Environmental and gene therapy approaches to improve glycemic control and promote healthy aging DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Travis Blaze McMurphy Graduate Program in Biomedical Sciences The Ohio State University 2017 Dissertation Committee: Dr. Lei Cao Ph.D., Advisor Dr. A. Courtney DeVries Ph.D. Dr. Denis C. Guttridge Ph.D. Dr. F. Kay Huebner Ph.D. Copyright by Travis Blaze McMurphy 2017 Abstract The epidemic of obesity and associated complications comprising metabolic syndrome exact a monumental burden on global public health in both morbidity and cost of treatment. Stressful, sedentary lifestyles coupled with excessive caloric intake contribute to increasing rates of obesity and type II diabetes mellitus worldwide. Additionally, the likelihood of excess visceral adiposity progressing into metabolic syndrome grows dramatically with age. Obesity-associated insulin resistance commonly precedes the onset of type II diabetes and most treatments for the resulting hyperglycemia mimic, sensitize, or enhance secretion of insulin. Therefore, interventions that improve glycemic control independently of insulin signaling are appealing alternatives. The overall objective of this dissertation is to evaluate the efficacy of environmental enrichment and gene therapy models to improve glycemic control and promote healthy aging. The ability of pathogenic viruses to alter host metabolism has been recently characterized. Infection by the human adenovirus serotype 36 (AD36) promotes obesity in animal models and correlates to increased adiposity in humans, yet improves glycemic control. Based on in vitro studies, the E4ORF1 protein is responsible for both the adipogenic and insulin sparing properties of AD36 infection via insulin independent Akt activation. We generated a recombinant adeno-associated viral (rAAV) vector to express ii AD36E4ORF1. Through intravenous delivery we expressed AD36E4ORF1 in the livers of diabetic, insulin resistant, and wild-type mice. Hepatic AD36E4ORF1 improved glucose tolerance and attenuated hyperglycemia in obese diabetic Db -/- mice without improving insulin sensitivity. AD36E4ORF1 also reduced hyperinsulinemia and improved glucose tolerance in insulin resistant mice with diet induced obesity (DIO). Liver specific glucose uptake was increased without improving insulin sensitivity. Confirming the findings of previous in vitro studies, Akt activity was not only increased but also required for AD36E4ORF1 mediated glucose uptake. AD36E4ORF1 expression is a model of insulin independent AKT activation and provides a novel therapeutic mechanism to improve glycemic control in cases of insulin resistance. Next, we looked at how environmental factors might contribute to healthy aging and prevention of metabolic syndrome. Animals housed in a larger, more complex enriched environment (EE) are provided with increased somatosensory stimulation, physical exercise, and enhanced social interactions. Together, these stimuli increase expression of brain derived neurotrophic factor (BDNF) in the hypothalamus, activating a hypothalamic sympathoneural-adipocyte axis (HSA). In young animals, HSA activation has been shown to improve glycemic control and overall health but its impact in older animals has not been previously characterized. Middle-aged 10 month old female mice were housed in EE for 6 weeks and displayed HSA activation, improved glycemic control, and decreased adiposity without a reduction in overall body weight. In a long term study of middle-aged mice housed in EE for 12 months, we observed a metabolic phenotype characteristic of healthy aging and iii improved glycemic control. The animals housed in EE exhibited improved glucose tolerance, enhanced motor skills, reduced adiposity, increased mitochondrial biogenesis, remodeling and browning of the white adipose tissue, and prevention of age-associated decline in the brown adipose tissue. Remodeling of the adipose tissue was accompanied by an adipose-specific upregulation of the tumor suppressor phosphatase tensin homologue deleted on chromosome ten (PTEN). Activation of the HSA axis was necessary and sufficient to upregulate PTEN in the adipose tissue. Moreover, sympathetic activation of type 1 and 2 β-adrenergic receptors was responsible for increasing PTEN expression. This study is the first to identify a novel physiological mechanism of PTEN upregulation through sympathetic stimulation of the adipose tissue. EE initiated in middle-aged female mice provides a model of improved glycemic control, healthy aging, and physiologically induced PTEN expression in the adipose tissue. Based on these findings, living in enriched environments with increased social and physical interactions