Single cell transcriptomic profiling of large intestinal enteroendocrine cells in mice – Identification of selective stimuli for insulin-like peptide-5 and glucagon-like peptide-1 co-expressing cells

Abstract

ObjectiveEnteroendocrine cells (EECs) of the large intestine, found scattered in the epithelial layer, are known to express different hormones, with at least partial co-expression of different hormones in the same cell. Here we aimed to categorize colonic EECs and to identify possible targets for selective recruitment of hormones.MethodsSingle cell RNA-sequencing of sorted enteroendocrine cells, using NeuroD1-Cre x Rosa26-EYFP mice, was used to cluster EECs from the colon and rectum according to their transcriptome. G-protein coupled receptors differentially expressed across clusters were identified, and, as a proof of principle, agonists of Agtr1a and Avpr1b were tested as candidate EEC secretagogues in vitro and in vivo.ResultsEECs from the large intestine separated into 7 clear clusters, 4 expressing higher levels of Tph1 (enzyme required for serotonin (5-HT) synthesis; enterochromaffin cells), 2 enriched for Gcg (encoding glucagon-like peptide-1, GLP-1, L-cells), and the 7th expressing somatostatin (D-cells). Restricted analysis of L-cells identified 4 L-cell sub-clusters, exhibiting differential expression of Gcg, Pyy (Peptide YY), Nts (neurotensin), Insl5 (insulin-like peptide 5), Cck (cholecystokinin), and Sct (secretin). Expression profiles of L- and enterochromaffin cells revealed the clustering to represent gradients along the crypt-surface (cell maturation) and proximal-distal gut axes. Distal colonic/rectal L-cells differentially expressed Agtr1a and the ligand angiotensin II was shown to selectively increase GLP-1 and PYY release in vitro and GLP-1 in vivo.ConclusionEECs in the large intestine exhibit differential expression gradients along the crypt-surface and proximal-distal axes. Distal L-cells can be differentially stimulated by targeting receptors such as Agtr1a.

Selective stimulation of colonic L cells improves metabolic outcomes in mice

“…Our results suggest that enterochromaffin cells were activated by L distalDq cell stimulation. Direct activation of enterochromaffin cells seems unlikely, as only a small number of GFP-labelled cells stained positive for 5-HT, consistent with previous reports of only a small overlap of Gcg and Tph1-expressing cells in the small intestine [46] and colon [22]. Although we cannot exclude that direct CNO stimulation of rare cells positive for both INSL5 and 5-HT might have been sufficient to activate faecal expulsion, we speculate that L distalDq cell stimulation indirectly activated en tero ch roma ffin cell s via pa rac rine cro sstalk .…”

“…We showed previously that INSL5 is produced by the majority of L cells in the distal two-thirds of the mouse colon/rectum [22]. Despite previous reports of Insl5expression in the hypothalamus [23], we were unable to detect reporter gene expression in the CNS of DOX-induced Insl5rtTAxGCaMP6ΔCMV mice [4], which expressed the reporter in the colon (ESM Fig.…”

“…Enterochromaffin cells have been shown previously to express Glp1r [47], but antagonism of GLP1R did not prevent the increased faecal output in our model. Other possible enterochromaffin targets for L cell products include Y1R and relaxin/insulin-like family peptide receptor 4 (RXFP4), the receptor for INSL5, mRNA for which we found to be expressed in colonic Tph1-positive cells [22]. Both Y1R and RXFP4 are predominantly G i -coupled, but pertussis toxinsensitive stimulation of 5-HT release has been shown downstream of α 2 -adrenergic stimulation [48].…”

Enteroendocrine cells sense bacterial tryptophan catabolites to activate enteric and vagal neuronal pathways

“…Whereas Trpa1+EECs are abundant in the small intestine of human and rodents (Yang et al, 2019, Nozawa et al, 2009, previous mouse studies demonstrated that Trpa1 in the colon is mainly expressed in mesenchymal cells but not in EECs (Yang et al, 2019, Billing et al, 2019. Conversely to Trpa1+EECs, microbially derived tryptophan metabolites are restricted to the colon and largely absent in small intestine under normal physiological conditions.…”

“…Many microbial tryptophan metabolites, including indole and IAld, are known to be produced by mammalian commensal microbes (Roager and Licht, 2018). Previous studies demonstrated that Trpa1+EECs are restricted to the mouse and human small intestine and not found in the colon (Yang et al, 2019, Billing et al, 2019. We therefore analyzed the tryptophan metabolite concentrations in the small intestine and colon from conventionally-reared mice.…”