Pharmacological approach to understanding the control of insulin secretion in human islets.

AIMS: To understand better the control of insulin secretion by human ß 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 ß cells were found to display more similarities than differences in the control of insulin secretion.

Dynamics of glucose-induced insulin secretion in normal human islets.

The biphasic pattern of glucose-induced insulin secretion is altered in type 2 diabetes. Impairment of the first phase is an early sign of ß-cell dysfunction, but the underlying mechanisms are still unknown. Their identification through in vitro comparisons of islets from diabetic and control subjects requires characterization and quantification of the dynamics of insulin secretion by normal islets. When perifused normal human islets were stimulated with 15 mmol/l glucose (G15), the proinsulin/insulin ratio in secretory products rapidly and reversibly decreased (∼50%) and did not reaugment with time. Switching from prestimulatory G3 to G6-G30 induced biphasic insulin secretion with flat but sustained (2 h) second phases. Stimulation index reached 6.7- and 3.6-fold for the first and second phases induced by G10. Concentration dependency was similar for both phases, with half-maximal and maximal responses at G6.5 and G15, respectively. First-phase response to G15-G30 was diminished by short (30-60 min) prestimulation in G6 (vs. G3) and abolished by prestimulation in G8, whereas the second phase was unaffected. After 1-2 days of culture in G8 (instead of G5), islets were virtually unresponsive to G15. In both settings, a brief return to G3-G5 or transient omission of CaCl2 restored biphasic insulin secretion. Strikingly, tolbutamide and arginine evoked immediate insulin secretion in islets refractory to glucose. In conclusion, we quantitatively characterized the dynamics of glucose-induced insulin secretion in normal human islets and showed that slight elevation of prestimulatory glucose reversibly impairs the first phase, which supports the view that the similar impairment in type 2 diabetic patients might partially be a secondary phenomenon.

Metabolic amplification of insulin secretion by glucose is independent of ß-cell microtubules.

Glucose-induced insulin secretion (IS) by ß-cells is controlled by two pathways. The triggering pathway involves ATP-sensitive potassium (K(ATP)) channel-dependent depolarization, Ca(2+) influx, and rise in the cytosolic Ca(2+) concentration ([Ca(2+)](c)), which triggers exocytosis of insulin granules. The metabolic amplifying pathway augments IS without further increasing [Ca(2+)](c). After exclusion of the contribution of actin microfilaments, we here tested whether amplification implicates microtubule-dependent granule mobilization. Mouse islets were treated with nocodazole or taxol, which completely depolymerized and polymerized tubulin. They were then perifused to measure [Ca(2+)](c) and IS. Metabolic amplification was studied during imposed steady elevation of [Ca(2+)](c) by tolbutamide or KCl or by comparing [Ca(2+)](c) and IS responses to glucose and tolbutamide. Nocodazole did not alter [Ca(2+)](c) or IS changes induced by the three secretagogues, whereas taxol caused a small inhibition of IS that is partly ascribed to a decrease in [Ca(2+)](c). When [Ca(2+)](c) was elevated and controlled by KCl or tolbutamide, the amplifying action of glucose was unaffected by microtubule disruption or stabilization. Both phases of IS were larger in response to glucose than tolbutamide, although triggering [Ca(2+)](c) was lower. This difference, due to amplification, persisted in nocodazole- or taxol-treated islets, even when IS was augmented fourfold by microfilament disruption with cytochalasin B or latrunculin B. In conclusion, metabolic amplification rapidly augments first and second phases of IS independently of insulin granule translocation along microtubules. We therefore extend our previous proposal that it does not implicate the cytoskeleton but corresponds to acceleration of the priming process conferring release competence to insulin granules.

Metabolic amplifying pathway increases both phases of insulin secretion independently of beta-cell actin microfilaments.

Two pathways control glucose-induced insulin secretion (IS) by beta-cells. The triggering pathway involves ATP-sensitive potassium (K(ATP)) channel-dependent depolarization, Ca(2+) influx, and a rise in the cytosolic Ca(2+) concentration ([Ca(2+)](c)), which triggers exocytosis of insulin granules. The metabolic amplifying pathway augments IS without further increasing [Ca(2+)](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(2+)](c) and IS. Metabolic amplification was studied during imposed steady elevation of [Ca(2+)](c) by tolbutamide or KCl or by comparing the magnitude of [Ca(2+)](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(2+)](c), which indicates a predominantly inhibitory function of microfilaments in exocytosis at a step distal to [Ca(2+)](c) increase. When [Ca(2+)](c) was elevated and controlled by KCl 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(2+)](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.

