Overnight culture unmasks glucose-induced insulin secretion in mouse islets lacking ATP-sensitive K+ channels by improving the triggering Ca2+ signal

A current model ascribes glucose-induced insulin secretion to the interaction of a triggering pathway (K(ATP) channel-dependent Ca(2+) influx and rise in cytosolic [Ca(2+)](c)) and an amplifying pathway (K(ATP) channel-independent augmentation of secretion without further increase of [Ca(2+)](c)). However, several studies of sulfonylurea receptor 1 null mice (Sur1KO) failed to measure significant effects of glucose in their islets lacking K(ATP) channels. We addressed this issue that challenges the model. Compared with controls, fresh Sur1KO islets showed slightly elevated basal [Ca(2+)](c) and insulin secretion. In 15 mm glucose, the absolute rate of secretion was approximately 3-fold lower in Sur1KO than control islets, with only poor increase above base line. Overnight culture of Sur1KO islets in 10 mm glucose (not in 5 mm) augmented basal insulin secretion and considerably improved the response to 15 mm glucose, which reached higher values than in control islets, in which culture had little impact. Glucose stimulation during KCl depolarization showed that the amplifying pathway is functional in fresh and cultured Sur1KO islets. The differences in insulin secretion between fresh and cultured Sur1KO islets and between Sur1KO and control islets were not attributable to differences in insulin content, glucose oxidation rate, or synchronization of [Ca(2+)](c) oscillations. The unmasking of glucose-induced insulin secretion in beta-cells lacking K(ATP) channels is paradoxically due to improvement in the production of a triggering signal (elevated [Ca(2+)](c)). The results show that K(ATP) channels are not the only transducer of glucose effects on [Ca(2+)](c) in beta-cells. They explain controversies in the literature and refute arguments raised against the model implicating an amplifying pathway in glucose-induced insulin secretion.     

Both triggering and amplifying pathways contribute to fuel-induced insulin secretion in the absence of sulfonylurea receptor-1 in pancreatic beta-cells

In normal beta-cells glucose induces insulin secretion by activating both a triggering pathway (closure of K(ATP) channels, depolarization, and rise in cytosolic [Ca(2+)](i)) and an amplifying pathway (augmentation of Ca(2+) efficacy on exocytosis). It is unclear if and how nutrients can regulate insulin secretion by beta-cells lacking K(ATP) channels (Sur1 knockout mice). We compared glucose- and amino acid-induced insulin secretion and [Ca(2+)](i) changes in control and Sur1KO islets. In 1 mm glucose (non-stimulatory for controls), the triggering signal [Ca(2+)](i) was high (loss of regulation) and insulin secretion was stimulated in Sur1KO islets. This "basal" secretion was decreased or increased by imposed changes in [Ca(2+)](i) and was dependent on ATP production, indicating that both triggering and amplifying signals are involved. High glucose stimulated insulin secretion in Sur1KO islets, by an unsuspected, transient increase in [Ca(2+)](i) and a sustained activation of the amplifying pathway. Unlike controls, Sur1KO islets were insensitive to diazoxide and tolbutamide, which rules out effects of either drug at sites other than K(ATP) channels. Amino acids potently increased insulin secretion by Sur1KO islets through both a further electrogenic rise in [Ca(2+)](i) and a metabolism-dependent activation of the amplifying pathway. After sulfonylurea blockade of their K(ATP) channels, control islets qualitatively behaved like Sur1KO islets, but their insulin secretion rate was consistently lower for a similar or even higher [Ca(2+)](i). In conclusion, fuel secretagogues can control insulin secretion in beta-cells without K(ATP) channels, partly by an unsuspected influence on the triggering [Ca(2+)](i) signal and mainly by the modulation of a very effective amplifying pathway.     

