The molecular and metabolic influence of long term agmatine consumption.

Agmatine (AGM), a product of arginine decarboxylation, influences multiple physiologic and metabolic functions. However, the mechanism(s) of action, the impact on whole body gene expression and metabolic pathways, and the potential benefits and risks of long term AGM consumption are still a mystery. Here, we scrutinized the impact of AGM on whole body metabolic profiling and gene expression and assessed a plausible mechanism(s) of AGM action. Studies were performed in rats fed a high fat diet or standard chow. AGM was added to drinking water for 4 or 8 weeks. We used (13)C or (15)N tracers to assess metabolic reactions and fluxes and real time quantitative PCR to determine gene expression. The results demonstrate that AGM elevated the synthesis and tissue level of cAMP. Subsequently, AGM had a widespread impact on gene expression and metabolic profiling including (a) activation of peroxisomal proliferator-activated receptor-α and its coactivator, PGC1α, and (b) increased expression of peroxisomal proliferator-activated receptor-γ and genes regulating thermogenesis, gluconeogenesis, and carnitine biosynthesis and transport. The changes in gene expression were coupled with improved tissue and systemic levels of carnitine and short chain acylcarnitine, increased ß-oxidation but diminished incomplete fatty acid oxidation, decreased fat but increased protein mass, and increased hepatic ureagenesis and gluconeogenesis but decreased glycolysis. These metabolic changes were coupled with reduced weight gain and a curtailment of the hormonal and metabolic derangements associated with high fat diet-induced obesity. The findings suggest that AGM elevated the synthesis and levels of cAMP, thereby mimicking the effects of caloric restriction with respect to metabolic reprogramming.

Regulation of glucagon secretion in normal and diabetic human islets by γ-hydroxybutyrate and glycine.

Paracrine signaling between pancreatic islet ß-cells and α-cells has been proposed to play a role in regulating glucagon responses to elevated glucose and hypoglycemia. To examine this possibility in human islets, we used a metabolomic approach to trace the responses of amino acids and other potential neurotransmitters to stimulation with [U-(13)C]glucose in both normal individuals and type 2 diabetics. Islets from type 2 diabetics uniformly showed decreased glucose stimulation of insulin secretion and respiratory rate but demonstrated two different patterns of glucagon responses to glucose: one group responded normally to suppression of glucagon by glucose, but the second group was non-responsive. The non-responsive group showed evidence of suppressed islet GABA levels and of GABA shunt activity. In further studies with normal human islets, we found that γ-hydroxybutyrate (GHB), a potent inhibitory neurotransmitter, is generated in ß-cells by an extension of the GABA shunt during glucose stimulation and interacts with α-cell GHB receptors, thus mediating the suppressive effect of glucose on glucagon release. We also identified glycine, acting via α-cell glycine receptors, as the predominant amino acid stimulator of glucagon release. The results suggest that glycine and GHB provide a counterbalancing receptor-based mechanism for controlling α-cell secretory responses to metabolic fuels.

Augmenting ureagenesis in patients with partial carbamyl phosphate synthetase 1 deficiency with N-carbamyl-L-glutamate.

Identical studies using stable isotopes were performed before and after a 3-day trial of oral N-carbamyl-l-glutamate (NCG) in 5 subjects with late-onset carbamyl phosphate synthetase deficiency. NCG augmented ureagenesis and decreased plasma ammonia in 4 of 5 subjects. There was marked improvement in nitrogen metabolism with long-term NCG administration in 1 subject.

Effects of a glucokinase activator on hepatic intermediary metabolism: study with 13C-isotopomer-based metabolomics.

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 (13)C-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 (13)C into the cycle (anaplerosis) and increased generation of (13)C 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.

Down-regulation of hepatic urea synthesis by oxypurines: xanthine and uric acid inhibit N-acetylglutamate synthase.

