Effect of the antiepileptic drug sodium valproate on glutamine and glutamate metabolism in isolated human kidney tubules

We studied the effects of sodium valproate, a widely used antiepileptic drug and a hyperammonemic agent, on L-[1-14C]glutamine and L-[1-14C]glutamate metabolism in isolated human kidney-cortex tubules. Valproate markedly stimulated glutamine removal as well as the formation of ammonia, 14CO2, pyruvate, lactate and alanine, but it inhibited glucose synthesis; the increase in ammonia formation was explained by a stimulation by valproate mainly of flux through glutaminase (EC 3.5.1.2) and to a much lesser extent of flux through glutamate dehydrogenase (EC 1.4.1.3). By contrast, valproate did not stimulate glutamate removal or ammonia formation, suggesting that the increase in flux through glutamate dehydrogenase observed with glutamine as substrate was secondary to the increase in flux through glutaminase. Accumulation of pyruvate, alanine and lactate in the presence of valproate was less from glutamate than from glutamine. Inhibition by aminooxyacetate of accumulation of alanine from glutamine caused by valproate did not prevent the acceleration of glutamine utilization and the subsequent stimulation of ammonia formation. It is concluded from these data, which are the first concerning the in vitro metabolism of glutamine and glutamate in human kidney-cortex tubules, that the stimulatory effect of valproate is primarily exerted at the level of glutaminase in human renal cortex.     

Calcium-ion transport by intact synaptosomes. Intrasynaptosomal compartmentation and the role of the mitochondrial membrane potential.

1. The pathways and the fate of glutamate carbon and nitrogen were investigated in isolated guinea-pig kidney-cortex tubules. 2. At low glutamate concentration (1 mM), the glutamate carbon skeleton was either completely oxidized or converted into glutamine. At high glutamate concentration (5 mM), glucose, lactate and alanine were additional products of glutamate metabolism. 3. At neither concentration of glutamate was there accumulation of ammonia. 4. Nitrogen-balance calculations and the release of 14CO2 from L-[1-14C]glutamate (which gives an estimation of the flux of glutamate carbon skeleton through alpha-oxoglutarate dehydrogenase) clearly indicated that, despite the absence of ammonia accumulation, glutamate metabolism was initiated by the action of glutamate dehydrogenase and not by transamination reactions as suggested by Klahr, Schoolwerth & Bourgoignie [(1972) Am. J. Physiol. 222, 813-820] and Preuss [(1972) Am. J. Physiol. 222, 1395-1397]. Additional evidence for this was obtained by the use of (i) amino-oxyacetate, an inhibitor of transaminases, which did not decrease glutamate removal, or (ii) L-methionine DL-sulphoximine, an inhibitor of glutamine synthetase, which caused an accumulation of ammonia from glutamate. 5. Addition of NH4Cl plus glutamate caused an increase in both glutamate removal and glutamine synthesis, demonstrating that the supply of ammonia via glutamate dehydrogenase is the rate-limiting step in glutamine formation from glutamate. NH4Cl also inhibited the flux of glutamate through glutamate dehydrogenase and the formation of glucose, alanine and lactate. 6. The activities of enzymes possibly involved in the glutamate conversion into pyruvate were measured in guinea-pig renal cortex. 7. Renal arteriovenous-difference measurements revealed that in vivo the guinea-pig kidney adds glutamine and alanine to the circulating blood.     

The regulation of glucose and pyruvate formation from glutamine and citric-acid-cycle intermediates in the kidney cortex of rats, dogs, rabbits and guinea pigs.

