Amino acid sequence of the trypsin-generated C3d fragment from human complement factor C3.

Energy metabolism in proliferating cultured rat thymocytes was compared with that of freshly prepared non-proliferating resting cells. Cultured rat thymocytes enter a proliferative cycle after stimulation by concanavalin A and Lymphocult T (interleukin-2), with maximal rates of DNA synthesis at 60 h. Compared with incubated resting thymocytes, glucose metabolism by incubated proliferating thymocytes was 53-fold increased; 90% of the amount of glucose utilized was converted into lactate, whereas resting cells metabolized only 56% to lactate. However, the latter oxidized 27% of glucose to CO2, as opposed to 1.1% by the proliferating cells. Activities of hexokinase, 6-phosphofructokinase, pyruvate kinase and aldolase in proliferating thymocytes were increased 12-, 17-, 30- and 24-fold respectively, whereas the rate of pyruvate oxidation was enhanced only 3-fold. The relatively low capacity of pyruvate degradation in proliferating thymocytes might be the reason for almost complete conversion of glucose into lactate by these cells. Glutamine utilization by rat thymocytes was 8-fold increased during proliferation. The major end products of glutamine metabolism are glutamate, aspartate, CO2 and ammonia. A complete recovery of glutamine carbon and nitrogen in the products was obtained. The amount of glutamate formed by phosphate-dependent glutaminase which entered the citric acid cycle was enhanced 5-fold in the proliferating cells: 76% was converted into 2-oxoglutarate by aspartate aminotransferase, present in high activity, and the remaining 24% by glutamate dehydrogenase. With resting cells the same percentages were obtained (75 and 25). Maximal activities of glutaminase, glutamate dehydrogenase and aspartate aminotransferase were increased 3-, 12- and 6-fold respectively in proliferating cells; 32% of the glutamate metabolized in the citric acid cycle was recovered in CO2 and 61% in aspartate. In resting cells this proportion was 41% and 59% and in mitogen-stimulated cells 39% and 65% respectively. Addition of glucose (4 mM) or malate (2 mM) strongly decreased the rates of glutamine utilization and glutamate conversion into 2-oxoglutarate by proliferating thymocytes and also affected the pathways of further glutamate metabolism. Addition of 2 mM-pyruvate did not alter the rate of glutamine utilization by proliferating thymocytes, but decreased the rate of metabolism beyond the stage of glutamate significantly. Formation of acetyl-CoA in the presence of pyruvate might explain the relatively enhanced oxidation of glutamate to CO2 (56%) by proliferating thymocytes.     

Pathways of glutamine and glutamate metabolism in resting and proliferating rat thymocytes: comparison between free and peptide-bound glutamine

Pathways of glutamine metabolism in resting and proliferating rat thymocytes were evaluated by in vitro incubations of freshly prepared or 60-h cultured cells for 1-2 h with [U14C]glutamine. Complete recovery of glutamine carbons utilized in products allowed quantification of the pathways of glutamine metabolism under the experimental conditions. Partial oxidation of glutamine via 2-oxoglutarate in a truncated citric acid cycle to CO2 and oxaloacetate, which then was converted to aspartate, accounted for 76 and 69%, respectively, of the glutamine metabolized beyond the stage of glutamate by resting and proliferating thymocytes. Complete oxidation to CO2 in the citric acid cycle via 2-oxoglutarate dehydrogenase and isocitrate dehydrogenase accounted for 25 and 7%, respectively. In proliferating cells a substantial amount of glutamine carbons was also recovered in pyruvate, alanine, and especially lactate. The main route of glutamine and glutamate entrance into the citric acid cycle via 2-oxoglutarate in both cells is transamination by aspartate aminotransferase rather than oxidative deamination by glutamate dehydrogenase. In the presence of glucose as second substrate, glutamine utilization and aspartate formation markedly decreased, but complete oxidation of glutamine carbons to CO2 increased to 37 and 23%, respectively, in resting and proliferating cells. The dipeptide, glycyl-L-glutamine, which is more stable than free glutamine, can substitute for glutamine in thymocyte cultures at higher concentrations.     

Glutamine and glucose metabolism in thymocytes from normal and spontaneously diabetic BB rats.