We previously reported that isobutylmethylxanthine (IBMX), a derivative of oxypurine, inhibits citrulline synthesis by an as yet unknown mechanism. Here, we demonstrate that IBMX and other oxypurines containing a 2,6-dione group interfere with the binding of glutamate to the active site of N-acetylglutamate synthetase (NAGS), thereby decreasing synthesis of N-acetylglutamate, the obligatory activator of carbamoyl phosphate synthase-1 (CPS1). The result is reduction of citrulline and urea synthesis. Experiments were performed with (15)N-labeled substrates, purified hepatic CPS1, and recombinant mouse NAGS as well as isolated mitochondria. We also used isolated hepatocytes to examine the action of various oxypurines on ureagenesis and to assess the ameliorating affect of N-carbamylglutamate and/or l-arginine on NAGS inhibition. Among various oxypurines tested, only IBMX, xanthine, or uric acid significantly increased the apparent K(m) for glutamate and decreased velocity of NAGS, with little effect on CPS1. The inhibition of NAGS is time- and dose-dependent and leads to decreased formation of the CPS1-N-acetylglutamate complex and consequent inhibition of citrulline and urea synthesis. However, such inhibition was reversed by supplementation with N-carbamylglutamate. The data demonstrate that xanthine and uric acid, both physiologically occurring oxypurines, inhibit the hepatic synthesis of N-acetylglutamate. An important and novel concept emerging from this study is that xanthine and/or uric acid may have a role in the regulation of ureagenesis and, thus, nitrogen homeostasis in normal and disease states.

N-carbamylglutamate enhancement of ureagenesis leads to discovery of a novel deleterious mutation in a newly defined enhancer of the NAGS gene and to effective therapy.

N-acetylglutamate synthase (NAGS) catalyzes the conversion of glutamate and acetyl-CoA to NAG, the essential allosteric activator of carbamyl phosphate synthetase I, the first urea cycle enzyme in mammals. A 17-year-old female with recurrent hyperammonemia attacks, the cause of which remained undiagnosed for 8 years in spite of multiple molecular and biochemical investigations, showed markedly enhanced ureagenesis (measured by isotope incorporation) in response to N-carbamylglutamate (NCG). This led to sequencing of the regulatory regions of the NAGS gene and identification of a deleterious single-base substitution in the upstream enhancer. The homozygous mutation (c.-3064C>A), affecting a highly conserved nucleotide within the hepatic nuclear factor 1 (HNF-1) binding site, was not found in single nucleotide polymorphism databases and in a screen of 1,086 alleles from a diverse population. Functional assays demonstrated that this mutation decreases transcription and binding of HNF-1 to the NAGS gene, while a consensus HNF-1 binding sequence enhances binding to HNF-1 and increases transcription. Oral daily NCG therapy restored ureagenesis in this patient, normalizing her biochemical markers, and allowing discontinuation of alternate pathway therapy and normalization of her diet with no recurrence of hyperammonemia. Inc.

Measuring in vivo ureagenesis with stable isotopes.

Stable isotopes have been an invaluable adjunct to biomedical research for more than 70years. Indeed, the isotopic approach has revolutionized our understanding of metabolism, revealing it to be an intensely dynamic process characterized by an unending cycle of synthesis and degradation. Isotopic studies have taught us that the urea cycle is intrinsic to such dynamism, since it affords a capacious mechanism by which to eliminate waste nitrogen when rates of protein degradation (or dietary protein intake) are especially high. Isotopes have enabled an appreciation of the degree to which ureagenesis is compromised in patients with urea cycle defects. Indeed, isotopic studies of urea cycle flux correlate well with the severity of cognitive impairment in these patients. Finally, the use of isotopes affords an ideal tool with which to gauge the efficacy of therapeutic interventions to augment residual flux through the cycle.

Effects of a single dose of N-carbamylglutamate on the rate of ureagenesis.

