The role of amino acids in appetite regulation A thesis submitted for the degree of Doctor of Philosophy in Imperial College London Anne McGavigan 2013 Section of Investigative Medicine Division of Diabetes, Endocrinology & Metabolism Department of Medicine Imperial College London Abstract There is currently a lack of safe and effective treatment options for obesity. A high protein diet is an effective weight loss and weight maintenance strategy. However, like many diets, high protein diets can be difficult to adhere to. The mechanisms by which protein exerts its weight-reducing effect remain unclear. However, it has been reported that different types of protein exert different effects on appetite. One possible explanation for these differences is the varied amino acid constituents of the protein. Preliminary data from our group investigated the effect of a range of amino acids on food intake in rodents. L-cysteine was identified as the most anorexigenic amino acid. This thesis has investigated the effect of L- cysteine on food intake and explored possible mechanisms by which it mediates this effect. L-cysteine dose dependently decreased food intake in both rats and mice following oral gavage and intraperitoneal administration. This reduction in food intake did not appear to be secondary to behavioural side effects or feelings of nausea. L-cysteine increased neuronal activation in the area postrema and nucleus tractus solitarius, delayed gastric emptying and suppressed plasma acyl-ghrelin levels. However, the anorectic effect of L- cysteine did not appear to depend on NMDA, GPRC6A or CCK-A receptors, nor on subdiaphragmatic vagal afferent signalling. Repeated administration of L-cysteine also decreased food intake in rats and diet-induced obese mice. The studies described in this thesis demonstrate the anorectic effects of L-cysteine and identify possible sites of action. It is likely that different amino acids exert different effects on appetite through a number of mechanisms, the combination of which contributes towards the success of high protein diets on body weight and appetite. This thesis provides a framework for future studies to investigate the therapeutic potential of combinations of amino acids that could provide a safe and practical therapeutic treatment for obesity. 2 Acknowledgements I would like to express my sincerest thanks and appreciation to my supervisors: Dr Kevin Murphy and Professor Anders Lehmann for sharing their knowledge and for their instrumental support, guidance and advice. I would also like to extend my gratitude to Professor Steve Bloom for giving me the opportunity to work in his department. I am also extremely grateful for the assistance I received from various members of the Section of Investigative Medicine, Imperial College London, with special thanks going to Dr Hannah Greenwood and Dr James Kinsey-Jones. I am also very grateful to members of the Cardiovascular and Metabolic Disease group, AstraZeneca for their help and for making my time there so enjoyable, particularly Gina Hyberg and Dr Carina Ämmäla. I would also like to specially thank Myrtha Arnold (Swiss Federal Institute of Technology) who kindly taught me the SDA technique. Finally, I would like to thank the BBSRC and AstraZeneca for funding my PhD. 3 Copyright Declaration The copyright of this thesis rests with the author and is made available under a Creative Commons Attribution Non-Commercial No Derivatives licence. Researchers are free to copy distribute or transmit the thesis on the condition that they attribute it, that they do not use it for commercial purposes and that they do not alter, transform or build upon it. For any reuse or redistribution, researchers must make clear to others the licence terms of this work. Declaration of contributors This thesis was composed entirely by the author and all of the work in this thesis was performed by the author with any collaboration and assistance described below: Chapter 2 Preliminary data presented in the introduction was carried out by Dr Hannah Greenwood. All in-house radioimmunoassays were established and maintained by Professor M. Ghatei. Brainstem c-Fos was counted by Miss Kathryn Miller who was blinded from treatment groups. Cannulation experiments were performed with assistance from Dr James Kinsey- Jones. Chapter 3 SDA surgeries were performed with assistance from Gina Hyberg (AstraZeneca). The GPRC6A knockout mouse colony was bred and maintained by Dr James Kinsey-Jones. 