The suppression by 3-mercaptopicolinate of gluconeogenesis from glutamine or 2-oxoglutarate in rat or dog kidney tubules did not affect the amount of these substrates undergoing complete oxidation. Furthermore, 3-mercaptopicolinate caused an accumulation of lactate in dog tubules. 3-Mercaptopicolinate abolished both gluconeogenesis and substrate oxidation in tubules from rabbit and guinea-pig kidney. These results imply the presence of an alternative pathway to phosphoenolpyruvate carboxykinase/pyruvate kinase for the production of pyruvate from citric-acid-cycle intermediates in the kidney cortex of rats and dogs but not in that of rabbits or guinea pigs. Oxaloacetate decarboxylase (present in the kidney cortex of all four species) or 'malic' enzyme (present in rat and dog but absent in rabbit and guinea-pig kidney cortex) could function in this role. Our observations indicate that 'malic' enzyme is probably implicated in this phenomenon. The lactate production observed in dog tubules in the presence of 3-mercaptopicolinate can be suppressed when aspartate formation is inhibited by 2-amino-4-methoxy-trans-but-3-enoic acid. This suggests that the provision of cytosolic NADH from citric-acid-cycle intermediates is facilitated by accumulation of aspartate acting as a 'sink' for cytosolic oxaloacetate.     

Inhibition of glutamine synthetase in the mouse kidney: a novel mechanism of adaptation to metabolic acidosis

As part of a study on the regulation of renal ammoniagenesis in the mouse kidney, we investigated the effect of chronic metabolic acidosis on glutamine synthesis by isolated mouse renal proximal tubules. The results obtained reveal that, in tubules from control mice, glutamine synthesis occurred at high rates from glutamate and proline and, to a lesser extent, from ornithine, alanine, and aspartate. A 48 h, metabolic acidosis caused a marked inhibition of glutamine synthesis from near-physiological concentrations of both alanine and proline that were avidly metabolized by the tubules; metabolic acidosis also greatly stimulated glutamine utilization and metabolism. These effects were accompanied by a large increase (i) in alanine, proline, and glutamine gluconeogenesis and (ii) in ammonia accumulation from proline and glutamine. In the renal cortex of acidotic mice, the activity of phosphoenolpyruvate carboxykinase increased 4-fold, but that of glutamate dehydrogenase did not change; in contrast with what is known in the rat renal cortex, metabolic acidosis markedly diminished the glutamine synthetase activity and protein level, but not the glutamine synthetase mRNA level in the mouse renal cortex. These results strongly suggest that, in the mouse kidney, glutamine synthetase is an important regulatory component of the availability of the ammonium ions to be excreted for defending systemic acid-base balance. Furthermore, they show that, in rodents, the regulation of renal glutamine synthetase is species-specific.     

A role for bicarbonate in the regulation of mammalian glutamine metabolism.

1. The concentration of HCO3- (independent of any change of pH) exerts different effects on glutamine metabolism in rat kidney-cortex tubules, hepatocytes and enterocytes.2. In kidney tubules HCO3- (10.5-50 MM) has no effect on glutaminase (EC 3.5.1.2), whereas glutamate dehydrogenase (EC 1.4.1.3) is inhibited as HCO3- concentration is increased. The result is that flux through the entire glutamate-to-glucose pathway is inhibited by increasing HCO3- concentrations. A large proportion (more than 30%) of the glutamine removed undergoes complete oxidation. 3. In hepatocytes, and to a smaller extent in enterocytes, HCO3- is an accelerator of glutaminase. Synthesis of glucose and urea from glutamine in hepatocytes increases as HCO3- concentration is increased. Calculations show that fumarate, formed via aspartate aminotransferase and arginino-succinate lyase, is the precursor of the glucose. There is no complete oxidation of the carbon skeleton of glutamine in hepatocytes. 4. Leucine at near-physiological concentrations (0.1-1 mM) is an accelerator of glutaminase in hepatocytes, but not in kidney tubules or in enterocytes. 5. The results are discussed in relation to regulation of acid/base balance in vivo.     

