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The Effects of Growth Hormone and Insulin- Like

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The Effects of Growth Hormone and Insulin- Like THE EFFECTS OF GROWTH HORMONE AND INSULIN- LIKE GROWTH FACTOR-1 TREATMENTS ON HEPATIC GENE EXPRESSION IN OBESE AND DIABETIC MICE WITH NONALCOHOLIC FATTY LIVER DISEASE A Thesis Presented to The Honors Tutorial College Ohio University In Partial Fulfillment of the Requirements for Graduation from the Honors Tutorial College with the degree of Bachelor of Science in Biological Sciences by John D. Blischak June 2010 Blischak 2 This thesis has been approved by The Honors Tutorial College and the Department of Biological Sciences __________________________ Dr. Edward List Scientist, Edison Biotechnology Institute Thesis Advisor ___________________________ Dr. Darlene Berryman Associate Professor, Human and Consumer Sciences Thesis Advisor ___________________________ Dr. Soichi Tanda Associate Professor, Honors Tutorial College, Director of Studies, Biological Sciences ___________________________ Dr. Jeremy Webster Dean, Honors Tutorial College Blischak 3 Acknowledgements I am extremely grateful for the opportunity to interact with so many great people during my time here at Ohio University. Dr. List has been my primary adviser for the last two years, and he has taught me everything from good laboratory technique to grant writing and working with collaborators. The best part about working with Dr. List was anytime I was frustrated or discouraged, he was always able to get me excited about the project again. Dr. Berryman was a great source of knowledge and fielded my many questions on fat metabolism, qPCR, and statistics. Dr. Kopchick provided me many great opportunities including presenting at group meetings, co-authoring a book chapter, and attending the ENDO conference twice. I am indebted to everyone in the Kopchick laboratory. Every one of them has contributed to my project in one form or another, whether it was offering advice, helping with dissections, or helping me learn a new technique. Furthermore, many people also had direct roles in my project. Jake Wright-Piekarski and I split all the mouse work, Hiro Tanda taught me RNA extraction and qPCR, Ellen Lubbers and Elizabeth Walcutt both performed the liver triglyceride assays, and Lucila Sackmann- Sala helped me with the statistics. Dr. Tanda, my academic adviser, has been a great mentor over the years assisting me not only with class schedules but also the Goldwater Scholarship and graduate school applications. Dr. Sarah Wyatt, my adviser for the Choose Ohio First for Bioinformatics scholarship, has been instrumental in my professional development and shaping my research interests. Blischak 4 The microarray studies could not have been performed without all the assistance and encouragement from the Ohio University Genomics Facility staff. Vijay Nadella assisted with the experimental design and overall management of the projects, Kaiyu Shen helped with the statistical analysis, and Tara Killen ran the many RNA nano chips for the Bioanalyzer. The many research experiences I have had would not have been possible without the financial support I have received. Many of the direct research costs were supported by a Student Enhancement Award and a Provost Undergraduate Research Fund. Travel assistance to conferences was generously provided by the Dean’s Discretionary Fund of the Honors Tutorial College and the Diabetes Research Initiative. I was able to receive financial support for working at the lab over the summers and during my junior year by support from the Honors Tutorial College Apprenticeship Program and the American Veterans (AMVETS). Lastly, I have received scholarship support over the years from an Ohio University Gateway Excellence Scholarship, Choose Ohio First for Bioinformatics Scholarship, a C. Paul and Beth K. Stocker Scholarship, and an Honors Tutorial College Scholarship. And of course I am thankful for the support of my friends and family. Specifically, the other HTC seniors in biology: Brooks Kohli, Hiro Tanda, Marie Braasch, and Sarah Gutzwiller; my long-time friends: Alex Al-Doory, Andrew Bader, Alex Douglas, and John Fulvimar; my girlfriend Melissa Feigi; and my parents Maggi and Dave. Blischak 5 Table of contents Approval page………………………………………………………………………….2 Acknowledgements…………………………………………………………………….3 Table of contents……………………………………………………………………….5 List of figures……………………………………………………………………..........7 List of tables……………………………………………………………………………9 List of abbreviations…………………………………………………………………..12 Abstract………………………………………………………………………..