We studied the effect on ureagenesis of a single dose of N-carbamylglutamate (NCG) in healthy young adults who received a constant infusion (300 min) of NaH(13)CO(3). Isotope ratio-mass spectrometry was used to measure the appearance of label in [(13)C]urea. At 90 min after initiating the H(13)CO3-infusion each subject took a single dose of NCG (50 mg/kg). In 5/6 studies the administration of NCG increased the formation of [(13)C]urea. Treatment with NCG significantly diminished the concentration of blood alanine, but not that of glutamine or arginine. The blood glucose concentration was unaffected by NCG administration. No untoward side effects were observed. The data indicate that treatment with NCG stimulates ureagenesis and could be useful in clinical settings of acute hyperammonemia of various etiologies.

Ketosis and brain handling of glutamate, glutamine, and GABA.

We hypothesize that one mechanism of the anti-epileptic 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.

Ifosfamide-induced nephrotoxicity: mechanism and prevention.

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 beta-oxidation, would prevent nephrotoxicity. Our model system was isolated mitochondria obtained from the kidney cortex of rats treated with IFO or IFO + AGM. Oxidative phosphorylation was determined with electron donors specific to complexes I, II, III, or IV (C-I, C-II, C-III, or C-IV, respectively). A parallel study was done with (13)C-labeled pyruvate to assess metabolic dysfunction. Ifosfamide treatment significantly inhibited oxidative phosphorylation with only C-I substrates. Inhibition of C-I was associated with a significant elevation of [NADH], depletion of [NAD], and decreased flux through pyruvate dehydrogenase and the TCA cycle. However, administration of AGM with IFO increased [cyclic AMP (cAMP)] and prevented IFO-induced inhibition of C-I. In vitro studies with various metabolites of IFO showed that only CAA inhibited C-I, even with supplementation with 2-mercaptoethane sulfonic acid. Following IFO treatment daily for 5 days with 50 mg/kg, the level of CAA in the renal cortex was approximately 15 micromol/L. Taken together, these observations support the hypothesis that CAA is accumulated in renal cortex and is responsible for nephrotoxicity. AGM may be protective by increasing tissue [cAMP], which phosphorylates NADH:oxidoreductase. The current findings may have an important implication for the prevention of IFO-induced nephrotoxicity and/or mitochondrial diseases secondary to defective C-I.

Evaluation of formulas for calculating total energy requirements of preadolescent children with cystic fibrosis.

BACKGROUND: To support age-appropriate growth and to prevent and treat malnutrition in children with cystic fibrosis (CF), energy requirements for those children are often set above the requirements for healthy children. Care providers use one of several empirically derived formulas to calculate energy requirements, yet the validity of these formulas has seldom been tested. OBJECTIVE: We evaluated 6 proposed formulas for calculating energy requirements in children with CF against a total energy requirement for children with CF (TER-CF) derived from measured total energy expenditure, fecal fat energy loss, and the theoretic energy required for age-appropriate tissue accretion. DESIGN: Subjects were children aged 6-8 y who had CF and pancreatic insufficiency. Calculated TERs from each formula were evaluated against TER-CF by using summary statistics, regression analysis, and residual plots. RESULTS: Subjects (n = 53) had suboptimal nutrition and growth status and mild-to-moderate lung disease. The formula that most closely (within 2%) approximated TER-CF was the estimated energy requirement (EER) formula at the active level (EERact). Regression analysis of TER-CF onto calculated TER from each formula yielded the best indexes of model fit for the EERact formula; residual plots of the EERact formula were tightly and normally distributed around zero. CONCLUSIONS: The EERact formula should be used to establish TER-CF in children in this age group who have mild-to-moderate CF. Changes in weight, height, and other indicators of nutritional status must be monitored to modify TER-CF as needed to support individual patient care goals.

Energy balance and the accuracy of reported energy intake in preadolescent children with cystic fibrosis.

