Investigating the effect of chronic activation of AMP-activated protein kinase in vivo Alice Pollard CASE Studentship Award A thesis submitted to Imperial College London for the degree of Doctor of Philosophy September 2017 Cellular Stress Group Medical Research Council London Institute of Medical Sciences Imperial College London 1 Declaration I declare that the work presented in this thesis is my own, and that where information has been derived from the published or unpublished work of others it has been acknowledged in the text and in the list of references. This work has not been submitted to any other university or institute of tertiary education in any form. Alice Pollard The copyright of this thesis rests with the author and is made available under a Creative Commons Attribution Non-Commercial No Derivatives license. 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 license terms of this work. 2 Abstract The prevalence of obesity and associated diseases has increased significantly in the last decade, and is now a major public health concern. It is a significant risk factor for many diseases, including cardiovascular disease (CVD) and type 2 diabetes. Characterised by excess lipid accumulation in the white adipose tissue, which drives many associated pathologies, obesity is caused by chronic, whole-organism energy imbalance; when caloric intake exceeds energy expenditure. Whilst lifestyle changes remain the most effective treatment for obesity and the associated metabolic syndrome, incidence continues to rise, particularly amongst children, placing significant strain on healthcare systems, as well as financial burden. AMP-activated protein kinase (AMPK) is widely regarded as a master regulator of energy homeostasis, acting as a cellular ‘fuel gauge’ to maintain intracellular ATP concentrations under conditions of metabolic stress. AMPK is known to promote catabolic pathways, including lipolysis, fatty acid oxidation and glycolysis, whilst inhibiting anabolic pathways in response to energy deprivation. AMPK is a strong therapeutic candidate for the treatment of obesity and the metabolic syndrome, with several studies providing evidence for the amelioration of type 2 diabetes in vivo. In collaboration with AstraZeneca, a novel transgenic mouse model was generated, expressing a gain-of-function mutation in the regulatory γ-subunit of AMPK; D316A, resulting in a constitutively active AMPK complex expressed globally. Initial observations revealed a polycystic kidney-like disease with a possible mouse strain effect, as well as cardiac glycogen accumulation, with no associated conductance defect. The subsequent characterisation of this mouse model revealed a novel role for AMPK activation in the protection from diet-induced obesity, conferred by a significant increase in exercise-independent energy expenditure, driven by UCP1-independent thermogenesis in the subcutaneous white adipose tissue (WATsc). It was found that, on a chow diet, the WATsc resembled classical brown adipose tissue which, when exposed to a high-fat diet, was subjected to transcriptional re-programming leading to the expression of many muscle-related genes associated with calcium/creatine futile cycling. This work highlights a role for AMPK in the protection from obesity, through the alteration of cell fate. 3 Acknowledgements I would sincerely like to thank my supervisors, Professor David Carling and Dr. Angela Woods, for their help, support and enthusiasm for this work over the course of this PhD project. I am grateful for the opportunity to work with such accomplished and passionate scientists, who have been so open and willing to share their knowledge and experience to make this work possible. I would like to thank Angela for her assistance in the laboratory, and for her dedication to helping not just myself but to everyone working in our group. It has been a pleasure to work in such an open and honest environment, facilitating many thought-provoking scientific discussions. I would very much like to thank AstraZeneca, the BBSRC and Imperial College London for their generous funding that has supported both this work and my development as a scientist. In addition, I would like to thank those at AstraZeneca, particularly my supervisor, Jon Read, for the support of this work, Maryam Clausen and Aurelie Bornot for their assistance with RNA sequencing, and Daniel Sutton for his assistance and technical expertise with immunohistochemistry. I would like to thank Phillip Muckett for his help, guidance and technical expertise with all animal studies. Without it, the work presented here could not have been achieved. In addition, I would like to thank the members of the animal physiology team for their help with in vivo studies. I would like to thank Dr. Luis Martins for his technical assistance with several key studies and for his expertise surrounding several aspects of this work. I would like to thank Laura Wilson for her work on the heart and kidney, for embracing this aspect of the project and taking it on as her own. Over the course of my PhD I have received invaluable support and friendship from every member of the Cell Stress Group, for which I am especially grateful. It has been an amazing experience to work in such a fun and supportive environment, with many laughs and engaging discussions over coffee. I would like to thank my family, my friends, my colleagues and my partner for their unwavering support over the years. Though no longer with us, I would like to thank my grandfather for passing on to me his passion for science, I am sure we would have had many enjoyable discussions. 4 Contents Declaration ................................................................................................................. 2 Abstract ...................................................................................................................... 3 Acknowledgements .................................................................................................... 4 Table of figures .......................................................................................................... 9 List of Abbreviations, Terms and Acronyms ............................................................. 13 1 CHAPTER 1: INTRODUCTION ......................................................................... 18 1.1 Pathogenesis of Obesity and the Metabolic Syndrome ............................... 20 1.1.1 Food Intake ........................................................................................... 25 1.1.2 Energy Expenditure .............................................................................. 35 1.2 Current Treatments and Therapeutic Strategies ......................................... 43 1.2.1 Current Treatments .............................................................................. 43 1.3 AMP-Activated Protein Kinase: A Master Regulator of Energy Homeostasis 45 1.3.1 A Highly Conserved Metabolic Regulator ............................................. 45 1.3.2 Structure and Regulation of AMPK ....................................................... 47 1.3.3 Downstream Targets of AMPK ............................................................. 52 1.4 Studying AMPK Activation in vivo ............................................................... 58 1.4.1 Activators and Inhibitors ....................................................................... 58 1.4.2 Mouse Models of AMPK Signalling ....................................................... 62 1.4.3 The Activating Mutant: An Novel Tool to Study Gain of Function in vivo 68 1.5 Project Aims ................................................................................................ 69 2 CHAPTER 2: MATERIALS AND METHODS ..................................................... 71 2.1 Materials...................................................................................................... 71 2.1.1 General Reagents ................................................................................ 71 2.1.2 Buffers .................................................................................................. 72 2.1.3 Antibodies ............................................................................................. 72 2.1.4 Primers ................................................................................................. 74 2.2 Animal Studies ............................................................................................ 76 2.2.1 Declaration ........................................................................................... 76 2.2.2 Animal Models ...................................................................................... 76 2.2.3 Diet Administration ............................................................................... 77 2.2.4 Blood sampling ..................................................................................... 77 2.2.5 Glucose Tolerance ............................................................................... 77 5 2.2.6 Comprehensive Laboratory Animal Monitoring System (CLAMS) ........ 78 2.2.7 Feeding studies (BioDAQ) .................................................................... 78 2.2.8 Ultrasound and Echocardiogram .........................................................