Prolonged culture of human pancreatic islets under glucotoxic conditions changes their acute beta cell calcium and insulin secretion glucose response curves from sigmoid to bell-shaped.

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.

The lack of functional nicotinamide nucleotide transhydrogenase only moderately contributes to the impairment of glucose tolerance and glucose-stimulated insulin secretion in C57BL/6J vs C57BL/6N mice.

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.

Metallothionein 1 negatively regulates glucose-stimulated insulin secretion and is differentially expressed in conditions of beta cell compensation and failure in mice and humans.

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.

A Triple-Mode Flexible E-Skin Sensor Interface for Multi-Purpose Wearable Applications.

This study presents a flexible wireless electronic skin (e-skin) sensor system that includes a multi-functional sensor device, a triple-mode reconfigurable readout integrated circuit (ROIC), and a mobile monitoring interface. The e-skin device's multi-functionality is achieved by an interlocked micro-dome array structure that uses a polyvinylidene fluoride and reduced graphene oxide (PVDF/RGO) composite material that is inspired by the structure and functions of the human fingertip. For multi-functional implementation, the proposed triple-mode ROIC is reconfigured to support piezoelectric, piezoresistance, and pyroelectric interfaces through single-type e-skin sensor devices. A flexible system prototype was developed and experimentally verified to provide various wireless wearable sensing functions-including pulse wave, voice, chewing/swallowing, breathing, knee movements, and temperature-while their real-time sensed data are displayed on a smartphone.

3D evaluation of the extracellular matrix of hypoxic pancreatic islets using light sheet fluorescence microscopy.

Pancreatic islet transplantation is a promising treatment for type 1 diabetes, but the survival and function of transplanted islets are hindered by the loss of extracellular matrix (ECM) during islet isolation and by low oxygenation upon implantation. This study aimed to evaluate the impact of hypoxia on ECM using a cutting-edge imaging approach based on tissue clearing and 3D microscopy. Human and rat islets were cultured under normoxic (O2 21%) or hypoxic (O2 1%) conditions. Immunofluorescence staining targeting insulin, glucagon, CA9 (a hypoxia marker), ECM proteins (collagen 4, fibronectin, laminin), and E-cadherin (intercellular adhesion protein) was performed on fixed whole islets. The cleared islets were imaged using Light Sheet Fluorescence Microscopy (LSFM) and digitally analyzed. The volumetric analysis of target proteins did not show significant differences in abundance between the experimental groups. However, 3D projections revealed distinct morphological features that differentiated normoxic and hypoxic islets. Under normoxic conditions, ECM could be found throughout the islets. Hypoxic islets exhibited areas of scattered nuclei and central clusters of ECM proteins, indicating central necrosis. E-cadherin was absent in these areas. Our results, demonstrating a diminution of islets' functional mass in hypoxia, align with the functional decline observed in transplanted islets experiencing low oxygenation after grafting. This study provides a methodology combining tissue clearing, multiplex immunofluorescence, Light Sheet Fluorescence Microscopy, and digital image analysis to investigate pancreatic islet morphology. This 3D approach allowed us to highlight ECM organizational changes during hypoxia from a morphological perspective.

Frequency-selective acoustic and haptic smart skin for dual-mode dynamic/static human-machine interface.

Accurate transmission of biosignals without interference of surrounding noises is a key factor for the realization of human-machine interfaces (HMIs). We propose frequency-selective acoustic and haptic sensors for dual-mode HMIs based on triboelectric sensors with hierarchical macrodome/micropore/nanoparticle structure of ferroelectric composites. Our sensor shows a high sensitivity and linearity under a wide range of dynamic pressures and resonance frequency, which enables high acoustic frequency selectivity in a wide frequency range (145 to 9000 Hz), thus rendering noise-independent voice recognition possible. Our frequency-selective multichannel acoustic sensor array combined with an artificial neural network demonstrates over 95% accurate voice recognition for different frequency noises ranging from 100 to 8000 Hz. We demonstrate that our dual-mode sensor with linear response and frequency selectivity over a wide range of dynamic pressures facilitates the differentiation of surface texture and control of an avatar robot using both acoustic and mechanical inputs without interference from surrounding noise.

In depth functional characterization of human induced pluripotent stem cell-derived beta cells in vitro and in vivo.

