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UCP2) Knockout Mouse Model Metabolic and Functional Characterization of an Inducible Pancreatic β Cell Specific Uncoupling Protein-2 (UCP2) Knockout Mouse Model By Qian-yu Guo A thesis submitted in conformity with the requirement for the degree of Masters of Science Graduate Department of Physiology University of Toronto © Copyright by Qian-yu Guo (2012) Abstract Metabolic and Functional Characterization of an Inducible Pancreatic β Cell Specific Uncoupling Protein-2 (UCP2) Knockout Mouse Model Qian-yu Guo Master of Science Thesis 2012 Department of Physiology University of Toronto In order to elucidate how uncoupling protein 2 (UCP2) influences pancreatic β cells and glucose homeostasis, I have generated and characterized an inducible β cell-specific UCP2 deletion model, MIPCreER×loxUCP2 mice. Male littermates were injected with tamoxifen to induce UCP2 deletion (UCP2 iBKO) or with corn oil (CO). The phenotypes of both short-term (3-4 weeks after the last injection) and long-term (8-9 weeks after the last injection) were determined: Short-term iBKO mice displayed no differences in glucose or insulin tolerance, but enhanced in vivo and in vitro insulin secretion and suppressed islet reactive oxygen species (ROS) levels; while long-term iBKO mice displayed no difference in glucose tolerance, but impaired in vivo and in vitro insulin secretion and enhanced islet ROS levels. In conclusion, short-term UCP2 deletion in β cells promotes insulin secretion, while long-term UCP2 deletion impairs insulin secretion, possibly due to the opposite background of islet ROS. ii Supervisor’s Declaration This is to certify that the thesis entitled “Inducible Deletion of Uncoupling Protein-2 (UCP2) in Pancreatic β cells Enhances Insulin Secretion”, submitted by Qian-yu Guo in fulfillment of the degree of Master of Science, is ready for submission. Professor Michael B. Wheeler, Ph.D August 31 st , 2012 Author Declaration I hereby declare that this thesis is entirely my own work. This thesis does not, to the best of my knowledge, contain any material from any other source, except where due reference is made. This thesis was written completely and solely for the degree of Master of Science, and has not been submitted for a higher degree or diploma at any other academic institution. Figure 1.1 has been published in “Basford CL, Prentice KJ, Hardy AB, Sarangi F, Micallef SJ, Li X, Guo Q, Elefanty AG, Stanley EG, Keller G, Allister EM, Nostro MC and Wheeler MB. The functional and molecular characterisation of human embryonic stem cell-derived insulin-positive cells compared with adult pancreatic beta cells. Diabetologia 55(2):358-71 (2012) .” Part of the data in Chapter 3 has been published as “Guo Q , Robson-Doucette CA, Allister EM, Wheeler MB. Inducible deletion of UCP2 in pancreatic β cells enhances insulin secretion. Canadian Journal of Diabetes (Accepted)”. Permissions to use the articles have been obtained from both journals and attached at the end. Qian-yu Guo August 31 st , 2012 iii Acknowledgement The thesis submission marks the end of an eventful journey for which there are many people that I would like to acknowledge for their support along the way. I would like to extent my gratitude to my primary supervisor, Prof. Michael B. Wheeler, firstly for taking me on, and then for his passing of wisdom and guidance. I would also like to thank Drs. Christine A. Robson-Doucette and Emma M. Allister for being my mentors and guiding me through every large and small endeavor. I am grateful for my supervisory committee members, Drs. Zhong-ping Feng and Tianru Jin for their important discussions. I also wish to thank my entire extended family and my friends for providing a loving environment and being supportive for me all the time. iv Table of Contents Abstract…………………………………………………………………..………………….…......i Supervisor’s declaration……………………………………………………………………….……ii Author declaration………………………………………………………………………….……….ii Acknowledgement……………………………………………….……………………………….....iii List of Abbreviations………………………………………………………………………..………v List of Figures…………………………………………………………………………….………...viii Chapter 1. Introduction.………………………………………………………….……………………1 Chapter 2. Methods……………………………………………………….………………………….17 Chapter 3. Results…………………………………………………………….……………………...23 Chapter 4. Discussion…………………………………………………………………..…………….36 Reference……………………………………………………………………………..