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In Search of a Physiological Role for Amine Oxidase,Copper Containing-3 (AOC3) in Adipocytes

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In search of a physiological role for amine oxidase,copper containing-3 (AOC3) in adipocytes By Sam Hsien-I Shen A thesis submitted in partial satisfaction of the requirements for the degree of Doctor of Philosophy in Chemistry in the Graduate Division of the University of California, Berkeley Committee in charge: Professor Judith P. Klinman, Chair Professor Christopher J. Chang Professor Gary Firestone Spring 2010 Dedication I would like to dedicate this Doctoral dissertation to both my father and mother, Dr. Thomas C. Shen and Mrs. Mayling C. Shen. Without their undying support and advice, this work would not have been possible. i Acknowledgements I would like to acknowledge the guidance and instruction of Professor Judith Klinman. Her keen insights, comments, and suggestions will undoubtedly be a part of all my future scientific endeavors. I would also like to acknowledge Professor Sirpa Jalkanen at the University of Turku, Finland for providing me an opportunity to work in her lab and giving me important insights into my project. Ann Fisher provided invaluable counsel on all things tissue culture. ii Table of Contents Dedication …………………………………………………………………………………... i Acknowledgments …………………………………………………………………………... ii Table of Contents ……………………………………………………………………………. iii Table of Figures ……………………………………………………………………………... v List of Abbreviations ………………………………………………………………………... vii Abstract ……………………………………………………………………………………… 1 Chapter 1: Introduction Introduction ……………………………………………………………………………… 1 References ………………………………………………………………………………... 11 Chapter 2: Entry of exogenously produced H2O2 by AOC3 into 3T3-L1 adipocytes Introduction ……………………………………………………………………………… 16 Methods ………………………………………………………………………………….. 20 Results ……………………………………………………………………………………. 21 Discussion ………………………………………………………………………………... 25 References ………………………………………………………………………………... 26 Chapter 3: Substrate kinetic profile of human AOC-3 Introduction……………………………………………………………………………… 41 Methods ………………………………………………………………………………….. 45 Results ……………………………………………………………………………………. 50 Discussion ………………………………………………………………………………... 55 References ………………………………………………………………………………... 57 Chapter 4: Isolation of mesenchymal stem cells from AOC3 knockout mice Introduction……………………………………………………………………………… 89 Methods ………………………………………………………………………………….. 92 Results ……………………………………………………………………………………. 94 Discussion ………………………………………………………………………………... 96 References ………………………………………………………………………………... 97 Chapter 5: Monocyte chemoattractant protein-1 (MCP-1) production by adipocytes Introduction……………………………………………………………………………… 105 Methods ………………………………………………………………………………….. 108 iii Chapter 5: Monocyte chemoattractant protein-1 (MCP-1) production by adipocytes Results ……………………………………………………………………………………. 110 Discussion ………………………………………………………………………………... 115 References ………………………………………………………………………………... 117 Chapter 6: Involvement of adipocyte-derived chemokine(s) in macrophage chemotaxis Introduction……………………………………………………………………………… 132 Methods ………………………………………………………………………………….. 135 Results ……………………………………………………………………………………. 137 Discussion ………………………………………………………………………………... 138 References ………………………………………………………………………………... 139 iv Table of Figures Figures 2.1 Visualization of intracellular H2O2 using PF1 probe ………………………………… 29 2.2 Treatment of adipocytes with H2O2 for 3.5 hours ……………………………………. 31 2.3 Treatment of adipocytes with lipophilic FM464 dye ………………………………… 32 2.4 Treatment of adipocytes for 2.5 hours with AOC3 substrate, isoamylamine ………... 33 2.5 Treatment of adipocytes for 3 hours with AOC3 substrate, isoamylamine ………….. 34 2.6 Treatment of adipocytes for 4 hours with AOC3 substrate, isoamylamine ………….. 35 2.7 Treatment of adipocytes with 10 uM PF1 probe and methylamine substrate ………... 36 2.8 Visualization of intracellular H2O2 using PF2 probe …………………………………. 37 2.9 Treatment of adipocytes with isoamylamine and PF2 ………………………………... 38 2.10 Treatment of adipocytes with methylamine and PF2 ………………………………… 39 2.11 Treatment of adipocytes with insulin and AOC3 substrates ………………………….. 40 3.1 Protein fractions after DEAE anion exchange, pH 7.2 ……………………………….. 60 3.2 Protein fractions after gel filtration …………………………………………………… 61 3.3 Immunoblot of protein fractions after anion exchange and gel filtration …………….. 62 3.4 Protein fractions after Q-sepharose …………………………………………………… 63 3.5 Protein fractions after DEAE anion exchange, pH 8.0 ……………………………….. 64 3.6 Immunoblot from test expressions of histidine-tagged AOC3 ……………………….. 65 3.7 Histidine-tagged AOC3 after EDTA wash ……………………………………………. 66 3.8 Protein fractions after TALON column ……………………………………………….. 67 3.9 AOC3 precipitation with variable percent ammonium sulfate ………………………... 