mRNA levels of oxidative stress-response genes like Metallothionein 1a (Mt1a). In this study, we tested the effect of ZnCl(2), a potent inducer of Mt1a, on apoptosis, mitochondrial oxidative stress and alterations of glucose-induced insulin secretion (GSIS) induced by prolonged exposure to low and high vs. intermediate glucose concentrations., Methods: Male Wistar rat islets were cultured in RPMI medium. Islet gene mRNA levels were measured by RTq-PCR. Apoptosis was quantified by measuring islet cytosolic histone-associated DNA fragments and the percentage of TUNEL-positive β-cells. Mitochondrial thiol oxidation was measured in rat islet cell clusters expressing "redox sensitive GFP" targeted to the mitochondria (mt-roGFP1). Insulin secretion was measured by RIA., Results: As observed for Mt1a mRNA levels, β-cell apoptosis and loss of GSIS, culture in either G5 or G30 vs. G10 significantly increased mt-roGFP1 oxidation. While TPEN decreased Mt1a/2a mRNA induction by G5, addition of 50-100 µM ZnCl(2) to the culture medium strongly increased Mt1a/2a mRNA and protein levels, reduced early mt-roGFP oxidation and significantly decreased late β-cell apoptosis after prolonged culture in G5 or G30 vs. G10. It did not, however, prevent the loss of GSIS under these culture conditions., Conclusion: ZnCl(2) reduces mitochondrial oxidative stress and improves rat β-cell survival during culture in the presence of low and high vs. intermediate glucose concentrations without improving their acute GSIS.

In vitro, survival and function of rat pancreatic β-cells are optimally preserved in the presence of 10 mmol/l glucose (G10) and markedly altered by prolonged culture at either 2 mmol/l glucose (G2) or 30 mmol/l glucose (G30). The increase in islet cell apoptosis in G2 and G30 vs. G10 is preceded by parallel increases in the mRNA levels of the integrated stress response (ISR) gene activating transcription factor 3 (Atf3) and its putative target and proapoptotic gene growth arrest- and DNA damage-inducible gene 153 (Gadd153/Chop). In this study, we used islets from Atf3 knockout (Atf3(-/-)) mice to test the role of ATF3 in the stimulation of islet cell apoptosis under conditions associated with ISR activation. The glucose sensitivity of Atf3(-/-) and WT islets for the stimulation of insulin secretion and Xbp1 mRNA splicing during 18h culture was similar, demonstrating that glucose metabolism was unaffected by Atf3 deletion. However, the stimulation of islet cell apoptosis by the SERCA pump inhibitor thapsigargin was slightly but significantly reduced in Atf3(-/-) vs. WT islets despite similar level of expression of Gadd153 and Gadd34 mRNA. Also, the stimulation of islet cell apoptosis by 7 days of culture in G2 was slightly but significantly reduced in Atf3(-/-) vs. WT islets, and this effect was accompanied by a significant reduction in Gadd153 mRNA expression. In conclusion, the increase in Atf3 gene expression induced by thapsigargin and low glucose concentrations slightly contributes to the stimulation of islet cell apoptosis under these culture conditions., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Alteration of pancreatic beta-cell survival and Preproinsulin gene expression by prolonged hyperglycemia may result from increased c-MYC expression. However, it is unclear whether c-MYC effects on beta-cell function are compatible with its proposed role in glucotoxicity. We therefore tested the effects of short-term c-MYC activation on key beta-cell stimulus-secretion coupling events in islets isolated from mice expressing a tamoxifen-switchable form of c-MYC in beta-cells (MycER) and their wild-type littermates. Tamoxifen treatment of wild-type islets did not affect their cell survival, Preproinsulin gene expression, and glucose stimulus-secretion coupling. In contrast, tamoxifen-mediated c-MYC activation for 2-3 days triggered cell apoptosis and decreased Preproinsulin gene expression in MycER islets. These effects were accompanied by mitochondrial membrane hyperpolarization at all glucose concentrations, a higher resting intracellular calcium concentration ([Ca(2+)](i)), and lower glucose-induced [Ca(2+)](i) rise and islet insulin content, leading to a strong reduction of glucose-induced insulin secretion. Compared with these effects, 1-wk culture in 30 mmol/l glucose increased the islet sensitivity to glucose stimulation without reducing the maximal glucose effectiveness or the insulin content. In contrast, overnight exposure to a low H(2)O(2) concentration increased the islet resting [Ca(2+)](i) and reduced the amplitude of the maximal glucose response as in tamoxifen-treated MycER islets. In conclusion, c-MYC activation rapidly stimulates apoptosis, reduces Preproinsulin gene expression and insulin content, and triggers functional alterations of beta-cells that are better mimicked by overnight exposure to a low H(2)O(2) concentration than by prolonged culture in high glucose.

