Physiological roles of the peptide Prokineticin 1 Thesis for the degree of Doctor of Philosophy Dr Ali Abbara MBBS BSc MRCP 2014 Department of Investigative Medicine Imperial College Abstract Prokineticin-1 (PK-1) and prokineticin-2 (PK-2) are two closely related cysteine-rich peptides which both bind to the prokineticin-1 and prokineticin-2 receptors (PKR-1 and PKR-2). The prokineticins were originally named due to their ability to stimulate gastrointestinal motility. Since that time, these peptides have been found to play a role in other biological processes including reproduction and nociception. Prokineticin-2 is known to be expressed in regions of the central nervous system involved in food intake. Consistent with this, work from our own department has shown that administration of prokineticin-2 to rodents reduces food intake both in lean and obese rodents. Prokineticin-2 is predominantly expressed within the central nervous system, whereas prokineticin-1 is heavily expressed in the gut. My pilot data showed that prokineticin 1 reduces food intake in rodents. I therefore hypothesised that prokineticin 1 may be a novel gut hormone. In this thesis, I investigate the role of prokineticin-1 on appetite in rodents and also compare the effects of prokineticin-1 with prokineticin-2. I also measured the changes in plasma levels of prokineticin in humans during feeding to establish the potential physiological relevance of prokineticin-1 as an anorectic gut hormone. Prokineticin-1 is also a potent angiogenic mitogen on endocrine vascular epithelium and hence it is also known as ‘Endocrine Gland-Vascular Endothelial Factor’. Placental angiogenesis plays an important role in placental function and risk of pregnancy complications such as miscarriage. I therefore hypothesised that prokineticin-1 is a biomarker of pregnancy complications in humans. In this thesis, I have measured circulating levels of prokineticin-1 found in pregnancy and correlated these with the occurrence of pregnancy complications. I have also compared the utility of prokineticin 1 with other potential novel markers of pregnancy complications. Together this body of work investigates the potential physiological roles of prokineticin-1. 2 Acknowledgements I would like to gratefully acknowledge Professor Waljit Dhillo and Dr Channa Jayasena for supervising me and supporting me throughout my PhD. I would also like to thank all members of our group for all their support and assistance with this work. I am also grateful to the Wellcome Trust for funding my Clinical Training Fellowship. 3 Declaration of contributors The majority of the work carried out in this thesis was performed by the author. All collaborations and assistance are described below: Chapter 2: Assistance with animal studies was provided by Dr Gardiner, Professor Dhillo and members of the hypothalamic group. Chapter 3: I would like to thank my colleagues Dr Izzi-Engbeaya, Ms Ganiyu-Dada for their help in collecting the pregnancy samples from the maternity unit. I would also like to thank my colleagues Dr Jayasena, Dr Comninos, Dr Sarang, Ms Malik, Dr Narayanaswamy, Ms Ng, Dr Buckley, for their help in carrying out the measurement of PK-1 and other pregnancy markers. In-house radioimmunoassay for the measurement of plasma kisspeptin used in this programme of research were established and maintained by Professor Mohammad Ghatei. 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 signature Ali Abbara . 4 Table of Contents: Abstract ............................................................................................................................ 2 Acknowledgements ......................................................................................................... 3 Declaration of contributors .............................................................................................. 4 Abbreviations ................................................................................................................ 14 Chapter 1 ...................................................................................................................... 17 The obesity epidemic ..................................................................................................... 18 The Hypothalamus ......................................................................................................... 20 The Arcuate Nucleus (ARC) ......................................................................................... 23 The Paraventricular nucleus (PVN) ............................................................................... 25 The Lateral Hypothalamus (LH) ................................................................................... 26 The Ventromedial Nucleus (VMN) ............................................................................... 27 The Dorsomedial Nucleus (DMN) ................................................................................ 29 Circulating Factors which act within the hypothalamus to regulate food intake and body weight ............................................................................................................................. 30 Prokineticins- a potential novel regulator of appetite .................................................... 33 Prokineticins (PKs): novel hypothalamic regulators of appetite and placental angiogenesis .................................................................................................................... 41 Chapter 2 ...................................................................................................................... 43 Introduction ................................................................................................................... 44 Hypothesis ..................................................................................................................... 47 Aims .............................................................................................................................. 47 Material and methods .................................................................................................... 48 5 1. Effect of ICV administration of 15, 50, 150 pmol of PK-1 during Dark Phase in male Wistar rats ....................................................................................................................... 48 Materials ........................................................................................................................ 48 Rodent models ............................................................................................................... 48 2. Effect of ICV administration of 15, 150 and 1500 pmol PK-1 ................................. 50 3. Comparison of peripheral administration of IP PK-1 and PK-2 at doses of 20, 60 and 540 nmol/kg between 0-24hrs in male C57BL/6 mice. .................................................. 51 Materials ........................................................................................................................ 51 4. Effect of IP PK-1 7 nmol/kg for up to 1 hour in mice (n=6 per group). ................... 52 5. Determination of the effect of fasting on expression of PK-1 in the stomach of Fed and Fasted mice ..................................................................................................................... 53 Total RNA extraction .................................................................................................... 54 Reverse Transcription (RT) ........................................................................................... 56 6. Human plasma PK1 levels fed fasted ........................................................................ 58 Methods: Plasma PK-1 measurement by ELISA kit (ABNOVA) ................................ 61 Reconstitution of the human PK-1 standard: ................................................................. 61 Preparation of 0.01 M PBS wash buffer: ....................................................................... 62 1. Results: ...................................................................................................................... 63 2. Results: ...................................................................................................................... 65 3. Results: ...................................................................................................................... 67 4. Results: ...................................................................................................................... 71 5. Results: ...................................................................................................................... 74 6. Results: ...................................................................................................................... 76 6 Discussion: .................................................................................................................... 80 Chapter 3 ...................................................................................................................... 83 Introduction: .................................................................................................................