title: The M3-muscarinic acetylcholine receptor stimulates glucose uptake in L6 skeletal muscle cells by a CaMKK-AMPK-dependent mechanism.
abstract: The role of muscarinic acetylcholine receptors (mAChRs) in regulating glucose uptake in L6 skeletal muscle cells was investigated. [(3)H]-2-Deoxyglucose uptake was increased in differentiated L6 cells by insulin, acetylcholine, oxotremorine-M and carbachol. mAChR-mediated glucose uptake was inhibited by the AMPK inhibitor Compound C. Whole cell radioligand binding using [(3)H]-N-methyl scopolamine chloride identified mAChRs in differentiated but not undifferentiated L6 cells and M(3) mAChR mRNA was detected only in differentiated cells. M(3) mAChRs are Gq-coupled, and cholinergic stimulation by the mAChR agonists acetylcholine, oxotremorine-M and carbachol increased Ca(2+) in differentiated but not undifferentiated L6 cells. This was due to muscarinic but not nicotinic activation as responses were antagonised by the muscarinic antagonist atropine but not the nicotinic antagonist tubocurarine. Western blotting showed that both carbachol and the AMPK activator AICAR increased phosphorylation of the AMPKalpha subunit at Thr172, with responses to carbachol blocked by Compound C and the CaMKK inhibitor STO609 but not by the PI3K inhibitor wortmannin. AICAR-stimulated AMPK phosphorylation was not sensitive to STO-609, confirming that this compound inhibits CaMKK but not the classical AMPK kinase LKB1. The TAK1 inhibitor (5Z)-7-oxozeaenol and the G(i) inhibitor pertussis toxin both failed to block AMPK phosphorylation in response to carbachol. Using CHO-K1 cells stably expressing each of the mAChR subtypes (M(1)-M(4)), it was determined that only the M(1) and M(3) mAChRs phosphorylate AMPK, confirming a G(q)-dependent mechanism. This study demonstrates that activation of M(3) mAChRs in L6 skeletal muscle cells stimulates glucose uptake via a CaMKK-AMPK-dependent mechanism, independent of the insulin-stimulated pathway.
authors.affiliation: Department of Pharmacology, Monash Institute of Pharmaceutical Sciences, Monash University, Victoria, 3800, Australia.
authors.forename: Jon
authors.initials: J
authors.lastname: Merlin
authors.forename: Bronwyn A
authors.initials: BA
authors.lastname: Evans
authors.forename: Robert I
authors.initials: RI
authors.lastname: Csikasz
authors.forename: Tore
authors.initials: T
authors.lastname: Bengtsson
authors.forename: Roger J
authors.initials: RJ
authors.lastname: Summers
authors.forename: Dana S
authors.initials: DS
authors.lastname: Hutchinson
mesh_terms: D000091162:AMP-Activated Protein Kinase Kinases; D000249:Adenosine Monophosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D001692:Biological Transport; D016466:CHO Cells; D002118:Calcium / Q000378:metabolism; D054737:Calcium-Calmodulin-Dependent Protein Kinase Kinase / Q000378:metabolism*; D002454:Cell Differentiation; D006224:Cricetinae; D003412:Cricetulus; D003847:Deoxyglucose / Q000378:metabolism; D005947:Glucose / Q000378:metabolism*; D018482:Muscle, Skeletal / Q000378:metabolism*; D011494:Protein Kinases / Q000378:metabolism*; D051381:Rats; D043587:Receptor, Muscarinic M3 / Q000378:metabolism*; D015398:Signal Transduction
chemical_list: D043587:Receptor, Muscarinic M3; D000249:Adenosine Monophosphate; D000255:Adenosine Triphosphate; D003847:Deoxyglucose; D011494:Protein Kinases; D054737:Calcium-Calmodulin-Dependent Protein Kinase Kinase; D000091162:AMP-Activated Protein Kinase Kinases; D005947:Glucose; D002118:Calcium