Vitamin B3 salvage and NAD+ metabolism in skeletal muscle By Rachel Fletcher A thesis submitted to the University of Birmingham for the degree of DOCTOR OF PHILOSOPHY Institute of Metabolism and Systems Research College of Medical and Dental Sciences University of Birmingham June 2017 University of Birmingham Research Archive e-theses repository This unpublished thesis/dissertation is copyright of the author and/or third parties. The intellectual property rights of the author or third parties in respect of this work are as defined by The Copyright Designs and Patents Act 1988 or as modified by any successor legislation. Any use made of information contained in this thesis/dissertation must be in accordance with that legislation and must be properly acknowledged. Further distribution or reproduction in any format is prohibited without the permission of the copyright holder. Abstract Nicotinamide adenine dinucleotide (NAD+) is both an essential redox coenzyme and a substrate for NAD+-consuming enzymes, such as the sirtuins, which adapt transcriptional programmes to increase energy availability. Skeletal muscle is a major regulator of energy metabolism and its function is impaired with ageing. Uncovering the key routes regulating NAD+ availability may provide valuable insight into novel aspects of skeletal muscle metabolic health. Data presented here identifies a limited set of enzymes important for skeletal muscle NAD+-biosynthesis namely; nicotinamide phosphoribosyltransferase (NAMPT) and nicotinamide riboside kinases (NMRK) 1 and 2, which salvage vitamin B3s nicotinamide (NAM) and nicotinamide riboside (NR) to NAD+. NAMPT was confirmed vital for recycling of NAM, with NAD+ depleted in myotubes following NAMPT inhibition. Single and double NMRK knockout mouse models found NMRK activity non-essential for maintaining basal NAD+, with activity restricted by NR availability. Exogenous NR delivery enhanced NAD+ and recovered the effects of NAD+ depletion following NAMPT inhibition. NMRK2 was determined highly muscle-specific; although energy signalling was mostly unperturbed in NMRK2KOs, in vivo data indicated impaired metabolic flexibility following high fat diet. While the muscle-specific role of NMRK2 requires further investigation, this thesis identifies NMRK1/2 as important therapeutic targets for enhancing NAD+ by NR supplementation. I Acknowledgements Firstly, and most importantly, I would like to thank my supervisor Prof. Gareth Lavery for all of his help, support, encouragement and time over the past 4 years. I am truly grateful and I know that his excellence as a scientist, innovator and mentor has enabled and inspired me to grow as a researcher. He has listened (a great deal), motivated me through the project difficulties and given me the confidence to become independent with my research ideas. I couldn’t have asked for anything more. Equally I would like to thank all the current and past members of the Lavery lab / Molecular Metabolism Research Group. Together (Craig, Agnieszka, Lucy, Yasir, Antje, Dave and Ganesh) have all contributed to my PhD experience, they have helped to make the difficult times easier and the good times better, so I am truly grateful to them all. They have not only been great colleagues but have also become great friends and I have so many fond memories. I would like to especially thank Craig, as he not only taught me everything I know in the lab (including what not to do) but endlessly supported me throughout this project. His invaluable advice, calm and in lab banter has been much appreciated. He also inspired (or challenged rather) me to take up running, I’m yet to decide how happy I feel about this, but without the ‘Shadow Ninjas’ I wouldn’t have managed to complete two marathons during my PhD. I would also like to thank everyone in CEDAM and the IMSR for their support and guidance. I feel incredibly lucky to have been part of such a great institute and work alongside such friendly, helpful and exceptional peers. II During my PhD I had the opportunity to partake in two placements at the University of Iowa and the Nestlé Institute of Health Sciences. I would like to thank Prof. Charles Brenner for the time I spent in Iowa City. I am grateful to Charlie and all of the Brenner group for their help and training during my visit. Equally, I would like to thank Dr Carles Canto and Nestlé for providing me with the opportunity to spend time researching in Lausanne. I am truly indebted as this work put the final pieces of my PhD research together, something I could never have done without this opportunity. I am particularly grateful to Joanna for all of her help during my time in Lausanne, we made a great team and I know I have found a true friend through this collaboration. Finally, I would like to thank my family for a lifetime of love, care and encouragement. Their endless support has allowed me to achieve so much already. I’m incredibly proud to have such amazing parents who have always given me the freedom to thrive in whatever I choose. I’m also extremely lucky to have my brothers, James and Gary, who have inspired me since I can remember. Their successes have always motivated me to follow suit and encouraged me to strive to be the best that I can be. Last, but not least, I would like to thank Chris. We met at the beginning of my PhD journey and I couldn’t think of a better person to share this experience with; he is the most hardworking and focused person I have ever met and has made me more determined than ever to succeed. III Table of Contents Chapter 1 General Introduction ......................................................................... 1 1.1 Skeletal muscle metabolism .......................................................................... 2 1.1.1 Skeletal muscle structure........................................................................ 2 1.1.2 Myogenesis ............................................................................................ 4 1.1.3 Skeletal muscle function ......................................................................... 6 1.1.4 Skeletal muscle fibre types ..................................................................... 8 1.1.5 Skeletal muscle energy metabolism ....................................................... 9 1.1.6 Metabolic adaptation to exercise .......................................................... 12 1.1.7 Mitochondrial biogenesis and metabolism ............................................ 14 1.2 NAD+: History and classic functions ............................................................ 16 1.3 NAD+ signalling and consumption ............................................................... 22 1.3.1 Sirtuins .................................................................................................. 22 1.3.2 PARP .................................................................................................... 25 1.3.3 Cyclic ADP-Ribose Synthases .............................................................. 26 1.4 NAD+ Biosynthesis ...................................................................................... 27 1.4.1 De novo synthesis ................................................................................ 27 1.4.2 Vitamin B3 Salvage pathways .............................................................. 29 1.4.3 Preiss – Handler pathway ..................................................................... 30 1.4.4 NAMPT salvage pathway ..................................................................... 31 1.4.5 NMRK salvage pathway ....................................................................... 32 1.4.6 NAD+ biosynthesis in skeletal muscle ................................................... 33 IV 1.5 NAD+ metabolism in health and disease ..................................................... 34 1.6 NAD+ in Ageing and Sarcopenia ................................................................. 36 1.7 Strategies to boost NAD+ signalling ............................................................ 39 1.7.1 Vitamin B3 supplementation ................................................................. 39 1.8 Project rationale .......................................................................................... 45 1.9 Hypothesis .................................................................................................. 47 1.10 Project Aims ............................................................................................. 48 Chapter 2 Materials and Methods .................................................................... 49 2.1 C2C12 cell line growth and maintenance .................................................... 50 2.1.1 C2C12 cell line ..................................................................................... 50 2.1.2 Proliferation .......................................................................................... 50 2.1.3 Differentiation ....................................................................................... 51 2.1.4 Cryopreservation .................................................................................. 51 2.2 Primary muscle cell isolation and culture .................................................... 52 2.2.1 Primary murine satellite cell isolation .................................................... 52 2.2.2 Proliferation .......................................................................................... 53 2.2.3 Differentiation ......................................................................................