title: Cooperative enhancement of insulinotropic action of GLP-1 by acetylcholine uncovers paradoxical inhibitory effect of beta cell muscarinic receptor activation on adenylate cyclase activity.
abstract: The cooperative effect of glucagon-like peptide 1 (GLP-1) and acetylcholine (ACh) was evaluated in a beta cell line model (BRIN BD11). GLP-1 (20 nM) and ACh (100 microM) increased insulin secretion by 24-47%, whereas in combination there was a further 89% enhancement of insulin release. Overnight culture with 100 ng/mL pertussis toxin (PTX) or 10nM PMA significantly reduced the combined insulinotropic action (P<0.05 and P<0.001, respectively) and the sole stimulatory effects of GLP-1 (PTX treatment; P<0.01) or ACh (PMA treatment; P<0.05). Under control conditions, ACh (50nM-1mM) concentration-dependently inhibited by up to 40% (P<0.001) the 10-fold (P<0.001) elevation of cyclic 3',5'-adenosine monophosphate (cAMP) induced by 20 nM GLP-1. The paradoxical inhibitory action of ACh was abolished by PTX pre-treatment, suggesting involvement of G(i) and/or G(o) G protein alpha subunit. Effects of selective muscarinic receptor antagonists on the concentration-dependent insulinotropic actions of ACh (50 nM-1 mM) on 20 nM GLP-1 induced insulin secretion revealed inhibition by rho-FHHSiD (M3 antagonist, P<0.05), stimulation with pirenzepine (M1 antagonist, P<0.001) and no significant effects of either methoctramine (M2 antagonist) or MT-3 (M4 antagonist). Antagonism of M2, M3 and M4 muscarinic receptor effects with methoctramine (3-100 nM), rho-FHHSiD (3-30 nM) or MT-3 (10-300 nM) did not significantly affect the inhibitory action of ACh on GLP-1 stimulated cAMP production. In contrast, M1 receptor antagonism with pirenzepine (3-30 0nM) resulted in a concentration-dependent decrease in the inhibitory action of ACh on GLP-1 stimulated cAMP production (P<0.001). These data indicate an important functional cooperation between the cholinergic neurotransmitter ACh and the incretin hormone GLP-1 on insulin secretion mediated through the M3 muscarinic receptor subtype. However, the insulinotropic action of ACh was associated with a paradoxical inhibitory effect on GLP-1 stimulated cAMP production, achieved through a novel PTX- and pirenzepine-sensitive M1 muscarinic receptor activated pathway. An imbalance between these pathways may contribute to dysfunctional insulin secretion.
authors.affiliation: School of Biomedical Sciences, University of Ulster, Coleraine, Co Londonderry, Northern Ireland, BT52 1SA, UK. jcmiguel@uem.br
authors.forename: João C
authors.initials: JC
authors.lastname: Miguel
authors.forename: Yasser H A
authors.initials: YH
authors.lastname: Abdel-Wahab
authors.forename: Brian D
authors.initials: BD
authors.lastname: Green
authors.forename: Paulo C F
authors.initials: PC
authors.lastname: Mathias
authors.forename: Peter R
authors.initials: PR
authors.lastname: Flatt
mesh_terms: D000109:Acetylcholine / Q000494:pharmacology*; D000262:Adenylyl Cyclases; D000242:Cyclic AMP / Q000378:metabolism; D005934:Glucagon / Q000494:pharmacology*; D052216:Glucagon-Like Peptide 1; D006801:Humans; D007328:Insulin / Q000378:metabolism; D000078790:Insulin Secretion; D007515:Islets of Langerhans / Q000187:drug effects* / Q000378:metabolism; D010446:Peptide Fragments / Q000494:pharmacology*; D011498:Protein Precursors / Q000494:pharmacology*; D011976:Receptors, Muscarinic / Q000378:metabolism*
chemical_list: D007328:Insulin; D010446:Peptide Fragments; D011498:Protein Precursors; D011976:Receptors, Muscarinic; D052216:Glucagon-Like Peptide 1; D005934:Glucagon; D000242:Cyclic AMP; D000262:Adenylyl Cyclases; D000109:Acetylcholine