Beta(2)-Adrenergic activation increases glycogen synthesis in L6 skeletal muscle cells through a signalling pathway independent of cyclic AMP.
OBJECTIVE
In skeletal muscle, the storage of glycogen by insulin is regulated by glycogen synthase, which is regulated by glycogen synthase kinase 3 (GSK3). Here we examined whether adrenergic receptor activation, which can increase glucose uptake, regulates glycogen synthesis in L6 skeletal muscle cells.


METHODS
We used L6 cells and measured glycogen synthesis (as incorporation of D: -[U-(14)C]glucose into glycogen) and GSK3 phosphorylation following adrenergic activation.


RESULTS
Insulin (negative logarithm of median effective concentration [pEC(50)] 8.2 +/- 0.3) and the beta-adrenergic agonist isoprenaline (pEC(50) 7.5 +/- 0.3) induced a twofold increase in glycogen synthesis in a concentration-dependent manner. The alpha(1)-adrenergic agonist cirazoline and alpha(2)-adrenergic agonist clonidine had no effect. Both insulin and isoprenaline phosphorylated GSK3. The beta-adrenergic effect on glycogen synthesis is mediated by beta(2)-adrenoceptors and not beta(1)-/beta(3)-adrenoceptors, and was not mimicked by 8-bromo-cyclic AMP or cholera toxin, and also was insensitive to pertussis toxin, indicating no involvement of cyclic AMP or inhibitory G-protein (G(i)) signalling in the beta(2)-adrenergic effect on glycogen synthesis. 12-O-tetra-decanoylphorbol-13-acetate (TPA) increased glycogen synthesis 2.5-fold and phosphorylated GSK3 fourfold. Inhibition of protein kinase C (PKC) isoforms with 12-(2-cyanoethyl)-6,7,12,13-tetrahydro-13-methyl-5-oxo-5H-indolo(2,3-a)pyrrollo(3,4-c)-carbazole (Gö6976; inhibits conventional and novel PKCs) or 2-[1-(3-dimethylaminopropyl)-5-methoxyindol-3-yl]-3-(1H-indol-3-yl)maleimide (Gö6983; inhibits conventional, novel and atypical PKCs) inhibited the stimulatory TPA effect, but did not significantly inhibit glycogen synthesis mediated by insulin or isoprenaline. Inhibition of phosphatidylinositol 3-kinase (PI3K) with wortmannin inhibited the effects of insulin and isoprenaline on glycogen synthesis.


CONCLUSIONS
These results demonstrate that in L6 skeletal muscle cells adrenergic stimulation through beta(2)-adrenoceptors, but not involving cyclic AMP or G(i), activates a PI3K pathway that stimulates glycogen synthesis through GSK3.
Yamamoto
D L
DL
Department of Physiology, The Wenner-Gren Institute, Arrhenius Laboratories F3, Stockholm University, SE 10691, Stockholm, Sweden.
Hutchinson
D S
DS
Bengtsson
T
T
D055372:AMP-Activated Protein Kinases; D000318:Adrenergic beta-Agonists / Q000494:pharmacology; D000730:Androstadienes / Q000494:pharmacology; D000818:Animals; D002460:Cell Line; D000242:Cyclic AMP / Q000502:physiology*; D005947:Glucose / Q000378:metabolism; D006003:Glycogen / Q000378:metabolism*; D038362:Glycogen Synthase Kinase 3 / Q000378:metabolism; D007328:Insulin / Q000494:pharmacology; D007545:Isoproterenol / Q000494:pharmacology; D009097:Multienzyme Complexes / Q000378:metabolism; D018482:Muscle, Skeletal / Q000166:cytology / Q000378:metabolism*; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D000081082:Phosphoinositide-3 Kinase Inhibitors; D010766:Phosphorylation / Q000187:drug effects; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D051381:Rats; D018343:Receptors, Adrenergic, beta-2 / Q000378:metabolism*; D015398:Signal Transduction / Q000502:physiology*; D000077191:Wortmannin
D055372:AMP-Activated Protein Kinases; D000318:Adrenergic beta-Agonists; D000730:Androstadienes; D000242:Cyclic AMP; D005947:Glucose; D006003:Glycogen; D038362:Glycogen Synthase Kinase 3; D007328:Insulin; D007545:Isoproterenol; D009097:Multienzyme Complexes; D019869:Phosphatidylinositol 3-Kinases; D000081082:Phosphoinositide-3 Kinase Inhibitors; D047428:Protein Kinase Inhibitors; D017346:Protein Serine-Threonine Kinases; D018343:Receptors, Adrenergic, beta-2; D000077191:Wortmannin