CARBOXYLESTERASE 1 PLAYS A PROTECTIVE ROLE AGAINST METABOLIC DISEASE A dissertation submitted to Kent State University in partial fulfillment of the requirements for the degree of Doctor of Philosophy By Jiesi Xu May 2016 © Copyright All rights reserved Except for previously published materials Dissertation written by Jiesi Xu B.S., Jilin University 2008 Ph.D., Kent State University 2016 Approved by ___________________________________ , Chair, Doctoral Dissertation Committee Dr. Yanqiao Zhang, M.D., Associate Professor, NEOMED ___________________________________ , Member, Doctoral Dissertation Committee Dr. John Y.L. Chiang Ph.D., Distinguished Professor, NEOMED ___________________________________, Member, Doctoral Dissertation Committee Dr. Colleen M. Novak, Ph.D., Associate Professor, Kent State University ___________________________________, Member, Doctoral Dissertation Committee Dr. Min You, Ph.D., Professor, NEOMED ___________________________________, Member, Doctoral Dissertation Committee Dr. Eric M. Mintz, Ph.D., Professor, Associate dean, Kent State University Accepted by ___________________________________ , Director, School of Biomedical Sciences Dr. Ernest Freeman, Ph.D. ___________________________________ , Dean, College of Arts and Sciences Dr. James L. Blank, Ph.D ii TABLE OF CONTENTS TABLE OF CONTENTS LIST OF FIGURES ............................................................................................................v LIST OF ABBREVIATIONS .........................................................................................viii ACKNOWLEDGEMENTS ...............................................................................................x ABSTRACT ......................................................................................................................xi CHAPTER 1 THE ROLE OF CARBOXYLESTERASE 1 IN NON-ALCOHOLIC FATTY LIVER DISEASE AND CARBOHYDRATE METABOLISM………………...1 1.1 INTRODUCTION……………........................................................................ 1 1.2 METHODS…………........................................................................................7 1.3 RESULTS .......................................................................................................14 1.3.1 Hepatic Carboxylesterase1 is Induced by Glucose and Regulates Postprandial Glucose Levels................................................................................. 14 1.3.2 Hepatic Carboxylesterase 1 is Essential for Normal and Farnesoid X Receptor-Controlled Lipid Homeostasis................................................................25 1.4 DISCUSSION……………………..................................................................50 CHPTER 2 THE ROLE OF CARBOXYLESTERASE 1 IN ALCOHOLIC LIVER DISEASE………………………………...…................................................................... 56 2.1 INTRODUCTION.......................................................................................... 56 2.2 METHODS..................................................................................................... 59 2.3 RESULTS....................................................................................................... 67 iii 2.4 DISCUSSION ................................................................................................ 96 CHAPTER 3 THE ROLE OF CARBOXYLESTERASE 1 IN ATHEROSCLEROSIS…………………………............................................................100 3.1 INTRODUCTION.........................................................................................100 3.2 METHODS....................................................................................................103 3.3 RESULTS......................................................................................................105 3.4 DISCUSSION ...............................................................................................111 CHAPTER 4 CONCLUSION…….……………............................................................113 REFERENCES…………………………………………………………………………116 iv LIST OF FIGURES Figure 1. Hepatic CES1 is regulated by nutritional status……………………………….15 Figure 2. Hepatic CES1 is regulated by glucose but not insulin………………………...18 Figure 3. ACL is required for glucose-induced hepatic CES1 expression………………21 Figure 4. ACL is required for glucose-mediated acetylation of histones (H3, H4) in the CES1 chromatin………………………………………………………….