Wayne State University Wayne State University Dissertations 1-1-2017 Protein Phosphatase 2a Interactions In Islet And Human Skeletal Muscle In Diabetes Divyasri Damacharla Wayne State University, Follow this and additional works at: https://digitalcommons.wayne.edu/oa_dissertations Part of the Medicinal Chemistry and Pharmaceutics Commons Recommended Citation Damacharla, Divyasri, "Protein Phosphatase 2a Interactions In Islet And Human Skeletal Muscle In Diabetes" (2017). Wayne State University Dissertations. 1794. https://digitalcommons.wayne.edu/oa_dissertations/1794 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. PROTEIN PHOSPHATASE 2A INTERACTIONS IN ISLET AND HUMAN SKELETAL MUSCLE IN DIABETES by DIVYASRI DAMACHARLA DISSERTATION Submitted to the Graduate School of Wayne State University, Detroit, Michigan in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY 2017 MAJOR: PHARMACEUTICAL SCIENCES Approved By: Advisor Date © COPYRIGHT BY DIVYASRI DAMACHARLA 2017 All Rights Reserved DEDICATION This work is dedicated to my father Dr. Srinivas Rao Damacharla, who has been selflessly working very hard for the past 26 years and dreaming about our future (my brother and me). I am forever indebted to him. ii ACKNOWLEDGEMENTS I would like to first thank the department of pharmaceutical sciences for accept- ing me into this program and giving me a chance to discover my strengths through this program. I would also like to thank my advisor Dr.Zhengping Yi for believing in me by accepting me into his lab and assigning me a great project that contributes to the progress in science. I am grateful to work under Dr.Anjan Kowluru, my co-mentor. His confidence in me has always been a huge support. Working with his lab members has broadened my exposure and that will always be a added strength to my career. I will also cherish the support from my committee members Dr.Fei Chen and Dr.Kyle Burghardt for their constant support throughout the program, for their sugges- tions that helped strengthen my project. This project wouldn’t have been possible without the support of my lab mem- bers Dr.Xiangmin Zhang, Dr.Danjun Ma, Dr.Michael Caruso, Yue Qi, and many oth- ers. Their suggestions and guidance throughout helped me progress one step at a time. I would like to specially thank Dr.Xiangmin Zhang who has always been my mentor for the past 5 years and for inspiring me to work hard every day. I am very much indebted for the love and affection of my mother, Padmavathy Damacharla and my brother, Sivaram Damacharla Lastly, my heartfelt thanks to all my friends who helped. Achieving this degree wouldn’t have been possible without my seniors, Anusha Mannepalli and Rahul Guru- raj. They have supported me in all possible ways ever since I came to USA and continue to do so even now. I would also like to specially thank my friend Ratna Karthik who has been the pillar of my strength during the tough times I had and for just standing with me no matter what. iii TABLE OF CONTENTS DEDICATION……………………………………………………………………………..…ii ACKNOWLEDGEMENT…………………………………………………………….…….iii LIST OF FIGURES…………………………………………………………..