Copyright by Karrie E. Wheatley 2010
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Copyright by Karrie E. Wheatley 2010 The Dissertation Committee for Karrie Elizabeth Wheatley Certifies that this is the approved version of the following dissertation: Energy Balance and Cancer Risk: Metabolic and Molecular Studies Committee: Stephen D. Hursting, Supervisor Kimberly Kline Michelle A. Lane Jon M. Huibregtse Robin Fuchs-Young Energy Balance and Cancer Risk: Metabolic and Molecular Studies by Karrie Elizabeth Wheatley, B.A. Dissertation Presented to the Faculty of the Graduate School of The University of Texas at Austin in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy The University of Texas at Austin May 2010 Acknowledgements I would like to first thank my mentor, Stephen D. Hursting for his years of patience, for his willingness to pursue unfamiliar avenues of research, and for his enthusiasm. I know that I will continue to rely on him for advice on my professional development and am grateful to know that he will gladly continue to offer whatever support he can. I would also like to thank my committee members for their time and service. They each contributed a unique attribute to the process. Dr. Kline was adamant in her support of realistic and achievable timelines. Dr. Lane encouraged me to publish as much as I could. Dr. Fuchs-Young rigorously questioned me about data and experimental design. Dr. Huibregtse, as he was less familiar with nutritional sciences, asked the most interesting questions that made me consider a broader audience as I wrote this dissertation. I want to thank Dr. Susan Perkins for her many thoughtful and careful revisions over the years. Finally, I would like to thank Dr. Lashinger and Dr. Smith for their daily contributions to my development. iv Energy Balance and Cancer Risk: Metabolic and Molecular Studies Publication No._____________ Karrie Elizabeth Wheatley, Ph.D The University of Texas at Austin, 2010 Supervisor: Stephen D. Hursting Over 60% of U.S. adults are either overweight or obese. The effect of obesity on cancer mortality is striking: approximately 90,000 deaths per year from cancer may be avoided if Americans could maintain a BMI of <25.0 throughout adulthood. The aim of this research was to employ and evaluate energy balance interventions designed to reverse obesity-related risk factors. The overarching hypothesis was that energy balance interventions would reduce cancer risk. To test this hypothesis, we used mostly animal models of diet-induced obesity and tested the effect of a low carbohydrate diet, calorie restriction, and exercise on adiposity, levels of circulating hormones, insulin resistance, and oxidative stress. To extend on in vivo findings from the final animal study, we utilized a human breast cancer cell line to further characterize the gene expression of thioredoxin-interacting protein in the context of p53 deficiency. Calorie restriction was v the most potent energy balance intervention. It caused weight loss, slowed tumor growth, reduced circulating IGF-1 and leptin levels, improved insulin resistance, and elicited a robust transcriptional response in visceral white adipose tissue following weight loss. Although a low carbohydrate diet and exercise did decrease hormones associated with obesity, (IGF-1 and leptin respectively) calorie restriction proved to be the most effective at reducing multiple obesity-related factors. Finally, from our studies analyzing the effect of obesity and exercise on oxidative stress in the context of p53-deficiency, we discovered that thioredoxin-interacting protein is transcriptionally upregulated in response to increased glucose flux associated with metabolic dysregulation that occurs as a consequence of loss of p53. vi Table of Contents List of Tables ...........................................................................................................x List of Figures........................................................................................................ xi List of Illustrations................................................................................................ xii Chapter 1: Introduction...........................................................................................1 1.1 Obesity Defined ..............................................................................1 1.2 The U.S. Obesity Epidemic ............................................................1 1.3 Obesity and Cancer.........................................................................2 1.4 Possible Mechanisms Underlying the Obesity-Cancer Connection 5 1.4.1 Chronic Hyperinsulinemia and IGF-1 ................................6 1.4.2 Estrogen ..............................................................................7 1.4.3 Oxidative Stress ...................................................................8 1.4.4 Inflammation.......................................................................9 1.5. The effect of calorie restriction on cancer risk .............................11 1.6. Studying Cancer Risk and Exercise..............................................11 1.6.1 Breast cancer and exercise.................................................13 1.6.2 Colon cancer and exercise .................................................14 1.7 Possible mechanisms underlying physical activity and cancer......14 1.7.1 Effects on circulating estrogen..........................................14 1.7.2 Effects on hyperinsulinemia and IGF-1............................15 1.7.3 Oxidative stress and inflammation.....................................17 1.8 Obesity and tumor metabolism .....................................................18 1.9 Dissertation objectives...................................................................19 Chapter 2: Low-Carbohydrate Diet versus Caloric Restriction: Effects on Weight Loss, Hormones and Colon Tumor Growth in Obese Mice .........................23 2.1 Introduction...................................................................................23 2.2 Materials and methods ................................................................25 2.2.1 Animals and Diets...........................................................25 vii 2.2.2 Serum IGF-1 and Leptin Levels ...................................29 2.2.3 Percent Body Fat Analysis............................................29 2.2.4 Statistical Analysis.......................................................29 2.3 Results...........................................................................................30 2.3.1 Body Weight Analysis ....................................................30 2.3.2 Serum IGF-1 .....................................................................34 2.3.3 Serum Leptin.....................................................................34 2.3.5 Tumor Latency..................................................................36 2.3.6 Final Tumor Burden..........................................................36 2.4 Discussion.....................................................................................38 Chapter 3: Differential effects of calorie restriction and exercise on the adipose transcriptome in diet-induced obese mice.....................................................41 3.1 Introduction....................................................................................41 3.2 Materials and Methods...................................................................42 3.2.1 Animal Study Design.........................................................42 3.2.2 Glucose Tolerance Test......................................................44 3.2.3 Serum Hormones ...............................................................45 3.2.4 Microarray Analysis...........................................................45 3.2.5 Chromatin immunoprecipitation (ChIP) assay ..................46 3.2.6 Statistics ......................................................................................47 3.3 Results............................................................................................47 3.4 Discussion......................................................................................63 Chapter 4: p53, oxidative stress, and metabolism.................................................69 4.1 Introduction...................................................................................69 4.2 Materials and Methods...................................................................70 4.2.1 Animal Study Design.........................................................70 4.2.2 Glucose Tolerance Test......................................................73 4.2.3 Quantitation of lipid and DNA oxidation ..........................73 4.2.4 Quantitative real-time PCR analysis..................................73 4.2.5 Cell culture.........................................................................74 4.2.6 Small interfering RNA constructs and expression vectors 74 viii 4.2.7 Measurement of glucose consumption and assay production ...................................................................................75 4.2.8 SDS-PAGE, Western blotting, and immunodetection.......75 4.2.9 Statistics .............................................................................76 4.3 Results............................................................................................76 4.4 Discussion......................................................................................93 Chapter 5: Concluding Remarks...........................................................................96