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Congenital hyperinsulinism causes persistent hypoglycemia in neonates and infants. Most often, uncontrolled insulin secretion (IS) results from a lack of functional [K.sub.ATP] channels in all β-cells or only in β-cells within a resectable focal lesion. In more rare cases, without [K.sub.ATP] channel mutations, hyperfunctional islets are confined within few lobules, whereas hypofunctional islets are present throughout the pancreas. They also can be cured by selective partial pancreatectomy; however, unlike those with a [K.sub.ATP] focal lesion, they show clinical sensitivity to diazoxide. Here, we characterized in vitro IS by fragments of pathological and adjacent normal pancreas from six such cases. Responses of normal pancreas were unremarkable. In pathological region, IS was elevated at 1 mmol/L and was further increased by 15 mmol/L glucose. Diazoxide suppressed IS and tolbutamide antagonized the inhibition. The most conspicuous anomaly was a large stimulation of IS by 1 mmol/L glucose. In five of six cases, immunohistochemistry revealed undue presence of 1ow-[K.sub.m] hexokinase-I in β-cells of hyperfunctional islets only. In one case, an activating mutation of glucokinase (I211F) was found in pathological islets only. Both abnormalities, attributed to somatic genetic events, may account for inappropriate IS at low glucose levels by a subset of β-cells. They represent a novel cause of focal congenital hyperinsulinism., Congenital hyperinsulinism (CHI) is the major cause of severe and persistent hypoglycemia in neonates and infants and is a brain-damaging and potentially life-threatening condition (1-3). Excessive secretion of insulin by [...]

Pancreatic islets were isolated from 16 nondiabetic organ donors and, after culture for ~2 days in 5 mmol/l glucose, were perifused to characterize nutrient-induced insulin secretion in human islets. Stepwise increases from 0 to 30 mmol/l glucose (eight 30-min steps) evoked concentration-dependent insulin secretion with a threshold at 3-4 mmol/l glucose, [K.sub.m] at 6.5 mmol/l glucose, and [V.sub.max] at 15 mmol/l glucose. An increase from 1 to 15 mmol/l glucose induced biphasic insulin secretion with a prominent first phase (peak increase of ~18-fold) and a sustained, flat second phase (~10-fold increase), which were both potentiated by forskolin. The central role of ATP-sensitive [K.sup.+] channels in the response to glucose was established by abrogation of insulin secretion by diazoxide and reversible restoration by tolbutamide. Depolarization with tolbutamide or KCl (plus diazoxide) triggered rapid insulin secretion in 1 mmol/l glucose. Subsequent application of 15 mmol/l glucose further increased insulin secretion, showing that the amplifying pathway is operative. In control medium, glutamine alone was ineffective, but its combination with leucine or nonmetabolized 2-amino-bicyclo [2,2,1]-heptane-2-carboxylic acid (BCH) evoked rapid insulin secretion. The effect of BCH was larger in low glucose than in high glucose. In contrast, the insulin secretion response to arginine or a mixture of four amino acids was potentiated by glucose or tolbutamide. Palmitate slightly augmented insulin secretion only at the supraphysiological palmitate-to-albumin ratio of 5. Inosine and membrane-permeant analogs of pyruvate, glutamate, or succinate increased insulin secretion in 3 and 10 mmol/l glucose, whereas lactate and pyruvate had no effect. In conclusion, nutrient-induced insulin secretion in normal human islets is larger than often reported. Its characteristics are globally similar to those of insulin secretion by rodent islets, with both triggering and amplifying pathways. The pattern of the biphasic response to glucose is superimposable on that in mouse islets, but the concentration-response curve is shifted to the left, and various nutrients, in particular amino acids, influence insulin secretion within the physiological range of glucose concentrations., Glucose homeostasis requires precise control of insulin secretion by pancreatic β-cells. This control is primarily ensured by glucose itself, but it also involves other nutrients and numerous neural and hormonal [...]

