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© 2014 Jonathan Guo-Han Mun © 2014 Jonathan Guo-Han Mun HYPOGLYCEMIA-INDUCED CHANGES IN GLUCOSE METABOLISM IN THE HYPOTHALAMUS BY JONATHAN GUO-HAN MUN DISSERTATION Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Nutritional Sciences in the Graduate College of the University of Illinois at Urbana-Champaign, 2014 Urbana, Illinois Doctoral Committee: Professor Rodney W. Johnson, Chair Professor J. Lee Beverly, Director of Research Associate Professor Justin S. Rhodes Associate Professor Manabu T. Nakamura ABSTRACT Hypoglycemia is the most common acute complication associated with insulin-dependent diabetes mellitus (Type 1 and advanced Type 2), which affects 300,000-500,000 individuals of all ages in the United States, with 30,000 new cases each year (1). It is characterized by low blood glucose concentration (less than 70 mg/dL or 4 mM) that can be attributed to a mismatch of insulin, food intake, sleep, and physical activity and can result in serious morbidity or death (2). The glucose counterregulatory response is the primary defense against hypoglycemia and involves (A) secretion of epinephrine and glucagon to elevate blood glucose concentration and (B) sympathetic activation to prompt the individual to take action against hypoglycemia through symptoms that include hunger, shaking, rapid heart rate, and sweating (2). Recurrent episodes of hypoglycemia result in failure of the counterregulatory response, which increases the frequency and severity of subsequent hypoglycemia. The underlying physiology by which this occurs is not well understood (3), but changes occurring in the ventromedial hypothalamus (VMH), a brain region that is responsible for initiating the counterregulatory response to hypoglycemia (4) is the most promising target. In the following studies, we utilized metabolomic (Chapter 2), transcriptomic (Chapter 3), and isotope tracer approaches (Chapter 4) to identify changes in glucose metabolism in the VMH. We did not see significant changes in glucose metabolite concentrations, but we observed altered glycogen metabolism through changes in expression of genes involved in glycogen synthesis and mobilization. Impact of altered brain glycogen metabolism in the development of HAAF is underway, but research to gain a robust understanding brain glycogen kinetics will be necessary before investigating the impact of recurrent hypoglycemia on its turnover. Many genes were identified to be associated with the ii hypoglycemia treatments and among them, were advanced glycosylation end product-specific receptor and Vgf nerve growth factor, which are promising candidate genes for future research due to their functions and associations with diabetes. We also identified changes in arachidonate 15-lipoxygenase (Alox15) gene expression, which is an enzyme that oxidizes polyunsaturated fatty acids, phospholipids, and other complex substrates. Alox15 expression was highly correlated with the hypoglycemia treatments and was complemented by changes in cholesterol and phospholipid metabolites in response to recurrent hypoglycemia. We demonstrate in these studies a role of altered VMH glucose metabolism in response to hypoglycemia and propose a novel role of altered VMH lipid metabolism in the development of impaired glucose counterregulation. LITERATURE CITED 1. LaPorte RE, Matsushima M, Chang Y-F. Prevalence and incidence of insulin-dependent diabetes. Diabetes in America. 1995;2:37-46. 2. Cryer PE, Davis SN, Shamoon H. Hypoglycemia in diabetes. Diabetes Care. 2003;26(6):1902-12. 3. Dagogo-Jack S. Hypoglycemia in type 1 diabetes mellitus: pathophysiology and prevention. Treatments in Endocrinology. 2004;3(2):91-103. 4. Routh VH. Glucose sensing neurons in the ventromedial hypothalamus. Sensors. 2010;10(10):9002-25. iii To My Mother and Father iv ACKNOWLEDGMENTS As I reflect upon my time as a graduate student in the Division of Nutritional Sciences, I am reminded of how fortunate I have been to have crossed paths with the brilliant minds and the kind hearts of individuals that I now call my mentors, my mentees, my colleagues, or more simply, my friends. Many of these individuals have contributed to my development and deserve to be recognized. First and foremost, I am immensely grateful to my advisor, Lee Beverly, for taking me under his guidance as a PhD student in his lab, for training me to think more critically, for offering me the opportunity to work with students in his classroom, and for providing me with the resources to acquire an expertise in a broad range of techniques and technologies. Dr. Beverly’s mentorship has been integral to shaping my approach to addressing research questions and I would surely not be the same person that I am today without his invested efforts. By sheer coincidence, Dr. Beverly’s