Cellular expression and Function of CCK in the Mouse Duodenum A thesis submitted to the University of Manchester for the degree of PhD Physiology in The Faculty of Life Sciences 2013 Claire Demenis Faculty of Life Sciences Table of Contents TITLE PAGE .......................................................................................................................................1 TABLE OF CONTENTS.....................................................................................................................2 LIST OF FIGURES, GRAPHS AND TABLES .................................................................................7 ABSTRACT ...................................................................................................................................... 10 DECLARATION............................................................................................................................... 11 COPYRIGHT STATEMENT .......................................................................................................... 12 ACKNOWLEDGEMENTS .............................................................................................................. 13 ABBREVIATIONS........................................................................................................................... 14 CHAPTER ONE INTRODUCTION ............................................................................................................................ 17 1.0. OVERVIEW ............................................................................................................................................... 18 1.1. THE GASTROINTESTINAL TRACT......................................................................................................... 18 1.1.1. Enteroendocrine Cells (EEC).....................................................................................................22 1.1.2. Epithelial Cell Development ......................................................................................................25 1.2. NUTRIENT HANDLING IN THE GI TRACT............................................................................................ 27 1.2.1. Nutrient Digestion .........................................................................................................................27 1.2.2. Nutrient handling in the Small intestine .............................................................................29 1.2.3. Nutrient Sensing by EECs ...........................................................................................................31 1.2.3.1. G‐Protein CoupleD Receptors ..........................................................................................................................31 1.2.3.2. Fatty aciD sensing GPCRs...................................................................................................................................32 1.2.3.3. Fat‐DeriveD compounDs as liganDs for intestinal GPCRs.....................................................................33 1.2.3.4. Amino AciD sensing by GPCRs.........................................................................................................................33 1.2.3.5 CarbohyDrate sensing by GPCRs .....................................................................................................................35 1.2.3.6. Non‐GPCR meDiateD nutrient Detection......................................................................................................35 1.2.4. Systemic Signals influencing EEC activity...........................................................................38 1.2.4.1. EECs anD nutritional status ..............................................................................................................................38 1.2.4.2. EECs anD resiDent gut microflora ..................................................................................................................40 1.2.4.3. EECs anD intestinal immunity .........................................................................................................................40 1.2.4.4. Intra‐EEC signalling .............................................................................................................................................41 1.2.5. Intestinal Nutrient absorption.................................................................................................42 1.2.5.1. Membrane proteins involveD in intestinal fat uptake...........................................................................44 1.2.5.2. The Fatty AciD Transport Protein (FATP) family....................................................................................44 1.2.5.3. The Fatty AciD Translocase CD36 ..................................................................................................................45 1.2.5.4. The Plasma Membrane AssociateD ‘Fatty AciD BinDing Protein’ (FABPpm)...............................46 1.2.5.5. Peroxisome Proliferator‐ActivateD Receptor‐ α (PPAR‐α).................................................................47 1.3. THE GUT‐BRAIN AXIS............................................................................................................................ 49 1.4. ENTEROENDOCRINE GASTROINTESTINAL HORMONES .................................................................... 54 1.4.1. Cholecystokinin...............................................................................................................................54 1.4.2. Peptide tyrosine tyrosine (PYY)...............................................................................................54 1.4.3. Proglucagon­derived peptides .................................................................................................55 1.4.4. Gastric Inhibitory Peptide (GIP)..............................................................................................57 1.4.5. Neurotensin ......................................................................................................................................57 1.4.6. Secretin...............................................................................................................................................58 1.4.7. Preproghrelin...................................................................................................................................58 1.5. CHOLECYSTOKININ AND THE ENTEROENDOCRINE I‐CELL............................................................. 61 1.5.1. What are I­cells?.............................................................................................................................61 1.5.2. Cholecystokinin; Discovery and Functions..........................................................................63 2 1.5.3. CCK gene and structure...............................................................................................................65 1.5.4. CCK receptors...................................................................................................................................67 1.6. I‐CELL RESEARCH .................................................................................................................................. 70 1.7. THESIS AIMS AND HYPOTHESES.......................................................................................................... 73 CHAPTER TWO MATERIALS & METHODS ........................................................................................................... 75 2.1. ANIMALS .................................................................................................................................................. 76 2.1.1. eGFP­CCK mice ................................................................................................................................76 2.1.2. CCK Knockout (KO) mice.............................................................................................................76 2.1.3. CD1 and C57 mice controls........................................................................................................77 2.2 IMMUNOSTAINING OF PARAFFIN EMBEDDED TISSUE SECTIONS..................................................... 77 2.2.1. Tissue Preparation ........................................................................................................................77 2.2.2. Tissue Fixation and Paraffin Embedding ............................................................................78 2.2.3. Immuno­staining using paraffin sections............................................................................78 2.2.4. Double staining using two antisera raised in the same species.................................81 2.3. MICROSCOPY ........................................................................................................................................... 82 2.3.1. Imaging of dual­labelled sections...........................................................................................82 2.3.2. High magnification microscopic imaging of sections that were double­stained for two hormones.......................................................................................................................................82 2.3.2.1. Co‐localisation analysis of pixel fluorescence ..........................................................................................83 2.4. CONTROL EXPERIMENTS AIMED AT ASSESSING ANTISERA SPECIFICITY ....................................... 86 2.4.1. Verification