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Title: Cellular expression and function of CCK in the mouse duodenum
Author: Demenis, Claire
ISNI:       0000 0004 5356 2437
Awarding Body: University of Manchester
Current Institution: University of Manchester
Date of Award: 2014
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Enteroendocrine cells (EECs) express key gastrointestinal (GI) hormones including cholecystokinin (CCK), gastric inhibitory peptide (GIP), peptide tyrosine tyrosine(PYY), glucagon‐like peptide‐1 (GLP‐1) and ghrelin. EECs are characterised to contain the hormones derived from one precursor protein. Of these, CCK‐cells are typically concentrated in the proximal small intestine and release CCK upon stimulation by nutrient ligands and in so doing signal to multiple tissues to co‐ordinate and optimise digestive, absorptive functions, and instil hunger or satiety. The aims of this study were to establish whether EECs co‐expressed CCK alongside other key GI peptides and to determine a paracrine role for CCK to increase FA uptake in intestinal cells. These studies utilised an eGFP‐CCK transgenic mouse model. Tissue sections frome GFP‐CCK mice were paraffin embedded and immunostained against an array of targets. Firstly, an anti‐GFP antiserum was employed to visualise eGFP‐cells along the GI tract, and duodenal sections were dual stained for anti‐GFP and an anti‐proCCK antiserum to confirm eGFP‐cells represented CCK‐cells. A series of dual immunostaining experiments ensued to probe duodenal eGFP‐cells for a range of different hormonal targets and demonstrated that a significant number of eGFP‐CCK cells contained GIP (37%), PYY (45%), proglucagon (14%) and ghrelin (50%). Further dual‐staining experiments were carried out to stain for CCK alongside PYY, GIP or ghrelin and enabled analysis of the intracellular localisation of co‐expressing peptides, which indicated that these peptides were packaged in the same and also with indistinctly separate vesicles. These data demonstrate CCK‐cells can co‐express more than one peptide and analysis of intracellular labelling indicates they may have the ability to co‐release CCK alongside other peptides. To investigate a potential paracrine‐signalling pathway for CCK a FA uptake assay was performed using a fluorescent C12‐fatty acid (FA) analogue (Bodipy‐FA) that was analysed using fluorescent activated cell sorting (FACS). Single small intestinal cells of eGFP‐CCK mice were prepared using an EDTA chemical/mechanical dissociation method. Cell samples were either non‐treated (control) or pre‐treated with a targeted compound prior to incubation with Bodipy‐FA. Treatment of cells witholeoylethanolamide, glucagon‐like peptide‐2 (GLP‐2) or CCK increased FA uptake 2 to3‐fold and this increase was demonstrated to be carrier‐mediated. Experiments ensued employing CCK‐cell ligands to implicate activity of CCK‐cells in this process. Bombesin and L‐amino acids induced a dynamic increase in FA uptake comparable to that achieved by pre‐treatment with CCK. However, implementation of the protocol using cells from a CCK KO model achieved replicate data and therefore demonstrated these effects were not exclusive to CCK‐cells. In conclusion, data presented in this thesis establish that a spectrum of key gut hormones is expressed in individual EECs. Furthermore, a paracrine action of CCK signalling is implicated to increase the absorptive ability of neighbouring enterocytes. These data suggest that CCK‐cells have the ability to integrate nutrient signals and secrete a cocktail of hormones in response. These findings imply an increased complexity to the enteroendocrine system whereby GI peptides may work together to potentiate a desired response without requirement of signals from higher centres.
Supervisor: Case, Maynard; Smith, Craig Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: Cholecystokinin ; Enteroendocrine