promotes healthy aging through activation of a brain-adipocyte connection which reduces risk factors contributing to age-associated pathologies. iv Dedication I dedicate this document to my girlfriend, Stephanie Steiger, and my family. v Acknowledgments I am extremely grateful to everyone who has supported me as I have worked towards this point. To my advisor Dr. Lei Cao, I cannot thank you enough for your guidance, and the opportunity to succeed in your lab. To my committee, Drs. Courtney DeVries, Denis Guttridge, and Kay Huebner, I thank you for providing invaluable guidance and direction. I would also like to thank my undergraduate mentor, Dr. Matthew Foradori for granting me my first research opportunity and inspiring me to continue my education. To my colleagues I thank you for your contributions, insight and intelligent discussions. To my friends and family, I greatly appreciate the positive impact you have on my life. Above all I am grateful to my exceedingly supportive girlfriend Stephanie Steiger, and to my parents Jack and Marcia McMurphy. vi Vita 2004................................................................Grand Valley High School 2009................................................................B.S. Marketing Edinboro University of PA 2011................................................................B.S. Biology Edinboro University of PA 2012 to present ..............................................Graduate Research Associate, Biomedical Sciences Graduate Program, The Ohio State University Publications McMurphy, T. B., Huang, W., Xiao, R., Liu, X., Dhurandhar, N. V., & Cao, L. (2017). Hepatic Expression of Adenovirus 36 E4ORF1 Improves Glycemic Control and Promotes Glucose Metabolism Through AKT Activation. Diabetes, 66(2), 358- 371. doi: 10.2337/db16-0876 McMurphy, T., Xiao, R., Magee, D., Slater, A., Zabeau, L., Tavernier, J., & Cao, L. (2014). The anti-tumor activity of a neutralizing nanobody targeting leptin receptor in a mouse model of melanoma. PLoS One, 9(2), e89895. doi: 10.1371/journal.pone.0089895 Huang, W., McMurphy, T., Liu, X., Wang, C., & Cao, L. (2016). Genetic Manipulation of Brown Fat Via Oral Administration of an Engineered Recombinant Adeno- associated Viral Serotype Vector. Mol Ther, 24(6), 1062-1069. doi: 10.1038/mt.2016.34 vii During, M. J., Liu, X., Huang, W., Magee, D., Slater, A., McMurphy, T., . Cao, L. (2015). Adipose VEGF Links the White-to-Brown Fat Switch With Environmental, Genetic, and Pharmacological Stimuli in Male Mice. Endocrinology, 156(6), 2059-2073. doi: 10.1210/en.2014-1905 Liu, X., McMurphy, T., Xiao, R., Slater, A., Huang, W., & Cao, L. (2014). Hypothalamic gene transfer of BDNF inhibits breast cancer progression and metastasis in middle age obese mice. Mol Ther, 22(7), 1275-1284. doi: 10.1038/mt.2014.45 Liu, X., Magee, D., Wang, C., McMurphy, T., Slater, A., During, M., & Cao, L. (2014). Adipose tissue insulin receptor knockdown via a new primate-derived hybrid recombinant AAV serotype. Mol Ther Methods Clin Dev, 1. doi: 10.1038/mtm.2013.8 Field of Study Major Field: Biomedical Sciences Program viii Table of Contents Abstract ............................................................................................................................... ii Dedication ........................................................................................................................... v Acknowledgments.............................................................................................................. vi Vita .................................................................................................................................... vii List of Tables ..................................................................................................................... xi List of Figures ................................................................................................................... xii Chapter 1: A review of insulin signaling and resistance.................................................... 1 The global epidemics of obesity and metabolic syndrome ............................................. 2 Insulin mediated glucose uptake ..................................................................................... 4 The role of the akt in insulin signaling ........................................................................ 5 Mechanisms of insulin resistance .................................................................................... 8 AD36E4ORF1, a potent activator of Akt ...................................................................... 10 PTEN as a regulator of glucose metabolism ................................................................