Inhibition of contraction-stimulated AMP-activated protein kinase inhibits contraction-stimulated increases in PAS-TBC1D1 and glucose transport without altering PAS-AS160 in rat skeletal muscle.
OBJECTIVE
Phosphorylation of two members of the TBC1 domain family of proteins, Akt substrate of 160 kDa (AS160, also known as TBC1D4) and TBC1D1, has been implicated in the regulation of glucose transport in skeletal muscle. Insulin-stimulated phosphorylation (measured using the phospho-Akt substrate [PAS] antibody) of AS160 and TBC1D1 appears to occur in an Akt-dependent manner, but the kinases responsible for contraction-stimulated PAS-AS160 and PAS-TBC1D1 remain unclear. AMP-activated protein kinase (AMPK) and Akt, both activated by contraction, can each phosphorylate AS160 and TBC1D1 in cell-free assays.


METHODS
To evaluate the roles of AMPK and Akt on insulin- or contraction-stimulated PAS-AS160, PAS-TBC1D1, and glucose transport, rat epitrochlearis was incubated with and without compound C (inhibitor of AMPK) or Wortmannin (inhibitor of phosphatidylinositol [PI] 3-kinase, which is upstream of Akt) before and during insulin stimulation or contraction.


RESULTS
Insulin-stimulated glucose transport and phosphorylation of both AS160 and TBC1D1 were completely inhibited by Wortmannin. Wortmannin eliminated contraction stimulation of phospho-Ser(21/9)glycogen synthase kinase 3alpha/beta (pGSK3; Akt substrate) and PAS-AS160 but did not significantly alter pAMPK, phospho-Ser79acetyl CoA carboxylase (pACC; AMPK substrate), PAS-TBC1D1, or glucose transport in contraction-stimulated muscle. Compound C completely inhibited contraction-stimulated pACC and PAS-TBC1D1 and partially blocked glucose transport, but it did not significantly alter pAkt, pGSK3, or PAS-AS160.


CONCLUSIONS
These data suggest that 1) insulin stimulates glucose transport and phosphorylation of AS160 and TBC1D1 in a PI 3-kinase/Akt-dependent manner, 2) contraction stimulates PAS-AS160 (but not PAS-TBC1D1 or glucose transport) in a PI 3-kinase/Akt-dependent manner, and 3) contraction stimulates PAS-TBC1D1 and glucose transport (but not PAS-AS160) in an AMPK-dependent manner.
Funai
Katsuhiko
K
Muscle Biology Laboratory, School of Kinesiology, University of Michigan, Ann Arbor, Michigan, USA.
Cartee
Gregory D
GD
D016475:3T3 Cells; D055372:AMP-Activated Protein Kinases / Q000037:antagonists & inhibitors*; D017667:Adipocytes / Q000166:cytology / Q000187:drug effects / Q000502:physiology; D000730:Androstadienes / Q000494:pharmacology; D000818:Animals; D004791:Enzyme Inhibitors / Q000494:pharmacology; D020690:GTPase-Activating Proteins / Q000187:drug effects / Q000378:metabolism; D005947:Glucose / Q000378:metabolism; D006801:Humans; D007328:Insulin / Q000494:pharmacology; D008297:Male; D051379:Mice; D009119:Muscle Contraction / Q000187:drug effects / Q000502:physiology*; D018482:Muscle, Skeletal / Q000187:drug effects / Q000502:physiology*; D009687:Nuclear Proteins / Q000187:drug effects / Q000378:metabolism* / Q000502:physiology; D000081082:Phosphoinositide-3 Kinase Inhibitors; D051381:Rats; D017208:Rats, Wistar; D011994:Recombinant Proteins / Q000494:pharmacology; D000077191:Wortmannin
D000730:Androstadienes; D004791:Enzyme Inhibitors; D020690:GTPase-Activating Proteins; D007328:Insulin; D009687:Nuclear Proteins; D000081082:Phosphoinositide-3 Kinase Inhibitors; D011994:Recombinant Proteins; C095906:Tbc1d1 protein, mouse; C505647:Tbc1d4 protein, mouse; D055372:AMP-Activated Protein Kinases; D005947:Glucose; D000077191:Wortmannin