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Impact of Ergot Alkaloid and Estradiol 17B on Whole-Body Protein Turnover and Expression of Mtor Pathway Proteins in Muscle of Cattle

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Impact of Ergot Alkaloid and Estradiol 17B on Whole-Body Protein Turnover and Expression of Mtor Pathway Proteins in Muscle of Cattle University of Kentucky UKnowledge Theses and Dissertations--Animal and Food Sciences Animal and Food Sciences 2020 Impact of Ergot Alkaloid and Estradiol 17B on Whole-Body Protein Turnover and Expression of mTOR Pathway Proteins in Muscle of Cattle Taylor Dawn Ferguson University of Kentucky, [email protected] Author ORCID Identifier: https://orcid.org/0000-0001-6598-4133 Digital Object Identifier: https://doi.org/10.13023/etd.2020.459 Right click to open a feedback form in a new tab to let us know how this document benefits ou.y Recommended Citation Ferguson, Taylor Dawn, "Impact of Ergot Alkaloid and Estradiol 17B on Whole-Body Protein Turnover and Expression of mTOR Pathway Proteins in Muscle of Cattle" (2020). Theses and Dissertations--Animal and Food Sciences. 122. https://uknowledge.uky.edu/animalsci_etds/122 This Master's Thesis is brought to you for free and open access by the Animal and Food Sciences at UKnowledge. It has been accepted for inclusion in Theses and Dissertations--Animal and Food Sciences by an authorized administrator of UKnowledge. For more information, please contact [email protected]. STUDENT AGREEMENT: I represent that my thesis or dissertation and abstract are my original work. Proper attribution has been given to all outside sources. I understand that I am solely responsible for obtaining any needed copyright permissions. I have obtained needed written permission statement(s) from the owner(s) of each third-party copyrighted matter to be included in my work, allowing electronic distribution (if such use is not permitted by the fair use doctrine) which will be submitted to UKnowledge as Additional File. I hereby grant to The University of Kentucky and its agents the irrevocable, non-exclusive, and royalty-free license to archive and make accessible my work in whole or in part in all forms of media, now or hereafter known. I agree that the document mentioned above may be made available immediately for worldwide access unless an embargo applies. I retain all other ownership rights to the copyright of my work. I also retain the right to use in future works (such as articles or books) all or part of my work. I understand that I am free to register the copyright to my work. REVIEW, APPROVAL AND ACCEPTANCE The document mentioned above has been reviewed and accepted by the student’s advisor, on behalf of the advisory committee, and by the Director of Graduate Studies (DGS), on behalf of the program; we verify that this is the final, approved version of the student’s thesis including all changes required by the advisory committee. The undersigned agree to abide by the statements above. Taylor Dawn Ferguson, Student Dr. Kyle R. McLeod, Major Professor Dr. David L. Harmon, Director of Graduate Studies IMPACT OF ERGOT ALKALOID AND ESTRADIOL 17B ON WHOLE-BODY PROTEIN TURNOVER AND EXPRESSION OF MTOR PATHWAY PROTEINS IN MUSCLE OF CATTLE ________________________________________ THESIS ________________________________________ A thesis submitted in partial fulfillment of the requirements for the degree of Master of Science in the College of Agriculture, Food and Environment at the University of Kentucky By Taylor Dawn Ferguson Lexington, Kentucky Director: Dr. Kyle McLeod, Professor of Animal Science Lexington, Kentucky 2020 Copyright © Taylor Dawn Ferguson 2020 https://orcid.org/0000-0001-6598-4133 ABSTRACT OF THESIS IMPACT OF ERGOT ALKALOID AND ESTRADIOL 17B ON WHOLE-BODY PROTEIN TURNOVER AND EXPRESSION OF MTOR PATHWAY PROTEINS IN MUSCLE OF CATTLE Beef cattle consuming endophyte infected tall fescue typically exhibit reduced performance in terms of both decreased dry matter intake (DMI) and growth rates. It has been suggested that lower concentrations of circulating IGF-1 (an important stimulator of the mTOR pathway and ultimately protein synthesis), as observed with consumption of ergot alkaloids, contribute to reduced growth rates. The objective of the current study was to determine if fescue-derived alkaloids decrease muscle protein synthesis though inhibitory action on the mTOR pathway via a direct effect on signal proteins, and if these negative effects can be alleviated by implantation with anabolic agents. Thirty-two Holstein steers were used in a 2x2 factorial design, and treatments consisted of intramuscular administration of bromocriptine (vehicle or 0.1 mg/kg BW) and a subdermal estradiol implant (with or without). Throughout the 35-day experiment, steers were fed a corn silage-based diet, with intake restricted to 1.5 times maintenance energy requirement. Bromocriptine injections were given every three days for 34 days. On days 27 through 32, steers were moved to metabolism