Wayne State University Wayne State University Dissertations 1-1-2012 Use of methionine sulfoximine to dissect the role of glutamine synthetase and glutamine in progression of acute liver failure Amruta Anil Jambekar Wayne State University, Follow this and additional works at: http://digitalcommons.wayne.edu/oa_dissertations Part of the Biochemistry Commons, and the Immunology and Infectious Disease Commons Recommended Citation Jambekar, Amruta Anil, "Use of methionine sulfoximine to dissect the role of glutamine synthetase and glutamine in progression of acute liver failure" (2012). Wayne State University Dissertations. Paper 378. This Open Access Dissertation is brought to you for free and open access by DigitalCommons@WayneState. It has been accepted for inclusion in Wayne State University Dissertations by an authorized administrator of DigitalCommons@WayneState. USE OF METHIONINE SULFOXIMINE TO DISSECT THE ROLE OF GLUTAMINE SYNTHETASE AND GLUTAMINE IN PROGRESSION OF ACUTE LIVER FAILURE by AMRUTA A. JAMBEKAR DISSERTATION Submitted to the Graduate School of Wayne State University Detroit, Michigan in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOHPY 2011 MAJOR: BIOCHEMISTRY Approved by: Advisor Date © COPYRIGHT BY AMRUTA A JAMBEKAR 2011 All Rights Reserved DEDICATION This work is dedicated to my parents. I would never have attained this degree without their unconditional love, support and encouragement. Aai and baba, I love you. Translation: Guru Brahma, Guru Vishnu Guru Devo Maheshwara, Guru sakshath Parabrahma, Tasmai Shri Gurave Namah The Guru (Teacher) is the creator Brahma, The Guru is the preserver Vishnu and The Guru is the destroyer Shiva. Guru is the supreme spirit and I bow to such a Guru (Teacher). ii ACKNOWLEDGEMENTS Thank you Dr. Brusilow. Under Dr. William S. A. Brusilow’s mentorship, I have understood what scientific research stands for. He gave me complete independence to plan and conduct various experiments. He continuously motivated and guided me. His conversations, help, creativity and support made my PhD easy and enjoyable. I would especially thank him for the respect he has shown me and his calmness – I have never seen him get angry or say a cross word. I would like to acknowledge Dr. Richard Needleman’s help in my project. His help in literature search was invaluable to me. He has asked me some tough questions and helped me to better my research. He has also helped me in scientific writing and reviewed my work. He has been a friend as well as an advisor. I would like to thank my other committee members; Dr. Joyce Benjamins and Dr. Maik Hüttemann. My conversations with Dr. Benjamins have always helped me put my results into perspective. I have used her numerous lab equipments, and without those I would not be able to complete my research. Dr. Hütteman has asked me the toughest questions in committee meetings. His input every year has given a new dimension to my work. I would like to acknowledge the help of Dr. Paolo Bernardi and Dr. Andrea Rasola in writing the first paper. Also, they allowed me to continue a project which originated in their lab. I would like to thank my lab members and friends Ms. Monica Bame and Mrs. Wei ping Mattingly. I would also like to acknowledge Dr. Doscher and the support staff of Biochemistry department – Ms. Yanna Marsh, Ms. Roselle Cooper and Mr. Joe Fiore. iii I thank my parents; Mr. Anil M. Jambekar and Mrs. Shubhada A. Jambekar. They have always pushed me to do my best and have experienced every day of my PhD. I thank my sister Mrs. Archana V. Khandkar. She is my best friend and staunchest supporter. Without her my life would have been impossible. I would like to thank my grandparents, Mrs. Vijaya C. Deshpande, Mr. Chintamani Despande and Mr. Manohar D. Jambekar, and my father – in law Mr. Raghvendra Rao for their blessings and love. I thank Pujya Vishwanath Avdhootji Maharaj for his blessings. Lastly, I would like to thank my friend and husband Mr. Krishna R. Rao for his support and love. He has allowed me to fulfill my wishes and suffered anxiety, tensions, stress, and JOYS of this work. iv TABLE OF CONTENTS Dedication .................................................................................................................................................. ii Acknowledgements ................................................................................................................................... iii List of Tables ............................................................................................................................................ vi List of Figures .......................................................................................................................................... vii Chapter 1 – Understanding liver, disease pathogenesis and basic immunology involved in acute liver failure ............................................................................................................................1 Chapter 2 – Glutamine synthetase as a potential therapeutic target and use of methionine sulfoximine to understand mechanisms involved in progression of acute liver failure ................................................................................................................................38 Chapter 3 – A Glutamine Synthetase inhibitor increases survival and decreases cytokine response in a mouse model of Acute Liver Failure ............................................................................57 Chapter 4 – Methionine sulfoximine’s prevention of acute liver failure: understanding the involvement of glutamine synthetase and glutamine ........................................................83 Chapter 5 – Conclusions and future directions .......................................................................................110 Appendix A .............................................................................................................................................117 Appendix B .............................................................................................................................................118 References ...............................................................................................................................................120 Abstract ...................................................................................................................................................141 Autobiographical Statement ....................................................................................................................143 v LIST OF TABLES Table 1.1 Classification terms used to characterize various forms of liver failure ....................................5 Table 2.1 Glutamine synthetase activity in brains of 70 day wild type (WT) and ALS mice with and without MSO treatment .....................................................................................................53 Table 2.2 GS activity in brain tissue of 120 day old mice, treated with MSO starting at 85 days in wild type (WT) and ALS mice .............................................................................................54 Table 4.2 The table describes the glutamine conditions to which cells of all experiments were exposed .....................................................................................................................................99 vi LIST OF FIGURES Figure 1.1 Cellular classification of liver ...................................................................................................3 Figure 1.2 A flow chart diagram to show disease mechanism, symptoms and complication associated with acute liver failure .......................................................................................13 Figure 1.3 Cell death pathway activated by TNF ....................................................................................31 Figure 1.4 The signal transduction pathways leading to cell survival, activated via TNFR1 and TNFR2 ...................................................................................................................................32 Figure 1.5 Signal transduction pathways are activated by IL-6 ...............................................................34 Figure 1.6 IFN-γ activated signal transduction pathways – activation of STAT1 ...................................35 Figure 2.1 Glutamine synthetase catalyses reaction between glutamate and ammonia to form glutamine ...............................................................................................................................41 Figure 2.2 A cartoon of glutamine synthetase structure ..........................................................................42 Figure 2.3 Glutamine synthetase distribution in hepatic tissue ...............................................................45 Figure 2.4 A schematic diagram of glutamine – glutamate cycle in liver ...............................................46 Figure 2.5 Structure of methionine sulfoximine and methionine sulfoximine phosphate .......................49 Figure 3.1 Survival of male and female CD1 mice pretreated with either MSO or saline three hours prior to injection with LPS and D-Galactosamine (D-GalN) at time zero to induce liver failure ...............................................................................................................64 Figure 3.2 Glutamine synthetase activities measured in liver extracts from mice treated with LPS