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Aims/hypothesis: The rapid remission of type 2 diabetes by a diet very low in energy correlates with a marked improvement in glucose-stimulated insulin secretion (GSIS), emphasising the role of beta cell dysfunction in the early stages of the disease. In search of novel mechanisms of beta cell dysfunction after long-term exposure to mild to severe glucotoxic conditions, we extensively characterised the alterations in insulin secretion and upstream coupling events in human islets cultured for 1–3 weeks at ~5, 8, 10 or 20 mmol/l glucose and subsequently stimulated by an acute stepwise increase in glucose concentration. Methods: Human islets from 49 non-diabetic donors (ND-islets) and six type 2 diabetic donors (T2D-islets) were obtained from five isolation centres. After shipment, the islets were precultured for 3–7 days in RPMI medium containing ~5 mmol/l glucose and 10% (vol/vol) heat-inactivated FBS with selective islet picking at each medium renewal. Islets were then cultured for 1–3 weeks in RPMI containing ~5, 8, 10 or 20 mmol/l glucose before measurement of insulin secretion during culture, islet insulin and DNA content, beta cell apoptosis and cytosolic and mitochondrial glutathione redox state, and assessment of dynamic insulin secretion and upstream coupling events during acute stepwise stimulation with glucose [NAD(P)H autofluorescence, ATP/(ATP+ADP) ratio, electrical activity, cytosolic Ca2+ concentration ([Ca2+]c)]. Results: Culture of ND-islets for 1–3 weeks at 8, 10 or 20 vs 5 mmol/l glucose did not significantly increase beta cell apoptosis or oxidative stress but decreased insulin content in a concentration-dependent manner and increased beta cell sensitivity to subsequent acute stimulation with glucose. Islet glucose responsiveness was higher after culture at 8 or 10 vs 5 mmol/l glucose and markedly reduced after culture at 20 vs 5 mmol/l glucose. In addition, the [Ca2+]c and insulin secretion responses to acute stepwise stimulation with glucose were no longer sigmoid but bell-shaped, with maximal stimulation at 5 or 10 mmol/l glucose and rapid sustained inhibition above that concentration. Such paradoxical inhibition was, however, no longer observed when islets were acutely depolarised by 30 mmol/l extracellular K+. The glucotoxic alterations of beta cell function were fully reversible after culture at 5 mmol/l glucose and were mimicked by pharmacological activation of glucokinase during culture at 5 mmol/l glucose. Similar results to those seen in ND-islets were obtained in T2D-islets, except that their rate of insulin secretion during culture at 8 and 20 mmol/l glucose was lower, their cytosolic glutathione oxidation increased after culture at 8 and 20 mmol/l glucose, and the alterations in GSIS and upstream coupling events were greater after culture at 8 mmol/l glucose. Conclusions/interpretation: Prolonged culture of human islets under moderate to severe glucotoxic conditions markedly increased their glucose sensitivity and revealed a bell-shaped acute glucose response curve for changes in [Ca2+]c and insulin secretion, with maximal stimulation at 5 or 10 mmol/l glucose and rapid inhibition above that concentration. This novel glucotoxic alteration may contribute to beta cell dysfunction in type 2 diabetes independently from a detectable increase in beta cell apoptosis. [ABSTRACT FROM AUTHOR]

Aims/hypothesis: Nicotinamide nucleotide transhydrogenase (NNT) is involved in mitochondrial NADPH production and its spontaneous inactivating mutation (NntTr [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 NntTr from that of other genetic variants between C57BL/6J and C57BL/6N mice on mitochondrial glutathione redox state (EGSH), glucose-stimulated insulin secretion (GSIS) and glucose tolerance. Methods: Male and female N5BL/6J mice that express wild-type Nnt (NntWT) or NntTr (N5-WT and N5-Tr mice) on the C57BL/6J genetic background were obtained by crossing N5BL/6J NntWT/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 EGSH 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) EGSH 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 NntWT genotype in C57BL/6 mice does not provide proof that their glucose tolerance is as good as in C57BL/6N mice. [ABSTRACT FROM AUTHOR]

