Targeted Expression of Plasminogen Activator Inhibitor (PAI)-1 to the Stomach Inhibits Gut-Brain Signalling by the Satiety Hormone Cholecystokinin (CCK) Thesis submitted in accordance with the requirements of the University of Liverpool for the degree of Doctor in Philosophy By Joanne Gamble October 2013 I For Lily, you are the sunshine in my life…. II Table of Contents Figures and tables VII Acknowledgements XI Publications XIII Abstract XIV Chapter 1 ................................................................................................................................ 1 1.1 Overview ........................................................................................................................... 2 1.2 The Gastrointestinal Tract and Digestive Function ...................................................... 4 1.2.1 Distribution, Structure and Biology of Enteroendocrine (EEC) Cells ........................ 5 1.2.2 Luminal Sensing ......................................................................................................... 6 1.3 Energy Homeostasis ......................................................................................................... 7 1.3.1 Gut Hormones ........................................................................................................... 10 1.3.1.1 The Gastrin Family ............................................................................................ 11 1.3.2 PP-fold Family .......................................................................................................... 16 1.3.2.1 Peptide Tyrosine Tyrosine (PYY) ...................................................................... 16 1.3.2.2 Pancreatic polypeptide (PP) ............................................................................... 19 1.3.2.3 Neuropeptide Y (NPY) ...................................................................................... 20 1.3.3 Secretin/Glucagon Superfamily ................................................................................ 21 1.3.3.1 Secretin .............................................................................................................. 21 1.3.3.2 Glucagon ............................................................................................................ 22 1.3.3.3 Products of Preproglucagon Cleavage ............................................................... 23 1.3.3.4 GIP ..................................................................................................................... 26 1.3.3.5 Vasoactive intestinal peptide ............................................................................. 27 1.3.4 Gut Hormone Interactions ......................................................................................... 28 1.3.4.1 Synergistic Interactions - CCK & Leptin ........................................................... 28 1.3.4.2 Orexigenic & Anorexigenic Interactions ........................................................... 30 1.4 The Cannabinoid System .............................................................................................. 33 1.4.1 Lipid Amides ............................................................................................................ 33 1.4.1.2 Oleoylethanolamide (OEA) ............................................................................... 33 1.4.1.3 Anandamide (AEA) ........................................................................................... 34 III 1.5 Obesity ............................................................................................................................ 35 1.5.1 Background ............................................................................................................... 35 1.5.2 Comorbidities ............................................................................................................ 36 1.5.3 Obesity - A Multifactorial Condition ........................................................................ 36 1.5.4 Gut Microbiota .......................................................................................................... 37 1.5.5 Obesity - An Inflammatory Disease? ........................................................................ 39 1.5.6 Treating Obesity ........................................................................................................ 41 1.5.6.1 Drugs and Targets .............................................................................................. 42 1.5.6.2 Surgery ............................................................................................................... 43 1.5.7 Animal Models of Obesity ........................................................................................ 44 1.6 The Urokinase Plasminogen Activator System ........................................................... 46 1.6.1 Urokinase and the uPA Receptor .............................................................................. 46 1.6.1.1 Plasminogen Activator Inhibitor (PAI)-1........................................................... 49 1.7 Aims and objectives ....................................................................................................... 53 Chapter 2 .............................................................................................................................. 57 2.1 Chemical reagents .......................................................................................................... 58 2.2 Equipment ...................................................................................................................... 59 2.3 Animals ........................................................................................................................... 60 2.4 Animal treatments ......................................................................................................... 61 2.4.1 Whole animal fixation via transcardial perfusion ..................................................... 62 2.5 Immunohistochemistry for the detection of fos in the brainstem and hypothalamus ............................................................................................................................................... 63 2.5.1 Brainstem .................................................................................................................. 63 2.5.2 Quantification of fos ................................................................................................. 64 2.5.3 Hypothalamus ........................................................................................................... 64 2.6 Gastric Emptying Assays............................................................................................... 64 2.7 Feeding studies ............................................................................................................... 65 2.7.1 Anorexigenic peptides .............................................................................................. 65 2.7.2 Orexigenic peptides .................................................................................................. 65 2.7.3 Short term fasting studies .......................................................................................... 66 2.8 Elisa studies .................................................................................................................... 66 2.9 Statistical analyses ......................................................................................................... 66 Chapter 3 .............................................................................................................................. 67 3.1 Introduction .................................................................................................................... 68 3.1.1 Aims .......................................................................................................................... 70 IV 3.2 Methods ........................................................................................................................... 71 3.2.1 Animals (C57BL/6, PAI-1-HKβ , uPAR-/- and PAI-1 -/-) ......................................... 71 3.2.2 Brainstem fos labelling ............................................................................................. 71 3.2.3 H.felis infection ......................................................................................................... 71 3.3 Results ............................................................................................................................. 72 3.3.1 Mouse brainstem ....................................................................................................... 72 3.3.2 PAI-1 suppresses neuronal activation by CCK ......................................................... 74 3.3.3 PAI-1-H/Kβ have decreased brainstem fos associated with neuronal activation by exogenous CCK8s .............................................................................................................. 76 3.3.4 Exogenous PAI-1 suppresses neuronal activation following feeding ....................... 79 3.3.5 uPAR is required for the action of PAI-1 ................................................................. 81 3.3.6 Helicobacter felis infection does not have a significant effect on brainstem fos expression .......................................................................................................................... 83 3.4 Discussion.......................................................................................................................