ADIPOCYTE PROTEIN CARBONYLATION AND OXIDATIVE STRESS IN OBESITY-LINKED MITOCHONDRIAL DYSFUNCTION AND INSULIN RESISTANCE A DISSERTATION SUBMITTED TO THE FACULTY OF THE GRADUATE SCHOOL OF THE UNIVERSITY OF MINNESOTA BY JESSICA MARIE CURTIS IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DR. DAVID A BERNLOHR, ADVISOR SEPTEMBER 2011 © JESSICA MARIE CURTIS 2011 ACKNOWLEDGEMENTS I would like to thank my graduate advisor Dr. Bernlohr for his guidance and support. His positive attitude and patience make him a remarkable teacher, and he has played a pivotal role in my development as a scientist. I especially would like to thank the members of my committee, Drs. Alex Lange, Howard Towle, Edgar Arriaga and Doug Mashek, for their guidance during the last 5 years. Their metabolic expertise and critical reviews of my research has been incredibly helpful. To the members of the Bernlohr Lab, past and present, thank you for the camaraderie and help through the years. You have been an essential part of my graduate experience, creating an environment that is friendly and rewarding of hard work. I must thank Paul Grimsrud for developing the initial GSTA4 studies and for his mentorship. Wendy Hahn has been an essential mentor and GSTA4 project collaborator as well as a great friend. Thank you also to Ann Hertzel for your critical eye and always knowing where everything is; Tina Hellberg for your amazing attention to detail; Rocio Foncea for your fun spirit and antioxidant knowledge; Marissa Lee, the caretaker of cells. Past lab members who shaped my graduated career include Anne Smith, Brian Thomspon, Brian Wiczer, Sandra Lobo, Jason Fowler, and honorary lab member Sri Bandhakavi. Thank you to my graduate student support network, especially Katie Furniss, Becca Pulver and Andrea Yoder, and other faculty mentors, including Michele Sanders, Anja Bielinsky, and Tim Griffin. i DEDICATION This thesis is dedicated to the people I love and who love me, namely my family and friends that have supported me throughout this journey. To my mother and father for teaching me to believe in myself, the importance of balance, and for their constant love and support. I am no doubt a product of my environment. To my brothers, Dale and Kevin, for instilling in me the competitive desire to always be better. To my extended family, even if youʼre all over the country its impossible not to recognize the support you have given me over the years. To my partner Eric, for the incredible amount of support and perspective youʼve provided when life grew challenging. And to my best friends Katie Zwolski and Cherisse Kellen, who for the past 10 years have never failed me when Iʼve needed them, even in the face of their own struggles. ii ABSTRACT Carbonylation is the covalent, non-reversible modification of the side chains of cysteine, histidine and lysine residues by lipid peroxidation end products such as 4-hydroxy- and 4-oxononenal. The antioxidant enzyme glutathione S-transferase A4 (GSTA4) catalyzes a major detoxification pathway for such reactive lipids but its expression was selectively down regulated in the obese, insulin resistant adipocyte resulting in increased protein carbonylation. The effects of such modifications are associated with increased oxidative stress and metabolic dysregulation centered on mitochondrial energy metabolism. Mitochondrial functions in adipocytes of lean or obese GSTA4 null mice were significantly compromised compared to wild type controls and were accompanied by an increase in superoxide anion. Silencing GSTA4 mRNA in cultured adipocytes resulted in increased protein carbonylation, increased mitochondrial ROS, dysfunctional state 3 respiration and altered glucose transport and lipolysis. To address the role of protein carbonylation in the pathogenesis of mitochondrial dysfunction quantitative proteomics was employed to identify specific targets of carbonylation in GSTA4-silenced or overexpressing 3T3-L1 adipocytes. GSTA4- silenced adipocytes displayed elevated carbonylation of several key mitochondrial proteins including the phosphate carrier protein, NADH dehydrogenase 1 alpha subcomplexes 2 and 3, translocase of inner mitochondrial membrane 50, and valyl-tRNA synthetase. Elevated protein carbonylation is accompanied by diminished complex I activity, impaired respiration, increased superoxide production and a reduction in membrane potential without changes in mitochondrial number, area or density. These results suggest protein carbonylation plays a major instigating role in mitochondrial dysfunction and may be a linked to the development of insulin resistance in the adipocyte. iii TABLE OF CONTENTS Acknowledgements……………………………………………………………………i Dedication……………………………………………………………………………….ii Abstract…………………………………………………………………………………iii List of Tables…………………………………………………………………………..vi List of Figures………………………………………………………………………...vii Chapter 1 Adipocyte Metabolism, Insulin Resistance & Mitochondrial Function……………..1 I. Metabolic Syndrome and Insulin Action in Obesity-linked Type II Diabetes………………………………………..