Islet ATP Content Research Articles

Metabolic Role of GABA in the Secretory Function of Pancreatic β-Cells: Its Hypothetical Implication in β-Cell Degradation in Type 2 Diabetes.

The stimulus-secretion coupling of a glucose-induced release is generally attributed to the metabolism of the hexose in the β-cells in the glycolytic pathway and the citric acid cycle. Glucose metabolism generates an increased cytosolic concentration of ATP and of the ATP/ADP ratio that closes the ATP-dependent K+-channel at the plasma membrane. The resultant depolarization of the β-cells opens voltage-dependent Ca2+-channels at the plasma membrane that triggers the exocytosis of insulin secretory granules. The secretory response is biphasic with a first and transient peak followed by a sustained phase. The first phase is reproduced by a depolarization of the β-cells with high extracellular KCl maintaining the KATP-channels open with diazoxide (triggering phase); the sustained phase (amplifying phase) depends on the participation of metabolic signals that remain to be determined. Our group has been investigating for several years the participation of the β-cell GABA metabolism in the stimulation of insulin secretion by three different secretagogues (glucose, a mixture of L-leucine plus L-glutamine, and some branched chain alpha-ketoacids, BCKAs). They stimulate a biphasic secretion of insulin accompanied by a strong suppression of the intracellular islet content of gamma-aminobutyric acid (GABA). As the islet GABA release simultaneously decreased, it was concluded that this resulted from an increased GABA shunt metabolism. The entrance of GABA into the shunt is catalyzed by GABA transaminase (GABAT) that transfers an amino group between GABA and alpha-ketoglutarate, resulting in succinic acid semialdehyde (SSA) and L-glutamate. SSA is oxidized to succinic acid that is further oxidized in the citric acid cycle. Inhibitors of GABAT (gamma-vinyl GABA, gabaculine) or glutamic acid decarboxylating activity (GAD), allylglycine, partially suppress the secretory response as well as GABA metabolism and islet ATP content and the ATP/ADP ratio. It is concluded that the GABA shunt metabolism contributes together with the own metabolism of metabolic secretagogues to increase islet mitochondrial oxidative phosphorylation. These experimental findings emphasize that the GABA shunt metabolism is a previously unrecognized anaplerotic mitochondrial pathway feeding the citric acid cycle with a β-cell endogenous substrate. It is therefore a postulated alternative to the proposed mitochondrial cataplerotic pathway(s) responsible for the amplification phase of insulin secretion. It is concluded the new postulated alternative suggests a possible new mechanism of β-cell degradation in type 2 (perhaps also in type 1) diabetes.

HYPOXIC PRECONDITIONING AMELIORATES ISCHEMIA-INDUCED DAMAGE OF ISOLATED RAT ISLETS

Aim: Islet isolation results in disruption of the native islet vascular network until posttransplant islet revascularisation is completed. Prolonged exposure to a hypoxic and nutrient-depleted environment has detrimental effects on morphological and functional integrity of isolated islets. In solid organ transplantation it was shown that short sublethal periods of anoxia can induce adaptive mechanisms in the affected tissue offering protection against subsequent potentially lethal hypoxic insults. This study was aimed to explore the potential of hypoxic preconditioning for isolated islets. Methods: Rat islets, isolated from 20 non-heartbeating donors, were subjected either to 2 x 2 min-periods of anoxia, intermitted by a 2 min-period of oxygen supply, or to sham-treatment. Hypoxic preconditioning and sham-treatment were followed by 4 hours of recovery in normoxia prior to 24 hr-culture at 1.5% oxygen. Islet characterisation included yield of islet equivalents (IEQ) or islet particle number (IN), viability (FDA-PI), DNA (pico-green) and ATP (luciferase assay) content, intraislet reactive oxygen species (ROS) production (DCFH-DA), gene expression (qRT-PCR), and glucose stimulation index (2 vs 20 vs 2 mM). Data were normalised to islet DNA content and are presented as mean ± SEM. Results: Hypoxic preconditioning had no effect on islet survival when compared with sham-treatment (64 ± 7 vs 61 ± 7%). Compared with preconditioned islets, sham-treated controls were characterised by increased fragmentation when determined as IN/IEQ ratio (1.51 ± 0.18 vs 1.24 ± 0.13, p<0.05). Fragmentation corrrelated inversely with viability of sham-treated and preconditioned islets (61 ± 3 vs 66 ± 3 %, p<0.01). ATP (0.66 ± 0.14 vs 0.73 ± 0.20 pM/ng DNA) and insulin content (51.1 ± 12.5 vs 46.3 ± 11.7 ng/ng) were not altered by hypoxic preconditioning compared with sham-treatment. ROS production was reduced after hypoxic preconditioning compared with sham-treatment (645 ± 73 vs 858 ± 88 AU/ng, p<0.01). Islets of both experimental groups responded with insulin release after glucose challenge, but the stimulation index of preconditioned islets was significantly larger compared with sham-treated islets (2.39 ± 0.37 vs 2.09 ± 0.33, p<0.05). Whilst pro-apoptotic BAX and CHOP gene expression was reduced by 54 ± 15% (p<0.05) and 49 ± 8% (p<0.05), hypoxic preconditioning increased HIF-1 alpha and VEGF-A gene expression by 90 ± 41% (p<0.01) and 184 ± 65% (p<0.05), respectively. Conclusion: Although hypoxic preconditioning could not ameliorate ischaemia-induced loss of islets, islet morphology and function was significantly improved presumably as result of reduced intraislet ROS production. qRT-PCR-determined gene expression suggests that hypoxic preconditioning induces rescue mechanisms islets and mediates a significant anti-apoptotic effect. This study describes a new option for pretransplant treatment of isolated human islets. European Union’s Cooperation Programme FP7 (HEALTH-F2-2012-305746). Oxford NIHR Biomedical Research Centre.

