β-Adrenoceptor stimulation potentiates insulin-stimulated PKB phosphorylation in rat cardiomyocytes via cAMP and PKA

Abstract

Background and purpose: Genetic approaches have documented protein kinase B (PKB) as a pivotal regulator of heart function. Insulin strongly activates PKB, whereas adrenaline is not considered a major physiological regulator of PKB in heart. In skeletal muscles, however, adrenaline potentiates insulin-stimulated PKB activation without having effect in the absence of insulin. The purpose of the present study was to investigate the interaction between insulin and β-adrenergic stimulation in regulation of PKB phosphorylation. Experimental approach: Cardiomyocytes were isolated from adult rats by collagenase, and incubated with insulin, isoprenaline, and other compounds. Protein phosphorylation was evaluated by Western blot and phospho-specific antibodies. Key results: Isoprenaline increased insulin-stimulated PKB Ser 473 and Thr 308 phosphorylation more than threefold in cardiomyocytes. Isoprenaline alone did not increase PKB phosphorylation. Isoprenaline also increased insulin-stimulated GSK-3β Ser 9 phosphorylation approximately twofold, supporting that PKB phosphorylation increased kinase activity. Dobutamine (β 1-agonist) increased insulin-stimulated PKB phosphorylation as effectively as isoprenaline (more than threefold), whereas salbutamol (β 2-agonist) only potentiated insulin-stimulated PKB phosphorylation by approximately 80%. Dobutamine, but not salbutamol, increased phospholamban Ser 16 phosphorylation and glycogen phosphorylase activation (PKA-mediated effects). Furthermore, the cAMP analogue that activates PKA (dibutyryl-cAMP and N 6-benzoyl-cAMP) increased insulin-stimulated PKB phosphorylation by more than threefold without effect alone. The Epac-specific activator 8-(4-chlorophenylthio)-2′-O- methyl-cAMP (007) increased insulin-stimulated PKB phosphorylation by approximately 50%. Db-cAMP and N 6-benzoyl-cAMP, but not 007, increased phospholamban Ser 16 phosphorylation. Conclusions and implications: β-adrenoceptors are strong regulators of PKB phosphorylation via cAMP and PKA when insulin is present. We hypothesize that PKB mediates important signalling in the heart during β-adrenergic receptors stimulation. © 2010 The British Pharmacological Society.