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The Physiological Role of Glucocorticoid and Mineralocorticoid Receptor Activation in Zebrafish

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The Physiological Role of Glucocorticoid and Mineralocorticoid Receptor Activation in Zebrafish University of Calgary PRISM: University of Calgary's Digital Repository Graduate Studies The Vault: Electronic Theses and Dissertations 2019-06-25 The Physiological Role of Glucocorticoid and Mineralocorticoid Receptor Activation in Zebrafish Faught, Leslie Erin Faught, L. E. (2019). The Physiological Role of Glucocorticoid and Mineralocorticoid Receptor Activation in Zebrafish (Unpublished doctoral thesis). University of Calgary, Calgary, AB. http://hdl.handle.net/1880/110575 doctoral thesis University of Calgary graduate students retain copyright ownership and moral rights for their thesis. You may use this material in any way that is permitted by the Copyright Act or through licensing that has been assigned to the document. For uses that are not allowable under copyright legislation or licensing, you are required to seek permission. Downloaded from PRISM: https://prism.ucalgary.ca UNIVERSITY OF CALGARY The Physiological Role of Glucocorticoid and Mineralocorticoid Receptor Activation in Zebrafish by Leslie Erin Faught A THESIS SUBMITTED TO THE FACULTY OF GRADUATE STUDIES IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY GRADUATE PROGRAM IN BIOLOGICAL SCIENCES CALGARY, ALBERTA JUNE, 2019 © Leslie Erin Faught 2019 Abstract Glucocorticoids are key mediators of the vertebrate stress response. In teleosts, the primary glucocorticoid, cortisol, is a ligand for two corticosteroid receptors (CRs), the glucocorticoid receptor (GR) and the mineralocorticoid receptor (MR). The affinity of cortisol for these receptors is markedly different, with MR having an almost 10-fold higher affinity for the ligand compared to GR. This led to the hypothesis 30 years ago in mammals, that MR is responsible for basal cortisol function, while GR is active only when cortisol levels are high. In zebrafish (Danio rerio), the role of cortisol-GR signalling during stress is well characterized and is primarily involved in energy substrate mobilization to cope with stress. However, despite the persistence of MR in vertebrate evolution, there is no known physiological role for MR in ray-finned fishes. Ubiquitous knockout of MR (MRKO) and GR (GRKO) in mammals results in death, in utero or postnatally, due to dehydration or delayed lung maturation, respectively. This makes zebrafish an attractive model to study the physiological impacts of GR and MR activation at the systems level. Here we tested the hypothesis that both MR and GR activation are necessary for mediating the effects of cortisol in zebrafish. During embryogenesis (0-5 days post-fertilization [dpf]), the presence of both GR and MR are necessary for the activation of the hypothalamus-pituitary- interrenal axis, and stress-related behaviour. When treated with cortisol, postnatally (5-15 dpf) larvae at 15 dpf are significantly smaller compared to the wildype, and this response was abolished in both the GRKO and MRKO zebrafish larvae. MR activation is necessary for lipid accumulation, while GR activation is required for lipolysis. Both MR and GR activation are also required for growth hormone/insulin-like growth factor 1 axis by GR and MR. However, only GR activation results in an increase in transcript abundance of proteolytic genes. We then tested the hypothesis that under basal cortisol conditions, a loss of GR would have a substantial impact on muscle growth. Indeed, adult GRKO fish had larger body mass, more total muscle protein, increased phosphorylation of eIF4B (protein translation), and a decrease in genes involved in protein catabolism. Taken together this thesis highlights the dichotomy of GR and MR receptor activation; MR acting to promote anabolic processes, while GR is a potent initiator of catabolism during stress. Overall, this thesis establishes a physiological role for MR in ray-finned fishes and indicates that the mode of action may involve either MR activation alone and/or an interaction with GR in modulating metabolism during stress. 1 Preface Chapter 1: Portions of the introductory text are reprinted with permission from: [Faught, E., Aluru, N. and Vijayan, M.M. 2016. The Molecular Stress Response. Fish Physiology vol 35: Biology of Stress in Fish. Edited by C.B. Schreck, L. Tort, Farrell, A. and Brauner, C.J. Elsevier, New York. Pp 113-166] [Faught E, Vijayan MM. 2018 Maternal stress and fish reproduction: The role of cortisol revisited. Fish Fisheries 19, 1016–1030] and [Faught, E., Hernandez-Perez, J., Wilson, J.M. and Vijayan, M.M. Stress in Response to Environmental Changes. Climate Change and Non-infectious Fish disorders. Edited by PTK Woo, and GK Iwama. CAB International – in press] Chapter 3: This chapter is reprinted with permission from [Faught E., and Vijayan M.M. 2018. The mineralocorticoid receptor is essential for stress axis regulation. Scientific Reports 8, Article number: 18081] Chapter 4: This chapter has been accepted in the Journal of Physiology and is currently in press [Faught E., and Vijayan M.M. 2019. Postnatal triglyceride accumulation is regulated by the mineralocorticoid receptor activation under basal and stress conditions. Journal of Physiology - 10.1113/JP278088] Chapter 6: This chapter is reprinted with permission from [Faught, E., and Vijayan M.M. 2019. Loss of the glucocorticoid receptor in zebrafish improves muscle glucose availability and increases growth. American Journal of Physiology: Endocrinology and Metabolism, 316(6): E1093-1104]. 