The mechanisms underlying biphasic insulin secretion have not been completely elucidated. We compared the pattern of plasma insulin changes during hyperglycemic clamps in mice to that of glucose-induced insulin secretion and cytosolic calcium concentration ([[[Ca.sup.2+]].sub.c]) changes in perifused mouse islets. Anesthetized mice were infused with glucose to clump blood glucose at 8.5 (baseline), 11.1, 16.7, or 30 mmol/l. A first-phase insulin response consistently peaked at 1 min, and a slowly ascending second phase occurred at 16.7 and 30 mmol/l glucose. Glucose-induced insulin secretion in vivo is thus biphasic, with a similarly increasing second phase in the mouse as in humans. In vitro, square-wave stimulation from a baseline of 3 mmol/; glucose induced similar biphasic insulin secretion and [[[Ca.sup.2+]].sub.c] increases, with sustained and flat second phases. The glucose dependency (3-30 mmol/l) of both changes was sigmoidal with, however, a shift to the right of the relation for insulin secretion compared with that for [[[Ca.sup.2+]].sub.c]. The maximum [[Ca.sup.2+]].sub.c] increase was achieved by glucose concentrations, causing half-maximum insulin secretion. Because this was true for both phases, we propose that contrary to current concepts, amplifying signals are also implicated in first-phase glucose-induced insulin secretion. To mimic in vivo conditions, islets were stimulated with high glucose after being initially perifused with 8.5 instead of 3.0 mmol/l glucose. First-phase insulin secretion induced by glucose at 11.1, 16.7, and 30 mmol/l was decreased by ~50%, an inhibition that could not be explained by commensurate decreases in [[[Ca.sup.2+]].sub.c] or in the pool of readily releasable granules. Also unexpected was the gradually ascending pattern of the second phase, now similar to that in vivo. These observations indicated that variations in prestimulatory glucose can secondarily affect the magnitude and pattern of subsequent glucose-induced insulin secretion., Biphasic insulin secretion is the normal response of β-cells to a rapid and sustained increase in glucose concentration (1,2). The first phase corresponds to a prompt and marked, but transient [...]

Two sarcoendoplasmic reticulum [Ca.sup.2+]-ATPases, SERCA3 and SERCA2b, are expressed in pancreatic islets. Immunocytochemistry showed that SERCA3 is restricted to β-cells in the mouse pancreas. Control and SERCA3-deficient mice were used to evaluate the role of SERCA3 in β-cell cytosolic-free [Ca.sup.2+] concentration ([[Ca.sup.2+]].sub.c) regulation, insulin secretion, and glucose homeostasis. Basal [[[Ca.sup.2+]].sub.c] was not increased by SERCA3 ablation. Stimulation with glucose induced a transient drop in basal [[[Ca.sup.2+]].sub.2+] that was suppressed by inhibition of all SERCAs with thapsigargin (TG) but unaffected by selective SERCA3 ablation. [Ca.sup.2+] mobilization by acetylcholine was normal in SERCA3-deficient β-cells. In contrast, [[[Ca.sup.2+]].sub.c] oscillations resulting from intermittent glucose-stimulated [Ca.sup.2+] influx and [[[Ca.sup.2+]].sub.c] transients induced by pulses of high [K.sup.+] were similarly affected by SERCA3 ablation or TG pretreatment of control islets; their amplitude was increased and their slow descending phase suppressed. This suggests that, during the decay of each oscillation, the endoplasmic reticulum releases [Ca.sup.2+] that was pumped by SERCA3 during the upstroke phase. SERCA3 ablation increased the insulin response of islets to 15 mmol/l glucose. However, basal and postprandial plasma glucose and insulin concentrations in SERCA3-deficient mice were normal. In conclusion, SERCA2b, but not SERCA3, is involved in basal [[[Ca.sup.2+]].sub.c] regulation in β-cells. SERCA3 becomes operative when [[Ca.sup.2+]].sub.c] rises and is required for normal [[[Ca.sup.2+]].sub.c] oscillations in response to glucose. However, a lack of SERCA3 is insufficient in itself to alter glucose homeostasis or impair insulin secretion in mice., The sarcoendoplasmic reticulum [Ca.sup.2+]-ATPase (SERCA) transfers [Ca.sup.2+] from the cytosol to the lumen of the reticulum. Three SERCA genes (SERCA1-3) have been identified, all of which can generate several alternatively [...]

