Glutamate metabolism in a human intestinal epithelial cell layer model

Plasma glutamate concentrations are constant despite dynamic changes in diets. Most likely, virtually all the dietary glutamate is metabolized in the gut. The present study investigated permeability and metabolism of dietary glutamate in a Caco-2 intestinal epithelial cell layer model by tracing the fate of [U-¹³C] or [¹⁵N]glutamate added to the apical medium. For comparison, several other labelled essential and non-essential amino acids were tested as well. Almost all the labelled glutamate in the apical medium (98% and 96% at 24 h of the culture, respectively) was incorporated in the cell layer, while it barely appeared at the basolateral side, indicating an almost complete utilization of glutamate. Indeed, the ¹³C was incorporated into alanine, proline, ornithine, and glutamine, and the ¹⁵N was incorporated into alanine, glutamine, ornithine, proline, branched chain amino acids and also found as ammonia indicative of oxidation. In contrast, substantial apical-to-basolateral transport of amino acids (8–85% of uptake) other than glutamate and aspartate was evident in studies using amino acid tracers labelled with ¹³C, ¹⁵N or D. These results suggest that the intestinal epithelial cell monolayer utilizes dietary glutamate which adds to maintaining glutamate homeostasis in the body.

     

Glutamine limited fed-batch culture reduces the overflow metabolism of amino acids in myeloma cells

The murine myeloma cell line Sp 2/0-Ag 14 was cultured in an ordinary batch culture and in a glutamine limited fed-batch culture. In batch culture, the overflow metabolism of glutamine ends in excess production of ammonium and the amino acids alanine, proline, ornithine, asparagine, glutamate, serine and glycine. This pattern was dramatically changed in the fed-batch culture. Glutamine limitation halved the cellular ammonium production and reduced the ratio of NH₄ ⁺/glutamine. The excess production of alanine, proline and ornithine was reduced by a factor of 2–6 while asparagine was not produced at all. In contrary to the other amino acids glycine production was increased. These results are discussed in view of the different nature of glutamine metabolism in the mitochondrial compartment vs. the cytosolic. Furthermore, essential amino acids were used more efficiently in the fed-batch as judged by the increase in the cellular yield coefficients in the range of 1.3–2.6 times for seven of the 11 consumed ones. In all, this leads to a more efficient use of the energy sources glucose and glutamine as revealed by an increase in the cellular yield coefficient for glucose by 70% and for glutamine by 61%.     

Growth of Certain Aquatic Oomycetes on Amino Acids II. Apodachlya, Aphanomyces, and Pythium

Four aquatic fungi —Apodachlya brachynema and A. minima (Leptomitales), Aphanomyces laevis (Saprolegniales), and Pythium ultimum (Peronosporales) —were tested for growth in synthetic media containing one of a variety of carbon sources. Apodachlya brachynema readily utilized five amino acids — alanine, glutamate, aspartate, proline and leucine — as well as glucose and acetate. Growth on sucrose as a carbon source was slight. Apodachlya minima differed from A. brachynema in that it could not utilize proline and leucine. Aphanomyces laevis grew well on only three of the substrates tested — glucose, alanine and glutamate. Pythium ultimum utilized glucose, sucrose, maltose, cellobiose, alanine, glutamate, aspartate, proline, asparagine, ornithine, and serine, but not eight other amino acids. All of these fungi hydrolyzed gelatin. Radioactively labeled carbon dioxide was released during incubation of Aphanomyces laevis in media containing labeled leucine, proline, or phenylalanine. These data provide evidence of some catabolism of the three substrates although none of these substrates can support the growth of Aphanomyces laevis as a sole source of carbon and nitrogen.     

Role of the global regulator Rex in control of NAD+‐regeneration in Clostridioides (Clostridium) difficile

For the human pathogen Clostridioides (also known as Clostridium) difficile, the ability to adapt to nutrient availability is critical for its proliferation and production of toxins during infection. Synthesis of the toxins is regulated by the availability of certain carbon sources, fermentation products and amino acids (e.g. proline, cysteine, isoleucine, leucine and valine). The effect of proline is attributable at least in part to its role as an inducer and substrate of D‐proline reductase (PR), a Stickland reaction that regenerates NAD⁺ from NADH. Many Clostridium spp. use Stickland metabolism (co‐fermentation of pairs of amino acids) to generate ATP and NAD⁺. Synthesis of PR is activated by PrdR, a proline‐responsive regulatory protein. Here we report that PrdR, in the presence of proline, represses other NAD⁺‐generating pathways, such as the glycine reductase and succinate‐acetyl CoA utilization pathways leading to butyrate production, but does so indirectly by affecting the activity of Rex, a global redox‐sensing regulator that responds to the NAD⁺/NADH ratio. Our results indicate that PR activity is the favored mechanism for NAD⁺ regeneration and that both Rex and PrdR influence toxin production. Using the hamster model of C. difficile infection, we revealed the importance of PrdR‐regulated Stickland metabolism in the virulence of C. difficile.     