4 Abbreviations 3-MST 3-mercaptopyruvate sulfurtransferase 5-HT 5-hydroxytryptamine (serotonin) 5-HT2c Serotonin 2c receptor 7TM 7 transmembrane AAV Adeno-associated virus AC Adenylate cyclase AgRP Agouti related peptide AHA Anterior hypothalamic area ANOVA Analysis of variance AP Area postrema APC Anterior piriform cortex ARC Arcuate nucleus ATP Adenosine trisphosphate BBB Blood brain barrier BMI Body mass index BSA Bovine serum albumin BSO Buthionine sulfoximine cAMP Cyclic adenosine monophosphate CART Cocaine and amphetamine regulated transcript CaSR Calcium sensing receptor CAT Cysteine amino transferase CB1R Cannabinoid 1 receptor CBS Cystathionine beta synthetase CCK Cholecystokinin CCK-1R Cholecystokinin 1 receptor CCK-2R Cholecystokinin 2 receptor CDO Cysteine dioxygenase cFLI c-Fos like immunoreactivity c-Fos Cellular Fos CHH O-carboxymethyl hydroxylamine hemihydrochloride CL Cysteine lyase CNS Central nervous system CSD Cysteine sulfinate decarboxylase CSE Cystathionine gamma lyase CTA Conditioned taste aversion DA Dopamine DAG Diacylglycerol DIO Diet induced obese DIT Diet induced thermogenesis DMC Dorsal vagal complex DMN Dorsomedial nucleus DMX Dorsal motor nucleus of the vagus eIF2α Eukaryotic initiation factor 2 α EMEA European Medicines Agency ENS Enteric nervous system 5 ER Endoplasmic reticulum ERK1/2 Extracellular signal regulated kinase 1/2 FDA Food and Drugs Administration FTO Fat mass and obesity associated protein GCGR Glucagon receptor GCN2 General control nonrepressed 2 GDP Guanosine diphosphate GDW Glass distilled water GHRL Ghrelin gene GHSR Growth hormone secretagogue receptor GI Gastrointestinal GIP Glucose-dependent insulinotropic peptide GLP-1 Glucagon-like peptide-1 GLP-1R Glucagon-like peptide-1 recetor GOAT Ghrelin o-acyltransferase GPCR G-protein coupled receptor GPRC6A G-protein coupled receptor group C member 6A GSH Glutathione GSSG Glutathione disulphide GTP Guanosine trisphosphate HFD High fat diet HPLC High pressure liquid chromatography IAA Indispensible amino acid ICV Intracerebroventricular IHC Immunohistochemistry IP Intraperitoneal IP3 Inositol trisphosphate IP3R Inositol trisphosphate receptor IQR Interquartile range IR Insulin receptor KO Knockout Lep-R Leptin receptor LHA Lateral hypothalamic area LTP Long term potentiation MBH Mediobasal hypothalamus MC3R Melanocortin 3 receptor MC4R Melanocortin 4 receptor MCH Melanin-concentrating hormone MCHR1 Melanin-concentrating hormone receptor 1 MCHR2 Melanin-concentrating hormone receptor 2 MEF Mouse embryonic fibroblasts mTOR Mammalian target of rapamycin mTORC1 Mammalian target of rapamycin complex 1 NA Noradrenaline NAcc Nucleus Accumbens NDP-MSH [Nle4,D-Phe7] alpha-melanocyte stimulating hormone NMDA N-methyl-D-aspartate 6 NPY Neuropeptide Y NTS Nucleus tractus solitarius Ob-R Leptin receptor OG Oral Gavage OX1R Orexin receptor 1 OX2R Orexin receptor 2 OXM Oxyntomodulin PBS Phosphate buffered saline Pe Periventricular nucleus PIP2 Phosphotidylinositol-4,5 bisphosphate PLC Phospholipase C PLP Pyridoxal 5’ phosphate POMC Pro-opiomelanocortin PPG Proparagylglycine PVN Paraventricular nucleus PYY Peptide Tyrosine Tyrosine RIA Radio-immunoassay RNS Reactive nitrogen species ROS Reactive oxygen species SC Subcutaneous SDA Subdiaphragmatic vagal deafferentation SEM Standard error of the mean SLC Solute-linked carrier SNAC Sodium N-[8-(2-hydroxybenzoyl) amino caprylate SNP Single nucleotide polymorphism SON Supraoptic nucleus T1R1 Taste receptor type 1 member 1 T1R2 Taste receptor type 1 member 2 T1R3 Taste receptor type 1 member 3 T2DM Type II Diabetes Mellitus TG Triglyceride TRPM5 Transient receptor potential cation channel subfamily M member 5 TSC Tuberosclerosis complex VFT Venus fly trap VMN Ventromedial nucleus VTA Ventral tegmental area α-MSH Alpha-melanocyte stimulating hormone γGCS Gamma-glutamyl cysteine synthetase 7 Table of Contents Abstract ...................................................................................................... 2 Acknowledgements .................................................................................... 3 Copyright Declaration ................................................................................. 4 Declaration of contributors ......................................................................... 4 Abbreviations ............................................................................................. 5 Table of Contents ........................................................................................ 8 List of Figures ............................................................................................ 15 List of Tables ............................................................................................. 17 CHAPTER 1: GENERAL INTRODUCTION .......................................... 18 1.1 The Obesity Epidemic .......................................................................... 19 1.1.1 Current treatment options ..................................................................................... 19 1.2 Energy