Impaired glucagon secretory responses in mice lacking the type 1 sulfonylurea receptor

Pancreatic alpha-cells, like beta-cells, express ATP-sensitive K(+) (K(ATP)) channels. To determine the physiological role of K(ATP) channels in alpha-cells, we examined glucagon secretion in mice lacking the type 1 sulfonylurea receptor (Sur1). Plasma glucagon levels, which were increased in wild-type mice after an overnight fast, did not change in Sur1 null mice. Pancreas perfusion studies showed that Sur1 null pancreata lacked glucagon secretory responses to hypoglycemia and to synergistic stimulation by arginine. Pancreatic alpha-cells isolated from wild-type animals exhibited oscillations of intracellular free Ca(2+) concentration ([Ca(2+)](i)) in the absence of glucose that became quiescent when the glucose concentration was increased. In contrast, Sur1 null alpha-cells showed continuous oscillations in [Ca(2+)](i) regardless of the glucose concentration. These findings indicate that K(ATP) channels in alpha-cells play a key role in regulating glucagon secretion, thereby adding to the paradox of how mice that lack K(ATP) channels maintain euglycemia.     

Sur1 knockout mice. A model for K(ATP) channel-independent regulation of insulin secretion

Sur1 knockout mouse beta-cells lack K(ATP) channels and show spontaneous Ca(2+) action potentials equivalent to those seen in patients with persistent hyperinsulinemic hypoglycemia of infancy, but the mice are normoglycemic unless stressed. Sur1(-/-) islets lack first phase insulin secretion and exhibit an attenuated glucose-stimulated second phase secretion. Loss of the first phase leads to mild glucose intolerance, whereas reduced insulin output is consistent with observed neonatal hyperglycemia. Loss of K(ATP) channels impairs the rate of return to a basal secretory level after a fall in glucose concentration. This leads to increased hypoglycemia upon fasting and contributes to a very early, transient neonatal hypoglycemia. Whereas persistent hyperinsulinemic hypoglycemia of infancy underscores the importance of the K(ATP)-dependent ionic pathway in control of insulin release, the Sur1(-/-) animals provide a novel model for study of K(ATP)-independent pathways that regulate insulin secretion.     

INGAP-PP up-regulates the expression of genes and proteins related to K+ ATP channels and ameliorates Ca2+ handling in cultured adult rat islets

Islet Neogenesis Associated Protein (INGAP) increases pancreatic beta-cell mass and potentiates glucose-induced insulin secretion. Here, we investigated the effects of the pentadecapeptide INGAP-PP in adult cultured rat islets upon the expression of proteins constitutive of the K(+)(ATP) channel, Ca(2+) handling, and insulin secretion. The islets were cultured in RPMI medium with or without INGAP-PP for four days. Thereafter, gene (RT-PCR) and protein expression (Western blotting) of Foxa2, SUR1 and Kir6.2, cytoplasmic Ca(2+) ([Ca(2+)](i)), static and dynamic insulin secretion, and (86)Rb efflux were measured. INGAP-PP increased the expression levels of Kir6.2, SUR1 and Foxa2 genes, and SUR1 and Foxa2 proteins. INGAP-PP cultured islets released significantly more insulin in response to 40 mM KCl and 100 muM tolbutamide. INGAP-PP shifted to the left the dose-response curve of insulin secretion to increasing concentrations of glucose (EC(50) of 10.0+/-0.4 vs. 13.7+/-1.5 mM glucose of the controls). It also increased the first phase of insulin secretion elicited by either 22.2 mM glucose or 100 microM tolbutamide and accelerated the velocity of glucose-induced reduction of (86)Rb efflux in perifused islets. These effects were accompanied by a significant increase in [Ca(2+)](i) and the maintenance of a considerable degree of [Ca(2+)](i) oscillations. These results confirm that the enhancing effect of INGAP-PP upon insulin release, elicited by different secretagogues, is due to an improvement of the secretory function in cultured islets. Such improvement is due, at least partly, to an increased K(+)(ATP) channel protein expression and/or changing in the kinetic properties of these channels and augmented [Ca(2+)](i) response. Accordingly, INGAP-PP could potentially be used to maintain the functional integrity of cultured islets and eventually, for the prevention and treatment of diabetes.     