support of my interest in keeping fish in our grad office has not only shaped the way that I think about dynamic biological systems (and by distant association, systems physiology), but has also led me to working with Justin Rhodes, my co- advisor. Dr. Rhodes has been tremendously helpful with reaching the end of my PhD by providing deep insights into my hypotheses, lending his expertise in statistics to my massively daunting data sets, editing this lengthy document, and keeping me from burning out on this arduous journey by drawing me into the fish lab to take breaks and reminding me to get some rest. The DNS faculty and administration have been truly remarkable in their willingness to help contribute ideas and facilitate the development of their students. In particular, I want to v recognize my committee members, Mani Nakamura, Rodney Johnson, and Peter Garlick. Dr. Nakamura instructs the most challenging course I have ever taken, entitled “Regulation of Metabolism” and from it I gained a bigger picture of human health as it relates to homeostatic mechanisms. I could not have completed the work presented here without a thorough understanding of the material from his class and for that I am thankful. Dr. Johnson has taught me a lot about the critical evaluation of scientific literature and has been a huge motivating force for getting me to finish this dissertation. Dr. Garlick has helped with developing my ideas for isotope tracers in metabolic research and offered his entire lab space for me to work with the GC/MS. The DNS administration – Jessica Hartke and Ashley Browning in particular – have been a highly efficient team for helping me get through my exit seminar, defense, and dissertation deposit and for coordinating the DNS events that I enjoy. Generously offering their expertise in biochemical analyses, Lucas Li and Alex Ulanov from the Roy J. Carver Biotechnology Center have been great with helping me understand the internal workings of GC/MS and the identification and quantification of metabolic tracers. Jenny Zadeh from HPCBio and Bruce Southey were tremendously helpful for learning to understand how to properly analyze RNAseq data and Jennifer Rytych taught me all about running real time- PCR. My labmates, past and present, have been vitally important to my progress through grad school. In particular, Chiara Barbieri and Kristy Du have been incredibly helpful by assisting with daily lab activities and providing the emotional support of having a first-hand understanding of my frustrations when the techniques that we use don’t work. Chiara was tremendously helpful for getting me back on track when the stress of making progress led to inevitable breakdowns and Kristy has been (and continues to be) a great help with keeping me on task. John Cavaretta vi and Andrew Cooper were techs that kept the lab running smoothly and John was particularly helpful with the metabolomics and RNAseq work in this thesis. Briana Grymonprez and Katrina Pioli were exceptional undergrads in our lab and helped with making microdialysis probes and assisting with experiments. The members of the Rhodes lab have also made huge contributions to this research by critically evaluating my work and my ability to present it. Most notably, Gillian Hamilton, Martina Mustroph, Petra Majdak, Ross DeAngelis, Tushar Bhattacharya, and Dan Miller have asked tough questions about my work that have helped me identify areas where my explanations are weakest. Ross has also been a reliable friend with a shared enthusiasm for reef aquarium keeping, which has helped relieve some of the pressures of research. Several other close friends that I have made here in grad school have made my time in Illinois truly enjoyable. Tristan Kraft was my first friend in Urbana-Champaign and has been one of the greatest influences in my life. Chris Moulton and Naiman Khan are two solid friends who have made the entire journey through grad school with me from day one and have been incredible sources of inspiration. Hannah Holscher and Jen Barnes are two brilliant scientists, whose opinions I highly respect, who helped me get over the major DNS hurdles of NUTR 511 and the qualifying exam. Matt Panasevich and Josh Smith are also incredibly talented scientists, who have shaped my perspectives in research and are always looking out for me. Finally, my family has always been immensely supportive of my education. The following work has been dedicated to my parents, who have made incredible sacrifices to provide me with the best education possible. vii TABLE OF CONTENTS CHAPTER 1: Introduction ……………………………………………………………………… 1 CHAPTER 2: Biochemical analysis of the ventromedial hypothalamus (VMH) and CA1 region of the hippocampus in response to acute and recurrent hypoglycemia
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