University of North Dakota UND Scholarly Commons Theses and Dissertations Theses, Dissertations, and Senior Projects January 2013 Acetate Increases Brain Purine Metabolism And Glial Cell Culture Fatty Acid Content: Studies Investigating Glyceryl Triacetate's Therapeutic Mechanism Of Action Dhaval Paresh Bhatt Follow this and additional works at: https://commons.und.edu/theses Recommended Citation Bhatt, Dhaval Paresh, "Acetate Increases Brain Purine Metabolism And Glial Cell Culture Fatty Acid Content: Studies Investigating Glyceryl Triacetate's Therapeutic Mechanism Of Action" (2013). Theses and Dissertations. 1504. https://commons.und.edu/theses/1504 This Dissertation is brought to you for free and open access by the Theses, Dissertations, and Senior Projects at UND Scholarly Commons. It has been accepted for inclusion in Theses and Dissertations by an authorized administrator of UND Scholarly Commons. For more information, please contact [email protected]. ACETATE INCREASES BRAIN PURINE METABOLISM AND GLIAL CELL CULTURE FATTY ACID CONTENT: STUDIES INVESTIGATING GLYCERYL TRIACETATE’S THERAPEUTIC MECHANISM OF ACTION by Dhaval Paresh Bhatt Bachelor of Pharmacy, (B. Pharm.), Gujarat University, India, 2005 Master of Pharmacy (M. Pharm., Pharmacology), Gujarat University, India, 2007 A Dissertation Submitted to the Graduate Faculty of the University of North Dakota in partial fulfillment of the requirements for the degree of Doctor of Philosophy Grand Forks, North Dakota December 2013 Copyright© 2013 Dhaval Paresh Bhatt ii PERMISSION Title: Acetate Increases Brain Purine Metabolism and Glial Cell Culture Fatty Acid Content: Studies Investigating Glyceryl Triacetate’s Therapeutic Mechanism of Action Department: Pharmacology, Physiology and Therapeutics Degree: Doctor of Philosophy In presenting this dissertation in partial fulfillment of the requirements for a graduate degree from the University of North Dakota, I agree that the library of this University shall make it freely available for inspection. I further agree that permission for extensive copying for scholarly purposes may be granted by the professor who supervised my dissertation work or, in their absence, by the chairperson of the department or the dean of the Graduate School. It is understood that any copying or publication or other use of this dissertation or part thereof for financial gain shall not be allowed without my written permission. It is also understood that due recognition shall be given to me and to the University of North Dakota in any scholarly use which may be made of any material in my dissertation. Dhaval Paresh Bhatt November 25, 2013 iv TABLE OF CONTENTS LIST OF FIGURES ......................................................................................................... viii LIST OF TABLES .............................................................................................................. x ACKNOWLEDGMENTS ................................................................................................. xi ABSTRACT ..................................................................................................................... xiii CHAPTER I. INTRODUCTION ............................................................................................ 1 Acetate Supplementation ............................................................................ 1 Disruption of brain energy metabolism and available energy sources ....... 2 Ketogenic diet and brain energy metabolism.............................................. 3 Neuroinflammation: link to energy metabolism ......................................... 4 Rat model of neuroinflammation ................................................................ 6 Purinergic signaling and neuroinflammation .............................................. 6 Lipid synthesis and metabolism ................................................................ 10 Overall hypothesis, approach, and outcomes ............................................ 11 II. METHODS ..................................................................................................... 15 Reagents .................................................................................................... 15 Animals ..................................................................................................... 17 Rat Model of Neuroinflammation ............................................................. 18 Nucleotide and Phosphagen Extraction from Rat Brain ........................... 20 v Preparation of Nucleotide and Phosphagen Standards ............................. 20 Quantification of Brain Nucleotides ......................................................... 21 Quantification of Brain Phosphagens ....................................................... 22 Glycogen Analysis .................................................................................... 23 Cardiolipin Analysis ................................................................................. 24 Electron Microscopy ................................................................................. 25 Rat Brain Extraction for Ecto-5’-nucleotidase Activity ........................... 26 Ecto-5’-nucleotidase Activity Assay ........................................................ 27 Cell culture ................................................................................................ 27 Lactate Dehydrogenase Assay for Cell Death Determination .................. 30 Nucleotide Extraction from Cell Cultures ................................................ 30 Quantification of Cell Culture Nucleotides .............................................. 31 Western Blot Analysis .............................................................................. 33 Lipid Extraction from BV2 Cell Culture .................................................. 34 Quantification of Fatty Acids from BV2 Cell Cultures ............................ 35 TLC Separation and Phospholipid Analysis ............................................. 36 Cholesterol Assay ..................................................................................... 37 Protein Analysis ........................................................................................ 37 Statistical Analysis .................................................................................... 38 III. RESULTS ....................................................................................................... 39 Single-dose acetate supplementation reduces brain AMP levels without altering other nucleotides .......................................................................... 39 Single-dose acetate supplementation increases brain phosphocreatine levels ......................................................................................................... 40 vi Single-dose and long-term acetate supplementation do not alter brain glycogen content ....................................................................................... 44 Long-term acetate supplementation does not alter hippocampal neuron mitochondrial number or whole brain cardiolipin content ....................... 44 Long-term acetate supplementation does not alter nucleotide or phosphocreatine levels in normal rats ....................................................... 45 Long-term acetate supplementation does not alter nucleotide or phosphocreatine levels in rats subjected to LPS-induced neuroinflammation .................................................................................... 48 Long-term acetate supplementation alters the levels of adenosine metabolizing enzymes and receptors in a rat model of neuroinflammation .................................................................................... 50 Acetate alters the levels of adenosine metabolizing enzymes and adenosine receptors in LPS-stimulated BV2 microglia but not in primary astrocytes ..................................................................................... 57 Acetate increases astrocyte energy reserves but does not alter extracellular adenosine levels in primary astrocyte cultures or BV2 microglia ................................................................................................... 66 Acetate treatment increases fatty acid content in BV2 microglia ............. 72 Acetate does not alter total phospholipid content in BV2 microglia ........ 74 Acetate increases cholesterol levels in BV2 microglia ............................. 77 Acetate increases histone acetylation in a time- and concentration- dependent manner ..................................................................................... 77 IV. DISCUSSION ................................................................................................. 83 Summary and Significance ..................................................................... 107 APPENDICES ................................................................................................................ 113 Abbreviations .......................................................................................... 114 REFERENCES ............................................................................................................... 118 vii LIST OF FIGURES Figure Page 1. Adenosine metabolism across cell membrane. .......................................................9 2. Single-dose acetate supplementation reduces brain AMP levels without altering ATP levels. ..............................................................................................41