Molecular and cellular mechanisms of energy homeostasis in birds Yang Xiao Dissertation submitted to the Graduate Faculty of the Virginia Polytechnic Institute and State University in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Animal and Poultry Sciences Elizabeth R. Gilbert, Chair Mark A. Cline Sally E. Johnson Dongmin Liu Paul B. Siegel March, 2020 Blacksburg, VA Key Words: chicks, anorexia, obesity, adipose tissue, DNA methylation Copyright 2020, Yang Xiao Molecular and cellular mechanisms of energy homeostasis in birds Yang Xiao Abstract (Academic) Hypothalamus and adipose tissue are essential central and peripheral sites regulating energy homeostasis. Disruption of energy homeostasis can lead to diseases like anorexia and obesity in humans and reduced productivity in animals. Therefore, integrating knowledge in hypothalamic appetite regulation and adipose tissue metabolism is essential to maintain homeostasis. The aim of this dissertation was to elucidate molecular and cellular mechanisms of energy homeostasis in birds. We determined adipose tissue physiological changes during the first two weeks post-hatch in chickens from lines selected for low (LWS) and high (HWS) body weight. LWS was more dependent on yolk and subcutaneous fat mobilization for growth from hatch to day 4 post-hatch, with hyperplasia-predominated replenishment of the reservoir. In contrast, HWS was more dependent on feed for growth and maintained depot mass through hyperplasia and hypertrophy. From day 4 to 14 post-hatch, compared to maintenance of depot weight and adipocyte size in LWS, HWS accumulated clavicular and abdominal fat with minimal lipolysis. There was greater expression of precursor and proliferation markers in LWS with more apoptotic cells in the abdominal stromal vascular fraction on day 14 post-hatch, suggesting that apoptosis contributed to lower adipogenic potential and lack of abdominal fat in LWS. Exposure to thermal and nutritional stressors at hatch impaired growth by reducing yolk utilization and lowering body weight, lean and fat masses in LWS. Stress exposure resulted in increased global DNA methylation and DNA methyltransferase activity in the arcuate nucleus of the hypothalamus in LWS. Moreover, there was decreased binding to methyl-CpG-binding domain protein 2 in the promoter of corticotropin-releasing factor (CRF) because of hypomethylation in one CpG site at its core binding site in stressed LWS, which explains the increased CRF expression in the paraventricular nucleus of the hypothalamus. We next determined effects of nutritional status on adipose tissue physiology in Japanese quail, a less-intensively selected avian species. Six-hour fasting promoted lipolysis and gene expression changes in 7-day old quail with some changes restored to original levels within 1 hour of refeeding. Overall, our results reveal novel cellular and molecular mechanisms regulating appetite and adiposity in birds early post-hatch. ii Abstract (Public) Hypothalamus and adipose tissue are essential for regulating energy homeostasis in central and peripheral body sites, respectively. Disruption of energy homeostasis can lead to diseases like anorexia and obesity in humans and reduced productivity in animals. Therefore, integrating knowledge in hypothalamic appetite regulation and adipose tissue metabolism is essential to maintain energy homeostasis in both humans and animals. The aim of this dissertation was to elucidate molecular and cellular mechanisms of energy homeostasis in birds. We first determined adipose tissue physiological changes in chickens during the first two weeks post-hatch from lines selected for low (LWS) and high (HWS) body weight. These chickens have been selected for juvenile body weight for over 60 generations. The LWS are lean and anorexic, while HWS eat compulsively and develop obesity and metabolic syndrome. Such characteristics make the body weight line chickens good animal models to study physiological changes under anorexia and obesity. We found that LWS was more dependent on yolk reserves and subcutaneous fat mobilization for growth from hatch to day 4 post-hatch, with replenishment of the fat reservoir by increases in cell number. By contrast, HWS was more dependent on feed for growth and maintained depot mass through increased cell number and cell size. From day 4 to 14 post-hatch, HWS accumulated fat throughout the body, with less fat breakdown as compared to LWS. There was greater expression of cellular precursor and proliferation markers in LWS, with more dying cells in their abdominal fat on day 14 post-hatch, suggesting that programmed cell death is responsible for the lack of fat cell development in LWS. Exposure to thermal and nutritional stressors at hatch impaired growth by reducing yolk utilization and lowering body weight, lean and fat masses in LWS. There were many molecular changes in the hypothalamus, including changes in DNA that led to increased activation of