Help for hypertrophy: One way that cardiomyocytes respond to injury or stress is by increasing in size (hypertrophy). Montiel et al. studied the role of oxidative stress in myocardial hypertrophy. They saw that the water channel aquaporin-1 (AQP1) was increased in mouse and human hypertrophic heart tissue and that AQP1 regulated hydrogen peroxide transport within myocytes. Deletion or pharmacological blockade of AQP1 abrogated hypertrophy in response to different stressors in cells and in a mouse model of angiotensin II–induced cardiac hypertrophy, identifying a potential therapeutic target for hypertrophic cardiomyopathy. Pathological remodeling of the myocardium has long been known to involve oxidant signaling, but strategies using systemic antioxidants have generally failed to prevent it. We sought to identify key regulators of oxidant-mediated cardiac hypertrophy amenable to targeted pharmacological therapy. Specific isoforms of the aquaporin water channels have been implicated in oxidant sensing, but their role in heart muscle is unknown. RNA sequencing from human cardiac myocytes revealed that the archetypal AQP1 is a major isoform. AQP1 expression correlates with the severity of hypertrophic remodeling in patients with aortic stenosis. The AQP1 channel was detected at the plasma membrane of human and mouse cardiac myocytes from hypertrophic hearts, where it colocalized with NADPH oxidase-2 and caveolin-3. We show that hydrogen peroxide (H2O2), produced extracellularly, is necessary for the hypertrophic response of isolated cardiac myocytes and that AQP1 facilitates the transmembrane transport of H2O2 through its water pore, resulting in activation of oxidant-sensitive kinases in cardiac myocytes. Structural analysis of the amino acid residues lining the water pore of AQP1 supports its permeation by H2O2. Deletion of Aqp1 or selective blockade of the AQP1 intrasubunit pore inhibited H2O2 transport in mouse and human cells and rescued the myocyte hypertrophy in human induced pluripotent stem cell–derived engineered heart muscle. Treatment of mice with a clinically approved AQP1 inhibitor, Bacopaside, attenuated cardiac hypertrophy. We conclude that cardiac hypertrophy is mediated by the transmembrane transport of H2O2 by the water channel AQP1 and that inhibitors of AQP1 represent new possibilities for treating hypertrophic cardiomyopathies. [ABSTRACT FROM AUTHOR]

This work reports on synchrotron X-ray fluorescence (SR-XRF) imaging of vitrified islets of Langerhans in two-dimensional and computed tomography (CT) mode and serves as a proof-of-principle for the preparation and SR-XRF imaging of cryoloops in a cryogenic environment. Selection of suited cryoloops and cryoprotectant solution enabled vitrification of the islets. The SR-XRF experimental setup was adapted with a cryogenic gas stream, enabling analysis of cryofrozen islets and a dual silicon drift detector (SDD) configuration, providing an increased solid angle for XRF collection. Element distributions of K, Ca, Fe and Zn within single, cryofrozen islets could be obtained with 500 nm spatial resolution using X-ray compound refractive lenses (CRLs) with an adaptive phase plate. Titanium, present as a trace element in the cryoloops, was used as a marker signal to efficiently define the XRF scan area. Preliminary 2D cryo-analysis of wild type (n = 1) and Mt1-Mt2-knock-out (n = 1) islets under low/high glucose conditions did not reveal changes in the distribution and concentration of Ca, Fe and Zn. Cryotomography XRF scans indicated aspherical islets within the cryoloop, likely due to capillary effects in the cryoloop before plunge freezing. The presence of diffracting ice crystals hampered CT reconstruction, indicating the need for further development of a routine workflow ensuring perfect vitrification. Collectively, this novel multidisciplinary approach proved to be valid and can be used for future studies to assess elemental compositions of islets of Langerhans or other cell clusters/tissues under different pathophysiological conditions. [ABSTRACT FROM AUTHOR]

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 Ca2+ concentration with Fura-2 leakage resistant (Fura-2 LR), cytosolic free Zn2+ 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 Ca2+ concentration and NAD(P)H levels, or intracellular Zn2+ 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. [ABSTRACT FROM AUTHOR]

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]

