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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+]crather 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+]cduring acute or steady-state glucose stimulation, whereas Epac activator had no effect alone or in combination. Neither activator affected [Ca2+]cresponse 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+]crise. The influence of Epac seems more important than that of PKA during first phase.

Glucose increases insulin secretion by raising cytoplasmic Ca2+([Ca2+]i) in β-cells (triggering pathway) and augmenting the efficacy of Ca2+on exocytosis (amplifying pathway). It has been suggested that glutamate formed from α-ketoglutarate is a messenger of the amplifying pathway (Maechler, P., and Wollheim, C. B. (1999) Nature402, 685–689). This hypothesis was tested with mouse islets depolarized with 30 mmKCl (+ diazoxide) or with a saturating concentration of sulfonylurea. Because [Ca2+]iwas elevated under these conditions, insulin secretion was stimulated already in 0 mmglucose. The amplification of secretion produced by glucose was accompanied by an increase in islet glutamate. However, glutamine (0.5–2 mm) markedly augmented islet glutamate without affecting insulin secretion, whereas glucose augmented secretion without influencing glutamate levels when these were elevated by glutamine. Allosteric activation of glutamate dehydrogenase by BCH (2-amino 2-norbornane carboxylic acid) lowered islet glutamate but increased insulin secretion. Similar insulin secretion thus occurred at very different cellular glutamate levels. Glutamine did not affect islet [Ca2+]iand pHi, whereas glucose and BCH slightly raised pHiand either slightly decreased (30 mmKCl) or increased (tolbutamide) [Ca2+]i. The general dissociation between changes in islet glutamate and insulin secretion refutes a role of β-cell glutamate in the amplification of insulin secretion by glucose.

Glucose stimulation of pancreatic β cells induces oscillations of the membrane potential, cytosolic Ca2+([Ca2+]i), and insulin secretion. Each of these events depends on glucose metabolism. Both intrinsic oscillations of metabolism and repetitive activation of mitochondrial dehydrogenases by Ca2+have been suggested to be decisive for this oscillatory behavior. Among these dehydrogenases, mitochondrial glycerol-3-phosphate dehydrogenase (mGPDH), the key enzyme of the glycerol phosphate NADH shuttle, is activated by cytosolic [Ca2+]i. In the present study, we compared different types of oscillations in β cells from wild-type and mGPDH−/−mice. In clusters of 5–30 islet cells and in intact islets, 15 mmglucose induced an initial drop of [Ca2+]i, followed by an increase in three phases: a marked initial rise, a partial decrease with rapid oscillations and eventually large and slow oscillations. These changes, in particular the frequency of the oscillations and the magnitude of the [Ca2+] rise, were similar in wild-type and mGPDH−/−mice. Glucose-induced electrical activity (oscillations of the membrane potential with bursts of action potentials) was not altered in mGPDH−/−β cells. In single islets from either type of mouse, insulin secretion strictly followed the changes in [Ca2+]iduring imposed oscillations induced by pulses of high K+or glucose and during the biphasic elevation induced by sustained stimulation with glucose. An imposed and controlled rise of [Ca2+]iin β cells similarly increased NAD(P)H fluorescence in control and mGDPH−/−islets. Inhibition of the malate-aspartate NADH shuttle with aminooxyacetate only had minor effects in control islets but abolished the electrical, [Ca2+]iand secretory responses in mGPDH−/−islets. The results show that the two distinct NADH shuttles play an important but at least partially redundant role in glucose-induced insulin secretion. The oscillatory behavior of β cells does not depend on the functioning of mGPDH and on metabolic oscillations that would be generated by cyclic activation of this enzyme by Ca2+.

Although insulin secretion is usually regarded as a Ca2+‐dependent mechanism, recent studies have suggested the existence of a Ca2+‐independent pathway of regulation by glucose. Here, mouse islets were used to compare the contribution of Ca2+‐dependent and ‐independent pathways. Glucose increased insulin release in a concentration‐dependent manner both in a control medium, when it depolarizes β cells and raises [Ca2+]i(triggering signal), and in the presence of 30 mM K+and diazoxide, when it does not further raise [Ca2+]ibut increases its efficacy on exocytosis. Both Ca2+‐dependent responses were amplified by glucagon‐like peptide‐1+acetylcholine, and were strongly potentiated by forskolin+PMA. Under conditions of mild or stringent Ca2+deprivation, glucose had no effect either alone or with GLP‐1 and acetylcholine, and was poorly effective even during pharmacological activation of protein kinases A and C. Similar results were obtained with rat islets. It is concluded that physiological regulation of insulin release by glucose is essentially achieved through the two Ca2+‐dependent pathways without significant contribution of a Ca2+‐independent mechanism.

Although insulin secretion is usually regarded as a Ca 2+-dependent mechanism, recent studies have suggested the existence of a Ca 2+-independent pathway of regulation by glucose. Here, mouse islets were used to compare the contribution of Ca 2+-dependent and -independent pathways. Glucose increased insulin release in a concentration-dependent manner both in a control medium, when it depolarizes β cells and raises [Ca 2+] i(triggering signal), and in the presence of 30 mM K +and diazoxide, when it does not further raise [Ca 2+] ibut increases its efficacy on exocytosis. Both Ca 2+-dependent responses were amplified by glucagon-like peptide-1+acetylcholine, and were strongly potentiated by forskolin+PMA. Under conditions of mild or stringent Ca 2+deprivation, glucose had no effect either alone or with GLP-1 and acetylcholine, and was poorly effective even during pharmacological activation of protein kinases A and C. Similar results were obtained with rat islets. It is concluded that physiological regulation of insulin release by glucose is essentially achieved through the two Ca 2+-dependent pathways without significant contribution of a Ca 2+-independent mechanism.