MMiicchhiiggaann TTeecchhnnoollooggiiccaall UUnniivveerrssiittyy DDiiggiittaall CCoommmmoonnss @@ MMiicchhiiggaann TTeecchh Dissertations, Master's Theses and Master's Dissertations, Master's Theses and Master's Reports - Open Reports 2012 IInnhhiibbiittiioonn ooff LL--ttyyppee aammiinnoo aacciidd ttrraannssppoorrtt wwiitthh nnoonn--pphhyyssiioollooggiiccaall aammiinnoo aacciiddss iinn tthhee PPaahheennuu22 mmoouussee mmooddeell ooff pphheennyyllkkeettoonnuurriiaa Kara R. Vogel Michigan Technological University Follow this and additional works at: https://digitalcommons.mtu.edu/etds Part of the Biology Commons Copyright 2012 Kara R. Vogel RReeccoommmmeennddeedd CCiittaattiioonn Vogel, Kara R., "Inhibition of L-type amino acid transport with non-physiological amino acids in the Pahenu2 mouse model of phenylketonuria", Master's Thesis, Michigan Technological University, 2012. https://doi.org/10.37099/mtu.dc.etds/193 Follow this and additional works at: https://digitalcommons.mtu.edu/etds Part of the Biology Commons INHIBITION OF L-TYPE AMINO ACID TRANSPORT WITH NON- PHYSIOLOGICAL AMINO ACIDS IN THE Pahenu2 MOUSE MODEL OF PHENYLKETONURIA By Kara R. Vogel A THESIS Submitted in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE (Biological Sciences) MICHIGAN TECHNOLOGICAL UNIVERSITY 2012 © 2012 Kara R. Vogel This thesis, “Inhibition of L-Type Amino Acid Transport with Non-Physiological Amino Acids in the Pahenu2 Mouse Model of Phenylketonuria,” is hereby approved in partial fulfillment of the requirements for the Degree of MASTER OF SCIENCE IN BIOLOGICAL SCIENCES. Department of Biological Sciences Signatures: Thesis Advisor ________________________________ Dr. K. Michael Gibson Committee Member ________________________________ Dr. Thomas Werner Committee Member ________________________________ Dr. Ashutosh Tiwari Department Chair ________________________________ Dr. K. Michael Gibson Date ________________________________ Dedication To my Beautifuls. Table of Contents List of Figures .......................................................................................................vi List of Tables ....................................................................................................... vii Acknowledgements ............................................................................................ viii List of Abbreviations .............................................................................................ix Abstract ................................................................................................................xi 1. Introduction .................................................................................................... 1 1.1. Phenylketonuria................................................................................................ 1 1.2. Current treatment ............................................................................................. 2 1.3. Neurological dysfunction .................................................................................. 4 1.4. L-type amino acid transporter, LAT1................................................................. 4 1.5. Non-physiological amino acids, NPAAs ............................................................ 8 1.6. Pahenu2 mouse ................................................................................................10 2. Objective ...................................................................................................... 11 3. Materials and methods ................................................................................. 12 3.1. Husbandry and subject number .......................................................................12 3.2. NPAA administration .......................................................................................12 3.3. High performance liquid chromatography, HPLC .............................................13 3.4. Liquid chromatography tandem mass spectrometry ........................................13 3.5. Statistical analysis ...........................................................................................14 4. Results ......................................................................................................... 15 4.1. Health and consumption ..................................................................................15 4.2. Amino acids in brain ........................................................................................15 4.3. Amino acids in sera .........................................................................................18 4.4. Catecholamine levels in brain ..........................................................................20 4.5. NPAA levels in brain and blood .......................................................................22 iv 4.6. Effect of NL feeding on blood chemistries........................................................23 5. Discussion .................................................................................................... 23 6. Future directions .......................................................................................... 26 7. Conclusion ................................................................................................... 30 8. Bibliography ................................................................................................. 31 v List of Figures Figure 1.1. Phenylalanine metabolism. ................................................................. 2 Figure 1.2. LNAA trafficking. ................................................................................. 7 Figure 1.3. Dopamine (DA) and Serotonin (5-HT) synthesis and metabolism ...... 7 Figure 1.4. Non-physiological amino acids ........................................................... 9 Figure 4.1.a. Brain LNAAs and SAMe ................................................................ 17 Figure 4.2.a Sera LNAAs and SAH .................................................................... 19 Figure 4.3.a. Brain catecholamines .................................................................... 21 Figure 6.1. Supplements .................................................................................... 27 Figure 6.2. Open field testing paradigm .............................................................. 29 vi List of Tables Table 1.1. System L-type amino acid transporters ............................................... 5 Table 4.1. Consumption and weight ................................................................... 15 Table 5.1. Phe and Tyr effects ............................................................................ 24 Table 6.1. NPAA supplements............................................................................ 27 vii Acknowledgements Thank you very much to my advisor K. Michael Gibson, Ph.D., who I admire and respect. I am very lucky for the opportunity to know and to learn from you- not enough can be said. Thank you very much to my committee members, Thomas Werner, Ph.D. and Ashutosh Tiwari, Ph.D; I am grateful to have your intellectual contribution to my project. I don’t know that I could have survived it all without such a great mentor and friend as Dr. Werner. Thank you for allowing me to use your imager, microscope, for all the homemade taq polymerase, and for your continuous help and evaluation of my writing and presentation skills. Thank you so much to our collaborators in this work Teodoro Bottiglieri, Ph.D., Brandi Wasek and Erland Arning for running my samples and for training me in analytical chemistry and regional brain dissections. I had a lovely time in Dallas. Thank you to Jonathan Gruber, Ph.D. for teaching me laboratory technique, etc. Thank you to David Talaga II for help weighing animals and making chow. Thank you to the Gibson laboratory members Matt Helgen, Andrew Kennedy, Aparna Deshpande, and Faith Lambert. Partial support for these studies was obtained from NIH HD 58553 and a grant from the National PKU Alliance (NPKUA), which is gratefully acknowledged. viii List of Abbreviations 3-MT 3- methoxytyramine 5-HIAA 5-hydroxyindoleacetic acid 5-HT serotonin 5-HTP 5-hydroxytryptophan AA amino acids AIB aminoisobutyric acid ASCT alanine-serine-cysteine transporter BBB blood brain barrier BCAA branched-chain amino acid BH tetrahydrobiopterin 4 CNS central nervous system Cys; C cysteine DA dopamine DOPAC 3,4-dihydroxyphenylacetic acid Glu; E glutamate Gln; Q glutamine His; H histidine HMGCR 3-hydroxy-3-methyl-glutaryl-CoA reductase HVA homovanillic acid Ile; I isoleucine L-DOPA L-dihydroxyphenylalanine LAT1 L-type amino acid transporter Leu; L leucine ix