Like all the cells of an organism, pancreatic ß-cells originate from embryonic stem cells through a complex cellular process termed differentiation. Differentiation involves the coordinated and tightly controlled activation/repression of specific effectors and gene clusters in a time-dependent fashion thereby giving rise to particular morphological and functional cellular features. Interestingly, cellular differentiation is not a unidirectional process. Indeed, growing evidence suggests that under certain conditions, mature ß-cells can lose, to various degrees, their differentiated phenotype and cellular identity and regress to a less differentiated or a precursor-like state. This concept is termed dedifferentiation and has been proposed, besides cell death, as a contributing factor to the loss of functional ß-cell mass in diabetes. ß-cell dedifferentiation involves: (1) the downregulation of ß-cell-enriched genes, including key transcription factors, insulin, glucose metabolism genes, protein processing and secretory pathway genes; (2) the concomitant upregulation of genes suppressed or expressed at very low levels in normal ß-cells, the ß-cell forbidden genes; and (3) the likely upregulation of progenitor cell genes. These alterations lead to phenotypic reconfiguration of ß-cells and ultimately defective insulin secretion. While the major role of glucotoxicity in ß-cell dedifferentiation is well established, the precise mechanisms involved are still under investigation. This review highlights the identified molecular mechanisms implicated in ß-cell dedifferentiation including oxidative stress, endoplasmic reticulum (ER) stress, inflammation and hypoxia. It discusses the role of Foxo1, Myc and inhibitor of differentiation proteins and underscores the emerging role of non-coding RNAs. Finally, it proposes a novel hypothesis of ß-cell dedifferentiation as a potential adaptive mechanism to escape cell death under stress conditions. [ABSTRACT FROM AUTHOR]

The ability of certain cancer cells to maintain a highly reduced intracellular environment is correlated with aggressiveness and drug resistance. Since the glutathione (GSH) and thioredoxin (TRX) systems cooperate to a tight regulation of ROS in cell physiology, and to a stimulation of tumour initiation and progression, modulation of the GSH and TRX pathways are emerging as new potential targets in cancer. In vivo methods to assess changes in tumour redox status are critically needed to assess the relevance of redox-targeted agents. The current study assesses in vitro and in vivo biomarkers of tumour redox status in response to treatments targeting the GSH and TRX pathways, by comparing cytosolic and mitochondrial redox nitroxide electron paramagnetic resonance (EPR) probes, and cross-validation with redox dynamic fluorescent measurement. For that purpose, the effect of the GSH modulator buthionine sulfoximine (BSO) and of the TRX reductase inhibitor auranofin were measured in vitro using both cytosolic and mitochondrial EPR and roGFP probes in breast and cervical cancer cells. In vivo, mice bearing breast or cervical cancer xenografts were treated with the GSH or TRX modulators and monitored using the mito-TEMPO spin probe. Our data highlight the importance of using mitochondria-targeted spin probes to assess changes in tumour redox status induced by redox modulators. Further in vivo validation of the mito-tempo spin probe with alternative in vivo methods should be considered, yet the spin probe used in vivo in xenografts demonstrated sensitivity to the redox status modulators. [ABSTRACT FROM AUTHOR]

Aims/hypothesis: Hypoxia may contribute to beta cell failure in type 2 diabetes and islet transplantation. The adaptive unfolded protein response (UPR) is required for endoplasmic reticulum (ER) homeostasis. Here we investigated whether or not hypoxia regulates the UPR in beta cells and the role the adaptive UPR plays during hypoxic stress. Methods: Mouse islets and MIN6 cells were exposed to various oxygen (O) tensions. DNA-damage inducible transcript 3 (DDIT3), hypoxia-inducible transcription factor (HIF)1α and HSPA5 were knocked down using small interfering (si)RNA; Hspa5 was also overexpressed. db/db mice were used. Results: Hypoxia-response genes were upregulated in vivo in the islets of diabetic, but not prediabetic, db/db mice. In isolated mouse islets and MIN6 cells, O deprivation (1-5% vs 20%; 4-24 h) markedly reduced the expression of adaptive UPR genes, including Hspa5, Hsp90b1, Fkbp11 and spliced Xbp1. Coatomer protein complex genes ( Copa, Cope, Copg [also known as Copg1], Copz1 and Copz2) and ER-to-Golgi protein trafficking were also reduced, whereas apoptotic genes ( Ddit3, Atf3 and Trb3 [also known as Trib3]), c-Jun N-terminal kinase (JNK) phosphorylation and cell death were increased. Inhibition of JNK, but not HIF1α, restored adaptive UPR gene expression and ER-to-Golgi protein trafficking while protecting against apoptotic genes and cell death following hypoxia. DDIT3 knockdown delayed the loss of the adaptive UPR and partially protected against hypoxia-induced cell death. The latter response was prevented by HSPA5 knockdown. Finally, Hspa5 overexpression significantly protected against hypoxia-induced cell death. Conclusions/interpretation: Hypoxia inhibits the adaptive UPR in beta cells via JNK and DDIT3 activation, but independently of HIF1α. Downregulation of the adaptive UPR contributes to reduced ER-to-Golgi protein trafficking and increased beta cell death during hypoxic stress. [ABSTRACT FROM AUTHOR]

