Gq/11-coupled muscarinic acetylcholine receptors (mAChRs) expressedin peripheral tissues have been recently reported to regulate energy metabolism and maintenance of physiological blood glucose levels.However, relatively little is known on the ability of these receptors tocontrol energy homeostasis in neuronal cells. Previous studies haveshown that in human SH-SY5Y neuroblastoma cells mAChRs stimu-late the metabolic sensor AMP-activated protein kinase (AMPK)through Ca2+/calmodulin-dependent protein kinase kinase-b (CaM-KKb), but the source of Ca2+required for this response has not yetbeen elucidated. The present study investigates the involvement ofstore-operated Ca2+entry (SOCE) in mAChRs-induced AMPK activa-tion. We found that in SH-SY5Y cells AMPK activation was mediatedby pharmacologically defined M3mAChRs, strictly dependent onextracellular Ca2+and blocked by the SOCE inhibitor 2-amin-oethoxydiphenyl borate (2-APB). Stimulation of M3mAChR activatedthe endoplasmic reticulum Ca2+sensor stromal interaction molecule(STIM) 1, as indicated by the redistribution of STIM1 immunofluores-cence into puncta, and promoted the association of STIM1 with twocomponents of SOCE channels, transient receptor potential canonical 1(TRPC1) and Orai1. Either STIM1 or TRPC1 knockdown curtailed M3mAChR-induced AMPK activation. Moreover, Ca2+store depletion bythapsigargin or cyclopiazonic acid activated AMPK in a manner simi-lar to M3-mAChRs. We also found that activation of M3mAChRsstimulated glucose uptake and this response required extracellularCa2+, was inhibited by 2-APB and reduced by knockdown of STIM1.Moreover, pharmacological inhibition of either CaMKKß withSTO609 or AMPK with Compound C prevented M3mAChR-stimu-lated glucose uptake. The data provide evidence that in a neuronal-likecell system activation of M3mAChRs stimulate AMPK and down-stream glucose uptake by triggering SOCE and suggest that SOCE is acritical process linking M3AChRs to the control of neuronal energy metabolism
M3 MUSCARINIC ACETYLCHOLINE RECEPTORSACTIVATE THE METABOLIC SENSOR AMP-ACTIVATEDPROTEIN KINASE AND STIMULATE GLUCOSE UPTAKETHROUGH STORE-OPERATED CA2+ ENTRY IN SH-SY5YHUMAN NEUROBLASTOMA CELLS
OLIANAS, MARIA CONCETTA;DEDONI, SIMONA;ONALI, PIER LUIGI
2014-01-01
Abstract
Gq/11-coupled muscarinic acetylcholine receptors (mAChRs) expressedin peripheral tissues have been recently reported to regulate energy metabolism and maintenance of physiological blood glucose levels.However, relatively little is known on the ability of these receptors tocontrol energy homeostasis in neuronal cells. Previous studies haveshown that in human SH-SY5Y neuroblastoma cells mAChRs stimu-late the metabolic sensor AMP-activated protein kinase (AMPK)through Ca2+/calmodulin-dependent protein kinase kinase-b (CaM-KKb), but the source of Ca2+required for this response has not yetbeen elucidated. The present study investigates the involvement ofstore-operated Ca2+entry (SOCE) in mAChRs-induced AMPK activa-tion. We found that in SH-SY5Y cells AMPK activation was mediatedby pharmacologically defined M3mAChRs, strictly dependent onextracellular Ca2+and blocked by the SOCE inhibitor 2-amin-oethoxydiphenyl borate (2-APB). Stimulation of M3mAChR activatedthe endoplasmic reticulum Ca2+sensor stromal interaction molecule(STIM) 1, as indicated by the redistribution of STIM1 immunofluores-cence into puncta, and promoted the association of STIM1 with twocomponents of SOCE channels, transient receptor potential canonical 1(TRPC1) and Orai1. Either STIM1 or TRPC1 knockdown curtailed M3mAChR-induced AMPK activation. Moreover, Ca2+store depletion bythapsigargin or cyclopiazonic acid activated AMPK in a manner simi-lar to M3-mAChRs. We also found that activation of M3mAChRsstimulated glucose uptake and this response required extracellularCa2+, was inhibited by 2-APB and reduced by knockdown of STIM1.Moreover, pharmacological inhibition of either CaMKKß withSTO609 or AMPK with Compound C prevented M3mAChR-stimu-lated glucose uptake. The data provide evidence that in a neuronal-likecell system activation of M3mAChRs stimulate AMPK and down-stream glucose uptake by triggering SOCE and suggest that SOCE is acritical process linking M3AChRs to the control of neuronal energy metabolism
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