Secretory phospholipase A2 is released from pancreatic beta-cells and stimulates insulin secretion via inhibition of ATP-dependent K+ channels

The release of sPLA(2) from single mouse pancreatic beta-cells was monitored using a fluorescent substrate of the enzyme incorporated in the outer leaflet of the plasma membrane. Stimulation of beta-cells with agents that increased cytosolic free Ca(2+) concentration ([Ca(2+)](i)) induced a rapid release of sPLA(2) to the extracellular medium. Exogenous sPLA(2) strongly stimulated insulin secretion in mouse pancreatic islets at both basal and elevated glucose concentrations. The stimulation of insulin secretion by sPLA(2) was mediated via inhibition of ATP-dependent K(+) channels and an increase in [Ca(2+)](i). Measurements of cell capacitance in single beta-cells revealed that sPLA(2) did not modify depolarisation-induced exocytosis. Our data suggest that a positive feedback regulation of insulin secretion by co-released sPLA(2) is operational in pancreatic beta-cells and point to this enzyme as an autocrine regulator of insulin secretion.     

5-amino-imidazole carboxamide riboside acutely potentiates glucose-stimulated insulin secretion from mouse pancreatic islets by KATP channel-dependent and -independent pathways

AMP-activated protein kinase (AMPK) is an important signaling effector that couples cellular metabolism and function. The effects of AMPK activation on pancreatic beta-cell function remain unresolved. We used 5-amino-imidazole carboxamide riboside (AICAR), an activator of AMPK, to define the signaling mechanisms linking the activation of AMPK with insulin secretion. Application of 300 microM AICAR to mouse islets incubated in 5-14 mM glucose significantly increased AMPK activity and potentiated insulin secretion. AICAR inhibited ATP-sensitive K(+) (K(ATP)) channels and increased the frequency of glucose-induced calcium oscillations in islets incubated in 8-14 mM glucose. At lower glucose concentration (5mM) AICAR did not affect K(ATP) activity or intracellular ([Ca(2+)](i)). AICAR also did not inhibit (86)Rb(+) efflux from islets isolated from Sur1(-/-) mice that lack K(ATP) channels yet significantly potentiated glucose stimulated insulin secretion. Our data suggest that AICAR stimulates insulin secretion by both K(ATP) channel-dependent and -independent pathways.     

Oleic acid interacts with GPR40 to induce Ca2+ signaling in rat islet beta-cells: mediation by PLC and L-type Ca2+ channel and link to insulin release

It has long been thought that long-chain free fatty acids (FFAs) stimulate insulin secretion via mechanisms involving their metabolism in pancreatic beta-cells. Recently, it was reported that FFAs function as endogenous ligands for GPR40, a G protein-coupled receptor, to amplify glucose-stimulated insulin secretion in an insulinoma cell line and rat islets. However, signal transduction mechanisms for GPR40 in beta-cells are little known. The present study was aimed at elucidating GPR40-linked Ca(2+) signaling mechanisms in rat pancreatic beta-cells. We employed oleic acid (OA), an FFA that has a high affinity for the rat GPR40, and examined its effect on cytosolic Ca(2+) concentration ([Ca(2+)](i)) in single beta-cells by fura 2 fluorescence imaging. OA at 1-10 microM concentration-dependently increased [Ca(2+)](i) in the presence of 5.6, 8.3, and 11.2 mM, but not 2.8 mM, glucose. OA-induced [Ca(2+)](i) increases at 11.2 mM glucose were inhibited in beta-cells transfected with small interfering RNA targeted to rat GPR40 mRNA. OA-induced [Ca(2+)](i) increases were also inhibited by phospholipase C (PLC) inhibitors, U73122 and neomycin, Ca(2+)-free conditions, and an L-type Ca(2+) channel blocker, nitrendipine. Furthermore, OA increased insulin release from isolated islets at 8.3 mM glucose, and it was markedly attenuated by PLC and L-type Ca(2+) channel inhibitors. These results demonstrate that OA interacts with GPR40 to increase [Ca(2+)](i) via PLC- and L-type Ca(2+) channel-mediated pathway in rat islet beta-cells, which may be link to insulin release.     