Sex effect on insulin secretion and mitochondrial function in pancreatic beta cells of elderly Wistar rats

ABSTRACTAims/hypothesis: Glucose tolerance progressively declines with age, and there is a high prevalence of type 2 diabetes in the elderly people. Previous studies have reported the sex differences in risk for type 2 diabetes, especially in the elderly people, whereas reasons for these sex differences remain poorly understood. This study aims to evaluate the effect of sex on glucose-stimulated insulin secretion and mitochondrial function in pancreatic beta cells of Wistar rats. Methods: 3-month-old and 18-month-old Wistar rats of both sexes were used. Insulin secretion of islets was analyzed by glucose-stimulated insulin secretion and islet perifusion assays; ATP content and oxygen consumption rate of islets were determined to evaluate the mitochondrial function. Results: Insulin secretion of islets under high glucose conditions declined significantly with age in both sexes. Glucose-stimulated insulin secretion of elderly female groups was markedly higher than that of male groups under high glucose conditions. Importantly, islets from elderly female groups showed higher mitochondrial function compared with male counterparts, evidenced by higher ATP content and oxygen consumption rate under high glucose conditions. It was also noted that mitochondrial biogenesis of islets from elderly female rats was significant higher compared with male rats. There were notable increases in expression of genes involved in mitochondrial biogenesis in islets from elderly female rats compared with male rats. Conclusion: This study demonstrates a sex dimorphism in the age-associated impairment of pancreatic beta cell function in elderly rats, while the potential mechanism may be related to the sexual differences in mitochondrial biogenesis and function.

KCl -Permeabilized Pancreatic Islets: An Experimental Model to Explore the Messenger Role of ATP in the Mechanism of Insulin Secretion.

Our previous work has demonstrated that islet depolarization with KCl opens connexin36 hemichannels in β-cells of mouse pancreatic islets allowing the exchange of small metabolites with the extracellular medium. In this study, the opening of these hemichannels has been further characterized in rat islets and INS–1 cells. Taking advantage of hemicannels’opening, the uptake of extracellular ATP and its effect on insulin release were investigated. 70 mM KCl stimulated light emission by luciferin in dispersed rat islets cells transduced with the fire-fly luciferase gene: it was suppressed by 20 mM glucose and 50 μM mefloquine, a specific connexin36 inhibitor. Extracellular ATP was taken up or released by islets depolarized with 70 mM KCl at 5 mM glucose, depending on the external ATP concentration. 1 mM ATP restored the loss of ATP induced by the depolarization itself. ATP concentrations above 5 mM increased islet ATP content and the ATP/ADP ratio. No ATP uptake occurred in non-depolarized or KCl-depolarized islets simultaneously incubated with 50 μM mefloquine or 20 mM glucose. Extracellular ATP potentiated the secretory response induced by 70 mM KCl at 5 mM glucose in perifused rat islets: 5 mM ATP triggered a second phase of insulin release after the initial peak triggered by KCl-depolarization itself; at 10 mM, it increased both the initial, KCl-dependent, peak and stimulated a greater second phase of secretion than at 5 mM. These stimulatory effects of extracellular ATP were almost completely suppressed by 50 μM mefloquine. The magnitude of the second phase of insulin release due to 5 mM extracellular ATP was decreased by addition of 5 mM ADP (extracellular ATP/ADP ratio = 1). ATP acts independently of KATP channels closure and its intracellular concentration and its ATP/ADP ratio seems to regulate the magnitude of both the first (triggering) and second (amplifying) phases of glucose-induced insulin secretion.

