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Omentin Activates AMP-Activated Protein Kinase and Plays a Role in Energy Metabolism and Immune Response

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Omentin Activates AMP-Activated Protein Kinase and Plays a Role in Energy Metabolism and Immune Response Omentin Activates AMP-activated Protein Kinase and Plays a Role in Energy Metabolism and Immune Response Item Type dissertation Authors Yu, Daozhan Publication Date 2011 Abstract Obesity is a risk factor for type 2 diabetes, cardiovascular diseases, and other diseases as well. It has been recognized that adipose tissue is an active endocrine organ that secrets many adipokines to regulate energy metabolism and inflammation. Ou... Keywords AMPK; fatty acid oxidation; omentin; AMP-Activated Protein Kinases; Diabetes Mellitus, Type 2; Inflammation; Insulin Resistance Download date 24/09/2021 17:49:22 Link to Item http://hdl.handle.net/10713/542 Abstract Title of Dissertation: Omentin Activates AMP-activated Protein Kinase and Plays a Role in Energy Metabolism and Immune Response Name of Candidate: Daozhan Yu ，Doctor of Philosophy, 2011 Dissertation Directed By: Da-Wei Gong, M.D., Ph.D. Associate Professor of Medicine Division of Endocrinology, Diabetes and Nutrition University of Maryland, Baltimore Obesity is a risk factor for type 2 diabetes, cardiovascular diseases, and other diseases as well. It has been recognized that adipose tissue is an active endocrine organ that secrets many adipokines to regulate energy metabolism and inflammation. Our group has identified omentin as among the first omental fat-specific adipokine in humans and has shown that omentin can enhance insulin-mediated glucose transport and AKT phosphorylation in adipocytes. Here, I have further characterized the role of omentin in metabolism and inflammation. In this study, I first expressed omentin protein in High-Five insect cell expression system and then purified it with a galactose-conjugated affinity column. In C2C12 myocytes, omentin increases AMP-activated protein kinase (AMPK) phosphorylation, which is known to be important for energy metabolism. Downstream target of AMPK, acetyl-CoA carboxylase (ACC), is also phosphorylated in a dose-dependent manner. Accordingly, fatty acid oxidation was increased with omentin treatment dose-dependently. Omentin inhibits phosphorylation of another downstream target of AMPK, p70S6K, and increases insulin-mediated glucose uptake, indicating that omentin may enhance insulin activity by this mechanism. Moreover, administration of omentin in mice increases ACC phosphorylation in muscle and liver, and improves glucose disposal in an intra-peritoneal glucose tolerance test. In endothelial cells, omentin activates AMPK and eNOS phosphorylation. I also demonstrate that omentin increases cell migration with human primary mesothelial cells and breast cancer cell line MCF-7. Finally, I show that IL-6 induces omentin secretion in endothelial cells and mesothelial cells. These findings suggest that omentin may participate in immune response. To explore possible mechanism of omentin signaling, I used yeast two hybrid system and identified a possible omentin interactive protein, Loc401397. The full length gene has not been cloned yet, probably due to the upstream GC-rich region which might jeopardize the reverse transcription. In summary, in this study I have demonstrated that omentin activates AMPK and regulates energy metabolism in vitro and in vivo . Moreover, omentin activates AMPK-eNOS pathway and promotes migration of mesothelial cells to defend host during infection. These findings suggest that omentin is a multifunctional protein that plays a role in both energy metabolism and inflammatory response. Omentin Activates AMP-activated Protein Kinase and Plays a Role in Energy Metabolism and Immune Response By Daozhan Yu Dissertation submitted to the Faculty of the Graduate School of the University of Maryland Baltimore in partial fulfillment of the requirements for the degree of Doctor of Philosophy 2011 Acknowledgements This dissertation would not have been possible without the outstanding mentorship of Dr. Da-Wei Gong. I am extremely grateful for his excellent guidance, receptivity to my ideas and generous support throughout this work in the lab and beyond. His enthusiasm and optimistic outlook on science, and life in general, enlightened my life in the past years and will benefit my entire career in the future. I am lucky and honored to be his graduate student. I would also like to extend my gratitude to members of my thesis committee: Dr. Alan R. Shuldiner for his immense support and advices; Dr. Susan K. Fried for her critical suggestions and help with experiments and thesis writing; Dr. John C. McLenithan for his instruction on experiment design, cell culture and other lab techniques, and valuable advice on thesis writing; Dr. James A. Mixson for his time, insights and full