68 3.10 Protein fractions after cation exchange eluted with salt gradient ……………………... 69 3.11 Protein fractions after SP Sepharose cation exchange ………………………………… 70 3.12 Protein fractions after gel filtration ……………………………………………………. 71 3.13 Benzylamine turnover to measure AOC3 enzyme activity ……………………………. 72 3.14 Active purified human AOC3 determined by phenylhydrazine assay ………………… 73 3.15 Human AOC3 Michaelis-Menton kinetic profile ……………………………………... 74 3.16 Relationship between substrate hydrophobicity and AOC3 efficiency ……………….. 76 3.17 Relationship between substrate volume and AOC3 efficiency ……………………….. 77 3.18 Comparison of catalytic efficiency between human and mouse AOC3 ………………. 78 3.19 AOC3 kinetic parameters measured using whole cells ……………………………….. 79 4.1 Marrow derived cells from AOC3 KO mice ………………………………………….. 99 4.2 FACS sorting of marrow derived cells labeled with WGA ………………………….... 100 4.3 Differentiated marrow derived cells from AOC3 KO mice ……………………............ 101 4.4 Differentiated marrow derived cells show foam cell phenotype ……………………… 102 4.5 Differentiated AOC3 KO marrow derived cells after adherence selection …………… 103 4.6 Differentiated AOC3 WT marrow derived cells after adherence selection …………… 104 5.1 Millipore MAP mouse 22-plex cytokine/chemokine ELISA …………………………. 120 5.2 IL-13 production by 3T3-L1 adipocytes ………………………………………………. 121 5.3 MCP-1 release by isoamylamine-treated adipocytes ………………………………….. 122 5.4 MCP-1 release by adipocytes treated with variable H2O2 …………………………….. 123 5.5 MCP-1 release by adipocytes after 4 - 24 hour H2O2 treatment ………………………. 124 v Figures 5.6 Percent MCP-1 release relative to 24 hour H2O2 treatment …………………………… 125 5.7 Adipocyte-released Cytochrome c immunoblot after H2O2 treatment ………………... 126 5.8 Intracellular MCP-1 levels in adipocytes after H2O2 treatment ……………………….. 127 5.9 3T3-L1 pre-adipocytes ………………………………………………………………… 128 5.10 MCP-1 release by 3T3-L1 pre-adipocytes …………………………………………….. 129 5.11 MCP-1 release by U20S osteocytes and C2C12 myoblasts after H2O2 treatment …….. 130 5.12 MCP-1 release by adipocytes after isoamylamine treatment …………………………. 131 6.1 RAW 264.7 macrophages stained with DAPI after chemotaxis ………………………. 141 6.2 Positive and negative controls of RAW 264.7 macrophage chemotaxis ……………… 142 6.3 Macrophage chemotaxis to adipocyte-conditioned at high [substrate] ……………….. 143 6.4 Macrophage chemotaxis to adipocyte-conditioned at low [substrate] ………………... 144 6.5 Comparison of macrophage chemotaxis with and without AOC3 inhibition ………… 145 vi List of Abbreviations AOC3 Amine oxidase, copper containing-3 DMEM Dulbecco’s modified Eagle medium FACS Fluorescence-activated cell sorting FBS Fetal bovine serum IL Interleukin KO Knockout KPi Potassium phosphate MSC Mesenchymal stem cells NCS Newborn calf serum NEAA Non-essential amino acid PBS Phosphate buffered saline PF1 Peroxyfluor-1 P/S Penicillin/ Streptomycin s2 Schneider 2 Drosophila cells shAOC3 soluble human AOC3 TC Tissue culture TPQ 2,4,5 trihydroxyphenylalanine quinone WGA Wheat germ agglutinin WT Wild-type vii Abstract In search of a physiological role for amine oxidase,copper containing-3 (AOC3) in adipocytes by Sam Hsien-i Shen Doctor of Philosophy in Chemistry University of California, Berkeley Professor Judith P. Klinman, Chair In search of a physiological role for amine oxidase,copper containing-3 (AOC3) in adipocytes The function of adipocytes was initially thought to be fat storage, thermal insulation, and organ protection. With the discovery of adipokine, cytokine, and chemokine production by adipocytes, they are now considered to have endocrine function with a major role in homeostasis and energy balance. Amine oxidase, copper containing 3 (AOC3) is a major ectoenzyme expressed by adipocytes and catalyzes the oxidative deamination of amines to produce hydrogen peroxide, ammonia, and aldehyde. The physiological function of AOC3 in adipocytes and its endogenous substrate(s) are currently unknown. First, a signaling role for the hydrogen peroxide product was investigated. Using an intracellular probe specific for H2O2, it was found that the plasma membrane of adipocytes is permeable to H2O2 introduced exogenously. However, it was not possible to detect an increase in intracellular H2O2 even after long incubation times with AOC3 substrates. Adipocytes were found to produce extracellular H2O2 at an approximate rate of 0.078 uM/min/well upon incubation with AOC3 substrate from whole cell kinetic studies. Due to this slow rate of H2O2 production, the probe may not be sensitive enough to show an increase in intracellular H2O2 originating from AOC3 turnover. Adipocytes may have an immune function, with possible involvement in chemokine production. Using chemotaxis assays, the migration of RAW264.7 macrophages was found to be independent of media components released by adipocytes
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