Treatment with CNX-011-67, a novel GPR40 agonist, delays onset and progression of diabetes and improves beta cell preservation and function in male ZDF rats

BackgroundThe role of G protein-coupled receptor (GPR40), which is highly expressed in pancreatic beta cells, has been studied extensively in the amelioration of beta cell dysfunction in T2D using rat and mouse islets, beta cell lines and in animal models of diabetes. But its potential as a therapeutic target has not been fully explored. This aim of the study is to evaluate the therapeutic potential of CNX-011-67, a highly selective, potent and orally bioavailable GPR40 agonist, in controlling diabetes and other metabolic parameters.MethodsSeven week old male ZDF rats were treated with either vehicle or CNX-011-67, 5 mg/kg twice daily, for seven weeks. The animals were subjected to oral glucose tolerance and insulin tolerance tests. Plasma glucose, insulin, triglyceride, HbA1c, fructosamine and free fatty acids were measured at selected time points. Pancreas from control and treated animals were subjected to insulin and pancreatic and duodenal homeobox 1 (PDX1) immunohistochemistry and were also evaluated by electron microscopy. Also the potential impact of CNX-011-67 on islet insulin secretion, content, ATP levels and markers of both glucose oxidation, beta cell health in rat islets under chronic glucolipotoxic conditions was evaluated.ResultsTreatment of male ZDF rats with CNX-011-67 for 7 weeks significantly enhanced insulin secretion in response to oral glucose load, delayed the onset of fasting hyperglycemia by 3 weeks, reduced nonfasting glucose excursions, fasting free fatty acids and triglyceride levels. A significant increase in PDX1 expression and insulin content and reduction in plasma fructosamine, HOMA-IR, and beta cell apoptosis were observed. CNX-011-67 improves glucose mediated insulin secretion, insulin gene transcription and islet insulin content in cultured rat islets under chronic glucolipotoxic condition. Also enhanced glucose oxidation in the form of increased islet ATP content and overall improvement in beta cell health in the form of reduced expression of stress markers (TXNIP and CHOP mRNA) were observed.ConclusionsThese findings, suggest that long-term oral therapy with CNX-011-67 could be of clinical value to provide good glycemic control and improve islet beta cell function.

The Amphiphilic Oxygen Carrier Perfluorohexyloctane Improves Oxygenation of Ischemic Pig Pancreases and Increases the Quality of Isolated Islets Compared to Perfluorodecalin

Introduction: Ischemia is one of the most critical variables for success or failure of islet isolation and islet transplantation. Prolonged cold storage is associated with reduced yield and decreased viability of isolated islets. Technical equipment for continuous vascular oxygen supply during cold storage of retrieved organs is expensive and requires trained personnel for operation. In the present study we assessed the efficiency of the amphiphilic compound perfluorohexyloctane (F6H8) to provide oxygen during static cold storage of pancreases retrieved from adult non-heartbeating pigs for subsequent islet isolation. Methods: Pancreases (n=10) were explanted after 15 min of warm ischemia and dissected ex-situ into the connecting and spleenic lobe. After intraductal perfusion with cold UW-solution the pancreatic segments were immersed in oxygen-precharged F6H8 or perfluorodecalin (PFD) and stored on ice for 9-10 hours until processing for islet isolation and purification. After culture at 37°C for 2-3 days islets were subjected to extensive quality assessment. All data are presented as mean ± SEM and were analysed by Wilcoxon test. Results: Pancreatic segments stored in oxygen-precharged F6H8 had a significantly higher intrapancreatic pO2 compared to preoxygenated PFD (10.1±3.9 vs. 1.6±1.1 mm Hg, p< 0.05). This finding corresponds to the significantly increased ATP content in islets isolated from segments stored in F6H8 (290±60 vs 203±31 pg/ng DNA, p< 0.01). Although freshly isolated islet yield per gram trimmed tissue did not significantly differ between F6H8 and PFD (3020±330 vs. 2940±270 IEQ/g) significantly more islets were recovered after culture when isolated from F6H8-stored lobes (57±6 vs. 39±6%, p< 0.01). Lower viability (76±3 vs. 82±3%, p< 0.05) measured in islets after PFD storage correlated with their inability to downregulate insulin release after switching from high (20 mmol/L) to basal (2 mmol/L) glucose concentration during static glucose incubation (Fig. 1).[Figure 1]Figure 1. Insulin release of islets isolated from ischemic porcine pancreatic segments after 9-10 hours of cold storage in oxygenprecharged PFD or F6H8. Islets were sequentially stimulated by 2 (white bars), 20 (grey bars) and again 2 mmol/L of glucose. *p< 0.05, **p< 0.01 as indicated. Conclusions: In the case that pig races, suitable for successful islet isolation, are locally not available, pancreas preservation for subsequent islet isolation can be significantly improved utilizing oxygen-precharged F6H8 for organ shipment. The same applies if pathogen-free pig pancreases have to be processed in institutions highly experienced in pig islet isolation.

