Molecular mechanisms involved in the regulation of agouti-related peptide and neuropeptide Y by endocrine disrupting chemical bisphenol A in hypothalamic neurons by Neruja Loganathan A thesis submitted in conformity with the requirements for the degree of Doctor of Philosophy Department of Physiology University of Toronto © Copyright by Neruja Loganathan 2021 Molecular mechanisms involved in the regulation of agouti-related peptide and neuropeptide Y by endocrine disrupting chemical bisphenol A in hypothalamic neurons Neruja Loganathan Doctor of Philosophy Department of Physiology University of Toronto 2021 Abstract Bisphenol A (BPA), a ubiquitous endocrine disrupting chemical found in plastics and receipts, is a disruptor of reproductive function and is a known ‘obesogen’ as it is linked to increased body mass index in humans and leads to weight gain in animal models. The hypothalamus houses orexigenic NPY/AgRP neurons, which integrate peripheral hormones and nutritional signals, to increase food intake and decrease energy expenditure. NPY neurons are also afferent regulators of the hypothalamic-pituitary gonadal axis, and thus reproductive function. This thesis investigated whether the NPY/AgRP neurons, and particularly Npy and Agrp expression, are altered by BPA. We hypothesized that BPA increases Npy and Agrp gene expression in hypothalamic neurons and that this effect is mediated through nuclear receptor activation, induction of cellular stress and subsequent transcription factor activation or circadian dysregulation. We demonstrated that BPA increased Agrp mRNA expression in mHypoA-59 and mHypoE-41 cells. Inhibition of AMPK and knock-down of transcription factor ATF3 prevented the BPA-mediated increase in Agrp expression in the mHypoA-59 cells. ATF3 was also required for BPA-mediated increase in Npy in the mHypoE-41 cells. We also described subpopulation- specific changes in Npy expression in response to BPA. While BPA induced Npy expression in ii mHypoA-59, -2/12 and mHypoE-41, -42 neurons, Npy expression was downregulated in mHypoE-46 and -44 neurons. Inhibition of AMPK with compound C or oxidative stress with antioxidants and vitamin B6 prevented the BPA-mediated induction in Npy in the mHypoA-59 cells, whereas antagonism of ERß or GPER prevented the decrease in Npy mRNA in the mHypoE-46 cells. Finally, we showed that circadian gene dysregulation occurred with BPA exposure and using hypothalamic cell lines lacking BMAL1, demonstrated that the BPA- mediated induction of Npy, but not Agrp, required BMAL1. Accordingly, treatment with BPA increased BMAL1 binding to the Npy promoter. These findings illustrate distinct mechanisms responsible for the BPA-mediated changes in appetite-increasing Npy and Agrp gene expression, suggesting that NPY/AgRP neurons are susceptible to the endocrine disrupting effects of BPA. Furthermore, we describe potential targetable pathways to combat the obesogenic or reproductive dysfunction-inducing effects of BPA at the hypothalamic level. iii Acknowledgements First and most importantly, I would like to thank my supervisor, Dr. Denise Belsham. You have helped me develop my skills as a scientist, taught me the importance of always asking questions and instilled in me a love for neuroendocrinology. You have been compassionate and motivating and I cannot imagine having done my PhD anywhere else. Your passion for encouraging women in science is truly remarkable, and I will forever be grateful for the opportunities you have given me and the continuous support you have provided me. I would also like to thank my supervisory committee members - Dr. Mark Palmert, Dr. Carolyn Cummins and Dr. Amira Klip. The challenging questions you asked and the careful input you provided was essential for the development and completion of my project. Thank you for helping me become a better scientist. I also wish to thank Dr. Debby Kurrasch, Dr. Patricia Brubaker and Dr. Andy Babwah for their comments and edits on my thesis. Thank you to the Department of Physiology - from the administrative staff that have been nothing but kind and helpful to the neighbouring labs that have always lent support when I needed to borrow equipment. The last five years would not have made such an impact on my life if it were not for all the members of the Belsham Lab. You have helped me with my experiments, you have provided ample suggestions for my project and you have made me laugh a million times in between. I am very thankful for having gotten to know each one of you. To Jenn - thank you for teaching me every technique I know and helping me troubleshoot my experiments. You always made time to help someone when they needed it, which is so admirable. To Erika - thank you for navigating the beginning stages of graduate school with me. Your enthusiasm for figuring things out and learning something new made working with you so fun. To Andy and Calvin - thank you for making everyday in the lab so joyful. To Emma - from the countless experiments, to looking up the answer to a question or editing my writing, you have been a tremendous support throughout my entire degree. Thank you for always helping me, but most importantly, thank you for your friendship. Finally, I would like to thank my friends and family for their constant encouragement and emotional support. Thank you for believing in me. To my mother, I cannot imagine where I would be without you. Thank you for absolutely everything. iv Table of Contents Acknowledgements ........................................................................................................................ iv Table of Contents .............................................................................................................................v List of Tables and Figures.............................................................................................................. xi List of Abbreviations ................................................................................................................... xiv List of Appendices ..................................................................................................................... xviii Chapter 1 Introduction....................................................................................................................1 1 Introduction .................................................................................................................................2 1.1 Obesity and reproductive dysfunction .................................................................................2 1.2 Endocrine disrupting chemicals ...........................................................................................3 1.2.1 General characteristics .............................................................................................3 1.2.2 Bisphenol A: Sources and Concentrations ...............................................................5 1.2.3 BPA disrupts energy homeostasis ............................................................................7 1.2.3.1 Humans ......................................................................................................7 1.2.3.2 Animal and in vitro models .......................................................................8 1.2.4 BPA induces reproductive dysfunction .................................................................10 1.3 Hypothalamic control of energy homeostasis and reproduction........................................10 1.3.1 Energy homeostasis is controlled by NPY/AgRP and POMC neurons .................10 1.3.1.1 NPY and AgRP are potent orexigens ......................................................11 1.3.1.2 NPY and AgRP regulate metabolism independent of food intake ..........12 1.3.2 Reproduction is controlled by GnRH neurons .......................................................13 1.3.2.1 Afferent regulators of GnRH neurons .....................................................14 1.3.3 Regulation of Npy and Agrp transcription and secretion .......................................15 1.3.3.1 Regulation of Npy ....................................................................................16 1.3.3.2 Regulation of Agrp ..................................................................................18 1.4 Hypothalamic control of circadian rhythms ......................................................................21 v 1.4.1 Circadian involvement in Npy regulation ..............................................................23 1.5 Effects of BPA in the hypothalamus ..................................................................................23 1.6 Signaling pathways activated by BPA ...............................................................................25 1.6.1 Hormone and nuclear receptors .............................................................................26 1.6.2 Non-nuclear receptor pathways including induction of cellular stress ..................27 1.7 Hypothalamic cell models to study BPA-induced dysregulation ......................................29 1.8 Hypothesis and Aims .........................................................................................................32 Chapter 2 Materials and Methods ................................................................................................34 2 Materials and Methods ..............................................................................................................35 2.1 Cell culture and reagents ....................................................................................................35