Metabolic effects of pent-4-enoate in isolated perfused rat heart.

The metabolic effects of the hypoglycaemic agent pent-4-enoate were studied in isolated, beating or potassium-arrested rat hearts. The addition of 0.8mM-pent-4-enoate to the perfusion fluid increased O2 consumption by 76% in the arrested heart and by 14% in the beating heart; the concentration ratio of phosphocreatine/creatine increase concomitantly by 47% and 27% respectively. Perfusion of the heart with pent-4-enoate resulted in a 30-fold increase in the concentration of the pool of tricarboxylic acid-cycle intermediates in the tissue, about 90% of this increase being due to malate. The sum of the concentrations of the myocardial free amino acids remained virtually unchanged during the accumulation of the tricarboxylic acid-cycle intermediates. It was concluded that pent-4-enoate can be effectively metabolized in the myocardium and that its metabolism probably proceeds via propionyl-CoA, since pent-4-enoate reproduces many of the metabolic characteristics of propionate in the cardiac muscle. The accumulation of the tricarboxylic acid-cycle intermediates is probably due to carboxylation of propionyl-CoA. The response pattern of the metabolite concentrations in the cardiac muscle is quite different from that in the liver, in which decrease of the concentrations of the tricarboxylic acid-cycle intermediates has been observed previously [Williamson, Rostand & Peterson (1970) J. Biol. Chem. 245, 3242-3251].     

Pyruvate carboxylation as an anaplerotic mechanism in the isolated perfused rat heart.

1. The role of pyruvate carboxylation in the net synthesis of tricarboxylic acid-cycle intermediates during acetate metabolism was studied in isolated rat hearts perfused with [1-14C]pyruvate. 2. The incorporation of the 14C label from [1-14C]pyruvate into the tricarboxylic acid-cycle intermediates points to a carbon input from pyruvate via enzymes in addition to pyruvate dehydrogenase and citrate synthase. 3. On addition of acetate, the specific radioactivity of citrate showed an initial maximum at 2 min, with a subsequent decline in labelling. The C-6 of citrate (which is removed in the isocitrate dehydrogenase reaction) and the remainder of the molecule showed differential labelling kinetics, the specific radioactivity of C-6 declining more rapidly. Since this carbon is lost in the isocitrate dehydrogenase reaction, the results are consistent with a rapid inactivation of pyruvate dehydrogenase after the addition of acetate, which was confirmed by measuring the 14CO2 production from [1-14C]pyruvate. 4. The results can be interpreted to show that carboxylation of pyruvate to the C4 compounds of the tricarboxylic acid cycle occurs under conditions necessitating anaplerosis in rat myocardium, although the results do not identify the enzyme involved. 5. The specific radioactivity of tissue lactate was too low to allow it to be used as an indicator of the specific radioactivity of the intracellular pyruvate pool. The specific radioactivity of alanine was three times that of lactate. When the hearts were perfused with [1-14C]lactate, the specific radioactivity of alanine was 70% of that of pyruvate. The results suggest that a subcompartmentation of lactate and pyruvate occurs in the cytosol.     

Metabolism of propionate by sheep liver: Pathway of propionate metabolism in aged homogenate and mitochondria

Experiments were conducted with aged nuclear-free homogenate of sheep liver and aged mitochondria in an attempt to measure both the extent of oxidation of propionate and the distribution of label from [2-¹⁴C]propionate in the products. With nuclear-free homogenate, propionate was 44% oxidized with the accumulation of succinate, fumarate, malate and some citrate. Recovery of ¹⁴C in these intermediates and respiratory carbon dioxide was only 33%, but additional label was detected in endogenous glutamate and aspartate. With washed mitochondria 30% oxidation of metabolized propionate occurred, and proportionately more citrate and malate accumulated. Recovery of ¹⁴C in dicarboxylic acids, citrate, α-oxoglutarate, glutamate, aspartate and respiratory carbon dioxide was 91%. The specific activities of the products and the distribution of label in the carbon atoms of the dicarboxylic acids were consistent with the operation solely of the methylmalonate pathway together with limited oxidation of the succinate formed by the tricarboxylic acid cycle via pyruvate. In a final experiment with mitochondria the label consumed from [2-¹⁴C]propionate was entirely recovered in the intermediates of the tricarboxylic acid cycle, glutamate, aspartate, methylmalonate and respiratory carbon dioxide.     

13C-NMR study of acetate assimilation in Thermoproteus neutrophilus

Acetate assimilation into amino acids and the functioning of central biosynthetic pathways in the extremely thermophilic anaerobic archaebacterium Thermoproteus neutrophilus was investigated using 13C NMR as the method for determination of the labelling patterns. Acetate was assimilated via reductive carboxylation of acetyl-CoA to pyruvate and pyruvate conversion to phosphoenolpyruvate which was further carboxylated to oxaloacetate. 2-Oxoglutarate was mainly formed via citrate. However, the labelling patterns of glutamic acid and alanine were in agreement with the concurrent synthesis of about 15% 2-oxoglutarate and 5% pyruvate through the reductive citric acid cycle. A scrambling phenomenon occurring in aspartate and all amino acids derived through oxaloacetate was observed. The labelling patterns of amino acids were in agreement with their standard biosynthetic pathways, with two remarkable exceptions: isoleucine was synthesized via the citramalate pathway and lysine was synthesized via the 2-aminoadipate pathway which has previously been reported only in eukaryotic microorganisms.     