Pharmacological properties and functional role of Kslow current in mouse pancreatic beta-cells: SK channels contribute to Kslow tail current and modulate insulin secretion

The pharmacological properties of slow Ca(2+)-activated K(+) current (K(slow)) were investigated in mouse pancreatic beta-cells and islets to understand how K(slow) contributes to the control of islet bursting, [Ca(2+)](i) oscillations, and insulin secretion. K(slow) was insensitive to apamin or the K(ATP) channel inhibitor tolbutamide, but UCL 1684, a potent and selective nonpeptide SK channel blocker reduced the amplitude of K(slow) tail current in voltage-clamped mouse beta-cells. K(slow) was also selectively and reversibly inhibited by the class III antiarrythmic agent azimilide (AZ). In isolated beta-cells or islets, pharmacologic inhibition of K(slow) by UCL 1684 or AZ depolarized beta-cell silent phase potential, increased action potential firing, raised [Ca(2+)](i), and enhanced glucose-dependent insulin secretion. AZ inhibition of K(slow) also supported mediation by SK, rather than cardiac-like slow delayed rectifier channels since bath application of AZ to HEK 293 cells expressing SK3 cDNA reduced SK current. Further, AZ-sensitive K(slow) current was extant in beta-cells from KCNQ1 or KCNE1 null mice lacking cardiac slow delayed rectifier currents. These results strongly support a functional role for SK channel-mediated K(slow) current in beta-cells, and suggest that drugs that target SK channels may represent a new approach for increasing glucose-dependent insulin secretion. The apamin insensitivity of beta-cell SK current suggests that beta-cells express a unique SK splice variant or a novel heteromultimer consisting of different SK subunits.     

Glucose-induced cytosolic pH changes in beta-cells and insulin secretion are not causally related: studies in islets lacking the Na+/H+ exchangeR NHE1

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

Glucose induces synchronous mitochondrial calcium oscillations in intact pancreatic islets

Mitochondria shape Ca(2+) signaling and exocytosis by taking up calcium during cell activation. In addition, mitochondrial Ca(2+) ([Ca(2+)](M)) stimulates respiration and ATP synthesis. Insulin secretion by pancreatic beta-cells is coded mainly by oscillations of cytosolic Ca(2+) ([Ca(2+)](C)), but mitochondria are also important in excitation-secretion coupling. Here, we have monitored [Ca(2+)](M) in single beta-cells within intact mouse islets by imaging bioluminescence of targeted aequorins. We find an increase of [Ca(2+)](M) in islet-cells in response to stimuli that induce either Ca(2+) entry, such as extracellular glucose, tolbutamide or high K(+), or Ca(2+) mobilization from the intracellular stores, such as ATP or carbamylcholine. Many cells responded to glucose with synchronous [Ca(2+)](M) oscillations, indicating that mitochondrial function is coordinated at the whole islet level. Mitochondrial Ca(2+) uptake in permeabilized beta-cells increased exponentially with increasing [Ca(2+)], and, particularly, it became much faster at [Ca(2+)](C)>2 microM. Since the bulk [Ca(2+)](C) signals during stimulation with glucose are smaller than 2 microM, mitochondrial Ca(2+) uptake could be not uniform, but to take place preferentially from high [Ca(2+)](C) microdomains formed near the mouth of the plasma membrane Ca(2+) channels. Measurements of mitochondrial NAD(P)H fluorescence in stimulated islets indicated that the [Ca(2+)](M) changes evidenced here activated mitochondrial dehydrogenases and therefore they may modulate the function of beta-cell mitochondria. Diazoxide, an activator of K(ATP), did not modify mitochondrial Ca(2+) uptake.     