Regulation of leucine catabolism by metabolic fuels in mammary epithelial cells

Lactation is associated with elevated catabolism of branched-chain amino acids (BCAA) in mammary glands to produce glutamate, glutamine, alanine, aspartate, and asparagine. This study determined effects of metabolic fuels on the catabolism of leucine (a representative BCAA) in bovine mammary epithelial cells. Cells were incubated at 37 °C for 2 h in Krebs buffer containing 0.5 mM l-leucine and either l-[1-¹⁴C]leucine or l-[U-¹⁴C]leucine. The medium also contained 0–5 mM d-glucose, 0–2 mM l-glutamine, 0–4 mM dl-β-hydroxybutyrate, or 0–2 mM oleic acid. Rates of leucine decarboxylation were 60 % lower, but rates of α-ketoisocaproate production were 34 % higher, in the presence of 2 mM glucose than in its absence. All variables of leucine catabolism did not differ between 2 and 5 mM glucose or between 0 and 4 mM dl-β-hydroxybutyrate. Compared with 0–0.25 mM glutamine, 0.5 and 2 mM l-glutamine reduced leucine transport, transamination, and decarboxylation. In contrast, increasing the concentration of oleic acid from 0 to 2 mM dose-dependently stimulated leucine transamination, decarboxylation, and oxidation of carbons 2–6. Oleic acid also enhanced the abundance of cytosolic BCAA transaminase, while reducing the phosphorylated level (inactive state) of the E1α subunit of the mitochondrial branched-chain α-ketoacid dehydrogenase complex. Thus, hypoglycemia or ketosis in early lactation does not likely affect BCAA metabolism in mammary epithelial cells. Increasing circulating levels of BCAA and oleic acid may have great potential to increase the syntheses of glutamate, glutamine, aspartate, alanine, and asparagine by lactating mammary glands, thereby leading to enhanced production of milk for suckling neonates.     

Fatty acids and glycerol or lactate are required to induce gluconeogenesis from alanine in isolated rabbit renal cortical tubules

Summary In isolated rabbit renal cortical tubules, glucose synthesis from 1 mM alanine is negligible, while the amino acid is metabolized to glutamine and glutamate. The addition of 0.5 mM octanoate plus 2 mM glycerol induces incorporation of [U-¹⁴C]Alnine into glucose and decreases glutamine synthesis, whereas oleate and palmitate in the presence of glycerol are less potent than octanoate. Gluconeogenesis is also significantly accelerated when glycerol is substituted by lactate. In view of an increase in¹⁴CO₂ fixation and elevation of both cytosolic and mitochondrial NADH/NAD⁺ ratios, the activation of glucose formation from alanine upon the addition of glycerol and octanoate is likely due to (i) stimulation of pyruvate carboxylation, (ii) increased availability of NADH for glyceraldehyde-3-phosphate dehydrogenase and (iii) elevation of mitochondrial redox state causing a diminished provision of ammonium for glutamine synthesis. The induction of gluconeogenesis in the presence of alanine, glycerol and octanoate is not related to cell volume changes. The results presented in this paper show the importance of free fatty acids and glycerol for regulation of renal gluconeogenesis from alanine. The possible physiological significance of the data is discussed.     

l-proline dehydrogenase of Triticum vulgare germ: Purification,properties and cofactor interactions

The enzyme catalysing the l-proline-dependent reduction of NAD⁺has been purified over 600-fold from wheat germ acetone powder extracts. l-Proline, 3,4 dehydro-dl-proline, thiazolidine-4-carboxylate were the only substrates utilized readily. The K_m for l-proline was 1·0 mM and for NAD⁺ 0·8 mM. The enzyme was highly specific for NAD⁺ with NADP⁺ and NADPH acting as effective competitive inhibitors with a K_i of 1·8 and 0·4 μM, respectively. All ribonucleoside triphosphates tested were good non-competitive inhibitors, in particular UTP. The purified enzyme could reduce pyrroline-5-carboxylate, either chemically synthesized or generated in a linked assay system from ornithine by a highly-purified ornithine transaminase. In the latter case both NADH and NADPH were utilized equally well as the reductant. With chemically synthesized dl-pyrroline-5-carboxy-late as the substrate. NADPH was used at only 25% the rate of NADH, and NADPH strongly inhibited the oxidation of NADH.     

