N(epsilon)-(gamma-L-glutamyl)-L-lysine (GGEL) is increased in cerebrospinal fluid of patients with Huntington's disease.

Pathological-length polyglutamine (Q(n)) expansions, such as those that occur in the huntingtin protein (htt) in Huntington's disease (HD), are excellent substrates for tissue transglutaminase in vitro, and transglutaminase activity is increased in post-mortem HD brain. However, direct evidence for the participation of tissue transglutaminase (or other transglutaminases) in HD patients in vivo is scarce. We now report that levels of N(epsilon)-(gamma-L-glutamyl)-L-lysine (GGEL)--a 'marker' isodipeptide produced by the transglutaminase reaction--are elevated in the CSF of HD patients (708 +/- 41 pmol/mL, SEM, n = 36) vs. control CSF (228 +/- 36, n = 27); p < 0.0001. These data support the hypothesis that transglutaminase activity is increased in HD brain in vivo.

Ketogenic diet, amino acid metabolism, and seizure control.

The ketogenic diet has been utilized for many years as an adjunctive therapy in the management of epilepsy, especially in those children for whom antiepileptic drugs have not permitted complete relief. The biochemical basis of the dietary effect is unclear. One possibility is that the diet leads to alterations in the metabolism of brain amino acids, most importantly glutamic acid, the major excitatory neurotransmitter. In this review, we explore the theme. We present evidence that ketosis can lead to the following: 1) a diminution in the rate of glutamate transamination to aspartate that occurs because of reduced availability of oxaloacetate, the ketoacid precursor to aspartate; 2) enhanced conversion of glutamate to GABA; and 3) increased uptake of neutral amino acids into the brain. Transport of these compounds involves an uptake system that exchanges the neutral amino acid for glutamine. The result is increased release from the brain of glutamate, particularly glutamate that had been resident in the synaptic space, in the form of glutamine. These putative adaptations of amino acid metabolism occur as the system evolves from a glucose-based fuel economy to one that utilizes ketone bodies as metabolic substrates. We consider mechanisms by which such changes might lead to the antiepileptic effect.

Brain amino acid metabolism and ketosis.

The relationship between ketosis and brain amino acid metabolism was studied in mice that consumed a ketogenic diet (>90% of calories as lipid). After 3 days on the diet the blood concentration of 3-OH-butyrate was approximately 5 mmol/l (control = 0.06-0.1 mmol/l). In forebrain and cerebellum the concentration of 3-OH-butyrate was approximately 10-fold higher than control. Brain [citrate] and [lactate] were greater in the ketotic animals. The concentration of whole brain free coenzyme A was lower in ketotic mice. Brain [aspartate] was reduced in forebrain and cerebellum, but [glutamate] and [glutamine] were unchanged. When [(15)N]leucine was administered to follow N metabolism, this labeled amino acid accumulated to a greater extent in the blood and brain of ketotic mice. Total brain aspartate ((14)N + (15)N) was reduced in the ketotic group. The [(15)N]aspartate/[(15)N]glutamate ratio was lower in ketotic animals, consistent with a shift in the equilibrium of the aspartate aminotransferase reaction away from aspartate. Label in [(15)N]GABA and total [(15)N]GABA was increased in ketotic animals. When the ketotic animals were injected with glucose, there was a partial blunting of ketoacidemia within 40 min as well as an increase of brain [aspartate], which was similar to control. When [U-(13)C(6)]glucose was injected, the (13)C label appeared rapidly in brain lactate and in amino acids. Label in brain [U-(13)C(3)]lactate was greater in the ketotic group. The ratio of brain (13)C-amino acid/(13)C-lactate, which reflects the fraction of amino acid carbon that is derived from glucose, was much lower in ketosis, indicating that another carbon source, i.e., ketone bodies, were precursor to aspartate, glutamate, glutamine and GABA.

Alanine metabolism in the perfused rat liver. Studies with (15)N.

