GENETIC RESISTANCE TO DIET-INDUCED OBESITY IN MICE By LINDSAY CATHERINE BURRAGE Submitted in partial fulfillment of the requirements For the degree of Doctor of Philosophy Thesis Advisors: Dr. Joseph H. Nadeau Dr. Colleen M. Croniger Department of Genetics CASE WESTERN RESERVE UNIVERSITY August, 2006 CASE WESTERN RESERVE UNIVERSITY SCHOOL OF GRADUATE STUDIES We hereby approve the dissertation of ______________________________________________________ candidate for the Ph.D. degree *. (signed)_______________________________________________ (chair of the committee) ________________________________________________ ________________________________________________ ________________________________________________ ________________________________________________ ________________________________________________ (date) _______________________ *We also certify that written approval has been obtained for any proprietary material contained therein. Copyright © 2006 by Lindsay Catherine Burrage All rights reserved 1 TABLE OF CONTENTS 2 LIST OF TABLES. 4 LIST OF FIGURES. 8 ACKNOWLEDGEMENTS . 12 LIST OF ABBREVIATIONS. 14 ABSTRACT. 19 CHAPTER I: INTRODUCTION AND SUMMARY OF RESEARCH AIMS. 21 A. Obesity: An introduction. 22 1. Historical perspective. .22 2. Definition and prevalence of human obesity. 22 3. Causes of human obesity. 26 4. Pathological consequences of obesity. 28 5. Physiologic control of energy balance and body weight. 32 6. Treatment of human obesity. 35 B. Genetics of obesity. 36 1. An introduction to obesity genetics. 36 2. Genetic forms of obesity. 37 C. Methods for investigating the genetics of obesity. 42 1. Genetic studies of obesity in humans. 42 2. Genetic studies of obesity in mouse. 43 D. C57BL/6J and A/J inbred strains: Mouse models for obesity. 52 1. Differential susceptibility to diet-induced obesity in C57BL/6J and A/J male mice. 52 2. Energy metabolism in C57BL/6J and A/J male mice. 54 3. Genetic studies of obesity in C57BL/6J and A/J inbred strains. 55 4. C57BL/6J and A/J: Advantages for complex trait studies. 56 5. B6 –ChrA CSSs accelerate QTL mapping studies in C57BL/6J and A/J strains. 57 E. Summary and research aims. 63 CHAPTER II: GENETIC DISSECTION OF OBESITY IN CHROMOSOME SUBSTITUTION STRAINS. 66 A. Introduction. 67 B. Materials and Methods. 69 C. Results. 78 D. Discussion. 110 CHAPTER III: GENETIC DISSECTION OF OBESITY RESISTANCE ON A/J-DERIVED CHROMOSOME 6. 118 A. Introduction. 119 B. Materials and Methods. 123 C. Results. 129 D. Discussion. 147 3 CHAPTER IV: PHENOTYPIC DISSECTION OF RESISTANCE TO DIET-INDUCED OBESITY IN B6-CHR 6A CHROMOSOME SUBSTITUTION STRAIN MICE. 153 A. Introduction. 154 B. Materials and Methods. 156 C. Results. 160 D. Discussion. 181 CHAPTER V: DISCUSSION AND FUTURE DIRECTIONS. .188 A. Discussion. 189 1. Multiple genes and gene interactions influence obesity resistance. 189 2. CSSs provide models for studying obesity resistance and related traits. 192 3. Evaluation of CSS intercrosses as a method for QTL localization . 193 B. Future Directions. 196 1. How do we identify obesity resistance genes detected in CSSs?. 196 2. How do we identify obesity resistance genes which did not produce peaks in the CSS intercrosses?. 198 3. What mechanism explains the resistance to diet-induced obesity in CSSs and congenic strains?. 202 APPENDICES. 205 A. Appendix I: Genetic markers used in CSS intercross mapping studies. 205 B. Appendix II: Correlations among traits in CSS intercross progeny. 212 B. Appendix III: Multiple QTL analysis in CSS intercross progeny. 233 C. Appendix IV: Genetic and phenotypic analyses of Obrq2. 242 REFERENCES. 264 4 LIST OF TABLES 5 CHAPTER II II-1. Composition of high-fat and low-fat diets. 72 II-2. Three-way ANOVA tables for C57BL/6J and A/J fed diets with varied fat and carbohydrate composition. 81 II-3. Initial HFSC diet CSS survey. 83 II-4. LFCC diet CSS survey. 85 II-5. HFSC vs. LFCC diet CSS survey analysis. 88 II-6. Comparison of replicate HFSC diet CSS surveys. 91 II-7. HFSC diet replicate CSS survey #1. 92 II-8. HFSC diet replicate CSS survey #2. 93 II-9. Trait correlations in B6-ChrA CSS F2 crosses. 96 II-10. LOD scores for B6-ChrA F2 genome scan with p values adjusted for traits analyzed. 