Metabolism of glutamine and glucose was studied in thymocytes from normal rats and BB rats with the spontaneous autoimmune diabetic syndrome to assess their potential roles as fuels. The major measured products from glucose were lactate and, to a lesser extent, CO2, and pyruvate. Glutamine had no effect on the rates of their production from glucose. Glutamine was metabolized to ammonia, aspartate, glutamate, and CO2, with aspartate being the major product of carbons from glutamine in the absence of glucose. Glucose markedly decreased the formation of ammonia, aspartate, and CO2 from glutamine, but increased that of glutamate, with an overall decrease in glutamine utilization by 55%. More glutamate than aspartate was produced from glutamine in the presence of glucose. The potential production of ATP from glucose was similar to that when glutamine was present alone. However, glucose markedly decreased production of ATP from glutamine, but not vice versa. This resulted in ATP production from glucose being 2.5 times that from glutamine when both substrates were present. The oxidation of glucose to CO2 via the Krebs cycle accounts for 75-80% of glucose-derived ATP production. Cellular ATP levels markedly decreased in the absence of exogenous substrates, but were constant throughout a 2-h incubation in the presence of glutamine, glucose, or both. There were no differences in thymocyte glucose or glutamine metabolism between normal and diabetic BB rats, in contrast to previous findings in peripheral lymphoid organs. Our results suggest that glucose is a more important fuel than glutamine for "resting" thymocytes, again in contrast to the cells of peripheral lymphoid organs in which glutamine is as important as glucose as a fuel.(ABSTRACT TRUNCATED AT 250 WORDS)     

Glutamine and glucose metabolism in bovine blood lymphocytes.

1. Glutamine and glucose metabolism was studied in bovine blood lymphocytes incubated at 37 degrees C in the presence of Krebs-Ringer bicarbonate buffer (pH 7.4) containing 1 mM [U-14C]glutamine and 5 mM [U-14C]glucose, respectively. 2. The major metabolic products from glutamine were ammonia, glutamate, and to a lesser extent, aspartate and CO2. Glucose was metabolized mainly to lactate and, to a lesser extent, pyruvate and CO2. These findings indicate incomplete oxidation of glutamine and glucose carbons in bovine blood lymphocytes. 3. Glucose provided three-fold greater amounts of energy to bovine blood lymphocytes than did glutamine on the basis of their measured end-products. Glycolysis accounted for 50% of glucose-derived ATP production. 4. Our findings suggest similar metabolic patterns of glutamine and glucose in lymphocytes between ruminants and non-ruminant species (e.g. rats). However, in contrast to rat peripheral lymphocytes, glucose, rather than glutamine, was a major energy substrate for bovine blood lymphocytes.     

Metabolism of glucose into glutamate via the hexose monophosphate shunt and its inhibition by 6-aminonicotinamide in rat brain in vivo

The treatment of rats for 4 h with 6-aminonicotinamide (60 mg kg-1) resulted in an 180-fold increase in the concentration of 6-phosphogluconate in their brains; glucose increased 2.6-fold and glucose 6-phosphate, 1.7-fold. Moreover, lactate decreased by 20%, glutamate by 8% and gamma-aminobutyrate by 12%, and aspartate increased by 10%. No significant changes were found in glutamine and citrate. In blood, 6-phosphogluconate increased 5-fold; glucose, 1.4-fold and glucose 6-phosphate, 1.8-fold. The metabolism of glucose in the rat brain, via both the Embden-Meyerhof pathway and the hexose monophosphate shunt, was investigated by injecting [U-14C]glucose or [2-14C]glucose, and that via the hexose monophosphate shunt alone by injecting [3,4-14C]glucose. The total radioactive yield of amino acids in the rat brain was 5.63 mumol at 20 min after injection of [U-14C]glucose, or 5.82 mumol after injection of [2-14C]glucose; by contrast, it was 0.62 mumol after injection of [3,4-14C]glucose. The treatment of rats with 6-aminonicotinamide showed significant decreases in these values, owing to decreases in the radioactive yields of glutamate, glutamine, aspartate, gamma-aminobutyrate, and alanine+glycine+serine. Glutamate isolated from the brain contained approximately 43% of its radioactivity in carbon 1 after injection of [3,4-14C]glucose, in contrast to 13% and 18% after injection of [U-14C]glucose and [2-14C]glucose, respectively, in both the control and treated rats. The calculations based on these findings showed that approximately 69% of the 14C-labelled glutamate was formed from [14C]acetyl coenzyme A (acetyl CoA) and the residual 31% by 14CO2 fixation of pyruvate after injection of [3,4-14C]glucose in both control and treated rats. The results gave direct evidence that glutamate and gamma-aminobutyrate in the brain were formed by metabolism of glucose via the hexose monophosphate shunt as well as via the Embden-Meyerhof pathway. From the radioactive yields of glutamate formed via [14C]acetyl CoA it was estimated that approximately 7.8% of the total glucose utilized was channelled via the hexose monophosphate shunt. Assuming that [14C]glutamate formed by carbon-dioxide fixation of pyruvate was also dependent on the metabolism of glucose through the hexose monophosphate shunt, the estimated value was approximately 9.5% of the total glucose converted into glutamate. The results of the present investigation, taken in conjunction with other findings, suggest that the utilization of glucose via the hexose monophosphate shunt is functionally important in the rat brain.     