β-Cell Uncoupling Protein 2 Regulates Reactive Oxygen Species Production, Which Influences Both Insulin and Glucagon Secretion

OBJECTIVEThe role of uncoupling protein 2 (UCP2) in pancreatic β-cells is highly debated, partly because of the broad tissue distribution of UCP2 and thus limitations of whole-body UCP2 knockout mouse models. To investigate the function of UCP2 in the β-cell, β-cell–specific UCP2 knockout mice (UCP2BKO) were generated and characterized.RESEARCH DESIGN AND METHODSUCP2BKO mice were generated by crossing loxUCP2 mice with mice expressing rat insulin promoter-driven Cre recombinase. Several in vitro and in vivo parameters were measured, including respiration rate, mitochondrial membrane potential, islet ATP content, reactive oxygen species (ROS) levels, glucose-stimulated insulin secretion (GSIS), glucagon secretion, glucose and insulin tolerance, and plasma hormone levels.RESULTSUCP2BKO β-cells displayed mildly increased glucose-induced mitochondrial membrane hyperpolarization but unchanged rates of uncoupled respiration and islet ATP content. UCP2BKO islets had elevated intracellular ROS levels that associated with enhanced GSIS. Surprisingly, UCP2BKO mice were glucose-intolerant, showing greater α-cell area, higher islet glucagon content, and aberrant ROS-dependent glucagon secretion under high glucose conditions.CONCLUSIONSUsing a novel β-cell–specific UCP2KO mouse model, we have shed light on UCP2 function in primary β-cells. UCP2 does not behave as a classical metabolic uncoupler in the β-cell, but has a more prominent role in the regulation of intracellular ROS levels that contribute to GSIS amplification. In addition, β-cell UCP2 contributes to the regulation of intraislet ROS signals that mediate changes in α-cell morphology and glucagon secretion.

Glucose promotion of GABA metabolism contributes to the stimulation of insulin secretion in β-cells

We have demonstrated recently that branched-chain α-keto acid stimulation of insulin secretion is dependent on islet GABA (γ-aminobutyric acid) metabolism: GABA transamination to succinic semialdehyde is increased by 2-oxoglutarate, generated in α-keto acid transamination to its corresponding α-amino acid. The present work was aimed at investigating whether glucose also promotes islet GABA metabolism and whether the latter contributes to the stimulation of insulin secretion. Glucose (20 mM) decreased both the content and release of islet GABA. Gabaculine (1 mM), a GABA transaminase inhibitor, partially suppressed the secretory response of rat perifused islets to 20 mM glucose at different L-glutamine concentrations (0, 1 and 10 mM), as well as the glucose-induced decrease in islet GABA. The drug also reduced islet ATP content and the ATP/ADP ratio at 20 mM glucose. Exogenous succinic semialdehyde induced a dose-dependent increase in islet GABA content by reversal of GABA transamination and a biphasic insulin secretion in the absence of glucose. It depolarized isolated β-cells and triggered action potential firing, accompanied by a reduction of membrane currents through ATP-sensitive K(+) channels. The gene expression and enzyme activity of GABA transaminase were severalfold higher than that of 2-oxoglutarate dehydrogenase in islet homogenates. We conclude that, at high glucose concentrations, there is an increased diversion of glucose metabolism from the citric acid cycle into the 'GABA shunt'. Semialdehyde succinic acid is a cell-permeant 'GABA-shunt' metabolite that increases ATP and the ATP/ADP ratio, depolarizes β-cells and stimulates insulin secretion. In summary, an increased islet GABA metabolism may trigger insulin secretion.