97 II-11. LOD scores from B6-ChrA F2 genome scan with p values adjusted for number of crosses and number of traits analyzed. 99 II-12. Support intervals for significant and suggestive QTLs. 100 II-13. QTL inheritance patterns. 103 CHAPTER III III-1. Markers used for congenic panel construction. 126 III-2. FW in the HFSC diet congenic strain survey. 135 III-3. Final BMI for the HFSC diet congenic strain survey. 136 III-4. 62.9-A and 62-BL congenic strain replicate analyses. 137 6 III-5. 62-BL reciprocal cross analysis. 146 CHAPTER IV IV-1. HFSC diet consumption in C57BL/6J, A/J, and B6-Chr 6A males. 167 IV-2. Blood chemistry and liver triglycerides in C57BL/6J (B6), A/J, and B6-Chr 6A at 85 days of age (50 days of high-fat diet consumption). 169 IV-3. Blood chemistry and liver triglycerides in C57BL/6J (B6), A/J, and B6-Chr 6A at 135 days of age (100 days of high-fat diet consumption). 170 IV-4. Metabolic phenotyping in 62-B congenic strain. 175 IV-5. Sterol measurements in C57BL/6J, A/J, B6-Chr 6A (CSS-6), and 62-B congenic strain. 177 IV-6. Amino acid measurements in C57BL/6J, A/J, B6-Chr 6A (CSS-6), and 62-B congenic strain. 179 IV-7. Acylcarnitine profile in C57BL/6J, A/J, B6-Chr 6A (CSS-6), and 62-B congenic strain. 180 APPENDIX III AIII-1. Two dimensional genome scan in B6-ChrA CSS F2 crosses. 236 APPENDIX IV AIV-1. Composition of LabDiet 5010. 246 AIV-2. FW and BMI for 92-A vs. 62-BL after 100 days of 5010 or HFSC diet consumption. 253 7 AIV-3. HFSC diet consumption in 92-A vs. 62-BL males. 256 AIV-4. Blood chemistry in 92-A vs. 62-BL male mice fed the HFSC diet for 28 or 100 days. 257 AIV-5. 62-BL subcongenic HFSC diet survey. 262 8 LIST OF FIGURES 9 CHAPTER I I-1. Energy balance. 24 I-2. Leptin and the melanocortin pathway. 34 I-3. B6-ChrA CSS panel construction. 58 I-4. QTL mapping with CSSs. 60 CHAPTER II II-1. Time course for body weight studies. 73 II-2. C57BL/6J (B6) and A/J weight gain on diets that differ in fat and carbohydrate composition. 79 II-3. C57BL/6J (B6) and A/J body weight (MW, FW, EWG, FWG, and WG) when fed diets that differ in fat and carbohydrate composition. 80 II-4. Correlations between replicate HFSC diet survey traits. 89 II-5. B6-ChrA CSS whole genome scan. 101 II-6. Comparison of CSS surveys and CSS whole genome scan analysis . 104 II-7. FW for B6-ChrA CSS F2 progeny vs. C57BL/6J (B6). 106 II-8. Maternal inheritance was not detected in B6-MitoA . 109 CHAPTER III III-1. Congenic panel derived from B6-Chr 6A. 124 III-2. FW and BMI in the F1 [B6-Chr 6A (CSS-6) x C57BL/6J (B6)] male mice. 131 III-3. FW and BMI in the HFSC diet congenic strain survey. 134 III-4. 62-BL congenic strain HFSC diet replicate analysis. 138 10 III-5. Obrq1 critical interval. 141 III-6. Obrq2 critical interval. 143 III-7. Obrq3 critical interval. 144 III-8. Summary of QTLs discovered on chromosome 6 . 145 CHAPTER IV IV-1. Gonadal fat pad weights and BMI in C57BL/6J (B6) relative to A/J and B6-Chr 6A (CSS-6) fed the HFSC diet for 100 days. 161 IV-2. Pearson’s correlation coefficients for fat pad weight vs. final body weight and fat pad weight vs. BMI in B6-Chr 6A males fed the HFSC diet. 162 IV-3. HFSC diet consumption in C57BL/6J (B6), A/J, and B6-Chr 6A (CSS-6) males. 164 IV-4. B6-Chr 6A (CSS-6) is resistant to the development of fatty liver after 50 days of HFSC diet consumption. 172 IV-5. B6-Chr 6A (CSS-6) is resistant to the development of fatty liver after 100 days of HFSC diet consumption. 173 IV-6. Summary of adiposity, food intake, blood chemistry, and liver triglyceride analyses in C57BL/6J (B6) and B6-Chr 6A male mice fed the HFSC diet for 100 days. 183 IV-7. Summary of adiposity, food intake, blood chemistry, and liver triglyceride analyses in C57BL/6J (B6) and 62-B male mice fed the HFSC diet for 100 days. 185 11 CHAPTER V V-1. Methods for investigating the mechanism of obesity resistance. 204 APPENDIX III AIII-1. Two dimensional genome scan in the B6-ChrA F2 crosses. 240 APPENDIX IV AIV-1. 62-B subcongenic panel. 249 AIV-2. 92-A vs. 62-BL growth curves on 5010 and HFSC diets. .252 AIV-3. HFSC diet consumption in 92-A vs. 62-BL male mice. 255 AIV-4. 62-BL subcongenic HFSC diet survey. ..