Exploring the Function of G6PC2 in Pancreatic Islet Beta Cells By Kayla Ann Boortz 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 Molecular Physiology and Biophysics December, 2016 Nashville, Tennessee Approved: Owen P. McGuinness, Ph.D. Roger D. Cone, Ph.D. David A. Jacobson, Ph.D. Fiona E. Yull, D.Phil Acknowledgments Primarily I need to thank the O’Brien Lab and all of its members. Specifically, I need to thank Ken for always keeping things entertaining and keeping me on my toes. Not to mention doing my tails and always supporting me with whatever help I may need at the time. I can truly say I will never work with an individual like Ken again and I am thankful for the time I got to spend with him. Next I need to thank Kristen who not only has become one of my dear friends but has been a constant support system since she joined. Her willingness to answer my dumb questions, help me with math or encouragement during times of stress is invaluable and I will miss her dearly. Next, Karin’s support in a time of transition and help in all things is very much appreciated. Finally, none of this work would be possible without the support, guidance and aid of Richard. His training and knowledge has created an environment that is both challenging but encouraging. I am a better student, scientist, writer, speaker and soccer enthusiast because of him. I also need to thank my committee, department and support staff at Vanderbilt. My committee was crucial to guiding me through my project and making me a better problem solver and scientist. There, often critical, evaluation was necessary to making me better and I appreciate them for always pushing me to improve and think about my data harder. I also need to thank MP&B for the support and opportunities I was given while I was here. Being in such a collaborative and friendly department definitely makes a difference on the day to day of graduate school. I will miss the Halloween Parties and relay races and just general environment that was cultivated within our ranks. Finally I need to thank Colette Bosley and Karen Gieg for making our lives as students just a little bit easier. Colette always has a smile and just calming effect when you are around her, while Karen was crucial to making everything simpler for us. Next I need to thank my family. My parent’s support throughout my life gave me the courage and will to not only pursue my PhD but also to be successful. While they always stressed that I pursue something I was good at, I am sure they did not anticipate me staying in school this long. From the emotional phone calls to the excited ones, they have been by my side from the start. Next I need to thank all my friends, both new and old that have been along for this ride. Having a support system disconnected ii from graduate school was such a necessary thing for me to stay balanced and focused. While they didn’t understand what I was talking about half the time, I love them for pretending like they did and knowing when to ask questions and when to just listen. Last but not least, I need to thank Jesse. Not only did he survive organic chemistry in undergraduate with me but, also, every single day of graduate school. Day in and day out he was someone to lean on and a driving force for when things weren’t exactly going my way in lab. He was supportive and provided a constant relief from the stresses of graduate school, always making me laugh and smile regardless of the situation. I can’t imagine having gone through my PhD without his presence and support. As they say “it takes a village” and, as evidence by this lengthy acknowledgments section, that couldn’t be truer. Acknowledgments of Support This research was supported by the following grants: R.O’B., DK92589; D.A.J., DK081666 and DK20593; J.-C.W., DK083591; O.P.M., DK043748 and DK078188; and A.C.P., DK72473, DK89572, DK104211, the Department of Veterans Affairs and the JDRF. The Vanderbilt Hormone Assay & Analytical Services Core and the Vanderbilt Islet Procurement and Analysis Core are both supported by NIH grant P60 DK20593, to the Vanderbilt Diabetes Research Training Center and NIH grant DK59637, to the Vanderbilt Mouse Metabolic Phenotyping Center. K. E. S. and L. D. P. were supported by the Vanderbilt Molecular Endocrinology Training Program grant 5T32 DK07563. iii Table of Contents Acknowledgments ........................................................................................................................................................... ii Acknowledgments of Support .................................................................................................................................... iii Abbreviations .................................................................................................................................................................. vi List of Tables .................................................................................................................................................................. viii List of Figures .................................................................................................................................................................. ix Chapter I. Introduction .............................................................................................................................................................. 1 Diabetes Mellitus ...................................................................................................................................................................... 1 Type 1 Diabetes ................................................................................................................................................................... 1 Type 2 Diabetes ................................................................................................................................................................... 4 Discovery of Hepatic Glucose-6-phosphatase .............................................................................................................. 7 The Glucose-6-Phosphatase Gene Family ...................................................................................................................... 8 G6PC1 ...................................................................................................................................................................................... 8 G6PC3 .................................................................................................................................................................................... 11 G6PC2 .................................................................................................................................................................................... 12 The Identification of G6PC2 Single Nucleotide Polymorphisms (SNPs) that Regulate Fasting Blood Glucose in Humans ................................................................................................................................................................ 12 G6pc2 Function In Vivo ........................................................................................................................................................ 13 Characterization of Common G6PC2 SNPs ................................................................................................................... 23 Identification and Characterization of Rare SNP Variants..................................................................................... 27 Glucocorticoid Biology ......................................................................................................................................................... 28 Glucocorticoids Effect Glucose Metabolism in the Liver, Skeletal Muscle and White Adipose Tissue . 32 Glucocorticoids Effect Glucose Metabolism in Pancreatic Islets ......................................................................... 36 Hypothesis: Glucocorticoids Modulate G6pc2 Expression and Glucose Metabolism .................................. 39 Hypothesis: Rare G6PC2 SNPs Exist and Affect Protein Expression and Enzyme Activity ....................... 40 II. Materials and Methods ...................................................................................................................................... 41 Generation of G6pc2 KO Mice ............................................................................................................................................ 41 PCR Genotyping of G6pc2 KO Mice .................................................................................................................................. 42 Animal Care .............................................................................................................................................................................. 42 Phenotypic Analysis of Fasted WT and G6pc2 KO Mice .......................................................................................... 42 Intraperitoneal Glucose Tolerance Tests ..................................................................................................................... 43 Analysis of Glucose-Stimulated Insulin Secretion In Vivo ...................................................................................... 43 Dexamethasone Injection Paradigm .............................................................................................................................