A New Oxygen Carrier for Improved Long-Term Storage of Human Pancreata Before Islet Isolation

Pancreas oxygenation during cold storage has been established in islet isolation and transplantation to prevent ischemic tissue damage using perfluorodecalin (PFD) as hyperoxygen carrier. However, studies in humans and pigs provided conflicting results about the efficiency of PFD for pancreas oxygenation. The aim of this study was to compare PFD with a newly developed oxygen carrier composed of perfluorohexyloctane and polydimethylsiloxane 5 (F6H8S5) for long-term storage of human pancreata. After 24-hr storage in preoxygenated PFD or F6H8S5, pancreata were processed using Liberase HI for pancreas dissociation and a Ficoll gradient for islet purification. Islet quality assessment was performed measuring glucose-stimulated insulin release, viability, islet ATP content, and posttransplant function in diabetic nude mice. Compared with PFD, F6H8S5 significantly increased the intrapancreatic partial oxygen pressure and islet ATP content. This corresponded to an increase of islet yield, recovery after culture, glucose stimulation index, viability, and improved graft function in diabetic nude mice. The present findings indicate clearly that F6H8S5 improves isolation outcome after prolonged ischemia compared with PFD. This observation seems to be related to the significant lipophilicity and almost pancreas-specific density of F6H8S5. Moreover, these characteristics facilitate pancreas shipment without using custom-made transport vessels as required for PFD.

Senescence Marker Protein-30/Gluconolactonase Deletion Worsens Glucose Tolerance through Impairment of Acute Insulin Secretion

Senescence marker protein-30 (SMP30) is an androgen-independent factor that decreases with age. We recently identified SMP30 as the lactone-hydrolyzing enzyme gluconolactonase (GNL), which is involved in vitamin C biosynthesis in animal species. To examine whether the age-related decrease in SMP30/GNL has effects on glucose homeostasis, we used SMP30/GNL knockout (KO) mice treated with L-ascorbic acid. In an ip glucose tolerance test at 15 wk of age, blood glucose levels in SMP30/GNL KO mice were significantly increased by 25% at 30 min after glucose administration compared with wild-type (WT) mice. Insulin levels in SMP30/GNL KO mice were significantly decreased by 37% at 30 min after glucose compared with WT mice. Interestingly, an insulin tolerance test showed a greater glucose-lowering effect in SMP30/GNL KO mice. High-fat diet feeding severely worsened glucose tolerance in both WT and SMP30/GNL KO mice. Morphometric analysis revealed no differences in the degree of high-fat diet-induced compensatory increase in beta-cell mass and proliferation. In the static incubation study of islets, insulin secretion in response to 20 mm glucose or KCl was significantly decreased in SMP30/GNL KO mice. On the other hand, islet ATP content at 20 mm in SMP30/GNL KO mice was similar to that in WT mice. Collectively, these data indicate that impairment of the early phase of insulin secretion due to dysfunction of the distal portion of the secretion pathway underlies glucose intolerance in SMP30/GNL KO mice. Decreased SMP30/GNL may contribute to the worsening of glucose tolerance that occurs in normal aging.

Pig Pancreas Oxygenation at 20°C Increases Islet ATP Generation but Deteriorates Islet Function

