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

Specificity and versatility in cAMP signalling are governed by the spatial localization and temporal dynamics of the signal. Phosphodiesterases (PDEs) are important for shaping cAMP signals by hydrolyzing the nucleotide. In pancreatic β-cells, glucose triggers sub-plasma membrane cAMP oscillations important for insulin secretion, but the mechanisms underlying the oscillations are poorly understood. Here, we investigated the role of different PDEs for generating cAMP oscillations by monitoring the sub-membrane cAMP concentration ([cAMP]pm) with ratiometric evanescent wave microscopy in MIN6-cells or mouse pancreatic β-cells expressing a fluorescent translocation biosensor. The general PDE inhibitor IBMX increased [cAMP]pm, and while oscillations were frequently observed at 50 µM IBMX, 300 µM–1 mM of the inhibitor caused stable [cAMP]pm elevation. [cAMP]pm was nevertheless markedly suppressed by the adenylyl cyclase inhibitor 2′,5′-dideoxyadenosine, indicating also IBMX-insensitive cAMP degradation. Among IBMX-sensitive PDEs, PDE3 was most important for maintaining a low basal [cAMP]pm in unstimulated cells. After glucose induction of [cAMP]pm oscillations, PDE1, -3 and -4 inhibitors increased the average cAMP level, often without disturbing the [cAMP]pm rhythmicity. Knockdown of the IBMX-insensitive PDE8B by shRNA in MIN6-cells increased basal [cAMP]pm and prevented the [cAMP]pm-lowering effect of 2′,5′-dideoxyadenosine after IBMX exposure. 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 β-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.