ANALYSIS OF GENETIC SUSCEPTIBILITY TO TYPE II DIABETES IN MICE By SOHA N. YAZBEK Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy Dissertation advisor: Dr. Joseph H. Nadeau Department of Genetics CASE WESTERN RESERVE UNIVERSITY August 2010 CASE WESTERN RESREVER UNIVERSITY SCHOOL OF GRADUATE STUDIES We hereby approve the thesis/dissertation of Soha N. Yazbek ________________________________________________________ candidate for the PhD degree* Mark Adams (signed)__________________________________________________ (chair of the committee) Joseph Nadeau __________________________________________________ Colleen Croniger ____________________________________________________ Mitchell Drumm ____________________________________________________ May 4, 2010 (date) _______________________ *We also certify that written approval has been obtained for any proprietary material contained therein. 2 Dedication I dedicate this work to the soul of my late grandmother, Mountaha Harb, who died two years short of seeing what I have become. I am sure she is watching over me and I hope she is proud. 3 Table of Contents Dedication ............................................................................................................................................. 3 TABLE OF CONTENTS.......................................................................................................... 4 List of tables ......................................................................................................................................... 7 List of figures ........................................................................................................................................ 8 Acknowledgement ............................................................................................................................... 9 Abstract ................................................................................................................................................. 11 1 CHAPTER 1: BACKGROUND AND SIGNIFICANCE .................................................. 13 1.1 Type II diabetes: An introduction ..................................................................................................... 14 1.2 Overview of Insulin Action on Glucose Homeostasis ........................................................................ 16 1.2.1 Insulin ............................................................................................................................................... 16 1.2.2 Insulin Secretion ............................................................................................................................... 19 1.2.3 Insulin Signaling ............................................................................................................................... 21 1.2.4 Glucose homeostasis ....................................................................................................................... 22 1.3 Genetics of Type 2 diabetes .............................................................................................................. 25 1.3.1 Genetic studies in humans ............................................................................................................... 26 1.3.2 Missing Heritability ......................................................................................................................... 30 1.3.3 Genetic studies in mouse models .................................................................................................... 32 1. Spontaneous mutant mouse models of T2D ............................................................................... 32 2. Single gene Knock-out and transgenic models of T2D ................................................................. 33 3. Inbred strains (mouse models of polygenic T2D) ........................................................................ 36 1.4 C57BL/6J and A/J Inbred Strains: Alternative model ........................................................................ 38 1.4.1 Advantage of B6 and A/J inbred strains ........................................................................................... 38 1.4.2 B6-chrA CSS development and advantage ........................................................................................ 40 1.4.3 Choosing CSS-6 ................................................................................................................................. 45 1.6 Summary and Research Aims ........................................................................................................... 46 2 CHAPTER 2: INCREASED MITOCHONDRIAL OXIDATIVE PHOSPHORYLATION IN THE LIVER IS ASSOCIATED WITH OBESITY AND INSULIN RESISTANCE ............................................................................................................ 49 2.1 Abstract ............................................................................................................................................ 50 2.2 Introduction ..................................................................................................................................... 52 4 2.3 Materials and Methods .................................................................................................................... 54 2.3.1 Mice. ................................................................................................................................................ 54 2.3.2 RNA Isolation. ................................................................................................................................... 54 2.3.3 Microarray hybridization. ................................................................................................................ 55 2.3.4 Microarray data analysis. ................................................................................................................. 55 2.3.5 Glucose tolerance test (GTT) and insulin secretion ......................................................................... 55 2.3.6 Insulin tolerance test (ITT). .............................................................................................................. 56 2.3.7 Statistics. .......................................................................................................................................... 56 2.4 Results .............................................................................................................................................. 57 2.4.1 Localization of Obrq2 to a 30 Mb interval on chromosome 6 ......................................................... 57 2.4.2 6C1 mice are insulin resistant relative to 6C2 .................................................................................. 59 2.4.3 Global gene expression analysis ...................................................................................................... 61 2.4.4 Genes involved in oxidative phosphorylation are over-expressed in the liver of 6C1 ..................... 63 2.5 Discussion ........................................................................................................................................ 67 3 CHAPTER 3: SOLUTE RECEPTOR SLC35B4 OVER-EXPRESSION LINKED TO DIET-INDUCED OBESITY AND INSULIN RESISTANCE ................................................. 74 3.1 Abstract ............................................................................................................................................ 75 3.2 Introduction ..................................................................................................................................... 77 3.3 Materials and Methods .................................................................................................................... 80 3.3.1 Husbandry ........................................................................................................................................ 80 3.3.2 Generation of subcongenic and subsubcongenic strains ................................................................. 80 3.3.3 Sequence Analysis. ........................................................................................................................... 81 3.3.4 Fasting insulin, fasting glucose and HOMA-IR .................................................................................. 81 3.3.5 Glucose Tolerance Test. ................................................................................................................... 82 3.3.6 Tissue Collection and RNA extraction .............................................................................................. 82 3.3.7 Real-time quantitative PCR. ............................................................................................................. 82 3.3.8 Hyperinsulinemic-Euglycemic Clamp. .............................................................................................. 82 3.3.9 Determination of glucose uptake in peripheral tissues. .................................................................. 84 3.3.10 Statistics. ........................................................................................................................................ 85 3.4 Results ............................................................................................................................................. 85 3.4.1 Obrq2a regulates body weight and IR ............................................................................................