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Stimulation of motilin secretion by bile, free fatty acids and acidification in human duodenal organoids

ABSTRACT: Characterision of the bioactive peptide content of M-cells. M-cells derived from organoids were sorted using FAC sorting techniques and were lysed and analysed by LC-MS/MS using a peptidomics approach.

INSTRUMENT(S):

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Duodenum, Type M Enteroendocrine Cell

DISEASE(S): Type 2 Diabetes Mellitus

SUBMITTER: Richard Kay

LAB HEAD: Professor Fiona Gribble

PROVIDER: PXD026621 | Pride | 2021-11-19

REPOSITORIES: Pride

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			Action	DRS
	21031706.mgf	Mgf
	21031706.raw	Raw
	21031707.mgf	Mgf
	21031707.raw	Raw
	21031708.mgf	Mgf

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Stimulation of motilin secretion by bile, free fatty acids, and acidification in human duodenal organoids.

Project description:ObjectiveMotilin is a proximal small intestinal hormone with roles in gastrointestinal motility, gallbladder emptying, and hunger initiation. In vivo motilin release is stimulated by fats, bile, and duodenal acidification but the underlying molecular mechanisms of motilin secretion remain poorly understood. This study aimed to establish the key signaling pathways involved in the regulation of secretion from human motilin-expressing M-cells.MethodsHuman duodenal organoids were CRISPR-Cas9 modified to express the fluorescent protein Venus or the Ca2+ sensor GCaMP7s under control of the endogenous motilin promoter. This enabled the identification and purification of M-cells for bulk RNA sequencing, peptidomics, calcium imaging, and electrophysiology. Motilin secretion from 2D organoid-derived cultures was measured by liquid chromatography-tandem mass spectrometry (LC-MS/MS), in parallel with other gut hormones.ResultsHuman duodenal M-cells synthesize active forms of motilin and acyl-ghrelin in organoid culture, and also co-express cholecystokinin (CCK). Activation of the bile acid receptor GPBAR1 stimulated a 3.4-fold increase in motilin secretion and increased action potential firing. Agonists of the long-chain fatty acid receptor FFA1 and monoacylglycerol receptor GPR119 stimulated secretion by 2.4-fold and 1.5-fold, respectively. Acidification (pH 5.0) was a potent stimulus of M-cell calcium elevation and electrical activity, an effect attributable to acid-sensing ion channels, and a modest inducer of motilin release.ConclusionsThis study presents the first in-depth transcriptomic and functional characterization of human duodenal motilin-expressing cells. We identify several receptors important for the postprandial and interdigestive regulation of motilin release.

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