corticotropin-releasing factor (CRF), a iii signaling molecule that is known to regulate the body’s stress and appetite responses. Stress exposure increased global DNA methylation and DNA methyltransferase activity in the arcuate nucleus of the hypothalamus in LWS. Moreover, there was less methylation at the core binding site of methyl-CpG-binding domain protein 2 (MBD2), a protein that binds to methylated DNA to repress gene expression, in the CRF gene, in stressed LWS. In response to stress, there was decreased binding of MBD2 to the promoter region of CRF, which may explain increased expression of CRF in the paraventricular nucleus of LWS. These results demonstrate that early- life stressful events can cause epigenetic changes (like DNA methylation) that lead to alterations in physiology and behavior that persist to later in life. We next determined effects of nutritional status on adipose tissue physiology in Japanese quail, which have undergone less artificial selection than chickens and are more representative of a wilder-type bird. Six-hour fasting promoted lipolysis and gene expression changes in 7-day old quail with some changes restored to original levels within 1 hour of refeeding. Overall, our results provide novel perspectives on cellular and molecular mechanisms regulating appetite and adiposity in birds during early post- hatch development. iv Acknowledgements Dr. Gilbert: I am so lucky and thankful to have you as my supervisor. You are always so kind and provides me generous help. Your passion towards teaching, patience towards the students, strictness towards research and optimism towards life influenced me a lot and set a fantastic model for me. I always wish that one day I could be a super woman like you. I greatly enjoy and appreciate the times you share your stories with me and encourage and inspire me with your life experience. Without you, I would not have opportunities to do such interesting research, learn this many techniques, present research at conferences, or meet the amazing people here at Virginia Tech. Forrest Gump said “Life is like a box of chocolates. You never know what you’re gonna get.” You are definitely the most treasured piece of chocolate that made my academic life sweeter and enjoyable. Dr. Cline: You are one of the smartest persons I have ever met. You can always think out of the box and bring up creative and new ideas. I cannot convey in words how much I appreciate your help and guidance in the past four years. You are also a great teacher that whenever you are in front of the class, I see a fun person who is talkative and attracts students’ attention with his knowledge well mixed in the adventurous life stories. Dr. Siegel: I cannot express how grateful I am to have you as my committee member. You are definitely the most wisdom person in my mind who is also so fun and easy to go along with. Though tough at the start, your rigorousness towards research, especially when working at the farm allowed me to learn the logics behind even tiny things and always think which is the most effective way to achieve goals. I am so glad that after years of training we form a great team now. Your life experience is so colorful and enlightening. You can never imagine how much I iv enjoy our talks and how much your words inspired and encouraged me. I wish to be a wisdom and charming person like you in the future. Dr. Liu: You are a nice and thoughtful person. During the committee meetings, you always provide insightful suggestions to help me find approaches and tackle the problems. You generously shared the resources and opinions with me on job seeking, which I greatly appreciated. Thank you for being such a supportive committee member. Dr. Johnson: Every time I saw you, you always gave me a big smile and talked to me so gently like a breeze in the beautiful spring. I still remember how patient you were and the kind words in your email when I was preparing for the Ph.D. application. I also remember the warm encouragements and insightful suggestions you gave me during my first committee meeting which calmed me down from depression and stress because of the failures in the experiments. Thank you for being this kind to me. Dr. Wong: Thank you for organizing Poultry Research Group. It provides me a great opportunity to acquire knowledge about birds from different aspects and communicate with professionals and colleagues. Special thanks to you for reminding me that a person who is writing dissertation should not take a nap. Sasha Simpkins: Thank you so much for being so patient and helpful for the countless questions I asked. Thank you for being this responsible and efficient as a graduate coordinator. Patricia Williams: You are such a kindhearted, selfless and sweet person providing tremendous help to the students and faculties in the department. I would not be able to follow the schedule of the experiments without you these years. And thank you so much for your kind care and help of my life.