We have utilized [(15)N]alanine or (15)NH(3) as metabolic tracers in order to identify sources of nitrogen for hepatic ureagenesis in a liver perfusion system. Studies were done in the presence and absence of physiologic concentrations of portal venous ammonia in order to test the hypothesis that, when the NH(4)(+):aspartate ratio is >1, increased hepatic proteolysis provides cytoplasmic aspartate in order to support ureagenesis. When 1 mm [(15)N]alanine was the sole nitrogen source, the amino group was incorporated into both nitrogens of urea and both nitrogens of glutamine. However, when studies were done with 1 mm alanine and 0.3 mm NH(4)Cl, alanine failed to provide aspartate at a rate that would have detoxified all administered ammonia. Under these circumstances, the presence of ammonia at a physiologic concentration stimulated hepatic proteolysis. In perfusions with alanine alone, approximately 400 nmol of nitrogen/min/g liver was needed to satisfy the balance between nitrogen intake and nitrogen output. When the model included alanine and NH(4)Cl, 1000 nmol of nitrogen/min/g liver were formed from an intra-hepatic source, presumably proteolysis. In this manner, the internal pool provided the cytoplasmic aspartate that allowed the liver to dispose of mitochondrial carbamyl phosphate that was rapidly produced from external ammonia. This information may be relevant to those clinical situations (renal failure, cirrhosis, starvation, low protein diet, and malignancy) when portal venous NH(4)(+) greatly exceeds the concentration of aspartate. Under these circumstances, the liver must summon internal pools of protein in order to accommodate the ammonia burden.

Compartmentation of brain glutamate metabolism in neurons and glia.

Intrasynaptic [glutamate] must be kept low in order to maximize the signal-to-noise ratio after the release of transmitter glutamate. This is accomplished by rapid uptake of glutamate into astrocytes, which convert glutamate into glutamine. The latter then is released to neurons, which, via mitochondrial glutaminase, form the glutamate that is used for neurotransmission. This pattern of metabolic compartmentation is the "glutamate-glutamine cycle." This model is subject to the following two important qualifications: 1) brain avidly oxidizes glutamate via aspartate aminotransferase; and 2) because almost no glutamate crosses from blood to brain, it must be synthesized in the central nervous system (CNS). The primary source of glutamate carbon is glucose, and a major source of glutamate nitrogen is the branched-chain amino acids, which are transported rapidly into the CNS. This arrangement accomplishes the following: 1) maintenance of low external [glutamate], thereby maximizing signal-to-noise ratio upon depolarization; 2) the replenishing of the neuronal glutamate pool; 3) the "trafficking" of glutamate through the extracellular fluid in a nonneuroactive form (glutamine); 4) the importation of amino groups from blood, thus maintaining brain nitrogen homeostasis; and 5) the oxidation of glutamate/glutamine, a process that confers an additional level of control in terms of the regulation of brain glutamate, aspartate and gamma-aminobutyric acid.

Acidosis and astrocyte amino acid metabolism.

The relationship between acidosis and the metabolism of glutamine and glutamate was studied in cultured astrocytes. Acidification of the incubation medium was associated with an increased formation of aspartate from glutamate and glutamine. The rise of the intracellular content of aspartate was accompanied by a significant decline in the extracellular concentration of both lactate and citrate. Studies with either [2-(15)N]glutamine or [15N]glutamate indicated that there occurred in acidosis an increased transamination of glutamate to aspartate. Studies with L-[2,3,3,4,4-(2)H5]glutamine indicated that in acidosis glutamate carbon was more rapidly converted to aspartate via the tricarboxylic acid cycle. Acidosis appears to result in increased availability of oxaloacetate to the aspartate aminotransferase reaction and, consequently, increased transamination of glutamate. The expansion of the available pool of oxaloacetate probably reflects a combination of: (a) Restricted flux through glycolysis and less production from pyruvate of acetyl-CoA, which condenses with oxaloacetate in the citrate synthetase reaction; and (b) Increased oxidation of glutamate and glutamine through a portion of the tricarboxylic acid cycle and enhanced production of oxaloacetate from glutamate and glutamine carbon. The data point to the interplay of the metabolism of glucose and that of glutamate in these cells.

