The obesogenic potential of high fat/high protein diets is determined by both amino acid- and fatty acid composition of the protein source used KARIANNE KORSMO MASTER THESIS IN HUMAN NUTRITION DEPARTMENT OF CLINICAL MEDICINE FACULTY OF MEDICINE AND DENTISTRY, UNIVERSITY OF BERGEN MAY 2016 The obesogenic potential of high fat/high protein diets is determined by both amino acid- and fatty acid composition of the protein source used MASTER THESIS IN HUMAN NUTRITION KARIANNE KORSMO MAY 2016 ACKNOWLEDGEMENTS The work presented in this thesis was performed at the National Institute of Nutrition and Seafood Research (NIFES) in Bergen from autumn 2015 to spring 2016. First, I would like to thank my main supervisor Dr. Philos Even Fjære for introducing me to the interesting field of nutritional research, for his guidance and optimism throughout the year and for always making the time to answer my numerous questions. Further, I would like to thank my co-supervisor Dr. Philos Lise Madsen for encouragement and advice along the way and my co-supervisor Dr. Philos Livar Frøyland for reviewing my thesis and giving me useful and positive feedback. I would also like to thank Dr. Philos Lene Secher Myrmel for her help with animal testing and handling data from qPCR and for always making herself available for assistance. A thank you also goes to Astrid Elise Hasselberg for her superb assistance with animal care and valuable tutoring along the way. Additionally, I would like to express my gratitude to Hui-Shan Tung, Synnøve Wintertun and Eva Mykkeltvedt for teaching me RT-qPCR. A thank you also goes to the Molecular Imaging Center at Haukeland University Hospital for conducting sectioning of my histology samples. To my fellow master students I would like to say thank you for lots of laughter and support. Especially thanks to Anita for great cooperation with the animal experiment and for always solving my IT-problems. The final thanks goes to than Hans Petter for his great patience and for crucial support, comfort and motivation through the entire year. Bergen, May 2016 Karianne Korsmo TABLE OF CONTENTS LIST OF FIGURES .................................................................................................................... 1 LIST OF TABLES ..................................................................................................................... 2 LIST OF ABBERIVATIONS .................................................................................................... 3 ABSTRACT ............................................................................................................................... 4 1.0 Introduction .......................................................................................................................... 5 1. 1 Overweight and obesity ................................................................................................... 5 1.1.1 Causes ........................................................................................................................ 5 1.1.2 Health consequences ................................................................................................. 6 1.1.3 High protein- and high fat diets ................................................................................. 6 1.2 Omega-6 and omega-3 essential fatty acids ..................................................................... 7 1.2.1 Changes in consumption of omega-6 and omega-3 and possible consequences ...... 8 1.2.2 The endocannabinoid system .................................................................................. 10 1.3 The adipose tissue .......................................................................................................... 11 1.3.1 White adipose tissue ................................................................................................ 11 1.3.2 Brown adipose tissue ............................................................................................... 11 1.3.3 Beige adipocytes – a third type of fat cell ............................................................... 12 1.3.4 Origin of adipocytes ................................................................................................ 13 1.3.5 Brown adipose tissue as a target of obesity prevention .......................................... 14 1.4 Introduction to the study ................................................................................................ 15 1.5 Aims of the study ........................................................................................................... 16 2.0 Materials and methods ....................................................................................................... 17 2.1 The animal experiment ................................................................................................... 17 2.1.1 Ethical statement ..................................................................................................... 18 2.1.2 Diets ........................................................................................................................ 18 2.1.3 Preparation of the diets ............................................................................................ 19 2.1.4 Housing and feeding ................................................................................................ 19 2.1.5 Measurements .......................................................................................................... 19 2.1.6 Insulin tolerance test ................................................................................................ 20 2.1.7 Oral glucose tolerance test ...................................................................................... 20 2.1.8 Termination ............................................................................................................. 20 2.2 Histology ........................................................................................................................ 21 2.2.1 Fixation .................................................................................................................... 21 2.2.2 Dehydration and paraffin infiltration ...................................................................... 21 2.2.3 Embedding in paraffin ............................................................................................. 21 2.2.4 Sectioning and staining ........................................................................................... 22 2.2.5 Immunohistochemistry ............................................................................................ 22 2.2.6 Microscopy .............................................................................................................. 23 2.3 Ultra Sensitive Mouse ELISA Insulin Kit ...................................................................... 23 2.4 Reverse Transcription qPCR .......................................................................................... 