Stimulation of glucagon-like peptide-1 secretion by muscarinic agonist in a murine intestinal endocrine cell line

Abstract

Studies on the cholinergic regulation of intestinal L-cells have been focused on the release of enteroglucagon, but the signal transduction pathways were not defined. These were here investigated by using as index the release of immunoreactive glucagon-like peptide-1 (GLP-1) from the endocrine cell line STC-1, that has been shown to contain proglucagon mRNA transcripts. Abundant GLP-1 immunoreactivity was revealed in STC-1 cells at immunocytochemistry and by RIA. The cell content was 4927 +/- 689 pg/10(6) cells, as measured with antiserum 199D that recognizes specifically the C-terminal amidated forms of GLP-1. The secretion of GLP-1 over a 2-h incubation period amounted to 1.4 +/- 0.3% of the total GLP-1 cell content and was significantly increased by 10 microM forskolin and 100 nM 12-O-tetradecanoylphorbol 13-acetate to 206% and 574% of control values, respectively. The cholinergic agonist carbachol stimulated GLP-1 secretion in a concentration-dependent manner, maximal release was observed at 1 mM carbachol (228% of the control value). Binding of the muscarinic antagonist [N-methyl-]scopolamine ([3H]NMS) on cell homogenates was time dependent, specific, and saturable. Scatchard analysis revealed one class of receptors (Kd, 14 pM; binding capacity, 20 fmol/mg protein). Carbachol (0.1 microM to 1 mM) dose dependently displaced [3H] NMS binding and increased the intracellular calcium concentration without modification of adenylate cyclase activity. The order of potency of different antagonists, showing a preferential affinity for M1, M2, and M3 muscarinic receptor subtypes, to inhibit [3H]NMS binding, the carbachol-induced increase in intracellular calcium, and carbachol-stimulated GLP-1 secretion, was as follows: atropine (nonselective) > 4-diphenylacetoxy-N-methylpiperidine methiodide (M3) > pirenzepine (M1) > AF-DX 116 (M2). The results of the present study, therefore, demonstrate that secretion of GLP-1 induced by cholinergic agonist depends on muscarinic M3-subtype receptors in the endocrine intestinal cell line STC-1. This system may prove useful to study the cellular mechanisms of GLP-1 secretion.

A stereologic evaluation of glucagon-like peptide-1 (GLP-1) mucosal cells in the small intestine of the developing pig

“…Probably this occurs in addition to indirect means of stimulation, e.g. by the release of gastric inhibitory peptide (GIP) from endocrine cells located in the proximal small intestine (Herrmann-Rinke et al 1996;Damholt et al 1999) or through the activation of muscarinic receptors that are located on the intestinal L-cells (Abello et al 1994;Balks et al 1997). The indirect mechanism is supposed to predominate in the ileum under normal conditions.…”

Inhibition of carbohydrate-mediated glucagon-like peptide-1 (7‒36)amide secretion by circulating non-esterified fatty acids

“…The stimulus for the early postprandial release of GLP-1 secretion is not clearly understood. Studies in animals have indicated a possible role for GIP and\or the parasympathetic system in release of GLP-1 from the ileum in response to the presence of food in the duodenum and jejunum [27,28], but no such mechanism has been demonstrated in man [1]. We suggest that the fall in plasma NEFA levels after ingestion of food may be an important mechanism by which GLP-1 release is either stimulated or enhanced and that failure of NEFA suppression may be partly responsible for the impaired GLP-1 secretion seen in obesity.…”

Comparison of the postprandial release of peptide YY and proglucagon-derived peptides in the rat

“…The GLP-1 assay was performed as described elsewhere [1]. Briefly, an antiserum against GLP-1(7-36) amide was raised in a rabbit by immunization with synthetic GLP-1(7-36) amide conjugated to bovine serum albumin (BSA).…”