Rapid method for determining the rate of DNA synthesis and cellular proliferation.

A new method has been developed for determination of DNA synthesis during cell proliferation. The method is based on the metabolism of [U-(13)C(6)]glucose to deoxyribose (DR) and then incorporation of [U-(13)C(5)]DR into newly synthesized DNA. Extracted cellular DNA is subjected to HCl hydrolysis (2 h at 100 degrees C), which converts DR into levulinic acid. The (13)C enrichment in DR is determined in the trimethylsilyl derivative of levulinate using gas chromatography-mass spectrometry. The method is rapid and sensitive. It can precisely determine (13)C enrichment below 1 at.% excess in as little as 4 ng DNA. We have used this method to determine the rate of cell proliferation in vitro and the level of DR in a given amount of DNA. The current approach has significant advantages over previously described methods and overcomes several difficulties related to the determination of DNA synthesis both in vivo and in vitro.

Correction of ureagenesis after gene transfer in an animal model and after liver transplantation in humans with ornithine transcarbamylase deficiency.

We report effects of gene transfer and liver transplantation on urea synthesis in ornithine transcarbamylase deficiency (OTCD). We measured the formation of [15N] urea after oral administration of 15NH4Cl in two girls with partial OTCD before and after liver transplantation. Ureagenesis was less than 20% of that observed in controls before transplantation, and was normalized afterward. Studies performed on the OTCD sparse fur (spf/Y) mouse showed discordance between OTC enzyme activity and ureagenesis with modest increases in OTC enzyme activity after gene transfer resulting in significant improvement in ureagenesis. This study suggests that both liver transplantation and gene therapy may be effective in improving ureagenesis in OTCD.

Decreased intraplatelet Ca2+ release and ATP secretion in pediatric nephrotic syndrome.

Platelets play an important role in the natural history of idiopathic nephrotic syndrome (NS). Although thromboembolic events are rare, the activation of circulating platelets is generally considered an important factor in the prethrombotic state in children with NS. Platelet-activating factor (PAF), a potent endogenous phospholipid mediator of inflammation, stimulates intracellular free calcium (Ca2+) mobilization, aggregation, and release reactions in platelets obtained from normal donors. Platelet-related effects of PAF in children with NS are unknown. We studied PAF-induced intracellular Ca2+ mobilization in washed platelets and ATP secretion in platelet-rich plasma in 34 children with idiopathic NS and in 7 healthy children. There was a significant decrease in ATP secretion: 0.021+/-0.011 microg/10(7) cells with 20 nM PAF and 0.089+/-0.017 microg/10(7) platelets with 200 nM PAF versus control values (0.195+/-0.004 microg/10(7) and 0.813+/-0.09 microg/10(7), respectively). Moreover, PAF-evoked increase in intracellular free Ca2+ concentration was twofold lower in NS patients than in control subjects (230.1+/-22.4 nM versus 455.6+/-15.3 nM). Also, thrombin-induced intracellular free Ca2+ mobilization was diminished in children with NS compared with the control group. Thus, contrary to expectations, a decrease of platelet reactivity in response to PAF in vitro was observed in children with idiopathic NS. We suggest that this decreased platelet reactivity may reflect a period refractory to PAF and may be considered as platelet desensitization to PAF released in vivo in children with NS.

Ketone bodies and brain glutamate and GABA metabolism.