The kinetics of inhibition of Vigna catjang cotyledon and buffalo liver arginase by L-proline and branched-chain amino acids

The effect of proline, isoleucine, leucine, valine, lysine and ornithine under standard physiological conditions, on purified Vigna catjang cotyledon and buffalo liver arginases was studied. The results showed that V. catjang cotyledon arginase is inhibited by proline at a lower concentration than buffalo liver arginase and the inhibition was found to be linear competitive for both enzymes. Buffalo liver arginase was more sensitive to inhibition by branched-chain amino acids than V. catjang cotyledon. Leucine, lysine, ornithine and valine are competitive inhibitors while isoleucine is a mixed type of inhibitor of liver arginase. We have also studied the effect of manganese concentration which acts as a cofactor and leads to activation of arginase. The optimum Mn^{2 +} concentration for Vigna catjang cotyledon arginase is 0.6 mM and liver arginase is 2.0 mM. The preincubation period required for liver arginase is 20 min at 55°C, the preincubation period and temperature required for activation of cotyledon arginase was found to be 8 min at 35°C. The function of cotyledon arginase in polyamine biosynthesis and a possible role of branched chain amino acids in hydrolysis of arginine in liver are discussed.     

Inhibition of gluconeogenesis from proline by ethanol oxidation

The effect of ethanol oxidation on proline metabolism in the perfused rat liver was studied. Ethanol oxidation inhibited proline consumption by about 80%, glucose production by 92% and urea formation by 60%. The mechanism in the [NADH]/[NAD⁺]-ratio.     

Alanine metabolism in skeletal muscle in tissue culture.

Alanine production by skeletal muscle in tissue culture was studied using an established myogenic line (L6) of rat skeletal muscle cells. Correlation analyses were performed on rates of metabolism of alanine, glucose, lactate and pyruvate over incubation periods up to 96 h. Alanine production did not correlate significantly with glucose utilization (r = 0.24, P less than 0.20). Alanine production, however, did correlate with lactate production (r = 0.72, P less than 0.0005) as well as medium (r = 0.50, P less than 0.025) and intracellular (r = 0.85, P less than 0.0005) pyruvate concentrations. The intercepts of the latter two correlation analyses indicated that when medium or cell pyruvate fell below 0.28 mM or 1 nmol/mg protein, respectively, net alanine consumption occurred. Alanine synthesis also correlated (r = 0.71, P less than 0.0005) with the percent change in the cell mass action ratio for the sum of the alanine and aspartate aminotransferase reactions, i.e., [alanine] [malate]/[aspartate] [lactate]. These results suggest that alanine production is not necessarily linked to the rate of glucose utilization but rater to pyruvate overflow above a critical intracellular level; under conditions of pyruvate overflow, alanine synthesis is driven by the tendency to establish equilibrium between metabolites of the linked amino acid transaminases in skeletal muscle.     

Production of nadh by microbial reduction of added nad

Summary Of thirteen bacterial strains and four strains of yeast-like organisms, permeabilized cells of two bacterial and one yeast strain effectively converted added NAD⁺ into NADH in the presence of glucose as substrate.Arthrobacter ureafaciens reduced more than 90% of 10 mM NAD⁺ into NADH during 1h.     

Nitrogen metabolism in tumor bearing mice

In experiments with whole animals infested with a highly malignant strain of Ehrlich ascites tumor cells, serial concentrations of amino acids were determined for host plasma, ascitic fluid, and tumor cells, throughout tumor development. Concentration gradients of glutamine, asparagine, valine, leucine, isoleucine, phenylalanine, tyrosine, histidine, tryptophan, arginine, serine, methionine, and taurine from the host plasma toward the ascitic liquid were established; while on the other hand, concentration gradients from the ascitic liquid toward the plasma were established for glutamate, aspartate, glycine, alanine, proline, and threonine. With the exception of aspartate the concentrations of these amino acids were highest inside the cells. Arginine was the only amino acid not detected in tumor cells. In vitro incubations of tumor cells in the presence of glutamine and/or glucose, as the energy and nitrogen sources, confirmed the amino acid fluxes previously deduced from the observed relative concentrations of amino acids in plasma, ascitic liquid, and tumor cells, suggesting that glutamate, alanine, aspartate, glycine, and serine can be produced by tumors. These findings support that changes in amino acid patterns occurring in the host system are related to tumor development.     

