Glutamic acid is an important excitatory neurotransmitter of the brain. Two key goals of brain amino acid handling are to maintain a very low intrasynaptic concentration of glutamic acid and also to provide the system with precursors from which to synthesize glutamate. The intrasynaptic glutamate level must be kept low to maximize the signal-to-noise ratio upon the release of glutamate from nerve terminals and to minimize the risk of excitotoxicity consequent to excessive glutamatergic stimulation of susceptible neurons. The brain must also provide neurons with a constant supply of glutamate, which both neurons and glia robustly oxidize. The branched-chain amino acids (BCAAs), particularly leucine, play an important role in this regard. Leucine enters the brain from the blood more rapidly than any other amino acid. Astrocytes, which are in close approximation to brain capillaries, probably are the initial site of metabolism of leucine. A mitochondrial branched-chain aminotransferase is very active in these cells. Indeed, from 30 to 50% of all alpha-amino groups of brain glutamate and glutamine are derived from leucine alone. Astrocytes release the cognate ketoacid [alpha-ketoisocaproate (KIC)] to neurons, which have a cytosolic branched-chain aminotransferase that reaminates the KIC to leucine, in the process consuming glutamate and providing a mechanism for the "buffering" of glutamate if concentrations become excessive. In maple syrup urine disease, or a congenital deficiency of branched-chain ketoacid dehydrogenase, the brain concentration of KIC and other branched-chain ketoacids can increase 10- to 20-fold. This leads to a depletion of glutamate and a consequent reduction in the concentration of brain glutamine, aspartate, alanine, and other amino acids. The result is a compromise of energy metabolism because of a failure of the malate-aspartate shuttle and a diminished rate of protein synthesis.

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N-acetylglutamate (NAG) is an endogenous essential cofactor for conversion of ammonia to urea in the liver. Deficiency of NAG causes hyperammonemia and occurs because of inherited deficiency of its producing enzyme, NAG synthase (NAGS), or interference with its function by short fatty acid derivatives. N-carbamylglutamate (NCG) can ameliorate hyperammonemia from NAGS deficiency and propionic and methylmalonic acidemia. We developed a stable isotope 13 C tracer method to measure ureagenesis and to evaluate the effect of NCG in humans. Seventeen healthy adults were investigated for the incorporation of 13 C label into urea. [ 13 C]urea appeared in the blood within minutes, reaching maximum by 100 min, whereas breath 13 CO 2 reached a maximum by 60 min. A patient with NAGS deficiency showed very little urea labeling before treatment with NCG and normal labeling thereafter. Correspondingly, plasma levels of ammonia and glutamine decreased markedly and urea tripled after NCG treatment. Similarly, in a patient with propionic acidemia, NCG treatment resulted in a marked increase in urea labeling and decrease in glutamine, alanine, and glycine. These results provide a reliable method for measuring the effect of NCG on nitrogen metabolism and strongly suggest that NCG could be an effective treatment for inherited and secondary NAGS deficiency.

Nitric oxide (NO) is a signaling intermediate during glutamatergic neurotransmission in the central nervous system (CNS). NO signaling is in part accomplished through cysteine S-nitrosylation, a posttranslational modification by which NO regulates protein function and signaling. In our investigation of the protein targets and functional impact of S-nitrosylation in the CNS under physiological conditions, we identified 269 S-nitrosocysteine residues in 136 proteins in the wild-type mouse brain. The number of sites was significantly reduced in the brains of mice lacking endothelial nitric oxide synthase (eNOS−/−) or neuronal nitric oxide synthase (nNOS−/−). In particular, nNOS−/− animals showed decreased S-nitrosylation of proteins that participate in the glutamate/glutamine cycle, a metabolic process by which synaptic glutamate is recycled or oxidized to provide energy. 15N-glutamine–based metabolomic profiling and enzymatic activity assays indicated that brain extracts from nNOS−/− mice converted less glutamate to glutamine and oxidized more glutamate than those from mice of the other genotypes. GLT1 [also known as EAAT2 (excitatory amino acid transporter 2)], a glutamate transporter in astrocytes, was S-nitrosylated at Cys373 and Cys561 in wild-type and eNOS−/− mice, but not in nNOS−/− mice. A form of rat GLT1 that could not be S-nitrosylated at the equivalent sites had increased glutamate uptake compared to wild-type GLT1 in cells exposed to an S-nitrosylating agent. Thus, NO modulates glutamatergic neurotransmission through the selective, nNOS-dependent S-nitrosylation of proteins that govern glutamate transport and metabolism.

The efficacy of ifosfamide (IFO), an antineoplastic drug, is severely limited by a high incidence of nephrotoxicity of unknown etiology. We hypothesized that inhibition of complex I (C-I) by chloroacetaldehyde (CAA), a metabolite of IFO, is the chief cause of nephrotoxicity, and that agmatine (AGM), which we found to augment mitochondrial oxidative phosphorylation and B-oxidation, would prevent nephrotoxicity. Our model system was isolated mitochondria obtained from the kidney cortex of rats treated with IFO or IFO + AGM.