Glucose controls cytosolic Ca2+ and insulin secretion in mouse islets lacking adenosine triphosphate-sensitive K+ channels owing to a knockout of the pore-forming subunit Kir6.2.

Glucose-induced insulin secretion is classically attributed to the cooperation of an ATP-sensitive potassium (K ATP) channel-dependent Ca2+ influx with a subsequent increase of the cytosolic free Ca2+ concentration ([Ca2+]c) (triggering pathway) and a K ATP channel-independent augmentation of secretion without further increase of [Ca2+]c (amplifying pathway). Here, we characterized the effects of glucose in beta-cells lacking K ATP channels because of a knockout (KO) of the pore-forming subunit Kir6.2. Islets from 1-yr and 2-wk-old Kir6.2KO mice were used freshly after isolation and after 18 h culture to measure glucose effects on [Ca2+]c and insulin secretion. Kir6.2KO islets were insensitive to diazoxide and tolbutamide. In fresh adult Kir6.2KO islets, basal [Ca2+]c and insulin secretion were marginally elevated, and high glucose increased [Ca2+]c only transiently, so that the secretory response was minimal (10% of controls) despite a functioning amplifying pathway (evidenced in 30 mm KCl). Culture in 10 mm glucose increased basal secretion and considerably improved glucose-induced insulin secretion (200% of controls), unexpectedly because of an increase in [Ca2+]c with modulation of [Ca2+]c oscillations. Similar results were obtained in 2-wk-old Kir6.2KO islets. Under selected conditions, high glucose evoked biphasic increases in [Ca2+]c and insulin secretion, by inducing K ATP channel-independent depolarization and Ca2+ influx via voltage-dependent Ca2+ channels. In conclusion, Kir6.2KO beta-cells down-regulate insulin secretion by maintaining low [Ca2+]c, but culture reveals a glucose-responsive phenotype mainly by increasing [Ca2+]c. The results support models implicating a K ATP channel-independent amplifying pathway in glucose-induced insulin secretion, and show that K ATP channels are not the only possible transducers of metabolic effects on the triggering Ca2+ signal.

Immaturity of insulin secretion by pancreatic islets isolated from one human neonate.

Human ß-cells are functionally mature by the age of 1 year. The timeline and mechanisms of this maturation are unknown owing to the exceptional availability of testable tissue. Here, we report the first in vitro study of insulin secretion by islets from a 5-day-old newborn. Glucose was inefficient alone, but induced insulin secretion, which was concentration-dependent, showed a biphasic time-course and was of similar magnitude as in infant islets when ß-cell cyclic adenosine monophosphate was raised by forskolin. Tolbutamide alone was effective in low glucose, but its effect was not augmented by high glucose. Metabolic amplification by glucose was thus inoperative, in contrast to amplification by cyclic adenosine monophosphate. Newborn islets showed high basal insulin secretion that could be inhibited by diazoxide or omission of CaCl2 . Postnatal acquisition of functional maturity by human ß-cells implicates control of basal secretion and production of metabolic signals able to activate both triggering and amplifying pathways of insulin secretion.

Nutrient control of insulin secretion in isolated normal human islets.

Pancreatic islets were isolated from 16 nondiabetic organ donors and, after culture for approximately 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(m) at 6.5 mmol/l glucose, and V(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 approximately 18-fold) and a sustained, flat second phase ( approximately 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.

In vivo and in vitro glucose-induced biphasic insulin secretion in the mouse: pattern and role of cytoplasmic Ca2+ and amplification signals in beta-cells.

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(2+)](c)) changes in perifused mouse islets. Anesthetized mice were infused with glucose to clamp 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/l glucose induced similar biphasic insulin secretion and [Ca(2+)](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(2+)](c). The maximum [Ca(2+)](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 approximately 50%, an inhibition that could not be explained by commensurate decreases in [Ca(2+)](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.

Dynamics and Regulation of Insulin Secretion in Pancreatic Islets from Normal Young Children.

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 ß-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 ß-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 ß-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.

Signals and pools underlying biphasic insulin secretion.