……….…....vii LIST OF TABLES………………………………………………………….….……....…..viii CHAPTER 1 INTRODUCTION……………………………………………………….……1 1.1 INTRODUCTION TO DIABETES AND INSULIN SIGNALING PATHWAY........1 1.1.1 INTRODUCTION TO DIABETES……………………..……………...…......1 1.1.2 NORMAL GLUCCOSE HOMEOSTASIS….…………………….….….…...2 1.1.3 INSULIN PRODUCTION AND RELEASE INPANCREAS………………..3 1.1.4 INSULIN SIGNALING PATHWAYS……………………………………….4 1.1.5 PATHOGENESIS OF TYPE 2 DIABETES………………………………….6 1.2 KINASE AND PHOSPHATASES………………....……………………..….…….....9 1.3 PROTEIN PHOSPHATASE 2A (PP2A), REGULATION AND EFFECT OF INSU- LIN……………………………………………………….….....................................11 1.3.1 SUBUNITS OF PP2A……………………………………...…….……........ 11 1.3.2 REGULATION OF PP2A……………………………..…..……...…..……..13 1.3.3 INHIBITORS OF PP2A………………………………………………..…....14 1.3.4 ROLE OF PP2A………………………………………………………..…….14 1.3.5 PP2A IN DIABETES……..……………….....................................................15 1.4 INSULIN SENSITIVITY; PROTEIN INTERACTIONS AND MASS SPEC- TROMTRY...............................................................................................…..............17 1.4.1 METHODS TO MEASURE INSULIN SENSITIVITY…………..………...17 1.4.2 IMPORTANCE OF PROTEIN-PROTEIN INTERACTIONS………..….…17 1.4.3 MASS SPECTROMETRY…………………………………………………..18 1.5 SPECIFIC AIMS…………………………………………………………………….20 1.5.1 SPECIFIC AIM 1: INS-1 832/13 CELLS…………………………………...20 1.5.2 SPECIFIC AIM 2: HUMAN SKELETAL MUSCLE BIOPSY……………..20 iv CHAPTER 2 RESEARCH DESIGN AND METHODS.....................................................21 2.1 REAGENTS………………………………………………………………………….21 2.2 SPECIFIC AIM 1: INS-1 832/13 CELLS…………………………………………...21 2.2.1 CELL CULTURE AND HIGH GLUCOSE TREATMENT………...…...... 21 2.2.2 PROTEOMICS SAMPLE PREPARATION AND ANALYSIS……………22 2.2.3 STATISTICAL ANALYSIS………………………………………………..22 2.2.4 VALIDATION THROUGH WESTERN BLOT ANALYSIS……………...23 2.3 SPECIFIC AIM 2 : HUMAN SKELETAL MUSCLE BIOPSIES…………………..23 2.3.1 SUBJECTS………………………………………………………………….23 2.3.2 HYPERINSULENEMIC EUGLYCEMIC CLAMP WITH MUSCLE BIOP- SIES...…………………………………………………………………….....23 2.3.3 OUTLINE...………………………………………………………………...24 2.3.4 PROTEOMIC SAMPLE PREPARATION…………………………………25 2.3.5 STATISTICAL ANALYSIS FOR PP2A……………………...……………27 CHAPTER 3 RESULTS…………………………………………………………………….29 3.1 SPECIFIC AIM 1: DETERMINE THE INTERACTION PARTNERS OF PP2AC IN HUMAN Β-ISLET CELLS UNDER BASAL AND HYPERGLYCEMIC CONDI- TIONS…..…………………………………………………………………………….….29 3.1.1 PP2AC INTERACTION PARTNERS IN INS-1 832/13 CELLS……………...29 3.1.2 GLUCOSE RESPONSIVE PP2AC INTERACTION PARTNERS…………...30 3.1.3 GLUCOSE RESPONSIVE PP2AC INTERACTION PARTNERS RELATED TO INSULIN SECRETION…………………………………………………………31 3.1.4 EXPERIMENTAL VALIDATION OF PPP22R1B AS A GLUCOSE RESPON- SIVE PP2AC INTERACTION PARTNER………………………………………….32 3.2 SPECIFIC AIM 2: DETERMINE INTERACTION PARTNERS OF PP2A IN HU- MAN SKELETAL MUSCLE UNDER BASAL AND HYPERINSULENEMIC CONDI- TIONS IN LEAN, OBESE/OVERWEIGHT NON-DIABETICS AND TYPE 2 DIA- BETIC SUBJECTS………………………………………………………………………32 3.2.1 PP2AC INTERACTION PARTNERS IN SKELETAL MUSCLE FROM LEAN, OVERWEIGHT/OBESE, AND TYPE 2 DIABETIC HUMAN PARTICIPANTS…32 3.2.2 PARTNERS WITH SIGNIFICANT DIFFERENCE AMONG LEAN CON TROL, OBESE/OVERWEIGHT CONTROL, AND TYPE 2 DIABETIC GROUPS..