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Investigations of Anaplerosis from Propionyl-Coa

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Investigations of Anaplerosis from Propionyl-Coa INVESTIGATIONS OF ANAPLEROSIS FROM PROPIONYL-COA PRECURSORS AND FATTY ACID OXIDATION IN THE BRAIN OF VLCAD AND CONTROL MICE by XIAO WANG Submitted in partial fulfillment of the requirements for the Degree of Doctor of Philosophy Thesis Advisor: Henri Brunengraber, M.D., Ph.D. Department of Nutrition CASE WESTERN RESERVE UNIVERISITY May, 2009 CASE WESTERN RESERVE UNIVERSITY SCHOOL OF GRADUATE STUDIES We hereby approve the thesis/dissertation of _____________________________________________________ candidate for the ______________________degree *. (signed)_______________________________________________ (chair of the committee) ________________________________________________ ________________________________________________ ________________________________________________ ________________________________________________ ________________________________________________ (date) _______________________ *We also certify that written approval has been obtained for any proprietary material contained therein. Table of Contents Table of Contents.……………………………………………………………….………i List of Table...……………………………………………………………………………v List of Figures.……………………………………………………………………..….…vi Acknowledgements …………………………………..…………………………..….…xi List of Abbreviations……………………………………………………………………xii Abstract………………………………………………………………………………....xvi CHAPTER 1: Substrate Utilization In The Brain 1.1 Overview of brain energy metabolism..…….………………………………….....1 1.2 The blood-brain barrier ……..……….…….………………………………….…... 2 1.3 Utilization of glucose in the brain………………………………………………..…4 1.3.1 Regional metabolism of glucose..……………………………………….…...5 1.3.2 Overview of astrocytes correlated with the brain energy metabolism……6 1.3.3 Metabolic trafficking between astrocytes and neurons...…………….……9 1.4 Utilization of fatty acids in the brain.………………………………………….….10 1.4.1 Overview of fatty acids in the brain ……………………………………......10 1.4.2 The mechanisms of fatty acid transport across the blood brain barrier ………………………………………………………….……………..…11 1.4.3 The uptake of polyunsaturated fatty acids ………..………………………12 1.4.4 The uptake of saturated fatty acids…………………………………….…..12 1.5 Utilization of ketone bodies in the brain ……..………………………………....14 1.5.1 Metabolic roles……….……………………………………………………....14 1.5.2 Metabolism and regulation…………….…………………………………....16 i 1.5.3 Therapeutic implications of cerebral ketone bodies ….…………………………………………………………………..…....20 CHAPTER 2: Anaplerosis And Cataplerosis 2.1 Overview……………………………………………………………………..……23 2.2 Functions of anaplerosis in the liver, muscle, and heart………………..…….26 2.3 Anaplerosis in the brain…………………………………………………..……...29 2.3.1 Overview……………………………………………………………...………30 2.3.2 The significance of the brain anaplerosis…………………………..……..30 2.4 Major anaplerotic pathways…..……………………………………......…….….37 2.4.1 From pyruvate……………………………………………………..………....37 2.4.2 From propionyl-CoA…………….……………………………..…………….40 CHAPTER 3: Fatty Acid Oxidation And Its Disorders 3.1 The definition and function of fatty acids..………...…..……………….………46 3.2 Fatty acid transport system and cellular uptake..…..………………...…..…..48 3.3 Fatty acid oxidation (β-oxidation)……………..………………………………..49 3.4 Malonyl-CoA metabolism……………………………………………………..…54 3.5 Regulation of fatty acid oxidation..……………………………………………..54 3.5.1 Overview……………………………………………..………………………56 3.5.2 Regulation by malonyl-CoA via the carnitine palmitoyl transferase system ..….……………………………………………………………………..58 3.5.3 Regulation by acetyl-CoA carboxylase .…………………………….……59 3.5.4 Regulation of PPARα……………..………………………………………...62 3.5.5 Regulation by malonyl-CoA decarboxylase ..……………………………64 ii 3.5.6 Regulation by L-carnitine and CoA availability…………………………..65 3.6 Fatty acid oxidation disorders (FOD) ….…………………………………...….65 3.6.1 Pathophysiology of FOD ..…...…………………………………………….66 3.6.2 Very-long-chain acyl-CoA dehydrogenase (VLCAD) deficiency the VLCAD knock-out mouse mode .…….………………………………………66 3.6.3 Traditional treatment of long-chain FOD …………………………………68 3.6.4 Limitations of traditional therapy call out for new available treatments …...………..……………………..………………………………...69 3.6.5 Triheptanoin: a novel treatment of FOD …...………………………….…71 3.6.6 Triheptanoin: Treatment for pyruvate carboxylase deficiency………….74 CHAPTER 4: Research Proposal 4.1 Project 1. Fatty acid oxidation and anaplerosis from propionyl-CoA precursors ………………………………………………………………………...76 4.2 Project 2. Assay of the activity of malonyl-CoA decarboxylase …………….84 4.3 Publications ………………………………………………..……………………..86 4.3.1 Wang X, Zhang GF, Puchowicz MA, Kasumov T, Allen F Jr, Rubin A, Lu M, Yu X, Roe CR, Brunengraber H. Fatty acid oxidation and anaplerosis from propionyl-CoA precursors in the brain of mice deficient in very-long-chain acyl-CoA dehydrogenase and control mice. To be submitted to the Journal of Biological chemistry. 