Investigating the Enteroendocrine – Brain Axis: Ghrelin Cell and Ecl Cell
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INVESTIGATING THE ENTEROENDOCRINE – BRAIN AXIS: GHRELIN CELL AND ECL CELL PHYSIOLOGY AND GHRELIN ACTION ON MOOD AND COMPLEX EATING APPROVED BY SUPERVISORY COMMITTEE Jeffrey M. Zigman, M.D.,Ph.D. Amelia J. Eisch, Ph.D. Craig M. Powell, M.D.,Ph.D. Philipp Scherer, Ph.D. DEDICATION I would first of all like to dedicate this work to my loving parents for their never-ending support and belief that I can accomplish anything. My mother has always provided me a shoulder to lean on when I am struggling, a hand to wipe away my tears when tragedy strikes, and a heart that generously gives more love than I could ever ask for. As I have grown and matured, she has become more than just a mother—she has become my best friend. I am also forever indebted to my father. He has always encouraged me to follow my dreams, never pressuring me with demands, but rather nurturing my strengths with positive reinforcement and tenderness that can only come from a devoted parent. My father served as the perfect model for a hard-working, responsible individual, selflessly working hard to provide our family with every opportunity possible. My parents have instilled in me the motivation, desire, and dedication to excel at every task at hand. I could never have become the person I am today without them. Secondly, I would like to thank David Barry for supporting me throughout my graduate school career. He has been a calming source during stressful times and is the first person to celebrate any of my accomplishments. Sharing his unwavering strength with me, David has made me more resilient when dealing with hardships. He has also shared with me such a compelling passion for science that is has opened my eyes to the beauty inside a whole range of scientific topics. I could not write this dedication without including a deep thanks to my dog, Hugo. Even after enduring lonely nights when I have to spend long hours in the lab, Hugo is always ready to greet me with a wagging tail upon my arrival home. He has been a great source of happiness and joy in my life, and I cannot picture these past years without him. I would also like to recognize Dr. Jeffrey Zigman and Dr. Amelia Eisch. As my mentor, Dr. Zigman has pushed me to exceed all expectations and become a productive, independent scientist. He has allowed me to expand my knowledge on a diverse range of subjects and techniques, thoroughly developing my scientific skills and teaching me how to overcome obstacles. Dr. Eisch has been very helpful as well, serving as my committee chair and advising me throughout different stages of graduate school. I truly appreciate the support I have received from her as well as from the members of her lab. INVESTIGATING THE ENTEROENDOCRINE – BRAIN AXIS: GHRELIN CELL AND ECL CELL PHYSIOLOGY AND GHRELIN ACTION ON MOOD AND COMPLEX EATING by ANGELA KAY WALKER DISSERTATION/THESIS Presented to the Faculty of the Graduate School of Biomedical Sciences The University of Texas Southwestern Medical Center at Dallas In Partial Fulfillment of the Requirements For the Degree of DOCTOR OF PHILOSOPHY The University of Texas Southwestern Medical Center Dallas, Texas April, 2014 INVESTIGATING THE ENTEROENDOCRINE – BRAIN AXIS: GHRELIN CELL AND ECL CELL PHYSIOLOGY AND GHRELIN ACTION ON MOOD AND COMPLEX EATING ANGELA KAY WALKER, Ph.D. The University of Texas Southwestern Medical Center at Dallas, 2014 JEFFREY M. ZIGMAN, M.D.,Ph.D. Abstract The mechanisms and neurochemical pathways through which the orexigenic peptide hormone ghrelin act to regulate homeostatic feeding is fairly well documented. However, less understood are the mechanisms and brain regions that mediate ghrelin’s effects on mood and complex eating behaviors. At the cellular level, little is known about the ghrelin cell’s transcriptional profile, its secretory products other than ghrelin, and its relationship to other gastric endocrine cells, such as the histamine producing enterochromaffin-like cell. My doctoral research encompasses multiple aspects of the ghrelin system, from physiological assessments of the ghrelin cell to evaluations of ghrelin action on cue-potentiated feeding and stress-induced iv depressive-like behavior. Ghrelin has antidepressant effects, which become obvious following chronic stress. In the first part of my thesis, I found that this effect was mediated by neurogenesis. I observed that chronic stress reduces neurogenesis more severely in the ventral dentate gyrus of Ghsr-null mice, suggesting ghrelin provides a level of neuroprotection in the stress environment. Administration of anti-apoptotic P7C3-related compounds not only blocked stress-induced reductions in neurogenesis, but also minimized the severity of depressive-like behavior in mice. Focal