Contraction-related stimuli regulate GLUT4 traffic in C2C12-GLUT4myc skeletal muscle cells.
Muscle contraction stimulates glucose uptake acutely to increase energy supply, but suitable cellular models that faithfully reproduce this complex phenomenon are lacking. To this end, we have developed a cellular model of contracting C(2)C(12) myotubes overexpressing GLUT4 with an exofacial myc-epitope tag (GLUT4myc) and explored stimulation of GLUT4 traffic by physiologically relevant agents. Carbachol (an acetylcholine receptor agonist) induced a gain in cell surface GLUT4myc that was mediated by nicotinic acetylcholine receptors. Carbachol also activated AMPK, and this response was sensitive to the contractile myosin ATPase inhibitor N-benzyl-p-toluenesulfonamide. The gain in surface GLUT4myc elicited by carbachol or by the AMPK activator 5-amino-4-carboxamide-1 beta-ribose was sensitive to chemical inhibition of AMPK activity by compound C and partially reduced by siRNA-mediated knockdown of AMPK catalytic subunits or LKB1. In addition, the carbachol-induced gain in cell surface GLUT4myc was partially sensitive to chelation of intracellular calcium with BAPTA-AM. However, the carbachol-induced gain in cell surface GLUT4myc was not sensitive to the CaMKK inhibitor STO-609 despite expression of both isoforms of this enzyme and a rise in cytosolic calcium by carbachol. Therefore, separate AMPK- and calcium-dependent signals contribute to mobilizing GLUT4 in response to carbachol, providing an in vitro cell model that recapitulates the two major signals whereby acute contraction regulates glucose uptake in skeletal muscle. This system will be ideal to further analyze the underlying molecular events of contraction-regulated GLUT4 traffic.
Program in Cell Biology, The Hospital for Sick Children, 555 University Ave., Toronto, ON, M5G 1X8, Canada.
Wenyan
W
Niu
Philip J
PJ
Bilan
Shuhei
S
Ishikura
Jonathan D
JD
Schertzer
Ariel
A
Contreras-Ferrat
Zhengxiang
Z
Fu
Jie
J
Liu
Shlomit
S
Boguslavsky
Kevin P
KP
Foley
Zhi
Z
Liu
Jinru
J
Li
Guilan
G
Chu
Thomas
T
Panakkezhum
Gary D
GD
Lopaschuk
Sergio
S
Lavandero
Zhi
Z
Yao
Amira
A
Klip
D055372:AMP-Activated Protein Kinases / Q000378:metabolism; D000109:Acetylcholine / Q000378:metabolism; D000818:Animals; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism*; D020013:Calcium Signaling / Q000187:drug effects; D002217:Carbachol / Q000494:pharmacology; D002460:Cell Line; D002478:Cells, Cultured; D018679:Cholinergic Agonists / Q000494:pharmacology; D005455:Fluorescent Antibody Technique; D005947:Glucose / Q000378:metabolism; D051275:Glucose Transporter Type 4 / Q000378:metabolism*; D051379:Mice; D009119:Muscle Contraction / Q000187:drug effects / Q000502:physiology*; D018485:Muscle Fibers, Skeletal / Q000187:drug effects / Q000378:metabolism*; D021381:Protein Transport / Q000187:drug effects / Q000502:physiology; D051381:Rats; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology
D018679:Cholinergic Agonists; D051275:Glucose Transporter Type 4; D002217:Carbachol; D055372:AMP-Activated Protein Kinases; D005947:Glucose; D000109:Acetylcholine; D002118:Calcium