EXPERIMENTAL STUDY Inhibition of insulin secretion via distinct signaling pathways in a2-adrenoceptor knockout mice

Objective: Adrenaline inhibits insulin secretion through activation of a2-adrenoceptors (ARs). These receptors are linked to pertussis toxin-sensitive G proteins. Agonist binding leads to inhibition of adenylyl cyclase, inhibition of Ca2þ channels and activation of Kþ channels. Recently, three distinct subtypes of a2-AR were described, a2A-AR, a2B-AR and a2C-AR. At present, it is unknown which of these a2-AR subtype(s) may regulate insulin secretion. We used mice deficient in a2-ARs to analyze the coupling and role of individual a2-AR subtypes in insulin-secreting b cells. Methods: The inhibitory effect of adrenaline on insulin secretion was measured in freshly isolated and cultured wild type (wt) and a2-AR knockout (KO) mouse islets in order to examine the receptor subtypes which mediate adrenaline-induced inhibition of insulin secretion. Adenylyl cyclase activity was measured in isolated cultured islets. Membrane potential was measured using the amphotericin B permeabilized patch clamp method in isolated and cultured single islet cells. Results: In wt, a2Aand a2C-AR KO mouse islets, adrenaline, 1mmol/l, inhibited secretion by 83, 80 and 100% respectively. In contrast, in a2A/2C-AR double KO mouse islets, adrenaline had no effect on stimulated secretion indicating that both a2A-AR and a2C-AR, but not a2B-AR, are functionally expressed in mouse islets. Surprisingly, glucose (16.7 mmol/l)-induced secretion in the presence of 1mmol/l forskolin was greatly impaired in a2A-AR KO islets. However, when cAMP levels were increased further by the combination of forskolin (5mmol/l) and 3-isobutyl-1-methylxanthine (100mmol/l), secretion was stimulated 2.7-fold (8.5-fold in wt islets). Adrenaline lowered the concentration of cAMP in wt and a2C-AR KO mouse islets by 74%. Adrenaline also hyperpolarized wt and a2C-AR KO b cells. In contrast, adrenaline did not inhibit adenylyl cyclase in islets of a2A-AR KO mice, nor did it hyperpolarize a2A-AR KO b cells. Conclusion: Adrenaline inhibits insulin release through a2Aand a2C-ARs via distinct intracellular signaling pathways. European Journal of Endocrinology 149 343–350