The contribution of Na(+)/H(+) exchange (achieved by NHE proteins) to the regulation of beta-cell cytosolic pH(c), and the role of pH(c) changes in glucose-induced insulin secretion are disputed and were examined here. Using real-time PCR, we identified plasmalemmal NHE1 and intracellular NHE7 as the two most abundant NHE isoforms in mouse islets. We, therefore, compared insulin secretion, cytosolic free Ca(2+) ([Ca(2+)](c)) and pH(c) in islets from normal mice and mice bearing an inactivating mutation of NHE1 (Slc9A1-swe/swe). The experiments were performed in HCO(-)(3)/CO(2) or HEPES/NaOH buffers. PCR and functional approaches showed that NHE1 mutant islets do not express compensatory pH-regulating mechanisms. NHE1 played a greater role than HCO(-)(3)-dependent mechanisms in the correction of an acidification imposed by a pulse of NH(4)Cl. In contrast, basal pH(c) (in low glucose) and the alkalinization produced by high glucose were independent of NHE1. Dimethylamiloride, a classic blocker of Na(+)/H(+) exchange, did not affect pH(c) but increased insulin secretion in NHE1 mutant islets, indicating unspecific effects. In control islets, glucose similarly increased [Ca(2+)](c) and insulin secretion in HCO(-)(3) and HEPES buffer, although pH(c) changed in opposite directions. The amplification of insulin secretion that glucose produces when [Ca(2+)](c) is clamped at an elevated level by KCl was also unrelated to pH(c) and pH(c) changes. All effects of glucose on [Ca(2+)](c) and insulin secretion proved independent of NHE1. In conclusion, NHE1 protects beta-cells against strong acidification, but has no role in stimulus-secretion coupling. The changes in pH(c) produced by glucose involve HCO(-)(3)-dependent mechanisms. Variations in beta-cell pH(c) are not causally related to changes in insulin secretion.

Stimulatory concentrations of glucose induce two patterns of cytosolic Ca2+ concentration ([Ca2+]c) oscillations in mouse islets: simple or mixed. In the mixed pattern, rapid oscillations are superimposed on slow ones. In the present study, we examined the role of the membrane potential in the mixed pattern and the impact of this pattern on insulin release. Simultaneous measurement of [Ca2+]c and insulin release from single islets revealed that mixed [Ca2+]c oscillations triggered synchronous oscillations of insulin secretion. Simultaneous recordings of membrane potential in a single beta-cell within an islet and of [Ca2+]c in the whole islet demonstrated that the mixed pattern resulted from compound bursting (i.e., clusters of membrane potential oscillations separated by prolonged silent intervals) that was synchronized in most beta-cells of the islet. Each slow [Ca2+]c increase during mixed oscillations was due to a progressive summation of rapid oscillations. Digital image analysis confirmed the good synchrony between subregions of an islet. By contrast, islets from sarco(endo)plasmic reticulum Ca2+-ATPase isoform 3 (SERCA3)-knockout mice did not display typical mixed [Ca2+]c oscillations in response to glucose. This results from a lack of progressive summation of rapid oscillations and from altered spontaneous electrical activity, i.e., lack of compound bursting, and membrane potential oscillations characterized by lower-frequency but larger-depolarization phases than observed in SERCA3+/+ beta-cells. We conclude that glucose-induced mixed [Ca2+]c oscillations result from compound bursting in all beta-cells of the islet. Disruption of SERCA3 abolishes mixed [Ca2+]c oscillations and augments beta-cell depolarization. This latter observation indicates that the endoplasmic reticulum participates in the control of the beta-cell membrane potential during glucose stimulation.