The effects of ketone bodies on brain metabolism of glutamate and GABA were studied in three different systems: synaptosomes, cultured astrocytes and the whole animal. In synaptosomes the addition of either acetoacetate or 3-OH-butyrate was associated with diminished consumption of glutamate via transamination to aspartate and increased formation of labelled GABA from either L-[2H5-2,3,3,4, 4]glutamine or L-[15N]glutamine. There was no effect of ketone bodies on synaptosomal GABA transamination. An increase of total forebrain GABA and a diminution of aspartate was noted when mice were injected intraperitoneally with 3-OH-butyrate. In cultured astrocytes the addition of acetoacetate to the medium was associated with a significantly enhanced rate of citrate production and with a diminution in the rate of conversion of [15N]glutamate to [15N]aspartate. These data are consistent with the hypothesis that the metabolism of ketone bodies to acetyl-CoA results in a diminution of the pool of brain oxaloacetate, which is consumed in the citrate synthetase reaction (oxaloacetate + acetyl-CoA --> citrate). As less oxaloacetate is available to the aspartate aminotransferase reaction, thereby lowering the rate of glutamate transamination, more glutamate becomes accessible to the glutamate decarboxylase pathway, thereby favoring the synthesis of GABA.

In vivo measurement of ureagenesis with stable isotopes.

We have utilized stable isotopes to measure in vivo rates of ureagenesis. In one testing procedure, 15NH4Cl was administered orally to controls and to heterozygotes for ornithine transcarbamylase deficiency. Controls produced [15N]urea at a rate that was greater than that of symptomatic carriers, but indistinguishable from that of asymptomatic carriers. In contrast, both symptomatic and asymptomatic heterozygotes produced [5-15N]glutamine more rapidly than the controls. Ureagenesis could also be measured by administering sodium [1-13C]acetate to a healthy adult and measuring subsequent formation of [13C]urea. The latter approach involves the use of isotope ratio mass spectrometry to determine isotopic abundance. This technique is much more sensitive than gas chromatography-mass spectrometry for the measurement of isotopic label, a consideration that makes the method more suitable for the study of subjects in whom ureagenesis is severely compromised, for example the human male neonate with a near complete deficiency of ornithine transcarbamylase.

Effects of ketone bodies on astrocyte amino acid metabolism.

The effects of acetoacetate and 3-hydroxybutyrate on glial amino acid metabolism were studied in primary cultures of astrocytes. The exchange of nitrogen among amino acids was measured with 15N as a metabolic probe and gas chromatography-mass spectrometry as a tool with which to quantify isotope abundance. Addition of either acetoacetate or 3-hydroxybutyrate (5 mM) to the incubation medium did not alter the initial rate of appearance of [15N]glutamate in the glia, but it did inhibit transamination of glutamate to [15N]aspartate. Addition of acetoacetate also inhibited formation of [2-(15)N]glutamine, but 3-hydroxybutyrate had a stimulatory effect. The presence in the medium of sodium acetate (5 mM) was also associated with diminished production of [15N]aspartate and [2-(15)N]glutamine with [15N]glutamate as precursor. Studies with [2-(15)N]glutamine as precursor indicated that treatment of the astrocytes with ketone bodies did not alter flux through the glutaminase pathway. Nor did the presence of the ketone bodies reduce significantly the flux of nitrogen from [15N]GABA to [2-(15)N]glutamine when the former species served as a metabolic tracer. The concentration of internal citrate increased in the presence of acetoacetate, 3-hydroxybutyrate, and acetate. Studies with purified sheep brain glutamine synthetase showed that citrate inhibited this enzyme. These findings are considered in terms of the known anticonvulsant effect of a ketogenic diet.

Regulation of GABA level in rat brain synaptosomes: fluxes through enzymes of the GABA shunt and effects of glutamate, calcium, and ketone bodies.