In vitro differentiation of human induced pluripotent stem cells (iPSCs) into beta cells represents an important cell source for diabetes research. Here, we fully characterized iPSC-derived beta cell function in vitro and in vivo in humanized mice. Using a 7-stage protocol, human iPSCs were differentiated into islet-like aggregates with a yield of insulin-positive beta cells comparable to that of human islets. The last three stages of differentiation were conducted with two different 3D culture systems, rotating suspension or static microwells. In the latter, homogeneously small-sized islet-like aggregates were obtained, while in rotating suspension size was heterogeneous and aggregates often clumped. In vitro function was assessed by glucose-stimulated insulin secretion, NAD(P)H and calcium fluctuations. Stage 7 aggregates slightly increased insulin release in response to glucose in vitro. Aggregates were transplanted under the kidney capsule of NOD-SCID mice to allow for further in vivo beta cell maturation. In transplanted mice, grafts showed glucose-responsiveness and maintained normoglycemia after streptozotocin injection. In situ kidney perfusion assays showed modulation of human insulin secretion in response to different secretagogues. In conclusion, iPSCs differentiated with equal efficiency into beta cells in microwells compared to rotating suspension, but the former had a higher experimental success rate. In vitro differentiation generated aggregates lacking fully mature beta cell function. In vivo, beta cells acquired the functional characteristics typical of human islets. With this technology an unlimited supply of islet-like organoids can be generated from human iPSCs that will be instrumental to study beta cell biology and dysfunction in diabetes.

Ticks collected from selected mammalian hosts surveyed in the Republic of Korea during 2008-2009.

A tick survey was conducted to determine the relative abundance and distribution of ticks associated with selected mammals in the Republic of Korea (ROK) during 2008-2009. A total of 918 ticks were collected from 76 mammals (6 families, 9 species) captured at 6 provinces and 3 Metropolitan Cities in ROK. Haemaphysalis longicornis (54.4%) was the most frequently collected tick, followed by Haemaphysalis flava (28.5%), Ixodes nipponensis (7.6%), Ixodes pomerantzevi (4.8%), Ixodes persulcatus (4.6%), and Haemaphysalis japonica (0.1%). Adults (57.0%) and nymphs (28.7%) of Ixodes and Haemaphysalis spp. were collected most frequently from medium or large mammals in this survey, while few larvae (14.3%) were collected. Hydropotes inermis was the most frequently captured mammal (52.6%), with a 16.4 tick index and 5 of 6 species of ticks collected during this survey. H. longicornis (69.7%) was the predominant tick collected from H. inermis, followed by H. flava (22.2%), I. persulcatus (6.1%), I. nipponensis (1.8%), and H. japonica (0.2%).

KATP channel blockers control glucagon secretion by distinct mechanisms: A direct stimulation of α-cells involving a [Ca2+]c rise and an indirect inhibition mediated by somatostatin.

