Bioactivity of Naringenin in Metabolic Dysregulation and Obesity-Associated Breast
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Bioactivity of Naringenin in Metabolic Dysregulation and Obesity-Associated Breast Cancer in a Mouse Model of Postmenopause DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Jia-Yu Ke Ohio State University Nutrition Graduate Program The Ohio State University 2015 Dissertation Committee: Dr. Martha A. Belury, Advisor Dr. Earl Harrison Dr. Kichoon Lee Dr. Lisa D Yee ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! Copyright by Jia-Yu Ke 2015 ! ! ! ! ! Abstract Loss of ovarian function after menopause is associated with accumulation of body fat in the waist region. These changes in body composition increase the risk of developing central obesity, metabolic syndrome, and other chronic diseases. Additionally, breast cancer is the second leading cause of cancer deaths in women, with the majority occurring in women past the age of menopause. Obesity is associated with increased risk of postmenopausal breast cancer and the underlying mechanism(s) likely involves obesity-related metabolic dysregulation. Therefore, improvement of metabolic status may be a useful approach to decrease the risk of breast cancer. Naringenin is a flavonone found in citrus fruits and tomatoes. It possesses anti- tumor properties as well as ameliorates obesity-associated metabolic dysregulation. Therefore, we hypothesized that dietary naringenin not only improves metabolic disturbances resulting from loss of ovarian function but also ameliorates metabolic disturbances associated with postmenopausal obesity. Furthermore, we hypothesized that naringenin inhibits mammary tumor growth induced by postmenopausal obesity. The first objective of this research was to evaluate the effect of naringenin on metabolic changes resulting from loss of ovarian function. Ovariectomized C57BL/6J female mice were fed a control diet (10% calories from fat) for 11 weeks. Mice were either continued on the control diet or switched to the control diet supplemented with 3% naringenin for the next 11 weeks. Ovariectomized mice, even fed a control diet, exhibited ii elevated fasting glucose levels and increased adiposity. Plasma leptin and leptin mRNA in adipose depots as well as adipose tissue inflammation were decreased in mice supplemented with naringenin. We also observed that mice fed a naringenin diet had less hepatic lipid accumulation with corresponding alterations of hepatic gene expression associated with de novo lipogenesis, fatty acid oxidation, and gluconeogenesis. In summary, dietary naringenin attenuated many of the metabolic disturbances associated with ovariectomy in female mice. The second objective of this research was to examine the effect of naringenin on metabolic disturbances in a mouse model of postmenopausal obesity. First, we measured naringenin concentrations in plasma, liver, perigonadal and subcutaneous adipose tissues, and muscle of ovariectomized C57BL/6J female mice after 11 weeks of naringenin supplementation. Naringenin accumulated 5-12 times more in mice fed a 3% naringenin diet than in mice fed a 1% naringenin diet. Then we fed ovariectomized mice a high-fat diet (60% kcal fat) for 11 weeks to induce obesity and half of the mice were then supplemented with 3% naringenin for another 11 weeks. Supplementation with dietary naringenin decreased weight gain, hyperglycemia, and intra-abdominal adiposity. Naringenin-fed mice exhibited elevated locomotor activity, maintained muscle mass, and reduced muscle diacylglycerol content. Real-time PCR analysis in skeletal muscle revealed decreased mRNA level for genes involved in de novo lipogenesis, lipolysis, and triglyceride synthesis/storage. In conclusion, naringenin supplementation attenuated metabolic dysregulation in obese ovariectomized mice. iii The third objective of this research was to evaluate the effects of naringenin on the growth of breast tumors in a mouse model of postmenopausal obesity. Ovariectomized mice were fed a high fat diet for 3 weeks to induce obesity and then supplemented with 1% or 3 % naringenin, or metformin as a positive control. After 2 weeks of experimental diets, E0771 murine breast cancer cells were inoculated into one mammary fat pad and the tumor size was monitored for 3 weeks. Naringenin supplementation significantly decreased body weight, adipose depot mass, as well as mRNA expression of inflammatory cytokines in both mammary and perigonadal adipose tissues. Naringenin supplementation suppressed tumor growth in the early stage but final tumor weight was not significantly different from the high fat group. Metformin reduced tumor growth and weight, without affecting body weight, tissue weights, and adipose tissue inflammation. Collectively, our