Organic Anion Transporter Blockade to Improve Beta Cell Function in Diabetes by Judith Andrea Eversley A thesis submitted in conformity with the requirements for the degree of Master of Science in Physiology Department of Physiology University of Toronto © Judith Andrea Eversley (2016) Organic Anion Transporter Blockade to Improve Beta Cell Function in Diabetes Judith Andrea Eversley Department of Physiology University of Toronto 2016 ABSTRACT The furan fatty acid metabolite 3-carboxy-4-methyl-5-propyl-2-furanpropanoic acid (CMPF) is elevated in Type 2 Diabetes. CMPF delivery to diabetic levels in mice causes glucose intolerance, impairing glucose-stimulated insulin secretion (GSIS). CMPF enters the β cell through Organic Anion Transporters (OATs). Here, we investigate whether OAT blockade represents a therapeutic target for diabetes prevention, blocking CMPF action on the β cell. First, we block CMPF transport in vivo using pharmacological OAT inhibition. CMPF impairs glucose tolerance, and islets isolated from CMPF-treated mice showed impaired GSIS, while co-treatment with pan-OAT inhibitor probenecid rescues glucose tolerance and islet function. Next, we developed Oat3 knockout (Oat3KO) mice which were also protected against the negative effects of CMPF in vivo, and remarkably, Oat3KO mice were more glucose tolerant than Wildtype controls. These novel studies demonstrate the requirement of OAT transport for CMPF to alter β cell function, and highlight potential for OAT inhibition to improve diabetes. ii ACKNOWLEDGEMENTS I owe the productivity and success I enjoyed during my Master’s degree to many people who have helped guide and support me along the way, and I would like to take this opportunity to acknowledge these people. First to my supervisor, Mike: thanks for providing me with endless opportunities to explore my scientific interests. You took a chance on me—even though you thought I was too timid to be a Master’s Student in the Wheeler Lab—and I’m so proud of how much I have grown under your supervision. Next to my colleagues in the Wheeler Lab: how lucky am I to have been surrounded by so many caring, intellectual individuals during my two years in the lab? Alpana and Elena, you are both so kind and have been so much help. Ying and Tseegii thank you for your guidance throughout these two years, and for being my bonus bosses. Sean and Rida, thank you for being great friends and keeping me going. Finally Dr. Prentice, thank you for teaching me literally everything I know about anything from western blots to intracellular signalling pathways to appropriate conference attire. To my Graduate Committee, Drs. Bazinet, Cummins, and Ng: it was a pleasure to work with all of you. Thank you for your insights, patience, and support! To my friends: it’s such a gift to have an amazing support network in Toronto and London. My AGD family, Western Phys/Pharm friends, and MCI friends deserve special recognition here. Importantly, I can’t write acknowledgements without mentioning my best friends Julia and Reid: thinking about the love and encouragement you’ve shown me makes me an emotional wreck. I couldn’t have done this without you. Finally to my family: Jude, John, and Jenn. Growing up in a family with a bunch of geniuses wasn’t easy at first but it certainly paid off in the end. I’ve always aspired to be as motivated, hardworking, and successful as you. It’s going to be a tight race to be the next Dr. J. A. Eversley, so I’ll have to keep on track. I want Grandpa’s sign now. iii TABLE OF CONTENTS Section Title Page Abstract ii Acknowledgements iii Table of Contents iv List of Tables viii List of Figures ix Abbreviations xi Chapter 1 – Introduction 1 1.1 Diabetes Mellitus 1 1.1.1 Classification of Diabetes 1 1.1.1a Type 1 Diabetes 1 1.1.1b Type 2 Diabetes 2 1.1.1c Gestational Diabetes 4 1.1.2 Complications of Diabetes 4 1.1.2a Macrovascular Complications 4 1.1.2b Microvascular Complications 5 1.1.2c GDM Complications 5 1.1.2d Managing Diabetes 5 1.1.3 The Pancreatic β Cell 7 1.1.3a Insulin 7 1.1.3b Glucose-Stimulated Insulin Secretion 8 1.1.3c Other Receptors Regulating Insulin Secretion 9 1.1.4 β Cell Dysfunction 10 1.1.4a Glucotoxicity, Lipotoxicity, Glucolipotoxicity 11 1.1.4b Oxidative Stress 11 1.1.5 Discovery-based Screening to Identify Factors Involved in T2D 12 1.1.5a Metabolomics 13 1.2 3-carboxy-4-methyl-5-propyl-2-furanpropanoic acid (CMPF) 15 1.2.1 Discovery