The synthesis of glucose and ammonia by kidney tubules isolated from suckling and early-weaned lambs

Glucose and ammonia production were examined in kidney tubules isolated from suckling and early-weaned lambs, on days 10-30 after birth, with abrupt weaning occurring at day 14. There were no differences in the rates of glucose or ammonia production for a given substrate by tubules isolated from any of the lambs, regardless of age or stage of weaning. The preferred substrates for gluconeogenesis were glycerol = lactate greater than propionate = pyruvate = fructose = proline greater than alanine greater than glutamate greater than glutamine greater than aspartate greater than glycine greater than serine, and for ammoniagenesis were glutamine much greater than alanine greater than aspartate much greater than serine greater than glycine = glutamate = proline.     

The conversion of alanine into glutamine in guinea-pig renal cortex. Essential role of pyruvate carboxylase.

1. The metabolism of L-alanine was studied in isolated guinea-pig kidney-cortex tubules. 2. In contrast with previous conclusions of Krebs [(1935) Biochem. J. 29, 1951-1969], glutamine was found to be the main carbon and nitrogenous product of the metabolism of alanine (at 1 and 5 mM). Glutamate and ammonia were only minor products. 3. At neither concentration of alanine was there accumulation of glucose, glycogen, pyruvate, lactate, aspartate or tricarboxylic acid-cycle intermediates. 4. Carbon-balance calculations and the release of 14CO2 from [U-14C]alanine indicate that oxidation of the alanine carbon skeleton occurred at both substrate concentrations. 5. A pathway involving alanine aminotransferase, glutamate dehydrogenase, glutamine synthetase, pyruvate dehydrogenase, pyruvate carboxylase and enzymes of the tricarboxylic acid cycle is proposed for the conversion of alanine into glutamine. 6. Strong evidence for this pathway was obtained by: (i) suppressing alanine removal by amino-oxyacetate, and inhibitor of transaminases, (ii) measuring the release of 14CO2 from [1-14C]alanine, (iii) the use of L-methionine DL-sulphoximine, an inhibitor of glutamine synthetase, which induced a large increase in ammonia release from alanine, and (iv) the use of fluoroacetate, an inhibitor of aconitase, which inhibited glutamine synthesis with concomitant accumulation of citrate from alanine. 7. In this pathway, the central role of pyruvate carboxylase, which explains the discrepancy between our results and those of Krebs (1935), was also demonstrated.     

The relationship between glutamate deamination and gluconeogenesis in kidney.

The effect of 3-mercaptopicolinate, an inhibitor of phosphoenolpyruvate carboxykinase [GTP:oxaloacetate carboxy-lyase (transphosphorylating), EC 4.1.1.32], was tested on NH3 formation via the purine nucleotide cycle and glutamate dehydrogenase (EC 1.4.1.2). NH3 excretion in rats increased 70-fold after 48 h of NH4Cl feeding, from 12.2 +/- 4.5 to 862 +/- 190 mumol/mg of creatinine. At 4 h after a single intraperitoneal injection of 3-mercaptopicolinate into NH4Cl-fed rats, NH3 excretion was inhibited by 93%. Kidneys of NH4Cl-fed plus 3-mercaptopicolinate-treated rats, compared with those of NH4Cl-fed rats, showed a 3.5-fold increase in the content of IMP, 5-fold increase in adenylosuccinate, 4-fold increase in aspartate, and a 30% increase in AMP. 3-Mercaptopicolinate completely inhibited NH3 and glucose formation from glutamate in tubules from acidotic rats and NH3 formation from aspartate in kidney perfusion experiments. When transamination in tubules was prevented by 2-amino-4-methoxy-trans-but-3-enoic acid, formation of glucose, but not of NH3, from glutamate was inhibited. 3-Mercaptopicolinate completely inhibited NH3 formation from aspartate in the presence of the aminotransferase inhibitor in kidney tubules. The data show that NH3 can be formed via glutamate dehydrogenase and the purine nucleotide cycle at significant and approximately equal rates. 3-Mercaptopicolinate has no direct effect on NH3 formation via glutamate dehydrogenase, but inhibits that via the purine nucleotide cycle. We conclude that gluconeogenesis is not regulatory for NH3 formation in kidney.     