…….22 Figure 5. CES1 regulates postprandial levels…………………………………………....24 Figure 6. Hepatic expression of CES1 lowers hepatic triglyceride levels and improves glucose homeostasis………………………………………………………...26,27 Figure 7. Hepatic expression of CES1 selectively regulates gene expression and has no effect on lipogenesis or VLDL secretion………………………………………30 Figure 8. Hepatic expression of CES1 increases triglyceride hydrolase activity and activates PPARα……………………………………………………………….32 Figure 9. Loss of hepatic CES1 causes fatty liver and increased plasma cholesterol level…………………………………………………………………………….35 Figure 10. Loss of hepatic CES1 induces de novo lipogenesis………………………….37 Figure 11. Hepatic CES1 is regulated by FXR…………………………………………..40 Figure 12. CES1 is a direct FXR target gene…………………………………………….43 Figure 13. Essential roles of hepatic CES1 in FXR-regulated lipid homeostasis………..45 Figure 14. Effects of the FXR agonist OCA (INT-747) on lipid and glucose homeostasis in C57BL/6 mice……………………………………………………………….47 Figure 15. Effects of the FXR agonist OCA on lipid homeostasis in ob/ob mice……….49 v Figure 16. CES1 and HNF4 expressions are reduced in patients with alcoholic steatohepatitis and in mouse and mouse primary hepatocytes treated with ethanol………………………………………………………………………….68 Figure 17. CES1 is regulated by HNF4………………………………………………...71 Figure 18. CES1 is a direct target of HNF4……………………………………………72 Figure 19. Over-expression of hepatic CES1 protects against alcohol-induced triglyceride accumulation in AML12 cells………………………………………………….74 Figure 20. Hepatic CES1 deficiency alters plasma lipid levels in response to alcohol challenge……………………………………………………………………….75 Figure 21. Hepatic CES1 deficiency exacerbates alcohol-induced hepatic steatosis……77 Figure 22. Hepatic CES1 deficiency exacerbates alcohol-induced liver inflammation…………………………………………………………………...80 Figure 23. Global deletion of CES1 does not exacerbate alcohol-induced hepatic steatosis...………………………………………………………………………83 Figure 24. Global deletion of CES1 exacerbates alcohol-induced liver inflammation….85 Figure 25. Global deletion of CES1 increases MCD diet-induced liver inflammation………………………………………………………..………….88 Figure 26. CES1 deficiency does not significantly change fibrogenic gene expressions……………………………………………………………………..88 Figure 27. Global deletion of CES1 does not exacerbate MCD-diet induced fibrosis….............................................................................................................89 Figure 28. Global deletion of CES1 increases hepatic acetaldehyde level and oxidative stress……………………………………………………………………………92 vi Figure 29. Global deletion of CES1 does not change mRNA levels of genes involved in fatty acid metabolism…………………………………………………………..95 Figure 30. Macrophage cholesterol efflux……………………………………………...102 Figure 31. Global deletion of CES1 results in increased lipid accumulation in macrophages………………………………………………………………….105 Figure 32. Loss of hepatic CES1 increases lipid contents in ApoE mice……………106 Figure 33. Loss of hepatic CES1 shows atherosclerotic lipid profile…………………..108 Figure 34. Loss of hepatic CES1 aggravates atherosclerosis in ApoE mice…………110 vii LIST OF ABBREVIATIONS ABCA1 ATP binding cassette sub-family A1 ABCG5 ATP binding cassette sub-family G5 ACC acetyl-CoA carboxylase ACL ATP citrate lyase AKT protein kinase B ALD alcoholic liver disease APOB apolipoprotein B CD36 cluster of differentiation 36 CES1 carboxylesterase 1 ChIP chromatin immunoprecipitation CPT carnitine palmitoyltransferase DGAT diacylglycerol O-acyltransferase EMSA electrophoretic mobility shift assay FAS fatty acid synthase FFA free fatty acid FPLC fast protein liquid chromatography FXR farnesoid X receptor GCK glucose kinase G6Pase glucose-6 phosphatase HDL high density lipoprotein HMGCS HMG-CoA synthase viii HMGCR HMG-CoA reductase HNF4 hepatocyte nuclear factor 4 IL-1 interleukin 1 IL-6 interleukin 6 LDL low density lipoprotein L-PK liver pyruvate kinase MCP monocyte chemoattractant protein MDA malondialdehyde MTP microsomal triglyceride transfer protein NAFLD non-alcoholic fatty liver disease PPAR peroxisome proliferator-activated receptor PDK pyruvate dehydrogenase kinase PEPCK phosphoenolpyruvate carboxykinase PGC1 peroxisome proliferator-activated receptor coactivator 1 ROS reactive oxygen species SREBP1C sterol regulatory binding protein 1c TC total cholesterol TNF tumor necrosis factor TG triglyceride VLDL very low density lipoprotein ix ACKNOWLEDGEMENTS I would like to express my gratitude to all the people who gave me help and support throughout the course of this work. I would especially like to thank my advisor Dr. Yanqiao Zhang for his invaluable guidance, caring and immense knowledge. I would also like to thank him for pushing me father than I thought I could go and providing financial support for my research. I wish to express my sincere thank to Dr. John Chiang, Dr. Colleen Novak and Dr. Min You for giving me insightful suggestions regarding my research. Their guidance and encouragements help my research from various perspectives. I would like to acknowledge and thank Dr. Novak for the help she has given with CLAMS analysis.