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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 Ca(2+) concentration ([Ca(2+)]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 [Ca(2+)]c rather 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 [Ca(2+)]c during acute or steady-state glucose stimulation, whereas Epac activator had no effect alone or in combination. Neither activator affected [Ca(2+)]c response 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 Ca(2+) on exocytosis and, for PKA only, slightly increasing glucose-induced [Ca(2+)]c rise. The influence of Epac seems more important than that of PKA during first phase.

Insulin secretion has only exceptionally been investigated in pancreatic islets from healthy young children. It remains unclear whether those islets behave like adult islets despite substantial differences in cellular composition and higher β-cell replication rates. Islets were isolated from 5 infants/toddlers (11–36 month-old) and perifused to characterize their dynamics of insulin secretion when subjected to various stimuli and inhibitors. Their insulin responses were compared to those previously reported for similarly treated adult islets. Qualitatively, infant islets responded like adult islets to stimulation by glucose, tolbutamide, forskolin (to increase cAMP), arginine and the combination of leucine and glutamine, and to inhibition by diazoxide and CaCl2 omission. This similarity included the concentration-dependency and biphasic pattern of glucose-induced insulin secretion, the dynamics of the responses to non-glucose stimuli and metabolic amplification of these responses. The insulin content was not different, but fractional insulin secretion rates were lower in infant than adult islets irrespective of the stimulus. However, the stimulation index was similar because basal secretion rates were also lower in infant islets. In conclusion, human β-cells are functionally mature by the age of one year, before expansion of their mass is complete. Their responsiveness (stimulation index) to all stimuli is not smaller than that of adult β-cells. Yet, under basal and stimulated conditions, they secrete smaller proportions of their insulin stores in keeping with smaller in vivo insulin needs during infancy. [ABSTRACT FROM AUTHOR]

Insulinomas are β-cell tumors that cause hypoglycemia through inappropriate secretion of insulin. Characterization of the in vitro dynamics of insulin secretion by perifused fragments of 10 human insulinomas permitted their subdivision into three functional groups with similar insulin content. Group A (four patients with fasting and/or postprandial hypoglycemic episodes) showed qualitatively normal responses to glucose, leucine, diazoxide, tolbutamide, and extracellular CaCl2 omission or excess. The effect of glucose was concentration dependent, but, compared with normal islets, insulin secretion was excessive in both low- and high-glucose conditions. Group B (three patients with fasting hypoglycemic episodes) was mainly characterized by large insulin responses to 1 mmol/L glucose, resulting in very high basal secretion rates that were inhibited by diazoxide and restored by tolbutamide but were not further augmented by other agents except for high levels of CaCl2. Group C (three patients with fasting hypoglycemic episodes) displayed very low rates of insulin secretion and virtually no response to stimuli (including high CaCl2 concentration) and inhibitors (CaCl2 omission being paradoxically stimulatory). In group B, the presence of low-Km hexokinase-I in insulinoma β-cells (not in adjacent islets) was revealed by immunohistochemistry. Human insulinomas thus show distinct, though not completely heterogeneous, defects in insulin secretion that are attributed to the undue expression of hexokinase-I in 3 of 10 patients. [ABSTRACT FROM AUTHOR]

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. Diffuse 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. [ABSTRACT FROM AUTHOR]

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+ 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. Diabetes 55: 3470-3477, 2006 [ABSTRACT FROM AUTHOR]

Sulfonylureas stimulate insulin secretion by blocking ATP-sensitive K+ channels (K+-ATP channels) of the beta-cell membrane, thereby causing depolarization, Ca2+ influx, and rise in cytoplasmic Ca2+ concentration ([Ca2+]i), whereas diazoxide inhibits insulin secretion by opening K+-ATP channels. It has been suggested recently that these drugs also respectively increase and decrease the efficacy of Ca2+ on exocytosis. This hypothesis was tested here with intact islets or single beta-cells from normal mice. Depolarizing islet cells by raising extracellular K+ from 4.8 to 15, 30, and 60 mmol/l progressively raised [Ca2+]i and stimulated insulin secretion. The magnitude of the [Ca2+]i rise produced by a subsequent addition of 100 micromol/l tolbutamide decreased as the concentration of K+ was increased. The effect on insulin secretion paralleled that on [Ca2+]i. Similarly, the magnitudes of the [Ca2+]i drop and of the inhibition of insulin secretion produced by 250 micromol/l diazoxide were inversely related to the concentration of K+. Either drug was effective on secretion only when it increased or decreased [Ca2+]i. Exocytosis of insulin granules from single, voltage-clamped beta-cells was also studied by measuring cell capacitance changes. In the perforated patch configuration, exocytosis was evoked by depolarizing pulses. Addition of tolbutamide to the extracellular medium did not affect the Ca2+ current and the resulting change in cell capacitance. In the whole-cell configuration, cell capacitance increased with the concentration of free Ca2+ in the solution diffusing from the pipette into the cell. It was markedly potentiated by cAMP, was inhibited by activation of alpha2-adrenoceptors with clonidine, and was strongly augmented by acetylcholine. In contrast, tolbutamide was ineffective whether applied intra- or extracellularly, at low or high free Ca2+, and with or without cAMP. Diazoxide also failed to interfere directly with exocytosis. These results indicate that tolbutamide and diazoxide affect insulin secretion by changing the concentration, not the action, of Ca2+ in beta-cells. [ABSTRACT FROM AUTHOR]

Aims To understand better the control of insulin secretion by human β cells and to identify similarities to and differences from rodent models. Methods Dynamic insulin secretion was measured in perifused human islets treated with pharmacological agents of known modes of action. Results Glucokinase activation ( Ro28-1675) lowered the glucose threshold for stimulation of insulin secretion to 1 mmol/ L ( G1), augmented the response to G3-G5 but not to G8-G15, whereas tolbutamide remained active in G20, which indicates that not all KATP channels were closed by high glucose concentrations. An almost 2-fold greater response to G15 than to supramaximal tolbutamide in G3 or to KCl+diazoxide in G15 vs G3 quantified the contribution of metabolic amplification to insulin secretion. Both disruption (latrunculin- B) and stabilization (jasplakinolide) of microfilaments augmented insulin secretion without affecting metabolic amplification. Tolbutamide-induced insulin secretion was consistently greater in G10 than G3, with a threshold at 1 and maximum at 10 µmol/ L tolbutamide in G10, vs 10 and 25 µmol/ L in G3. Sulphonylurea effects were thus clearly glucose-dependent. Insulin secretion was also increased by inhibiting K channels other than KATP channels: Kv or BK channels (tetraethylammonium), TASK-1 channels ( ML-365) and SK4 channels ( TRAM-34). Opening KATP channels with diazoxide inhibited glucose-induced insulin secretion with half maximum inhibitory concentrations of 9.6 and 24 µmol/ L at G7 and G15. Blockade of L-type Ca channels (nimodipine) abolished insulin secretion, whereas a blocker of T-type Ca channels ( NNC-55-0396) was ineffective at specific concentrations. Blockade of Na channels (tetrodotoxin) did not affect glucose-induced insulin secretion. Conclusions In addition to sharing a KATP channel-dependent triggering pathway and a metabolic amplifying pathway, human and rodent β cells were found to display more similarities than differences in the control of insulin secretion. [ABSTRACT FROM AUTHOR]