View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Vanderbilt Electronic Thesis and Dissertation Archive THE ROLE OF INSULIN SIGNALING ON DOPAMINE TRANSPORTER TRAFFICKING By Nicole K. Speed Dissertation Submitted to the Faculty of the Graduate School of Vanderbilt University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY in Pharmacology August, 2010 Nashville, Tennessee Approved: Dr. Aurelio Galli Dr. Randy Blakely Dr. Kevin Niswender Dr. Gregg Stanwood Dr. Brian Wadzinski ii To my amazing, supportive parents, for always keeping me smiling iii ACKNOWLEDGMENTS This work would not have been possible without the funding that helped support it. This financial support included an individual predoctoral NRSA fellowship (F31 DA024885) from the National Institute on Drug Abuse, as well as support awarded to my mentor, Dr. Galli, from the Vanderbilt Diabetes Research and Traning Center (DK020593), and grants from the National Institute of Health (DA14684 and DK085712). I would first like to acknowledge my advisor, Dr. Aurelio Galli. His support throughout my graduate career has been invaluable. He has encouraged my development scientifically and beyond. Thank you for pushing me, even when I pushed back. I hope to embrace science and life with the enthusiasm that he does. I would also like to thank my thesis committee members Drs. Gregg Stanwood, Brian Wadzinski, Kevin Niswender, and Randy Blakely for their helpful guidance and support throughout my graduate career. Special thanks are extended to my chair, Dr. Randy Blakely, for his help and advice, and to Dr. Kevin Niswender for his collaborative efforts on my project. I would also like to acknowledge my collaborators including Dr. Lynette Daws and Anthony Owens, who provided much time and effort to my project and always put a smile on my face, and Dr. Scott Russo, who provided the IRS-2 virus and helpful guidance with manuscript preparations. I am also grateful for the assistance I received from several people at Vanderbilt, including Dr. Fang Yu, Dr. Nicole Herring and Dr. Heather Gosnell, who helped me with learning iv techniques. Also I owe a debt of gratitude to Sanaz Saadat, who may be the most pleasant and helpful person I have ever encountered. I would also like to thank the Pharmacology Department for their help and support throughout graduate school. I am grateful to the members of the Galli lab for their support, scientifically and otherwise. I would especially like to thank Dr. Heiner Matthies for taking the time to provide training with laboratory techniques and answer my many questions, and Nicole Bibus-Christianson for her help, her patience at my disorganization and, most importantly, her friendship. She truly has a heart of gold. I would also like to thank Dr. Adeola Davis for her help with feeding rats. Most of all, I am grateful to my incredibly supportive family and friends. My parents have always given me faith in my abilities, and their constant encouragement and love is the reason I push myself to accomplish my goals. I cannot express how much their support means to me; it is the foundation to all of my successes. I also would like to thank my sister Alexa, who always lends an ear, and my crazy family, who always believe in me and are my rock of support. I want to thank Leda Ramoz and Jessica Moore for their friendship while in graduate school, which kept me sane, and Laurie and Ben Ott for their friendship and hospitality. Lastly I would like to thank my fiancé, Brandon Lute. His love and faith in me are endless, and for that I am extremely grateful. v TABLE OF CONTENTS Page DEDICATION ....................................................................................................... ii ACKNOWLEDGMENTS...................................................................................... iii LIST OF FIGURES ............................................................................................. vii LIST OF ABBREVIATIONS ................................................................................ ix Chapter I. INTRODUCTION .............................................................................................. 1 The Neurotransmitter Dopamine and Dopaminergic Pathways .......... 1 Dopaminergic Neurotransmission and the Dopamine Transporter .... 4 The Dopamine Transporter Structure and Function ........................... 10 Psychostimulants and the Dopamine Transporter ............................. 17 DAT Regulation by Interacting Proteins .............................................. 20 Regulation of Dopamine Transporter Surface Expression ................ 23 Insulin and the Dopamine Transporter ................................................ 30 Dopamine and Feeding Behavior ......................................................... 33 Insulin Regulation of Feeding Behavior .............................................. 37 Insulin, Diabetes, and DA-Related Diseases ....................................... 40 Specific Aims ......................................................................................... 42 II. ISOFORM SPECIFIC REGULATION OF DAT CELL SURFACE EXPRESSION BY AKT2 .................................................................................... 45 Introduction ............................................................................................ 45 Methods .................................................................................................. 47 Results .................................................................................................... 52 Discussion .............................................................................................. 60 III. DIET-INDUCED CHANGES IN INSULIN SIGNAILNG REGULATES THE TRAFFICKING AND FUNCTION OF THE DOPAMINE TRANSPORTER ........ 63 Introduction ............................................................................................ 63 Methods .................................................................................................. 67 Results .................................................................................................... 73 vi Discussion .............................................................................................. 93 IV. GENERAL DISCUSSION AND FUTURE DIRECTIONS ........................... 100 Summary .............................................................................................. 100 Obesity and Diabetes .......................................................................... 102 Alternative Interpretations .................................................................. 106 Future Directions ................................................................................. 109 V. REFERENCES ............................................................................................ 117 vii LIST OF FIGURES Figure Page 1. Pathways of dopamine signaling in the brain .................................................... 2 2. Biosynthesis of dopamine .................................................................................. 5 3. Diagram of a dopaminergic synapse .................................................................. 7 4. Enzymatic degradation of dopamine. ................................................................. 9 5. Illustration of the dopamine transporter ......................................................... 12 6. Dopamine transporter uptake action ................................................................ 15 7. Schematic of activation of Akt by insulin......................................................... 27 8. Schematic of insulin regulation of the dopamine transporter. ....................... 44 9. Structures of isoform selective, allosteric Akt inhibitors. .............................. 53 10. Inhibition of Akt2 reduces hDAT cell surface expression in hDAT cells ....... 55 11. Inhibition of Akt2 reduces rDAT cell surface expression in rat striatal tissue .................................................................................................................. 57 12. Inhibition of Akt2 reduces DAT-mediates reverse transport of DA. ............... 59 13. High fat feeding results in increased food intake and weight gain ................ 76 14. High fat feeding results in insulin resistance .................................................. 77 15. Tyrosine hydroxylase levels are unchanged by high fat feeding ................... 78 16. Dopamine (DA) levels in striatum are unchanged by high fat feeding ........... 79 17. DIO induces a decrease in p-Akt in striatum and substantia nigra ................ 81 18. DIO induces a decrease in Akt activity in the striatum ................................... 82 19. DIO induces a decrease in p-Akt2 .................................................................... 83 20. Dopamine transporter cell surface expression is reduced in striatal slices of high fat fed rats. .................................................................................. 85 21. Dopamine clearance rate is reduced in DIO animals....................................... 86 viii 22. AMPH-induced locomotor activity is reduced in DIO animals ........................ 88 23. AMPH levels in striatum after I.P. injection of AMPH remain the same between LF and HF fed rats .............................................................................. 89 24. Viral-mediated expression of HSV-GFP .......................................................... 91 25.