24 2.4.1 Homogenization and RNA-purification .................................................................. 24 2.4.2 RNA-precipitation ................................................................................................... 25 2.4.3 Measuring RNA integrity, Bioanalyzer................................................................... 25 2.4.4 Reverse transcription reaction ................................................................................. 26 2.4.5 Quantitative real time polymerase chain reaction ................................................... 27 2.5 Statistical analysis .......................................................................................................... 27 3.0 Results ................................................................................................................................ 28 3.1 Body weight development and body composition ......................................................... 28 3.1.1 Body weight development ....................................................................................... 28 3.1.2 Body composition ................................................................................................... 29 3.2 Energy intake, feed efficiency and digestibility ............................................................. 31 3.3 Insulin sensitivity and glucose tolerance ........................................................................ 33 3.3.1 Insulin tolerance test ................................................................................................ 33 3.3.2 Oral glucose tolerance test ...................................................................................... 35 3.4 White adipose tissue ....................................................................................................... 37 3.4.1 Weight of WAT ....................................................................................................... 37 3.4.2 Adipocyte size in iWAT .......................................................................................... 38 .......................................................................................................................................... 39 3.5 Brown adipose tissue ...................................................................................................... 40 3.5.1 Adipocyte size and weight of iBAT ........................................................................ 40 3.5.2 Gene expression in iBAT ........................................................................................ 42 3.5.3 Immunohistochemistry ............................................................................................ 43 4.0 Discussion .......................................................................................................................... 45 4.1 A HF/HP diet based on chicken is obesogenic relative to one based on cod, whereas a pangasius-based diet has an intermediate effect .................................................................. 45 4.1.1 Differences in body weight is partially caused by variations in energy intake ....... 45 4.1.2 The protein- and fat source in the diet varied in their effect on feed efficiency ..... 47 4.1.3 Low lean mass in chicken-fed mice may increase feed efficiency ......................... 49 4.2 Body mass determines glucose tolerance and insulin sensitivity ................................... 50 4.3 Calorie restriction attenuates whitening of iBAT, but changes are not solemnly decided by body weight ..................................................................................................................... 50 4.3.1 The whitening effect of chicken on iBAT morphology is weight related ............... 50 4.3.2 Pangasius attenuates obesity-induced whitening of iBAT ...................................... 51 4.3.3 Increased iBAT activation is not the main cause of differences in feed efficiency 52 4.4 The animal model and relevance to humans .................................................................. 53 4.5 Future perspectives ......................................................................................................... 54 5.0 Conclusion .......................................................................................................................... 55 REFERENCES ......................................................................................................................... 56 APPENDIX .............................................................................................................................. 61 Appendix I – Diets ............................................................................................................... 61 Appendix II – Histology ....................................................................................................... 66 Appendix III - Immunohistochemistry ................................................................................. 67 Appendix IV – Ultra Sensitive Mouse Insulin ELISA Kit ................................................... 68 Appendix V – RT-qPCR ...................................................................................................... 68 Appendix VI – Weight of liver, kidneys and m. Tibialis anterior ....................................... 70 LIST OF FIGURES Figure 1.1 Metabolism of essential fatty acids………………………………………………...8 Figure 1.2 Origins of adipocytes……………………………………………………………...13 Figure 2.1 Private photo of a C57BL/6J BomTac mouse…………………………………….17 Figure 2.2 Grouping…………………………………………………………………………..18 Figure 2.3 Preparation of working mouse insulin standards………………………………….23 Figure 2.4 Preparation of solutions for standard curve……………………………………….26 Figure 3.1 Body weight development and total weight gain...…………………………….....29 Figure 3.2 Body composition………………………………………………………………....30 Figure 3.3 Energy intake, feed efficiency and apparent fat- and nitrogen digestibility............32 Figure 3.4 Insulin tolerance test................................................................................................34 Figure 3.5 Oral glucose tolerance test.......................................................................................36 Figure 3.6 Weight of iWAT, eWAT and rWAT.......................................................................37 Figure 3.7 Adipocyte size in iWAT..........................................................................................39 Figure 3.8 Adipocyte size and weight of iBAT........................................................................41 Figure 3.9 Gene expression in iBAT........................................................................................42 Figure 3.10 Expression of UCP1 in iBAT................................................................................44 Appendix Figure A.1 Weight of liver, kidneys and m. Tibialis anterior..................................................70 1