METABOLISM OF GLUCOSE AND GLUTAMATE BY SYNAPTOSOMES FROM MAMMALIAN CEREBRAL CORTEX

—(1) Synaptosomes incubated in high sodium, low potassium media showed high linear respiration in the presence of glucose which was converted into lactate, aspartate, glutamate, glutamine, alanine and GABA during 1 hr incubation periods. (2) Total conversion of glucose into most of these substrates over the incubation period was similar in synaptosomes and cortex slices. Half the lactate and only a small fraction of the glutamine made by slices was formed by synaptosomes. (3) Pool sizes of amino acids in cortex slices after incubation with glucose were, in general, higher than in synaptosomes, glutamate and glutamine being four-fold higher in slices. (4) Most of the amino acids made from glucose by synaptosomes were contained within their structure and not lost to the medium. (5) Glutamate was actively metabolized by synaptosomes to aspartate, glutamine, alanine and GABA. The specific radioactivities of the amino acids (except glutamine) after 1 hr incubation, approached that of the glutamate. (6) Pyridoxal phosphate added to the incubation medium increased GABA production from glutamate but not from glucose.     

Proline metabolism in isolated rat liver cells

The metabolism of proline was studied in liver cells isolated from starved rats. The following observations were made. 1. Consumption of proline could be largely accounted for by production of glucose, urea, glutamate and glutamine. 2. At least 50% of the total consumption of oxygen was used for proline catabolism. 3. Ureogenesis and gluconeogenesis from proline could be stimulated by partial uncoupling of oxidative phosphorylation. 4. Addition of ethanol had little effect on either proline uptake or oxygen consumption, but strongly inhibited the production of both urea and glucose and caused further accumulation of glutamate and lactate. Accumulation of glutamine was not affected by ethanol. 5. The effects of ethanol could be overcome by partial uncoupling of oxidative phosphorylation. 6. The apparent K_m values of argininosuccinate synthetase (EC 6.3.4.5) for aspartate and citrulline in the intact hepatocyte are higher than those reported for the isolated enzyme. 7. 3-Mercaptopicolinate, an inhibitor of phosphoenolpyruvate carboxykinase (EC 4.1.1.32), greatly enhanced cytosolic aspartate accumulation during proline metabolism, but inhibited urea synthesis. 8. It is concluded that when proline is provided as a source of nitrogen to liver cells, production of ammonia by oxidative deamination of glutamate is inhibited by the highly reduced state of the nicotinamide nucleotides within the mitochondria. 9. Conversion of proline into glucose and urea is a net-energy-yielding process, and the high state of reduction of the nicotinamide nucleotides is presumably maintained by a high phosphorylation potential. Thus when proline is present as sole substrate, the further oxidation of glutamate by glutamate dehydrogenase (EC 1.4.1.3) is limited by the rate of energy expenditure of the cell.     