2 Acknowledgements First and foremost, I would like to thank my supervisor Dr. Matt Vijayan. After a decade in his lab, I never fail to be impressed with his creativity, enthusiasm, and drive to produce the best work possible. I will always be grateful for the time he took to teach me. I would also like to thank my committee members, Dr. Joe Harrison and Dr. Doug Muench, for their patience and support. A further thank you to my external examiners, Dr. Dickmeis and Dr. Chelikani for taking the time to critically review this thesis. I would next like to acknowledge my lab members both past and present. In particular, Dr. Oana Birceanu and Andrew Thompson who have always been a source of encouragement and friendship. Other members, Chinmayee Das, Marwa Thraya, Carol Best, Analisa Lazaro-Cote and Dr. Patrick Gauthier have also been an important source of support in the lab. I would also like to thank Sophia George, Warren Fitch and Rob Hampton for their help with the many administrative tasks that made the lab set-up, function and fish care manageable. Finally, I would like to thank my family for their patience and love, and to Penny, my constant companion. 3 Dedication To my grandfather, Douglas James Gerrard. 4 Table of Contents Abstract ................................................................................................................................1 Preface ..................................................................................................................................2 Acknowledgements ..............................................................................................................3 Dedication ............................................................................................................................4 Table of Contents .................................................................................................................5 List of Tables .......................................................................................................................8 List of Figures and Illustrations ...........................................................................................9 List of key Symbols, Abbreviations and Nomenclature ....................................................11 CHAPTER 1: GENERAL INTRODUCTION ..................................................................13 Introduction ....................................................................................................................14 The HPI axis: .................................................................................................................15 Cortisol signalling ..........................................................................................................18 Glucocorticoid Receptor ...........................................................................................18 Mineralocorticoid Receptor ......................................................................................20 Metabolic adjustments during stress ..............................................................................22 Glucose as fuel: ........................................................................................................23 Energy substrates for oxidation and gluconeogenesis: .............................................26 Development of the HPI axis .........................................................................................30 Glucocorticoid and Mineralocorticoid Morphants and Mutants: .............................33 Hypothesis and Objectives: ...........................................................................................36 CHAPTER 2: GENERATING AND GENOTYPING GR AND MR KNOCKOUTS IN ZEBRAFISH WITH CRISPR/CAS9 ........................................................................37 Introduction: ...................................................................................................................38 Materials and Methods: .................................................................................................40 Zebrafish maintenance ..............................................................................................40
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