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Quantitative Proteomic Investigation of Disease Models of Type 2 Diabetes Mark Gabriel Athanason University of South Florida, [email protected]

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Quantitative Proteomic Investigation of Disease Models of Type 2 Diabetes Mark Gabriel Athanason University of South Florida, Athanason@Mail.Usf.Edu University of South Florida Scholar Commons Graduate Theses and Dissertations Graduate School 11-17-2016 Quantitative Proteomic Investigation of Disease Models of Type 2 Diabetes Mark Gabriel Athanason University of South Florida, [email protected] Follow this and additional works at: http://scholarcommons.usf.edu/etd Part of the Biochemistry Commons, and the Biology Commons Scholar Commons Citation Athanason, Mark Gabriel, "Quantitative Proteomic Investigation of Disease Models of Type 2 Diabetes" (2016). Graduate Theses and Dissertations. http://scholarcommons.usf.edu/etd/6460 This Dissertation is brought to you for free and open access by the Graduate School at Scholar Commons. It has been accepted for inclusion in Graduate Theses and Dissertations by an authorized administrator of Scholar Commons. For more information, please contact [email protected]. Quantitative Proteomic Investigation of Disease Models of Type 2 Diabetes by Mark Gabriel Athanason A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy Department of Cell Biology, Microbiology, and Molecular Biology College of Arts and Sciences University of South Florida Major Professor: Brant R. Burkhardt, Ph.D. Stanley M. Stevens, Jr, Ph.D. Meera Nanjundan, Ph.D. Patrick Bradshaw, Ph.D. Date of Approval: November 4th, 2016 Keywords: PANDER, proteomics, phosphoproteomics, stable-isotope labeling Copyright © 2016, Mark Gabriel Athanason Dedication The work contained in this dissertation is dedicated to my love and companion, Tori. Her presence illuminates paths through the most challenging endeavors, which I myself could not navigate alone. Additionally, I also dedicate this work to my loving mother, Despina, and father, Gary, for their unrelenting and constant support. Acknowledgments I would like to thank my mentor, Dr. Brant Burkhardt for his guidance and support not only through the chaos of the scientific world, but also for providing guidance for every day challenges, too. I also thank my mentors and committee members: Dr. Meera Nanjundan, Dr. Patrick Bradshaw, and especially Dr. Stanley Stevens for his expert knowledge and mentorship that made this project possible. From Dr. Stevens’s lab I thank my colleagues and friends, Dr. Dale Chaput and Jenny Guergues. From my own lab I thank Dr. Whitney Ratliff, Dr. Melanie Kuehl, Katie MarElia, Tiffany Shemwell, Arielle Sharp, Ramsey Grace, and Amanda Fernandez for their friendship and comradery, I wish them all the best. Table of Contents List of Tables................................................ iv List of Figures............................................... vi Abstract...................................................... ix Chapter 1 – Introduction....................................... 1 Discovery and Characterization of Pancreatic Derived Factor .................................................... 1 The Pander Gene ...................................... 4 Structure of the Pander Protein ...................... 6 Physiological Role of Pander in the Pancreas .............. 7 Pander Expression, Localization and Regulation in Comparision to Insulin ............................... 8 Physiological Role of Pander in the Liver ................. 9 Liver Derived Pander Impacts Hepatic Insulin Signaling in Murine Models .......................... 10 Pancreatic Derived Pander Impacts Hepatic Insulin Sensitivity and Lipid Metabolism .................... 12 Initial Proteomic Analysis of The Pander Transgenic Mouse Liver using Super Silac Aml-12 Cells ............................................... 16 Liver X Receptor Modulates Pander Induced Hepatic Lipogensis .......................................... 21 Hepatic Insulin Resistance in Type 2 Diabetes ............ 25 Proteomics and Type 2 Diabetes Research .................. 29 Gel-Based Proteomics ................................ 29 Quantitative Mass Spectrometry Based Proteomics ..... 32 Innovation ............................................... 41 i Chapter 2 – Generation of The Pander Transgenic Model for Identification of Pander-Induced Hepatic Proteome............. 44 Abstract ................................................. 44 Materials and Methods .................................... 45 Generation of the Pander Transgenic Animal Colony ... 45 Metabolic Treatment of The Pander Transgenic Mouse ............................................... 46 Hepatic Protein Isolation ........................... 47 Sample Digestion, Desalt, and Scx Fractionation ..... 47 Acquisition of Hepatic Proteome by Lc-Ms/Ms ......... 48 Database Search for Identification and Quantification of Proteins .......................... 