Calcium influx activates adenylyl cyclase 8 for sustained insulin secretion in rat pancreatic beta cells.

Aims/hypothesis: Insulin is a key metabolic regulator in health and diabetes. In pancreatic beta cells, insulin release is regulated by the major second messengers Ca and cAMP: exocytosis is triggered by Ca and mediated by the cAMP/protein kinase A (PKA) signalling pathway. However, the causal link between these two processes in primary beta cells remains undefined. Methods: Time-resolved confocal imaging of fluorescence resonance energy transfer signals was performed to visualise PKA activity, and combined membrane capacitance recordings were used to monitor insulin secretion from patch-clamped rat beta cells. Results: Membrane depolarisation-induced Ca influx caused an increase in cytosolic PKA activity via activating a Ca-sensitive adenylyl cyclase 8 (ADCY8) subpool. Glucose stimulation triggered coupled Ca oscillations and PKA activation. ADCY8 knockdown significantly reduced the level of depolarisation-evoked PKA activation and impaired replenishment of the readily releasable vesicle pool. Pharmacological inhibition of PKA by two inhibitors reduced depolarisation-induced PKA activation to a similar extent and reduced the capacity for sustained vesicle exocytosis and insulin release. Conclusions/interpretation: Our findings suggest that depolarisation-induced Ca influx plays dual roles in regulating exocytosis in rat pancreatic beta cells by triggering vesicle fusion and replenishing the vesicle pool to support sustained insulin release. Therefore, Ca influx may be important for glucose-stimulated insulin secretion. [ABSTRACT FROM AUTHOR]