Hyperglycaemic activity and metabolic effects of 3-aminopicolinic acid

Administering 3-aminopicolinate to rats starved for 24h immediately initiated a progressive increase in blood glucose concentration. Hyperglycaemia was not the result of glycogenolysis, nor was it due to an inhibition of insulin release, since it caused marked hyperinsulinaemia. The rate of [6-³H]glucose disappearance from the blood of the intact rat was not altered by 3-aminopicolinate, indicating that it does not cause hyperglycaemia by inhibiting glucose utilization or by causing a redistribution of total body glucose. 3-Aminopicolinate increased the rate of fall in the specific radioactivity of blood [6-³H]-glucose, indicating dilution of the glucose pool by newly synthesized glucose. The rate of ¹⁴C incorporation into blood glucose from [¹⁴C]alanine and [¹⁴C]lactate was increased 90 and 35% respectively, whereas that from [¹⁴C]glycerol and [¹⁴C]xylitol was either unaffected or slightly decreased by 3-aminopicolinate administration. Liver phosphoenolpyruvate of rats was increased to four to seven times the normal concentration 10min to 1h after injections of 50–300mg of 3-aminopicolinate/kg body wt. and the amounts of 2-phosphoglycerate and 3-phosphoglycerate were increased to three to four times normal. The high concentrations of liver phosphoenolpyruvate, 2-phosphoglycerate and 3-phosphoglycerate, as well as the enhancement of gluconeogenesis from lactate and alanine, but not from glycerol or xylitol, is compatible with an enhancement of gluconeogenesis at a step between pyruvate and the triose phosphates. After injections of 3-aminopicolinate, liver malate, citrate, aspartate, alanine, lactate and pyruvate were also increased, but to lesser extents than was phosphoenolpyruvate. The increases in some of these metabolites were approximated after an intravenous infusion of glucose, so their elevated concentration after 3-aminopicolinate administration could have been, in part, a consequence of the hyperglycaemia. The possibility is considered that 3-aminopicolinate stimulates gluconeogenesis in vivo by facilitating Fe²⁺ activation of phosphoenolpyruvate carboxykinase as it does with the purified enzyme in vitro [MacDonald & Lardy (1978) J. Biol. Chem. 253, 2300–2307]. In this effect 3-aminopicolinate may simulate the physiological role of the naturally occurring ferroactivator protein [Bentle & Lardy (1977) J. Biol. Chem. 252, 1431–1440].     

ALANINE METABOLISM IN RAT CORTEX IN VITRO

Abstract— (1) The metabolism of [U-¹⁴C]alanine was followed in slices of rat cerebral cortex and its interaction with glucose, pyruvate and the metabolic inhibitors fluoracetate and malonate was studied.
(2) Alanine did not stimulate respiration above endogenous levels or affect the rate of oxygen uptake with glucose or pyruvate as cosubstrate. Radioactivity found in CO₂ from labelled alanine was only 6 per cent of that from labelled pyruvate. Lactate was not formed from alanine.
(3) After 2 h incubation with [U-¹⁴C]alanine the specific activities of glutamate, glutamine and GABA were 20–30 per cent that of alanine. All these specific activities except glutamate were lowered by addition of glucose, but with pyruvate as cosubstrate the specific activity of glutamate was increased by 87 per cent above the level with alanine alone.
(4) The effect of alanine as cosubstrate with [U-¹⁴C]pyruvate was to reduce the specific activity of GABA and of glutamine, but not glutamate or lactate; thus there was not an equal dilution of all the metabolites of pyruvate.
(5) Fluoracetate diminished respiration and the production of CO₂ from [U-¹⁴C]-alanine only slightly; the addition of malonate as well practically abolished both. Fluoracetate lowered incorporation from alanine into all the amino acids, and radioactivity could not be detected in glutamine at all; addition of malonate lowered the specific activity of glutamate to 25 per cent but increased that into aspartate, GABA and glutamine.
(6) The interpretation of these data in terms of known pathways of alanine metabolism is discussed.     

The pathway of glutamine and glutamate oxidation in isolated mitochondria from mammalian cells

1. Pyruvate strongly inhibited aspartate production by mitochondria isolated from Ehrlich ascites-tumour cells, and rat kidney and liver respiring in the presence of glutamine or glutamate; the production of (14)CO(2) from l-[U-(14)C]glutamine was not inhibited though that from l-[U-(14)C]glutamate was inhibited by more than 50%. 2. Inhibition of aspartate production during glutamine oxidation by intact Ehrlich ascites-tumour cells in the presence of glucose was not accompanied by inhibition of CO(2) production. 3. The addition of amino-oxyacetate, which almost completely suppressed aspartate production, did not inhibit the respiration of the mitochondria in the presence of glutamine, though the respiration in the presence of glutamate was inhibited. 4. Glutamate stimulated the respiration of kidney mitochondria in the presence of glutamine, but the production of aspartate was the same as that in the presence of glutamate alone. 5. The results suggest that the oxidation of glutamate produced by the activity of mitochondrial glutaminase can proceed almost completely through the glutamate dehydrogenase pathway if the transamination pathway is inhibited. This indicates that the oxidation of glutamate is not limited by a high [NADPH]/[NADP(+)] ratio. 6. It is suggested that under physiological conditions the transamination pathway is a less favourable route for the oxidation of glutamate (produced by hydrolysis of glutamine) in Ehrlich ascites-tumour cells, and perhaps also kidney, than the glutamate dehydrogenase pathway, as the production of acetyl-CoA strongly inhibits the first mechanism. The predominance of the transamination pathway in the oxidation of glutamate by isolated mitochondria can be explained by a restricted permeability of the inner mitochondrial membrane to glutamate and by a more favourable location of glutamate-oxaloacetate transaminase compared with that of glutamate dehydrogenase.     

