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University of Florida Thesis Or Dissertation Formatting Template PHYSIOLOGICAL ROLE OF INTESTINAL COPPER TRANSPORTER ATP7A IN IRON METABOLISM By SUKRU GULEC A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2013 1 © 2013 Sukru Gulec 2 To my wife and parents 3 ACKNOWLEDGMENTS This dissertation is the result of great collaboration of work with many people over four years. I am sincerely and heartily grateful to my advisor, Dr. Collins, for the support and guidance that he showed me throughout my Doctor of Philosophy (PhD) work and dissertation writing. I learned to be independent during my PhD carrier from him and this brought to me many skills which are important for my scientific carrier. I am sure that it would have not been possible without his help to complete this doctoral thesis. I would like to thank to my committee members Drs. Mitch Knutson, Robert Cousins, and Bruce Stevens for their invaluable support, advice and guidance during my dissertation work and I am greatly appreciated having them as a my committee members. I am obliged to many of my colleagues who supported me during my PhD studies in The Food Science and Human Nutrition (FSHN) Department. I would like to also thank to my lab members, Dr. Ranganathan, Caglar Doguer, Lingli Jiang, Yan Lu, Liwei Xie, April Kim for helpful discussion about my dissertation project and my friend Tolunay Beker Aydemir to help for experimental approaches. Lastly, I would like to thank my family and friends for their kind support that they provided me through my PhD life. I am also truly indebted and thankful to my wife, Afife, to share my life and support me during every step of my PhD studies. 4 TABLE OF CONTENTS Page ACKNOWLEDGMENTS .................................................................................................. 4 LIST OF TABLES ............................................................................................................ 7 LIST OF FIGURES .......................................................................................................... 8 LIST OF ABBREVIATIONS ............................................................................................. 9 ABSTRACT ................................................................................................................... 11 LITERATURE REVIEW ................................................................................................. 13 Main Functions of Iron ............................................................................................ 13 Iron in Food and Iron Deficiency ............................................................................. 14 Iron Metabolism ...................................................................................................... 14 Intestinal Iron Absorption and Utilization .......................................................... 15 Regulation of Iron Homeostasis ....................................................................... 17 Factors that Influence Iron Absorption .............................................................. 19 Iron Excretion ................................................................................................... 20 Main Functions of Copper ....................................................................................... 20 Copper in Food and Copper Deficiency .................................................................. 21 Copper Metabolism ................................................................................................. 21 Intestinal Copper Absorption ............................................................................ 22 Copper Excretion .............................................................................................. 23 Iron and Copper Interactions ............................................................................ 23 The Menkes Atp7a Gene and Protein ..................................................................... 24 Regulation of Atp7a ................................................................................................ 25 MATERIALS AND METHODS ...................................................................................... 30 Rescue of the Cu-Deficient Phenotype of Brindled Mice .................................. 30 Induction of Iron Deficiency .............................................................................. 30 Blood and Tissue Collections ........................................................................... 31 Non-heme Iron and Copper Determination....................................................... 31 RNA Isolation and Quantitative RT-PCR .......................................................... 32 Protein Isolation ................................................................................................ 33 Immunoblotting ................................................................................................. 34 Gavage Study ................................................................................................... 35 In Vivo and in Vitro Oxidase Activity Assay for Cp and Heph ........................... 36 Creating Atp7a Knockdown Rat Intestinal Cells ............................................... 36 Differentiation of the Rat Intestinal Cells .......................................................... 37 Induction of Iron Deficiency and CuCl2 Treatment ............................................ 38 Iron and Copper Measurements in Rat Intestinal Cells .................................... 38 Uptake and Transport of 59Fe ........................................................................... 38 5 Statistical Analysis ............................................................................................ 39 INVESTIGATION OF IRON METABOLISM IN A MOUSE MODEL OF MENKES DISEASE (BRINDLED; MoBr/y) ................................................................................ 44 Introduction ............................................................................................................. 44 Results .................................................................................................................... 46 Copper Rescue of Mutant Mice ........................................................................ 46 Blood Parameters and Iron Levels in Experimental Mice ................................. 47 Quantification of Gene Expression in Mouse Duodenal Mucosa and Liver ...... 47 Copper Levels in Serum and Tissues of Mice, and Serum Cp Activity ............. 48 59Fe Absorption and Utilization after Dietary Iron Deprivation with or without Concurrent Phlebotomy ................................................................................ 48 Discussion .............................................................................................................. 49 SMALL HAIRPIN RNA (shRNA) KNOCK-DOWN OF THE MENKES COPPER- TRANSPORTING ATPASE (ATP7A) ALTERS IRON HOMEOSTASIS IN RAT INTESTINAL EPITHELIAL (IEC-6) CELLS ............................................................. 65 Introduction ............................................................................................................. 65 Results .................................................................................................................... 68 Atp7a KD in IEC-6 Cells ................................................................................... 68 Quantification of Gene Expression in IEC-6 Cells ............................................ 68 Membrane and Cytosolic Fractionation of IEC-6 Cells ..................................... 69 Membrane and Cytosolic FOX Activity ............................................................. 69 59Fe Transport in IEC-6 Cells ........................................................................... 69 Discussion .............................................................................................................. 70 CONCLUSIONS AND FUTURE DIRECTIONS ............................................................. 82 Conclusions ............................................................................................................ 82 Future Directions .................................................................................................... 86 LIST OF REFERENCES ............................................................................................... 90 BIOGRAPHICAL SKETCH .......................................................................................... 105 6 LIST OF TABLES Table Page 2-1 List of mouse qPCR primers ............................................................................... 41 2-2 List of rat qPCR primers ..................................................................................... 42 2-3 Atp7a mRNA target shRNA and negative control (NC) shRNA sequences ........ 43 3-1 Analysis of blood collected from WT and Brindled (MoBr/y) mice ........................ 55 3-2 Summary of iron/copper related parameters in experimental mice ..................... 64 7 LIST OF FIGURES Figure Page 1-1 Intestinal non-heme and heme iron absorption................................................... 27 1-2 Intracellular regulation of iron metabolism .......................................................... 28 1-3 Intestinal copper absorption ............................................................................... 29 3-1 Phenotypic response of MoBr/y mice to copper injection ....................................
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