49 Results .................................................. 50 Discussion ............................................... 76 Chapter 3 – Investigation of The Pander Transgenic Hepatic Proteome...................................................... 78 Introduction ............................................. 78 Materials and Methods .................................... 81 Statistics And Pathway Analysis ..................... 81 Hepatic Phosphoproteomic Analysis of The Insulin Stimulated PANTG Mouse .............................. 84 Immunoblotting ...................................... 86 Cell Culture and Transient Transfection ............. 87 Results .................................................. 88 Network and Pathway Analysis ........................ 88 Proteomic Identification of Pander-Induced Hepatic LXR Pathway ................................. 93 Validation of Pander-Induced Hepatic LXR Pathway and Fatty Acid Synthase Expression .................. 94 Pander Stimulates LXR Transcriptional Activity In-Vitro ............................................ 97 Phosphoproteomic Analysis Reveals Increased Phosphorylation of Glycogen Synthase ............... 106 Discussion .............................................. 109 ii Chapter 4 – Exploring Proteomic Changes in the Fatty Liver... 116 Abstract ................................................ 116 Introduction ............................................ 117 Materials and Methods ................................... 120 Animals and Diet ................................... 120 Glucose and Insulin Tolerance Testing .............. 120 Proteomic Experimental Design ...................... 121 Statistical and Pathway Analysis ................... 121 Results ................................................. 122 Metabolic Phenotyping .............................. 122 Proteomic Characterization of the HFD Liver and Network Analysis ................................... 129 Proteomic Analysis: Effects After 2 Weeks HFD ...... 129 Proteomic Analysis: Effects After 9 Weeks HFD ...... 135 Discussion .............................................. 139 Chapter 5 – Conclusions and Future Directions................ 147 Overview and Summary of Major Findings .................. 147 Hepatic Proteome Characterization of the PANTG Mouse .............................................. 147 Pander Affects LXR Promoter Activity ............... 149 Hepatic Phosphoproteome Analysis of the PANTG Mouse Liver ........................................ 150 Metabolic and Proteomic Evaluation of High Fat Diet Fed Mice ...................................... 154 Future Directions ....................................... 157 Unexplored Information In The PANTG Proteomic Dataset ............................................ 157 Improvement of the Experimental Design ............. 159 Proteomic Evaluation of Other Tissues in the PANTG Mouse ........................................ 161 Proteomic Evaluation of the PANKO Mouse ............ 161 Pander as a Therapeutic Target For T2D ............. 161 iii References................................................... 163 Appendix I: Permissions...................................... 183 Appendix II: Additional Data................................. 196 iv List of Tables Table 1: Differentially expressed proteins in the fasted state ............................................... 54 Table 2: Differentially expressed proteins in the fasted state ............................................... 62 Table 3: Differentially expressed proteins in the fasted state ............................................... 69 Table 4: Affected metabolic functions in the insulin stimulated state .................................... 91 Table 5: Differentially expressed proteins in the fasted state .............................................. 107 Table 6: Proteins modulated by LXR activation after 2 weeks HFD .......................................... 134 v List of Figures Figure 1: North analysis of PANDER mRNA in human tissues ..... 2 Figure 2: Immunogold electron microscopy localization of PANDER in secretory granules ....................... 3 Figure 3: DNA sequence from -829 to +29 ...................... 5 Figure 4: PANDER structure ................................... 6 Figure 5: Metabolic phenotype of the PANTG mouse ............ 13 Figure 6: Mechanism of PANDER induced diabetes .............. 15 Figure 7: Proteomic analysis of PANTG mouse ................. 19 Figure 8: Classical LXR activation .......................... 24 Figure 9: Pathophysiology of T2D ............................ 28 Figure 10: Typical mass spectrometry based proteomic workflow .......................................... 36 Figure 11: SILAC proteomic experimental approach for investigation of PANTG ............................ 52 Figure 12: Distribution of proteins across metabolic conditions .......................................
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