Aims/hypothesis The mechanisms by which glucose regulates glucagon release are poorly understood. The present study aimed to clarify the direct effects of glucose on the glucagon-releasing alpha cells and those effects mediated by paracrine islet factors. Materials and methods Glucagon, insulin and somatostatin release were measured from incubated mouse pancreatic islets and the cytoplasmic Ca 2+ concentration ([Ca 2+ ] i ) recorded in isolated mouse alpha cells. Results Glucose inhibited glucagon release with maximal effect at 7 mmol/l. Since this concentration corresponded to threshold stimulation of insulin secretion, it is unlikely that inhibition of glucagon secretion is mediated by beta cell factors. Although somatostatin secretion data seemed consistent with a role of this hormone in glucose-inhibited glucagon release, a somatostatin receptor type 2 antagonist stimulated glucagon release without diminishing the inhibitory effect of glucose. In islets exposed to tolbutamide plus 8 mmol/l K + , glucose inhibited glucagon secretion without stimulating the release of insulin and somatostatin, indicating a direct inhibitory effect on the alpha cells that was independent of ATP-sensitive K + channels. Glucose lowered [Ca 2+ ] i of individual alpha cells independently of somatostatin and beta cell factors (insulin, Zn 2+ and γ-aminobutyric acid). Glucose suppression of glucagon release was prevented by inhibitors of the sarco(endo)plasmic reticulum Ca 2+ -ATPase, which abolished the [Ca 2+ ] i -lowering effect of glucose on isolated alpha cells. Conclusions/interpretation Beta cell factors or somatostatin do not seem to mediate glucose inhibition of glucagon secretion. We instead propose that glucose has a direct inhibitory effect on mouse alpha cells by suppressing a depolarising Ca 2+ store-operated current.

Cyclic AMP (cAMP) and Ca(2+) are key regulators of exocytosis in many cells, including insulin-secreting beta cells. Glucose-stimulated insulin secretion from beta cells is pulsatile and involves oscillations of the cytoplasmic Ca(2+) concentration ([Ca(2+)](i)), but little is known about the detailed kinetics of cAMP signaling. Using evanescent-wave fluorescence imaging we found that glucose induces pronounced oscillations of cAMP in the submembrane space of single MIN6 cells and primary mouse beta cells. These oscillations were preceded and enhanced by elevations of [Ca(2+)](i). However, conditions raising cytoplasmic ATP could trigger cAMP elevations without accompanying [Ca(2+)](i) rise, indicating that adenylyl cyclase activity may be controlled also by the substrate concentration. The cAMP oscillations correlated with pulsatile insulin release. Whereas elevation of cAMP enhanced secretion, inhibition of adenylyl cyclases suppressed both cAMP oscillations and pulsatile insulin release. We conclude that cell metabolism directly controls cAMP and that glucose-induced cAMP oscillations regulate the magnitude and kinetics of insulin exocytosis.

Pulsatile insulin secretionSince insulin is the only blood glucose-lowering hormone, its secretion is essential for glucose homeostasis, and disturbances are associated with glucose intolerance and diabetes.Glucose is the major stimulus for insulin release but secretion is enhanced also by other nutrients and is under stimulatory and inhibitory control by hormones and neurotransmitters.Although the external factors are essential determinants of insulin secretion, there are also input-independent variations in hormone release. Regular oscillations of the circulating insulin concentrations in normal subjects consequently occur without accompanying changes

OBJECTIVEcAMP is a critical messenger for insulin and glucagon secretion from pancreatic β- and α-cells, respectively. Dispersed β-cells show cAMP oscillations, but the signaling kinetics in cells within intact islets of Langerhans is unknown.RESEARCH DESIGN AND METHODSThe subplasma-membrane cAMP concentration ([cAMP]pm) was recorded in α- and β-cells in the mantle of intact mouse pancreatic islets using total internal reflection microscopy and a fluorescent translocation biosensor. Cell identification was based on the opposite effects of adrenaline on cAMP in α- and β-cells.RESULTSIn islets exposed to 3 mmol/L glucose, [cAMP]pm was low and stable. Glucagon and glucagon-like peptide-1(7-36)-amide (GLP-1) induced dose-dependent elevation of [cAMP]pm, often with oscillations synchronized among β-cells. Whereas glucagon also induced [cAMP]pm oscillations in most α-cells, <20% of the α-cells responded to GLP-1. Elevation of the glucose concentration to 11–30 mmol/L in the absence of hormones induced slow [cAMP]pm oscillations in both α- and β-cells. These cAMP oscillations were coordinated with those of the cytoplasmic Ca2+ concentration ([Ca2+]i) in the β-cells but not caused by the changes in [Ca2+]i. The transmembrane adenylyl cyclase (AC) inhibitor 2′5′-dideoxyadenosine suppressed the glucose- and hormone-induced [cAMP]pm elevations, whereas the preferential inhibitors of soluble AC, KH7, and 1,3,5(10)-estratrien-2,3,17-β-triol perturbed cell metabolism and lacked effect, respectively.CONCLUSIONSOscillatory [cAMP]pm signaling in secretagogue-stimulated β-cells is maintained within intact islets and depends on transmembrane AC activity. The discovery of glucose- and glucagon-induced [cAMP]pm oscillations in α-cells indicates the involvement of cAMP in the regulation of pulsatile glucagon secretion.

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