BACKGROUND: Suboptimal growth and nutritional status are common among children with cystic fibrosis (CF) and pancreatic insufficiency (PI). A better understanding of energy balance is required to improve prevention and treatment of malnutrition. OBJECTIVE: Our objective was to characterize energy balance and the reporting accuracy of dietary intake in children with CF by evaluating the relations between energy intake (EI), energy expenditure (EE), fecal energy loss, nutritional status, and growth. DESIGN: The subjects were participants of a 24-mo prospective study of children 6-10 y of age with CF and PI. EE, EI, fecal energy loss, and anthropometric measures were obtained at baseline and at 24 mo. RESULTS: The children (n = 69) had suboptimal growth at baseline (x +/- SD: weight-for-age z score, -0.53 +/- 1.19; adjusted height-for-age z score, -0.67 +/- 1.06; body mass index z score, -0.29 +/- 1.12), and these variables remained suboptimal at 24 mo. The median ratios of EI to EE at baseline and 24 mo were 1.15 and 1.18, respectively, which decreased to 1.09 and 1.10, respectively, when adjusted for fecal energy loss (EI(-FL):EE). At baseline, 7% of subjects were underreporters, 64% were accurate reporters, and 23% were overreporters of energy intake; the percentages were similar at 24 mo. CONCLUSIONS: Although EI(-FL):EE ratios were higher than expected at both baseline and 24 mo, this cohort showed only age-appropriate weight gain. Self-reported dietary intake data at the individual level should be interpreted with caution, and weight gain velocity may serve as an objective measure of long-term energy balance.

Short-term fasting, seizure control and brain amino acid metabolism.

The ketogenic diet is an effective treatment for seizures, but the mechanism of action is unknown. It is uncertain whether the anti-epileptic effect presupposes ketosis, or whether the restriction of calories and/or carbohydrate might be sufficient. We found that a relatively brief (24 h) period of low glucose and low calorie intake significantly attenuated the severity of seizures in young Sprague-Dawley rats (50-70 gms) in whom convulsions were induced by administration of pentylenetetrazole (PTZ). The blood glucose concentration was lower in animals that received less dietary glucose, but the brain glucose level did not differ from control blood [3-OH-butyrate] tended to be higher in blood, but not in brain, of animals on a low-glucose intake. The concentration in brain of glutamine increased and that of alanine declined significantly with low-glucose intake. The blood alanine level fell more than that of brain alanine, resulting in a marked increase ( approximately 50%) in the brain:blood ratio for alanine. In contrast, the brain:blood ratio for leucine declined by about 35% in the low-glucose group. When animals received [1-(13)C]glucose, a metabolic precursor of alanine, the appearance of (13)C in alanine and glutamine increased significantly relative to control. The brain:blood ratio for [(13)C]alanine exceeded 1, indicating that the alanine must have been formed in brain and not transported from blood. The elevated brain(alanine):blood(alanine) could mean that a component of the anti-epileptic effect of low carbohydrate intake is release of alanine from brain-to-blood, in the process abetting the disposal of glutamate, excess levels of which in the synaptic cleft would contribute to the development of seizures.

Biosynthesis of agmatine in isolated mitochondria and perfused rat liver: studies with 15N-labelled arginine.

An important but unresolved question is whether mammalian mitochondria metabolize arginine to agmatine by the ADC (arginine decarboxylase) reaction. 15N-labelled arginine was used as a precursor to address this question and to determine the flux through the ADC reaction in isolated mitochondria obtained from rat liver. In addition, liver perfusion system was used to examine a possible action of insulin, glucagon or cAMP on a flux through the ADC reaction. In mitochondria and liver perfusion, 15N-labelled agmatine was generated from external 15N-labelled arginine. The production of 15N-labelled agmatine was time- and dose-dependent. The time-course of [U-15N4]agmatine formation from 2 mM [U-15N4]arginine was best fitted to a one-phase exponential curve with a production rate of approx. 29 pmol x min(-1) x (mg of protein)(-1). Experiments with an increasing concentration (0- 40 mM) of [guanidino-15N2]arginine showed a Michaelis constant Km for arginine of 46 mM and a Vmax of 3.7 nmol x min(-1) x (mg of protein)(-1) for flux through the ADC reaction. Experiments with broken mitochondria showed little changes in Vmax or Km values, suggesting that mitochondrial arginine uptake had little effect on the observed Vmax or Km values. Experiments with liver perfusion demonstrated that over 95% of the effluent agmatine was derived from perfusate [guanidino-15N2]arginine regardless of the experimental condition. However, the output of 15N-labelled agmatine (nmol x min(-1) x g(-1)) increased by approx. 2-fold (P<0.05) in perfusions with cAMP. The findings of the present study provide compelling evidence that mitochondrial ADC is present in the rat liver, and suggest that cAMP may stimulate flux through this pathway.