Amino acid synthesis from glucose-U-14C in Glossina morsitans

Amino acid synthesis from glucose-U-¹⁴C was investigated in 2 day post-emergent and pregnant females of Glossina morsitans. This insect can synthesize alanine, aspartic acid, cystine, glutamic acid, glycine, proline, and serine from glucose. Arginine, histidine, hydroxyproline, isoleucine, leucine, lysine, methionine, phenylalanine, taurine, threonine, and valine showed no radioactivity and hence may be classified as nutritionally indispensable amino acids. Although tyrosine and hydroxyproline were not synthesized from glucose, they are at least partially dispensable nutrients for this insect because their synthesis from phenylalanine has been demonstrated. After the labelled glucose injection the highest radioactivity was recovered in the proline fraction. This is probably related to its rôle as an important energy reserve for flight. The radioactive amino acids recovered from females and from their offspring following glucose-U-¹⁴C injection were similar to those recovered from younger females. Radioactivity was also detected in the expired CO₂ and the excreta. The amino acids alanine, arginine, cystine, glycine, histidine, leucine/isoleucine, lysine, methionine, proline, and valine were identified in the excreta, of which arginine and histidine were in the largest amounts. Only excreted alanine, glycine, and proline showed radioactivity.     

A combination of intracellular leucine with either glutamate or aspartate inhibits autophagic proteolysis in isolated rat hepatocytes

It has been shown previously that the inhibition of autophagic proteolysis in liver by a physiological mixture of amino acids can be mimicked completely by addition of leucine in combination with alanine [Leverve, X. M., Caro, L. H. P., Plomp, P. J. A. M. and Meijer, A. J. (1987) FEBS Lett. 219, 455-458]. We have now further defined conditions which lead to this inhibition. Isolated rat hepatocytes were incubated in the perifusion system in which the cells can be maintained at a steady state in the presence of low amino acid concentrations. Combinations of leucine (0.5 mM) with either alanine, glutamine, asparagine or proline (2 mM) inhibited proteolysis by 40-50%. Under these conditions, both in the absence and presence of the transaminase inhibitor, aminooxyacetate, a correlation was found between the extent of inhibition of proteolysis and the sum of the total intracellular amounts of aspartate and glutamate. Inhibition of proteolysis by leucine and leucine analogues did not correlate with their ability to activate glutamate dehydrogenase.     

Content of amino acids in axons from the CNS of the lobster

The contents of alanine, proline, glycine, GABA, glutamate, and aspartate were measured in four bundles of axons (designated areas A through D) from the circumesophageal connective of the lobster (Homarus americanus). The contents of these amino acids were also determined in individual axons within specific bundles and in the external sheath covering the circumesophageal connective. Within the nerve bundles the levels of aspartate were highest of the amino acids measured, ranging from 1.95 ± 0.12 m̈mol/mg protein in area C to 7.55 ± 0.54 m̈mol/mg protein in area B. On the other hand, GABA had the lowest value in the four bundles; its highest level was found in area C (0.083 ± 0.006 m̈mol/mg protein) and the lowest in area B (none detected). The content of glycine ranged from 1.63 ± 0.14 (area C) to 2.52 ± 0.32 m̈mol/mg protein in area A; that for glutamate ranged from 0.390 ± 0.019 (area C) to 1.01 ± 0.103 (area B). The contents of alanine and proline changed relatively little from bundle-to-bundle. The content of aspartate was the highest of any of the amino acids assayed in individual axons (with diameters in the range of 40 to 65 m̈) dissected from areas B and C. Glycine had the next highest content followed in order by glutamate, proline, and alanine. GABA was not detected in these axons. With the exception of GABA (which could not be detected), aspartate had the lowest level (0.066 ± 0.017) and glycine had the highest level (2.00 ± 0.498 m̈mol/mg protein) in the external sheath covering the the circumesophageal connective.     

