title: Domain-dependent modulation of insulin-induced AS160 phosphorylation and glucose uptake by Ca2+/calmodulin-dependent protein kinase II in L6 myotubes.
abstract: In skeletal muscle, the molecular mechanisms by which insulin stimulates glucose transport remains incompletely understood. Our study investigated the cellular dynamics of intracellular Ca(2+) mobilisation and Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) activation on insulin-induced skeletal muscle glucose transport. L6 myotubes were treated without or with insulin [100 nM] for 15 min and subsequently monitored for glucose uptake using isotope-labelled 2-deoxyglucose (I-2DOG), intracellular Ca(2+) (Ca(i)(2+)) release using Fluo-4AM and protein phosphorylation using Western blotting. Acute exposure of myotubes to insulin increased both Akt substrate-160 kDa (AS160) phosphorylation and I-2DOG uptake. Insulin concurrently increased Ca(i)(2+) and activated CaMKII. Exposing myotubes to either BAPTA/AM to sequester Ca(i)(2+) or KN-93 to inhibit CaMKII activity, decreased insulin-induced glucose uptake without affecting AS160 phosphorylation. On the other hand, blocking either calmodulin or the autoregulatory domain of CaMKII blocked the effect of insulin on both AS160 phosphorylation and glucose transport. Likewise, genetic knockdown of CaMKII in myotubes using siRNA completely abolished insulin-mediated glucose uptake. These results illustrate impairments in Ca(i)(2+) mobilisation and CaMKII activation are sufficient to negatively influence insulin-dependent glucose transport by L6 myotubes. Additionally, our results show for the first time that Ca(i)(2+) and domain-dependent CaMKII signalling differentially affect insulin-induced AS160 phosphorylation, and establish that Ca(2+) and CaMKII are components of the insulin signalling pathway in L6 myotubes.
authors.affiliation: Human Nutritional Sciences, University of Manitoba, Winnipeg, MB, Canada.
authors.forename: Suresh K
authors.initials: SK
authors.lastname: Mohankumar
authors.forename: Carla G
authors.initials: CG
authors.lastname: Taylor
authors.forename: Peter
authors.initials: P
authors.lastname: Zahradka
mesh_terms: D000818:Animals; D001596:Benzylamines / Q000494:pharmacology; D020013:Calcium Signaling / Q000187:drug effects; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2 / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D002614:Chelating Agents / Q000494:pharmacology; D003847:Deoxyglucose / Q000378:metabolism*; D004533:Egtazic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D020690:GTPase-Activating Proteins / Q000378:metabolism*; D055785:Gene Knockdown Techniques; D006801:Humans; D007328:Insulin / Q000494:pharmacology* / Q000502:physiology; D018485:Muscle Fibers, Skeletal / Q000187:drug effects / Q000378:metabolism*; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation; D017434:Protein Structure, Tertiary; D034622:RNA Interference; D051381:Rats; D013449:Sulfonamides / Q000494:pharmacology; D000096926:Benzenesulfonamides
chemical_list: D001596:Benzylamines; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D002614:Chelating Agents; D003847:Deoxyglucose; D004533:Egtazic Acid; D020690:GTPase-Activating Proteins; D007328:Insulin; D010750:Phosphoproteins; D013449:Sulfonamides; C533101:TBC1D4 protein, rat; C072105:KN 93; D000096926:Benzenesulfonamides; C070379:1,2-bis(2-aminophenoxy)ethane N,N,N',N'-tetraacetic acid acetoxymethyl ester