hippocampal irradiation prevented the anti-depressant efficacy of P7C3-related compounds, indicating that P7C3 regulates mood directly through neurogenesis. In the second part of my thesis, I designed a novel protocol for studying cue-potentiated feeding behaviors in mice. Absence of ghrelin signaling in Ghsr-null mice, or administration of a ghrelin receptor antagonist in wild-type mice, disrupted the development of normal cue-food associations. Additionally, I discovered Ghsr expression in the basolateral amygdala (BLA), and BLA neuronal activation in response to a food-associated positive cue significantly correlated with amount of food intake. Thus, ghrelin signaling in the BLA may be responsible for its mediation of cue-potentiated feeding behaviors. The third part of my thesis examined the ghrelin cell transcriptome for potential secretory proteins and revealed significant expression of Rbp4, Ttr, and Nucb2, along with RBP4 protein secretion. Lastly, I characterized a novel HDC- Cre mouse model that may be advantageous in future studies to determine potential interactions between histaminergic and ghrelin signaling pathways. The full range of these discoveries advances our comprehensive understanding of ghrelin. v TABLE OF CONTENTS DEDICATION ............................................................................................. ii ABSTRACT ............................................................................................... iv TABLE OF CONTENTS .......................................................................... vi PRIOR PUBLICATIONS ......................................................................... xiii LIST OF FIGURES ................................................................................... xv LIST OF TABLES .................................................................................. xvii LIST OF DEFINITIONS ..........................................................................xviii CHAPTER ONE...........................................................................................1 Introduction The discovery of ghrelin and its relationship to other enteroendocrine cell types……………………………………………………………………………………………...1 Ghrelin’s role in homeostatic feeding and glucose metabolism...………………………….6 Ghrelin’s role in more complex, hedonic feeding behaviors……………………………....12 The obesity epidemic: a struggle between homeostatic feeding and hedonic feeding……………………………………………………………...……….12 Pharmacologic manipulations in ghrelin and food reward………………………..13 Endogenous elevations in ghrelin and food reward……………………………....15 Ghrelin in human weight regulation and feeding behavior………………………………...16 Ghrelin stimulates human appetite………………………………………………….16 Involvement of ghrelin signaling in human body weight regulation……………..16 Ghrelin regulates human food reward behavior…………………………………...19 Ghrelin as a mediator of dopaminergic signaling in the reward pathway………………..20 vi Ghrelin can regulate feeding behavior through the VTA………………………...20 Ghrelin enhances dopamine release……………………………………………...21 Interaction of ghrelin receptors and dopamine receptors……………………….22 The influence of ghrelin on mood………………………………………………………….23 Neurogenesis and depression……………………………………………………………..26 References…………………………………………………………………………………..30 CHAPTER TWO.........................................................................................50 The P7C3-class of neuroprotective compounds exerts antidepressant efficacy in mice by increasing hippocampal neurogenesis. Abstract….….……………………………………………………………………………....50 Introduction………………………………………………………………………………….51 Materials and methods………………………………………………………………….....53 Animals……………………………………………………………………...…….54 Behavioral testing…………………………………………………………...…...54 P7C3 compounds………………………………………………………………...54 Chronic social defeat stress (CSDS)……………………………………....…..54 Hippocampus-directed cranial irradiation………………………………...…...54 Caloric restriction…………………………………………………………...……54 Immunohistochemistry and stereology……………………………….............55 Quantitative RT-PCR…………………………………………………………….55 P7C3 brain penetration study…………………………………………………..55 Comparison of P7C3 compounds and antidepressant drugs……………….55 Statistical analysis………………………………………………………...….....55 Results………………………………………………………………………………………56 vii Chronic stress severely reduces DG cell proliferation and survival in Ghsr- null mice………………………………………………………………...56 P7C3 compounds augment neurogenesis in chronic stress-exposed mice……………………………………………………………………………..…58 P7C3 compounds reduce depression in chronic stress-exposed mice…....59 P7C3 blocks apoptosis and restores the anti-depressant response to caloric restrion in Ghsr-null mice……………...……………………………..…61 P7C3 has greater proneurogenic efficacy than current antidepressants......62 P7C3 has no effect on anxiety-like behaviors…...…………………………....63