stalls for urine collection and whole-body protein turnover was determined using a single pulse dose of [15N] glycine into the jugular vein on day 28. On day 35, muscle samples were collected from the musculus obliquus externus abdominis before (basal state) and 60 mins after (stimulated state) an i.v. glucose challenge (0.25 g glucose/kg). Blood samples were collected at regular intervals before and after glucose infusion for determination of circulating concentrations of glucose and insulin. Bromocriptine reduced insulin and glucose clearance following the glucose challenge, indicating decreased insulin sensitivity and possible disruption of glucose uptake and metabolism in the skeletal muscle. This suggests that fescue-derived alkaloids are detrimental to growing cattle in terms of overall glucose homeostasis and energy metabolism. Conversely, analysis of whole-body protein turnover demonstrated that bromocriptine does not appear to affect protein synthesis or N retention and western immunoblot analysis of skeletal muscle showed that it did not affect abundance of S6K1 or 4E-BP1, so does not appear to inhibit activation of the mTOR pathway or protein synthesis. Implantation improved N retention, decreased protein turnover, and had no effect on protein synthesis, suggesting that steroidal implants promote protein accretion through unchanged rates of synthesis and decreased degradation, even in the presence of bromocriptine, resulting in improved daily gains. Implanted steers likely experienced increased IGF-1 signaling, but downstream activation of mTOR, S6K and 4E-BP1, and thus increased protein synthesis did not occur as expected. Overall, this data suggests that fescue derived alkaloids do not have a negative impact on muscle protein synthetic pathways, independent of DMI. KEYWORDS: ruminant, bovine, ergot alkaloids, mtor pathway, protein metabolism Taylor Dawn Ferguson (Name of Student) 10/30/2020 Date IMPACT OF ERGOT ALKALOID AND ESTRADIOL 17B ON WHOLE-BODY PROTEIN TURNOVER AND EXPRESSION OF MTOR PATHWAY PROTEINS IN MUSCLE OF CATTLE By Taylor Dawn Ferguson Dr. Kyle R. McLeod Director of Thesis Dr. David L. Harmon Director of Graduate Studies 10/30/2020 Date ACKNOWLEDGMENTS I would like to thank my advisor, Dr. Kyle McLeod, for giving me this opportunity; I have appreciated his guidance, expertise, and patience through this long process. I also want to thank my committee members Dr. Kristine Urschel and Dr. Eric Vanzant for their time and guidance. Especially, Dr. Urschel for the use of her lab and assistance with western blotting, and Dr. Vanzant for all his help with my nemesis — statistics. Further thanks goes to Dr. James Matthews for letting me pick his brain about blotting on occasion. I never would have managed my thesis work without the help of our lab technicians Adam Bohannon, Cynthia Roberts, and Winston Lin, who are always willing to answer questions and help when needed. Adam’s expertise in the lab and assistance with animal care was invaluable and I literally could not have completed my study without him. Additional thanks go to the University of Kentucky Beef Unit farm staff, especially Lauren Clark and Kirk Vanzant, for their assistance with day to day care of my animals and completion of my experiment. Thank you to Kyle McLean for assistance on biopsy day and Hannah Herzing for running ion exchange columns. I have been lucky to have the support of many other people during my time at UK and have made invaluable friendships. I am grateful for the guidance of Amanda Pesqueira Schiff and the continued support I have received from Miriam Snider, Sophie Stratton, and Ashley Gerritsen — I never would have made it to this point without you guys. Special thanks go to Dr. Caroline Loos for teaching me the art of western blotting and for all her help troubleshooting when it invariably went wrong. iii Lastly, I would like to thank my brother, Nathan, for always keeping me honest and being the only person in my family science-y enough to understand what this thesis is about, and his wife Kaitlyn who definitely doesn’t care about cows but listens to me talk about rumens nonetheless. Thank you to my ever supportive and long-suffering parents, Joel and Cathy, for their love and encouragement. iv TABLE OF CONTENTS ACKNOWLEDGMENTS ...................................................................................................................... iii LIST OF TABLES ................................................................................................................................ vii LIST OF FIGURES .............................................................................................................................. viii FREQUENTLY USED ABBREVIATIONS ......................................................................................... ix CHAPTER 1. Introduction .....................................................................................................................
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