……...14 I. Introduction………………………………………………………………………...16 1. Epidemiology: The pandemic of metabolic disorders……………………..16 2. Pathogenesis of NAFLD…………………………………………………...19 3. Treatment of nonalcoholic fatty liver disease……………………………...25 4. Growth Hormone: Over a century of research…………………………….25 5. Growth hormone physiology and regulation………………………………26 6. GH and IGF-1 experimental therapy for NAFLD…………………………29 7. Goals of the study………………………………………………………….31 II. Materials and Methods…………………………………………………………….32 1. Mice and treatment regimen #1……………………………………………32 2. Mice and treatment regimen #2……………………………………………34 3. Liver triglycerides and histology…………………………………………..37 4. Plasma IGF-1………………………………………………………………37 5. Intraperitoneal glucose tolerance test………………………………………38 6. RNA extraction…………………………………………………………….38 7. Microarray sample preparation, hybridization, washing , and scanning…...38 8. Microarray design experiment #1………………………………………….39 9. Microarray design experiment #2………………………………………….39 10. Microarray data analysis………………………………………………….42 11. Statistics for physiological measurements………………………………..43 III. Results…………………………………………………………………………….43 1. Microarray experiment #1………………………………………………….43 Blischak 6 A. Physiological measurements………………………………………44 B. Differentially expressed genes…………………………………….54 C. Hierarchical clustering…………………………………………….54 2. Microarray experiment #2………………………………………………….56 A. Physiological measurements………………………………………56 B. Liver histology…………………………………………………….70 C. Differentially expressed genes…………………………………….72 D. Hierarchical clustering…………………………………………….72 3. Combined analysis of the two microarray experiments……………………74 A. Gene ontology classification and pathway analysis……………….74 B. Hierarchical clustering…………………………………………….87 C. Venn diagrams……………………………………………………..89 D. Genes of interest…………………………………………………...93 IV. Discussion………………………………………………………………………...95 1. Summary…………………………………………………………………...95 2. GH and IGF-1 treatments…………………………………………………..95 3. IGF-1 mechanism…………………………………………………………..98 4. Patterns of gene expression………………………………………………...99 5. Genes of interest………………………………………………………….101 6. Limitations………………………………………………………………..112 7. Future directions………………………………………………………….115 8. Conclusion………………………………………………………………..118 V. References……………………………………………………………………..…118 VI. Appendix………………………………………………………………………...132 Blischak 7 List of figures Figure 1. The survival rate of 420 individuals with NAFLD was significantly lower than the general population (Adams et al., 2005). .......................... 18 Figure 2. Diagram of normal hepatic lipid metabolism (Jou et al., 2008). .................. 20 Figure 3. Potential molecular pathways associated with ectopic fat deposition in the liver (Chavez-Tapia et al., 2009).. ....................................................... 22 Figure 4. Progression from NAFLD to NASH. Adapted from Jou et al., 2008. ........ 24 Figure 5. Regulation of growth hormone secretion. (Okada & Kopchick, 2001).. ........................................................................................................ 28 Figure 6. Study design for treatment regimen #1. ....................................................... 33 Figure 7. Study design for treatment regimen #2. ....................................................... 36 Figure 8. Design of microarray experiment #1.. .......................................................... 40 Figure 9. Design of microarray experiment #2.. .......................................................... 41 Figure 10. Body mass and body composition for all mice in treatment regimen #1. .............................................................................................................. 46 Figure 11. Body mass and body composition for smaller sets of mice used for microarray experiment #1.. ....................................................................... 47 Figure 12. Liver mass and triglyceride content for all mice in treatment regimen #1. .............................................................................................................. 49 Figure 13. Liver mass and triglyceride content for smaller sets of mice used for microarray experiment #1. ........................................................................ 49 Figure 14. Plasma IGF-1 and intraperitoneal glucose tolerance test for all mice in treatment regimen #1. ............................................................................ 53 Figure 15. Plasma IGF-1 and intraperitoneal glucose tolerance test for smaller sets of mice used for microarray experiment #1.. ..................................... 53 Figure 16. Hierarchical clustering. The 16 arrays from microarray experiment #1.. ............................................................................................................. 55 Figure 17. Body mass for mice in treatment regimen #2. ............................................ 60 Figure 18. Fat mass for mice in treatment regimen #2.. .............................................. 61 Blischak 8 Figure 19. Lean mass for mice in treatment regimen #2.. ..........................................
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