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Copyright: Das, Abhijit
The homeostatic regulation of amino acid concentrations is crucial for optimal brain function and development. Different amino acid transporters at cell membranes work together to facilitate the movement of amino acids into and out of the brain. Despite countless in vitro and in vivo research on these amino acids' activities, many fundamental concerns about their metabolic function in different brain areas and pathophysiological conditions remain unanswered. In the framework of this thesis, the effects of exogenous administration of several non-essential amino acids and the participation of their specific transporters in brain metabolism were investigated in Guinea pig cortical brain slices and mouse brain tissues using a targeted neuropharmacological and metabolomic strategy. Alterations in brain metabolism were analyzed using 1H and 13C nuclear magnetic resonance spectroscopy to evaluate changes in metabolite pools and 13C-enriched substrates. All the amino acid transporters mentioned in this study were addressed by the existing solute carrier (SLC) gene nomenclature system for amino acid transporters. The effect of exogenous L-aspartate, L-ornithine, and their salt, L-aspartate-L-ornithine (LOLA), on brain metabolism was investigated with or without an intact blood-brain barrier (BBB). The results indicated that neither L-aspartate, L-ornithine, nor LOLA, affected brain metabolism with an intact BBB. In cortical tissue slices L-aspartate increased brain metabolism concentration-dependently, L-ornithine significantly slowed it at higher concentrations (100 μmol/L), and the effects of LOLA was largely dependent on the balance of its two constituent amino acids. D-aspartate, another isoform of aspartate, produced a range of metabolic impacts, particularly on glutamatergic and GABAergic systems, with varying concentrations. In principal component analysis, the effects of D-aspartate were clearly distinguished from those of L-aspartate, indicating a metabolic pattern distinct from that of excitatory mechanisms. L-Proline administration significantly inhibited brain metabolism in Guinea pig cortical tissue slices, indicating a GABA-like effect; however, it was not a significant metabolic substrate. While it was actively taken up by cells in a concentration-dependent manner but was not completely metabolized. The metabolic pattern revealed that L-proline's effects clustered with 3-aminopropyl(methyl)phosphinic acid (SKF 97541), GABA, 1,5,6-tetrahydropyridin-4-yl)methylphosphinic acid (TPMPA) and (5,6-dihydro-5-methyl-6-oxo-4H-imidazo[1,5-a]thieno[2,3-f][1,4]diazepine-3-carboxylic acid) 1,1-dimethylethyl ester (RO194603) at lower concentrations (10 μmol/L) and with vigabatrin and RO194603 at higher concentrations (100 μmol/L); indicating that proline may act as a GABAB receptor agonist or GABAArho antagonist. Deletion of SLC6A17/NTT4 (neurotransmitter transporter 4) gene significantly impaired glutamate-glutamine cycle, reduced incorporation of 13C into Krebs cycle intermediates, and increased incorporation into lactate in the brain of mice lacking the gene. NTT4 knockout also altered several important metabolites in glutamatergic neurones, implying that it is a crucial transporter for maintaining brain amino acid homeostasis. Investigation of glutamine transport in cerebellum demonstrated that system A dominates glutamine transport in the cerebellum, with contributions from system N, which is inhibited by histidine and 2-(Methylamino)-2-methylpropionic acid (MeAIB) exerting the most metabolic influence. Inhibition of systems A and L by L-γ-Glutamyl-p-nitroanilide (GPNA) and 2-amino-4-bis(aryloxybenzyl)aminobutanoic acid (AABA) did not influence glutamine transport due to their low affinity for the transporters. Inhibition of systems L and B0 by 2-Aminobicyclo[2.2.1]heptane-2-carboxylic acid (BCH) showed little effect on fluxes from [1-13C]D-glucose but increased the flux of [1,2-13C]acetate into Glu C4,5 and Gln C4,5. Effects of cycloleucine were comparable to BCH but less powerful. This study provided new insight into the role of several non-essential amino acids in brain metabolism and also showed how brain metabolism is regulated in different brain regions.
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PhD Doctorate
UNSW Faculty