Enteroendocrine cells (EECs) are chemosensitive cells of the gastrointestinal epithelium that exert a wide range of physiological effects via production and secretion of hormones in response to ingested nutrients, bacterial metabolites and systemic signals. Glucagon-like peptide-1 (GLP-1) is one such hormone secreted from so-called L-cells found in both the small and large intestines. GLP-1 exerts an anorexigenic effect and together with glucose- dependent insulinotropic polypeptide (GIP), restores postprandial normoglycaemia through the incretin effect. These effects are exploited by GLP-1 analogues in the treatment of type 2 diabetes. GLP-1 may also contribute to weight-loss and remission of type 2 diabetes following bariatric surgery which increases postprandial GLP-1 excursions.

Here we investigated stimulus secretion coupling in L-cells. A novel 2D culture system from murine small intestinal organoids was established as an in vitro model. This was used to characterise synergistic stimulation of GLP-1 secretion in response to concomitant stimulation by bile acids through the Gs-protein coupled receptor GPBAR1 and free fatty acids through the Gq-coupled receptor FFAR1.

Roughly half of colonic, but not small intestinal, L-cells co-produce the orexigenic peptide insulin-like peptide 5 (INSL5). This hitherto poorly examined subpopulation of L-cells was characterised through transcriptomic analysis, intracellular calcium imaging (using a novel GCaMP6F-based transgenic mouse model), LC/MS peptide quantification and 3D super resolution microscopy (3D-SIM). Based on the observed prevalent co-storage of GLP-1 and INSL5 in secretory vesicles and similar secretory responses of both hormones to a range of different stimuli strengths (including short chain fatty acids, angiotensin II and arginine vasopressin (AVP)) it was concluded that GLP-1 and INSL5 are co-secreted, rather than being selectively recruited by different stimuli.

To further characterise the diversity of colonic EECs, single cell RNA-sequencing (scRNA-seq) was performed on cells isolated from mice with a pan-EEC fluorescent marker (NeuroD1- Cre:Rosa26-EYFP). This illustrated that INSL5-producing L-cells form one of two transcriptomically distinct subpopulations of L-cells in the murine colon, with the other distinguished by expression of neurotensin (Nts). Another major EEC subpopulation, enterochromaffin (EC) cells could be split into three groups, mechanosensitive and pro- inflammatory EC cells distinguished by Piezo2 and Tac1 expression, respectively and a third Sct-expressing group. Immunofluorescent labelling and RT-qPCR analysis revealed that the Nts-expressing and Insl5-expressing L-cell subpopulations are proximally and distally enriched in the murine colon, respectively. In primary cultures, angiotensin II and AVP stimulated INSL5, GLP-1 and PYY but not NTS secretion, correlating with selective expression profiles of the cognate receptors in the L-cell subpopulations.

In summary, the work presented suggests that different L-cell subpopulations exist that respond to different stimuli, but that hormones co-expressed in individual L-cells are co- released upon stimulation. Differences in receptor expression between these subpopulations and other EEC-populations might be exploitable for selective hormone recruitment for the therapy of diabetes, obesity and other diseases.