Stable isotopes were used to measure both the rate of GABA formation by glutamic acid decarboxylase (GAD) and the rate of utilization by GABA-transaminase (GABA-T). The initial rate of GABA accumulation, determined with either [2-15N]glutamine or [2H5]glutamine as precursor, was 0.3-0.4 nmol/min/mg of protein. Addition of the calcium ionophore A23187 enhanced GAD activity, whereas changes in levels of inorganic phosphate and H+ were without influence. Flux through GABA-T (GABA--> glutamate), measured with [15N]GABA as precursor, was 0.82 nmol/min/mg of protein, whereas the reamination of succinic acid semialdehyde (reverse flux through GABA-T) was almost sixfold faster, 4.8 nmol/min/mg of protein. The rate of GABA metabolism via the tricarboxylic acid cycle was very slow, with the upper limit on flux being 0.03 nmol/min/mg of protein. Addition of either acetoacetate or beta-hydroxybutyrate raised the internal content of glutamate and reduced that of aspartate; the GABA concentration and the rate of its formation increased. It is concluded that in synaptosomes (a) GABA-T is a primary factor in regulating the turnover of GABA, (b) a major regulator of GAD activity is the concentration of internal calcium, (c) GAD in nerve endings may not be saturated with its substrate, glutamate, and the concentration of the latter is a determinant of flux through this pathway, and (d) levels of ketone bodies increase, and maintain at a higher value, the synaptosomal content of GABA, a phenomenon that may contribute to the beneficial effect of a ketogenic diet in the treatment of epilepsy.

In vivo nitrogen metabolism in ornithine transcarbamylase deficiency.

We developed a new technique that monitors metabolic competency in female heterozygotes for ornithine transcarbamylase deficiency (OTCD). The method uses mass spectrometry to measure conversion of (15)NH4Cl to [15N]urea and [5-(15)N]glutamine following an oral load of (15)NH4Cl. We found that heterozygotes converted significantly less NH3 nitrogen to urea, with this difference being particularly obvious for symptomatic carriers, in whom the blood [15N]urea concentration (mM) was significantly less than control values at most time points. The blood concentration of [5-(15)N]-glutamine (microM) was significantly higher in both asymptomatic and symptomatic heterozygotes than it was in the control subjects. The administration of a test dose of sodium phenylbutyrate to the control group did not affect the rate of [15N]urea formation. We conclude: (a) This test effectively monitors in vivo N metabolism and might obviate the need for liver biopsy to measure enzyme activity in OTCD; (b) Asymptomatic OTCD carriers form urea at a normal rate, indicating that ureagenesis can be competent even though enzyme activity is below normal; (c) Although ostensibly asymptomatic OTCD carriers form urea at a normal rate, their nitrogen metabolism is still abnormal, as reflected in their increased production of [5-(15)N]glutamine; and (d) This new test may be important for monitoring the efficacy of novel treatments for OTCD, e.g., liver transplantation and gene therapy.

Neuronal metabolism of branched-chain amino acids: flux through the aminotransferase pathway in synaptosomes.

The metabolism of branched-chain amino acids (BCAAs) was studied in cortical synaptosomes. With [15N]leucine (1 mM) as precursor, the cumulative appearance of 15N in [15N]glutamate and [15N]aspartate was 0.2 nmol/min/mg of protein without supplemental alpha-ketoglutarate and 0.3 nmol/min/mg of protein in the presence of alpha-ketoglutarate (0.5 mM). The BCAA amino-transferase reaction also proceeded in the "reverse" direction [alpha-ketoisocaproate (KIC) + glutamate-->leucine + alpha-ketoglutarate]. This was documented by incubating synaptosomes with [15N]glutamate and measuring the formation of [15N]leucine. Without KIC in the medium, the rate of [15N]leucine production was 0.13 nmol/min/mg of protein. In the presence of 25 microM KIC the rate was 0.79 nmol/min/mg of protein and even greater (1.0 nmol/ min/mg of protein) in the presence of 500 microM KIC. The reamination of KIC was two- to threefold faster with [2-15N]glutamine as precursor compared with [15N]-glutamate. The ketoacid of valine, alpha-ketoisovalerate (KIV), was reaminated to [15N]valine at a rate comparable to that observed with respect to KIC. The BCAA transaminase mediated not only the bidrectional transfer of amino groups between leucine or valine and glutamate, but also the direct transfer of nitrogen between leucine and valine. This was ascertained in studies in which the incubation medium was supplemented with either [15N]leucine and KIV or [15N]valine and KIC (amino acids at 1 mM and ketoacids at 25 or 500 microM). The rate was faster in the direction of leucine formation at both the lower (6.1-fold) and higher (1.7-fold) KIC concentration. It is suggested that in synaptosomes the BCAA transaminase (a) functions predominantly in the direction of leucine formation and (b) maintains a constant ratio of BCAAs and ketoacids to one other.