OBJECTIVE: Glucagon is secreted by pancreatic α-cells in response to hypoglycemia and its hyperglycemic effect helps to restore normal blood glucose. Insulin and somatostatin (SST) secretions from ß- and δ-cells, respectively, are stimulated by glucose by mechanisms involving an inhibition of their ATP-sensitive K+ (KATP) channels, leading to an increase in [Ca2+]c that triggers exocytosis. Drugs that close KATP channels, such as sulfonylureas, are used to stimulate insulin release in type 2 diabetic patients. α-cells also express KATP channels. However, the mechanisms by which sulfonylureas control glucagon secretion are still largely debated and were addressed in the present study. In particular, we studied the effects of KATP channel blockers on α-cell [Ca2+]c and glucagon secretion in the presence of a low (1 mM) or a high (15 mM) glucose concentration and evaluated the role of SST in these effects. METHODS: Using a transgenic mouse model expressing the Ca2+-sensitive fluorescent protein, GCaMP6f, specifically in α-cells, we measured [Ca2+]c in α-cells either dispersed or within whole islets (by confocal microscopy). By measuring [Ca2+]c in α-cells within islets and glucagon secretion using the same perifusion protocols, we tested whether glucagon secretion correlated with changes in [Ca2+]c in response to sulfonylureas. We studied the role of SST in the effects of sulfonylureas using multiple approaches including genetic ablation of SST, or application of SST-14 and SST receptor antagonists. RESULTS: Application of the sulfonylureas, tolbutamide, or gliclazide, to a medium containing 1 mM or 15 mM glucose increased [Ca2+]c in α-cells by a direct effect as in ß-cells. At low glucose, sulfonylureas inhibited glucagon secretion of islets despite the rise in α-cell [Ca2+]c that they triggered. This glucagonostatic effect was indirect and attributed to SST because, in the islets of SST-knockout mice, sulfonylureas induced a stimulation of glucagon secretion which correlated with an increase in α-cell [Ca2+]c. Experiments with exogenous SST-14 and SST receptor antagonists indicated that the glucagonostatic effect of sulfonylureas mainly resulted from an inhibition of the efficacy of cytosolic Ca2+ on exocytosis. Although SST-14 was also able to inhibit glucagon secretion by decreasing α-cell [Ca2+]c, no decrease in [Ca2+]c occurred during sulfonylurea application because it was largely counterbalanced by the direct stimulatory effect of these drugs on α-cell [Ca2+]c. At high glucose, i.e., in conditions where glucagon release was already low, sulfonylureas stimulated glucagon secretion because their direct stimulatory effect on α-cells exceeded the indirect effect by SST. Our results also indicated that, unexpectedly, SST-14 poorly decreased the efficacy of Ca2+ on exocytosis in ß-cells. CONCLUSIONS: Sulfonylureas exert two opposite actions on α-cells: a direct stimulation as in ß-cells and an indirect inhibition by SST. This suggests that any alteration of SST paracrine influence, as described in diabetes, will modify the effect of sulfonylureas on glucagon release. In addition, we suggest that δ-cells inhibit α-cells more efficiently than ß-cells.

SGLT2 is not expressed in pancreatic α- and ß-cells, and its inhibition does not directly affect glucagon and insulin secretion in rodents and humans.

OBJECTIVE: Sodium-glucose cotransporter 2 (SGLT2) inhibitors (SGLT2i), or gliflozins, are anti-diabetic drugs that lower glycemia by promoting glucosuria, but they also stimulate endogenous glucose and ketone body production. The likely causes of these metabolic responses are increased blood glucagon levels, and decreased blood insulin levels, but the mechanisms involved are hotly debated. This study verified whether or not SGLT2i affect glucagon and insulin secretion by a direct action on islet cells in three species, using multiple approaches. METHODS: We tested the in vivo effects of two selective SGLT2i (dapagliflozin, empagliflozin) and a SGLT1/2i (sotagliflozin) on various biological parameters (glucosuria, glycemia, glucagonemia, insulinemia) in mice. mRNA expression of SGLT2 and other glucose transporters was assessed in rat, mouse, and human FACS-purified α- and ß-cells, and by analysis of two human islet cell transcriptomic datasets. Immunodetection of SGLT2 in pancreatic tissues was performed with a validated antibody. The effects of dapagliflozin, empagliflozin, and sotagliflozin on glucagon and insulin secretion were assessed using isolated rat, mouse and human islets and the in situ perfused mouse pancreas. Finally, we tested the long-term effect of SGLT2i on glucagon gene expression. RESULTS: SGLT2 inhibition in mice increased the plasma glucagon/insulin ratio in the fasted state, an effect correlated with a decline in glycemia. Gene expression analyses and immunodetections showed no SGLT2 mRNA or protein expression in rodent and human islet cells, but moderate SGLT1 mRNA expression in human α-cells. However, functional experiments on rat, mouse, and human (29 donors) islets and the in situ perfused mouse pancreas did not identify any direct effect of dapagliflozin, empagliflozin or sotagliflozin on glucagon and insulin secretion. SGLT2i did not affect glucagon gene expression in rat and human islets. CONCLUSIONS: The data indicate that the SGLT2i-induced increase of the plasma glucagon/insulin ratio in vivo does not result from a direct action of the gliflozins on islet cells.

A comparison of non-viral vectors for gene delivery to pancreatic beta-cells: delivering a hypoxia-inducible vascular endothelial growth factor gene to rat islets.

