• Abstract and Figures
• Public Full-text

EXAMINING THE REGULATION OF ADIPOCYTOKINE EXPRESSION DURING CHRONIC INSULIN RESISTANCE by EDITH ELIZABETH HAYDEN (Under the Direction of Lance Wells) ABSTRACT Type II diabetes, characterized by hyperglycemia and hyperinsulinemia, results in many costly and debilitating patient complications. A broad range of tissues respond to insulin and help to mediate its effects. In particular, adipose tissue secretes proteins known as adipocytokines, which play an important role in whole body energy homeostasis and have been implicated in the pathogenesis of diabetes. Increased flux through the hexosamine biosynthetic pathway and the corresponding increase in intracellular glycosylation of proteins via O-GlcNAc is sufficient to induce insulin resistance (IR) in multiple systems. Previously, our group used shotgun proteomics to identify rodent adipocytokines whose levels are modulated upon the induction of IR by indirectly and directly modulating O-GlcNAc levels. Since adipocytokines levels are regulated primarily at the level of transcription and O-GlcNAc alters the function of many transcription factors, we hypothesized that elevated O-GlcNAc levels on key transcription factors are modulating adipocytokine expression. Here, we show that upon the elevation of O- GlcNAc levels and the induction of IR in mature 3T3-F442a adipocytes, the transcript levels of multiple adipocytokines, as measured by quantitative RT-PCR, reflect the modulation observed at the protein level. We have gone on to validate the adipocytokine transcript levels in mouse models of diabetes. Using inguinal fat pads from the db/db mouse model and the diet-induced obesity mouse model, we have confirmed that the adipocytokines regulated by O-GlcNAc modulation in cell culture are likewise modulated in the whole animal upon a shift to IR. We find that Sp1 is a common cis-acting element on the promoters of co-regulated genes. Sp1 O- GlcNAc modification increases during IR. As measured by chromatin immunoprecipitation, Sp1 and O-GlcNAc modified proteins are enriched on the LPL and SPARC promoters during insulin resistance. These data lead us to the conclusion that adipocytokine expression is modulated by global O-GlcNAc levels and potentially by Sp1. We go on to characterize the primary human adipocyte secretome in order to identify more adipocytokines whose expression is modulated by O-GlcNAc and find that many of the rodent adipocytokines are likewise regulated by O-GlcNAc levels in homo sapiens. INDEX WORDS: O-GlcNAc, insulin resistance, adipocytes, adipokines, adipocytokines EXAMINING THE REGULATION OF ADIPOCYTOKINE EXPRESSION DURING CHRONIC INSULIN RESISTANCE by EDITH ELIZABETH HAYDEN BS, Saint Vincent College, 2006 A Dissertation Submitted to the Graduate Faculty of The University of Georgia in Partial Fulfillment of the Requirements for the Degree DOCTOR OF PHILOSOPHY ATHENS, GEORGIA 2012 © 2012 Edith Elizabeth Hayden All Rights Reserved EXAMING THE REGULATION OF ADIPOCYTOKINE EXPRESSION DURING CHRONIC INSULIN RESISTANCE by EDITH ELIZABETH HAYDEN Major Professor: Lance Wells Committee: Stephen Dalton Shaying Zhao Ruth Harris Electronic Version Approved: Maureen Grasso Dean of the Graduate School The University of Georgia August 2012 DEDICATION I would like to dedicate this work to my life partner, Paul, and my friends and family. Thank you for your patience and support. iv ACKNOWLEDGEMENTS Many people have helped to guide me through my time in graduate school. I would like to thank the members of of the Wells’ lab past and present for their helpful discussions and support. Special thanks goes to Lance Wells for helping me so much with my science and professional development and for dealing with my frequent stress-induced tantrums. My committee members, Steve, Ruth, and Shaying, have continually given me helpful advice. Thank you also to the many members of BCMB department and the CCRC who have helped me over the years, especially Alison Nairn and Mitche de la Rosa. For technical advice about sonication, thank you to James Chappell and Kenneth Lyon. Thank you to Bingqiang Liu for assistance with bioinformatics. Thank you to Ruth Harris for kindly providing us with mouse tissues and helping us to develop a diet-induced insulin resistant mouse model. Thank you to Dorothy Hausman for helping us to collect and culture primary adipocytes. I would also like to extend thanks to the administrative staff of the BCMB department and the CCRC. Finally, thank you to my friends, family, and pets whom have continuously provided love and support. v TABLE OF CONTENTS Page ACKNOWLEDGEMENTS .............................................................................................................v LIST OF TABLES ....................................................................................................................... viii LIST OF FIGURES .........................................................................................................................x CHAPTER 1 INSULIN RESISTANCE, ADIPOCYTOKINES, AND O-GLCNAC .........................1 Insulin Resistance ....................................................................................................1 White Adipose Tissue and Adipocytokines .............................................................2 Hexosamine Biosynthetic Pathway..........................................................................7 O-GlcNAc ................................................................................................................9 2 THE ROLE OF THE O-GLCNAC MODIFICATION IN REGULATING EUKARYOTIC GENE EXPRESSION .......................................................................27 Abstract ..................................................................................................................28 Introduction ............................................................................................................29 O-GlcNAc Detection and Site Mapping ................................................................31 O-GlcNAc Regulation of Eukaryotic Gene Expression ........................................33 OGT/OGA Targeting to Substrates: A Special Case of Protein/Protein Interactions .............................................................................................................46 Summary ................................................................................................................49 Acknowledgements ................................................................................................50 vi 3 GLOBAL O-GLCNAC LEVELS MODULATE ADIPOKINE TRANSCRIPTION DURING CHRONIC INSULIN RESISTANCE .........................................................57 Abstract ..................................................................................................................58 Introduction ............................................................................................................59 Materials and Methods ...........................................................................................61 Results ....................................................................................................................66 Discussion ..............................................................................................................71 Acknowledgements ................................................................................................77 4 QUANTITATIVE SECRETOME AND GLYCOME OF PRIMARY HUMAN ADIPOCYTES DURING INSULIN RESISTANCE ..................................................91 Abstract ..................................................................................................................92 Introduction ............................................................................................................94 Experimental Procedures .......................................................................................97 Results ..................................................................................................................106 Discussion ............................................................................................................111 Acknowledgements ..............................................................................................117 DISCUSSION .............................................................................................................................157 REFERENCES ............................................................................................................................161 vii LIST OF TABLES Page Table 2.1: Putative OGA-interacting proteins identified by yeast two-hybrid screen ...................56 Table 3.1: Adipokine transcript levels are proportional to obesity severity ..................................82 Supplementary Table S3.1: Common motifs for adipokine promoters .........................................87 Table 4.1: Total secreted proteins from human adipose tissues by LC-MS/MS .........................129 Table 4.2: Human adipocytokines regulated a minimum of 150% under both insulin resistant conditions .........................................................................................................................135 Table 4.3: Identification of N-linked glycosylation sites using PNGase F with the incorporation of 18O water in human adipocytokines.............................................................................136 Supplementary Table S4.1: The list of secreted proteins for a single peptide detection from human

Load more

  226

Copy Link