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Human β-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 β-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 CaCl 2 . Postnatal acquisition of functional maturity by human β-cells implicates control of basal secretion and production of metabolic signals able to activate both triggering and amplifying pathways of insulin secretion., (© 2017 The Authors. Journal of Diabetes Investigation published by Asian Association for the Study of Diabetes (AASD) and John Wiley & Sons Australia, Ltd.)

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 β-cells or only in β-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 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.

Insulin secretion (IS) triggered by β-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 β-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., (Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.)

Insulin secretion is triggered by an increase in the cytosolic calcium concentration ([Ca(2+)](c)) in β-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.

Glucose-induced insulin secretion (IS) by β-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.

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.

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.

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.

In adult beta-cells glucose-induced insulin secretion involves two mechanisms (a) a K(ATP) channel-dependent Ca(2+) influx and rise of cytosolic [Ca(2+)](c) and (b) a K(ATP) channel-independent amplification of secretion without further increase of [Ca(2+)](c). Mice lacking the high affinity sulfonylurea receptor (Sur1KO), and thus K(ATP) channels, have been developed as a model of congenital hyperinsulinism. Here, we compared [Ca(2+)](c) and insulin secretion in overnight cultured islets from 2-week-old normal and Sur1KO mice. Control islets proved functionally mature: the magnitude and biphasic kinetics of [Ca(2+)](c) and insulin secretion changes induced by glucose, and operation of the amplifying pathway, were similar to adult islets. Sur1KO islets perifused with 1 mm glucose showed elevation of both basal [Ca(2+)](c) and insulin secretion. Stimulation with 15 mm glucose produced a transient drop of [Ca(2+)](c) followed by an overshoot and a sustained elevation, accompanied by a monophasic, 6-fold increase in insulin secretion. Glucose also increased insulin secretion when [Ca(2+)](c) was clamped by KCl. When Sur1KO islets were cultured in 5 instead of 10 mm glucose, [Ca(2+)](c) and insulin secretion were unexpectedly low in 1 mm glucose and increased following a biphasic time course upon stimulation by 15 mm glucose. This K(ATP) channel-independent first phase [Ca(2+)](c) rise was attributed to a Na(+)-, Cl(-)-, and Na(+)-pump-independent depolarization of beta-cells, leading to Ca(2+) influx through voltage-dependent calcium channels. Glucose indeed depolarized Sur1KO islets under these conditions. It is suggested that unidentified potassium channels are sensitive to glucose and subserve the acute and long-term metabolic control of [Ca(2+)](c) in beta-cells without functional K(ATP) channels.

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.