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Flavin-Containing Monooxygenases: Regulation, Endogenous Roles and Dietary Supplements

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Flavin-Containing Monooxygenases: Regulation, Endogenous Roles and Dietary Supplements Flavin-containing monooxygenases: Regulation, endogenous roles and dietary supplements A thesis submitted to the University of London in partial fulfilment of the requirements for the degree of Doctor of Philosophy by Lyndsey Houseman Department of Biochemistry and Molecular Biology University College London February 2008 1, Lyndsey Houseman, confirm that the work presented in this thesis is my own. Where information has been derived from other sources, I confirm that this has been indicated in the thesis. 1 UMI Number: U59122B All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. Dissertation Publishing UMI U591223 Published by ProQuest LLC 2013. Copyright in the Dissertation held by the Author. Microform Edition © ProQuest LLC. All rights reserved. This work is protected against unauthorized copying under Title 17, United States Code. ProQuest LLC 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106-1346 Abstract To investigate the regulation of flavin-containing monooxygenase 5 (FM05) by xenobiotics in mouse liver, a method for isolating primary mouse hepatocytes was designed. Fmo5 expression was up-regulated by hormones (estradiol, progesterone), dietary supplements (lipoic acid) and other xenobiotics (rifampicin, ethanol) in primary mouse hepatocytes and HepG2 cells. Gene reporter assays showed that Fmo5 expression in HepG2 cells is mediated by pregnane-x-receptor (PXR) ligands. To uncover more about the endogenous role of FMOs, two knockout mouse models, Fmol, 2, 4 (-/-) and Fmo5 (-/-), were studied. Fmol, 2, 4 (-/-) males weighed less and had less gonadal fat than their wild-type counterparts. The bodyweight of the Fmol, 2, 4 (-/-) females was greater than that of the wild-type females. Histology shows that the adipocytes in the Fmol, 2, 4 (-/-) male mice are 50% smaller than wild-type male adipocytes, and the liver histology shows the Fmol, 2, 4 (-/-) males had less fat in the liver compared to wild-type males. Fmol, 2, 4 (-/-) mice had higher plasma total-, HDL- and LDL-cholesterol than the wild-type mice. On a high-fat diet, the male Fmol, 2, 4 (-/- ) mice gain weight and gonadal fat weight. The Fmo5 (-/-) mice have a similar bodyweight and fat weight to the wild-type animals, but Fmo5 (-/-) mice have lower plasma HDL cholesterol and lower plasma iron than WT mice. The results therefore show that a deficiency in FMOs interferes with endogenous fat metabolism. Lipoic acid is an endogenous substrate of FMOl, so to see if the inability to metabolise lipoic acid is the reason for the lower bodyweight and fat weight in Fmol, 2, 4 (-/-) males, the mice were fed a 0.1% lipoic acid diet. All knock-out and wild-type mice lose a similar amount of weight. This implies that the metabolism of lipoic acid is not the fundamental endogenous role of FMOs. 2 Contents 1 Introduction____________________________________________________________17 1.1 Liver structure and function ___________________________________________17 1.1.1 Vasculature of the liver ________________________________________________________ 18 1.2 The acinus-the functional unit of the liver ________________________________19 1.2.1 Liver lobule and acinus _______________________________________________________ 20 1.3 Xenobiotic Metabolism ________________________________________________21 1.4 Cytochromes P450 ___________________________________________________21 1.5 Flavin-containing monooxygenases (FMOs) ______________________________22 1.6 Expression of FMOs _________________________________________________23 1.6.1 FMO catalytic cycle__________________________________________________________ 25 1.7 FMO developmental expression________________________________________26 1.8 Sex differences and hormonal effects on FMO expression ___________________26 1.9 The effects of growth hormone on FMO expression ________________________28 1.10 FMOs in diabetes ____________________________________________________29 1.11 FMOs role in iron homeostasis _________________________________________30 1.12 Instances of FMO induction ___________________________________________30 1.13 FMOs and dietary components _________________________________________31 1.14 FMO, endogenous substrates and redox state _____________________________33 1.15 FM05 and its limited substrate portfolio _________________________________34 1.15.1 Substrates and inducers of FM 05 _____________________________________________ 36 1.16 FM05 regulation by hormones _________________________________________37 1.17 Orphan Nuclear Receptors ___________________________________________37 