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Abstract: Insulin secretion (IS) triggered by β-cell [Ca2+]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 [Ca2+]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 [Ca2+]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 &y& Elsevier]

The contribution of Na+/H+ exchange (achieved by NHE proteins) to the regulation of β-cell cytosolic pHc, and the role of pHc 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 Ca2+ ([Ca2+]c) and pHc in islets from normal mice and mice bearing an inactivating mutation of NHE1 (Slc9A1-swe/swe). The experiments were performed in HCO-3/CO2 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 NH4Cl. In contrast, basal pHc (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 pHc but increased insulin secretion in NHE1 mutant islets, indicating unspecific effects. In control islets, glucose similarly increased [Ca2+]c and insulin secretion in HCO-3 and HEPES buffer, although pHc changed in opposite directions. The amplification of insulin secretion that glucose produces when [Ca2+]c is clamped at an elevated level by KCl was also unrelated to pHc and pHc changes. All effects of glucose on [Ca2+]c and insulin secretion proved independent of NHE1. In conclusion, NHE1 protects -cells against strong acidification, but has no role in stimulus-secretion coupling. The changes in pHc produced by glucose involve HCO-3-dependent mechanisms. Variations in β-cell pHc are not causally related to changes in insulin secretion. [ABSTRACT FROM AUTHOR]

A current model ascribes glucose-induced insulin secretion to the interaction of a triggering pathway (KATP channel-dependent Ca2+ influx and rise in cytosolic [Ca2+]c) and an amplifying pathway (KATP channel-independent augmentation of secretion without further increase of [Ca2+]c). However, several studies of sulfonylurea receptor 1 null mice (Sur1KO) failed to measure significant effects of glucose in their islets lacking KATP channels. We addressed this issue that challenges the model. Compared with controls, fresh Sur1KO islets showed slightly elevated basal [Ca2+]c and insulin secretion. In 15 mM glucose, the absolute rate of secretion was ~3-fold lower in Sur1KO than control islets, with only poor increase above base line. Overnight culture of Sur1KO islets in 10 mc 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 [Ca2+]c oscillations. The unmasking of glucose-induced insulin secretion in β-cells lacking KATP channels is paradoxically due to improvement in the production of a triggering signal (elevated [Ca2+]c). The results show that KATP channels are not the only transducer of glucose effects on [Ca2+]c in β-cells. They explain controversies in the literature and refute arguments raised against the model implicating an amplifying pathway in glucose-induced insulin secretion. [ABSTRACT FROM AUTHOR]

In adult β-cells glucose-induced insulin secretion involves two mechanisms (a) a KATP channel-dependent Ca2+ influx and rise of cytosolic [Ca2+]c and (b) a KATP channel-independent amplification of secretion without further increase of [Ca2+]c. Mice lacking the high affinity sulfonylurea receptor (Sur1KO), and thus KATP channels, have been developed as a model of congenital hyperinsulinism. Here, we compared [Ca2+]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 [Ca2+]c and insulin secretion changes induced by glucose, and operation of the amplifying pathway, were similar to adult islets. Sur1KO islets perifused with 1 mc glucose showed elevation of both basal [Ca2+]c and insulin secretion. Stimulation with 15 mM glucose produced a transient drop of [Ca2+]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 [Ca2+}c was clamped by KCl. When Sur1KO islets were cultured in 5 instead of 10 mM glucose, [Ca2+]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 KATP channel-independent first phase [Ca2+]c rise was attributed to a Na+-, Cl--, and Na+-pump-independent depolarization of β-cells, leading to Ca2+ 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 [Ca2+]c in β-cells without functional KATP channels. [ABSTRACT FROM AUTHOR]

In normal β-cells glucose induces insulin secretion by activating both a triggering pathway (closure of KATP channels, depolarization, and rise in cytosolic [Ca2+]i) and an amplifying pathway (augmentation of Ca2+ efficacy on exocytosis). It is unclear if and how nutrients can regulate insulin secretion by β-cells lacking KATP channels (Sur1 knockout mice). We compared glucose-and amino acid-induced insulin secretion and [Ca2+]i changes in control and Sur1KO islets. In 1 mM glucose (non-stimulatory for controls), the triggering signal [Ca2+]i was high (loss of regulation) and insulin secretion was stimulated in Sur1KO islets. This "basal" secretion was decreased or increased by imposed changes in [Ca2+]i and was dependent on ATP production, indicating that both triggering and amplifying signals are involved. High glucose stimulated insulin secretion in Sur1KO islets, by an unsuspected, transient increase in [Ca2+]i and a sustained activation of the amplifying pathway. Unlike controls, Sur1KO islets were insensitive to diazoxide and tolbutamide, which rules out effects of either drug at sites other than KATP channels. Amino acids potently increased insulin secretion by Sur1KO islets through both a further electrogenic rise in [Ca2+]i and a metabolism-dependent activation of the amplifying pathway. After sulfonylurea blockade of their KATP channels, control islets qualitatively behaved like Sur1KO islets, but their insulin secretion rate was consistently lower for a similar or even higher [Ca2+]i. In conclusion, fuel secretagogues can control insulin secretion in β-cells without KATP channels, partly by an unsuspected influence on the triggering [Ca2+]i signal and mainly by the modulation of a very effective amplifying pathway. [ABSTRACT FROM AUTHOR]