title: Beta-adrenoceptor stimulation potentiates insulin-stimulated PKB phosphorylation in rat cardiomyocytes via cAMP and PKA.
abstract: OBJECTIVE
  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 beta-adrenergic stimulation in regulation of PKB phosphorylation.
  
  
  METHODS
  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.
  
  
  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-3beta Ser(9) phosphorylation approximately twofold, supporting that PKB phosphorylation increased kinase activity. Dobutamine (beta(1)-agonist) increased insulin-stimulated PKB phosphorylation as effectively as isoprenaline (more than threefold), whereas salbutamol (beta(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
  beta-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 beta-adrenergic receptors stimulation.
authors.affiliation: Department of Physiology, National Institute of Occupational Health, Oslo, Norway.
authors.forename: Jorid T
authors.initials: JT
authors.lastname: Stuenaes
authors.forename: Astrid
authors.initials: A
authors.lastname: Bolling
authors.forename: Ada
authors.initials: A
authors.lastname: Ingvaldsen
authors.forename: Camilla
authors.initials: C
authors.lastname: Rommundstad
authors.forename: Emina
authors.initials: E
authors.lastname: Sudar
authors.forename: Fang-Chin
authors.initials: FC
authors.lastname: Lin
authors.forename: Yu-Chiang
authors.initials: YC
authors.lastname: Lai
authors.forename: Jørgen
authors.initials: J
authors.lastname: Jensen
mesh_terms: D000318:Adrenergic beta-Agonists / Q000494:pharmacology*; D000818:Animals; D002135:Calcium-Binding Proteins / Q000378:metabolism; D000242:Cyclic AMP / Q000502:physiology*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000502:physiology*; D004280:Dobutamine / Q000494:pharmacology; D004357:Drug Synergism; D004789:Enzyme Activation; D024981:Glycogen Phosphorylase / Q000378:metabolism; D007328:Insulin / Q000494:pharmacology* / Q000502:physiology; D007545:Isoproterenol / Q000494:pharmacology; D008297:Male; D032383:Myocytes, Cardiac / Q000187:drug effects* / Q000378:metabolism; D010766:Phosphorylation; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism*; D051381:Rats; D017208:Rats, Wistar; D011943:Receptors, Adrenergic, beta / Q000502:physiology*; D000099211:Phospholamban
chemical_list: D000318:Adrenergic beta-Agonists; D002135:Calcium-Binding Proteins; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases; D004280:Dobutamine; D024981:Glycogen Phosphorylase; D007328:Insulin; D007545:Isoproterenol; D051057:Proto-Oncogene Proteins c-akt; D011943:Receptors, Adrenergic, beta; D000099211:Phospholamban
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