Search

Gastrointestinal hormones regulate a diverse range of physiological processes including digestion, metabolism, and food intake, as well as maintaining mucosal integrity and mounting defensive mechanism in response to pathogens or toxins. Disruptions of these regulatory processes are associated with disorders that include diabetes, obesity, irritable bowel syndrome, and coeliac disease. Despite this clinical significance, many aspects of the cells that contain gut hormones, enteroendocrine cells (EEC), remain poorly characterised. Historically, EEC were categorised based on their hormone content, with the assumption that each cell contained a single hormone. However, hidden in some early studies were examples that the simple ‘one cell‐one hormone’ classification system was not correct. A major focus of this thesis is the characterising of coexpression patterns of hormones in mouse and human intestine. Remarkably, examples of overlap could be found for all hormones investigated, demonstrating a need for a revised classification system for EEC. Moreover, species differences in EEC expression patterns occur, highlighting a further level of complexity in the field of research. The coexpression of EEC hormones raises new questions about how these hormones are stored at a subcellular level. Super‐resolution microscopy allowed the visualisation and quantification of vesicular stores in EEC. Overlapping and non‐overlapping vesicular stores were observed. Furthermore, the relative abundance of hormone expression varied considerably between cells and, consequentially, small amounts of hormones sometimes could not be detected with low‐resolution methods. In addition, a monoclonal antioxyntomodulin antibody was characterised, expanding the examination of hormone colocalisation patterns to the investigation of proglucagon‐derived peptides. Several hormones, including 5‐HT, influence electrolyte transport, which can be measured as the shortcircuit current (Isc) in an Ussing chambe

Recent studies have shown that patterns of colocalisation of hormones in enteroendocrine cells are more complex than previously appreciated and that the patterns differ substantially between species. In this study, the human sigmoid colon is investigated by immunohistochemistry for the presence of gastrointestinal hormones and their colocalisation. The segments of colon were distant from the pathology that led to colectomy and appeared structurally normal. Only four hormones, 5-hydroxytryptamine (5-HT), glucagon-like peptide 1 (GLP-1), peptide YY (PYY) and somatostatin, were common in enteroendocrine cells of the human colon. Cholecystokinin, present in the colon of some species, was absent, as were glucose-dependent insulinotropic peptide, ghrelin and motilin. Neurotensin cells were extremely rare. The most numerous cells were 5-HT cells, some of which also contained PYY or somatostatin and very rarely GLP-1. Almost all GLP-1 cells contained PYY. It is concluded that enteroendocrine cells of the human colon, like those of other regions and species, exhibit overlapping patterns of hormone colocalisation and that the hormones and their patterns of expression differ between human and other species.

There is general consensus that enteroendocrine cells, EEC, containing the enteric hormone cholecystokinin (CCK) are confined to the small intestine and predominate in the duodenum and jejunum. Contrary to this, EEC that express the gene for CCK have been isolated from the large intestine of the mouse and there is evidence for EEC that contain CCK-like immunoreactivity in the mouse colon. However, the human and rat colons do not contain CCK cells. In the current study, we use immunohistochemistry to investigate CCK peptide presence in endocrine cells, PCR to identify cck transcripts and chromatography to identify CCK peptide forms in the mouse small and large intestine. The colocalisation of CCK and 5-HT, hormones that have been hypothesised to derive from cells of different lineages, was also investigated. CCK immunoreactivity was found in EEC throughout the mouse small and large intestine but positive cells were rare in the rectum. Immunoreactive EEC were as common in the caecum and proximal colon as they were in the duodenum and jejunum. CCK gene transcripts were found in the mucosa throughout the intestine but mRNA for gastrin, a hormone that can bind some anti-CCK antibodies, was only found in the stomach and duodenum. Characterisation of CCK peptides of the colon by extraction, chromatographic separation and radioimmunoassay revealed bioactive amidated and sulphated forms, including CCK-8 and CCK-33. Moreover, CCK-containing EEC in the large intestine bound antibodies that target the biologically active sulfated form. Colocalisation of CCK and 5-HT occurred in a proportion of EEC throughout the small intestine and in the caecum but these hormones were not colocalised in the colon, where there was CCK and PYY colocalisation. It is concluded that authentic, biologically active, CCK occurs in EEC of the mouse large intestine.

TRPA1 is a ligand-activated cation channel found in the intestine and other tissues. Components of food that stimulate TRPA1 receptors (phytonutrients) include allyl isothiocyanate, cinnamaldehyde and linalool, but these may also act at other receptors. Cells lining the intestinal mucosa are immunoreactive for TRPA1 and Trpa1 mRNA occurs in mucosal extracts, suggesting that the TRPA1 receptor is the target for these agonists. However, in situ hybridisation reveals Trpa1 expression in 5-HT containing enteroendocrine cells, not enterocytes. TRPA1 agonists evoke mucosal secretion, which may be indirect (through release of 5-HT) or direct by activation of enterocytes. We investigated effects of the phytonutrients on transmucosal ion currents in mouse duodenum and colon, and the specificity of the phytonutrients in cells transfected with Trpa1, and in Trpa1-deficient mice. The phytonutrients increased currents in the duodenum with the relativepotencies: allyl isothiocyanate (AITC) > cinnamaldehyde > linalool (0.1 to 300 µM). The rank order was similar in the colon, but linalool was ineffective. Responses to AITC were reduced by the TRPA1 antagonist HC-030031 (100 µM), and were greatly diminished in Trpa1-/- duodenum and colon. Responses were not reduced by tetrodotoxin, 5-HT receptor antagonists, or atropine, but inhibition of prostaglandin synthesis reduced responses. Thus, functional TRPA1 channels are expressed by enterocytes of the duodenum and colon. Activation of enterocyte TRPA1 by food components has the potential to facilitate nutrient absorption.