Prolonged culture in low-glucose concentrations (

When fed a high-energy (HE) diet, diabetes-prone (DP) Psammomys obesus develop type 2 diabetes with altered glucose-stimulated insulin secretion (GSIS). Beta-cell stimulus-secretion coupling was investigated in islets isolated from DP P. obesus fed a low-energy (LE) diet (DP-LE) and after 5 days on a HE diet (DP-HE). DP-LE islets cultured overnight in 5 mmol/l glucose displayed glucose dose-dependent increases in NAD(P)H, mitochondrial membrane potential, ATP/(ATP + ADP) ratio, cytosolic calcium concentration ([Ca(2+)](c)), and insulin secretion. In comparison, DP-HE islets cultured overnight in 10 mmol/l glucose were 80% degranulated and displayed an increased sensitivity to glucose at the level of glucose metabolism, [Ca(2+)](c), and insulin secretion. These changes in DP-HE islets were only marginally reversed after culture in 5 mmol/l glucose and were not reproduced in DP-LE islets cultured overnight in 10 mmol/l glucose, except for the 75% degranulation. Diabetes-resistant P. obesus remain normoglycemic on HE diet. Their beta-cell stimulus-secretion coupling was similar to that of DP-LE islets, irrespective of the type of diet. Thus, islets from diabetic P. obesus display an increased sensitivity to glucose at the level of glucose metabolism and a profound beta-cell degranulation, both of which may affect their in vivo GSIS.

The mechanisms of control of glucagon secretion are largely debated. In particular, the paracrine role of somatostatin (SST) is unclear. We studied its role in the control of glucagon secretion by glucose and KATP channel blockers, using perifused islets and the in situ perfused pancreas. The involvement of SST was evaluated by comparing glucagon release of control tissue or tissue without paracrine influence of SST (pertussis toxin-treated islets, or islets or pancreas from Sst-/- mice). We show that removal of the paracrine influence of SST suppresses the ability of KATP channel blockers or KATP channel ablation to inhibit glucagon release, suggesting that in control islets, the glucagonostatic effect of KATP channel blockers/ablation is fully mediated by SST. By contrast, the glucagonostatic effect of glucose in control islets is mainly independent of SST for low glucose concentrations (0-7 mmol/L) but starts to involve SST for high concentrations of the sugar (15-30 mmol/L). This demonstrates that the glucagonostatic effect of glucose only partially depends on SST. Real-time quantitative PCR and pharmacological experiments indicate that the glucagonostatic effect of SST is mediated by two types of SST receptors, SSTR2 and SSTR3. These results suggest that alterations of the paracrine influence of SST will affect glucagon release. [ABSTRACT FROM AUTHOR]

The glucose stimulation of insulin secretion by pancreatic ß- cells depends on increased production of metabolic coupling factors, among which changes in NADPH and ROS (reactive oxygen species) may alter the glutathione redox state (EGSH) and signal through changes in thiol oxidation. However, whether nutrients affect EGSH in ß-cell subcellular compartments is unknown. Using redox-sensitive GFP2 fused to glutaredoxin 1 and its mitochondria-targeted form, we studied the acute nutrient regulation of EGSH in the cytosol/nucleus or the mitochondrial matrix of rat islet cells. These probes were mainly expressed in ß-cells and reacted to low concentrations of exogenous H2O2 and menadione. Under control conditions, cytosolic/nuclear EGSH was close to -300 mV and unaffected by glucose (from 0 to 30 mM). In comparison, mitochondrial EGSH was less negative and rapidly regulated by glucose and other nutrients, ranging from -280mVin the absence of glucose to -299mVin 30 mM glucose. These changes were largely independent from changes in intracellular Ca2+ concentration and in mitochondrial pH. They were unaffected by overexpression of SOD2 (superoxide dismutase 2) and mitochondria-targeted catalase, but were inversely correlated with changes in NAD(P)H autofluorescence, suggesting that they indirectly resulted from increased NADPH availability rather than from changes in ROS concentration. Interestingly, the opposite regulation of mitochondrial EGSH and NAD(P)H autofluorescence by glucose was also observed in human islets isolated from two donors. In conclusion, the present study demonstrates that glucose and other nutrients acutely reduce mitochondrial, but not cytosolic/nuclear, EGSH in pancreatic ß- cells under control conditions. [ABSTRACT FROM AUTHOR]

