UNIVERSITY OF CALIFORNIA Los Angeles Ellagic Acid and Urolithin A Improve Insulin Sensitivity in Diet-Induced Insulin Resistant Mice and Reduce Detrimental Effects of Palmitate Administration in Differentiated C2C12 Myotubes. A thesis submitted in partial satisfaction of the requirements for the degree Master of Science in Biochemistry, Molecular and Structural Biology by Brenda Chan 2018 © Copyright by Brenda Chan 2018 ABSTRACT OF THE THESIS Ellagic Acid and Urolithin A Improve Insulin Sensitivity in Diet-Induced Insulin Resistant Mice and Reduce Detrimental Effects of Palmitate Administration in Differentiated C2C12 Myotubes. by Brenda Chan Master of Science in Biochemistry, Molecular and Structural Biology University of California, Los Angeles, 2018 Professor Catherine F. Clarke, Chair Insulin resistance has been spreading as food and sedentary lifestyles are becoming more common. Major bodily complications often result, necessitating a search for affordable solutions. Phytochemicals are commonly used in alternative medicine but less so in areas where obesity and T2D prevail. This thesis studies ellagic acid (EA) and its metabolite urolithin A (UA) as potential treatments for insulin resistance, focusing on the main site of glucose uptake, skeletal muscle. In diet-induced insulin-resistant mice, long-term dietary EA&UA administration reduced glucose levels in IPITTs, while UA alone also reduced serum FFA and fasting glucose levels. Mitochondrial turnover and ROS detoxification markers increased with treatment, indicating improved mitochondrial quality control. Differentiated C2C12 mouse myotubes were utilized to study cellular effects of EA and UA. In insulin resistant myotubes, treatment enhanced ATP ii production and reduced ROS, cytotoxicity, and apoptosis. With insulin, UA increased uncoupled respiration while decreasing ROS, implying increased fuel oxidation without adding oxidative stress. UA tended to decrease glucose uptake as it increased cellular respiration, suggesting a stronger utilization of fatty acids. The results of this thesis support EA and UA as promising treatments for insulin resistance and the metabolic syndrome. iii The thesis of Brenda Chan is approved. Zhaoping Li Steven G. Clarke Catherine F. Clarke, Committee Chair University of California, Los Angeles 2018 iv DEDICATION This thesis is dedicated to my family, other loved ones, and all my mentors along the way. I couldn’t have asked for a more supportive crew. v Table of Contents Abstract ........................................................................................................................................... ii Committee Members ...................................................................................................................... iv Dedication ........................................................................................................................................v Table of Contents ........................................................................................................................... vi List of Figures ............................................................................................................................... vii Acknowledgements ...................................................................................................................... viii Introduction ......................................................................................................................................1 Materials and Methods ...................................................................................................................14 Results ............................................................................................................................................23 In vivo mouse studies.....................................................................................................................23 In vitro C2C12 studies ...................................................................................................................34 Discussion ......................................................................................................................................51 Supplementary Data .......................................................................................................................64 References ......................................................................................................................................71 vi List of Figures and Tables Figure 1. Gut Microbiota Metabolism of ellagitannins and ellagic acid .......................................11 Figure 2. Experimental design of in vivo mouse study .................................................................23 Figure 3. Body weights and food intake at end of study ...............................................................24 Figure 4. Baseline and 8-week IGTTs and IPITTs ........................................................................25 Figure 5. IPITTs at end of study ....................................................................................................26 Figure 6. Serum FFA, Adiponectin, and Fasting Glucose .............................................................27 Figure 7. Fatty acid metabolism markers in skeletal muscle .........................................................28 Figure 8. Mitochondrial biogenesis and mitophagy markers in skeletal muscle ...........................31 Figure 9. ROS detoxification markers in skeletal muscle ..............................................................33 Figure 10. Experimental design of C2C12 culture assays ....................................................... 34-35 Figure 11. ATP Viability Assay ....................................................................................................36 Figure 12. MTT Viability Assay ....................................................................................................37 Figure 13. Cytotoxicity and Caspase Assay ..................................................................................38 Figure 14. Intramyocellular Lipid Assay .......................................................................................40 Figure 15. Glucose Uptake Assay .................................................................................................41 Figure 16. Reactive Oxygen Species Assay ..................................................................................42 Figure 17. Metabolic Function of C2C12 cells in BSA and palmitate (+ and – insulin)......... 44-45 Figure 18. Metabolic Function of C2C12 cells in BSA (- insulin) ................................................47 Figure 19. Metabolic Function of C2C12 cells in BSA (+ insulin) ...............................................48 Figure 20. Metabolic Function of C2C12 cells in palmitate (- insulin) .........................................49 Figure 21. Metabolic Function of C2C12 cells in palmitate (+ insulin) ........................................50 Supplementary Table S1. Macronutrient Compositions of LF/HS and HF/HS diets ....................64 Supplementary Table S2. Primers used for qPCR ................................................................... 64-65 Supplementary Figure S1. Inflammatory markers in mouse skeletal muscle ................................65 Supplementary Figure S2. Intramyocellular levels of fatty acids ..................................................66 Supplementary Figure S3. Histology of mice skeletal muscle ......................................................66 Supplementary Figure S4. Mitochondrial markers in differentiated C2C12 myotubes ...............67 Supplementary Figure S5. ROS detoxification and inflammatory markers in C2C12 myotubes 68 Supplementary Figure S6. ATP content in C2C12 myotubes ......................................................69 Supplementary Figure S7. Fatty acid markers in C2C12 myotubes .............................................70 vii Acknowledgements I would like to thank a number of individuals for their support and guidance throughout my education. I am greatly indebted to my mentor, Jieping Yang, who has opened my eyes to the field of research in countless ways. She has been incredibly understanding and patient, teaching me not only the necessary techniques required to work in a lab, but also how to think more critically. Thank you for always encouraging me to further my education and believing in me. I am also grateful for all the others in my lab who think I have yet to reach my full potential. Next, I want to thank my committee members for taking the time to provide me with feedback on my thesis. They have been the kindest guides, using their decades of experience to discuss possible future paths in this field. Third, my past mentors, Luo Mi and Joe Schinaman, deserve much appreciation for their patience in introducing me to research. Fourth, I want to thank my friends who have supported me through my endeavors and pushed me to work as hard as they do. My roommates have provided me with both an intellectually stimulating and relaxing environment, helping me through this arduous journey. Lastly, I would not have been able to complete, or even begin my education without my family. I must thank my grandparents for helping put my brother and me through college and stressing the importance of a good education. My parents have been the most instrumental in getting me to where I am today; they have been so supportive and caring in helping