GLUTAMINE UPTAKE AND METABOLISM BY THE ISOLATED TOAD BRAIN: EVIDENCE PERTAINING TO ITS PROPOSED ROLE AS A TRANSMITTER PRECURSOR

Abstract— Hemisections of toad brains, when incubated in a physiological medium containing no glutamine. released considerable amounts of this amino acid into the medium. When glutamine was included in the medium at a concentration of 0.2 mm the net efflux from the tissue was reduced but not totally prevented. Although there was no net uptake of glutamine, the tissue did accumulate [U-¹⁴C]glu-tamine and some of this labelled glutamine was rapidly metabolized to glutamate, GABA and aspartate. The precursor-product relationship for the metabolism of glutamine to glutamate differed from the classic single compartment model in that the specific radioactivity of glutamate rose very quickly to approx one-tenth that of glutamine, but increased slowly thereafter. These data suggest that the [¹⁴C]glutamine was taken up into two metabolically distinct compartments and/or that some of the [¹⁴C]glutamine was converted to [¹⁴C]glutamate during the uptake process. The uptake of [¹⁴C]glutamine was diminished when the tissue was incubated in a non-oxygenated medium or when Na⁺ was omitted (substituted with sucrose) and K⁺ was concomitantly elevated. However, on a relative basis, the incorporation of radioactivity into glutamate and GABA was increased by these incubation conditions. The metabolism of glutamine to aspartate was greatly depressed when the tissue was not oxygenated. The glutamate formed from [U-¹⁴C]glutamine taken up by the tissue was converted to GABA at a faster rate than was glutamate derived from [U-¹⁴C]glucose. [U-¹⁴C]gly-cerol or exogenous [U-¹⁴C]glutamate. This suggests that glutamine was metabolized to GABA selectively; i.e. on a relative basis, glutamine served as a better source of carbon for the synthesis of GABA than did glucose, glycerol or exogenous glutamate. When the brain hemisections were incubated in the normal physiological medium with or without glutamine. there was very little efflux of glutamate, GABA or aspartate from the tissue. However when NaCl was omitted from the medium (substituted with sucrose) and K⁺ was elevated to 29 miu. a marked efflux of these three amino acids into the medium did occur, and over a period of 160min, the content of each amino acid in the tissue was depleted considerably. When glutamine (0.2 mm ) was included in the Na⁺ deficient-high K.⁺ medium, the average amount of glutamate, GABA and aspartate in the tissue plus the medium was greater than when glutamine was not included in the medium. Such data indicate that CNS tissues can utilize glutamine for a net synthesis of glutamate, GABA and aspartate. The results of this study provide further evidence in support of the concept that the functional (transmitter) pools of glutamate and GABA are maintained and regulated in part via biosynthesis from glutamine. One specific mechanism instrumental in regulating the content of glutamate in nerve terminals may be a process of glutamine uptake coupled to deamidation.     

