INVESTIGATING THE NEUROENDOCRINE CONTROL OF METABOLISM AND ENERGY HOMEOSTASIS Thesis submitted for the degree of Doctor of Philosophy at Imperial College London CHIOMA NMEREOBASI IZZI-ENGBEAYA 2018 Section of Investigative Medicine Division of Diabetes, Endocrinology & Metabolism Department of Medicine Imperial College London Abstract Obesity and associated metabolic disorders are major causes of morbidity and mortality in both developed and developing countries, but the options for treating these conditions are limited. Energy homeostasis and metabolism are regulated by a complex network of neuroendocrine systems, neural pathways, peripheral signals and circuits. Consequently, our knowledge of the regulation of energy homeostasis and metabolism remains incomplete. Two neuroendocrine systems were examined in this thesis, one involving glucocorticoids (a peripherally produced hormone acting centrally) and one involving kisspeptin (a centrally produced hormone acting peripherally). Reduction of active glucocorticoids in the arcuate nucleus of post-pubertal male rats (via stereotactic injection of recombinant adeno-associated virus to reduce expression of 11βHSD1) resulted in less weight gain despite matched food intake to controls on normal chow diet, with higher brown adipose tissue weight. However, on a high fat diet, reduction in arcuate active glucocorticoids resulted in higher average daily food intake and a trend towards higher body weight than controls. No differences in body composition, plasma corticosterone, plasma insulin and plasma glucose were produced by reduction of arcuate glucocorticoids. Administration of kisspeptin to healthy men resulted in increased glucose-stimulated insulin secretion (GSIS) during hyperglycaemia but not during euglycaemia. Kisspeptin enhanced GSIS without affecting the levels of metabolically active gut hormones. Additionally, acute kisspeptin administration did not affect appetite and food intake in healthy men. 2 These results provide further insights into the neuroendocrine control of energy homeostasis and metabolism and may help guide the development of hormone-modulating therapies for the treatment of obesity and associated conditions. 3 Acknowledgements I would like to thank God, who made all this possible. I would like to thank my husband for his unwavering support and belief in me, and my family for their support and encouragement. I would like to thank Prof Waljit Dhillo for his invaluable mentorship, and for providing the springboard for my academic career. I would like to thank Dr James Gardiner for his supervision. I would like to thank the members of the Kisspeptin Research Team, the Section of Investigative Medicine and the Clinical Chemistry staff at Imperial College NHS Trust for their assistance with various aspects of my project. I would like to thank the MRC for awarding a Fellowship to me, which provided the funding for my PhD. 4 Declaration of Originality The work in this thesis was designed and performed by the author, with contributions from collaborators (detailed in Declaration of Contributors). All other work is appropriately referenced and a comprehensive list of references is located at the end of this thesis. 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 The work in this thesis was designed and performed by the author and assistance from collaborators is detailed below. Chapter 2 - Intra-arcuate injection of rAAV was performed with the assistance of Dr Yue (David) Ma and Dr Risheka R Ratnasabapathy. - Decapitation of animals was performed with the assistance of Prof Kevin Murphy. 5 - Dissection of animals was carried out with the assistance of members of the Section of Investigative Medicine (Miss Isabel Fernandes Freitas, Dr Yue (David) Ma, Miss Mariana Norton, Dr Risheka Ratnasabapathy, Dr Rebecca Scott and Miss Gala Farooq). - Brain punch biopsies were performed with the assistance of Dr Yue (David) Ma. - Commands for generalised estimating equation analyses were developed with assistance from Dr Ali Abbara and Miss Anne Jomard. Chapter 3 - IVGTT and MMTT studies were performed with the assistance of the Kisspeptin Research Team. - Analysis of serum LH, FSH, testosterone, insulin, C-peptide and plasma glucose (using the Abbott Architect machines in the Imperial College Healthcare NHS Trust Clinical Chemistry Department) was performed with the assistance of Dr Sophie Clarke, Dr Sophie Jones, Miss Anne Jomard and Dr Lisa Yang. - RIA for the measurement of plasma kisspeptin was performed with the assistance of Dr Ali Abbara, Dr Sophie Clarke, Dr Lisa Yang and Dr Sophie Jones. - RIAs for measurement of plasma glucagon and GLP-1 were performed with the assistance of Dr Paul Bech and Dr Sophie Jones. - IVGTT insulin sensitivity index was calculated by Dr Ian Godsland (Wynn Reader in Human Metabolism, Imperial College London) using MLAB software. - Multilevel linear regression was performed by Paul Bassett (Statistician) on IVGTT and MMTT glucose, insulin and C-peptide curves. 6 Table of Contents Abstract .................................................................................................................................. 2 Acknowledgements ................................................................................................................ 4 Declaration of Originality ....................................................................................................... 5 Copyright Declaration ............................................................................................................ 5 Declaration of Contributors ................................................................................................... 5 Index of Figures ........................................................................................................................ 12 Chapter 1 .............................................................................................................................. 12 Chapter 2 .............................................................................................................................. 12 Chapter 3 .............................................................................................................................. 15 Index of Tables ......................................................................................................................... 18 Chapter 2 .............................................................................................................................. 18 Chapter 3 .............................................................................................................................. 18 Abbreviations ....................................................................................................................... 19 1 Chapter 1 General Introduction ....................................................................................... 21 1.1 Physiology and Pathology of Metabolism and Energy Homeostasis ........................ 22 1.2 Central Regulation of Metabolism and Energy Homeostasis ................................... 24 1.2.1 Key Hypothalamic Areas .................................................................................... 25 1.2.2 Hormones of Interest and the Hypothalamus ................................................... 35 7 1.3 Summary ................................................................................................................... 38 2 Chapter 2 – The effects of glucocorticoids on the regulation of metabolism and energy homeostasis ............................................................................................................................. 39 2.1 Introduction............................................................................................................... 39 2.1.1 11betaHSD enzyme system ............................................................................... 39 2.1.2 11betaHSD1 ....................................................................................................... 41 2.1.3 Summary ............................................................................................................ 50 2.2 Hypothesis and aims ................................................................................................. 51 2.2.1 Hypothesis.......................................................................................................... 51 2.2.2 Aims and objectives ........................................................................................... 51 2.3 Materials and Methods ............................................................................................. 52 2.3.1 Recombinant adeno-associated virus ................................................................ 52 2.3.2 In vivo Methods ................................................................................................. 52 2.3.3 Saponification of carcasses for body composition analysis............................... 57 2.3.4 Assays ................................................................................................................