R-type Ca(2+)-channel-evoked CICR regulates glucose-induced somatostatin secretion

Pancreatic islets have a central role in blood glucose homeostasis. In addition to insulin-producing beta-cells and glucagon-secreting alpha-cells, the islets contain somatostatin-releasing delta-cells. Somatostatin is a powerful inhibitor of insulin and glucagon secretion. It is normally secreted in response to glucose and there is evidence suggesting its release becomes perturbed in diabetes. Little is known about the control of somatostatin release. Closure of ATP-regulated K(+)-channels (K(ATP)-channels) and a depolarization-evoked increase in cytoplasmic free Ca(2+) concentration ([Ca(2+)](i)) have been proposed to be essential. Here, we report that somatostatin release evoked by high glucose (>or=10 mM) is unaffected by the K(ATP)-channel activator diazoxide and proceeds normally in K(ATP)-channel-deficient islets. Glucose-induced somatostatin secretion is instead primarily dependent on Ca(2+)-induced Ca(2+)-release (CICR). This constitutes a novel mechanism for K(ATP)-channel-independent metabolic control of pancreatic hormone secretion.     

Normalization of intracellular Ca(2+) induces a glucose-responsive state in glucose-unresponsive beta-cells

Although intracellular Ca(2+) in pancreatic beta-cells is the principal signal for insulin secretion, the effect of chronic elevation of the intracellular Ca(2+) concentration ([Ca(2+)](i)) on insulin secretion is poorly understood. We recently established two pancreatic beta-cell MIN6 cell lines that are glucose-responsive (MIN6-m9) and glucose-unresponsive (MIN6-m14). In the present study we have determined the cause of the glucose unresponsiveness in MIN6-m14. Initially, elevated [Ca(2+)](i) was observed in MIN6-m14, but normalization of the [Ca(2+)](i) by nifedipine, a Ca(2+) channel blocker, markedly improved the intracellular Ca(2+) response to glucose and the glucose-induced insulin secretion. The expression of subunits of ATP-sensitive K(+) channels and voltage-dependent Ca(2+) channels were increased at both mRNA and protein levels in MIN6-m14 treated with nifedipine. As a consequence, the functional expression of these channels at the cell surface, both of which are decreased in MIN6-m14 without nifedipine treatment, were increased significantly. Contrariwise, Bay K8644, a Ca(2+) channel agonist, caused severe impairment of glucose-induced insulin secretion in glucose-responsive MIN6-m9 due to decreased expression of the channel subunits. Chronically elevated [Ca(2+)](i), therefore, is responsible for the glucose unresponsiveness of MIN6-m14. The present study also suggests normalization of [Ca(2+)](i) in pancreatic beta-cells as a therapeutic strategy in treatment of impaired insulin secretion.     

Tetracaine stimulates insulin secretion through the mobilization of Ca2+ from thapsigargin- and IP3-insensitive Ca2+ reservoir in pancreatic beta-cells

The effect of tetracaine on 45Ca efflux, cytoplasmic Ca2+ concentration [Ca2+]i, and insulin secretion in isolated pancreatic islets and beta-cells was studied. In the absence of external Ca2+, tetracaine (0.1-2.0 mM) increased the 45Ca efflux from isolated islets in a dose-dependentOFF efflux caused by 50 mM K+ or by the association of carbachol (0.2 mM) and 50 mM K+. Tetracaine permanently increased the [Ca2+]i in isolated beta-cells in Ca2+-free medium enriched with 2.8 mM glucose and 25 microM D-600 (methoxiverapamil). This effect was also observed in the presence of 10 mM caffeine or 1 microM thapsigargin. In the presence of 16.7 mM glucose, tetracaine transiently increased the insulin secretion from islets perfused in the absence and presence of external Ca2+. These data indicate that tetracaine mobilises Ca2+ from a thapsigargin-insensitive store and stimulates insulin secretion in the absence of extracellular Ca2+. The increase in 45Ca efflux caused by high concentrations of K+ and by carbachol indicates that tetracaine did not interfere with a cation or inositol triphosphate sensitive Ca2+ pool in beta-cells.     