Rapid and sustained stimulation of beta-cells with glucose induces biphasic insulin secretion. The two phases appear to reflect a characteristic of stimulus-secretion coupling in each beta-cell rather than heterogeneity in the time-course of the response between beta-cells or islets. There is no evidence indicating that biphasic secretion can be attributed to an intrinsically biphasic metabolic signal. In contrast, the biphasic rise in cytoplasmic Ca(2+) concentration ([Ca(2+)](i)) induced by glucose is important to shape the two phases of secretion. The first phase requires a rapid and marked elevation of [Ca(2+)](i) and corresponds to the release of insulin granules from a limited pool. The magnitude of the second phase is determined by the elevation of [Ca(2+)](i), but its development requires production of another signal. This signal corresponds to the amplifying action of glucose and may serve to replenish the pool of granules that are releasable at the prevailing [Ca(2+)](i). The species characteristics of biphasic insulin secretion and its perturbations in pathological situations are discussed.

SERCA3 ablation does not impair insulin secretion but suggests distinct roles of different sarcoendoplasmic reticulum Ca(2+) pumps for Ca(2+) homeostasis in pancreatic beta-cells.

Two sarcoendoplasmic reticulum Ca(2+)-ATPases, SERCA3 and SERCA2b, are expressed in pancreatic islets. Immunocytochemistry showed that SERCA3 is restricted to beta-cells in the mouse pancreas. Control and SERCA3-deficient mice were used to evaluate the role of SERCA3 in beta-cell cytosolic-free Ca(2+) concentration ([Ca(2+)](c)) regulation, insulin secretion, and glucose homeostasis. Basal [Ca(2+)](c) was not increased by SERCA3 ablation. Stimulation with glucose induced a transient drop in basal [Ca(2+)](c) that was suppressed by inhibition of all SERCAs with thapsigargin (TG) but unaffected by selective SERCA3 ablation. Ca(2+) mobilization by acetylcholine was normal in SERCA3-deficient beta-cells. In contrast, [Ca(2+)](c) oscillations resulting from intermittent glucose-stimulated Ca(2+) influx and [Ca(2+)](c) transients induced by pulses of high K(+) 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(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(2+)](c) regulation in beta-cells. SERCA3 becomes operative when [Ca(2+)](c) rises and is required for normal [Ca(2+)](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.

Human Insulinomas Show Distinct Patterns of Insulin Secretion In Vitro.

Insulinomas are ß-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 ß-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.

Activators of PKA and Epac distinctly influence insulin secretion and cytosolic Ca2+ in female mouse islets stimulated by glucose and tolbutamide.

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.

Amplification of insulin secretion by acetylcholine or phorbol ester is independent of ß-cell microfilaments and distinct from metabolic amplification.

Insulin secretion (IS) triggered by ß-cell [Ca(2+)](c) is amplified by metabolic and receptor-generated signals. Diacylglycerol largely mediates acetylcholine (ACh) effects through protein-kinase C and other effectors, which can be directly activated by phorbol-ester (PMA). Using mouse islets, we investigated the possible role of microfilaments in ACh/PMA-mediated amplification of IS. PMA had no steady-state impact on actin microfilaments. Although ACh slightly augmented and PMA diminished glucose- and tolbutamide-induced increases in ß-cell [Ca(2+)](c), both amplified IS in control islets and after microfilament disruption (latrunculin) or stabilization (jasplakinolide). Both phases of IS were larger in response to glucose than tolbutamide, although [Ca(2+)](c) was lower. This difference in secretion, which reflects metabolic amplification, persisted in presence of ACh/PMA and was independent of microfilaments. Amplification of IS by ACh/PMA is thus distinct from metabolic amplification, but both pathways promote acquisition of release competence by insulin granules, which can access exocytotic sites without intervention of microfilaments.

Congenital hyperinsulinism caused by hexokinase I expression or glucokinase-activating mutation in a subset of ß-cells.

Congenital hyperinsulinism causes persistent hypoglycemia in neonates and infants. Most often, uncontrolled insulin secretion (IS) results from a lack of functional K(ATP) channels in all ß-cells or only in ß-cells within a resectable focal lesion. In more rare cases, without K(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(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 low-K(m) hexokinase-I in ß-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 ß-cells. They represent a novel cause of focal congenital hyperinsulinism.

cAMP-mediated and metabolic amplification of insulin secretion are distinct pathways sharing independence of ß-cell microfilaments.

