Exploring the function and sensing mechanisms of human enteroendocrine cells using intestinal organoids
The gut is the largest endocrine organ in our body thanks to the presence of hormone-releasing enteroendocrine cells (EECs), which represent only around 1% of the total gut epithelium. Gut hormones are released in response to cues of different natures to regulate several physiological events, such as nutrient digestion and absorption, intestinal motility, food intake, inflammation, and energy homeostasis. This thesis aimed to characterise hormone-releasing sensing mechanisms adopted by two important types of EECs: K-cells, which secrete glucose-dependent insulinotropic polypeptide (GIP), and enterochromaffin (EC) cells, which secrete serotonin, or 5-hydroxytryptamine. GIP, largely known as one of the two gut-derived insulinotropic incretin hormones, alongside with glucagon-like peptide 1 (GLP-1), has recently regained popularity in the field of metabolic research due to its newfound relevance as an anorexigenic hormone in rodent models and the advent of weight loss-inducing GLP1R/GIPR dual incretin mimetics. 5-HT is a monoamine neurotransmitter and non-canonical gut hormone that has a wide impact on the gut and whole-body physiology. Most studies exploring EEC function have used either animal models or 2D cell lines. However, the development of human gut organoids, 3D structures that recapitulate the intestinal epithelial complexities, and gene-editing tools, such as CRISPR-Cas9, paired with the establishment of robust characterisation assays, have allowed us, for the first time, to explore human EEC function and hormone secretory mechanism in depth. Using CRISPR-Cas9 homology directed repair (HDR), we fluorescently labelled K-cells and EC cells in patient-derived duodenal organoids. These transgenic reporter lines were employed to characterise human EEC responsiveness and function by performing fluorescence-activated cell sorting (FACS), bulk RNA sequencing, hormone secretion assays and live single-cell Ca2+ and cAMP imaging. cAMP imaging was possible through the generation of K- and EC cell transgenic lines expressing genetically encoded cAMP reporters (Epac-S-H187). Bulk RNA sequencing and peptidomic analysis of organoid-derived human K-cells showed enrichment of other peptide hormones besides GIP, such as gastrin and CCK. In line with previous studies performed on murine K-cells, this subset of EECs showed differential expression of nutrient-sensing receptors, such as FFAR1, GPR142 and CaSR. Glucose induced a 1.8-fold increase in GIP secretion compared to glucose-free conditions. This effect was hampered by SGLT1/2 inhibitor sotagliflozin, as well as by knock-out of SGLT1 in the GIP-Venus human organoid line, indicating that this sodium-coupled glucose transported is needed for the secretion of GIP from human K-cells. Importantly, we showed for the first time that human K-cells are electrically excitable upon current injection and exhibit action potential firing in response to glucose. Furthermore, we knocked-out amino acid-sensing GPCRs CaSR and GPR142 by CRISPR-Cas9 NHEJ, demonstrating that both are needed for the release of GIP from organoid-derived human K-cells. Amino acid sensing and FFAR1-mediated fat sensing by K- and L-cells were further interrogated in mouse proximal small intestinal perfusions. Our studies show that, similarly to L-cells, aromatic amino acids phenylalanine and tryptophan induce GIP release upon basolateral stimulation, but not apical, likely via activation of CaSR and GPR142. Surprisingly, we did not observe GIP secretion upon basolateral stimulation with FFAR1 agonist AM1638, while secretion of GLP-1 was detected. EC cells were previously labelled in human duodenal organoids in our laboratory by inserting the Venus transgene at the end of the tryptophan hydroxylase 1 (TPH1) gene, encoding for the rate-limiting enzyme in the production of serotonin, or 5-HT, hence labelling 5-HT-producing enterochromaffin cells. Live-cell Ca2+ imaging of Venus-tagged EC cells showed that aromatic amino acids phenylalanine and tryptophan, as well as dietary compounds such as cinnamaldehyde and catecholamines, significantly increased intracellular Ca2+ levels. In this study, we also generated an EC cell-specific FRET-based cAMP organoid reporter line. This transgenic line was used in live-cell fluorescence microscopy to show that SCFAs, catecholamines and gut hormones (such as GIP) increase intracellular cAMP levels in human EC cells. Finally, preliminary results from 5-HT secretion assays revealed that some of the aforementioned stimuli, including tryptophan, GLP1R agonist exendin-4 and bile acid sensing receptor GPBAR agonist GPBAR-A, induce 5-HT release in vitro. Building upon previous work from our laboratory, which delved into the secretory properties of human GLP-1-secreting L-cells and motilin-producing M-cells, we profiled some key aspects of hormone-secreting mechanisms in human K-cells and EC cells. By characterising the stimulus-sensing hormone secretory machineries of human EECs using the tools developed in our laboratory and employed in this thesis to characterise a subset of EECs, we hope to systematically understand the physiology of the human enteroendocrine system. Ultimately, this may help to develop novel therapeutic strategies that may harness the power of endogenous gut hormones for the treatment of metabolic diseases and gastrointestinal disorders.
Enteroendocrine, Hormone, Intestinal, Metabolic disease, Organoids
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