Although non-viral vectors are relatively safe, they have very low gene transfection efficiency, especially in pancreatic islet cells. To provide information on the use of non-viral vectors for transfecting genes into pancreatic islet cells, a comparative evaluation of non-viral options was performed. In vitro experiments were used to compare the transfection efficiency of three classes of non-viral vectors: Effectene, polyethylenimine (PEI, 25 kDa) and hemagglutinating virus of Japan-envelope (HVJ-E), into insulinoma cells (INS-1) and rat islets. Vascular endothelial growth factor (VEGF) gene with hypoxia-inducible RTP801 promoter was delivered into rat islets with Effectene and VEGF secretion under hypoxia was measured in the culture media. Luciferase activity and GFP assays indicated that Effectene exhibited the highest transfection efficiency, and HVJ-E was not suitable for transfection into pancreatic beta-cells. The cytotoxicity of Effectene was found to be similar to that of 25-kDa PEI by 7-amino actinomycin D (7-AAD) flow cytometry and acridine orange/propidium iodide (AO/PI) assays. When RTP801 promoter-VEGF plasmid was delivered to rat islets with Effectene, VEGF secretion increased specifically in islets under hypoxia. In conclusion, Effectene showed higher gene-delivery efficiency for pancreatic islets compared with other classes of non-viral delivery systems and is promising as a gene delivery agent for pretransplant ex vivo gene therapy of islets.

Somatostatin Is Only Partly Required for the Glucagonostatic Effect of Glucose but Is Necessary for the Glucagonostatic Effect of KATP Channel Blockers.

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.

NNT reverse mode of operation mediates glucose control of mitochondrial NADPH and glutathione redox state in mouse pancreatic ß-cells.

OBJECTIVE: The glucose stimulation of insulin secretion (GSIS) by pancreatic ß-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, Ca2+ 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 ß-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 H2O2 at non-stimulating glucose. Surprisingly, the lack of NNT did not alter the glucose-stimulation of Ca2+ influx and upstream mitochondrial events, but it markedly reduced both phases of GSIS by altering Ca2+-induced exocytosis and its metabolic amplification. CONCLUSION: These results drastically modify current views on NNT operation and mitochondrial function in pancreatic ß-cells.

The favorable outcome of human islet transplantation in Korea: experiences of 10 autologous transplantations.

BACKGROUND: Cystic neoplasms of the pancreas are an increasingly diagnosed entity, and surgical resection of the pancreas is advocated. Islet autotransplantation is a therapeutic approach used to prevent diabetes in cases of pathologically benign neoplasm after major pancreatectomy. METHODS: A total of 10 patients underwent pancreatectomy with islet autotransplantation. To evaluate islet transplantation efficiency, the authors compared 23 subjects who did not undergo islet transplantation after partial pancreatectomy with 87 subjects with normal glucose tolerance and with 77 diabetic subjects that did not undergo pancreatectomy. RESULTS: Ten female patients with nine cystic neoplasms and one patient with pancreatic injury underwent transplantation. Their mean islet equivalents (IEQ) was 3,159 IEQ/kg. During follow-up, two recipients required insulin or oral agents. At the 12-month follow-up, homeostasis model assessment (HOMA)-beta was 77.36+/-17.68, the insulinogenic index (INSindex) was 0.49+/-0.11, and fasting C-peptide and hemoglobin A1c were 1.28+/-0.18 ng/mL and 5.73+/-0.26%, respectively. Islet replacement was found to increase HOMA-beta by approximately 17% compared with distal pancreatectomy in normal glucose tolerance subjects without islet autotransplantation and by 46% compared with distal pancreatectomy diabetes subjects without islet autotransplantation. Factors different in the two insulin and oral hypoglycemic agent (OHA)-requiring recipients and the eight insulin- and OHA-free recipients were pancreatectomy extent, preoperative glucose metabolism insufficiency, age, and underlying cystic neoplasm disease. CONCLUSIONS: Even partial islet graft function can have a beneficial metabolic effect on the recipient in terms of metabolic parameters such as HOMA-beta and INSindex. This study suggests that islet replacement should be considered for experimental procedures in benign pancreatic conditions.

Calcium signaling in pancreatic ß-cells in health and in Type 2 diabetes.