The metabolic role of isoleucine in detoxification of ammonia in cultured mouse neurons and astrocytes

Cerebral hyperammonemia is a hallmark of hepatic encephalopathy, a debilitating condition arising secondary to liver disease. Pyruvate oxidation including tricarboxylic acid (TCA) cycle metabolism has been suggested to be inhibited by hyperammonemia at the pyruvate and -ketoglutarate dehydrogenase steps. Catabolism of the branched-chain amino acid isoleucine provides both acetyl-CoA and succinyl-CoA, thus by-passing both the pyruvate dehydrogenase and the -ketoglutarate dehydrogenase steps. Potentially, this will enable the TCA cycle to work in the face of ammonium-induced inhibition. In addition, this will provide the -ketoglutarate carbon skeleton for glutamate and glutamine synthesis by glutamate dehydrogenase and glutamine synthetase (astrocytes only), respectively, both reactions fixing ammonium. Cultured cerebellar neurons (primarily glutamatergic) or astrocytes were incubated in the presence of either [U-¹³C]glucose (2.5 mM) and isoleucine (1 mM) or [U-¹³C]isoleucine and glucose. Cell cultures were treated with an acute ammonium chloride load of 2 (astrocytes) or 5 mM (neurons and astrocytes) and incorporation of ¹³C-label into glutamate, aspartate, glutamine and alanine was determined employing mass spectrometry. Labeling from [U-¹³C]glucose in glutamate and aspartate increased as a result of ammonium-treatment in both neurons and astrocytes, suggesting that the TCA cycle was not inhibited. Labeling in alanine increased in neurons but not in astrocytes, indicating elevated glycolysis in neurons. For both neurons and astrocytes, labeling from [U-¹³C]isoleucine entered glutamate and aspartate albeit to a lower extent than from [U-¹³C]glucose. Labeling in glutamate and aspartate from [U-¹³C]isoleucine was decreased by ammonium treatment in neurons but not in astrocytes, the former probably reflecting increased metabolism of unlabeled glucose. In astrocytes, ammonia treatment resulted in glutamine production and release to the medium, partially supported by catabolism of [U-¹³C]isoleucine. In conclusion, i) neuronal and astrocytic TCA cycle metabolism was not inhibited by ammonium and ii) isoleucine may provide the carbon skeleton for synthesis of glutamate/glutamine in the detoxification of ammonium.     

GLUCOSE AND AMINO ACID METABOLISM IN SOME INVERTEBRATE NERVOUS SYSTEMS

(1) The in vitro metabolism of [U-¹⁴C]glucose and [U-¹⁴C]glutamate was compared in snail, octopus and locust ganglia, and in rat cerebral cortex. (2) The metabolic patterns are quantitatively similar. The major labelled metabolites formed from glucose or glutamate by rat cortex and the invertebrate systems were CO₂, aspartate, glutamate, glutamine and alanine. γ-Aminobutyric acid (GABA) was formed in substantial amounts only by locust and rat. (3) A much larger proportion of labelled glucose and glutamate was converted to alanine by the invertebrates compared with rat cortex, although ¹⁴CO₂ production was lower. (4) The effect of glucose in reducing aspartate formation and stimulating glutamine formation from [U-¹⁴C]glutamate in mammalian cortex was observed in the locust but not in the molluscs. (5) Labelled citric acid cycle intermediates were formed in substantial quantities from glucose and glutamate only by snail and locust.     

