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

Abstract Objective Motilin 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 are poorly understood. This study aimed to establish the key signalling pathways involved in the regulation of secretion from human motilin-expressing M-cells. Methods Human 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 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. Results Human duodenal M-cells synthesise 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. Conclusions This study presents the first in-depth transcriptomic and functional characterisation of human duodenal motilin-expressing cells. We identify several receptors important for the postprandial and interdigestive regulation of motilin release.