Rapid and sustained stimulation of β-cells with glucose induces biphasic insulin secretion. The two phases appear to reflect a characteristic of stimulus-secretion coupling in each β-cell rather than heterogeneity in [...]

Sulfonylureas stimulate insulin secretion by blocking ATP-sensitive [K.sup.+] channels ([K.sup.+]-ATP channels) of the Β-cell membrane, thereby causing depolarization, [Ca.sup.2+] influx, and rise in cytoplasmic [Ca.sup.2+] concentration ([[[Ca.sup.2+]].sub.i]), whereas diazoxide inhibits [...]

The mechanisms by which fatty acids increase insulin release are not known. In this study, mouse islets were used as a model and palmitate as a reference compound to study [...]

Amplification of insulin secretion by cAMP is mediated by protein kinase A (PKA) and exchange protein directly activated by cAMP (Epac). Using selective activators, we determined how each effector influences the cytosolic free Ca2+concentration ([Ca2+]c) and insulin secretion in mouse islets. Alone PKA activator amplified glucose- and tolbutamide-induced insulin secretion, with a greater impact on second than first phase. Epac activator strongly amplified both phases in response to either secretagogue. Amplification was even greater when activators were combined. Although both activators similarly amplified glucose-induced insulin secretion, Epac activator was particularly efficient on tolbutamide-induced insulin secretion. That greater efficacy is attributed to higher [Ca2+]crather than interaction of tolbutamide with Epac, because it was also observed during KCl stimulation. Moreover, in contrast to Epac activator, tolbutamide was inactive when insulin secretion was increased by gliclazide, and its effect on glucose-induced insulin secretion was unaffected by an inhibitor of Epac2. PKA activator increased [Ca2+]cduring acute or steady-state glucose stimulation, whereas Epac activator had no effect alone or in combination. Neither activator affected [Ca2+]cresponse to tolbutamide or KCl. Metabolic (glucose-mediated) amplification of insulin secretion was unaffected by PKA activator. It was attenuated when insulin secretion was augmented by Epac activator but insensitive to Epac2 inhibitor, which suggests distinct although somewhat overlapping mechanisms. In conclusion, activators of PKA and Epac amplify insulin secretion by augmenting the action of Ca2+on exocytosis and, for PKA only, slightly increasing glucose-induced [Ca2+]crise. The influence of Epac seems more important than that of PKA during first phase.

Glucose increases insulin secretion by raising cytoplasmic Ca2+([Ca2+]i) in β-cells (triggering pathway) and augmenting the efficacy of Ca2+on exocytosis (amplifying pathway). It has been suggested that glutamate formed from α-ketoglutarate is a messenger of the amplifying pathway (Maechler, P., and Wollheim, C. B. (1999) Nature402, 685–689). This hypothesis was tested with mouse islets depolarized with 30 mmKCl (+ diazoxide) or with a saturating concentration of sulfonylurea. Because [Ca2+]iwas elevated under these conditions, insulin secretion was stimulated already in 0 mmglucose. 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 [Ca2+]iand pHi, whereas glucose and BCH slightly raised pHiand either slightly decreased (30 mmKCl) or increased (tolbutamide) [Ca2+]i. The general dissociation between changes in islet glutamate and insulin secretion refutes a role of β-cell glutamate in the amplification of insulin secretion by glucose.