Response of brain amino acid metabolism to ketosis.

Our objective was to study brain amino acid metabolism in response to ketosis. The underlying hypothesis is that ketosis is associated with a fundamental change of brain amino acid handling and that this alteration is a factor in the anti-epileptic effect of the ketogenic diet. Specifically, we hypothesize that brain converts ketone bodies to acetyl-CoA and that this results in increased flux through the citrate synthetase reaction. As a result, oxaloacetate is consumed and is less available to the aspartate aminotransferase reaction; therefore, less glutamate is converted to aspartate and relatively more glutamate becomes available to the glutamine synthetase and glutamate decarboxylase reactions. We found in a mouse model of ketosis that the concentration of forebrain aspartate was diminished but the concentration of acetyl-CoA was increased. Studies of the incorporation of 13C into glutamate and glutamine with either [1-(13)C]glucose or [2-(13)C]acetate as precursor showed that ketotic brain metabolized relatively less glucose and relatively more acetate. When the ketotic mice were administered both acetate and a nitrogen donor, such as alanine or leucine, they manifested an increased forebrain concentration of glutamine and GABA. These findings supported the hypothesis that in ketosis there is greater production of acetyl-CoA and a consequent alteration in the equilibrium of the aspartate aminotransferase reaction that results in diminished aspartate production and potentially enhanced synthesis of glutamine and GABA.

Role of the glutamate dehydrogenase reaction in furnishing aspartate nitrogen for urea synthesis: studies in perfused rat liver with 15N.

The present study was designed to determine: (i) the role of the reductive amination of alpha-ketoglutarate via the glutamate dehydrogenase reaction in furnishing mitochondrial glutamate and its transamination into aspartate; (ii) the relative incorporation of perfusate 15NH4Cl, [2-15N]glutamine or [5-15N]glutamine into carbamoyl phosphate and aspartate-N and, thereby, [15N]urea isotopomers; and (iii) the extent to which perfusate [15N]aspartate is taken up by the liver and incorporated into [15N]urea. We used a liver-perfusion system containing a physiological mixture of amino acids and ammonia similar to concentrations in vivo, with 15N label only in glutamine, ammonia or aspartate. The results demonstrate that in perfusions with a physiological mixture of amino acids, approx. 45 and 30% of total urea-N output was derived from perfusate ammonia and glutamine-N respectively. Approximately two-thirds of the ammonia utilized for carbamoyl phosphate synthesis was derived from perfusate ammonia and one-third from glutamine. Perfusate [2-15N]glutamine, [5-15N]glutamine or [15N]aspartate provided 24, 10 and 10% respectively of the hepatic aspartate-N pool, whereas perfusate 15NH4Cl provided approx. 37% of aspartate-N utilized for urea synthesis, secondary to the net formation of [15N]glutamate via the glutamate dehydrogenase reaction. The results suggest that the mitochondrial glutamate formed via the reductive amination of alpha-ketoglutarate may have a key role in ammonia detoxification by the following processes: (i) furnishing aspartate-N for ureagenesis; (ii) serving as a scavenger for excess ammonia; and (iii) improving the availability of the mitochondrial [glutamate] for synthesis of N -acetylglutamate. In addition, the current findings suggest that the formation of aspartate via the mitochondrial aspartate aminotransferase reaction may play an important role in the synthesis of cytosolic argininosuccinate.

