THE CONTROL OF APOLIPOPROTEIN C-I GENE EXPRESSION DURING ADIPOCYTE DIFFERENTIATION by John M. David A thesis submitted to the Faculty of the University of Delaware in partial fulfillment of the requirements for the degree of Master of Science in Biological Sciences Spring 2006 Copyright 2006 John M. David All Rights Reserved UMI Number: 1435920 UMI Microform 1435920 Copyright 2006 by ProQuest Information and Learning Company. All rights reserved. This microform edition is protected against unauthorized copying under Title 17, United States Code. ProQuest Information and Learning Company 300 North Zeeb Road P.O. Box 1346 Ann Arbor, MI 48106-1346 THE CONTROL OF APOLIPOPROTEIN C-I GENE EXPRESSION DURING ADIPOCYTE DIFFERENTIATION by John M. David Approved: David C. Usher, Ph.D. Professor in charge of thesis on behalf of the Advisory Committee Approved: Daniel D. Carson, Ph.D. Chair of the Department of Biological Sciences Approved: Thomas M. Apple, Ph.D. Dean of the College of Arts and Sciences Approved: Conrado M. Gempesaw II, Ph.D. Vice Provost for Academic and International Programs ACKNOWLEDGMENTS I would like to thank my advisor, Dr. David Usher, for providing me with the opportunity to begin my graduate career, his guidance and support of this research project, and the invaluable experience that I gained while working in his laboratory. I would like to thank my committee members, Dr. Roger Wagner and Dr. William Cain, for their criticism and support. I would like to thank the past and present members of the Usher laboratory group, including Dr. Donna Maslak, Dr. Robin Davis, and Amanda Peters, for their assistance in the laboratory. Finally, I would like to thank the other students in the Biological Sciences department that I have interacted with during my academic career, most notably Brady Redmond, Dianna Willis, and Brian Danysh, for their help and advice in both my research and coursework. This manuscript is dedicated to my parents, John and Marylou David, for their unconditional love and support. iii TABLE OF CONTENTS LIST OF TABLES....................................................................................................... vii LIST OF FIGURES .................................................................................................... viii ABSTRACT...................................................................................................................xi Chapter 1 INTRODUCTION ....................................................................................................1 1.1 Obesity and Disease ........................................................................................1 1.2 The 3T3-L1 Cell Line: An In Vitro Model of Adipogenesis ..........................2 1.3 Regulation of Energy Balance.........................................................................5 1.3.1 Adipokines ...........................................................................................6 1.3.2 Cholesterol Balance .............................................................................9 1.3.3 Inflammation......................................................................................11 1.4 Hypothesis and Experimental Approach.......................................................13 2 MATERIALS AND METHODS............................................................................15 2.1 HDL Preparation ...........................................................................................15 2.2 Cell Culture ...................................................................................................15 2.2.1 3T3-L1 Culture Conditions................................................................15 2.2.2 -cyclodextrin/HDL Treatment .........................................................17 2.2.3 Stimulation of PPAR Activity with Pioglitazone.............................17 iv 2.2.4 Stimulation of PPAR Activity with Rosiglitazone...........................17 2.2.5 Stimulation LXR Activity with T0901317 ........................................18 2.2.6 Trypan Blue Staining .........................................................................18 2.2.7 Oil Red-O Staining ............................................................................18 2.3 Total RNA Isolation and Treatment..............................................................19 2.4 cDNA Synthesis ............................................................................................20 2.5 Quantitative PCR...........................................................................................21 2.5.1 Data Analysis.....................................................................................21 2.6 Apolipoprotein C-I ELISA............................................................................22 3 RESULTS ...............................................................................................................24 3.1 SAGE Analysis of 3T3-L1 Preadipocytes and Adipocytes ..........................24 3.1.1 The Expression of Genes Important for Lipid Transport...................26 3.2 The Expression of Apolipoprotein C-I mRNA in 3T3-L1 Adipocytes.........27 3.3 Apolipoprotein C-I mRNA Expression in Mouse Tissues and Human Adipocytes.....................................................................................................29 3.4 The Expression of Apolipoprotein C-I Protein in Human Adipocytes .........31 3.5 The Effect of Cholesterol Depletion on 3T3-L1 Adipocyte Differentiation ...............................................................................................31 3.5.1 The Histological Effect of -cyclodextrin/HDL Treatment on 3T3-L1 Differentiation.......................................................................34 3.5.2 The Effect of -cyclodextrin/HDL Treatment on Apolipoprotein C-I mRNA Expression During Adipocyte Differentiation ................35 v 3.6 The Role of Transcription Factors in the Regulation of the Apoc1 Gene .....35 3.6.1 The Effect of -cyclodextrin/HDL Treatment on Pparg and its Target Genes ......................................................................................36 3.6.2 The Effect of PPAR Agonists on 3T3-L1 Adipocyte Differentiation....................................................................................37 3.6.3 The Effect of -cyclodextrin/HDL Treatment on Srebp1-c, Srebp2, and their Target Genes..........................................................39 3.6.4 The Effect of -cyclodextrin/HDL Treatment on Lxr and its Target Genes ......................................................................................40 3.6.5 The Effect of the Liver X Receptor Ligand T0901317 on 3T3-L1 Adipocyte Differentiation ..................................................................41 4 DISCUSSION .........................................................................................................43 REFERENCES .............................................................................................................87 vi LIST OF TABLES 1 Primer sequences for genes studied in quantitative PCR experiments ...................55 2 Relative quantitation of gene expression ................................................................56 3 SAGE analysis of 3T3-L1 preadipocytes and adipocytes.......................................57 4 Cluster analysis of 3T3-L1 preadipocyte and adipocyte SAGE libraries ...............58 5 SAGE identification of genes that encode for secreted proteins ............................59 6 SAGE identification of secreted proteins that function in lipid metabolism ..........60 7 Production of apolipoprotein C-I by cultured human adipocytes ...........................61 8 Viability test results for 3T3-L1 differentiation in the presence of - cyclodextrin and HDL ...........................................................................................62 9 Viability test results for 3T3-L1 differentiation in the presence of PPAR and LXR agonists .........................................................................................................63 vii LIST OF FIGURES 1 Phenotypic and genotypic events during 3T3-L1 differentiation ...........................64 2 Light microscope images of 3T3-L1 preadipocytes and differentiated adipocytes ..............................................................................................................65 3 mRNA expression of the reference genes Tbp and Hprt1 during 3T3-L1 differentiation ........................................................................................................66 4 Apolipoprotein E/C1/C4/C2 linkage group expression in 3T3-L1 preadipocytes and adipocytes .......................................................................................................67 5 mRNA expression time course of adipocyte marker genes during 3T3-L1 differentiation ........................................................................................................68 6 mRNA expression time course of apolipoprotein genes during 3T3-L1 differentiation ........................................................................................................69 7 Apolipoprotein and adipocyte marker gene expression in murine liver and adipose tissue.........................................................................................................70 8 Light microscope images of human preadipocytes and differentiated adipocytes in culture ................................................................................................................71