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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.

Sirtuins (SIRTs) comprise a family of nicotinamide adenine dinucleotide (NAD+)-dependent deacetylases, capable of affecting health-span and DNA expression. In cell-culture and peripheral-tissue models, researchers have identified that SIRT1 and SIRT2 are also capable of changing enzymatic activity in glycolysis and Krebs cycle. In brain, the impact of SIRT1 and SIRT2 deacetylase activity on metabolism is poorly understood. The aim of this project was to determine if metabolic pathways in brain could be regulated by SIRT1 and SIRT2-mediated deacetylation in mammalian systems. An established ex vivo reductionist model of brain metabolism was used to test the hypothesis that direct inhibition, activation or ablation of SIRT1 or SIRT2 deacetylase activity would result in significant changes in brain metabolism. Alterations in brain metabolism were assessed by examining changes in 13C-enriched substrates, and metabolite pools with 13C and 1H nuclear magnetic resonance spectroscopy. Chapter three provides evidence that approximately 30% of the GABA synthesized from [1,2-13C]acetate was made directly in neurons. Activation of neuronal specific SIRT1 caused an increase in the incorporation of [1,2-13C]acetate into brain, while activation of astrocytes with potassium depolarization caused a decrease in [1,2-13C]acetate incorporation. These results indicate that acetate is not a reliable marker, nor exclusively metabolised in astrocytes. Further, brain metabolism of acetate is modulated through enzyme acetylation regulated by SIRT1 deacetylase activity. Results in chapter four posit that activation of SIRT1 with SRT 1720 directly stimulated incorporation of 13C into Krebs cycle intermediates and reduced incorporation into lactate. Several off-target effects were observed for SIRT1 activator resveratrol and SIRT1 inhibitory EX-527 that questions their suitability for study of SIRT1 activity. Chapter five concludes that inhibition of SIRT deacetylase activity by AGK2 produced an effect consistent with glutamatergic AMPA receptor activation, in keeping with known SIRT2 targets. Potent SIRT2 inhibitor C64 increased 13C label incorporation into GABA from [1-13C]D-glucose in guinea pigs, and glutamine from [1,2-13C]acetate in WT mice, an effect that was also observed in SIRT2 KO mice. These results indicate that SIRT2 deacetylase activity may impact neurotransmitter systems. This thesis supports the theory that SIRT1 and SIRT2 deacetylase activity can influence brain metabolism in mammalian systems.

β-Hydroxybutyrate (3-hydroxybutyric acid; CH3CHOHCH2COOH; βOHB) is a ketone body oxidised as brain fuel with direct and collateral effects. It can be produced in astrocytes from the oxidation of fatty acids or catabolism of amino acids and is metabolised in the mitochondria of all brain cell types. The thesis presented here investigates βOHB brain metabolism using 1H/13C nuclear magnetic resonance spectroscopy in guinea pig cortical brain slice model. The thesis initially evaluates metabolism of increasing concentrations of [U-13C]D-βOHB in conjunction with [1-13C]D-glucose. The thesis then compares the effects of βOHB on brain metabolism to AMP-activated protein kinase (AMPK) activators. The thesis then investigates the effect of ATP-citrate lyase (ACLY) inhibition, a key enzyme generating cytosolic acetyl-CoA and oxaloacetate from citrate, on the metabolism of [U-13C]D-βOHB, [1-13C]D-glucose, [3-13C]pyruvate, [3-13C]L-lactate, and [1,2-13C]acetate. [U-13C]D-βOHB at low concentrations (0.25 and 1.25 mmol/L) stimulated mitochondrial metabolism but did not substitute for glucose as a substrate. The effect of both direct and indirect AMPK activators (A769662, PF-06409577, AICAR, and metformin) showed an overall increase in brain metabolism but was not similar to the effect of βOHB on brain metabolism. Inhibition of ACLY using BMS-303141 with pyruvate, lactate, or βOHB as substrates resulted in increased incorporation of 13C label into Krebs cycle intermediates. When [1-13C]D-glucose was the sole substrate, total metabolite pools, and net flux of 13C into Krebs cycle intermediates and glycolytic by-products were significantly reduced. This flux of [1-13C]D-glucose was “rescued” when brain slices were supplemented with [1,2-13C]acetate increasing incorporation of label from both glucose and acetate. These results indicate that the impact of ACLY activity on Krebs cycle flux is significant, although it is currently not a part of most models of brain metabolism. In summary, this research advances the understanding of βOHB metabolism in the brain and has ramifications for translational research around neurodegenerative, inflammatory, and traumatic brain disorders. Keywords: β-Hydroxybutyrate, βOHB, AMP-activated protein kinase, AMPK, ATP-citrate lyase, ACLY, brain metabolism.

The brain normally uses glucose as its primary fuel, but is able to use ketone bodies as an alternative fuel during fasting, starvation, or feeding of high-fat, low-carb diets. Ketosis, as a physiological state, has been shown to be neuroprotective since the 1920s. The biochemical links between ketosis and neuroprotection has been of interest to clinicians and scientists. To investigate the metabolic mechanism, we hypothesized that 1) the total energy demand (glucose + ketone bodies) is constant during ketosis 2) in chronic ketosis, ketone bodies spare glucose from oxidative metabolism and shunts towards neurotransmitters. Using Positron Emission Tomography (PET) and 2-tissue compartment modeling, we show that the cerebral metabolic rate of glucose (CMRglc) decreases linearly (9% per 1mM blood ketone body increase) in rats with diet-induced ketosis. In another study, using Liquid Chromatography and Gas-Chromatography Mass Spectrometry (LC-MS, GC-MS), we applied carbon-13 (13C) isotopic flux analysis in ketotic rat brains with either [U13C]-glucose or [U13C]-acetoacetate intravenous infusions. The data show that ketosis reduced glucose carbon flux into the citric acid cycle and ¿-aminobutyric acid (GABA), whereas ketone body carbon flux increased in these pathways. In conclusion, ketone bodies partition and spare glucose oxidative metabolism in ketotic rat brain. This may lead to further understanding to neuroprotection from changes of metabolic energy balances.