Cyclic AMP Oscillations in Insulin-Secreting Cells

Abstract: Cyclic AMP is an intracellular messenger that regulates numerous processes in various types of cells. In pancreatic ?-cells, cAMP potentiates the secretion of insulin by promoting Ca2+ signals and by amplifying Ca2+-triggered exocytosis. Whereas Ca2+ signals have been extensively characterized, little is known about the kinetics of cAMP signals. To enable measurements of the cAMP concentration beneath the plasma membrane ([cAMP]pm) of individual cells, a translocation biosensor was created based on fluorescent-protein-tagged subunits of protein kinase A (PKA). Evanescent wave microscopy imaging of biosensor-expressing clonal ?-cells revealed that the insulinotropic hormones glucagon and GLP-1 triggered pronounced oscillations in [cAMP]pm. Simultaneous measurements of the intracellular Ca2+ concentration showed that cAMP and Ca2+ oscillations were synchronized and interdependent. [cAMP]pm oscillations were also triggered in clonal and primary mouse ?-cells by an elevation of the glucose concentration from 3 to 11 mM. These oscillations were preceded and enhanced by elevations of Ca2+. However, conditions raising cytoplasmic ATP could trigger cAMP elevations also without accompanying Ca2+ changes, indicating that adenylyl cyclase activity may be directly controlled by the substrate concentration. Experiments with 3-isobutylmethylxanthine (IBMX) and various family-selective phosphodiesterase (PDE) inhibitors indicated that [cAMP]pm oscillations are generated by periodic formation of the messenger by adenylyl cyclases. PDE1 and PDE3 as well as IBMX-insensitive mechanisms shape [cAMP]pm, but no single PDE isoform was required for glucose generation of [cAMP]pm oscillations. Recordings of single-cell insulin secretion kinetics with a fluorescent biosensor that reports formation of the phospholipid PIP3 in the plasma membrane in response to autocrine insulin receptor activation showed that [cAMP]pm oscillations were paralleled by pulsatile insulin release. Whereas adenylyl cyclase inhibition suppressed both [cAMP]pm oscillations and pulsatile insulin release, elevation of [cAMP]pm enhanced secretion. Investigation of the effects of different temporal patterns of [cAMP]pm showed that brief [cAMP]pm elevation is sufficient to trigger cytoplasmic responses, whereas sustained elevation is required to induce translocation of the PKA catalytic subunit into the nucleus. In conclusion, these studies demonstrate for the first time in mammalian cells that [cAMP]pm oscillates in response to physiological stimuli. The glucose-induced [cAMP]pm oscillations are generated by periodic cAMP production mediated by interplay between ATP and Ca2+ in the sub-membrane space, and may contribute to both triggering and amplifying pathways of insulin secretion. Apart from regulating the precise kinetics of insulin exocytosis, temporal encoding of cAMP signals might constitute a basis for differential regulation of downstream cellular targets.