EARLY DEVELOPMENT OF BODY-WEIGHT REGULATION SYSTEMS IN THE NONHUMAN PRIMATE: IN UTERO EFFECTS OF HIGH-FAT DIET by Bernadette Elizabeth Grayson A DISSERTATION Presented to the Neuroscience Graduate Program and the Oregon Health & Science University School of Medicine in partial fulfillment of the requirements of the degree of Doctor of Philosophy April 2009 School of Medicine Oregon Health & Science University ___________________________________ CERTIFICATE OF APPROVAL ___________________________________ This is to certify that the Ph.D. dissertation of Bernadette Elizabeth Grayson has been approved ______________________________________ Mentor/Advisor - Kevin Grove, PhD ______________________________________ Chairman - Daniel Marks, PhD ______________________________________ Member - Cynthia Bethea, PhD ______________________________________ Member - M. Susan Smith, PhD TABLE OF CONTENTS TABLE OF CONTENTS ........................................................................................ i LIST OF FIGURES ................................................................................................ iii LIST OF TABLES .................................................................................................. v LIST OF ABBREVIATIONS ................................................................................. vi ACKNOWLEDGEMENTS .................................................................................... ix PREFACE…… ....................................................................................................... xii ABSTRACT..... ....................................................................................................... xiii CHAPTER 1….. ..................................................................................................... 1 INTRODUCTION Background ................................................................................................. 2 Overview of Body-Weight Regulation ....................................................... 7 Development of Homeostatic Circuits ........................................................ 22 Neurocircuitry Put to the Test ..................................................................... 28 Conclusion .................................................................................................. 34 Aims of Dissertation and Approach ............................................................ 37 CHAPTER 2 .......................................................................................................... 40 Prenatal Development of Hypothalamic Neuropeptide Systems in the Nonhuman Primate Abstract ....................................................................................................... 41 Introduction ................................................................................................. 42 Materials and Methods ................................................................................ 43 Results ......................................................................................................... 49 Discussion ................................................................................................... 54 CHAPTER 3 ........................................................................................................... 67 NHP Model of Maternal Obesity CHAPTER 4 ........................................................................................................... 83 Changes in Melanocortin Expression and Inflammatory Pathways in Fetal Offspring in Nonhuman Primates Fed a High-Fat Diet Abstract ....................................................................................................... 84 Introduction ................................................................................................. 85 Materials and Methods ................................................................................ 87 Results ......................................................................................................... 93 Discussion ................................................................................................... 96 CHAPTER 5 ........................................................................................................... 111 Central Changes in the Serotonergic System in Response to Maternal High- Fat Diet in the Fetal Nonhuman Primate Abstract ....................................................................................................... 112 Introduction ................................................................................................. 113 Materials and Methods ................................................................................ 116 Results ......................................................................................................... 121 Discussion ................................................................................................... 123 CHAPTER 6 137 Discussion and Significance i APPENDIX A ......................................................................................................... 195 Prenatal Development of Hypothalamic MCH and Orexin in the NHP APPENDIX B ......................................................................................................... 201 Heteronuclear RNA and Pro-opiomelanocortin Gene APPENDIX C ......................................................................................................... 206 Alterations in Neurotransmitter Expression in Fetal NHP Exposed to Maternal High-Fat Diet APPENDIX D ......................................................................................................... 212 Double-label of AgRP and CART in the Adult NHP APPENDIX E ......................................................................................................... 216 Reciprocal Innervation of CRH and 5-HT in the Fetal NHP ii LIST OF FIGURES Figure 1-1 Body-weight regulation neurocircuitry in the rodent ............................ 36 Figure 1-2 Summary of overall work encompassing the development of body- 39 weight regulation systems in the fetal NHP .......................................... Figure 2-1 Leptin levels during development. ....................................................... 60 Figure 2-2 Postnatal development of NPY ............................................................. 61 Figure 2-3 Development of NPY/AgRP neurons in the ARH ................................ 62 Figure 2-4 Development of NPY/AgRP neurons in the PVH ................................ 63 Figure 2-5 Schematic drawings of ARH-derived NPY projections in the 64 hypothalamus of the G130 Japanese macaque ...................................... Figure 2-6 Presence of NPY/DBH fibers in the brainstem. .................................... 65 Figure 2-7 Postnatal development of CART and αMSH in the PVH and ARH. .... 66 Figure 3-1 Maternal consumption of HFD results in increased adiposity, 72 hyperinsulinemia and hyperglycerolemia ............................................ Figure 3-2 Adiposity, hyperinsulinemia and hyperglycerolemia are further 73 exacerbated in pregnant females consuming HFD. .............................. Figure 3-3 Pregnancy results in insulin resistance in HFD-sensitive animals. ....... 75 Figure 3-4 F-HFD have hyperlipidemia and hyperglycerolemia in the presence of 77 reduced G130 body weight. .................................................................. Figure 3-5 Maternal consumption of HFD leads to accumulation of lipids and 80 activation of oxidative stress pathways within the fetal liver. .............. Figure 3-6 Partial block of developmental fatty liver disease in offspring of 81 mother switched to CTR diet after four years of HFD consumption. ... Figure 4-1 Effects of Maternal HFD on fetal POMC and αMSH expression. ....... 103 Figure 4-2 Effects of Maternal HFD on fetal AgRP and MC4R expression. ......... 104 Figure 4-3 Serum maternal and fetal cortisol measurements. ................................ 105 Figure 4-4 IL1RI immunoreactivity in the ventromedial hypothalamus. 106 Figure 4-5 Effects of maternal diet-reversal (DR) on fetal POMC, AgRP and 107 cortisol ................................................................................................... S.Figure 4-1 Preabsorption of antisera with control peptide. .................................... 110 Figure 5-1 Effects of Maternal HFD on fetal POMC and αMSH expression. ........ 129 Figure 5-2 TPH2 mRNA is up-regulated with maternal consumption of HFD. ..... 130 Figure 5-3 Similar numbers of TPH2 labeled neurons are present in rostral, 131 medial and caudal raphe regions. .......................................................... Figure 5-4 5-HTT levels are not altered by maternal consumption of HFD. ......... 132 Figure 5-5 5-HT1A mRNA levels are elevated in F-HFD ....................................... 133 Figure 5-6 Enzyme, transporter and receptor mRNA expression in the 134 hypothalamus ........................................................................................ Figure 5-7 5-HT, metabolite 5-HIAA levels and 5-HT turnover ............................ 135 Figure 5-8 Schematic representation of 5-HT action within the hypothalamus and 136 midbrain in the G130 NHP ................................................................... Figure 6-1 Schematic of putative changes in the hypothalamus as a consequence 152 of maternal consumption of HFD during pregnancy ............................ Figure 6-2 Juvenile animals continued on respective diets