Aim/Hypothesis: Rat pancreatic islet cell apoptosis is minimal after prolonged culture in 10 mmol/l glucose (G10), largely increased in 5 mmol/l glucose (G5) and moderately increased in 30 mmol/l glucose (G30). This glucose-dependent asymmetric V-shaped profile is preceded by parallel changes in the mRNA levels of oxidative stress-response genes like Metallothionein 1a (Mt1a). In this study, we tested the effect of ZnCl2, a potent inducer of Mt1a, on apoptosis, mitochondrial oxidative stress and alterations of glucose-induced insulin secretion (GSIS) induced by prolonged exposure to low and high vs. intermediate glucose concentrations. Methods: Male Wistar rat islets were cultured in RPMI medium. Islet gene mRNA levels were measured by RTq-PCR. Apoptosis was quantified by measuring islet cytosolic histone-associated DNA fragments and the percentage of TUNEL-positive β-cells. Mitochondrial thiol oxidation was measured in rat islet cell clusters expressing "redox sensitive GFP" targeted to the mitochondria (mt-roGFP1). Insulin secretion was measured by RIA. Results: As observed for Mt1a mRNA levels, β-cell apoptosis and loss of GSIS, culture in either G5 or G30 vs. G10 significantly increased mt-roGFP1 oxidation. While TPEN decreased Mt1a/2a mRNA induction by G5, addition of 50-100 μM ZnCl2 to the culture medium strongly increased Mt1a/2a mRNA and protein levels, reduced early mt-roGFP oxidation and significantly decreased late βb-cell apoptosis after prolonged culture in G5 or G30 vs. G10. It did not, however, prevent the loss of GSIS under these culture conditions. Conclusion: ZnCl2 reduces mitochondrial oxidative stress and improves rat β-cell survival during culture in the presence of low and high vs. intermediate glucose concentrations without improving their acute GSIS. [ABSTRACT FROM AUTHOR]

Background: Glucose increases the expression of glycolytic enzymes and other hypoxia-response genes in pancreatic betacells. Here, we tested whether this effect results from the activation of Hypoxia-Inducible-factors (HIF) 1 and 2 in a hypoxiadependent manner. Methodology/Principal Findings: Isolated rat islets and insulin-secreting INS-1E cells were stimulated with nutrients at various pO2 values or treated with the HIF activator CoCl2. HIF-target gene mRNA levels and HIF subunit protein levels were measured by real-time RT-PCR, Western Blot and immunohistochemistry. The formation of pimonidazole-protein adducts was used as an indicator of hypoxia. In INS-1E and islet beta-cells, glucose concentration-dependently stimulated formation of pimonidazole-protein adducts, HIF1 and HIF2 nuclear expression and HIF-target gene mRNA levels to a lesser extent than CoCl2 or a four-fold reduction in pO2. Islets also showed signs of HIF activation in diabetic Leprdb/db but not non-diabetic Leprdb/+ mice. In vitro, these glucose effects were reproduced by nutrient secretagogues that bypass glycolysis, and were inhibited by a three-fold increase in pO2 or by inhibitors of Ca2+ influx and insulin secretion. In INS-1E cells, small interfering RNA-mediated knockdown of Hif1α and Hif2α, alone or in combination, indicated that the stimulation of glycolytic enzyme mRNA levels depended on both HIF isoforms while the vasodilating peptide adrenomedullin was a HIF2-specific target gene. Conclusions/Significance: Glucose-induced O2 consumption creates an intracellular hypoxia that activates HIF1 and HIF2 in rat beta-cells, and this glucose effect contributes, together with the activation of other transcription factors, to the glucose stimulation of expression of some glycolytic enzymes and other hypoxia response genes. [ABSTRACT FROM AUTHOR]