Successful pancreas preservation during storage in oxygenated perfluorodecalin (PFD) is mainly related to oxidative ATP generation during storage. Increasing the storage temperature would accelerate this process essential for resuscitation of ischemically damaged pancreatic tissue. The present study aimed at comparing islet isolation outcome from adult pig pancreata preserved in oxygenated PFD by means of a one-layer method during storage on ice or at 20 degrees C. Resected pancreata were intraductally flushed with cold UW solution and promptly processed (n = 6) or stored for 3 h in continuously oxygenated PFD at 4 degrees C (n = 5) or 20 degrees C (n = 7). Prior to digestion-filtration pancreata were intraductally injected with UW supplemented with Serva collagenase NB8 and neutral protease. Islet quality assessment determined viability, glucose stimulation index, mitochondrial activity, intracellular ATP content, and transplant function in diabetic nude mice. Pancreata oxygenated for 3 h at 20 degrees C yielded islet numbers similar to organs oxygenated at 4 degrees C. Compared to a storage temperature of 20 degrees C, preservation at 4 degrees C reduced islet ATP content (p < 0.05) as well as islet viability (p < 0.05). Nevertheless, PFD storage at 20 degrees C decreased insulin response to glucose compared to unstored pancreata (p < 0.05). In contrast to unstored pancreata or cold-stored organs, transplantation of islets isolated after oxygenation at 20 degrees C was characterized by an early loss of transplant function in 50% of recipients (p < 0.05). The present study demonstrates that PFD storage at 20 degrees C enhances islet ATP synthesis within a short period of oxygenation but deteriorates islet function. We conclude that the present data reflect an equilibration between reduced depression of metabolic activity resulting in damage of islets and temperature-stimulated acceleration of ATP synthesis. Future studies are required to adjust the optimum storage temperature for pancreas oxygenation in different species.

ATP Measurement Predicts Porcine Islet Transplantation Outcome in Nude Mice

Current nude mice islet transplantation studies cannot be used prospectively. Therefore, to predict transplantation outcomes, reliable and rapid assays for islet quality assessment are warranted. This study evaluated the predictive power of the porcine islet ATP content on the outcomes of islet transplantation in nude mice. Here, we report that the ATP measurement using a small number of handpicked islets with a diameter of 100 to 150 mum is a good predictor of islet graft efficacy in nude mice. Using receiver-operator characteristic analysis, the area under the curve of the ATP content using a small number of handpicked islets was 0.867 (95% confidence interval 0.744-0.989, P<0.001). The sensitivity and the specificity measured were 83.3% and 73.3%, respectively. In conclusion, a simple and a rapid measurement of intraislet ATP content could be a promising substitute for current nude mice islet transplantation studies.

Possible role of an ischemic preconditioning-like response mechanism in K ATP channel opener-mediated protection against streptozotocin-induced suppression of rat pancreatic islet function

Potassium channel openers (KCOs) decrease insulin secretion from β-cells. Some KCOs also protect against damage to β-cell function and type 1 diabetes in animal models. Previously we have found that the KCO NNC 55-0118 counteracted islet cell dysfunction, and this was associated with a lowering of the mitochondrial membrane potential (Δψ). Presently we aimed to explore whether inhibition of insulin secretion per se or rather inhibition of mitochondrial function correlates to counteraction of β-cell suppression. For this we used two novel KCOs (NNC 55-0321 and NNC 55-0462), which at certain concentrations have different actions regarding insulin secretion and the Δψ, with NNC 55-0321 being a potent inhibitor of Δψ and NNC 55-0462 being a potent inhibitor of insulin secretion. At 10 μM NNC 55-0321, but not with NNC 55-0462, the islet ATP content and ATP/ADP ratio was acutely decreased. This was accompanied by a complete protection against streptozotocin-induced suppression of islet insulin secretion using the former KCO. In cardiac research KCOs have been used to induce an ischemic preconditioning (IPC) response. In line with an IPC-like mechanism we found that NNC 55-0321 induced an initial free oxygen radical formation, PKC-ɛ isoform activation and a subsequent phosphorylation of the survival promoting factor Akt. Thus, KCOs may elicit mitochondrial events that resemble classical IPC seen in cardiomyocytes, and this could explain the enhanced islet cell function observed. KCOs with this property may be particularly interesting compounds to study as a rescue therapy during acute episodes of β-cell suppression/destruction.

Impaired Insulin Secretion by Diphenyleneiodium Associated with Perturbation of Cytosolic Ca2+ Dynamics in Pancreatic β-Cells