Glutamate Metabolism in Rat Cortical Astrocyte Cultures

Glutamate metabolism in rat cortical astrocyte cultures was studied to evaluate the relative rates of flux of glutamate carbon through oxidative pathways and through glutamine synthetase (GS). Rates of 14CO2 production from [1-14C]glutamate were determined, as was the metabolic fate of [14C(U)]glutamate in the presence and absence of the transaminase inhibitor aminooxyacetic acid and of methionine sulfoximine, an irreversible inhibitor of GS. The effects of subculturing and dibutyryl cyclic AMP treatment of astrocytes on these parameters were also examined. The vast majority of exogenously added glutamate was converted to glutamine and exported into the extracellular medium. Inhibition of GS led to a sustained and greatly elevated intracellular glutamate level, thereby demonstrating the predominance of this pathway in the astrocytic metabolism of glutamate. Nevertheless, there was some glutamate oxidation in the astrocyte culture, as evidenced by aspartate production and labeling of intracellular aspartate pools. Inhibition of aspartate aminotransferase caused a greater than 70% decrease in 14CO2 production from [1-14C]glutamate. Inhibition of GS caused an increase in aspartate production. It is concluded that transamination of glutamate rather than oxidative deamination catalyzed by glutamate dehydrogenase is the first step in the entry of glutamate carbon into the citric acid cycle in cultured astrocytes. This scheme of glutamate metabolism was not qualitatively altered by subculturing or by treatment of the cultures with dibutyryl cyclic AMP.     

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.     

Compartmentation of glutamate metabolism in brain. Evidence for the existence of two different tricarboxylic acid cycles in brain

1. (14)C from [1-(14)C]glucose injected intraperitoneally into mice is incorporated into glutamate, aspartate and glutamine in the brain to a much greater extent than (14)C from [2-(14)C]glucose. This difference for [1-(14)C]glucose and [2-(14)C]glucose increases with time. The amount of (14)C in C-1 of glutamate increases steadily with time with both precursors. It is suggested that a large part of the glutamate and aspartate pools in brain are in close contact with intermediates of a fast-turning tricarboxylic acid cycle. 2. (14)C from [1-(14)C]acetate and [2-(14)C]acetate is incorporated to a much larger extent into glutamine than into glutamate. An examination of the time-course of (14)C incorporated into glutamine and glutamate reveals that glutamine is not formed from the glutamate pool, labelled extensively by glucose, but from a small glutamate pool. This small glutamate pool is not derived from an intermediate of a fast-turning tricarboxylic acid cycle. 3. It is proposed that two different tricarboxylic acid cycles exist in brain.     

Aerobic glycolysis and lymphocyte transformation

1. The role of enhanced aerobic glycolysis in the transformation of rat thymocytes by concanavalin A has been investigated. Concanavalin A addition doubled [U-(14)C]glucose uptake by rat thymocytes over 3h and caused an equivalent increased incorporation into protein, lipids and RNA. A disproportionately large percentage of the extra glucose taken up was converted into lactate, but concanavalin A also caused a specific increase in pyruvate oxidation, leading to an increase in the percentage contribution of glucose to the respiratory fuel. 2. Acetoacetate metabolism, which was not affected by concanavalin A, strongly suppressed pyruvate oxidation in the presence of [U-(14)C]glucose, but did not prevent the concanavalin A-induced stimulation of this process. Glucose uptake was not affected by acetoacetate in the presence or absence of concanavalin A, but in each case acetoacetate increased the percentage of glucose uptake accounted for by lactate production. 3. [(3)H]Thymidine incorporation into DNA in concanavalin A-treated thymocyte cultures was sensitive to the glucose concentration in the medium in a biphasic manner. Very low concentrations of glucose (25mum) stimulated DNA synthesis half-maximally, but maximum [(3)H]thymidine incorporation was observed only when the glucose concentration was raised to 1mm. Lactate addition did not alter the sensitivity of [(3)H]-thymidine uptake to glucose, but inosine blocked the effect of added glucose and strongly inhibited DNA synthesis. 4. It is suggested that the major function of enhanced aerobic glycolysis in transforming lymphocytes is to maintain higher steady-state amounts of glycolytic intermediates to act as precursors for macromolecule synthesis.     

Amino acid oxidation and alanine production in rat hemidiaphragm in vitro. Effects of dichloroacetate.

Dichloroacetate (an activator of pyruvate dehydrogenase) stimulates 14CO2 production from [U-14C]glucose, but not from [U-14C]glutamate, [U-14C]aspartate, [U-14C]- and [1-14C]-valine and [U-14C]- and [1-14C]-leucine. It is concluded (1) that pyruvate dehydrogenase is not rate-limiting in the oxidation to CO2 of amino acids that are metabolized to tricarboxylic acid-cycle intermediates, and (2) that carbohydrate (and not amino acids) is the main carbon precursor in alanine formation in muscle.     