Objective: Sarco-endoplasmic reticulum Ca(2+)-ATPase 2b (SERCA2b) and SERCA3 pump Ca(2+) in the endoplasmic reticulum (ER) of pancreatic β-cells. We studied their role in the control of the free ER Ca(2+) concentration ([Ca(2+)](ER)) and the role of SERCA3 in the control of insulin secretion and ER stress.Research Design and Methods: β-Cell [Ca(2+)](ER) of SERCA3(+/+) and SERCA3(-/-) mice was monitored with an adenovirus encoding the low Ca(2+)-affinity sensor D4 addressed to the ER (D4ER) under the control of the insulin promoter. Free cytosolic Ca(2+) concentration ([Ca(2+)](c)) and [Ca(2+)](ER) were simultaneously recorded. Insulin secretion and mRNA levels of ER stress genes were studied.Results: Glucose elicited synchronized [Ca(2+)](ER) and [Ca(2+)](c) oscillations. [Ca(2+)](ER) oscillations were smaller in SERCA3(-/-) than in SERCA3(+/+) β-cells. Stimulating cell metabolism with various [glucose] in the presence of diazoxide induced a similar dose-dependent [Ca(2+)](ER) rise in SERCA3(+/+) and SERCA3(-/-) β-cells. In a Ca(2+)-free medium, glucose moderately raised [Ca(2+)](ER) from a highly buffered cytosolic Ca(2+) pool. Increasing [Ca(2+)](c) with high [K] elicited a [Ca(2+)](ER) rise that was larger but more transient in SERCA3(+/+) than SERCA3(-/-) β-cells because of the activation of a Ca(2+) release from the ER in SERCA3(+/+) β-cells. Glucose-induced insulin release was larger in SERCA3(-/-) than SERCA3(+/+) islets. SERCA3 ablation did not induce ER stress.Conclusions: [Ca(2+)](c) and [Ca(2+)](ER) oscillate in phase in response to glucose. Upon [Ca(2+)](c) increase, Ca(2+) is taken up by SERCA2b and SERCA3. Strong Ca(2+) influx triggers a Ca(2+) release from the ER that depends on SERCA3. SERCA3 deficiency neither impairs Ca(2+) uptake by the ER upon cell metabolism acceleration and insulin release nor induces ER stress. [ABSTRACT FROM AUTHOR]

OBJECTIVE-Sarco-endoplasmic reticulum Ca2+-ATPase 2b (SERCA2b) and SERCA3 pump Ca2+ in the endoplasmic reticulum (ER) of pancreatic β-cells. We studied their role in the control of the free ER Ca2+ concentration ([Ca2+]ER) and the role of SERCA3 in the control of insulin secretion and ER stress. RESEARCH DESIGN AND METHODS-β-Cell [Ca2+]ER of SERCA3+/+ and SERCA3-/- mice was monitored with an adenovirus encoding the low Ca2+-affinity sensor D4 addressed to the ER (D4ER) under the control of the insulin promoter. Free cytosolic Ca2+ concentration ([Ca2+]c) and [Ca2+]ER were simultaneously recorded. Insulin secretion and mRNA levels of ER stress genes were studied. RESULTS-Glucose elicited synchronized [Ca2+]ER and [Ca2+]c oscillations. [Ca2+]ER oscillations were smaller in SERCA3-/- than in SERCA3+/+ β-cells. Stimulating cell metabolism with various [glucose] in the presence of diazoxide induced a similar dosedependent [Ca2+]ER rise in SERCA3+/+ and SERCA3-/- β-cells. In a Ca2+-free medium, glucose moderately raised [Ca2+]ER from a highly buffered cytosolic Ca2+ pool. Increasing [Ca2+]c with high [K] elicited a [Ca2+]ER rise that was larger but more transient in SERCA3+/+ than SERCA3-/- β-cells because of the activation of a Ca2+ release from the ER in SERCA3+/+ β-cells. Glucose-induced insulin release was larger in SERCA32/2 than SERCA3+/+ islets. SERCA3 ablation did not induce ER stress. CONCLUSIONS-[Ca2+]c and [Ca2+]ER oscillate in phase in response to glucose. Upon [Ca2+]c increase, Ca2+ is taken up by SERCA2b and SERCA3. Strong Ca2+ influx triggers a Ca2+ release from the ER that depends on SERCA3. SERCA3 deficiency neither impairs Ca2+ uptake by the ER upon cell metabolism acceleration and insulin release nor induces ER stress. [ABSTRACT FROM AUTHOR]