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 auto-fluorescence, 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 gluco-toxicity 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. from an effect of glucose per se. [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]

The female steroid, 17β-estradiol (E2), is important for pancreatic β-cell function and acts via at least three estrogen receptors (ER), ERα, ERβ, and the G-protein coupled ER (GPER). Using a pancreas-specific ERα knockout mouse generated using the Cre-lox-P system and a Pdx1-Cre transgenic line (PERαKO−/−), we previously reported that islet ERα suppresses islet glucolipotoxicity and prevents β-cell dysfunction induced by high fat feeding. We also showed that E2 acts via ERα to prevent β-cell apoptosis in vivo. However, the contribution of the islet ERα to β-cell survival in vivo, without the contribution of ERα in other tissues is still unclear. Using the PERαKO−/− mouse, we show that ERα mRNA expression is only decreased by 20% in the arcuate nucleus of the hypothalamus, without a parallel decrease in the VMH, making it a reliable model of pancreas-specific ERα elimination. Following exposure to alloxan-induced oxidative stress in vivo, female and male PERαKO−/− mice exhibited a predisposition to β-cell destruction and insulin deficient diabetes. In male PERαKO−/− mice, exposure to E2 partially prevented alloxan-induced β-cell destruction and diabetes. ERα mRNA expression was induced by hyperglycemia in vivo in islets from young mice as well as in cultured rat islets. The induction of ERα mRNA by hyperglycemia was retained in insulin receptor-deficient β-cells, demonstrating independence from direct insulin regulation. These findings suggest that induction of ERα expression acts to naturally protect β-cells against oxidative injury. [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]

Endoplasmic reticulum (ER) homeostasis alteration contributes to pancreatic β-cell dysfunction and death and favors the development of diabetes. In this study, we demonstrate that HDLs protect β-cells against ER stress induced by thapsigargin, cyclopiazonic acid, palmitate, insulin overexpression, and high glucose concentrations. ER stress marker induction and ER morphology disruption mediated by these stimuli were inhibited by HDLs. Using a temperature-sensitive viral glycoprotein folding mutant, we show that HDLs correct impaired protein trafficking and folding induced by thapsigargin and palmitate. The ability of HDLs to protect β-cells against ER stress was inhibited by brefeldin A, an ER to Golgi trafficking blocker. These results indicate that HDLs restore ER homeostasis in response to ER stress, which is required for their ability to promote β-cell survival. This study identifies a cellular mechanism mediating the beneficial effect of HDLs on β-cells against ER stress-inducing factors. [ABSTRACT FROM AUTHOR]

Endoplasmic reticulum (ER) homeostasis alteration contributes to pancreatic β-cell dysfunction and death and favors the development of diabetes. In this study, we demonstrate that HDLs protect β-cells against ER stress induced by thapsigargin, cyclopiazonic acid, palmitate, insulin overexpression, and high glucose concentrations. ER stress marker induction and ER morphology disruption mediated by these stimuli were inhibited by HDLs. Using a temperature-sensitive viral glycoprotein folding mutant, we show that HDLs correct impaired protein trafficking and folding induced by thapsigargin and palmitate. The ability of HDLs to protect β-cells against ER stress was inhibited by brefeldin A, an ER to Golgi trafficking blocker. These results indicate that HDLs restore ER homeostasis in response to ER stress, which is required for their ability to promote β-cell survival. This study identifies a cellular mechanism mediating the beneficial effect of HDLs on β-cells against ER stress-inducing factors. [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]