Oxidation of glucose, ribose, alanine, and glutamate by Leishmania braziliensis panamensis

The metabolism of [1-14C]- and [6-14C]glucose, [1-14C]ribose, [1-14C]- and [U-14C]alanine, and [1-14C]- and [5-14C]glutamate by the promastigotes of Leishmania braziliensis panamensis was investigated in cells resuspended in Hanks' balanced salt solution supplemented with ribose, alanine, or glutamate. The ratio of 14CO2 produced from [1-14C]glucose to that from [6-14C]glucose ranged from about two to six, indicating appreciable carbon flow through the pentose phosphate pathway. A functional pentose phosphate pathway was further demonstrated by the production of 14CO2 from [1-14C]ribose although the rate of ribose oxidation was much lower than the rate of glucose oxidation. The rate of 14CO2 production from [1-14C]glucose was almost linear with time of incubation, whereas that of [6-14C]glucose accelerated, consistent with an increasing rate of flux through the Embden-Meyerhof pathway during incubation. Increasing the assay temperature from 26 degrees C to 34 degrees C had no appreciable effect on the rates or time courses of oxidation of either [1-14C]- or [6-14C]glucose or of [1-14C]ribose. Both alanine and glutamate were oxidized by L. b. panamensis, and at rates comparable to or appreciably greater than the rate of oxidation of glucose. The ratios of 14CO2 produced from [1-14C]- to [U-14C]alanine and from [1-14C]- to [5-14C]glutamate indicated that these compounds were metabolized via a functioning tricarboxylic acid cycle and that most of the label that entered the tricarboxylic acid cycle was oxidized to carbon dioxide.(ABSTRACT TRUNCATED AT 250 WORDS)     

Transport and metabolism of acetate in rat brain cortex in vitro

1. [1-(14)C]Acetate undergoes metabolism when incubated aerobically at 37 degrees in the presence of rat brain-cortex slices, forming (14)CO(2) and (14)C-labelled amino acids (glutamate, glutamine, aspartate and relatively small quantities of gamma-aminobutyrate). In the absence of glucose the yield of (14)C-labelled aspartate exceeds that of (14)C-labelled glutamate and glutamine. The addition of glucose brings about a doubling of the rate of formation of (14)CO(2) and a greatly increased yield of (14)C-labelled glutamate or glutamine, whereas that of (14)C-labelled aspartate is diminished. 2. The addition of potassium chloride (100mm) to the incubation medium causes an increased rate of (14)CO(2) formation in the presence or absence of glucose and an increased rate of utilization of acetate. 3. The addition of 2,4-dinitrophenol (0.1mm) suppresses the rate of utilization of [1-(14)C]acetate. 4. The presence of ouabain (10mum) suppresses the rate of formation of (14)CO(2) from [1-(14)C]acetate and the rate of acetate utilization. Acetate conversion into carbon dioxide in the rat brain cortex is both Na(+)- and K(+)-dependent and controlled by operation of the active sodium-transport process. Only the Na(+)-stimulated rate is suppressed by ouabain. 5. Sodium fluoroacetate (1mm) decreases the rate of (14)CO(2) evolution from [1-(14)C]acetate in the presence of rat brain cortex without affecting the respiratory rate. The results are consistent with the conclusion that fluoroacetate competes with, or blocks, a transport carrier for acetate, so that in its presence only the passive diffusion rate of acetate takes place. 6. The presence of sodium propionate or sodium butyrate suppresses the utilization of [1-(14)C]acetate in rat brain cortex and leads to a concentration ratio (tissue/medium) of [1-(14)C]-acetate greater than unity. 7. The presence of NH(4) (+) diminishes acetate utilization, this being attributed to a diminished ATP concentration. Glycine is also inhibitory. It is concluded that acetate transport into the brain is carrier-mediated and dependent on the operation of the sodium pump.     

