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The Hormonal Control of Neuropeptide Y and Gonadotropin-Releasing Hormone Hypothalamic Neurons

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The Hormonal Control of Neuropeptide Y and Gonadotropin-Releasing Hormone Hypothalamic Neurons THE HORMONAL CONTROL OF NEUROPEPTIDE Y AND GONADOTROPIN-RELEASING HORMONE HYPOTHALAMIC NEURONS by Sandeep S. Dhillon A thesis submitted in conformity with the requirements for the degree of Doctor of Philosophy Department of Physiology University of Toronto © Copyright by Sandeep S. Dhillon 2010 The Hormonal Control of Neuropeptide Y and Gonadotropin- Releasing Hormone Hypothalamic Neurons Sandeep S. Dhillon Doctor of Philosophy Department of Physiology University of Toronto 2010 Abstract The physiological mechanisms that control energy homeostasis are reciprocally linked to reproduction. However, the neuroendocrine circuitry that registers endocrine cues to direct homeostatic responses in energy balance and reproduction remain unknown. Neuropeptide Y (NPY) neurons have emerged as a key central target of estrogen and leptin that are capable of modulating both reproduction and energy balance. The hypothesis was generated that NPY neuronal subpopulations act as an integration centre to regulate the effects of estrogen and leptin on these important physiological processes through specific signaling pathways. Using hypothalamic cell lines that express the leptin receptor (Ob-R), estrogen receptor (ER) and NPY, this hypothesis was tested in three aims. 17β-estradiol (E2) was previously demonstrated to biphasically regulate NPY mRNA in the mHypoE-38 neuronal cell line; where 24 h E2 exposure induced NPY gene expression that our group proposed may be involved in the gonadotropin-releasing hormone (GnRH) preovulatory surge. E2 also acts as an anorexigenic hormone through unknown hypothalamic targets. E2 directly decreased NPY secretion in the mHypoE-42 and mHypoA-2/12 neuronal cell lines through ER-α. The anorexigenic action of E2 was ii mediated through the energy sensing 5’ AMP-activated protein kinase (AMPK) and the phosphoinositide-3-kinase (PI3K) pathway. NPY secretion was also decreased by leptin in mHypoA-59 and NPY-GFP cell models through AMPK- and PI3K-dependent mechanisms. Prolonged exposure to leptin in NPY-GFP cell lines prevented AMPK signaling and the leptin-mediated reduction in NPY secretion, indicating NPY neuronal resistance with prolonged leptin exposure. Leptin also stimulated NPY secretion in mHypoE-38 neurons, which was blocked by pharmacological inhibitors of the mitogen- activated protein kinase (MAPK) and PI3K pathways. Importantly, conditioned medium from the mHypoE-38 NPY neuronal cells induced GnRH transcripts in GT1-7 neurons, which was inhibited by Y1-receptor antagonists. Pharmacological inhibitors of the MAPK and PKA signal transduction pathways attenuated the NPY-mediated increase in GnRH transcription. Based upon these findings, I propose NPY neurons in the hypothalamus consist of a heterogeneous population of neurons, and provide the first evidence of intrinsically different responses to function as physiological integrators for two different systems: NPY secretion can be suppressed to decrease food intake and induced to stimulate GnRH neurons. iii Acknowledgments I owe my deepest gratitude to Dr. Denise Belsham, who has been a significant presence in my life. I am indebted to Dr. Belsham whose patience, kindness and academic experience have allowed me to become the person I am today. Thank you Denise. I would also like to thank my committee members, Dr. Isabella Canniggia and Dr. Theodore Brown. I have benefited greatly from their guidance, mentorship and encouragement, which have been instrumental to the completion of this degree. I would like to thank the members of the Belsham lab for their valuable discussion, encouragement and making the lab such an enjoyable experience. I also want to specially thank Ginah Kim. You have become my best friend both inside and outside the laboratory. Thank you for always being there. Finally, I would like to thank my family for their constant support, patience and love. I would not have made it here with out them. This accomplishment is as much yours as it is mine. iv Table of Contents Acknowledgments ........................................................................................................................ iv Table of Contents ...........................................................................................................................v List of Tables ..................................................................................................................................x List of Figures............................................................................................................................... xi List of Abbreviations ................................................................................................................. xiv 1 Chapter 1 Relevant literature reviews ....................................................................................1 1.1 Introduction........................................................................................................................2 1.2 Reproductive function .......................................................................................................4 1.2.1 The hypothalamic-pituitary-gonadal axis ................................................................4 1.2.2 Gonadotropin-releasing hormone (GnRH) in the hypothalamus.............................6 1.2.3 Regulation of GnRH neurons...................................................................................7 1.3 Energy homeostasis and reproductive function..............................................................9 1.3.1 Hypothalamic nuclei associated with regulation of food intake..............................9 1.3.2 Energy homeostasis and reproductive function .....................................................10 1.4 Neuropeptide Y ................................................................................................................13 1.4.1 Synthesis ................................................................................................................13 1.4.2 NPY receptors........................................................................................................14 1.4.3 Signaling pathways activated by NPY...................................................................14 1.4.4 NPY effects on energy homeostasis and reproduction ..........................................15 1.5 Estrogen ............................................................................................................................18 1.5.1 Synthesis and metabolism......................................................................................18 1.5.2 Estrogen receptors..................................................................................................19 1.5.3 Signaling pathways activated by estrogen .............................................................22 1.5.4 Effects of estrogen on reproduction and feeding behaviour ..................................27 v 1.5.5 Estrogen-mediated regulation of NPY neurons .....................................................29 1.6 Leptin ................................................................................................................................30 1.6.1 Synthesis and metabolism......................................................................................30 1.6.2 Leptin receptors and signaling events....................................................................31 1.6.3 Effects of leptin on feeding behaviour and reproduction.......................................34 1.6.4 Leptin-mediated regulation of NPY neurons.........................................................35 1.6.5 Leptin Resistance...................................................................................................37 1.7 Cell models........................................................................................................................38 1.7.1 GnRH-expressing GT1-7 neurons .........................................................................39 1.7.2 Embryonic hypothalamic cell lines – mHypoE-xx................................................41 1.7.3 Adult hypothalamic cell lines – mHypoA-xx ........................................................42 1.7.4 NPY-GFP cell line .................................................................................................43 1.8 Hypothesis and aims ........................................................................................................44 2 Chapter 2 .................................................................................................................................47 Materials and methods ................................................................................................................47 2.1 Cell culture and reagents.................................................................................................48 2.2 Semi-quantitative RT-PCR.............................................................................................49 2.2.1 One step RT-PCR ..................................................................................................49 2.2.2 Two step RT-PCR..................................................................................................49 2.3 Real-Time RT-PCR .........................................................................................................51 2.4 Enzyme Immunoassay.....................................................................................................51 2.5 Fluorescence-activated cell sorting (FACS)...................................................................52
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