Characterization of the Ileal Lipid Binding Protein (FABP6) in Tissues Involved in Bile Acid and Steroid Metabolism in Poultry Rosanne Mcquaid Department of Animal Science McGill University, Montreal 21111 Lakeshore Rd., Ste Anne De Bellevue, QC, H9X 3V9 December 2011 A thesis submitted to McGill University in partial fulfillment of the requirements of the degree of Master of Science ©Rosanne McQuaid 2011 I Table of Contents Table of Contents Characterization of the Ileal Lipid Binding Protein (FABP6) in Tissues Involved in Bile Acid and Steroid Metabolism in Poultry ..............................I Table of Contents ....................................................................................................... II List of Tables............................................................................................................... IV List of Figures ............................................................................................................. IV Abstract ..........................................................................................................................V Résumé .........................................................................................................................VI Introduction..................................................................................................................1 Fatty Acid Binding Proteins Literature Review ................................................3 Fatty Acid Binding Proteins.............................................................................................3 FABP6......................................................................................................................................5 Evidence of Bile Acid Binding Proteins in Poultry................................................ 10 Cholesterol Homeostasis............................................................................................... 12 Cholesterol Homeostasis in Poultry..................................................................................... 13 FABP6 and Cholesterol .............................................................................................................. 15 Cytochrome P450 Enzymes .......................................................................................... 17 Bile Acids............................................................................................................................. 18 Bile Acid Biosynthesis ................................................................................................................ 18 Enterohepatic Circulation......................................................................................................... 19 Steroid Hormones............................................................................................................ 22 FABP6 in Steroid Producing Tissues ......................................................................... 23 Conclusion .......................................................................................................................... 25 Materials and Methods........................................................................................... 26 Animals................................................................................................................................ 26 Tissue Collection: ............................................................................................................. 26 RNA Isolation:.................................................................................................................... 26 cDNA Synthesis: ................................................................................................................ 27 Polymerase Chain Reaction:......................................................................................... 28 PCR Cloning:....................................................................................................................... 29 Sequencing: ........................................................................................................................ 29 Relative mRNA Quantitation by Quantitative Real­ Time PCR: ....................... 29 Protein Extraction: .......................................................................................................... 31 Antibodies. ......................................................................................................................... 32 Immunoblot: ...................................................................................................................... 32 Immunohistochemistry: ................................................................................................ 32 Statistical Analysis........................................................................................................... 34 Results ......................................................................................................................... 35 Validation of Primers...................................................................................................... 35 II Sequence Alignment........................................................................................................ 36 Broilers:............................................................................................................................... 41 mRNA................................................................................................................................................. 41 Protein............................................................................................................................................... 42 Layers................................................................................................................................... 44 mRNA................................................................................................................................................. 44 Protein............................................................................................................................................... 46 Turkey Embryo................................................................................................................. 51 Discussion: ................................................................................................................. 54 Tissue­Specific Distribution ......................................................................................... 56 Intestine ........................................................................................................................................... 56 Kidney ............................................................................................................................................... 58 Liver ................................................................................................................................................... 58 Testis.................................................................................................................................................. 59 Ovarian Follicles ........................................................................................................................... 59 Embryos ........................................................................................................................................... 62 Conclusion .................................................................................................................. 65 III List of Tables Table 1. Primers for Standard PCR Table 2. Quantitative PCR primers List of Figures Figure 1. FABP tertiary structure of the Beta‐Clam Figure 2. FABPs in the Small Intestine Figure 3. Regulation of FABP6 expression by sensing intracellular cholesterol and cholesterol derivatives Figure 4. Enterohepatic circulation Figure 5. Cholesterol metabolism into steroid hormones Figure 6. Location of FABP6 primers in the ORF of Gallus gallus FABP6 mRNA Figure 7. Location of FABP6 primers in the ORF of the Meleagris gallopavo FABP6 mRNA Figure 8. Chicken and Mouse FABP6 mRNA Sequence Alignment Figure 9. Chicken and Mouse FABP6 Protein Amino Acid Sequence Alignment Figure 10. Chicken and Turkey FABP6 mRNA Sequence Alignment Figure 11. Chicken and Turkey FABP6 Amino Acid Sequence Alignment Figure 12. Chicken and Turkey ASBT mRNA Sequence Alignment Figure 13. Chicken and Turkey ASBT Amino Acid Sequence Alignment Figure 14. Chicken and Turkey LXR Amino Acid Sequence Alignment Figure 15. Chicken and Turkey FXR Amino Acid Sequence Alignment Figure 16. FABP6 mRNA Abundance in Broilers Figure 17. ASBT Abundance in Broiler Tissues Figure 18. Immunoblot of FABP6 in Broilers Figure 19. FABP6 Female Intestine and Ovarian Follicles Figure 20. ASBT Female Intestine and Ovarian Follicles Figure 21. FABP6 and ASBT in the F1 Follicle Tissues Figure 22. FABP6 Immunoblot Analysis Figure 23. Immunoreactivity of the Duodenum Figure 24. Immunoreactivity of the Ileum Figure 25. Immunoreactivity of Follicles Figure 26. FABP6 in 24ED Turkey Embryo Tissues Figure 27. FABP6 in 28ED Turkey Embryo Tissues Figure 28. ASBT in 28ED Turkey Embryo Tissues IV Abstract The Characterization of Ileal Lipid Binding Protein (FABP6) in Tissues Involved in Bile Acid and Steroid Metabolism in Poultry Fatty acid binding proteins (FABPs) are a family of proteins involved in the transport of a wide variety of hydrophobic molecules within the cytosol of cells. Tissues with high levels