……………………………………………………………………………34 v CHAPTER 4 DISCUSSION……………………………………….………………….…….36 4.1 SPECIFIC AIM 1: DETERMINE THE INTERACTION PARTNERS OF PP2AC IN HUMAN Β-ISLET CELLS UNDER BASAL AND HYPERGLYCEMIC CONDI- TIONS………………………………………………………………………..………….36 4.1.1 PP2AC INTERACTION WITH SIGNALING PROTEINS IMPORTANT FOR PHYSIOLOGICAL INSULIN SECRETION……………………………………….36 4.1.2 PP2AC INTERACTION WITH KEY PROTEINS THAT REGULATE CELL DYSFUNCTION AND APOPTOSIS………………………………………...……..37 4.2 SPECIFIC AIM 2: DETERMINE INTERACTION PARTNERS OF PP2A IN HU- MAN SKELETAL MUSCLE UNDER BASAL AND HYPERINSULENEMIC CONDI- TIONS IN LEAN, OBESE/OVERWEIGHT NON-DIABETICS AND TYPE 2 DIA- BETIC SUBJECTS………………………………………………………………………38 4.2.1 PP2AC INTERACTION PARTNERS IN SKELETAL MUSCLE……………38 4.2.2 KNOWN PARTNERS………………………………………………………....39 4.2.3 PROTEINS INVOLVED IN INSULIN RECEPTOR AND MTORSIGNAL- ING………………………………………………………………………………..…42 4.2.4 INTERACTION PARTNERS WITH SIGNIFICANT CHANGES IN THEIR IN- TERACTION TO PP2AC IN SKELETAL MUSCLE IN LEAN, OVER- WEIGHT/OBESE, AND TYPE 2 DIABETIC HUMAN PARTICIPANTS………...45 4.2.5 PARTNERS WITH SIGNIFICANT CHANGE BETWEEN TYPE 2 DIABETIC AND LEAN SUBJECTS…...……………………………………………………..…46 4.2.6 PARTNERS WITH SIGNIFICANT CHANGE BETWEEN OBESE AND LEAN SUBJECTS.……………………………………………………………….…52 4.2.7 PARTNERS WITH SIGNIFICANT CHANGE BETWEEN TYPE 2 DIABETIC AND OBESE……………………………………………………………………..….55 4.3 SUMMARY AND FUTURE DIRECTIONS ………………………...……..............60 REFERENCES……………………………………………………………….................…146 ABSTRACT……………………………………………………………………………......173 AUTOBIOGRAPHICAL STATEMENT………………………………………………...175 vi LIST OF FIGURES Figure 1 Glucose homeostasis involving major tissues………………………………………..62 Figure 2 Insulin signaling pathway showing the signaling molecules involved and various ef- fects seen …………………………………………………………….……………………….63 Figure 3 Negative regulators of insulin signaling pathway ……...............................................64 Figure 4 Insulin signaling regulation by inhibitory serine/threonine phosphorylation ………………………………………………………………………………………………...65 Figure 5 Diagrammatic representation of heterotrimeric PP2A complex …....……………….66 Figure 6 Main steps in mass spectrometry-based proteomics studies ………………................67 Figure 7 Cell culture, proteomics data acquisition, and data analysis ……………....................68 Figure 8 Proteomic data analysis (INS-1 832/13 CELLS)…………………………................69 Figure 9 Summary of glucose-responsive PP2Ac interaction partners ……………………….70 Figure 10 Experimental validation of PPP2R1B as a glucose responsive PP2Ac interaction part- ner......………………………………………………………………………………………...71 Figure 11 Clinical and proteomics data acquisition and data analysis ………………………...72 Figure 12 Proteomic data analysis (Human skeletal muscle)………………………………….73 Figure 13 The significantly enriched pathway, Insulin Receptor signaling, for the 211 PP2Ac interaction partners and PP2Ac in human skeletal muscle ………………...............................74