THE ROLE of KYNURENINE, a TRYPTOPHAN METABOLITE THAT INCREASES with AGE, in MUSCLE ATROPHY and LIPID PEROXIDATION) by Helen Eliz
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THE ROLE OF KYNURENINE, A TRYPTOPHAN METABOLITE THAT INCREASES WITH AGE, IN MUSCLE ATROPHY AND LIPID PEROXIDATION) By Helen Elizabeth Kaiser Submitted to the Faculty of the Graduate School of Augusta University in partial fulfillment of the Requirements of the Degree of (Doctor of Philosophy in Cellular Biology and Anatomy) April 2020 COPYRIGHT© 2020 by Helen Kaiser ACKNOWLEDGEMENTS I wish to express my deepest gratitude to my mentor Dr. Mark Hamrick. His support and advice were invaluable to my learning and development as a scientist. I would like to especially recognize Dr. Hamrick’s honesty to data, and kindness to everyone around him. It was an honor to work as his student, and to be a part of this project. I would like to thank my committee members, Drs. Carlos Isales, Meghan McGee-Lawrence, and Yutao Liu, for their continued support, guidance, and helpful suggestions. They have helped to shape and focus this project, and have augmented my experience as a student during the development of my dissertation. Additionally, I want to recognize and thank Dr. Sadanand Fulzele for sharing his wealth of knowledge in techniques, and always keeping his door open for student’s questions. Next, a most heartfelt thanks to my lab mates: Andrew Khayrullin, Bharati Mendhe, Ling Ruan and Emily Parker. This project has been a team effort, all of them have been fundamental to its success. I further want to show my appreciation for the rest of the department of Cellular Biology and Anatomy at Augusta University. The histology core members Donna Kumiski and Penny Roon for their direct help in sectioning, and contributions to this project. Also Cotton Mitchell, Diane Riffe, and Donna Tuten for their administrative support, and their constant encouragement. I thank Drs. Watsky and Cameron and the Graduate School team for allowing me the opportunities and resources to grow as both a leader and professional over the years, aiding me with career-developing opportunities and travel support to present my work at Experimental Biology in 2018, and The International Conference of Frailty and Sarcopenia in 2019. I thank my family for their unwavering support as I've gone through the highs and lows of my professional degree. I am especially grateful to my husband, Jacob Kaiser. His unconditional love, support, and encouragement over the years have been vital to this journey. Finally, to my dogs: Gordon, Mica and Oti for their love and helpful suggestions while writing this thesis. ABSTRACT HELEN KAISER The Role of Kynurenine, a Tryptophan Metabolite that increases with age, in Muscle Atrophy and Lipid Peroxidation. (Under the direction of DR. MARK HAMRICK) Loss of mobility and independence are risk factors for falls and mortality, and drastically reduce the quality of life among older adults. The cellular and molecular mechanisms underlying loss of muscle mass and strength with age (sarcopenia) are not well- understood; however, heterochronic parabiosis experiments show that circulating factors are likely to play a role. Kynurenine (KYN) is a circulating tryptophan metabolite that is known to increase with age and is implicated in several age-related pathologies. Here I tested the hypothesis that KYN contributes directly to muscle loss with aging. Results indicate that that KYN treatment of mouse and human myoblasts increased levels of reactive oxygen species (ROS) two-fold, and significantly increased lipid peroxidation enzymes. Small-molecule inhibition of the Aryl hydrocarbon receptor (Ahr), an endogenous KYN receptor, in vitro did not prevent KYN-induced increases in ROS, and homozygous Ahr knockout in vivo did not protect mice from KYN-induced stress, suggesting that KYN can directly increase ROS independent of Ahr activation. In vivo, wild-type mice treated with KYN had reduced skeletal muscle strength, size, and increased oxidative stress and lipid peroxidation. Old wild-type mice treated with 1MT, a small molecule that suppresses KYN production by IDO1, showed an increase in muscle fiber size, peak muscle strength, and oxidative stress. Protein analysis identified mitochondrial lipid peroxidation as a downstream mechanism that is increased upon KYN treatment. Lipid peroxidation enzymes increased with KYN have been shown to produce H2O2 outside of the electron transport chain. Our data suggest that IDO inhibition may represent a novel therapeutic approach for the attenuation of sarcopenia and possibly other age-associated conditions associated with KYN accumulation such as bone loss and neurodegeneration KEY WORDS: (Muscle, sarcopenia, tryptophan, kynurenine, lipid peroxidation, muscle atrophy, aging, Table of Contents Chapter Page 1 Introduction and specific aims ...........................................................................1 1.1 Statement of a problem ............................................................................1 1.2 Specific aims ............................................................................................4 1.3 Innovation ................................................................................................5 2 Literature review ................................................................................................6 2.1 Healthspan vs. Lifespan ..........................................................................6 2.2 Sarcopenia ...............................................................................................7 2.3 ROS and muscle atrophy .......................................................................11 2.4 Circulating factors ................................................................................14 2.5 Kynurenine in health and disease .........................................................16 3 Materials and Methods .....................................................................................28 3.1 Cell culture ............................................................................................28 3.2 MTT assay .............................................................................................28 3.3 AmplexTM Red assay ..............................................................................29 3.4 Total Antioxidant Capacity assay..........................................................29 3.5 Animal experimental design ..................................................................30 3.6 Histological staining .............................................................................31 3.7 Proteomics .............................................................................................32 3.8 Western blots .........................................................................................32 3.9 PCR array plates ...................................................................................33 3.10 In vivo functional muscle testing .........................................................33 3.11 Ex vivo muscle testing .........................................................................34 3.12 Open field activity assays ....................................................................36 3.13 Cytokine array panels .........................................................................36 3.14 Statistical analysis. ..............................................................................37 4 Results ..............................................................................................................38 4.1 KYN treatment increases ROS levels in C2C12 mouse and primary human myoblasts. ..................................................................................38 4.2 Ahr inhibition did not rescue KYN effects in vitro. ...............................40 4.3 KYN treatment produces an aged-muscle phenotype in young female .43 4.4 Ahr knockout weakly atenuated KYN effects in female mice. ................46 4.5 1-MT treatment decreased oxidative stress and attenuates muscle atrophy old female mice ........................................................................48 4.6 Peak muscle strength is attenuated with KYN .......................................50 4.7 PCR arrays reveal structural muscle proteins are decreased by KYN .52 4.8 KYN did not change intracellular lipid accumulation ..........................54 4.9 Fiber type proteins are reduced with KYN treatment ...........................56 4.10 Proteomic analysis of KYN treated and 1-MT treated skeletal muscle reveals differential expression of mitochondrial very long chain acyl- coa dehydrogenase ................................................................................58 4.11 Old female mice treated with 1-MT reduced activity 30 days after 1- MT treatment .........................................................................................61 4.12 Muscle and serum cytokines were altered with 30 days of KYN treatment. ...............................................................................................63 5 Discussion ........................................................................................................66 6 Summary ..........................................................................................................74 References ..........................................................................................................................76 Appendices .........................................................................................................................99 A. Abbreviations .....................................................................................................99