Cellular expression and Function of CCK in the Mouse Duodenum

Abstract

Enteroendocrine cells (EECs) express key gastrointestinal (GI) hormones includingcholecystokinin (CCK), gastric inhibitory peptide (GIP), peptide tyrosine tyrosine(PYY), glucagon‐like peptide‐1 (GLP‐1) and ghrelin. EECs are characterised to containthe hormones derived from one precursor protein. Of these, CCK‐cells are typicallyconcentrated in the proximal small intestine and release CCK upon stimulation bynutrient ligands and in so doing signal to multiple tissues to co‐ordinate and optimisedigestive, absorptive functions, and instil hunger or satiety. The aims of this study wereto establish whether EECs co‐expressed CCK alongside other key GI peptides and todetermine a paracrine role for CCK to increase FA uptake in intestinal cells.These studies utilised an eGFP‐CCK transgenic mouse model. Tissue sections fromeGFP‐CCK mice were paraffin embedded and immunostained against an array oftargets. Firstly, an anti‐GFP antiserum was employed to visualise eGFP‐cells along theGI tract, and duodenal sections were dual stained for anti‐GFP and an anti‐proCCKantiserum to confirm eGFP‐cells represented CCK‐cells. A series of dualimmunostainingexperiments ensued to probe duodenal eGFP‐cells for a range ofdifferent hormonal targets and demonstrated that a significant number of eGFP‐CCKcellscontained GIP (37%), PYY (45%), proglucagon (14%) and ghrelin (50%). Furtherdual‐staining experiments were carried out to stain for CCK alongside PYY, GIP orghrelin and enabled analysis of the intracellular localisation of co‐expressing peptides,which indicated that these peptides were packaged in the same and also withindistinctly separate vesicles. These data demonstrate CCK‐cells can co‐express morethan one peptide and analysis of intracellular labelling indicates they may have theability to co‐release CCK alongside other peptides.To investigate a potential paracrine‐signalling pathway for CCK a FA uptake assay wasperformed using a fluorescent C12‐fatty acid (FA) analogue (Bodipy‐FA) that wasanalysed using fluorescent activated cell sorting (FACS). Single small intestinal cells ofeGFP‐CCK mice were prepared using an EDTA chemical/mechanical dissociationmethod. Cell samples were either non‐treated (control) or pre‐treated with a targetedcompound 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 ensuedemploying CCK‐cell ligands to implicate activity of CCK‐cells in this process. Bombesinand L‐amino acids induced a dynamic increase in FA uptake comparable to thatachieved by pre‐treatment with CCK. However, implementation of the protocol usingcells from a CCK KO model achieved replicate data and therefore demonstrated theseeffects were not exclusive to CCK‐cells.In conclusion, data presented in this thesis establish that a spectrum of key guthormones is expressed in individual EECs. Furthermore, a paracrine action of CCKsignallingis implicated to increase the absorptive ability of neighbouring enterocytes.These data suggest that CCK‐cells have the ability to integrate nutrient signals andsecrete a cocktail of hormones in response. These findings imply an increasedcomplexity to the enteroendocrine system whereby GI peptides may work together topotentiate a desired response without requirement of signals from higher centres.

Date of Award	1 Aug 2014
Original language	English
Awarding Institution	The University of Manchester
Supervisor	Craig Smith (Supervisor) & Richard Case (Supervisor)