………………………...2 II. Mitochondrial Metabolism in the Adipocyte…………….…………...11 III. Oxidative Stress and Antioxidants…………………………………...21 IV. Current Objectives……………………………………………………..32 V. References…………………………...………………………………...32 Chapter 2 Downregulation of Adipose Glutathione S-Transferase A4 Leads to Increased Protein Carbonylation, Oxidative Stress, and Mitochondrial Dysfunction………………………………………………………………………..……40 I. Introduction …………………………………………………………….42 II. Research Design and Methods………………………………………44 III. Results…………………………………………………………………..53 IV. Discussion……………...……………………………………………....76 V. References……………………………………………………………..82 Chapter 3 Protein Carbonylation and Adipocyte Mitochondria Function….………………….86 I. Introduction……………………………………………………………..88 II. Experimental Procedures……………………………………………..91 III. Results…………………………………………………………………..99 IV. Discussion……………………………………………………….....…116 V. Abbreviations………………………………………………………….124 VI. References……………………………………………………………125 iv Chapter 4 Perspectives…………………………………………………………………………..139 References……………………………………………………………………149 Bibliography…………………………………………………………………………..151 v LIST OF TABLES Chapter 3: Protein Carbonylation and Adipocyte Mitochondria Function Table 1: Pathway analysis of carbonylated mitochondrial proteins in adipocytes……………………………………………………………………..105 Table 2: Differentially Modified Targets of Carbonylation in Adipocyte Mitochondria…………………………………………………………………..106 Supplemental Table 1: Targets of Protein Carbonylation Categorized by Subcellular Localization……………………………………………………...128 vi LIST OF FIGURES Chapter 1 Adipocyte Metabolism, Insulin Resistance & Mitochondrial Function Figure 1: Adipocentric view of the development of obesity-linked insulin resistance………………………………………………………………………...5 Figure 2: Mitochondrial electron transport, superoxide generation and antioxidant detoxification….………………………………….……………….22 Figure 3: Peroxidation of polyunsaturated acyl chains of glycerophospholipids…………………………………………………………..25 Figure 4: Detoxification of lipid aldehydes and protein carbonylation......27 Chapter 2 Downregulation of Adipose Glutathione S-Transferase A4 Leads to Increased Protein Carbonylation, Oxidative Stress, and Mitochondrial Dysfunction Figure 1: Expression of oxidative stress response genes in adipose tissue in obesity………………………………………………………………………..55 Figure 2: Effect of TNFα treatment on GSTA4 expression in 3T3-L1 adipocytes………………………………………………………………………57 Figure 3: Expression of human GSTA4 in obesity and insulin resistance……………………………………………………………………… 59 Figure 4: GSTA4 silencing and protein carbonylation in 3T3-L1 adipoctyes………………………………………………………………………61 Figure 5. Glucose and lipid metabolism in GSTA4 Kd and Scr adipocytes……...……………………………………………………………….64 Supplemental Figure 1: Lipid Metabolism in GSTA4 Kd and Scr 3T3-L1 Adipcoytes………………………………………………………………………66 Supplemental Figure 2: Acyl-carnitine profiling…….......……...………...68 Figure 6: Mitochondria function in GSTA4 Kd and Scr 3T3-L1 adipocytes………………………………………………………………………70 Figure 7. Expression of genes and proteins linked to mitochondrial biogenesis………………………………………………………………………71 Figure 8. Mitochondrial function and expression in adipose tissue from C57Bl/6J and GSTA4 -/- mice………………………………………………..74 Chapter 3 Protein Carbonylation and Adipocyte Mitochondria Function Figure 1: Expression and functionality of aP2-HA-GSTA4 in 3T3-L1 adipocytes……………………………………………………………………..101 Supplemental Figure 1: Subcellular localization Summary……………103 vii Figure 2: Oxygen consumption rates in GSTA4 silenced and overexpressing adipocytes………………………………………………….107 Figure 3: Mitochondrial superoxide production and membrane potential………………………………………………………………………..109 Figure 4: Cytochrome c localization……………………………………….111 Figure 5: Quantification of mitochondrial number and area by electron microscopy…………………………………………………………………….113 Figure 6: Complex I activity and expression in isolated mitochondria…115 Chapter 4 Conclusions and Perspectives Figure 1: Insulin signaling and glucose tolerance in the absence of GSTA4…………………………………………………………………………142 Figure 2: FOF1 ATPase activity and AMPK activation…………………...145 viii CHAPTER ONE Adipocyte Metabolism, Insulin Resistance & Mitochondrial Function Jessica Curtis wrote this chapter in its entirety. 1 I. Metabolic Syndrome and Insulin Action in Obesity-linked Type II Diabetes Excess food consumption and physical inactivity inevitably lead to weight gain and in extreme cases, obesity. Increased adiposity associated