Pancreatic islets express the superoxide-producing nicotinamide adenine dinucleotide phosphate (NADPH) oxidase system, but its role remains unknown. To address this, we studied the mechanisms of impaired insulin secretion induced by diphenyleneiodium (DPI), an NADPH oxidase inhibitor. We investigated the effects of DPI on glucose- and nonfuel-stimulated insulin secretion, islet glucose metabolism, and intracellular Ca2+ concentration ([Ca2+]i) dynamics in rat islets and beta-cell line RINm5F cells. DPI did not affect insulin secretion at 3.3 mm glucose but totally suppressed insulin secretion stimulated by 16.7 mm glucose (percentage of control, 9.2 +/- 1.2%; P <0.001). DPI also inhibited insulin release by high K+-induced membrane depolarization (percentage of control, 36.0 +/- 5.3%; P <0.01) and protein kinase C activation (percentage of control, 30.2 +/- 10.6% in the presence of extracellular Ca2+, P <0.01; percentage of control, 42.0 +/- 4.7% in the absence of extracellular Ca2+, P <0.01). However, DPI had no effect on mastoparan-induced insulin secretion at 3.3 and 16.7 mm glucose under Ca2+-free conditions. DPI significantly suppressed islet glucose oxidation and ATP content through its known inhibitory action on complex I in the mitochondrial respiratory chain. On the other hand, DPI altered [Ca2+]i dynamics in response to high glucose and membrane depolarization, and DPI per se dose-dependently increased [Ca2+]i. The DPI-induced [Ca2+]i rise was associated with a transient increase in insulin secretion and was attenuated by removal of extracellular Ca2+, by L-type voltage-dependent Ca2+ channel blockers, by mitochondrial inhibitors, or by addition of 0.1 or 1.0 microm H2O2 exogenously. Our results showed that DPI impairment of insulin secretion involved altered Ca2+ signaling, suggesting that NADPH oxidase may modulate Ca2+ signaling in beta-cells.

Long-Term Exposure to Glucose and Lipids Inhibits Glucose-Induced Insulin Secretion Downstream of Granule Fusion With Plasma Membrane

Mouse beta-cells cultured at 15 mmol/l glucose for 72 h had reduced ATP-sensitive K+ (K(ATP)) channel activity (-30%), increased voltage-gated Ca2+ currents, higher intracellular free Ca2+ concentration ([Ca2+]i; +160%), more exocytosis (monitored by capacitance measurements, +100%), and greater insulin content (+230%) than those cultured at 4.5 mmol/l glucose. However, they released 20% less insulin when challenged with 20 mmol/l glucose. Glucose-induced (20 mmol/l) insulin secretion was reduced by 60-90% in islets cocultured at 4.5 or 15 mmol/l glucose and either oleate or palmitate (0.5 mmol/l). Free fatty acid (FFA)-induced inhibition of secretion was not associated with any major changes in [Ca2+]i or islet ATP content. Palmitate stimulated exocytosis by twofold or more but reduced K+-induced secretion by up to 60%. Basal (1 mmol/l glucose) K(ATP) channel activity was 40% lower in islets cultured at 4.5 mmol/l glucose plus palmitate and 60% lower in islets cultured at 15 mmol/l glucose plus either of the FFAs. Insulin content decreased by 75% in islets exposed to FFAs in the presence of high (15 mmol/l), but not low (4.5 mmol/l), glucose concentrations, but the number of secretory granules was unchanged. FFA-induced inhibition of insulin secretion was not associated with increased transcript levels of the apoptosis markers Bax (BclII-associated X protein) and caspase-3. We conclude that glucose and FFAs reduce insulin secretion by interference with the exit of insulin via the fusion pore.

Modulation of Insulin Secretion by Fatty Acyl Analogs

The secretagogue, the incretin-like, and the suppressive activities of long-chain fatty acids (LCFAs) in modulating insulin secretion in vivo and in cultured islets were simulated here by beta,beta'-tetramethyl-hexadecanedioic acid (M16) and alpha,alpha'-tetrachloro-tetradecanedioic acid (Cl-DICA). M16, but not Cl-DICA, serves as a substrate for ATP-dependent CoA thioesterification but is not further metabolized. M16, but not Cl-DICA, acted as a potent insulin secretagogue in islets cultured in basal but not high glucose. Short-term exposure to M16 or Cl-DICA resulted in activation of glucose- but not arginine-stimulated insulin secretion. Long-term exposure to M16, but not to Cl-DICA, resulted in suppression of glucose-, arginine-, and K(+)-stimulated insulin secretion; inhibition of glucose-induced proinsulin biosynthesis; and depletion of islets insulin. beta-Cell mass and islet ATP content remained unaffected. Hence, nonmetabolizable LCFA analogs may highlight discrete LCFA metabolites and pathways involved in modulating insulin secretion, which could be overlooked due to the rapid turnover of natural LCFA.

