The M3-muscarinic acetylcholine receptor stimulates glucose uptake in L6 skeletal muscle cells by a CaMKK-AMPK-dependent mechanism

Jon Merlin, Bronwyn Anne Evans, Robert I Csikasz, Tore Bengtsson, Roger James Summers, Dana S Hutchinson

Research output: Contribution to journalArticleResearchpeer-review

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.
Original languageEnglish
Pages (from-to)1104 - 1113
Number of pages10
JournalCellular Signalling
Volume22
Issue number7
DOIs
Publication statusPublished - 2010

Cite this

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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.",
author = "Jon Merlin and Evans, {Bronwyn Anne} and Csikasz, {Robert I} and Tore Bengtsson and Summers, {Roger James} and Hutchinson, {Dana S}",
year = "2010",
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language = "English",
volume = "22",
pages = "1104 -- 1113",
journal = "Cellular Signalling",
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The M3-muscarinic acetylcholine receptor stimulates glucose uptake in L6 skeletal muscle cells by a CaMKK-AMPK-dependent mechanism. / Merlin, Jon; Evans, Bronwyn Anne; Csikasz, Robert I; Bengtsson, Tore; Summers, Roger James; Hutchinson, Dana S.

In: Cellular Signalling, Vol. 22, No. 7, 2010, p. 1104 - 1113.

Research output: Contribution to journalArticleResearchpeer-review

TY - JOUR

T1 - The M3-muscarinic acetylcholine receptor stimulates glucose uptake in L6 skeletal muscle cells by a CaMKK-AMPK-dependent mechanism

AU - Merlin, Jon

AU - Evans, Bronwyn Anne

AU - Csikasz, Robert I

AU - Bengtsson, Tore

AU - Summers, Roger James

AU - Hutchinson, Dana S

PY - 2010

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N2 - 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.

AB - 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.

UR - http://www.ncbi.nlm.nih.gov/pubmed/20206685

U2 - 10.1016/j.cellsig.2010.03.004

DO - 10.1016/j.cellsig.2010.03.004

M3 - Article

VL - 22

SP - 1104

EP - 1113

JO - Cellular Signalling

JF - Cellular Signalling

SN - 0898-6568

IS - 7

ER -