Changes in cytosolic free Ca(2+) concentration ([Ca(2+)]c) play a crucial role in the control of insulin secretion from the electrically excitable pancreatic ß-cell. Secretion is controlled by the finely tuned balance between Ca(2+) influx (mainly through voltage-dependent Ca(2+) channels, but also through voltage-independent Ca(2+) channels like store-operated channels) and efflux pathways. Changes in [Ca(2+)]c directly affect [Ca(2+)] in various organelles including the endoplasmic reticulum (ER), mitochondria, the Golgi apparatus, secretory granules and lysosomes, as imaged using recombinant targeted probes. Because most of these organelles have specific Ca(2+) influx and efflux pathways, they mutually influence free [Ca(2+)] in the others. In this article, we review the mechanisms of control of [Ca(2+)] in various compartments and particularly the cytosol, the endoplasmic reticulum ([Ca(2+)]ER), acidic stores and mitochondrial matrix ([Ca(2+)]mito), focusing chiefly on the most important physiological stimulus of ß-cells, glucose. We also briefly review some alterations of ß-cell Ca(2+) homeostasis in Type 2 diabetes.

Tolbutamide controls glucagon release from mouse islets differently than glucose: involvement of K(ATP) channels from both α-cells and δ-cells.

We evaluated the role of ATP-sensitive K⁺ (K(ATP)) channels, somatostatin, and Zn²âº in the control of glucagon secretion from mouse islets. Switching from 1 to 7 mmol/L glucose inhibited glucagon release. Diazoxide did not reverse the glucagonostatic effect of glucose. Tolbutamide decreased glucagon secretion at 1 mmol/L glucose (G1) but stimulated it at 7 mmol/L glucose (G7). The reduced glucagon secretion produced by high concentrations of tolbutamide or diazoxide, or disruption of K(ATP) channels (Sur1(-/-) mice) at G1 could be inhibited further by G7. Removal of the somatostatin paracrine influence (Sst(-/-) mice or pretreatement with pertussis toxin) strongly increased glucagon release, did not prevent the glucagonostatic effect of G7, and unmasked a marked glucagonotropic effect of tolbutamide. Glucose inhibited glucagon release in the absence of functional K(ATP) channels and somatostatin signaling. Knockout of the Zn²âº transporter ZnT8 (ZnT8(-/-) mice) did not prevent the glucagonostatic effect of glucose. In conclusion, glucose can inhibit glucagon release independently of Zn²âº, K(ATP) channels, and somatostatin. Closure of K(ATP) channels controls glucagon secretion by two mechanisms, a direct stimulation of α-cells and an indirect inhibition via somatostatin released from δ-cells. The net effect on glucagon release results from a balance between both effects.

Molecular detection of Anaplasma, Bartonella, and Borrelia species in ticks collected from migratory birds from Hong-do Island, Republic of Korea.

Bird migration is a recurring annual and seasonal event undertaken by more than 100 species of birds in the southeast Asian and northeast Palearctic regions that pass through or remain for short periods from April to May and September to November at Hong-do Island, Republic of Korea (ROK). A total of 212 ticks (40 Haemaphysalis flava, 12 H. longicornis, 146 Ixodes turdus, 13 I. nipponensis, and 1 I. ornithophila) were collected from 65/2,161 (3.0%) migratory birds consisting of 21 species that were captured from January, 2008, through December, 2009, as part of the Migratory Birds Center, Hong-do bird banding program for studying bird migration patterns. Adult ticks were assayed individually while larvae and nymphs were pooled (1-22 and 1-6 ticks per pool, respectively) into 31 and 65 pools, respectively. Ticks were assayed for zoonotic pathogens by PCR using 16S rRNA, heat shock protein (groEL), and internal transcribed spacer (ITS) gene primers to amplify genera specific for Anapalsma, Bartonella, and Borrelia PCR amplicons. Using the 16S rRNA-based nested PCR, A. phagocytophilum (n=1) was detected in I. nipponensis collected from Zoothera sibirica and A. bovis (n=1) was detected in I. turdus collected from Emberiza chrysophrys. Borrelia turdi 16S rRNA genes (n=3) were detected in I. turdus and I. nipponensis collected from Turdus pallidus and Zoothera aurea. Borrelia spp. 16S rRNA genes (n=4) were detected in Ixodes ticks collected from Emberiza tristrami, T. pallidus, and Z. aurea. The Bartonella grahamii ITS gene (n=1) was detected by nested PCR assay in I. turdus collected from Z. aurea. These results provide insight into the potential role of migratory birds in the dispersal of ticks and associated tick-borne pathogens throughout their ranges in Asia.