of CMPF in Diabetes 15 1.2.1a CMPF is Elevated in populations with GDM and T2D 15 1.2.1b What is CMPF 15 1.2.1c CMPF is Elevated Prior to Overt Diabetes Onset 17 1.2.1d Rapid Elevation in Circulating CMPF may Accelerate T2D 17 Progression 1.2.1e CMPF Predicts AUCglucose in GDM women 18 1.2.2 CMPF Directly Impairs β cell Function 18 1.2.2a CMPF Impairs Glucose Tolerance in vivo 18 1.2.2b CMPF Impairs Insulin Secretion 19 1.2.2c CMPF Directly Impairs Insulin Biosynthesis and Secretion in 19 vitro 1.2.3 Rapid Elevation in CMPF Accelerates Diabetes Progression in vivo 20 1.2.3a CMPF Accelerates T2D in Diet Induced Obesity 20 iv 1.2.3b CMPF Accelerates T2D in Genetic Prediabetes Models 21 1.2.3c CMPF Reduces Glycolysis and Increases Oxidative Stress 21 1.2.4 CMPF enters the β cell through OATs 22 1.2.4a OAT3 is Present in Insulin-Positive Cells 22 1.2.4b OAT3 Inhibition Protects against CMPF in vivo 23 1.3 Organic Anion Transporters 24 1.3.1 Classification of Drug Transporters 24 1.3.1a Characteristics of Drug Transporters 24 1.3.1b Drug Transporter Gene Families 25 1.3.2 The Organic Anion Transporter Family 27 1.3.2a OAT Nomenclature 27 1.3.3 Organic Anion Transporter 3 28 1.3.3a OAT3 Structure and Function 28 1.3.3b Renal Elimination of Organic Anions by OAT3 31 1.3.3c OAT3 Substrates 33 1.3.3d OAT3 Inhibitors 37 1.3.3e Factors Influencing OAT3 Expression 38 Chapter 2 – Research Aims and Hypotheses 39 2.1 Rationale 39 2.2 Objective and Hypotheses 39 2.3 Scientific Aims 39 2.3.1 OAT Blockade to Improve β cell Function in vivo 39 2.3.1a Aim 1a: in vivo Genetic Elimination of Oat3 40 2.3.1b Aim 1b: in vivo Pharmacological OAT Inhibition 40 2.3.2 Screening Methods to Discover Novel OAT3 Inhibitors 40 Chapter 3 – OAT Blockade to Improve β cell Function in vivo 41 3.1 Introduction 41 3.2 Materials and Methods 42 3.2.1 CMPF, Probenecid Preparation 42 3.2.2 Human Islets 42 3.2.3 Mitochondrial Membrane Potential Measurements 42 3.2.4 Generation of Oat3KO mice 43 3.2.4a Oat3KO mice 43 3.2.4b Oat3BKO mice 43 3.2.4c Genotyping Slc22a8 floxed mice 44 3.2.5 Intraperitoneal Injection of CMPF, Probenecid 46 3.2.5a Oat3KO-CMPF experiments 46 3.2.5b Probenecid then CMPF experiments 47 3.2.5c Long-term CMPF experiments 47 3.2.6 SRM/MS for the Quantification of CMPF 48 3.2.7 Tolerance Tests 48 3.2.8 Islet Isolation, Glucose-Stimulated Insulin Secretion, Insulin 48 HTRF v 3.2.9 Islet Oxygen Consumption Measurement 49 3.2.10 Gene Expression & Western Blotting 49 3.2.11 Statistical Analysis 50 3.3 Results 50 3.3.1 OAT Blockade to protect against CMPF in vitro 50 3.3.1a Human Islet ROS Accumulation 50 3.3.1b Glucose-Stimulated Insulin Secretion 51 3.3.1c Islet CMPF Metabolism 52 3.3.1d CMPF causes a Metabolic Switch in vitro 53 3.3.2 Characterization of Oat3KO mice 54 3.3.3 Genetic Elimination of Oat3 to protect against CMPF in vivo 56 3.3.3a Measurement of CMPF in Oat3KO mice 56 3.3.3b Glucose Tolerance Testing 56 3.3.3c Glucose-Stimulated Insulin Secretion 58 3.3.3d Islet Glucose Metabolism 58 3.3.4 Pharmacological Oat Inhibition to protect against CMPF in vivo 59 3.3.4a Measurement of CMPF in Probenecid-treated mice 59 3.3.4b Glucose Tolerance Testing 60 3.3.4c Glucose-Stimulated Insulin Secretion 61 3.3.4d Islet ROS Accumulation 61 3.3.5 CMPF Causes Persistent Glucose Intolerance 62 3.3.5a CMPF is Eliminated from Circulation 24h Following 62 Injection 3.3.5b CMPF has Long-term Action on Glucose Tolerance 63 with an Insulin Resistant Background 3.3.5c Long-term Action of CMPF is Inhibited by Oat3 64 Elimination 3.3.5d Long-term Action of CMPF is Inhibited by 65 Pharmacological OAT Inhibition 3.4 Discussion 65 Chapter 4 – Screening Methods to Discover Novel OAT3 Inhibitors 66 4.1 Introduction 66 4.2 Materials and Methods 67 4.2.1 CMPF, Probenecid Preparation 67 4.2.2 HEK293 Cell Culture, Transfection 68 4.2.3 Reactive Oxygen Species Measurement 68 4.2.4 6-CF Measurement of OAT3 Transport 68 4.2.5 Statistical Analysis 68 4.3 Results 69 4.3.1 Overexpression of OAT3 in HEK293 Cells 69 4.3.2 OAT inhibitor Screening Assays 70 4.3.2a Using CMPF-induced ROS Accumulation in HEK293- 70 OAT3 Cells vi 4.3.2b Using CMPF-induced ROS Accumulation in HEK293 70 Cells 4.3.2c Using 6-CF Transport 71 4.4 Discussion 72 Chapter 5 – Discussion 74 5.1 Concluding Remarks and Future Directions 74 References 78 vii LIST OF TABLES Table 1 Canadian Diabetes Association Criteria for the Diagnosis of IGT and T2D.