4.3.2 Wang X, Stanley WC, Darrow CJ, Brunengraber H, Kasumov T. Assay of the activity of malonyl-coenzyme A decarboxylase by gas iii chromatography-mass spectrometry. Anal Biochem 2007 Apr 15; 363(2): 169-74. CHAPTER 5: Discussion And Future Directions 5.1 Fatty acid oxidation and anaplerosis from propionyl-CoA precursors in the brain of VLCAD mice and control mice ………………………………………178 5.1.1 Summary and implications ..………..…………………………………..178 5.1.2 Discussion and conclusions ………………..…………………………..182 5.1.3 Future directions ………..…………………………………………….…191 5.2 The assay of the activity of malonyl-CoA decarboxylase by GC-MS .…...195 5.2.1 Discussion and conclusions .…………………………………………...195 5.2.2 Future directions ...……..……………………………………….….…....196 LITERATURE CITED………………………………………………………………..198 iv List of Table Table 4.1 Acyl-CoA content of whole brain of control and VLCAD mice ……….118 v List of Figures Figure 1.1 Simplified diagram showing pathways of cerebral C4-ketone body metabolism ……………………………………………………….……………………18 Figure 2.1 Scheme of the main anaplerotic pathways ..….……………………... 28 Figure 2.2 Brain citric acid cycle and the pathways of anaplerosis and cataplerosis ………………………..…………………………………………………..36 Figure 4.1 Concentrations of octanoate and C4 ketone bodies in the blood of control and VLCAD mice from increasing infused doses of [1-13C]octanoate …………………………………………………………………………………………..130 Figure 4.2 Labeling of ketone bodies from increasing infused doses of [1-13C]octanoate or [1-13C]heptanoate ………..…………………………………….131 Figure 4.3 Concentrations of selected metabolites in the blood of control and 13 VLCAD mice infused with increasing amounts of [5,6,7- C3]heptanoate …….. 132 Figure 4.4 Mass isotopomer distribution of blood glucose in VLCAD mice infused 13 13 with increasing amounts of [3,4,5- C3]pentanoate or [3,4,5- C3]BKP………….133 Figure 4.5 Mole percent enrichment of the parent acyl-CoA and acetyl-CoA in brains of control and VLCAD mice infused with increasing amounts of [1-13C]octanoate , [1-13C]heptanoate, or [1-13C]pentanoate …………………..….134 Figure 4.6 Concentration of acetyl-CoA in the brain of control and VLCAD mice infused with increasing amounts of octanoate, heptanoate, pentanoate or β-ketopentanoate …………………………………………………………………..... 136 vi Figure 4.7 Mol percent enrichment of M3 isotopomers of pentanoyl-CoA, propionyl-CoA, methylmalonyl-CoA and succinyl-CoA in mice infused with 13 [3,4,5- C3]pentanoate …...………………………………………………………...138 Figure 4.8 Relative anaplerosis in brain of control and VLCAD mice infused with 13 13 increasing amounts of [5,6,7- C3]heptanoate, [3,4,5- C3]pentanoate, 13 13 [3,4,5- C3]BKP, or [ C3]propionate …………………………..………..…………139 Figure 4.9 Relative brain concentrations of CAC intermediates and related compounds in VLCAD mice vs control mice ………….…………………………..141 Figure 4.10 Concentrations of selected metabolites in the blood of control and 13 VLCAD mice infused with increasing amounts of [5,6,7- C3]pentanoate ……..142 Figure 4.11 Concentrations of total M3 C5-ketone bodies and of total C4-ketone bodies in the blood of mice infused with increasing amounts of 13 β-keto-[3,4,5- C3]pentanoate ……………………………………………………....143 Figure 4.12 Concentrations of M3 propionate, total M3 C5-ketone bodies and of total C4-ketone bodies in the blood of mice infused with increasing amounts of 13 [ C3]propionate ………………………………………………….…………………...144 Figure 4.13 Concentrations of pentanoyl-CoA and propionyl-CoA in the brains of control and VLCAD mice infused with increasing amounts of 13 [3,4,5- C3]pentanoate ……………………………………………………………….145 vii Figure 4.14 Concentrations of heptanoyl-CoA, pentanoyl-CoA and propionyl-CoA in the brains of control and VLCAD mice infused with increasing amounts of 13 [5,6,7- C3]heptanoate ........................................................................................146 Figure 4.15 Concentrations of octanoyl-CoA, hexanoyl-CoA and butyryl-CoA in the brains of control and VLCAD mice infused with increasing amounts of [1-13C]Octanoate…………………………………...………………………...……..…147 Figure 4.16 MPE of brain acetyl-CoA, average MPE of acetyls of brain octanoyl-CoA, and average MPE of acetyls of blood β-hydroxybutyrate in mice infused with increasing amounts of [1-13C]octanoate …………...........................148 Figure 4.17 MPE of brain acetyl-CoA, average MPE of acetyls of brain heptanoyl- CoA, average MPE of acetyls of blood β-hydroxybutyrate, and MPE of acetyl of blood β-hydroxypentanoate in mice infused with increasing amounts of [1-13C]heptanoate ………………………………..…………………..….…………....149 Figure 4.18 Ion chromatograph of acetyl-CoA (analyzed as the thiophenol derivative) formed from [13C]malonyl-CoA by MCD present in an extract of rat liver. ………………………………………………………………...…………………. 171 13 Figure 4.19 Calibration
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