Type 1 and type 2 diabetes are characterized by progressive β-cell failure. Apoptosis is probably the main form of β-cell death in both forms of the disease. It has been suggested that the mechanisms leading to nutrient- and cytokine-induced β-cell death in type 2 and type 1 diabetes, respectively, share the activation of a final common pathway involving interleukin (IL)-1β, nuclear factor (NF)-KB, and Fas. We review herein the similarities and differences between the mechanisms of β-cell death in type 1 and type 2 diabetes. In the insulitis lesion in type 1 diabetes, invading immune cells produce cytokines, such as IL-113, tumor necrosis factor (TNF)-α, and interferon (IFN)-γ. IL-1β and/or TNF-α plus IFN-γ induce β-cell apoptosis via the activation of β-cell gene networks under the control of the transcription factors NF-κB and STAT-1. NF-κB activation leads to production of nitric oxide (NO) and chemokines and depletion of endoplasmic reticulum (ER) calcium. The execution of β-cell death occurs through activation of mitogen-activated protein kinases, via triggering of ER stress and by the release of mitochondrial death signals. Chronic exposure to elevated levels of glucose and free fatty acids (FFAs) causes β-cell dysfunction and may induce β-cell apoptosis in type 2 diabetes. Exposure to high glucose has dual effects, triggering initially "glucose hypersensitization" and later apoptosis, via different mechanisms. High glucose, however, does not induce or activate IL-1β, NF-κB, or inducible nitric oxide synthase in rat or human β-cell in vitro or in vivo in Psammomys obesus. FFAs may cause β-cell apoptosis via ER stress, which is NF-κB and NO independent. Thus, cytokines and nutrients trigger β-cell death by fundamentally different mechanisms, namely an NF-κB-dependent mechanism that culminates in caspase-3 activation for cytokines and an NF-κB-independent mechanism for nutrients. This argues against a unifying hypothesis for the mechanisms of β-cell death in type 1 and type 2 diabetes and suggests that different approaches will be required to prevent β-cell death in type 1 and type 2 diabetes. Diabetes 54 (Suppl. 2): S97-S107, 2005 [ABSTRACT FROM AUTHOR]

That oscillations of the cytoplasmic free Ca(2+) concentration ([Ca(2+)](i)) in beta-cells induce oscillations of insulin secretion is not disputed, but whether metabolism-driven oscillations of secretion can occur in the absence of [Ca(2+)](i) oscillations is still debated. Because this possibility is based partly on the results of experiments using islets from aged, hyperglycemic, hyperinsulinemic ob/ob mice, we compared [Ca(2+)](i) and insulin secretion patterns of single islets from 4- and 10-month-old, normal NMRI mice to those of islets from 7- and 10-month-old ob/ob mice (Swedish colony) and their lean littermates. The responses were subjected to cluster analysis to identify significant peaks. Control experiments without islets and with a constant insulin concentration were run to detect false peaks. Both ob/ob and NMRI islets displayed large synchronous oscillations of [Ca(2+)](i) and insulin secretion in response to repetitive depolarizations with 30 mmol/l K(+) in the presence of 0.1 mmol/l diazoxide and 12 mmol/l glucose. Continuous depolarization with high K(+) steadily elevated [Ca(2+)](i) in all types of islets, with no significant oscillation, and caused a biphasic insulin response. In islets from young (4-month-old) NMRI mice and 7-month-old lean mice, the insulin profile did not show significant peaks when [Ca(2+)](i) was stable. In contrast, two or more peaks were detected over 20 min in the response of most ob/ob islets. Similar insulin peaks appeared in the insulin response of 10-month-old lean and NMRI mice. However, the size of the insulin peaks detected in the presence of stable [Ca(2+)](i) was small, so that no more than 10-13% of total insulin secretion occurred in a pulsatile manner. In conclusion, insulin secretion does not oscillate when [Ca(2+)](i) is stably elevated in beta-cells from young normal mice. Some oscillations are observed in aged mice and are seen more often in ob/ob islets. These fluctuations of the insulin secretion rate at stably elevated [Ca(2+)](i), however, are small compared with the large oscillations induced by [Ca(2+)](i) oscillations in beta-cells. [ABSTRACT FROM AUTHOR]