Glucose stimulation of pancreatic β cells induces oscillations of the membrane potential, cytosolic Ca2+([Ca2+]i), and insulin secretion. Each of these events depends on glucose metabolism. Both intrinsic oscillations of metabolism and repetitive activation of mitochondrial dehydrogenases by Ca2+have been suggested to be decisive for this oscillatory behavior. Among these dehydrogenases, mitochondrial glycerol-3-phosphate dehydrogenase (mGPDH), the key enzyme of the glycerol phosphate NADH shuttle, is activated by cytosolic [Ca2+]i. In the present study, we compared different types of oscillations in β cells from wild-type and mGPDH−/−mice. In clusters of 5–30 islet cells and in intact islets, 15 mmglucose induced an initial drop of [Ca2+]i, followed by an increase in three phases: a marked initial rise, a partial decrease with rapid oscillations and eventually large and slow oscillations. These changes, in particular the frequency of the oscillations and the magnitude of the [Ca2+] rise, were similar in wild-type and mGPDH−/−mice. Glucose-induced electrical activity (oscillations of the membrane potential with bursts of action potentials) was not altered in mGPDH−/−β cells. In single islets from either type of mouse, insulin secretion strictly followed the changes in [Ca2+]iduring imposed oscillations induced by pulses of high K+or glucose and during the biphasic elevation induced by sustained stimulation with glucose. An imposed and controlled rise of [Ca2+]iin β cells similarly increased NAD(P)H fluorescence in control and mGDPH−/−islets. Inhibition of the malate-aspartate NADH shuttle with aminooxyacetate only had minor effects in control islets but abolished the electrical, [Ca2+]iand secretory responses in mGPDH−/−islets. The results show that the two distinct NADH shuttles play an important but at least partially redundant role in glucose-induced insulin secretion. The oscillatory behavior of β cells does not depend on the functioning of mGPDH and on metabolic oscillations that would be generated by cyclic activation of this enzyme by Ca2+.

Although insulin secretion is usually regarded as a Ca2+‐dependent mechanism, recent studies have suggested the existence of a Ca2+‐independent pathway of regulation by glucose. Here, mouse islets were used to compare the contribution of Ca2+‐dependent and ‐independent pathways. Glucose increased insulin release in a concentration‐dependent manner both in a control medium, when it depolarizes β cells and raises [Ca2+]i(triggering signal), and in the presence of 30 mM K+and diazoxide, when it does not further raise [Ca2+]ibut increases its efficacy on exocytosis. Both Ca2+‐dependent responses were amplified by glucagon‐like peptide‐1+acetylcholine, and were strongly potentiated by forskolin+PMA. Under conditions of mild or stringent Ca2+deprivation, glucose had no effect either alone or with GLP‐1 and acetylcholine, and was poorly effective even during pharmacological activation of protein kinases A and C. Similar results were obtained with rat islets. It is concluded that physiological regulation of insulin release by glucose is essentially achieved through the two Ca2+‐dependent pathways without significant contribution of a Ca2+‐independent mechanism.

Although insulin secretion is usually regarded as a Ca 2+-dependent mechanism, recent studies have suggested the existence of a Ca 2+-independent pathway of regulation by glucose. Here, mouse islets were used to compare the contribution of Ca 2+-dependent and -independent pathways. Glucose increased insulin release in a concentration-dependent manner both in a control medium, when it depolarizes β cells and raises [Ca 2+] i(triggering signal), and in the presence of 30 mM K +and diazoxide, when it does not further raise [Ca 2+] ibut increases its efficacy on exocytosis. Both Ca 2+-dependent responses were amplified by glucagon-like peptide-1+acetylcholine, and were strongly potentiated by forskolin+PMA. Under conditions of mild or stringent Ca 2+deprivation, glucose had no effect either alone or with GLP-1 and acetylcholine, and was poorly effective even during pharmacological activation of protein kinases A and C. Similar results were obtained with rat islets. It is concluded that physiological regulation of insulin release by glucose is essentially achieved through the two Ca 2+-dependent pathways without significant contribution of a Ca 2+-independent mechanism.