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Congenital hyperinsulinism (CHI) is the major cause of persistent neonatal hypoglycemia. CHI most often occurs due to mutations in the ABCC8 (which encodes sulfonylurea receptor 1) or KCNJ11 (which encodes the potassium channel Kir6.2) gene, which result in a lack of functional KATP channels in pancreatic β cells. Dif-fuse forms of CHI (DiCHI), in which all β cells are abnormal, often require subtotal pancreatectomy, whereas focal forms (FoCHI), which are characterized by localized hyperplasia of abnormal β cells, can be cured by resection of the lesion. Here, we characterized the in vitro kinetics of insulin secretion by pancreatic fragments from 6 DiCHI patients and by focal lesion and normal adjacent pancreas from 18 FoCHI patients. Responses of normal pancreas were similar to those reported for islets from adult organ donors. Compared with normal pancreas, basal insulin secretion was elevated in both FoCHI and DiCHI tissue. Affected tissues were heterogeneous in their secretory responses, with increased glucose levels often producing a rapid increase in insulin secretion that could be followed by a paradoxical decrease below prestimulatory levels. The KATP channel blocker tolbutamide was consistently ineffective in stimulating insulin secretion; conversely, the KATP channel activator diazoxide often caused an unanticipated increase in insulin secretion. These observed alterations in secretory behavior were similar in focal lesion and DiCHI tissue, and independent of the specific mutation in ABCC8 or KCNJ11. They cannot be explained by classic models of β cell function. Our results provide insight into the excessive and sometimes paradoxical changes in insulin secretion observed in CHI patients with inactivating mutations of KATP channels., Introduction Congenital hyperinsulinism (CHI) is the major cause of persistent hypoglycemia in newborns and infants (1, 2). The underlying genetic etiology and biochemical mechanisms are multiple, resulting in heterogeneous clinical [...]

Two pathways control glucose-induced insulin secretion (IS) by [beta]-cells. The triggering pathway involves ATP-sensitive potassium (KATe) channel-dependent depolarization, [Ca.sup.2+] influx, and a rise in the cytosolic [Ca.sup.2+] concentration ([[[Ca.sup.2+]].sub.c]), which triggers exocytosis of insulin granules. The metabolic amplifying pathway augments IS without further increasing [[[Ca.sup.2+]].sub.c]. The underlying mechanisms are unknown. Here, we tested the hypothesis that amplification implicates actin microfilaments. Mouse islets were treated with latrunculin B and cytochalasin B to depolymerize actin or jasplakinolide to polymerize actin. They were then perifused to measure [[[Ca.sup.2+]].sub.c] and IS. Metabolic amplification was studied during imposed steady elevation of [[[Ca.sup.2+]].sub.c] by tolbutamide or KCI or by comparing the magnitude of [[[Ca.sup.2+]].sub.c] and IS changes produced by glucose and tolbutamide. Both actin polymerization and depolymerization augmented IS triggered by all stimuli without increasing (sometimes decreasing) [[[Ca.sup.2+]].sub.c], which indicates a predominantly inhibitory function of microfilaments in exocytosis at a step distal to [[[Ca.sup.2+]].sub.c] increase. When [[[Ca.sup.2+]].sub.c] was elevated and controlled by KC1 or tolbutamide, the amplifying action of glucose was facilitated by actin depolymerization and unaffected by polymerization. Both phases of IS were larger in response to high-glucose than to tolbutamide in low-glucose, although triggering [[[Ca.sup.2+]].sub.c] was lower. This difference in IS, due to amplification, persisted when the IS rate was doubled by actin depolymerization or polymerization. In conclusion, metabolic amplification is rapid and influences the first as well as the second phase of IS. It is a late step of stimulus-secretion coupling, which does not require functional actin microfilaments and could correspond to acceleration of the priming process conferring release competence to insulin granules. biphasic insulin release; cytosolic calcium; exocytosis; insulin granules; pancreatic islets doi: 10.1152/ajpcell.00138.2010.