The mechanisms driving the pulsatility of insulin secretion in vivo and in vitro are still unclear. Because glucose metabolism and changes in cytosolic free Ca(2+) ([Ca(2+)](c)) in beta-cells play a key role in the control of insulin secretion, and because oscillations of these two factors have been observed in single isolated islets and beta-cells, pulsatile insulin secretion could theoretically result from [Ca(2+)](c) or metabolism oscillations. We could not detect metabolic oscillations independent from [Ca(2+)](c) changes in beta-cells, and imposed metabolic oscillations were poorly effective in inducing oscillations of secretion when [Ca(2+)](c) was kept stable, which suggests that metabolic oscillations are not the direct regulator of the oscillations of secretion. By contrast, tight temporal and quantitative correlations between the changes in [Ca(2+)](c) and insulin release strongly suggest that [Ca(2+)](c) oscillations are the direct drivers of insulin secretion oscillations. Metabolism may play a dual role, inducing [Ca(2+)](c) oscillations (via changes in ATP-sensitive K(+) channel activity and membrane potential) and amplifying the secretory response by increasing the efficiency of Ca(2+) on exocytosis. The mechanisms underlying the oscillations of insulin secretion by the isolated pancreas and those observed in vivo remain elusive. It is not known how the functioning of distinct islets is synchronized, and the possible role of intrapancreatic ganglia in this synchronization requires confirmation. That pulsatile insulin secretion is beneficial in vivo, by preventing insulin resistance, is suggested by the greater hypoglycemic effect of exogenous insulin when it is infused in a pulsatile rather than continuous manner. The observation that type 2 diabetic patients have impaired pulsatile insulin secretion has prompted the suggestion that such dysregulation contributes to the disease and justifies the efforts toward understanding of the mechanism underlying the pulsatility of insulin secretion both in vitro and in vivo. [ABSTRACT FROM AUTHOR]

Rapid and sustained stimulation of beta-cells with glucose induces biphasic insulin secretion. The two phases appear to reflect a characteristic of stimulus-secretion coupling in each beta-cell rather than heterogeneity in the time-course of the response between beta-cells or islets. There is no evidence indicating that biphasic secretion can be attributed to an intrinsically biphasic metabolic signal. In contrast, the biphasic rise in cytoplasmic Ca(2+) concentration ([Ca(2+)](i)) induced by glucose is important to shape the two phases of secretion. The first phase requires a rapid and marked elevation of [Ca(2+)](i) and corresponds to the release of insulin granules from a limited pool. The magnitude of the second phase is determined by the elevation of [Ca(2+)](i), but its development requires production of another signal. This signal corresponds to the amplifying action of glucose and may serve to replenish the pool of granules that are releasable at the prevailing [Ca(2+)](i). The species characteristics of biphasic insulin secretion and its perturbations in pathological situations are discussed. [ABSTRACT FROM AUTHOR]

Investigates whether rat pancreatic beta cells can adapt to the increase in insulin demand during glucose or lipid infusion. Islet gene expression; Quantification of beta cell mass and mitotic rate; Assessment of postinfusion peripheral insulin clearance.

Aims: Whether H2O2 contributes to the glucose-dependent stimulation of insulin secretion (GSIS) by pancreatic β cells is highly controversial. We used two H2O2-sensitive probes, roGFP2-Orp1 (reduction/oxidation-sensitive enhanced green fluorescent protein fused to oxidant receptor peroxidase 1) and HyPer (hydrogen peroxide sensor) with its pH-control SypHer, to test the acute effects of glucose, monomethyl succinate, leucine with glutamine, and α-ketoisocaproate on β cell cytosolic and mitochondrial H2O2 concentrations. We then tested the effects of low H2O2 and menadione concentrations on insulin secretion. Results: RoGFP2-Orp1 was more sensitive than HyPer to H2O2 (response at 2–5 vs. 10 μM) and less pH-sensitive. Under control conditions, stimulation with glucose reduced mitochondrial roGFP2-Orp1 oxidation without affecting cytosolic roGFP2-Orp1 and HyPer fluorescence ratios, except for the pH-dependent effects on HyPer. However, stimulation with glucose decreased the oxidation of both cytosolic probes by 15 μM exogenous H2O2. The glucose effects were not affected by overexpression of catalase, mitochondrial catalase, or superoxide dismutase 1 and 2. They followed the increase in NAD(P)H autofluorescence, were maximal at 5 mM glucose in the cytosol and 10 mM glucose in the mitochondria, and were partly mimicked by the other nutrients. Exogenous H2O2 (1–15 μM) did not affect insulin secretion. By contrast, menadione (1–5 μM) did not increase basal insulin secretion but reduced the stimulation of insulin secretion by 20 mM glucose. Innovation: Subcellular changes in β cell H2O2 levels are better monitored with roGFP2-Orp1 than HyPer/SypHer. Nutrients acutely lower mitochondrial H2O2 levels in β cells and promote degradation of exogenously supplied H2O2 in both cytosolic and mitochondrial compartments. Conclusion: The GSIS occurs independently of a detectable increase in β cell cytosolic or mitochondrial H2O2 levels. [ABSTRACT FROM AUTHOR]