Metabolism of N-acetyl-L-aspartate in rat brain.

The abundance and developmental regulation of N-acetylaspartate (NAA) in brain suggest that it plays an important role in brain metabolism. Previous studies demonstrated that NAA transports acetate from the mitochondrion to the cytoplasm where it is utilized for lipid synthesis, however, the metabolic fate of NAA-derived aspartate is not established. To investigate NAA metabolism, rats were injected intracranially with N-([2H3]acetyl)-L-[15N]aspartate ([2H3,15N]NAA) and whole brain metabolites were analyzed using gas chromatography and mass spectrometry techniques (GC/MS). The rapid decline of [2H3,15N]NAA was associated with a rapid appearance of [15N]glutamate, indicating rapid transamination of the [15N]aspartate that was derived from the enzymatic hydrolysis of [2H3,15N]NAA. Inability to detect [15N]NAA in brain extracts in several experiments indicates that the 15N moiety is not reutilized for NAA synthesis and suggests one metabolic role of NAA may be the transport of amino nitrogen from the mitochondrion to the cytoplasm.

Astrocyte leucine metabolism: significance of branched-chain amino acid transamination.

We studied astrocytic metabolism of leucine, which in brain is a major donor of nitrogen for the synthesis of glutamate and glutamine. The uptake of leucine into glia was rapid, with a Vmax of 53.6 +/- 3.2 nmol/mg of protein/min and a Km of 449.2 +/- 94.9 microM. Virtually all leucine transport was found to be Na+ independent. Astrocytic accumulation of leucine was much greater (3x) in the presence of alpha-aminooxyacetic acid (5 mM), an inhibitor of transamination reactions, suggesting that the glia rapidly transaminate leucine to alpha-ketoisocaproic acid (KIC), which they then release into the extracellular fluid. This inference was confirmed by the direct measurement of KIC release to the medium when astrocytes were incubated with leucine. Approximately 70% of the leucine that the glia cleared from the medium was released as the keto acid. The apparent Km for leucine conversion to extracellular KIC was a medium [leucine] of 58 microM with a Vmax of approximately 2.0 nmol/mg of protein/min. The transamination of leucine is bidirectional (leucine+alpha-ketoglutarate<==>KIC+glutamate) in astrocytes, but flux from leucine-->glutamate is more active than that from glutamate-->leucine. These data underscore the significance of leucine handling to overall brain nitrogen metabolism. The release of KIC from glia to the extracellular fluid may afford a mechanism for the "buffering" of glutamate in neurons, which would consume this neurotransmitter in the course of reaminating KIC to leucine.

Tricarboxylic acid cycle in rat brain synaptosomes. Fluxes and interactions with aspartate aminotransferase and malate/aspartate shuttle.

The flux through different segments of the tricarboxylic acid cycle was measured in rat brain synaptosomes with gas chromatography-mass spectrometry using either deuterated glutamine or [13C]aspartate. The flux between 2-oxoglutarate and oxaloacetate was estimated to be 3.14 and 4.97 nmol/min/mg protein with and without glucose, respectively. These values were 3-5-fold faster than the flux between oxaloacetate and 2-oxoglutarate (0.92 nmol/min per mg protein) measured in the presence of glucose. The pattern of intermediates labeling suggests that the overall rate-controlling reaction involves either citrate synthase or pyruvate dehydrogenase but not 2-oxoglutarate or isocitrate dehydrogenase. The enrichment in [3,3,4,4-2H4]glutamate from [2,3,3,4,4-2H5]glutamine was as rapid as in [2,3,3,4,4-2H5]glutamate, which indicates that the aspartate aminotransferase reaction is severalfold faster than the flux through the tricarboxylic acid cycle. [13C]Aspartate was rapidly converted to [13C]malate, suggesting that in intact synaptosomes aspartate entry into the mitochondrion is very slow. The finding that aspartate is taken up by mitochondria as malate, along with the observed high enrichment in [3-2H]malate (from [2,3,3,4,4-2H5]glutamine), is consistent with the substantial synaptosomal activity of the malate/aspartate shuttle.

