THE EFFECTS OF MANGO ON GUT MICROBIAL POPULATION AND ITS IMPACT ON BODY COMPOSITION AND GLUCOSE HOMEOSTASIS IN MICE FED HIGH FAT DIET By BABAJIDE OJO Bachelor of Science in Biochemistry University of Ado-Ekiti, Ado-Ekiti, Ekiti State 2011 Submitted to the Faculty of the Graduate College of the Oklahoma State University in partial fulfillment of the requirements for the Degree of MASTER OF SCIENCE July, 2015 THE EFFECTS OF MANGO ON GUT MICROBIAL POPULATION AND ITS IMPACT ON BODY COMPOSITION AND GLUCOSE HOMEOSTASIS IN MICE FED HIGH FAT DIET Thesis Approved: Dr. Edralin Lucas Thesis Adviser Dr. Brenda Smith Dr. Stephen Clarke ii ACKNOWLEDGEMENTS I am grateful to the sponsors of this project, the Nutritional Science Department and National Mango Board for making funds and a conducive environment available for this research. I am especially grateful to my advisor, Dr. Edralin A. Lucas for her unique guidance all along my journey towards achieving this project. Other professors in my advisory committee Dr. Brenda Smith and Dr. Stephen Clarke have also been very helpful in every step of this research. Kindly accept my heartfelt gratitude. I also appreciate my lab colleagues: Heba, Sawanya, Jess, Arella, Joanna, Jenny, Traces, Lei, Xin, Erica, and Zach, for their readiness to help at one point or the other. My gratitude also goes out to the staff of Robert M. Kerr Food and Agricultural Products Center (Oklahoma State University, Stillwater, OK), especially Dr. Guadalupe Davila-El Rassi and Angie Lathrop for their help with the gas chromatography analysis. Furthermore, I appreciate other professors and staff of the Nutritional Science Department, Dr. Lin, Dr. Winyoo, Dr. Gates and Sandra for their open-mindedness and readiness to help with any questions I bring to them. I am also grateful to my family Mrs. L. Onisile, Mrs. C.A. Ojo, Mr. and Mrs. S.A. Fakunle and Mr. and Mrs. R. Aiyedogbon who provided financial and moral support for the completion of my master’s degree. I appreciate my friends, Alimamy, Josephus, Hasina, Blanchefort, Demola, and Members of Stillwater council of the Knights of St. Columbus, who made cooling off after laboratory and class work possible and interesting. Last but definitely not least, my unending appreciation goes to my Father, the Almighty God for just about everything! iii Acknowledgements reflect the views of the author and are not endorsed by committee members or Oklahoma State University. Name: BABAJIDE OJO Date of Degree: JULY, 2015 Title of Study: THE EFFECTS OF MANGO ON GUT MICROBIAL POPULATION AND ITS IMPACT ON BODY COMPOSITION AND GLUCOSE HOMEOSTASIS IN MICE FED HIGH FAT DIET Major Field: NUTRITIONAL SCIENCE Abstract The gut microbial population is significantly compromised in high fat feeding. These changes are associated with obesity and type 2 diabetes (T2D). Fermentation of non- digestible carbohydrates and fiber from plant food sources are suggested to prevent gut dysbiosis due to high fat feeding. Various parts of mango have been studied for their anti- obesogenic, immunomodulatory and gastroprotective abilities. This study investigated the effects of 12-week freeze-dried mango pulp supplementation on the gut microbiota and its impact on body composition, glucose homeostasis and inflammatory markers in C57BL/6 mice fed a high fat (HF) diet. Male C57BL/6 mice were randomly assigned to 4 dietary treatment groups: Control (AIN-93M, 10% kcal from fat), HF (60% kcal from fat), and HF+1% or 10% mango. Cecal sample analyses by 16S rDNA sequencing show that HF feeding resulted in a significant loss of bacteria, most notably Bifidobacteria and Akkermansia while mango supplementation prevented the loss of these bacteria in a dose-dependent manner similar to control. Mango supplementation did not reduce body weight or fasting blood glucose. Plasma lipids were elevated with HF feeding compared to control, with both mango doses lowering plasma triglyceride. The HF+10% mango significantly lowered plasma non-esterified fatty acids but increased plasma total cholesterol. In comparison to the HF group, a dose-dependent increase in microbial fermentation was observed with mango supplementation, as evident in increased fecal and cecal acetic and butyric acid but not propionic acid. Furthermore, mango supplementation modulated gut inflammation, as observed with an increase in ileal and colonic IL-10 gene expression compared to the HF group. These results demonstrate that mango supplementation in high fat feeding modulated some of the adverse effects that accompanies high fat diet-induced obesity. iv TABLE OF CONTENTS Chapter Page I. INTRODUCTION ......................................................................................................1 Purpose of the Study ................................................................................................4 Study Hypothesis .....................................................................................................4 Specific Aims ...........................................................................................................4 II. REVIEW OF LITERATURE....................................................................................6 The Growing Trend of Obesity and T2D .................................................................6 The Gut Microbiota..................................................................................................8 Methods for Assessing the Gut Microbiota ...........................................................10 Effect of Diet on the Gut Microbiota ....................................................................13 Non-digestible Food Components .................................................................13 Polyphenols ....................................................................................................15 v The Role of Gut Microbiota in Nutrition and Health ............................................16 Metabolism and Benefits of Short-Chain Fatty Acids ...........................................18 The Gut Microbiota in Obesity ..............................................................................20 Nutrition and Health Value of Mango ...................................................................23 III. METHODOLOGY ................................................................................................27 Animals and Treatment Groups .............................................................................27 Necropsy and Tissue Processing ............................................................................29 Measurements and Assays .....................................................................................30 Gut Microbiota ...............................................................................................30 Glucose Tolerance Test ..................................................................................32 Determination of Plasma Lipids ....................................................................32 Determination of Plasma Incretins, Cytokines and Adipokines ....................33 SCFA Analysis...............................................................................................34 Gene Expression Analysis by q-PCR ............................................................36 Western Blot Analysis ...................................................................................37 Liver Lipids Determination............................................................................38 Statistical Analysis .................................................................................................39 vi Chapter Page IV. RESULTS ..............................................................................................................41 Effects of Mango Supplementation on the Gut Microbiota ...................................41 Impact of Mango Supplementation on Body and Tissue Weights, Food Intake, Body Composition, Glucose Homeostasis and Lipid Metabolism ..................................42 Impact of Mango Supplementation on SCFA Production and GPR43 Expression ... ................................................................................................................................44 Impact of Mango Supplementation on Gut and Plasma Inflammatory Markers ...45 V. DISCUSSION ........................................................................................................60 REFERENCES ............................................................................................................69 vii LIST OF TABLES Table Page 1: Assessment techniques for gut microbiota characterization .................................12 2: Treatment Groups .................................................................................................27 3: Mango Composition ............................................................................................28 4: Diet Composition .................................................................................................29 5: Primer sequences for gene expression analysis ...................................................37 6: Effects of freeze-dried mango supplementation on food intake, body and tissue weights, and body composition...........................................................................51 7: Effects of freeze-dried mango supplementation on fasting blood glucose, glucose area under the curve (AUC), plasma insulin and incretin ......................52 8: Effects of freeze-dried mango supplementation on