support. All the members have been so kind and helpful during the whole journey, and without them the dissertation would not be achievable. I have been trying to inherit their curiosity and wisdom in science and also life in general. I would also like to thank current or previous members in the division. Dr. Rongze Yang has been of tremendous help with experiment design, lab techniques, presentation, grant and thesis writing, and beyond. I am thankful to his support both in the lab and life. Dr. MiJeong Lee has been very helpful with experiment design, techniques and reagents. I thank Dr. Carole Sztalryd Woodle for her expertise and help. Her craziness about fat cell is always inspiring. I am also thankful to Hong Hu for her supportive work and excellent management in the lab. I thank Dr. Celia Batista for her kind help and encouragement. I want to thank Nikki Glenn, Jie Liu, Mao Fu, Xiaochun Wang, Urmila Sreenivasan, Christy Chang, Yanbei Zhu, Keith Tanner, John Shelton and all the other members in the division who have been so helpful and supportive these years. Special thanks to Sara Sturgess, Karen Norton and Delores Anderson who helped so much with the appointments, presentations, meetings and managements. i I also want to thank my program directors, Dr. Paul Welling, Dr. Toni Antalis, Dr. Terez Shea-Donohue and program coordinator, Ms. Sharron Graves, for their directing, assistance and great help making my training possible and fluent. Last but not the least; I want to thank my families for their love and patience. My parents, Jiaxian Yu and Jinghua Zhu, have been tremendously supportive financially and spiritually since my birth. My lovely sister, Linlin Yu, has also been so considerate and helpful. I deeply thank my beloved wife, Dong Han, for her unconditional caring and love. Thanks for helping make this long challenging journey much easier, enjoying and more meaningful. I love you. I also want to thank my two-year old son, Jason Yu, for being so sweet and bringing me so much joy. Thank you all my friends! - ii - TABLE OF CONTENTS Acknowledgements..............................................................................................i Table of Contents ..............................................................................................iii List of Tables....................................................................................................Viii List of Figures ....................................................................................................ix List of Abbreviations...........................................................................................xi CHAPTER 1: GENERAL INTRODUCTION ...........................................................1 1.1 Insulin resistance syndrome ........................................................................2 1.1.1 Obesity is becoming an epidemic and is the major risk factor for insulin resistance………………….....................................................................2 1.1.2 Pathogenesis of insulin resistance….....................................................5 1.1.2.1 “Lipotoxicity” in insulin resistance ...................................................5 1.1.2.2 Inflammation in insulin resistance ...................................................6 1.2.1 Adipose tissue as a fat storage depot....................................................9 1.2.2 Adipose tissue as an endocrine organ.................................................10 1.3 Omentin is a new adipokine........................................................................13 1.3.1 Molecular and cell biology of omentin..................................................13 1.3.2 Role of omentin in energy metabolism.................................................13 1.3.3 Role of omentin in innate immune response........................................15 1.3.3.1 Introduction of immune system......................................................15 1.3.3.2 Regulation of omentin during infection...........................................16 1.3.3.3 Omentin belongs to lectin family. ..................................................17 1.3.3.4 Omenin as a lactoferrin receptor. ..................................................18 1.4 Goal of this dissertation...............................................................................19 CHAPTER 2: PRODUCTION AND PURIFICATION OF RECOMBINANT HUMAN OMENTIN FOR FUNCTIONAL STUDY ........................................21 2.1 Introduction..................................................................................................22 2.1.1 General considerations.........................................................................22 - iii - 2.1.2 Purification methods…………………..………………………………..….23 2.1.2.1 Size exclusion chromatography……………………………………...23 2.1.2.2 Affinity chromatography ……………………………………….……..24 2.1.3 Characterization of purified
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