data demonstrated that naringenin and metformin alter mammary tumorigenesis via different mechanisms. Overall, these experiments suggest that naringenin supplementation may correct metabolic dysregulation induced by excess caloric consumption and/or loss of ovarian function. iv ! ! ! ! ! Acknowledgments I would like to express my sincerest gratitude to my advisor, Dr. Martha Belury, for providing me the opportunity to conduct this research and numerous opportunities to learn new skills and collaborate with other researchers, for encouraging me to challenge myself academically, and for helping me with many fellowship/grant applications, abstracts, posters, and papers throughout the past 5 years of my Ph.D. study. I would like to extend my gratitude to Dr. Lisa Yee for teaching me everything about the breast cancer and for helping me with fellowship applications, abstracts, posters, and papers. I also would like to sincerely thank to other members of my committee, Dr. Earl Harrison and Dr. Kichoon Lee for their time and advice from start to finish. This research could not have been done without all the collaborators, Dr. Kimerly Powell, Dr. Rebecca Andridge, Dr. Steven Schwartz, Dr. Ken Riedl, Dr. Santosh K. Maurya, Dr. Muthu Periasamy, and Shana Straka. I would like to thank them for sharing their time and extensive knowledge and expertise. I would like to thank my colleagues Min, Mike, Sarah, Queenie, Josephine, Taylor, Sharon, and Brad for helping me with experiments. Without them, I would not have accomplished all the studies in my 5 years. I would like to especially thank Kara, Essam, and Rachel for the unwavering support, and their suggestions, help with experiments, and friendship have been invaluable throughout my Ph.D. study. v Additionally, I am grateful to have join OSUN and would like to thank to OSUN faculty and students, especially Dr. Irene Hatsu, Kom Kamonpatana, Rumana Yasmeen, Han-Yi Lin, Shirley Tan, Yi Guo, Eunice Mah, Jinhui Li, Yi-Hsuan Liu, Leanna Perez, and Kellie Weinhold, for their support and suggestions. Thank you to the organizations that contributed financially to support my education and research: The Graduate School University Fellowship, College of Education and Human Ecology (EHE) Dissertation Research Fellowships, Leta Gigax Duhamel Scholarship, Helen Sells CLARKSON Memorial Scholarship, Food Innovation Center (FIC) Doctoral Research Grant, SEEDS: The OARDC Research Enhancement Competitive Grants Program, Russell Klein Memorial Award, and travel awards from OSUN, FIC, EHE, and Department of Human Sciences. Finally, I would like to thank my Taiwanese friends in Columbus for bringing me joy and laughter. I am extremely grateful to Fabi and Kara’s family, my husband, my sister and my parents for countless encouragement and unconditional support. vi Vita 2006 ............................................................... B.S. Biochemical Science & Technology, National Taiwan University, Taipei, Taiwan 2008 ............................................................... M.S. Molecular Medicine, National Taiwan University, Taipei, Taiwan 2010 to present .............................................. The Ohio State University Ph.D. Program in Nutrition, The Ohio State University, Columbus, OH, USA ! ! Publications Abstracts Ke JY, Hsiao YH, Straka SR, Yee LD, Belury MA. Comparison of the citrus flavonoid naringenin and metformin for effects on breast cancer in obese ovariectomized mice. Proceedings: AACR Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Cotton B, Ke JY, Hsiao YH, Banh T, Belury MA. Naringenin attenuates muscle loss in ovariectomized mice fed a high-fat diet. Poster Presentation, EHE Student Research Forum, The Ohio State University, Columbus, OH, Feb 2015. Peer-reviewed conference. vii Ke JY, Tian M, Kliewer KL, Schwartz SJ, Reidl KM, Tsai SY, Yee LD, Belury MA Evaluation of a citrus flavonoid as a chemopreventive agent against breast cancer. Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Ke JY, Schwartz SJ, Riedl KM, Yee LD, Kliewer K, and Belury MA Accumulation of dietary naringenin and metabolites in mice. FASEB J April 9, 2013 27:636.2 Ke JY, Tian M, Kliewer KL, Belury MA The effect of naringenin on the phosphorylation of AMPK in diet-induced obese mice. FASEB J March 29, 2012 26:818.2 Research Publications Research Publications Ke JY, Kliewer KL, Hamad E, Cole RM, Powell KA, Andridge RR, Straka SR, Yee LD, Belury MA. The flavonoid, naringenin, decreases adipose tissue mass and attenuates ovariectomy-associated metabolic disturbances in mice. Nutrition & Metabolism 2015, 12:1 Kliewer KL, Ke JY, Lee HY, Stout MB, Cole RM, Samuel VT, Shulman GI, Belury MA Short-term food restriction followed by controlled refeeding promotes gorging behavior, enhances fat deposition,