Insulin secretion is triggered by an increase in the cytosolic calcium concentration ([Ca(2+)](c)) in ß-cells. Ca(2+)-induced exocytosis of insulin granules can be augmented by metabolic amplification (unknown signals generated through glucose metabolism) or neurohormonal amplification (in particular cAMP mediated). Functional actin microfilaments are not required for metabolic amplification, but their possible role in cAMP-mediated amplification is unknown. It is also uncertain whether cAMP (generated in response to glucose) is implicated in metabolic amplification. These questions were addressed using isolated mouse islets. cAMP levels were increased by phosphodiesterase inhibition (with isobutylmethylxanthine) and adenylate-cyclase stimulation (with forskolin or glucagon-like peptide-1, 7-36 amide). Raising cAMP levels had no steady-state impact on actin polymerization in control islets. Neither disruption (depolymerization by latrunculin) nor stabilization (polymerization by jasplakinolide) of actin microfilaments was counteracted by cAMP. Both changes increased both phases of glucose- or tolbutamide-induced insulin secretion but did not prevent further amplification by cAMP. These large changes in secretion were not caused by changes in [Ca(2+)](c), which was only slightly increased by cAMP. Both phases of insulin secretion were larger in response to glucose than tolbutamide, although [Ca(2+)](c) was lower. This difference in secretion, which reflects metabolic amplification, was independent of microfilaments, was not attributable to differences in cAMP, and persisted in presence of dibutyryl-cAMP or when cAMP levels were variably raised by isobutylmethylxanthine + forskolin or glucagon-like peptide-1, 7-36 amide. We conclude that metabolic and cAMP-mediated amplification of insulin secretion are distinct pathways that accelerate acquisition of release competence by insulin granules that can access exocytotic sites without intervention of microfilaments.

Disruption and stabilization of ß-cell actin microfilaments differently influence insulin secretion triggered by intracellular Ca2+ mobilization or store-operated Ca2+ entry.

Latrunculin depolymerizes and jasplakinolide polymerizes ß-cell actin microfilaments. Both increase insulin secretion when Ca(2+) enters ß-cells during depolarization by glucose, sulfonylureas or potassium. Mouse islets were held hyperpolarized with diazoxide, and stimulated with acetylcholine to test the role of microfilaments in insulin secretion triggered by intracellular Ca(2+) mobilization and store-operated Ca(2+) entry (SOCE). Jasplakinolide slightly attenuated Ca(2+) mobilization and did not affect SOCE, but consistently inhibited the attending insulin secretion. Latrunculin did not affect Ca(2+) changes induced by acetylcholine, but consistently increased insulin secretion, its effect being larger in response to Ca(2+) entry than to Ca(2+) mobilization. Microfilaments have thus a distinct impact on exocytosis of insulin granules depending on the source of triggering Ca(2+).

In vitro insulin secretion by pancreatic tissue from infants with diazoxide-resistant congenital hyperinsulinism deviates from model predictions.

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 ß cells. Diffuse forms of CHI (DiCHI), in which all ß cells are abnormal, often require subtotal pancreatectomy, whereas focal forms (FoCHI), which are characterized by localized hyperplasia of abnormal ß 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 ß 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.

Insulin secretion in islets from mice with a double knockout for the dense core vesicle proteins islet antigen-2 (IA-2) and IA-2beta.

Islet antigen-2 (IA-2 or ICA 512) and IA-2beta (or phogrin) are major autoantigens in type 1 diabetes. They are located in dense core secretory vesicles including insulin granules, but their role in beta-cell function is unclear. Targeted disruption of either IA-2 or IA-2beta, or both, impaired glucose tolerance, an effect attributed to diminution of insulin secretion. In this study, we therefore characterized the dynamic changes in cytosolic Ca2+([Ca2+](c)) and insulin secretion in islets from IA-2/IA-2beta double knockout (KO) mice. High glucose (15 mM) induced biphasic insulin secretion in IA-2/IA-2beta KO islets, with a similar first phase and smaller second phase compared with controls. Since the insulin content of IA-2/IA-2beta KO islets was approximately 45% less than that of controls, fractional insulin secretion (relative to content) was thus increased during first phase and unaffected during second phase. This peculiar response occurred in spite of a slightly smaller rise in [Ca2+](c), could not be attributed to an alteration of glucose metabolism (NADPH fluorescence) and also was observed with tolbutamide. The dual control of insulin secretion via the K(ATP) channel-dependent triggering pathway and K(ATP) channel-independent amplifying pathway was unaltered in IA-2/IA-2beta KO islets, and so were the potentiations by acetylcholine or cAMP (forskolin). Intriguingly, amino acids, in particular the cationic arginine and lysine, induced larger fractional insulin secretion in IA-2/IA-2beta KO than control islets. In conclusion, IA-2 and IA-2beta are dispensable for exocytosis of insulin granules, but are probably more important for cargo loading and/or stability of dense core vesicles.

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.