Neuropeptide W in the rat pancreas: potentiation of glucose-induced insulin release and Ca2+ influx through L-type Ca2+ channels in beta-cells and localization in islets

Neuropeptide W (NPW) is a regulatory peptide that acts via two subtypes of G protein-coupled receptors, GPR7 and GPR8. Evidence has been provided that NPW is involved in the central regulation of energy homeostasis and feeding behavior. In this study, we examined the effects of NPW on insulin release and localization of NPW in the rat pancreas. NPW (10-100 nM) significantly increased insulin release in the presence of 8.3 mM, but not 2.8 mM, glucose in the isolated rat islets. By fura-2 microfluorometry, NPW (1-100 nM) concentration-dependently increased cytosolic Ca(2+) concentration ([Ca(2+)](i)) at 8.3 mM glucose in rat single beta-cells. The NPW-induced [Ca(2+)](i) increase was abolished under external Ca(2+)-free conditions and by an L-type Ca(2+) channel blocker nifedipine (10 microM). RT-PCR analysis revealed that mRNA for NPW was expressed in the rat pancreas and hypothalamus. Double immunohistochemical analysis showed that NPW-immunoreactivity was found in islets and co-localized with insulin-containing beta-cells, but not glucagon-containing alpha-cells and somatostatin-containing delta-cells. These results suggest that NPW could serve as a local modulator of glucose-induced insulin release in rat islets. NPW directly activates beta-cells to enhance Ca(2+) influx through voltage-dependent L-type Ca(2+) channels and potentiates glucose-induced insulin release.     

The Ca2+ dynamics of isolated mouse beta-cells and islets: implications for mathematical models

[Ca(2+)](i) and electrical activity were compared in isolated beta-cells and islets using standard techniques. In islets, raising glucose caused a decrease in [Ca(2+)](i) followed by a plateau and then fast (2-3 min(-1)), slow (0.2-0.8 min(-1)), or a mixture of fast and slow [Ca(2+)](i) oscillations. In beta-cells, glucose transiently decreased and then increased [Ca(2+)](i), but no islet-like oscillations occurred. Simultaneous recordings of [Ca(2+)](i) and electrical activity suggested that differences in [Ca(2+)](i) signaling are due to differences in islet versus beta-cell electrical activity. Whereas islets exhibited bursts of spikes on medium/slow plateaus, isolated beta-cells were depolarized and exhibited spiking, fast-bursting, or spikeless plateaus. These electrical patterns in turn produced distinct [Ca(2+)](i) patterns. Thus, although isolated beta-cells display several key features of islets, their oscillations were faster and more irregular. beta-cells could display islet-like [Ca(2+)](i) oscillations if their electrical activity was converted to a slower islet-like pattern using dynamic clamp. Islet and beta-cell [Ca(2+)](i) changes followed membrane potential, suggesting that electrical activity is mainly responsible for the [Ca(2+)] dynamics of beta-cells and islets. A recent model consisting of two slow feedback processes and passive endoplasmic reticulum Ca(2+) release was able to account for islet [Ca(2+)](i) responses to glucose, islet oscillations, and conversion of single cell to islet-like [Ca(2+)](i) oscillations. With minimal parameter variation, the model could also account for the diverse behaviors of isolated beta-cells, suggesting that these behaviors reflect natural cell heterogeneity. These results support our recent model and point to the important role of beta-cell electrical events in controlling [Ca(2+)](i) over diverse time scales in islets.     

Contribution of the endoplasmic reticulum to the glucose-induced [Ca(2+)](c) response in mouse pancreatic islets