Characteristics of glutamine metabolism by rat kidney tubules: a carbon and nitrogen balance.

The metabolism of glutamine by a suspension of rat kidney tubules was studied in vitro. The influence of duration of incubation, glutamine concentration, and metabolic state of the donor animals was investigated. The relative importance of glucose synthesis, amino acid production, and oxidation to CO2 was estimated by drawing a complete balance of the nitrogens and the carbon chains of the extracted glutamine. It was found that the initial (first 15 min) rate of glutamine utilization was significantly greater than the subsequent rate due to an initial, but transient, extracellular accumulation of glutamate. This phenomenon was suppressed when a small amount of glutamate was added to the incubation medium. Glucose production constitutes the major fate for glutamine metabolism. No net oxidation of glutamine could be detected with 1 mM glutamine during the first 30 min. However, glutamine oxidation becomes significant after prolonged incubation (16% at 120 min). The metabolic fate of glutamine differs when 5 or 10 mM are presented to the tubules, glutamate production and oxidation to CO2 becoming more important. Metabolic acidosis or a 48-h fast increases glutamine extraction and enhances its utilization glucose synthesis while they depress glutamate accumulation and oxidation to CO2. Metabolic alkalosis has the opposite effect. It is concluded that the metabolism of glutamine in vitro is dependent on the conditions of the study. Furthermore, total oxidation to CO2 is not a major fate for glutamine metabolism at physiological concentration and is not enhanced by acidosis in the rat kidney in vitro.     

Metabolic fate of glutamate carbon in rat renal tubules. Studies with 13C nuclear magnetic resonance and gas chromatography-mass spectrometry.

13C-n.m.r. spectroscopy and g.c.-m.s. were used to determine the metabolic fate of glutamate carbon in rat kidney. The main purpose was to characterize the effect of chronic metabolic acidosis on the utilization of glutamate carbon. Renal tubules obtained from normal and chronically acidotic rats were incubated in Krebs buffer, pH 7.4, in the presence of 2.5 mM-[3-13C]glutamate. During the course of incubation the concentrations of total glucose and NH3 were significantly (P less than 0.05) higher in tissue from acidotic rats. The levels of some tricarboxylic-acid-cycle intermediates were higher (P less than 0.05) in control tissue. In control tissue, 13C-n.m.r. spectra demonstrated a significantly higher rate of 13C appearance of aspartate, glutamine and [2,4-13C]glutamate. However, in acidosis the resonances of [13C]glucose carbon atoms were significantly higher. In the control, approx. 15% of glutamate carbon was accounted for by [13C]glucose formation as against 30% in chronic acidosis. However, in control tissue, 44% of glutamate carbon utilization was accounted for by recycling to glutamate and formation of aspartate, glutamine and GABA. In acidosis, only 11% was so recovered. Analysis of 15NH3 formation during the course of incubation with 2.5 mM-[15N]glutamate demonstrated a positive association between the appearance of [13C]glucose and 15NH3 both in the control and in acidosis. The data suggest that the control of gluconeogenesis and ammoniagenesis in acidosis is, in part, referable to a diminution in the rate of the reductive amination of alpha-oxoglutarate, that of the transamination reaction and that of glutamine synthesis.     

Glucose and glutamine metabolism in rat thymocytes.

