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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 Ca2+ concentration ([Ca2+]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 [Ca2+]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 [Ca2+]c during acute or steady-state glucose stimulation, whereas Epac activator had no effect alone or in combination. Neither activator affected [Ca2+]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 Ca2+ on exocytosis and, for PKA only, slightly increasing glucose-induced [Ca2+]c rise. The influence of Epac seems more important than that of PKA during first phase.

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

Proteins with a short half-life are potential sites of pancreatic ss cell dysfunction under pathophysiological conditions. In this study, mouse islets were used to establish which step in the regulation of insulin secretion is most sensitive to inhibition of protein synthesis by 10 microM cycloheximide (CHX). Although islet protein synthesis was inhibited approximately 95% after 1 h, the inhibition of insulin secretion was delayed and progressive. After long (18-20 h) CHX-treatment, the strong (80%) inhibition of glucose-, tolbutamide-, and K(+)-induced insulin secretion was not due to lower insulin stores, to any marked impairment of glucose metabolism or to altered function of K(+)-ATP channels (total K(+)-ATP currents were however decreased). It was partly caused by a decreased Ca(2+) influx (whole-cell Ca(2+) current) resulting in a smaller rise in cytosolic Ca(2+) ([Ca(2+)](i)). The situation was very different after short (2-5 h) CHX-treatment. Insulin secretion was 50-60% inhibited although islet glucose metabolism was unaffected and stimulus-induced [Ca(2+)](i) rise was not (2 h) or only marginally (5 h) decreased. The efficiency of Ca(2+) on secretion was thus impaired. The inhibition of insulin secretion by 15 h of CHX treatment was more slowly reversible (>4 h) than that of protein synthesis. This reversibility of secretion was largely attributable to recovery of a normal Ca(2+) efficiency. In conclusion, inhibition of protein synthesis in islets inhibits insulin secretion in two stages: a rapid decrease in the efficiency of Ca(2+) on exocytosis, followed by a decrease in the Ca(2+) signal mediated by a slower loss of functional Ca(2+) channels. Glucose metabolism and the regulation of K(+)-ATP channels are more resistant. Proteins with a short half-life appear to be important to ensure optimal Ca(2+) effects on exocytosis, and are the potential Achille's heel of stimulus-secretion coupling.