Stimulatory concentrations of glucose induce two patterns of cytosolic [Ca.sup.2+] concentration ([[[Ca.sup.2+]].sub.c]) oscillations in mouse islets: simple or mixed. In the mixed pattern, rapid oscillations are superimposed on slow ones. In the present study, we examined the role of the membrane potential in the mixed pattern and the impact of this pattern on insulin release. Simultaneous measurement of [[[Ca.sup.2+]].sub.c] and insulin release from single islets revealed that mixed [[[Ca.sup.2+]].sub.c] oscillations triggered synchronous oscillations of insulin secretion. Simultaneous recordings of membrane potential in a single [beta]-cell within an islet and of [[[Ca.sup.2+]].sub.c] in the whole islet demonstrated that the mixed pattern resulted from compound bursting (i.e., clusters of membrane potential oscillations separated by prolonged silent intervals) that was synchronized in most [beta]-cells of the islet. Each slow [[[Ca.sup.2+]].sub.c] increase during mixed oscillations was due to a progressive summation of rapid oscillations. Digital image analysis confirmed the good synchrony between subregions of an islet. By contrast, islets from sarco(endo)plasmic reticulum [Ca.sup.2+]-ATPase isoform 3 (SERCA3)-knockout mice did not display typical mixed [[[Ca.sup.2+]].sub.c] oscillations in response to glucose. This results from a lack of progressive summation of rapid oscillations and from altered spontaneous electrical activity, i.e., lack of compound bursting, and membrane potential oscillations characterized by lower-frequency but larger-depolarization phases than observed in SERCA[3.sup.+/+] [beta]-cells. We conclude that glucose-induced mixed [[[Ca.sup.2+]].sub.c] oscillations result from compound bursting in all [beta]-cells of the islet. Disruption of SERCA3 abolishes mixed [[[Ca.sup.2+]].sub.c] oscillations and augments [beta]-cell depolarization. This latter observation indicates that the endoplasmic reticulum participates in the control of the [beta]-cell membrane potential during glucose stimulation. electrical activity; insulin-secreting cell; thapsigargin

Glucose induces insulin secretion (IS) and also potentiates the insulin-releasing action of secretagogues such as arginine and sulfonylureas. This potentiating effect is known to be impaired in type 2 diabetic patients, but its cellular mechanisms are unclear. IS and cytosolic [Ca.sup.2+] concentration ([[[Ca.sup.2+]].sub.i]) were measured in mouse islets during perifusion with 3-15 mmol/l glucose (G3-G15, respectively) and pulse or stepwise stimulation with 1-10 mmol/l arginine or 5-250 [micro]mol/l tolbutamide. In G3, arginine induced small increases in [[[Ca.sup.2+]].sub.i] but no IS. G7 alone only slightly increased [[[Ca.sup.2+]].sub.i]] and IS but markedly potentiated arginine effects on [[[Ca.sup.2+]].sub.i], which resulted in significant IS (already at 1 mmol/l). For each arginine concentration, both responses further increased at G10 and G15, but the relative change was distinctly larger for IS than [[[Ca.sup.2+]].sub.i]. At all glucose concentrations, tolbutamide dose dependently increased [[[Ca.sup.2+]].sub.i] and IS with thresholds of 25 [micro]mol/l for [[[Ca.sup.2+]].sub.i] and 100 [micro]mol/l for IS at G3 and of 5 [micro]mol/l for both at G7 and above. Between G7 and G15, the effect of tolbutamide on [[[Ca.sup.2+]].sub.i] increased only slightly, whereas that on IS was strongly potentiated. The linear relationship between IS and [[[Ca.sup.2+].sub.i] at increasing arginine or tolbutamide concentrations became steeper as the glucose concentration was raised. Thus glucose augmented more the effect of each agent on IS than that on [[Ca.sup.2+]].sub.i]. In conclusion, glucose potentiation of arginine- or tolbutamide-induced IS involves increases in both the rise of [[[Ca.sup.2+]].sub.i] and the action of [Ca.sup.2+] on exocytosis. This dual mechanism must be borne in mind to interpret the alterations of the potentiating action of glucose in type 2 diabetic patients. insulin release; sulfonylureas; glucose potentiation; [beta]-cell cytosolic calcium ion; type 2 diabetes