Trafficking of pancreatic KATP channels to the plasma membrane critically depends on masking the endoplasmic reticulum (ER) retention signals of the SUR1 and Kir6.2 subunits upon their proper assembly into functional hetero-octamers. When expressed in the absence of the partner protein, each subunit might accumulate in the ER and trigger β-cell ER stress and oxidative stress. To test this hypothesis, Kir6.2 localisation, ER ultra-structure and ER-stress- and oxidative-stress-response gene mRNA levels were evaluated in pancreatic endocrine cells from adult wild-type (WT) and Sur1 knockout ( Sur1 -/-) mice. As previously reported, Kir6.2 was mainly expressed on secretory granules and at the plasma membrane of WT islet cells. In contrast, like the ER chaperone calreticulin, Kir6.2 was primarily localised in the rough endoplasmic reticulum (RER) of Sur1 -/- islet cells. ER retention of Kir6.2 was demonstrated (electron microscopy) by a significant increase in the length and Kir6.2 density of RER in Sur1 -/- vs WT islet cells. Despite Kir6.2 retention in RER, Xbp1 mRNA splicing and mRNA levels of preproinsulin and ER-stress-response genes Bip, Edem and Gadd153 were similar in WT and Sur1 -/- islets. However, mRNA levels of the antioxidant enzymes Sod1, Sod2, Gpx2 and catalase were significantly up-regulated in Sur1 -/- islets. Sequestration of Kir6.2 in RER of Sur1 -/- islet cells is thus associated with an increase in RER length and mild oxidative stress without activation of the classical ER stress response. [ABSTRACT FROM AUTHOR]

Alteration of pancreatic β-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 β-cell function are compatible with its proposed role in glucotoxicity. We therefore tested the effects of short-term c-MYC activation on key β-cell stimulus-secretion coupling events in islets isolated from mice expressing a tamoxifen-switchable form of c-MYC in β-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 ([Ca2+]i), and lower glucose-induced [Ca2+]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 H2O2 concentration increased the islet resting [Ca2+]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 β-cells that are better mimicked by overnight exposure to a low H2O2 concentration than by prolonged culture in high glucose. [ABSTRACT FROM AUTHOR]

Task division in multicellular organisms ensures that differentiated cell types produce cell-specific proteins that fulfill tasks for the whole organism. In some cases, the encoded mRNA species is so abundant that it represents a sizeable fraction of total mRNA in the cell. In this study, we have used a probe- and primer-free technique to quantify such abundant mRNA species in order to assess regulatory effects of in vitro and in vivo conditions. As a first example, we were able to quantify the regulation of proinsulin mRNA abundance in β-cells by food intake or by the glucose concentration in tissue culture. The second example of application of this technique is the effect of corticosteroids on growth hormone mRNA in enriched somatrotrophs. It is anticipated that other examples exist in which measurement of very abundant mRNAs in dedicated cells will help to understand biological processes, monitor disease states, or assist biotechnological manufacturing procedures. Diabetes 55:3214-3220, 2006 [ABSTRACT FROM AUTHOR]

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 β-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 β-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+/+ [3-cells. We conclude that glucose-induced mixed [Ca2+]c oscillations result from compound bursting in all β-cells of the islet. Disruption of SERCA3 abolishes mixed [Ca2+]c oscillations and augments β-cell depolarization. This latter observation indicates that the endoplasmic reticulum participates in the control of the β-cell membrane potential during glucose stimulation. [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]

The contribution of Ca2+ release from intracellular stores to the rise in the free cytosolic Ca2+ concentration (Ca2+c) triggered by Ca2+ influx was investigated in mouse pancreatic β-cells. Depolarization of β-cells by 45 mM K+ (in the presence of 15 mM glucose and 0.1 mM diazoxide) evoked two types of [Ca2+c responses: a monotonic and sustained elevation; or a sustained elevation superimposed by a transient [Ca2+ peak (TCP) (40-120 s after the onset of depolarization). Simultaneous measurements of [Ca2+c and voltage-dependent Ca2+ current established that the TCP did not result from a larger Ca2+ current. Abolition of the TCP by thapsigargin and its absence in sarco-endoplasmic reticulum Ca2+-ATPase 3 (SERCA3) knockout mice show that it is caused by Ca2+ mobilization from the endoplasmic reticulum. A TCP could not be evoked by the sole depolarization of β-cells but required a rise in [Ca2+c pointing to a Ca2+-induced Ca2+ release (CICR). This CICR did not involve inositol 1,4,5-trisphosphate (IP3) receptors (IP3Rs) because it was resistant to heparin. Nor did it involve ryanodine receptors (RyRs) because it persisted after blockade of RyRs with ryanodine, and was not mimicked by caffeine, a RyR agonist. Moreover, RyR1 and RyR2 mRNA were not found and RyR3 mRNA was only slightly expressed in purified β-cells. A CICR could also be detected in a limited number of cells in response to glucose. Our data demonstrate, for the first time in living cells, the existence of an atypical CICR that is independent from the IP3R and the RyR. This CICR is prominent in response to a supraphysiological stimulation with high K+, but plays little role in response to glucose in non-obese... [ABSTRACT FROM AUTHOR]