Regulation of amino acid transport in L6 muscle cells: I. Stimulation of transport system a by amino acid deprivation

The regulation of amino acid transport in L6 muscle cells by amino acid deprivation was investigated. Proline uptake was Na⁺-dependent, saturable and concentrative, and was predominantly through system A. Proline uptake was inhibited by alanine, α-amino isobutyric acid (AIB), and by α-methylamino isobutyric acid, but not by lysine or valine. At 25°C, Km of proline uptake was 0.5 mM. Amino acid-deprivation resulted in a progressive increase in the rate of proline uptake, reaching up to 6-fold stimulation after 6 hours. The basal and stimulated transport were equally Na⁺-dependent, and both were inhibited by competition with the same amino acids. Kinetic analysis showed that Km decreased by a factor of 2.4 and Vmax increased 1.9-fold in deprived cells. Amino acid-deprivation did not stimulate amino acid uptake through systems other than system A. This suggests that the higher Km in proline-supplemented cells is not due to release of intracellular amino acids into unstirred layers surrounding the cells. The presence of amino acids which are substrates of system A (including AIB) during proline-deprivation, prevented stimulation of proline uptake, whereas those transported by systems Ly⁺ or L exclusively were ineffective. The stimulation of the transport-rate in deprived cells could be reversed by subsequent exposure to proline or other substrates of system A. L6 cells, deprived of proline for 6 hours, retained the stimulation of transport after detachment from the monolayers with trypsin. Uptake rates were comparable in suspended and attached cells in monolayer culture. Thus, amino acid-depreivation of L6 cells results in an adaptive increase in proline uptake, which is not due to unstirred layers but appears to be mediated by other mechanisms of selective transport regulation.     

Free amino acids and reducing sugars in the freshwater shrimp <Emphasis Type="Italic">Gammarus lacustris</Emphasis> (Crustacea,Amphipoda) at the initial stage of preparation to winter season

A peculiarity of amino acid profile of the cold-resistant freshwater shrimp Gammarus lacustris in the beginning of autumn is a high level of ornithine, the second after alanine (17.0 ± 1.4 and 22.6 ± 1.4 μmol/ml of homogenate, respectively). The amount of alanine and ornithine accounts for 39.8% of the total pool of free amino acids and together with lysine and leucine—55%. Free glucose is the major reducing sugar (6.42 ± 0.78 μmol/ml).     

Comparative study of extracellular amino acids in culture of human liver and fibroblastic cells

Summary Amino acid concentrations are studied in the extracellular media of ten series of human fibroblast and liver cell monolayer cultures. These two cell types consume and produce ostensively the same amino acids. Among the nonessential amino acids, the most significant variations involve serine and aspartate which are decreased; α-alanine, glutamate, ornithine and proline are, on the contrary, increased. Among the essential amino acids, leucine, isoleucine and glutamine are preferentially decreased. The variations of some amino acids are correlated with the cell density. The interrelations which may exist between the variations of these different amino acids are discussed. Furthermore, the glycolytic activity of the cells studied is very high: 85% of glucose consumed is found in the form of lactate. Unité d’Hépatologie Infantile I.N.S.E.R.M. (U56) Laboratoire Central de Biochimie This work was supported by grant: INSERM CRL 73-5-057-04 and 74-5-075-04.     

Alanine metabolism in skeletal muscle in tissue culture

Alanine production by skeletal muscle in tissue culture was studied using an established myogenic line (L₆) of rat skeletal muscle cells. Correlation analyses were performed on rates of metabolism of alanine, glucose, lactate and pyruvate over incubation periods up to 96 h. Alanine production did not correlate significantly with glucose utilization (r = 0.24, P < 0.20). Alanine production, however, did correlate with lactate production (r = 0.72, P < 0.0005) as well as medium (r = 0.50, P < 0.025) and intracellular (r = 0.85, P < 0.0005) pyruvate concentrations. The intercepts of the latter two correlation analyses indicated that when medium or cell pyruvate fell below 0.28 mM or 1 nmol/mg protein, respectively, net alanine consumption occurred. Alanine synthesis also correlated (r = 0.71, P < 0.0005) with the percent change in the cell mass action ratio for the sum of the alanine and aspartate aminotransferase reactions, i.e., [alanine] [malate]/[aspartate] [lactate]. These results suggest that alanine production is not necessarily linked to the rate of glucose utilization but rather to pyruvate overflow above a critical intracellular level; under conditions of pyruvate overflow, alanine synthesis is driven by the tendency to establish equilibrium between metabolites of the linked amino acid transaminases in skeletal muscle.