1.18 AhR, CAR and PPAR ________________________________________________38 1.19 Pregnane X Receptor (PXR) __________________________________________39 1.19.1 Nuclear receptor mode of action and PXR ligands _______________________________ 40 1.20 Drug-drug and drug-diet interactions ___________________________________41 3 1.21 Liquorice __________________________________________________________42 1.22 Objectives of this project _____________________________________________43 2 Materials and Methods ________________________________________________45 2.1.1 Chemicals and Reagents ______________________________________________________ 45 2.1.2 Culture of bacteria ___________________________________________________________ 45 2.1.3 Small-scale isolation of plasmid DN A from bacteria _______________________________ 45 2.1.4 Medium-scale isolation of plasmid DN A from bacteria _____________________________ 46 2.1.5 Quantification of DNA by UV spectrophotometry _________________________________ 47 2.1.6 Agarose gel electrophoresis of DNA fragments ___________________________________ 47 2.1.7 Gel extraction ________________________________________________________________ 48 2.1.8 Polymerase Chain Reaction (PCR) ______________________________________________ 48 2.1.9 TOPO®/TA cloning ___________________________ ;______________________________ 49 2.1.10 Bacterial transformation ____________________________________________________ 49 2.1.11 Restriction endonuclease digestion of DNA ____________________________________ 51 2.1.12 Ligation _________________________________________________________________ 51 2.1.13 Preparation of glycerol stocks ________________________________________________ 51 2.2 Isolation and maintenance of primary mouse hepatocytes ___________________52 2.2.1 Sterilisation of the perfusion apparatus ___________________________________________ 52 2.2.2 Matrigel coating of culture plates _______________________________________________ 52 2.2.3 Dissection __________________________________________________________________ 52 2.2.4 Two-step Perfusion of the Mouse Liver _________________________________________ 53 2.2.5 Purification of Mouse Hepatocytes ______________________________________________ 56 2.2.6 Hepatocyte dosing regime _____________________________________________________ 56 2.2.7 Isolation of primary mouse hepatocyte lysates ____________________________________ 57 2.2.8 Trypan Blue exclusion test _____________________________________________________ 57 2.3 Cell culture________________________________________________________59 2.3.1 HepG2 cell culture ___________________________________________________________ 59 2.3.2 Transient transfection of HepG2 cells ____________________________________________ 59 2.3.3 Isolation of total RNA from HepG2 cells or primary hepatocytes_____________________59 2.4 Down-stream analysis ________________________________________________60 2.4.1 Dual-Luciferase reporter assay and pGL3 vectors __________________________________ 60 2.4.2 Protein concentration determination _____________________________________________ 63 2.4.3 SDS-PAGE_________________________________________________________________ 63 2.4.4 Coomassie Blue staining ______________________________________________________ 64 2.4.5 Western blotting _____________________________________________________________ 64 4 2.5 Microarray ________________________________________________________65 2.5.1 Cell treatment and preparation of RNA for microarray ______________________________ 65 2.5.2 Microarray procedure ________________________________________________________ 65 2.5.3 Real-time RT P C R _ _________________________________________________________ 67 2.6 Animal Husbandry ___________________________________________________68 2.6.1 Mouse weighing ______________________________________________________________ 69 2.6.2 Mouse diets _________________________________________________________________ 69 2.6.3 Dissection _________________________________________________________________ 69 2.6.4 Blood collection ______________________________________________________________ 70 2.6.5 Blood plasma biochemical tests ________ 70 2.6.6 Liver histology _______________________________________________________________ 70 2.6.7 Adipose histology ____________________________________________________________ 71 3 FMOS promoter cloning and analysis_____________________________________74 3.1 Identification of FMOS leader sequence _________________________________74 3.2 Amplification of the FMOS promoter region _____________________________74 3.2.1 FM05 cDNAs_______________________________________________________________ 75 3.2.2 Gel picture of clone inserts ____________________________________________________
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