Regulation of brain glutamate metabolism by nitric oxide and S-nitrosylation.

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. (15)N-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 Cys(373) and Cys(561) 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.

Engineering the gut microbiota to treat hyperammonemia.

Increasing evidence indicates that the gut microbiota can be altered to ameliorate or prevent disease states, and engineering the gut microbiota to therapeutically modulate host metabolism is an emerging goal of microbiome research. In the intestine, bacterial urease converts host-derived urea to ammonia and carbon dioxide, contributing to hyperammonemia-associated neurotoxicity and encephalopathy in patients with liver disease. Here, we engineered murine gut microbiota to reduce urease activity. Animals were depleted of their preexisting gut microbiota and then inoculated with altered Schaedler flora (ASF), a defined consortium of 8 bacteria with minimal urease gene content. This protocol resulted in establishment of a persistent new community that promoted a long-term reduction in fecal urease activity and ammonia production. Moreover, in a murine model of hepatic injury, ASF transplantation was associated with decreased morbidity and mortality. These results provide proof of concept that inoculation of a prepared host with a defined gut microbiota can lead to durable metabolic changes with therapeutic utility.

Ketogenic diet, brain glutamate metabolism and seizure control.

We do not know the mode of action of the ketogenic diet in controlling epilepsy. One possibility is that the diet alters brain handling of glutamate, the major excitatory neurotransmitter and a probable factor in evoking and perpetuating a convulsion. We have found that brain metabolism of ketone bodies can furnish as much as 30% of glutamate and glutamine carbon. Ketone body metabolism also provides acetyl-CoA to the citrate synthetase reaction, in the process consuming oxaloacetate and thereby diminishing the transamination of glutamate to aspartate, a pathway in which oxaloacetate is a reactant. Relatively more glutamate then is available to the glutamate decarboxylase reaction, which increases brain [GABA]. Ketosis also increases brain [GABA] by increasing brain metabolism of acetate, which glia convert to glutamine. GABA-ergic neurons readily take up the latter amino acid and use it as a precursor to GABA. Ketosis also may be associated with altered amino acid transport at the blood-brain barrier. Specifically, ketosis may favor the release from brain of glutamine, which transporters at the blood-brain barrier exchange for blood leucine. Since brain glutamine is formed in astrocytes from glutamate, the overall effect will be to favor the release of glutamate from the nervous system.

Metabolism of brain amino acids following pentylenetetrazole treatment.

We studied the effects of pentylenetetrazole (PTZ) on brain amino acid metabolism in mice. Administration of this convulsant did not change forebrain concentrations of amino acids, but when treated animals also received an injection of [15N]leucine, which served as a tracer of brain nitrogen metabolism, total (14N+15N) forebrain [leucine] exceeded control and [glutamate] and [aspartate] were less than control, as were forebrain concentrations of [15N]glutamate and [2-15N]glutamine. These data suggest greater uptake of [15N]leucine but diminished transamination of leucine to glutamate in experimental mice. In contrast to the [15N]leucine studies, which were associated with increased brain [leucine], the administration of [15N]alanine did not alter levels of alanine, glutamate or glutamine. However, label appeared in [2-15N]glutamine much more readily with [15N]alanine than with [15N]leucine as precursor and the ratio of enrichment in [2-15N]glutamine/[15N]alanine was much higher than that in [2-15N]glutamine/[15N]leucine, a finding that is compatible with preferential metabolism of alanine in astrocytes, which are the primary site of brain glutamine synthetase. We conclude that PTZ treatment favors the uptake of selected amino acids such as leucine but also diminishes transamination of leucine to yield glutamate via branched-chain amino acid transaminase. PTZ treatment may favor the "reverse" transamination of 2-keto-isocaproate (KIC), the ketoacid of leucine, to form leucine and to consume glutamate. A net result of these processes may be to enable the brain more readily to dispose of the glutamate that is released from neurons during convulsive activity.