Hepatocyte heterogeneity in glutamate uptake by isolated perfused rat liver

Glutamate is simultaneously taken up and released by perfused rat liver, as shown by 14CO2 production from [1-14C]glutamate in the presence of a net glutamate release by the liver, turning to a net glutamate uptake at portal glutamate concentrations above 0.3 mM. 14CO2 production from portal [1-14C]glutamate is decreased by about 60% in the presence of ammonium ions. This effect is not observed during inhibition of glutamine synthetase by methionine sulfoximine. 14CO2 production from [1-14C]glutamate is not influenced by glutamine. Also, when glutamate accumulates intracellularly during the metabolism of glutamine (added at high concentrations, 5 mM), 14CO2 production from [1-14C]glutamate is not affected. If labeled glutamate is generated intracellularly from added [U-14C]proline, stimulation of glutamine synthesis by ammonium ions did not affect 14CO2 production from [U-14C]proline. After induction of a perivenous liver cell necrosis by CCL4, i.e. conditions associated with an almost complete loss of perivenous glutamine synthesis but no effect on periportal urea synthesis, 14CO2 production from [1-14C]glutamate is decreased by about 70%. The results are explained by hepatocyte heterogeneity in glutamate metabolism and indicate a predominant uptake of glutamate (that reaches the liver by the vena portae) by the small perivenous population of glutamine-synthesizing hepatocytes, whereas glutamate production from glutamine or proline is predominantly periportal. In view of the size of the glutamine synthetase-containing hepatocyte pool [Gebhardt, R. and Mecke, D. (1983) EMBO J. 2, 567-570], glutamate transport capacity of these hepatocytes would be about 20-fold higher as compared to other hepatocytes.     

Effects of Ammonia and β-Methylene-dl-Aspartate on the Oxidation of Glucose and Pyruvate by Neurons and Astrocytes in Primary Culture

Both ammonia and beta-methylene-DL-aspartate (beta-MA), an irreversible inhibitor of aspartate aminotransferase activity and thus of the malate-aspartate shuttle, were found previously to decrease oxidative metabolism in cerebral cortex slices. In the present work, the possibility that ammonia and beta-MA affect energy metabolism by a common mechanism (i.e., via inhibition of the malate-aspartate shuttle) was investigated using primary cultures of neurons and astrocytes. Incubation of astrocytes for 30 min with 5 mM beta-MA resulted in a decreased production of 14CO2 from [U-14C]glucose, but did not affect 14CO2 production from [2-14C]pyruvate. Conversely, incubation of astrocytes with 3 mM ammonium chloride resulted in decreased 14CO2 production from [2-14C]pyruvate, but 14CO2 production from [U-14C]glucose was not significantly affected. Ammonium chloride had no significant effect on 14CO2 production from either [U-14C]glucose or [2-14]pyruvate by neurons. However, incubation of neurons with beta-MA or beta-MA plus ammonium chloride resulted in a approximately 45% decrease of 14CO2 production from both [U-14C]glucose and [2-14C]pyruvate. A 2-h incubation of astrocytes with beta-MA resulted in no change in ATP levels, but a 35% decrease in phosphocreatine. Similar treatment of neurons resulted in greater than 50% decrease in ATP, but had little effect on phosphocreatine. beta-MA also caused a decrease in glutamate and aspartate content of neurons, but not of astrocytes. The different metabolic responses of neurons and astrocytes towards beta-MA were probably not due to a differential inhibition of aspartate aminotransferase which was inhibited by approximately 45% in astrocytes and by approximately 55% in neurons.     

Comparison between transport and degradation of leucine and glutamine by peripheral human lymphocytes exposed to concanavalin A

Transport and pathways of leucine and glutamine degradation were evaluated in resting human peripheral lymphocytes and compared with the changes induced by concanavalin A (ConA). Cells were incubated with [1-14C]leucine (0.15 mM), [U-14C]leucine (0.15 mM), or [U-14C]glutamine (0.4 mM) after culture with or without 2, 5, 7, or 10 micrograms/ml ConA for 2, 18, or 24 hours, respectively. Initial rates of transport of leucine and glutamine were augmented 2.7-fold and threefold by the mitogen. Leucine transamination, irreversible oxidation, and catabolism beyond isovaleryl-CoA were increased by 90%, 20%, and 60%, respectively. Glutamine utilization increased threefold; accumulation of glutamate, aspartate, and ammonia increased by 700%, 50%, and 100%, respectively, and 14CO2 production by about 400% in response to ConA. The results indicate that ConA stimulates to about the same extent transport of leucine and glutamine into lymphocytes. Glutamine is mainly channeled into catabolic pathways, while leucine remains largely preserved. It is suggested that these metabolic changes provide more leucine for incorporation into protein and more N- and C-atoms required for the synthesis of macromolecules and energy from glutamine.     

