Role of phosphodiesterases in the shaping of sub-plasma-membrane cAMP oscillations and pulsatile insulin secretion.

Specificity and versatility in cyclic AMP (cAMP) signalling are governed by the spatial localisation and temporal dynamics of the signal. Phosphodiesterases (PDEs) are important for shaping cAMP signals by hydrolyzing the nucleotide. In pancreatic ß-cells, glucose triggers sub-plasma-membrane cAMP oscillations, which are important for insulin secretion, but the mechanisms underlying the oscillations are poorly understood. Here, we investigated the role of different PDEs in the generation of cAMP oscillations by monitoring the concentration of cAMP in the sub-plasma-membrane space ([cAMP](pm)) with ratiometric evanescent wave microscopy in MIN6 cells or mouse pancreatic ß-cells expressing a fluorescent translocation biosensor. The general PDE inhibitor IBMX increased [cAMP](pm), and whereas oscillations were frequently observed at 50 µM IBMX, 300 µM-1 mM of the inhibitor caused a stable increase in [cAMP](pm). The [cAMP](pm) was nevertheless markedly suppressed by the adenylyl cyclase inhibitor 2',5'-dideoxyadenosine, indicating IBMX-insensitive cAMP degradation. Among IBMX-sensitive PDEs, PDE3 was most important for maintaining a low basal level of [cAMP](pm) in unstimulated cells. After glucose induction of [cAMP](pm) oscillations, inhibitors of PDE1, PDE3 and PDE4 inhibitors the average cAMP level, often without disturbing the [cAMP](pm) rhythmicity. Knockdown of the IBMX-insensitive PDE8B by shRNA in MIN6 cells increased the basal level of [cAMP](pm) and prevented the [cAMP](pm)-lowering effect of 2',5'-dideoxyadenosine after exposure to IBMX. Moreover, PDE8B-knockdown cells showed reduced glucose-induced [cAMP](pm) oscillations and loss of the normal pulsatile pattern of insulin secretion. It is concluded that [cAMP](pm) oscillations in ß-cells are caused by periodic variations in cAMP generation, and that several PDEs, including PDE1, PDE3 and the IBMX-insensitive PDE8B, are required for shaping the sub-membrane cAMP signals and pulsatile insulin release.

Distinct plasma-membrane PtdIns(4)P and PtdIns(4,5)P2 dynamics in secretagogue-stimulated beta-cells.

Phosphoinositides regulate numerous processes in various subcellular compartments. Whereas many stimuli trigger changes in the plasma-membrane PtdIns(4,5)P(2) concentration, little is known about its precursor, PtdIns(4)P, in particular whether there are stimulus-induced alterations independent of those of PtdIns(4,5)P(2). We investigated plasma-membrane PtdIns(4)P and PtdIns(4,5)P(2) dynamics in insulin-secreting MIN6 cells using fluorescent translocation biosensors and total internal reflection microscopy. Loss of PtdIns(4,5)P(2) induced by phospholipase C (PLC)-activating receptor agonists or stimulatory glucose concentrations was paralleled by increased PtdIns(4)P levels. In addition, glucose-stimulated cells regularly showed anti-synchronous oscillations of the two lipids. Whereas glucose-induced PtdIns(4)P elevation required voltage-gated Ca(2+) entry and was mimicked by membrane-depolarizing stimuli, the receptor-induced response was Ca(2+) independent, but sensitive to protein kinase C (PKC) inhibition and mimicked by phorbol ester stimulation. We conclude that glucose and PLC-activating receptor stimuli trigger Ca(2+)- and PKC-dependent changes in the plasma-membrane PtdIns(4)P concentration that are independent of the effects on PtdIns(4,5)P(2). These findings indicate that enhanced formation of PtdIns(4)P, apart from ensuring efficient replenishment of the PtdIns(4,5)P(2) pool, might serve an independent signalling function by regulating the association of PtdIns(4)P-binding proteins with the plasma membrane.

Oscillations of cyclic AMP in hormone-stimulated insulin-secreting beta-cells.

Cyclic AMP is a ubiquitous second messenger that transduces signals from a variety of cell surface receptors to regulate diverse cellular functions, including secretion, metabolism and gene transcription. In pancreatic beta-cells, cAMP potentiates Ca2+-dependent exocytosis and mediates the stimulation of insulin release exerted by the hormones glucagon and glucagon-like peptide-1 (GLP-1) (refs 4, 5-6). Whereas Ca2+ signals have been extensively characterized and shown to involve oscillations important for the temporal control of insulin secretion, the kinetics of receptor-triggered cAMP signals is unknown. Here we introduce a new ratiometric evanescent-wave-microscopy approach to measure cAMP concentration beneath the plasma membrane, and show that insulin-secreting beta-cells respond to glucagon and GLP-1 with marked cAMP oscillations. Simultaneous measurements of intracellular Ca2+ concentration revealed that the two messengers are interlinked and reinforce each other. Moreover, cAMP oscillations are capable of inducing rapid on-off Ca2+ responses, but only sustained elevation of cAMP concentration induces nuclear translocation of the catalytic subunit of the cAMP-dependent protein kinase. Our results establish a new signalling mode for cAMP and indicate that temporal encoding of cAMP signals might constitute a basis for differential regulation of downstream cellular targets.

Glucose-induced cyclic AMP oscillations regulate pulsatile insulin secretion.

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.