ENHANCEMENT OF BRAIN MELANOCORTIN SIGNALING IN LEAN, ACTIVE RATS A dissertation submitted to Kent State University in partial fulfillment of the requirements for the degree of Doctor of Philosophy By Charu Shukla April 2014 Dissertation written by Charu Shukla M.S., University of Alabama at Birmingham, USA, 2009 M.Sc., Panjab University, India, 2007 B.Sc., CCS University, India 2005 Approved by Dr. Colleen M. Novak , Chair, Doctoral Dissertation Committee Dr. Gail C. Fraizer, Member, Doctoral Dissertation Committee Dr. Edgar E. Kooijman, Member, Doctoral Dissertation Committee Dr. Aaron M. Jasnow, Member, Doctoral Dissertation Committee Dr. Soumitra Basu, Member, Doctoral Dissertation Committee Accepted by Dr. Laura G. Leff, Acting Chair, Department of Biological Sciences Dr. Jan Crowther, Associate Dean, College of Arts and Sciences ii TABLE OF CONTENTS Page Appendix 1: List of Figures ............................................................................................. vi Appendix 2: List of Tables ............................................................................................... xi Appendix 3: List of Abbreviations used ....................................................................... xii Acknowledgements………………………………………………………………......... xv CHAPTER 1 : Introduction ................................................................................................. 1 1.1 Obesity ................................................................................................................... 1 1.2 Physical Activity and its Relevance in the Obesity Epidemic ............................... 2 1.3 Energy Expenditure and non-exercise activity thermogenesis (NEAT) ................ 4 1.4 Proopiomelanocortin (POMC) processing in the hypothalamus: Regulation of energy balance ................................................................................................................. 6 1.5 Parallel regulation of Pomc transcription and peptide processing ....................... 10 1.6 Melanocortin Receptors (MCR) ........................................................................... 15 1.6.1 MC1R: ........................................................................................................... 18 1.6.2 MC2R: ........................................................................................................... 18 1.6.3 MC3R and MC4R ......................................................................................... 19 1.6.4 MC5R ............................................................................................................ 20 1.7 MCR mutation studies .......................................................................................... 21 1.7.1 Mc3r null mice .............................................................................................. 22 iii 1.7.2 Mc4r null mice and rats ................................................................................. 22 1.7.3 Mc5r null mice .............................................................................................. 24 1.8 MCR mutations and human obesity ..................................................................... 25 1.9 Identification of target proteins within MC system ............................................. 28 1.10 Hypothalamic brain regions and energy balance ................................................. 29 1.10.1 Arcuate nucleus ........................................................................................... 30 1.10.2 Paraventricular Nucleus of hypothalamus (PVN or PVH) ......................... 30 1.10.3 Lateral hypothalamus (LH) and perifornical nucleus of LH or orexin cell body containing area (PeFLH) .................................................................................. 34 1.10.4 Dorsomedial Nucleus of the hypothalamus (DMN) ................................... 35 1.10.5 Ventromedial nucleus of the hypothalamus (VMN) ................................... 35 1.11 Modeling physical activity ................................................................................... 36 1.12 Rat model of obesity and leanness ....................................................................... 37 1.13 Specific Aims ....................................................................................................... 41 CHAPTER 2 : Expression of Brain Melanocortin Receptors (MCRs) in HCR- LCR rats ............................................................................................................................. 43 2.1 STUDY 1: Relative quantitative mRNA expression in specific brain regions. ... 44 2.1.1 Methods: ........................................................................................................ 44 2.1.2 Results ........................................................................................................... 47 2.2 Study 2: Protein expression of MCRs in paraventricular nucleus (PVN) and the perifornical region of the hypothalamus (PeFLH) ........................................................ 53 2.2.1 Methods ......................................................................................................... 53 iv 2.2.2 Results ........................................................................................................... 55 2.3 Discussion: ........................................................................................................... 59 CHAPTER 3 : Laser Capture Microdissection (LCM): Precise Measurement of Brain MCR Expression in HCR LCR rats. .................................................................................. 62 3.1 Methods: ............................................................................................................... 65 3.2 Results: ................................................................................................................. 73 3.3 Discussion: ........................................................................................................... 80 CHAPTER 4 : Effects of MCR agonists/antagonists treatment on physical activity and Energy expenditure in HCR-LCR rats ............................................................................... 83 4.1 Methods ................................................................................................................ 83 4.2 STUDY 1: Non-specific MCR agonist melanotan II (MTII), and PVN .............. 92 4.3 Study 2: MCR subtype-specific agonist and antagonist treatments in PeFLH and PVN .............................................................................................................................. 98 4.4 Discussion .......................................................................................................... 127 CHAPTER 5 : Melanocortin Receptors (MCRs) and Food Intake ................................. 130 5.1 Experimental Design: ......................................................................................... 132 5.2 Results ................................................................................................................ 133 5.3 Discussion .......................................................................................................... 141 CHAPTER 6 : General discussion ................................................................................... 145 6.1 Future perspectives ............................................................................................. 153 v Appendix 1: List of Figures Figure 1: Schematic diagram indicating the endoproteolytic cleavage of the Proopiomelanocortin (POMC) molecule including the resulting peptide products .... 7 Figure 2: Parallel regulation of proopiomelanocortin (POMC) transcription and peptide processing .................................................................................................................. 12 Figure 3: Summary of some of the inter-related signals involved in energy balance and the control of energy homeostasis by neurons of the arcuate nucleus. ...................... 14 Figure 4: Illustrations from a rat brain highlighting the key areas involved in energy balance ....................................................................................................................... 33 Figure 5: Micropunches: Quantitative PCR- Arcuate nucleus .......................................... 49 Figure 6: Micropunches: Quantitative PCR in target brain regions .................................. 50 Figure 7: Quantitative PCR in the gastrocnemius muscle tissue: Melanocortin 5 Receptor (MC5R). ..................................................................................................................... 52 Figure 8: Protein expression of melanocortin receptors (MCRs) in the perifornical region of the hypothalamus (PeFLH) ................................................................................... 56 Figure 9: Protein expression of melanocortin receptors (MCRs) in the paraventricular nucleus (PVN) ........................................................................................................... 57 Figure 10: Western blot images showing the expression of melanocortin receptors 3, 4, and 5 (MC3R, MC4R, MC5R) along with actin as the loading control in PVN and the PeFLH. ................................................................................................................. 58 vi Figure 11: Schematic representation of infrared capture of tissue of interest from laser capture microdissection (LCM). ...............................................................................