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Understanding the Regulation of Adipogenesis and Adipocyte Metabolism in Obesity a Dissertation Submitted to the Faculty Of UNDERSTANDING THE REGULATION OF ADIPOGENESIS AND ADIPOCYTE METABOLISM IN OBESITY A DISSERTATION SUBMITTED TO THE FACULTY OF THE UNIVERSITY OF MINNESOTA BY MING ZHAO IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY Name of Advisor: XIAOLI CHEN AUGUST, 2015 © Ming Zhao 2015 ACKNOWLEDGMENTS First and foremost, I’d like to express my gratitude to my advisor Dr. Xiaoli Chen for her constant encouragement and support in the last five years. She has provided me great help in every possible way. I wouldn’t have completed the projects without her support. I would also like to thank to my committee members: Dr. Daniel Gallaher, Dr. David Bernlohr, and Dr. Douglas Mashek for their wisdoms and helpful commentaries on my research projects. I also want to thank my colleagues, especially Dr. Hong Guo and Dr. Yuanyuan Zhang and Jessica Deis, for their insightful comments and assistance. I also want to extend my gratitude to other faculty and staff in the Department of Food Science and Nutrition, who have offered help on my study and work. With all my heart I would like to thank my parents, for their spiritual support throughout my life. Finally, I’m grateful for having so many lovely friends here, who have turned life in the cold and lengthy winter in Minnesota into a colorful and unforgettable adventure. i ABSTRACT Inflammation in hypertrophic adipose tissue is a critical inducer of adipose tissue dysfunction, which ultimately leads to systemic insulin resistance and type 2 diabetes. Elevation of lipopolysaccharide (LPS) induced by high fat diet has been recently proposed to be one of the potential mechanisms contributing to the development of inflammation and metabolic dysfunction of adipose tissue in obesity. Therefore, it is of interest to expand our understanding of LPS effect on adipocyte metabolism and to identify the molecular mechanism by which LPS deteriorates adipose tissue metabolism. In the first study, we investigated the effect of LPS on the adipogenic capacity and cellular senescence of adipocyte progenitors by using stromal-vascular (SV) cells isolated from inguinal adipose tissue of C57BL/6 mice. We found that LPS treatment for 24-hour prior to the induction of differentiation led to the inhibition of adipogenesis. In addition, LPS induced features of premature senescence of SV cells. Further studies showed that LPS treatment caused a reduction in Zfp423 and PPARγ expression in SV cells, suggesting that LPS impairs pre-adipocyte differentiation. In the second study, we explored the role of endosomal/lysosomal protein NPC2 (Niemann-Pick disease, Type C2) in regulating lysosomal activity and in mediating LPS effect on adipocyte inflammation and function. NPC2 knockdown reduced lysosomal protease cathepsin B levels and impaired autophagy-lysosomal activity in 3T3-L1 adipocytes. Interestingly, NPC2 knockdown diminished LPS effect on inflammatory response and blunted LPS- induced glucose uptake in adipocytes. In the third study, we determined the effect of eicosapentaenoic acid (EPA) on promoting metabolic health of adipocytes, specifically the browning of subcutaneous white adipocytes. When added to SV cell cultures during ii 8-day adipocyte differentiation, EPA significantly increased the expression of thermogenic genes as well as mitochondrial DNA content. These results indicate that EPA enhances energy expenditure capacity by recruiting beige adipocytes. In summary, we have demonstrated a new mechanism by which LPS disrupts adipogenesis and adipocyte metabolism. We have also characterized the role of NPC2 as an important molecular mediator of LPS-induced adipocyte inflammation and the effect of EPA on promoting the browning of subcutaneous adipocytes. iii TABLE OF CONTENTS LIST OF TABLES.......................................................................................................... vi LIST OF FIGURES ....................................................................................................... vii LITERATURE REVIEW .................................................................................................. 1 1. Role of adipose tissue in metabolism ..............................................................................3 1.1 White adipose tissue (WAT)...............................................................................................3 1.2 Brown adipose tissue (BAT) ...............................................................................................3 1.3 Regulation of adipogenesis................................................................................................6 2. Adipose tissue dysfunction in obesity and insulin resistance............................................8 2.1 Inflammation links obesity and insulin resistance.............................................................9 2.2 TNFα as a major regulator of adipocyte metabolism and insulin resistance ..................13 2.3 Endotoxin (LPS) in obesity................................................................................................18 2.4 Inflammation inhibits the browning of white adipose tissue..........................................19 3. Mitochondrial function and insulin sensitivity ...............................................................20 3.1 Mitochondrial function in oxidative metabolism ............................................................21 3.2 Mitochondrial dysfunction and insulin resistance...........................................................22 3.3 Reactive oxygen species (ROS).........................................................................................22 EFFECT OF LIPOPOLYSACCHARIDES ON ADIPOGENIC POTENTIAL AND PRE-MATURE SENESCENCE OF ADIPOCYTE PROGENITORS .............................................................. 26 SUMMARY .......................................................................................................................27 INTRODUCTION................................................................................................................28 MATERIALS AND METHODS..............................................................................................31 RESULTS...........................................................................................................................35 Short-term exposure of inguinal SV cells to LPS reduces adipocyte differentiation .............35 LPS induces pre-mature senescence of adipose SV cells.......................................................36 LPS inhibition of adipogenesis is NF-κB independent............................................................37 LPS regulates the expression of preadipocyte differentiation genes in SV cells ...................39 DISCUSSION .....................................................................................................................40 FIGURES...........................................................................................................................46 FIGURE LEGEND................................................................................................................51 KNOCKING DOWN NPC2 EXPRESSION REDUCES LYSOSOMAL ACTIVITY AND IMPAIRS INFLAMMATORY EFFECT OF LIPOPOLYSACCHARIDE IN ADIPOCYTES.......................... 54 SUMMARY .......................................................................................................................55 INTRODUCTION................................................................................................................56 MATERIALS AND METHODS..............................................................................................60 RESULTS...........................................................................................................................63 Role of lysosomal activity in inflammation in adipocytes......................................................63 Generation of stable NPC2 knockdown 3T3-L1 cell line........................................................64 iv NPC2 knockdown impairs lysosomal activity in adipocytes ..................................................65 NPC2 knockdown reduces LPS effect on inflammatory response in adipocytes...................66 NPC2 knockdown diminishes LPS-stimulated glucose uptake, but does not affect insulin sensitivity in adipocytes.........................................................................................................67 DISCUSSION .....................................................................................................................68 FIGURES...........................................................................................................................73 FIGURE LEGEND................................................................................................................79 EICOSAPENTAENOIC ACID PROMOTES THERMOGENIC AND FATTY ACID STORAGE CAPACITY IN MOUSE SUBCUTANEOUS ADIPOCYTES.................................................. 82 SUMMARY .......................................................................................................................83 INTRODUCTION................................................................................................................84 METHODS AND MATERIALS..............................................................................................86 RESULTS...........................................................................................................................89 EPA regulates thermogenic gene expression in inguinal adipocytes
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