Inhibition of astrocyte glutamine production by alpha-ketoisocaproic acid.

We have evaluated the effect of alpha-ketoisocaproic acid (KIC), the ketoacid of leucine, on the production of glutamine by cultured astrocytes. We used 15NH4Cl as a metabolic tracer to measure the production of both [5-15N]glutamine, reflecting amidation of glutamate via glutamine synthetase, and [2-15N]glutamine, representing the reductive amination of 2-oxoglutarate via glutamate dehydrogenase and subsequent conversion of [15N]glutamate to [2-15N]glutamine. Addition of KIC (1 mM) to the medium diminished the production of [5-15N]glutamine and stimulated the formation of [2-15N]glutamine with the overall result being a significant inhibition of net glutamine synthesis. An external KIC concentration as low as 0.06 mM inhibited synthesis of [5-15N]glutamine and a level as low as 0.13 mM enhanced labeling (atom% excess) of [2-15N]glutamine. Higher concentrations of KIC in the medium had correspondingly larger effects. The presence of KIC in the medium did not affect flux through glutaminase, which was measured using [2-15N]glutamine as a tracer. Nor did KIC inhibit the activity of glutamine synthetase that was purified from sheep brain. Addition of KIC to the medium caused no increased release of lactate dehydrogenase from the astrocytes, suggesting that the ketoacid was not toxic to the cells. KIC treatment was associated with an approximately twofold increase in the formation of 14CO2 from [U-14C]glutamate, indicating that transamination of glutamate with KIC increases intraastrocytic alpha-ketoglutarate, which is oxidized in the tricarboxylic acid cycle. KIC inhibited glutamine synthesis more than any other ketoacid tested, with the exception of hydroxypyruvate.(ABSTRACT TRUNCATED AT 250 WORDS)

Cerebral alanine transport and alanine aminotransferase reaction: alanine as a source of neuronal glutamate.

Alanine transport and the role of alanine amino-transferase in the synthesis and consumption of glutamate were investigated in the preparation of rat brain synaptosomes. Alanine was accumulated rapidly via both the high- and low-affinity uptake systems. The high-affinity transport was dependent on the sodium concentration gradient and membrane electrical potential, which suggests a cotransport with Na+. Rapid accumulation of the Na(+)-alanine complex by synaptosomes stimulated activity of the Na+/K+ pump and increased energy utilization; this, in turn, activated the ATP-producing pathways, glycolysis and oxidative phosphorylation. Accumulation of Na+ also caused a small depolarization of the plasma membrane, a rise in [Ca2+]i, and a release of glutamate. Intra-synaptosomal metabolism of alanine via alanine amino-transferase, as estimated from measurements of N fluxes from labeled precursors, was much slower than the rate of alanine uptake, even in the presence of added oxoacids. The velocity of [15N]alanine formation from [15N]glutamine was seven to eight times higher than the rate of [15N]-glutamate generation from [15N]alanine. It is concluded that (a) overloading of nerve endings with alanine could be deleterious to neuronal function because it increases release of glutamate; (b) the activity of synaptosomal alanine aminotransferase is much slower than that of glutaminase and hence unlikely to play a major role in maintaining [glutamate] during neuronal activity; and (c) alanine amino-transferase might serve as a source of glutamate during recovery from ischemia/hypoxia when the alanine concentration rises and that of glutamate falls.