Single-cell RNA-sequencing reveals a distinct population of proglucagon-expressing cells specific to the mouse upper small intestine

Leslie L Glass,Fernando J Calero-Nieto,Wajid Jawaid,Pierre Larraufie,Richard G Kay,Berthold Göttgens,Frank Reimann,Fiona M Gribble

doi:10.1016/j.molmet.2017.07.014

Abstract

ObjectivesTo identify sub-populations of intestinal preproglucagon-expressing (PPG) cells producing Glucagon-like Peptide-1, and their associated expression profiles of sensory receptors, thereby enabling the discovery of therapeutic strategies that target these cell populations for the treatment of diabetes and obesity. MethodsWe performed single cell RNA sequencing of PPG-cells purified by flow cytometry from the upper small intestine of 3 GLU-Venus mice. Cells from 2 mice were sequenced at low depth, and from the third mouse at high depth. High quality sequencing data from 234 PPG-cells were used to identify clusters by tSNE analysis. qPCR was performed to compare the longitudinal and crypt/villus locations of cluster-specific genes. Immunofluorescence and mass spectrometry were used to confirm protein expression. ResultsPPG-cells formed 3 major clusters: a group with typical characteristics of classical L-cells, including high expression of Gcg and Pyy (comprising 51% of all PPG-cells); a cell type overlapping with Gip-expressing K-cells (14%); and a unique cluster expressing Tph1 and Pzp that was predominantly located in proximal small intestine villi and co-produced 5-HT (35%). Expression of G-protein coupled receptors differed between clusters, suggesting the cell types are differentially regulated and would be differentially targetable. ConclusionsOur findings support the emerging concept that many enteroendocrine cell populations are highly overlapping, with individual cells producing a range of peptides previously assigned to distinct cell types. Different receptor expression profiles across the clusters highlight potential drug targets to increase gut hormone secretion for the treatment of diabetes and obesity.

Highlights

Enteroendocrine preproglucagon-expressing PPG-cells secrete the gut hormones glucagon-like peptide 1 (GLP-1) and peptideYY (PYY) and are important regulators of glucose metabolism and appetite [1]
Visual inspection of hormone expression profiles across the clusters revealed differential distributions of Gcg, Gip, Pyy, Cck, and Tph1, suggesting that the tSNE analysis separated cells that differ in their expression of hormonal genes
RNAseq of PPG-cell populations isolated from different regions of the gut revealed that Pregnancy zone protein (Pzp) expression was found in PPG-cells from the proximal but not the distal small intestine, suggesting that Cl3 is specific to the upper small intestine (Figure 2C)

Summary

Introduction

Enteroendocrine preproglucagon-expressing PPG-cells (traditionally known as L-cells) secrete the gut hormones glucagon-like peptide 1 (GLP-1) and peptideYY (PYY) and are important regulators of glucose metabolism and appetite [1]. PYY promotes satiety and is under investigation as a potential basis for new anti-obesity therapeutics [2] These observations raised the prospect of developing treatments that promote the secretion of GLP-1 and other gut hormones. Recent studies investigating genetically tagged PPG-cells have shown that, as a population, they express gut hormones previously thought to be expressed in distinct enteroendocrine cell types, including Cck (cholecystokinin, I-cells), Sct (secretin, S-cells), and Gip (glucose-dependent insulinotropic polypeptide, K-cells) [4]. It remained unclear whether cells expressing different hormone combinations represent fundamentally distinct cell populations. The aim of the present study was to use single cell RNA sequencing to determine whether PPG-cells

Objectives

Results

Discussion

Conclusion