Inhibition of insulin secretion via distinct signaling pathways in α2-adrenoceptor knockout mice

Objective: Adrenaline inhibits insulin secretion through activation of alpha(2)-adrenoceptors (ARs). These receptors are linked to pertussis toxin-sensitive G proteins. Agonist binding leads to inhibition of adenylyl cyclase, inhibition of Ca2+ channels and activation of K+ channels. Recently, three distinct subtypes of alpha(2)-AR were described, alpha(2A)-AR, alpha(2B)-AR and alpha(2C)-AR. At present, it is unknown which of these alpha(2)-AR subtype(s) may regulate insulin secretion. We used mice deficient in alpha(2)-ARs to analyze the coupling and role of individual alpha(2)-AR subtypes in insulin-secreting beta cells. Methods: The inhibitory effect of adrenaline on insulin secretion was measured in freshly isolated and cultured wild type (wt) and alpha(2)-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, alpha(2A)- and alpha(2C)-AR KO mouse islets, adrenaline, 1 mumol/l, inhibited secretion by 83, 80 and 100% respectively. In contrast, in alpha(2A/2C)-AR double KO mouse islets, adrenaline had no effect on stimulated secretion indicating that both alpha(2A)-AR and U2c-AR, but not alpha(2B)-AR, are functionally expressed in mouse islets. Surprisingly, glucose (16.7 mmol/l)-induced secretion in the presence of 1 mumol/l forskolin was greatly impaired in alpha(2A)-AR KO islets. However, when cAMP levels were increased further by the combination of forskolin (5 Lmol/l) and 3-isobutyl-1-methylxanthine (100 mumol/l), secretion was stimulated 2.7-fold (8.5-fold in wt islets). Adrenaline lowered the concentration of cAMP in wt and alpha(2C)-AR KO mouse islets by 74%. Adrenaline also hyperpolarized wt and U2c-AR KO beta cells. In contrast, adrenaline did not inhibit adenylyl cyclase in islets of alpha(2A)-AR KO mice, nor did it hyperpolarize alpha(2A)-AR KO beta cells. Conclusion: Adrenaline inhibits insulin release through alpha(2A)- and alpha(2C)-ARs via distinct intracellular signaling pathways.