THE PREPROGLUCAGON DERIVED PEPTIDES AND ENERGY HOMEOSTASIS A thesis submitted for the degree of Doctor of Philosophy Jennifer Parker 2013 Department of Medicine Imperial College London Abstract Obesity is a major contributor to the development of chronic diseases, and there is a paucity of effective treatments. Recent studies suggest that co-agonists at the glucagon-like peptide-1 (GLP-1) and glucagon receptor efficaciously reduce body weight and improve glucose homeostasis. This thesis explores the effects of glucagon, GLP-1 and the endogenous GLP-1/glucagon receptor co-agonist oxyntomodulin, on appetite and glucose homeostasis and their mechanisms. As expected, peripheral injection of GLP-1 or glucagon to fasted mice transiently reduced food intake. Interestingly, subanorectic doses of GLP-1 and glucagon potently inhibited food intake when combined. Agonists at the GLP-1 (GLPAg) and glucagon (GCGAg) receptors designed in this laboratory were found to have receptor affinities comparable with those of GLP-1 and GCG, but with a prolonged duration of action. When administered chronically, individually and in combination, to an obese mouse model, the combination of these peptides appeared to cause superior reduction in body weight and improvement in glucose tolerance compared to the individual peptides. The receptors and central appetite regulating centres involved in the response to anorectic doses of GLP-1, glucagon and oxyntomodulin were investigated. The pattern of c-fos immunoreactivity in response to glucagon was examined for the first time and appeared indistinguishable from that induced by GLP-1. Oxyntomodulin appeared to induce greater c-fos activation in the nucleus tractus solitarius (NTS) than either glucagon or GLP-1 at equivalently anorectic doses. No difference in the activation of catecholaminergic, preproglucagon or POMC expressing neurons in the NTS was seen between the three peptides. The anorectic effects of both oxyntomodulin and glucagon appeared to depend on the presence of the GLP-1 receptor. The effects of both glucagon and oxyntomodulin on blood glucose and insulin release in mice were investigated and found to be highly dose dependant. Surprisingly, high doses of glucagon did not have a detectable hyperglycemic effect and some evidence suggested this may be due to cross reactivity with the GLP-1 receptor. The GLP-1 receptor antagonist EX 9-39 had an unexpected hyperglycemic effect, present in GLP-1 receptor knockout mice suggesting an alternative mechanism of action. Overall the work contained in this thesis has added mechanistic information to our knowledge about the anorectic and glucoregulatory effects of the preproglucagon derived peptides and supports development of synthetic agonists of the glucagon and GLP-1 receptors as treatments for obesity. 2 Declaration of contributors The author performed the majority of the work contained within this thesis. All collaboration and assistance is described below: Experimental chapter 1: Receptor Binding Assays for the screening of analogues were done in collaboration with James Plumer, Klara Hostomska, James Minnion and Joyceline Cuenco-Shillito. Feeding studies, pair-feeding studies, pharmacokinetic studies and glucose tolerance testing were carried out with the help of the ‘G series’ team, in particular James Minnion, Joyceline Cuenco-Shillito, James Plumer, Katherine McCullough, Sam Price and Tanya Stezhka. Experimental chapter 2: Dose response and combinatorial c-fos studies were carried out in collaboration with Katherine McCullough. The comparative glucagon, GLP-1, oxyntomodulin c-fos study was carried out with the assistance of John Tadross under whose guidance the dual immunohistochemistry was also carried out. GLP-1R knockout mice were bred with the kind permission of Dr Daniel Drucker (Samuel Lunenfeld Research Institute, University of Toronto). Experimental chapter 3: Studies measuring blood glucose carried out with the assistance of Joyceline Cuenco-Shillito, Sam Price, Jamie Plumer and in particular Tanya Stezhka. 3 Acknowledgements I would like firstly to thank my supervisors: Steve Bloom for allowing me to work in his laboratory, and for always thinking of the things I forget, and Ben Field for his thoughtful discussions, and inexhaustible supply of supporting and encouraging words. I also wish to thank James Minnion, for being an excellent supervisor in all but name and Mohammad Ghatei for his wisdom. I would like to thank all the members of the G-series team without whom this thesis would be a lot thinner and to whom I am grateful for every hour of shared toil in the BSU, enlightening thought on pH, and for laughing when no one else could. There’s nothing like spending three years alternately sweating and shivering at your desk