Thapsigargin (TG), a blocker of Ca(2+) uptake by the endoplasmic reticulum (ER), was used to evaluate the contribution of the organelle to the oscillations of cytosolic Ca(2+) concentration ([Ca(2+)](c)) induced by repetitive Ca(2+) influx in mouse pancreatic beta-cells. Because TG depolarized the plasma membrane in the presence of glucose alone, extracellular K(+) was alternated between 10 and 30 mM in the presence of diazoxide to impose membrane potential (MP) oscillations. In control islets, pulses of K(+), mimicking regular MP oscillations elicited by 10 mM glucose, induced [Ca(2+)](c) oscillations whose nadir remained higher than basal [Ca(2+)](c). Increasing the depolarization phase of the pulses while keeping their frequency constant (to mimic the effects of a further rise of the glucose concentration on MP) caused an upward shift of the nadir of [Ca(2+)](c) oscillations that was reproduced by raising extracellular Ca(2+) (to increase Ca(2+) influx) without changing the pulse protocol. In TG-pretreated islets, the imposed [Ca(2+)](c) oscillations were of much larger amplitude than in control islets and occurred on basal levels. During intermittent trains of depolarizations, control islets displayed mixed [Ca(2+)](c) oscillations characterized by a summation of fast oscillations on top of slow ones, whereas no progressive summation of the fast oscillations was observed in TG-pretreated islets. In conclusion, the buffering capacity of the ER in pancreatic beta-cells limits the amplitude of [Ca(2+)](c) oscillations and may explain how the nadir between oscillations remains above baseline during regular oscillations or gradually increases during mixed [Ca(2+)](c) oscillations, two types of response observed during glucose stimulation.     

Attenuation of insulin secretion by insulin-like growth factor binding protein-1 in pancreatic beta-cells

IGFBP-1 is involved in glucohomeostasis, but the direct action of IGFBP-1 on the beta-cell remains unclear. Incubation of dispersed mouse beta-cells with IGFBP-1 for 30min inhibited insulin secretion stimulated by glucose, glucagon-like peptide 1 (GLP-1) or tolbutamide without changes in basal release of insulin and in cytosolic free Ca(2+) concentration ([Ca(2+)](i)) and NAD(P)H evoked by glucose. In contrast, IGFBP-1 augmented glucose-stimulated insulin secretion in intact islets, associated with a reduced somatostatin secretion. These results suggest a suppressive action of IGFBP-1 on insulin secretion in isolated beta-cells through a mechanism distal to energy generating steps and not involving regulation of [Ca(2+)](i). In contrast, IGFBP-1 amplifies glucose-stimulated insulin secretion in intact islets, possibly by suppressing somatostatin secretion. These direct modulatory influences of IGFBP-1 on insulin secretion may imply an important regulatory role of IGFBP-1 in vivo and in the pathogenesis of type 2 diabetes, in which loss of insulin release is an early pathogenetic event.     

The elevation of glutamate content and the amplification of insulin secretion in glucose-stimulated pancreatic islets are not causally related

Glucose increases insulin secretion by raising cytoplasmic Ca(2+) ([Ca(2+)](i)) in beta-cells (triggering pathway) and augmenting the efficacy of Ca(2+) on exocytosis (amplifying pathway). It has been suggested that glutamate formed from alpha-ketoglutarate is a messenger of the amplifying pathway (Maechler, P., and Wollheim, C. B. (1999) Nature 402, 685-689). This hypothesis was tested with mouse islets depolarized with 30 mm KCl (+ diazoxide) or with a saturating concentration of sulfonylurea. Because [Ca(2+)](i) was elevated under these conditions, insulin secretion was stimulated already in 0 mm glucose. The amplification of secretion produced by glucose was accompanied by an increase in islet glutamate. However, glutamine (0.5-2 mm) markedly augmented islet glutamate without affecting insulin secretion, whereas glucose augmented secretion without influencing glutamate levels when these were elevated by glutamine. Allosteric activation of glutamate dehydrogenase by BCH (2-amino 2-norbornane carboxylic acid) lowered islet glutamate but increased insulin secretion. Similar insulin secretion thus occurred at very different cellular glutamate levels. Glutamine did not affect islet [Ca(2+)](i) and pH(i), whereas glucose and BCH slightly raised pH(i) and either slightly decreased (30 mm KCl) or increased (tolbutamide) [Ca(2+)](i). The general dissociation between changes in islet glutamate and insulin secretion refutes a role of beta-cell glutamate in the amplification of insulin secretion by glucose.