Two sarcoendoplasmic reticulum Ca(2+)-ATPases, SERCA3 and SERCA2b, are expressed in pancreatic islets. Immunocytochemistry showed that SERCA3 is restricted to beta-cells in the mouse pancreas. Control and SERCA3-deficient mice were used to evaluate the role of SERCA3 in beta-cell cytosolic-free Ca(2+) concentration ([Ca(2+)](c)) regulation, insulin secretion, and glucose homeostasis. Basal [Ca(2+)](c) was not increased by SERCA3 ablation. Stimulation with glucose induced a transient drop in basal [Ca(2+)](c) that was suppressed by inhibition of all SERCAs with thapsigargin (TG) but unaffected by selective SERCA3 ablation. Ca(2+) mobilization by acetylcholine was normal in SERCA3-deficient beta-cells. In contrast, [Ca(2+)](c) oscillations resulting from intermittent glucose-stimulated Ca(2+) influx and [Ca(2+)](c) transients induced by pulses of high K(+) were similarly affected by SERCA3 ablation or TG pretreatment of control islets; their amplitude was increased and their slow descending phase suppressed. This suggests that, during the decay of each oscillation, the endoplasmic reticulum releases Ca(2+) that was pumped by SERCA3 during the upstroke phase. SERCA3 ablation increased the insulin response of islets to 15 mmol/l glucose. However, basal and postprandial plasma glucose and insulin concentrations in SERCA3-deficient mice were normal. In conclusion, SERCA2b, but not SERCA3, is involved in basal [Ca(2+)](c) regulation in beta-cells. SERCA3 becomes operative when [Ca(2+)](c) rises and is required for normal [Ca(2+)](c) oscillations in response to glucose. However, a lack of SERCA3 is insufficient in itself to alter glucose homeostasis or impair insulin secretion in mice. [ABSTRACT FROM AUTHOR]

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. [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]

Hypertrophy is one mechanism of pancreatic beta-cell growth and is seen as an important compensatory response to insulin resistance. We hypothesized that the induction of protective genes contributes to the survival of enlarged (hypertrophied) beta-cells. Here, we evaluated changes in stress gene expression that accompany beta-cell hypertrophy in islets from hyperglycemic rats 4 weeks after partial pancreatectomy (Px). A variety of protective genes were upregulated, with markedly increased expression of the antioxidant genes heme oxygenase-1 and glutathione peroxidase and the antiapoptotic gene A20. Cu/Zn-superoxide dismutase (SOD) and Mn-SOD were modestly induced, and Bcl-2 was modestly reduced; however, several other stress genes (catalase, heat shock protein 70, and p53) were unaltered. The increases in mRNA levels corresponded to the degree of hyperglycemia and were reversed in Px rats by 2-week treatment with phlorizin (treatment that normalized hyperglycemia), strongly suggesting the specificity of hyperglycemia in eliciting the response. Hyperglycemia in Px rats also led to activation of nuclear factor-kappaB in islets. The profound change in beta-cell phenotype of hyperglycemic Px rats resulted in a reduced sensitivity to the beta-cell toxin streptozotocin. Sensitivity to the toxin was restored, along with the beta-cell phenotype, in islets from phlorizin-treated Px rats. Furthermore, beta-cells of Px rats were not vulnerable to apoptosis when further challenged in vivo with dexamethasone, which increases insulin resistance. In conclusion, beta-cell adaptation to chronic hyperglycemia and, hence, increased insulin demand is accompanied by the induction of protective stress genes that may contribute to the survival of hypertrophied beta-cells. [ABSTRACT FROM AUTHOR]

Presents information on a study which determined whether beta-cells can adapt to the increase in insulin demand during glucose or lipid infusion and the molecular mechanisms behind the adaptation. Background on adaptive beta-cells; Materials and methodology used; Results and discussion.