………………44 Copyright permissions………………………………………………………………………………..59 v List of Abbreviations ANT - adenine nucleotide translocase ATP - adenosine triphosphate CoA - coenzyme A CO - corn oil CPT-I - carnitine-palmitoyl transferase-I DM - diabetes mellitus DMEM - Dulbecco’s modified Eagle’s medium ESC - embryonic stem cells EGTA - ethylene glycol tetraaccetic acid FBS - fetal bovine serum FFA - free fatty acids GPx - glutathione peroxidase GSIS - glucose stimulated insulin secretion mETC - mitochondrial electron transporter chain MHC - melanin-concentrating hormone MMP - mitochondrial membrane potential MIP - mouse insulin 1 promoter MIPCreER - cre recombinase-estrogen receptor fusion protein driven by the mouse insulin promoter mNCX - mitochondrial Na +/Ca 2+ exchange NFκB - nuclear factor kappa-light-chain-enhancer of activated B cells NO - nitric oxide Nrf-2 –nuclear factor erythroid 2-related factor vi O2 - oxygen ORF - open reading frame OGTT - oral glucose tolerance test OXPHOS – oxidative phosphorylation H2O2 - hydrogen peroxide HO-1 - heme oxygenase-1 iAUC - incremental area under the curve IR - insulin resistance ITT - insulin tolerance test JNK - c-Jun N-terminal kinase KATP channel - ATP-sensitive potassium channel Keap-1 - kelch-like ECH-associated protein 1 KRB - Kreb’s Ringer buffer KV channel- voltage-gated potassium channel PBS - phosphate buffered solution PDX-1 - pancreatic and duodenal homeobox-1 PMF - proton motive force POMC - pro-opiomelanocortin PPAR - peroxisome proliferator activated receptor PGC-1 - PPAR-γ coactivator-1 RIP - rat insulin 2 promoter ROS - reactive oxygen species RyR - ryanodine receptor vii SOD - superoxide dismutases siRNA - small interference RNA SIRT-1 - Sirtuin-1 SRE - sterol regulatory element SREBP - sterol regulatory element binding protein T2DM - type 2 diabetes mellitus Tmx - tamoxifen UCP2- uncoupling protein 2 UCP2 -/- - whole-body UCP2 knockout model UCP2 BKO - β cell specific UCP2 knockout UCP2 iBKO - inducible β cell specific UCP2 knockout VDCC - voltage-dependent calcium channels viii List of Figures Chapter 1. Introduction Figure 1.1. The canonical GSIS pathway………………………………………………………..……3 Figure 1.2. The regulation of UCP2 expression………………………………………………………7 Figure 1.3. The mechanism of Nrf2-dependent antioxidant gene expression……………………….10 Figure 1.4. The influence of UCP2 on ROS production and insulin secretion……………………...14 Chapter 2. Methods Figure 2.1. The key genetic constructs and timeline of experiments…………………………...…...18 Chapter 3. Results Figure 3.1. Effective UCP2 deletion specifically from pancreatic β cells……………………...…...26 Figure 3.2. Co-localization of insulin and YFP in MIPCreER×ROSA26eYFP islet cells……...…..27 Figure 3.3. Tamoxifen treatment does not alter global glucose homeostasis…………………….…27 Figure 3.4. Body weight and fasting blood glucose……………………………..……………….….28 Figure 3.5. Short-term UCP2 iBKO mice exhibit normal glucose tolerance……………………......29 Figure 3.6. Short-term UCP2 iBKO mice exhibit in vivo glucagon secretion but enhanced in vivo insulin secretion during OGTT………………………………………………………………………30 Figure 3.7. Short-term UCP2 iBKO mice exhibit normal insulin sensitivity……………...………..31 Figure 3.8. Short-term UCP2 iBKO islets exhibit enhanced insulin secretion under the stimulation of high glucose…………………………………………………………………………………………..32 Figure 3.9. The mitochondrial membrane potential of short-term UCP2 deficient islet cells………33 Figure 3.10. Short-term UCP deleted iBKO islets exhibit decreased ROS levels…………………..34 Figure 3.11. Short-term UCP2 inhibited min-6 cells exhibit decreased ROS levels………………..35 Figure 3.12. ATP contents of UCP2 iBKO and CO islets were similar……………………………..36 ix Figure 3.13. Body weight and fasting blood glucose…………………………………………...…...36 Figure 3.14. Long-term UCP2 inducible BKO (iBKO) mice exhibited normal glucose tolerance but tended to have decreased plasma insulin levels during OGTT…………………………………..…..37 Figure 3.15. Long-term UCP2 iBKO islets exhibit impaired insulin secretion under the stimulation of high glucose……………………………………………………………………………….………38 Figure 3.16. Long-term UCP deleted iBKO islets exhibit increased ROS levels………………...…39 Figure 3.17. Long-term UCP2 inhibited min-6 cells tended to have increased ROS level………….39 x CHAPTER 1. INTRODUCTION 1 CHAPTER 1. INTRODUCTION 1. Type 2 diabetes mellitus (T2DM) The body maintains blood glucose levels within a relatively narrow range. The homeostatic system is consisted of of several components, of which hormone regulation is the most critical one. There are two types of mutually antagonistic metabolic hormones that regulate blood glucose levels: catabolic hormones, including glucagon, cortisol and catecholamines, which increase blood glucose, and one anabolic hormone, insulin, which decreases blood glucose. The islets of Langerhans are the region of the pancreas that plays a pivotal role in glucose homeostasis as they contain various endocrine cells, including α cells secreting glucagon, β cells producing insulin, δ cells generating somatostatin, PP cells secreting pancreatic polypeptide and ε cells producing ghrelin 1. Diabetes mellitus (DM) is a disorder primarily defined by the level of hyperglycaemia
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