In contrast to pancreatic islets, isolated [beta]-cells stimulated by glucose display irregular and asynchronous increases in cytoplasmic [Ca.sup.2+] concentration ([[[Ca.sup.2+]].sub.i]). Here, clusters of 5-30 cells were prepared from a single mouse islet or from pools of islets, loaded with fura-2, and studied with a camera-based system. [[[Ca.sup.2+]].sub.i] oscillations were compared in pairs of clusters by computing the difference in period and a synchronization index [lambda]. During perifusion with 12 mM glucose, the clusters exhibited regular [[[Ca.sup.2+]].sub.i] oscillations that were quasi-perfectly synchronized ([DELTA] period of 1.4% and index [lambda] close to 1.0) between cells of each cluster. In contrast, separate clusters were not synchronized, even when prepared from one single islet. Pairs of clusters neighboring on the same coverslip were not better synchronized than pairs of clusters examined separately (distinct coverslips). We next attempted to synchronize clusters perifused with 12 mM glucose by applying external signals. A single pulse of 20 mM glucose, 10 mM amino acids, or 10 [micro]M tolbutamide transiently altered [[[Ca.sup.2+]].sub.i] oscillations but did not reset the clusters to oscillate synchronously. On a background of 12 mM glucose, repetitive applications (1 min/5 min) of 10 [micro]M tolbutamide, but not of 20 mM glucose, synchronized separate clusters. Our results identity a level of [beta]-cell heterogeneity intermediate between single [beta]-cells and the whole islet. They do not support the idea that substances released by islet cells serve as paracrine synchronizers. However, synchronization can be achieved by an external signal, if this signal has a sufficient strength to overwhelm the intrinsic rhythm of glucose-induced oscillations and is repetitively applied. [beta]-cells; intracellular calcium; insulin secretion

Thapsigargin (TG), a blocker of [Ca.sup.2+] uptake by the endoplasmic reticulum (ER), was used to evaluate the contribution of the organelle to the oscillations of cytosolic [Ca.sup.2+] concentration ([[[Ca.sup.2+]].sub.c]) induced by repetitive [Ca.sup.2+] influx in mouse pancreatic [beta]-cells. Because TG depolarized the plasma membrane in the presence of glucose alone, extracellular [K.sup.+] was alternated between 10 and 30 mM in the presence of diazoxide to impose membrane potential (MP) oscillations. In control islets, pulses of [K.sup.+], mimicking regular MP oscillations elicited by 10 mM glucose, induced [[[Ca.sup.2+]].sub.c] oscillations whose nadir remained higher than basal [[[Ca.sup.2+]].sub.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.sup.2+]].sub.c] oscillations that was reproduced by raising extracellular [Ca.sup.2+] (to increase [Ca.sup.2+] influx) without changing the pulse protocol. In TG-pretreated islets, the imposed [[[Ca.sup.2+]].sub.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.sup.2+]].sub.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.sup.2+]].sub.c] oscillations and may explain how the nadir between oscillations remains above baseline during regular oscillations or gradually increases during mixed [[[Ca.sup.2+]].sub.c] oscillations, two types of response observed during glucose stimulation. cytosolic [Ca.sup.2+] concentration oscillations; endoplasmic reticulum; pancreatic [beta]-cell; thapsigargin

The role of calcium (Ca2+) currents in the initiation of insulin secretion in intact mouse pancreatic islets is investigated based on measurements of insulin release, cytoplasmic calcium concentration and B cell membrane potential. Data indicate that Ca2+ influx via reverse Na+-Ca2+ exchange has no effect on cytoplasmic calcium concentration. However, Ca2+ influx via voltage-dependent Ca2+ channels promote insulin release by increasing cytoplasmic calcium levels through removal of Na+.

Streptozotocin (STZ)-diabetic rats were orally treated with molybdate for eight weeks to determine the effect of molybdate (Mo) on the expression of gene coding for key enzymes involved in hepatic glucose metabolism. Hyperglycemia and glucosuria were reduced 75% but glycogen stores and level of plasma nonesterified fatty acids were restored. Acetyl-CoA carboxylase expression and activity in the liver increased but not in adipose tissue. It is suggested that Mo has an insulin-like action which could reverse impaired pretranslational regulatory mechanisms in hepatic glucose metabolism.