Beta-adrenoceptor stimulation potentiates insulin-stimulated PKB phosphorylation in rat cardiomyocytes via cAMP and PKA.
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.
Stuenaes
Jorid T
JT
Department of Physiology, National Institute of Occupational Health, Oslo, Norway.
Bolling
Astrid
A
Ingvaldsen
Ada
A
Rommundstad
Camilla
C
Sudar
Emina
E
Lin
Fang-Chin
FC
Lai
Yu-Chiang
YC
Jensen
Jørgen
J
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
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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