THE PENNSYLVANIA STATE UNIVERSITY SCHREYER HONORS COLLEGE DEPARTMENT OF KINESIOLOGY THE EFFECT OF STRAIN AND EXERCISE ON HYPOTHALMIC GENE EXPRESSION IN C57BL/6J AND DBA/2J MICE JAMES METKUS SPRING 2013 A thesis submitted in partial fulfillment of the requirements for a baccalaureate degree in Biology with honors in Kinesiology Reviewed and approved* by the following: Dena Lang, Ph. D. Research Associate in Kinesiology Thesis Supervisor Jinger Gottschall Assistant Professor of Kinesiology Honors Adviser * Signatures are on file in the Schreyer Honors College. i ABSTRACT Osteoporosis is a degenerative bone disease that affects a majority of Americans, specifically those of older age. Within the last few decades, researchers have begun to consider neurological pathways that contribute to osteoporosis and other bone disorders, including hormonal effects on osteoporosis or other bone disorders. There have been decades of studies focusing on exercise and it has been shown that exercise has positive effects on bone mass by increasing bone density and gene expression within bone. However, there is a lack of research on how exercise affects gene expression within the brain or specific parts of the brain, such as the hypothalamus. In this study, 77 480 day old C57BL/6 (B6) and DBA/2J(D2) female mice were divided into three treatment groups: a treadmill running group (14 B6 and 14 D2 mice), a tower climbing group (13 B6 and 12 D2) and finally a sedentary control group (14 B6 and 10 D2). After 5 weeks of exercise treatment, RNA was extracted from the hypothalamus of each mouse, homogenized and underwent Nanodrop and Bioanalyzer assessment for quality control. Two samples were pooled onto three gene chips for each treatment group in order to measure gene expression within the hypothalamus. Gene expression was then compared across to identify differential gene expression as a function of mouse strain and/or exercise. While the exercise treatment did not produce significant differences in gene expression, important differences that were suggested from this study include the expression of Insulin Growth factor-1(IGF-1) in B6 tower climbing as compared to B6 sedentary controls. In terms of strain differences, around 497 genes were differentially expressed between B6 and D2 mice. The identification of genes such as IGF-1 within the hypothalamuses of B6 and D2 mice may help provide greater insight into how differences in activity levels and genetics may lead to the elucidation of new pathways for the prevention as well as treatment of osteoporosis and other bone disorders. ii TABLE OF CONTENTS List of Figures .......................................................................................................................... iv List of Tables ........................................................................................................................... v Acknowledgements .................................................................................................................. vi Chapter 1 Introduction ............................................................................................................. 1 1.1 Study aims. ................................................................................................................. 2 1.2 Study limitations ......................................................................................................... 2 Chapter 2 Literature Review .................................................................................................... 3 2.1 Neurological Control of Bone Remodeling ................................................................ 3 2.1a Exercise Induced Neurogenesis........................................................................ 3 2.2 Hypothalmic-Pituitary-Gonadal-Skeletal Axis .......................................................... 4 2.2a Estrogen and Follicle Stimulating Hormone .................................................... 4 2.3 Hypothalmic-Pituitary-Adrenal Axis ......................................................................... 5 2.4 Leptin is correlated to high turnover in osteoporosis ................................................ 6 2.4a Cocaine and Amphetamine-Regulated Transcript ........................................... 7 2.4b Sympathetic Nervous System Pathway ............................................................. 7 2.4c Neuropeptide Y ................................................................................................. 8 2.4d Insulin like Growth Factor ............................................................................... 8 2.4e Insulin ............................................................................................................... 9 2.5 Strain Differences between B6 and D2 mice .............................................................. 9 Chapter 3 Materials and Methods ............................................................................................ 12 3.1 Animals and Experimental Design ............................................................................. 12 3.2 Tissue Harvesting ....................................................................................................... 14 3.3 RNA Extraction .......................................................................................................... 14 3.3a. Preparation ..................................................................................................... 14 3.3b. RNA Extraction ............................................................................................... 15 3.3c. RNA quality analysis ....................................................................................... 15 3.4 RNA Pooling and Labeling ........................................................................................ 17 3.5 Microarray Processing and Data Analysis ................................................................ 18 Chapter 4 Results ..................................................................................................................... 19 4.1 Differential Expression as a Function of Genetic Strain ........................................... 19 4.2 Differential Expression as a Function of Exercise Treatment ................................... 37 iii Chapter 5 Discussion ............................................................................................................... 39 5.2 Analysis of Treatment Effect ...................................................................................... 42 5.3 Recommendations for Future Investigation ............................................................... 44 Appendix A Treadmill Operator Protocol ....................................................................... 45 Appendix B Tower Design.............................................................................................. 47 References ........................................................................................................................ 48 iv LIST OF FIGURES Figure 2-1 Sun et al HPG axis [7] ............................................................................................ 4 Figure 2-2 Estrogen’s effects on bone resorption .................................................................... 5 Figure 2-3 Leptin Osteoblast Pathway [14] ............................................................................. 7 Figure 2-4 Insulin’s effects on bone resorption [24] ................................................................ 9 Figure 3-1 Schematic of experimental design. ......................................................................... 12 Figure 5-1 Design of Tower for Exercise Intervention ............................................................ 47 v LIST OF TABLES Table 3-1Protocol of settings used during treadmill running intervention .............................. 13 Table 3-2 Summary of Chip Data used for Microarray Analysis ............................................ 16 Table 4-1 Genes expressed in B6 mice over D2 with a FC of greater than 5 .......................... 19 Table 4-2 Genes expressed higher in B6 over D2 mice with a FC of 3 or 4 Part I .................. 21 Table 4-3 Genes expressed higher in B6 over D2 mice with a FC of 3 or 4 Part II ................ 22 Table 4-4 Genes expressed higher in B6 over D2 mice with a FC of 2 Part I ......................... 24 Table 4-5Genes expressed higher in B6 over D2 mice with a FC of 2 Part II ......................... 25 Table 4-6Genes expressed higher in B6 over D2 mice with a FC of 2 Part III ....................... 26 Table 4-7Genes expressed higher in D2 over B6 mice with a FC of 5 or greater ................... 28 Table 4-8Genes expressed higher in D2 over B6 mice with a FC of 3 or 4 Part I ................... 29 Table 4-9Genes expressed higher in D2 over B6 mice with a FC of 3 or 4 Part II ................. 30 Table 4-10Genes expressed higher in D2 over B6 mice with a FC of 3 or 4 Part III .............. 31 Table 4-11Genes expressed higher in D2 over B6 mice with a FC of 2 Part I ........................ 33 Table 4-12Genes expressed higher in D2 over B6 mice with a FC of 2 Part II ....................... 35 Table 4-13Genes expressed higher in D2 over B6 mice with a FC of 2 Part III ..................... 36 Table 4-14 Genes Involved in Hypothalmic Neurogenesis ..................................................... 38 Table 4-15 Genes Involved in HPG Axis ...............................................................................