Glucose and glutamine metabolism in rat macrophages: enhanced glycolysis and unaltered glutaminolysis in spontaneously diabetic BB rats.

Metabolism of glutamine (Gln, 2 mM) and glucose (5 mM) was studied in vitro in isolated resident peritoneal macrophages from both normal (BBn) and spontaneously diabetic BB (BBd) rats. The major products from Gln were ammonia, glutamate, CO2 and to a lesser extent aspartate. Glucose decreased (P less than 0.01) the production of ammonia, CO2 and aspartate from Gln by 34-60%, but had no effect on the amount of glutamate accumulated. The major products from glucose were lactate and to a much lesser extent pyruvate and CO2. Gln decreased (P less than 0.01) 14CO2 production from [U-14C]glucose by 19-28%, increased (P less than 0.01) pyruvate production by 35-49%, but had no effect on lactate production. The fraction of glucose metabolized via the pentose phosphate pathway (PC) was less than 5%. There were no significant differences in Gln metabolism between BBn and BBd macrophages. The production of lactate and pyruvate and the flux from glucose into the PC were increased (P less than 0.01) by 2.4, 1.8 and 1.5-fold, respectively, in BBd cells. Increased macrophage glucose metabolism was also observed in diabetes-prone BB (BBdp) rats at 75-80 days but not at 50 days of age. In the presence of both Gln and glucose, potential ATP production from glucose was 2- and 4-times that from Gln, respectively, in BBn and BBd cells. Lactate production was the major pathway for glucose-derived ATP generation. These results demonstrate (a) glycolysis and flux from glucose through the pentose phosphate pathway are enhanced with no alteration in glutaminolysis in BBd macrophages; and (b) glucose may be a more important fuel than Gln for macrophages, particularly in BBd rats. The increased glucose metabolism may be associated with functional activation of the macrophages that have been proposed to be involved in beta-cell destruction and the development of diabetes.     

Quantification of carbon fluxes through the tricarboxylic acid cycle in early germinating lettuce embryos

A method involving labeling to isotopic steady state and modeling of the tricarboxylic acid cycle has been used to identify the respiratory substrates in lettuce embryos during the early steps of germination. We have compared the specific radioactivities of aspartate and glutamate and of glutamate C-1 and C-5 after labeling with different substrates. Labeling with [U-14C]acetate and 14CO2 was used to verify the validity of the model for this study; the relative labeling of aspartate and glutamate was that expected from the normal operation of the tricarboxylic acid cycle. After labeling with 14CO2, the label distribution in the glutamate molecule (95% of the label at glutamate C-1) was consistent with an input of carbon via the phosphoenolpyruvate carboxylase reaction, and the relative specific radioactivities of aspartate and glutamate permitted the quantification of the apparent rate of the fumarase reaction. CO2 and intermediates related to the tricarboxylic acid cycle were labeled with [U-14C]acetate, [1-14C] hexanoate, or [U-14C]palmitic acid. The ratios of specific radioactivities of asparate to glutamate and of glutamate C-1 to C-5 indicated that the fatty acids were degraded to acetyl units, suggesting the operation of beta-oxidation, and that the acety-CoA was incorporated directly into citrate. Short-term labeling with [1-14C]hexanoate showed that citrate and glutamate were labeled earlier than malate and aspartate, showing that this fatty acid was metabolized through the tricarboxylic acid cycle rather than the glyoxylate cycle. This was in agreement with the flux into gluconeogenesis compared to efflux as respiratory CO2. The fraction of labeled substrate incorporated into carbohydrates was only about 5% of that converted to CO2; the carbon flux into gluconeogenesis was determined after labeling with 14CO2 and [1-14C]hexanoate from the specific radioactivity of aspartate C-1 and the amount of label incorporated into the carbohydrate fraction. It was only 7.4% of the efflux of respiratory CO2. The labeling of alanine indicates a low activity of either a malic enzyme or the sequence phosphoenolpyruvate carboxykinase/pyruvate kinase. After labeling with [U-14C]glucose, the ratios of specific radioactivities indicated that the labeled carbohydrates contributed less than 10% to the flux of acetyl-CoA. The model indicated that the glycolytic flux is partitioned one-third to pyruvate and two-thirds to oxalacetate and is therefore mainly anaplerotic. The possible role of fatty acids as the main source of acetyl-CoA for respiration is discussed.     

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.     

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.