to bring people together and thus I’d like to thank all the girls in fellows’ room 2, past, present and Jamie, for their friendship and frequently thought provoking discussion in and out of the lab. I’d also like to thank Anne and Ari, for putting up with me at home, at work and everywhere in between. I wish all of you the best of luck for the future and hope we’ll remain friends for many years to come. Finally I’d like to thank my friends and family. I’m extremely fortunate to have friends so talented in feigning interest in mouse obesity and spending time with you makes bad days in the lab seem utterly unimportant. I thank my family for being more interested, enthusiastic and supportive than I could have asked for and I wish to thank my father in particular, I hope you enjoyed learning about the gila monster. I’d also like to thank John, for the odd bit of help, and for making science fun. 4 Abbreviations ABC Avidin-biotin complex GABA γ-amino butyric acid ACh Acetyl choline GCGR Glucagon receptor ACTH Adrenocorticotrophic hormone GHRH Growth hormone releasing hormone AgRP Agouti-related protein GHSR Growth hormone secretagogue ANOVA Analysis of variance receptor AP Area postrema GLP-1 Glucagon-like peptide-1 ARC Arcuate nucleus of the hypothalamus GLP-2 Glucagon-like peptide-2 BBB Blood brain barrier GLP-1R Glucagon-like peptide-1 receptor BDNF Brain-derived neurotrophic factor GPCR G-protein coupled receptor BMI Body mass index GRP gastrin releasing peptide BSA Bovine serum albumin GWAS Genome-wide association studies CA catecholamine GTT Glucose tolerance test cAMP Cyclic adenosine monophosphate HPA Hypothalamic-pituitary-adrenal CART Cocaine-and amphetamine-regulated HRP Horseradish peroxidase transcript protein ICV Intracerebroventricular CCK Cholecystokinin IGF Insulin like growth factor CeA Central nucleus of the Amygdala IHC Immunohistochemistry CNS Central nervous system IP Intraperitoneal CRH Corticotrophin-releasing hormone IRS Insulin receptor substrate DAB 3’3’-diaminobenzidine KO Knockout tetrahydrochloride hydrate LHA Lateral hypothalamic area of the DMN Dorsomedial nucleus of the hypothalamus hypothalamus MC4R Melanocortin 4 receptor DMV Dorsal motor nucleus of vagus MCH Melanocortin-concentrating hormone DNA Deoxyribonucleic acid ME Median eminence DPP-IV Dipeptidyl peptidase-IV MEMRI Manganese enhanced magnetic EDTA Ethylenediaminetetraacetic acid resonace imaging ERK Extracellular signal-related kinase MFB Medial forebrain bundle EX-4 Exendin-4 mRNA Messenger ribonucleic acid EX 9-39 Exendin 9-39 MSH Melanocyte-stimulating hormone FFA Free Fatty Acid NPY Neuropeptide Y FI Food intake NTS Nucleus of the tractus solitarius fMRI Functional magnetic resonance imaging OXM Oxyntomodulin PACAP pituitary adenylate cyclase-activating RNA Ribonucleic acid poly-peptide s/c Subcutaneous PBS Phosphate buffered saline SCN Suprachiasmatic nucleus of the PBN Parabrachial nucleus hypothalamus PC Prohormone convertase SEM Standard error of the mean PCR Polymerase chain reaction SON Supraoptic nucleus of the hypothalamus PEPCK Phosphoenolpyruvate carboxykinase TAE Tris-acetate-ethylenediaminetetraacetic PLC Phospholipase C acid POMC Proopiomelanocortin TH Tyrosine hydroxylase PP Pancreatic polypeptide VIP Vasoactive intestinal peptide PVN Paraventricular nucleus of the VMN Ventromedial nucleus of the hypothalamus hypothalamus PYY Peptide YY VTA Ventral tegmental area RIA Radioimmunoassay WT Wild-type 6 Table of Contents Abstract…………………………………………………………………………………………………………………………………………………………….2 Declaration of contributors……………………………………………………………………………………………………………………………….3 Acknowledgements…………………………………………………………………………………………………………………………………………..4 Abbreviations……………………………………………………………………………………………………………………………………………………5 Table of figures………………………………………………………………………………………………………………………………………………..12 List of tables…………………………………………………………………………………………………………………………………………………….15 1 GENERAL INTRODUCTION .................................................................................................. 16 1.1 Introduction ...................................................................................................................................... 17 1.2 Energy Homeostasis .......................................................................................................................... 18 1.3 Overview of appetite control systems ............................................................................................... 21 1.3.1 Neurocircuitry ..................................................................................................................................... 21 1.3.1.1 The Hypothalamus ..................................................................................................................... 23