GKAs (glucokinase activators) are promising agents for the therapy of Type 2 diabetes, but little is known about their effects on hepatic intermediary metabolism. We monitored the fate of 13C-labelled glucose in both a liver perfusion system and isolated hepatocytes. MS and NMR spectroscopy were deployed to measure isotopic enrichment. The results demonstrate that the stimulation of glycolysis by GKA led to numerous changes in hepatic metabolism: (i) augmented flux through the TCA (tricarboxylic acid) cycle, as evidenced by greater incorporation of 13C into the cycle (anaplerosis) and increased generation of 13C isotopomers of citrate, glutamate and aspartate (cataplerosis); (ii) lowering of hepatic [Pi] and elevated [ATP], denoting greater phosphorylation potential and energy state; (iii) stimulation of glycogen synthesis from glucose, but inhibition of glycogen synthesis from 3-carbon precursors; (iv) increased synthesis of N-acetylglutamate and consequently augmented ureagenesis; (v) increased synthesis of glutamine, alanine, serine and glycine; and (vi) increased production and outflow of lactate. The present study provides a deeper insight into the hepatic actions of GKAs and uncovers the potential benefits and risks of GKA for treatment of diabetes. GKA improved hepatic bioenergetics, ureagenesis and glycogenesis, but decreased gluconeogenesis with a potential risk of lactic acidosis and fatty liver.

SUMMARYWe hypothesize that one mechanism of the antiepileptic effect of the ketogenic diet is to alter brain handling of glutamate. According to this formulation, in ketotic brain astrocyte metabolism is more active, resulting in enhanced conversion of glutamate to glutamine. This allows for:(a) more efficient removal of glutamate, the most important excitatory neurotransmitter; and (b) more efficient conversion of glutamine to GABA, the major inhibitory neurotransmitter. KEY WORDS: Glutamic acid, GABA, Ketosis, Brain amino acid metabolism.Our laboratory has addressed the relationship between ketosis and brain handling of amino acids, particularly glutamate (Erecinska et al., 1996;Yudkoff et al., 1997Yudkoff et al., , 2004Yudkoff et al., , 2005Yudkoff et al., , 2006Yudkoff et al., , 2007. Glutamate is the major excitatory neurotransmitter (Meldrum, 2000) but brain transports virtually no glutamate (or glutamine) from blood. Thus, brain must continually synthesize and deliver to neurons the glutamate that they release upon depolarization. It also is critically important that the brain rapidy and efficiently remove glutamate from the synaptic space, for two reasons: (a) maintaining low levels of glutamate in the synapse maximizes the signal-to-noise ratio upon release of this transmitter from nerve endings; and (b) a chronic elevation of glutamate in the synaptic space can excessively excite postdendritic glutamate receptors ("excitotoxicity") and injure susceptible neurons, a factor that may figure in phenomena such as hypoxic-ischemic brain injury, hypoglycemia, epilepsy, certain inborn errors of metabolism, and other neurologic disorders (Olney, 2003;Schousboe & Waagepetersen, 2005). The brain maintains a low level of external glutamate and a high intraneuronal concentration by assigning primarily to astrocytes the task of removing synaptic glutamate, a function they execute with remarkable efficiency. Astrocytes first convert glutamate to glutamine via glutamine synthetase, an exclusively glial enzyme, and then export glutamine to neurons. The latter cells hydrolyze glutamine to glutamate via the action of phosphate-dependent glutaminase, a mitochondrial enzyme that yields ammonia as well as glutamate. Such shuttling of glutamate and glutamine between astrocytes and neurons is termed the "glutamate-glutamine cycle" (Fig. 1). This cycle constitutes the fundamental conceputalization in our thinking about brain amino acid metabolism (Bak et al., 2006;Albrecht et al., 2007).We hypothesized that an alteration in the dynamics of the glutamate-glutamine cycle could constitute an important component of the antiepileptic effect of the ketogenic diet (KD) (Fig. 1, right side). Our data suggests that ketosis induced the following metabolic changes: (a) Flux through the astrocytic glutamine synthetase pathway is intensified, thereby favoring the "buffering" of synaptic glutamate that is taken up by the glia; (b) More glutamine becomes accessible to GABA-ergic neurons, which then have at their disposal a larger precursor p...

-acetylglutamate (NAG) and the synthesis of urea. However, the underlying mechanism is unknown. We have explored the hypothesis that agmatine, a metabolite of arginine, may stimulate NAG synthesis and, thereby, urea synthesis. We tested this hypothesis in a liver perfusion system to determine 1) the metabolism of L-[guanidino-15 N2]arginine to either agmatine, nitric oxide (NO), and/or urea; 2) hepatic uptake of perfusate agmatine and its action on hepatic N metabolism; and 3) the role of arginine, agmatine, or NO in regulating NAG synthesis and ureagenesis in livers perfused with 15 N-labeled glutamine and unlabeled ammonia or 15 NH4Cl and unlabeled glutamine. Our principal findings are 1) [guanidino-15 N2]agmatine is formed in the liver from perfusate L-[guanidino-15 N2]arginine (ϳ90% of hepatic agmatine is derived from perfusate arginine); 2) perfusions with agmatine significantly stimulated the synthesis of 15 N-labeled NAG and [15 N]urea from 15 N-labeled ammonia or glutamine; and 3) the increased levels of hepatic agmatine are strongly correlated with increased levels and synthesis of 15 N-labeled NAG and [ 15 N]urea. These data suggest a possible therapeutic strategy encompassing the use of agmatine for the treatment of disturbed ureagenesis, whether secondary to inborn errors of metabolism or to liver disease. arginine; N-acetylglutamate; carbamoyl phosphate synthetase I; hyperammonemia; nitric oxide