Nrg4 and Gpr120 Signalling in Brown Fat Anthony Chukunweike OKOLO Institute of Reproductive and Developmental Biology Department of Surgery and Cancer Faculty of Medicine Imperial College London A thesis submitted in fulfilment of the requirements for award of the degree of Doctor of Philosophy 1 Statement of Originality All experiments included in this thesis were performed by me unless otherwise stated in the text. 2 Copyright statement The copyright of this thesis rests with the author and is made available under a Creative Commons Attribution Non-Commercial No Directive Licence. Researchers are free to copy, distribute or transmit the thesis on the condition that they attribute it, and they do not use it for commercial purposes, and they do not alter, transform or build upon it. For any re-use or re-distribution, researchers must make clear to others the licence terms of this work. 3 Acknowledgments I would like to thank my supervisors Dr Aylin Hanyaloglu and Dr Mark Christian for giving me the great opportunity to work in their labs. Aylin put in a great deal of effort especially in area of Gpr120 signalling, including having to guide me through the critical imaging procedures. Aylin and Mark contributed a great deal towards the final edition of this thesis. I would also like to thank Dr Mark Christian for bringing me to Imperial College London to start off my PhD in his laboratory, and for being a great mentor and a continuous source of knowledge for me. I am grateful for your enduring patience in trying to bring out the best in me and ensuring that I develop the ‘critical thinking’ that is needed as a scientist. My special thanks go to Prof Malcolm Parker, who has always put in his special professorial touch to my work, and to Prof Ilpo Huhtaniemi for setting up my admission, and indeed my sojourn in Imperial College. I would especially like to thank Dr Kunal Shah and Dr Evanthia Nikolopoulou who were my senior PhD students. They took the pains to teach me the rudiment of Laboratory practices. I will also like to appreciate Dr David Barneda-Ciurana for guiding me through the rough beginning of my laboratory work. My special thanks also go to Dr Olayiwola Oduwole for our long friendly and scientific conversations, Dr Usi Oboh and Mr and Mrs Andy Evans for happily receiving me in the UK. I would also like to thank my lab mates Dr Jenny Steel, Dr Kim Jonas, Camilla West, Camilla Larson, Natarin Caegprasath, and Silvia Sposini for the years working and sharing together as a real team. Finally, I would like to express my appreciation to my Love, friend and confidant, Modupe and my jewels, Oyiye, Chineme, Okuolise and Chimamaka for their enduring love and affection. 4 This acknowledgement will not be complete without mentioning ETF, Nigerian for initiating the funding for my PhD, and the department of Surgery and Cancer, Imperial College London, for ensuring that my PhD was completed. 5 Abstract Brown adipose tissue (BAT) dissipates energy, whereas white adipose tissue (WAT) is an energy storage site. The worldwide increase in obesity and its associated metabolic complications represents a growing challenge for health care systems. With the recent demonstration of significant deposits of adult human BAT, and that WAT is dynamic and can gain BAT characteristics, there is therefore a possibility for manipulative control of energy homeostasis via the induction of specific genes to enhance BAT levels. Nrg4 and Gpr120 are two genes we identified to be highly expressed in BAT and regulated in a temperature- dependent manner. Nrg4 is a member of the Neuregulin family that activates the ErbB-4 (HER4) tyrosine kinase receptor. Nrg4 was secreted from brown adipocytes and promoted neurite outgrowth, a function that may improve BAT thermogenesis by enhancing sympathetic innervation. Gpr120 is a member of the superfamily of G protein-coupled receptors that is activated by long chain fatty acids. Although Gpr120 is known to mediate anti-inflammatory and insulin- sensitizing effects in humans and mice, its role in BAT is unknown. In order to identify the signalling pathways and downstream functions activated by this receptor in brown adipocytes, distinct known and novel Gpr120 ligands, GW9508, TUG891 and TUG1096, were employed. These ligands increased expression of genes associated with glucose and fatty acid metabolism and reduced inflammatory gene markers. All of these effects were reversed in Gpr120 knockout cells. However, the levels of gene expression varied considerably with each ligand. Although Gpr120 is a known Gαq/11-coupled receptor in other cell systems, these distinct ligands exhibited various degrees of bias towards Gαq/11 and distinct signalling pathways. These findings could provide insight into the requirements for Gpr120 function in BAT, the 6 exploitation of which could generate ligands for therapeutic benefit and represents a promising opportunity to develop safer and more efficacious drugs. 7 Table of content Statement of Originality ............................................................................................................. 2 Copyright statement ................................................................................................................... 3 Acknowledgments...................................................................................................................... 4 Abstract ...................................................................................................................................... 6 List of Figures…………………………………………………………………………………………………………………………………..14 List of Tables……………………………………………………………………………………………………………………………………17 List of Abbreviations ............................................................................................................. 148 Chapter 1 .................................................................................................................................. 23 Introduction .............................................................................................................................. 23 Introduction .......................................................................................................................... 24 1.1 Adipose tissue and adipocytes........................................................................................ 24 1.1.2 White adipose tissue (WAT) ................................................................................... 24 1.1.3 Brown adipose tissue (BAT) ................................................................................... 25 1.2 Development of adipose tissues ..................................................................................... 27 1.3 Beige/brite ...................................................................................................................... 28 1.4 Dynamic nature of adipose tissue................................................................................... 30 1.5 Human BAT ................................................................................................................... 31 1.6 Measurement of BAT ..................................................................................................... 33 1.7 BAT Thermogenesis ...................................................................................................... 33 1.7.1 Role of UCP1 in BAT thermogenesis ..................................................................... 34 1.7.2 Role of adrenergic signalling in thermogenesis ...................................................... 35 1.8 Transcriptional control of brown fat development and function .................................... 38 1.9 BAT and obesity............................................................................................................. 39 1.10 Free fatty acids (FFAs) ................................................................................................. 42 1.11 Fatty acid metabolism .................................................................................................. 42 1.11.1 Lipogenesis ............................................................................................................ 42 1.11.2 Lipolysis ................................................................................................................ 43 1.11.3 Regulation of fatty acid metabolism ...................................................................... 45 1.12 Free fatty acid receptors ........................................................................................... 46 1.13 G-proteins ..................................................................................................................... 49 8 1.14 Diversification and specification/regulation of GPCR signalling ................................ 51 1.15 Role of β-arrestins in GPCR signalling ........................................................................ 52 1.16 G-protein-coupled receptor 120 (Gpr120) ................................................................... 54 1.17 Signalling pathways activated by Gpr120 .................................................................... 56 1.18 Development of selective ligands for Gpr120.............................................................. 56 1.19 Hypothesis and Aims of the study................................................................................ 60 Chapter 2 .................................................................................................................................