Simple Summary: Defective insulin secretion by pancreatic beta cells is key for the development of type 2 diabetes but the precise mechanisms involved are poorly understood. Metallothioneins are metal binding proteins whose precise biological roles have not been fully characterized. Available evidence indicated that Metallothioneins are protective cellular effectors involved in heavy metal detoxification, metal ion homeostasis and antioxidant defense. This concept has however been challenged by emerging evidence in different medical research fields revealing novel negative roles of Metallothioneins, including in the context of diabetes. In this review, we gather and analyze the available knowledge regarding the complex roles of Metallothioneins in pancreatic beta cell biology and insulin secretion. We comprehensively analyze the evidence showing positive effects of Metallothioneins on beta cell function and survival as well as the emerging evidence revealing negative effects and discuss the possible underlying mechanisms. We expose in parallel findings from other medical research fields and underscore unsettled questions. Then, we propose some future research directions to improve knowledge in the field. Metallothioneins (MTs) are low molecular weight, cysteine-rich, metal-binding proteins whose precise biological roles have not been fully characterized. Existing evidence implicated MTs in heavy metal detoxification, metal ion homeostasis and antioxidant defense. MTs were thus categorized as protective effectors that contribute to cellular homeostasis and survival. This view has, however, been challenged by emerging evidence in different medical fields revealing novel pathophysiological roles of MTs, including inflammatory bowel disease, neurodegenerative disorders, carcinogenesis and diabetes. In the present focused review, we discuss the evidence for the role of MTs in pancreatic beta-cell biology and insulin secretion. We highlight the pattern of specific isoforms of MT gene expression in rodents and human beta-cells. We then discuss the mechanisms involved in the regulation of MTs in islets under physiological and pathological conditions, particularly type 2 diabetes, and analyze the evidence revealing adaptive and negative roles of MTs in beta-cells and the potential mechanisms involved. Finally, we underscore the unsettled questions in the field and propose some future research directions. [ABSTRACT FROM AUTHOR]

The loss of functional beta cell mass characterises all forms of diabetes. Beta cells are highly susceptible to stress, including cytokine, endoplasmic reticulum (ER) and oxidative stress. This study examined the role of pleckstrin homology-like, domain family A, member 3 (Phlda3) in beta cell survival under stress conditions and the regulatory basis. We found that the mRNA levels of Phlda3 were markedly upregulated in vivo in the islets of diabetic humans and mice. In vitro, exposure of MIN6 cells or islets to cytokines, palmitate, thapsigargin or ribose upregulated Phlda3 mRNA and protein levels, concurrent with the induction of ER stress (Ddit3 and Trb3) and antioxidant (Hmox1) genes. Furthermore, H2O2 treatment markedly increased PHLDA3 immunostaining in human islets. Phlda3 expression was differentially regulated by adaptive (Xbp1) and apoptotic (Ddit3) unfolded protein response (UPR) mediators. siRNA-mediated knockdown of Xbp1 inhibited the induction of Phlda3 by cytokines and palmitate, whereas knockdown of Ddit3 upregulated Phlda3. Moreover, knockdown of Phlda3 potentiated cytokine-induced apoptosis in association with upregulation of inflammatory genes (iNos, IL1β and IκBα) and NFκB phosphorylation and downregulation of antioxidant (Gpx1 and Srxn1) and adaptive UPR (Xbp1, Hspa5 and Fkbp11) genes. Knockdown of Phlda3 also potentiated apoptosis under oxidative stress conditions induced by ribose treatment. These findings suggest that Phlda3 is crucial for beta cell survival under stress conditions. Phlda3 regulates the cytokine, oxidative and ER stress responses in beta cells via the repression of inflammatory gene expression and the maintenance of antioxidant and adaptive UPR gene expression. Phlda3 may promote beta cell survival in diabetes. [ABSTRACT FROM AUTHOR]

Examines the role of response gene c-myc in pancreatic beta-cell adaptation to glucose concentration changes. Factors affecting the loss of beta-cell differentiation; Effect of chronic hyperglycemia on beta-cell function; Increase of island of Langerhans c-myc-to-TATA box binding protein.