Effect of Donor Age on Function of Isolated Human Islets

This study intended to evaluate the impact of donor age on the function of isolated islets. Analysis of human islets from cadaveric donors (age 16-70 years) was performed using glucose-stimulated insulin release (GSIR) (n = 93), islet ATP content (n = 27), diabetic nude mouse bioassay (n = 72), and the insulin secretory function after single-donor clinical islet allotransplantation (n = 7). The GSIR index was significantly higher in younger donors (age < or =40 years) than in older donors and negatively correlated with the donor age (r = -0.535). Islet ATP was higher in younger donors (115.7 +/- 17.7 vs. 75.7 +/- 6.6 pmol/microg DNA). The diabetes reversal rate of mice with 2,000 IE was significantly higher in younger donors (96 vs. 68%). C-peptide increment to glucose during intravenous glucose tolerance test at days 90-120 after clinical transplantation showed negative correlation with donor age (r = -0.872) and positive correlation with the islet mass (r = 0.832). On the other hand, acute insulin response to arginine only showed correlation with the islet mass and not with donor age. These results show that insulin secretory response to glucose deteriorates with increasing age and that it may be related to changes in ATP generation in beta-cells.

Adenine Nucleotide Pattern in Rat Pancreatic Islets Exposed to Nutrient Secretagogues

The effects of D-glucose, D-mannose, D-galactose, Dglyceraldehyde, pyruvate, L-lactate, 2-ketoisocaproate, L-leucine, and/or L-glutamine on the ATP and ADP content of rat isolated pancreatic islets were reevaluated in order to compare changes evoked by these nutrient secretagogues in the islet ATP content and ATP/ADP ratio to their effects upon insulin release. Although being compatible with the fuel concept for nutrient-stimulated insulin secretion, the results of this study also argue against the monolithic view that the adenine nucleotide pattern in islet cells represents the sole coupling factor between metabolic and more distal events in the process of nutrient-stimulated insulin release.

Evaluation of Energy State of Islet Independent of Size Using a Newly Developed ATP Bioluminescence Assay

ATP content or energy charge (EC) of islets may be a good parameter to assess viability. In this study, we examined adenine nucleotides and EC of freshly isolated or 24-hour cultured rat islets of various diameters using a novel bioluminescent enzymatic cycling assay system. For freshly isolated islets, ATP content and islet diameter showed a high correlation ( r = 0.842, P < .001), but a significant correlation was not observed for cultured islets ( r = 0.284) when all islets were included for the analysis. When only the cultured islets with a diameter <350 μm were included for analysis, a significant correlation was observed ( r = 0.719). EC of freshly isolated islets fluctuated widely irrespective of diameter, in contrast with results of 24-hour cultured islets, which showed a stable, high EC, regardless of diameter. These data suggest that the ATP content of islets correlates with the islet size and that EC of islets widely fluctuates following isolation, indicating a significant role of monitoring ATP and EC of islets before transplantation.

Pancreatic β-Cell Lipoprotein Lipase Independently Regulates Islet Glucose Metabolism and Normal Insulin Secretion

Lipid and glucose metabolism are adversely affected by diabetes, a disease characterized by pancreatic beta-cell dysfunction. To clarify the role of lipids in insulin secretion, we generated mice with beta-cell-specific overexpression (betaLPL-TG) or inactivation (betaLPL-KO) of lipoprotein lipase (LPL), a physiologic provider of fatty acids. LPL enzyme activity and triglyceride content were increased in betaLPL-TG islets; decreased LPL enzyme activity in betaLPL-KO islets did not affect islet triglyceride content. Surprisingly, both betaLPL-TG and betaLPL-KO mice were strikingly hyperglycemic during glucose tolerance testing. Impaired glucose tolerance in betaLPL-KO mice was present at one month of age, whereas betaLPL-TG mice did not develop defective glucose homeostasis until approximately five months of age. Glucose-simulated insulin secretion was impaired in islets isolated from both mouse models. Glucose oxidation, critical for ATP production and triggering of insulin secretion mediated by the ATP-sensitive potassium (KATP) channel, was decreased in betaLPL-TG islets but increased in betaLPL-KO islets. Islet ATP content was not decreased in either model. Insulin secretion was defective in both betaLPL-TG and betaLPL-KO islets under conditions causing calcium-dependent insulin secretion independent of the KATP channel. These results show that beta-cell-derived LPL has two physiologically relevant effects in islets, the inverse regulation of glucose metabolism and the independent mediation of insulin secretion through effects distal to membrane depolarization.