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 b-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-3b Ser 9 phosphorylation approximately twofold, supporting that PKB phosphorylation increased kinase activity. Dobutamine (b1-agonist) increased insulin-stimulated PKB phosphorylation as effectively as isoprenaline (more than threefold), whereas salbutamol (b2-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-methylcAMP (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: b-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 b-adrenergic receptors stimulation. Pharmacology (2010) 160, 116-129; doi:10.1111/j.1476-5381.2010 published online 23 March 2010 Keywords: Heart; Akt; GSK-3; phosphorylation; phosphatidylinositol 3-kinase; phoshodiesterase; phospholamban; hypertrophy; rolipram; ERK Abbreviations: DNA-PK, DNA-dependent protein kinase; Epac, exchange protein directly activated by cAMP; ERK, extracellular signal-regulated kinase; GSK-3, glycogen synthase kinase-3; IGF-1, insulin like growth factor-1; mAKAP, muscle-specific A-kinase anchoring protein; MEK, mitogen-activated protein kinase kinase; mTORC2, mammalian target of rapamyosin (mTOR) complex-2; PDK1, phosphoinositide-dependent kinase-1; PTEN, phosphatase and tensin homolog deleted on chromosome 10 British Journal of

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