Modulation of Noradrenaline-Induced cAMP Production in HEK-293 Cells by Insulin

Abstract— The first messengers regulate cellular functions by involving surface receptors coupled to diverse intracellular signaling pathways. Those do not function completely independently, and an increasing body of evidence argues for the existence of crosstalk between signaling systems and their coordination. Here we analyzed a potential contribution of the PI3K–Akt cascade to the regulation of cAMP signaling initiated by noradrenaline (NA) in HEK-293 cells that expressed Pink Flamindo, a genetically encoded cAMP sensor, at a constant and high level. This feature makes this cell line a sufficiently effective cellular model for the analysis of mechanisms involved in the generation of intracellular cAMP signals. As was shown, the stimulation of β‑adrenoreceptors (β-AR) by NA led to a rise in cAMP in HEK-293 cells. Being applied shortly for 100–150 s, NA initiated cAMP signals, the magnitude of which gradually increased with an agonist concentration, exhibiting the EC50 of about 1 µM. In the presence of 150 nM insulin, the magnitude of cAMP responses to 1 µM NA decreased by 31% on average compared to control. The inhibitors of PI3K and Akt, wortmannin (10 µM) and GSK690693 (5 µM), respectively, which as such did not affect cAMP responses of cells, canceled inhibitory effects of insulin on cAMP signaling triggered by NA. This indicated that insulin diminished NA-induced cAMP signals by involving insulin receptors coupled to the PI3K–Akt pathway, which, in turn, modulated efficacy of NA transduction mediated by the signaling cascade β-AR–Gs-protein–adenylate cyclase/phosphodiesterase (PDE). Reportedly, the phosphorylation of PDE3 by Akt stimulated this cAMP-specific PDE isoform, the phenomenon that could account for the decrease of cAMP signals observed in the presence of insulin. Supporting this idea was that selective PDE3 inhibitor Trequinsin (4 µM) per se gave rise to intracellular cAMP, indicating that the activity of PDE3 in non-stimulated cells was sufficiently high. At the same time, insulin decreased cAMP responses to Trequinsin by 36% compared to control. The overall data obtained here suggest that insulin can modulate cAMP responses of HEK-293 cells to NA largely due to coupling of the insulin receptor to the PI3K–Akt pathway that controlled activity of cAMP-hydrolyzing PDE3 in an insulin-dependent manner.