The Effect of β-Cell Specific ZnT8 Deficiency on Insulin Secretion Anne Wu A thesis submitted in conformity with the requirements for the degree of Masters of Physiology Graduate Department of Medicine University of Toronto © Copyright by Anne Wu (2020) The Effect of β-Cell Specific ZnT8 Deficiency on Insulin Secretion Anne Wu Master of Science Department of Physiology University of Toronto 2020 Abstract Zinc plays a role in insulin processing, storage, and secretion. Zinc transport processes may be linked to defects in insulin secretion associated with type 2 diabetes (T2D). Indeed, ZnT8, a zinc influx transporter highly expressed in pancreatic β-cells, is associated with T2D risk. Presently, the role ZnT8 plays in diabetes progression and whether altered ZnT8 activity affects T2D risk/treatment is controversial. The effects of ZnT8 knockdown in mouse are modestly to severely deleterious, whereas in humans, ZnT8 loss- of-function haploinsufficiency appears to reduce diabetes risk. A β-cell specific ZnT8 knockout and haploinsufficiency model were developed and examined under normal and metabolically stressed conditions. Mice with reduced ZnT8 did not have significantly impaired glucose homeostasis or β-cell function compared to controls under normal and conditions of acute insulin resistance. Overall, pancreatic β-cell ZnT8 deletion or knockdown did not impair β-cell function, nor was it protective of hyperglycaemia associated with insulin resistance. ii Acknowledgements Thank you to the entire Wheeler lab for supporting me. Thanks to Dr. Alpana Bhatacharjee, Dr. Feihan Dai, Dana Al Rijjal, Jie Xu, Ashley Untereiner, Dr. Mi Lai, Dr. Saifur Khan for their advice and training. They provided great examples of how to conduct research. Importantly, thanks to Dr. Michael B. Wheeler for providing me this opportunity to grow as both a scientist and as a person. I truly appreciate the adaptations taken to accommodate my learning style. I would be remiss not to thank the organizations who helped fund my schooling. Thanks to the Ontario Graduate Scholarship award (from the Estate of Gladys. A Fidlar), the Banting and Best Diabetes Centre Novo Nordisk Studentship, and the CIHR grant held by Dr. Michael B. Wheeler for providing me with the means to pursue diabetes research. A huge thank you to Dr. Jonathan Rocheleau and Dr. Cynthia Luk, my committee members. They provided valuable insight and offered considerations we would have otherwise missed. I’d like to thank my friends who kept me sane through this degree. They provided a great sounding board for me to talk through my confusion and gave me much needed reminders to take breaks to work more effectively. And finally, I’d like to thank the mice whose sacrifices contributed to further diabetic research. iii Table of Contents Abstract ................................................................................................................................................... ii Acknowledgements ............................................................................................................................... iii Awards and Publications ....................................................................................................................... vii List of Tables ........................................................................................................................................ viii List of Figures ......................................................................................................................................... ix Abbreviations .......................................................................................................................................... x Chapter 1: Introduction .......................................................................................................................... 1 1.1 Zinc Homeostasis .......................................................................................................................... 1 1.1.1 Zinc in Diabetes ...................................................................................................................... 1 1.2 Zinc Transporters .......................................................................................................................... 4 1.3 Zinc Transporter 8 (ZnT8) and Diabetes ....................................................................................... 5 1.3.1 GWAS studies of SLC30A8/ZnT8 ............................................................................................ 6 1.3.2 ZnT8 in Mice ........................................................................................................................... 8 1.3.3 ZnT8 Controversy ................................................................................................................. 10 1.3.4 Other functions of ZnT8 ....................................................................................................... 12 1.4 Rationale ..................................................................................................................................... 14 1.4.1 Hypothesis ........................................................................................................................... 14 1.4.2 Objectives ............................................................................................................................ 15 Chapter 2: Methods .............................................................................................................................. 16 2.1 Model Generation ....................................................................................................................... 16 2.1.1 PCR ....................................................................................................................................... 17 2.2 Islet Isolation ............................................................................................................................... 18 2.3 Knockdown Detection ................................................................................................................. 19 2.3.1 qPCR ..................................................................................................................................... 19 iv 2.3.2 Dithizone staining ................................................................................................................ 20 2.4 Validation of S961 ....................................................................................................................... 20 2.4.1 Daily S961 injection .............................................................................................................. 20 2.4.2 Acute S961 Injection Challenge ........................................................................................... 22 2.5 In vivo experiments ..................................................................................................................... 22 2.5.1 Oral Glucose Tolerance Tests............................................................................................... 22 2.5.2 Acute S961 Challenge .......................................................................................................... 22 2.6 Ex Vivo Experiments .................................................................................................................... 23 2.6.1 Glucose Stimulated Insulin Secretion .................................................................................. 23 2.6.2 Total Islet Insulin .................................................................................................................. 23 2.6.3 Insulin Concentration Measurements ................................................................................. 23 2.7 Statistical Analysis ....................................................................................................................... 24 Chapter 3: Results ................................................................................................................................. 25 3.1 Model Confirmation .................................................................................................................... 25 3.1.1 PCR ....................................................................................................................................... 25 3.1.2 qPCR ..................................................................................................................................... 25 3.2.3 Dithizone staining ................................................................................................................ 26 3.2 S961 Validation ........................................................................................................................... 27 3.2.1 Daily S961 Injections ............................................................................................................ 27 3.2.2 Acute S961 Challenge Validation ......................................................................................... 27 3.3 In vivo data .................................................................................................................................. 30 3.3.1 OGTT .................................................................................................................................... 31 3.3.2 S961 ..................................................................................................................................... 31 3.4