The metabolism of glucose and glutamine in freshly prepared resting and concanavalin A-stimulated rat thymocytes was studied. Concanavalin A addition enhanced uptake of both glucose and glutamine and led to an increase in oxidative degradation of both substrates to CO2. With variously labelled [14C]glucose, it was shown that the pathways of glucose dissimilation were equally stimulated by the mitogen. A disproportionately large percentage of the extra glucose taken up was converted into lactate, but concanavalin A also caused an increase in the oxidation of pyruvate as judged by the enhanced release of 14CO2 from [2-14C]-, [3,4-14C]- and [6-14C]-glucose. Addition of glutamine did not affect glucose metabolism. The major end products of glutamine metabolism by resting and mitogen-stimulated rat thymocytes were glutamate, aspartate, CO2 and NH3. Virtually no lactate was formed from glutamine. Concanavalin A enhanced the formation of all end products except glutamate, indicating that more glutamine was metabolized beyond the stage of glutamate in the mitogen-activated cells. Addition of glucose caused a significant decrease in the rates of glutamine utilization and conversion into aspartate and CO2 in the absence and in the presence of concanavalin A. In the presence of glucose, almost all nitrogen of the metabolized glutamine was accounted for as NH3 released via the glutaminase and/or glutamate dehydrogenase reactions. In the absence of glucose, part (18%) of the glutamine nitrogen was metabolized by the resting and to a larger extent (38%) by the mitogen-stimulated thymocytes via a transaminase or amidotransferase reaction.     

Neuronal Glutamine Utilization: Pathways of Nitrogen Transfer tudied with [15N]Glutamine

Gas chromatography-mass spectrometry was used to evaluate the metabolism of [15N]glutamine in isolated rat brain synaptosomes. In the presence of 0.5 mM glutamine, synaptosomes accumulated this amino acid to a level of 25-35 nmol/mg protein at an initial rate greater than 9 nmol/min/mg of protein. The metabolism of [15N]glutamine generated 15N-labelled glutamate, aspartate, and gamma-aminobutyric acid (GABA). An efflux of both [15N]glutamate and [15N]aspartate from synaptosomes to the medium was observed. Enrichment of 15N in alanine could not be detected because of a limited pool size. Elimination of glucose from the incubation medium substantially increased the rate and amount of [15N]aspartate formed. It is concluded that: (1) With 0.5 mM external glutamine, the glutaminase reaction, and not glutamine transport, determines the rate of metabolism of this amino acid. (2) The primary route of glutamine catabolism involves aspartate aminotransferase which generates 2-oxoglutarate, a substrate for the tricarboxylic acid cycle. This reaction is greatly accelerated by the omission of glucose. (3) Glutamine has preferred access to a population of synaptosomes or to a synaptosomal compartment that generates GABA. (4) Synaptosomes maintain a constant internal level of glutamate plus aspartate of about 70-80 nmol/mg protein. As these amino acids are produced from glutamine in excess of this value, they are released into the medium. Hence synaptosomal glutamine and glutamate metabolism are tightly regulated in an interrelated manner.     

The anaplerotic substrate alanine stimulates acetate incorporation into glutamate and glutamine in rabbit kidney tubules. A (13)C NMR study

Although acetate, the main circulating volatile fatty acid in humans and animals, is metabolized at high rates by the renal tissue, little is known about the precise fate of its carbons and about the regulation of its renal metabolism. Therefore, we studied the metabolism of variously labeled [(13)C]acetate and [(14)C]acetate molecules and its regulation by alanine, which is also readily metabolized by the kidney, in isolated rabbit renal proximal tubules. With acetate as the sole substrate, 72% of the C-1 and 49% of the C-2 of acetate were released as CO(2); with acetate plus alanine, the corresponding values were decreased to 49 and 25%. The only other important products formed from the acetate carbons were glutamine, and to a smaller extent, glutamate. By combining (13)C NMR and radioactive and enzymatic measurements with a novel model of acetate metabolism, fluxes through the enzymes involved were calculated. Thanks to its anaplerotic effect, alanine caused a stimulation of acetate removal and a large increase in fluxes through pyruvate carboxylase, citrate synthase, and the enzymes involved in glutamate and glutamine synthesis but not in flux through alpha-ketoglutarate dehydrogenase. We conclude that the anaplerotic substrate alanine not only accelerates the disposal of acetate but also prevents the wasting of the latter compound as CO(2).