Metabolism of propionate to acetate in the cockroach Periplaneta americana

Carbon-13 NMR and radiotracer studies were used to determine the precursor to methylmalonate and to study the metabolism of propionate in the cockroach Periplaneta americana. [3,4,5-13C3]Valine labeled carbons 3, 4, and 26 of 3-methylpentacosane, indicating that valine was metabolized via propionyl-CoA to methylmalonyl-CoA and served as the methyl branch unit precursor. Potassium [2-13C]propionate labeled the odd-numbered carbons of hydrocarbons and potassium [3-13C]propionate labeled the even-numbered carbons of hydrocarbons in this insect. This labeling pattern indicates that propionate is metabolized to acetate, with carbon-2 of propionate becoming the methyl carbon of acetate and carbon-3 of propionate becoming the carboxyl carbon of acetate. In vivo studies in which products were separated by HPLC showed that [2-14C]propionate was readily metabolized to acetate. The radioactivity from sodium [1-14C]propionate was not incorporated into succinate nor into any other tricarboxylic acid cycle intermediate, indicating that propionate was not metabolized via methylmalonate to succinate. Similarly, [1-14C]propionate did not label acetate. An experiment designed to determine the subcellular localization of the enzymes involved in converting propionate to acetate showed that they were located in the mitochondrial fraction. Data from both in vivo and in vitro studies as a function of time indicated that propionate was converted directly to acetate and did not first go through tricarboxylic acid cycle intermediates. These data demonstrate a novel pathway of propionate metabolism in insects.     

Acetate and CO2 assimilation by Methanothrix concilii.

Biosynthetic pathways in Methanothrix concilii, a recently isolated aceticlastic methanogen, were studied by 13C-nuclear magnetic resonance spectroscopy. Labeling patterns of amino acids, lipids, and carbohydrates were determined. Similar to other methanogens, acetate was carboxylated to pyruvate, which was further converted to amino acids by various biosynthetic pathways. The origin of carbon atoms in glutamate, proline, and arginine clearly showed that an incomplete tricarboxylic acid cycle operating in the oxidative direction was used for their biosynthesis. Isoleucine was synthesized via citramalate, which is a typical route for methanogens. As with Methanosarcina barkeri, an extensive exchange of the label between the carboxyl group of acetate and CO2 was observed. Lipids predominantly contained diphytanyl chains, the labeling of which indicated that biosynthesis proceeded through mevalonic acid. Labeling of the C-1,6 of glucose from [2-13C]acetate is consistent with a glucogenic route for carbohydrate biosynthesis. Except for the different origins of the methyl group of methionine, the metabolic properties of Methanothrix concilii are closely related to those of Methanosarcina barkeri.     

Amino acid biosynthesis and sodium-dependent transport in Methanococcus voltae, as revealed by 13C NMR

Of several methanogenic bacteria examined only Methanococcus voltae readily incorporated exogenous amino acids into cell protein. This was easily shown, since growth in the presence of exogenous amino acids resulted in a loss of signal intensities from those carbon atoms normally labelled by [13C]acetate during biosynthesis. From 80% to 95% of the Ser, Lys, Pro or Val incorporated into protein could be supplied directly from the growth medium. In contrast, Asp and Glu, if supplied to the medium, accounted for only a small percentage of the total acidic amino acid used in protein synthesis. Constitutive transport systems took up a wide range of amino acids at rates of 0.1-4.1 nmol min-1 mg-1. The transport systems required Na+, with the possible exception of the basic amino acid lysine, and were inhibited by N-ethylmaleimide or 3,3',4',5-tetrachlorosalicylanilide. No interconversion of Ile to other amino acids was detected when cells were given [13C]Ile during growth, whereas the expected labelling of the Asp and Glu families of amino acids resulted when [13C]Asp was provided to the culture. Mc. voltae synthesized its amino acids from acetate via routes fully consistent with those found in Methanospirillum hungatei [Ekiel, I., Smith, I.C.P. & Sprott, G.D. (1983) J. Bacteriol. 156, 316-326]. Propionate could substitute for an auxotrophic requirement for Ile, resulting in the synthesis of Ile with the beta-carbon originating from the carboxyl of acetate and the alpha-carbon from the carboxyl of propionate. No labelling of Ile from [13C]acetate could occur without the fatty acid. These results provide strong evidence for the carboxylation of propionate to form 2-oxobutyrate as intermediate in Ile biosynthesis, and show that the metabolic defect in Ile biosynthesis occurs prior to 2-oxobutyrate synthesis. The presence of constitutive amino acid transport systems and multiple routes for ile biosynthesis make Methanococcus voltae an attractive methanogen for genetic studies.     

Intramitochondrial reductive carboxylation of 2-oxoglutarate in adipose tissue and its contribution to fatty acid synthesis

• 1.1. The reductive carboxylation of 2-oxoglutarate was found to proceed in mitochondria of rat epididymal fat pads and rabbit perirenal adipose tissue at a rate similar to that in liver mitochondria.
• 2.2. In rat fat pads the incorporation of ¹⁴C from [5-¹⁴C]2-oxoglutarate into fatty acids via the carboxylation was suppressed by butylmalonate by 30%.
• 3.3. 2-Oxoglutarate and glutamate stimulated the incorporation into fatty acids of ¹⁴C from [2-¹⁴C]acetate in rat fat pads with the simultaneous reduction of tissue NADP. These effects persisted after inhibition of succinate dehydrogenase by malonate.
• 4.4. It is concluded that in adipose tissue 2-oxoglutarate carboxylation proceeds in both the cytoplasm and mitochondria. Therefore, it can supply carbon atoms as well as NADPH for fatty acid synthesis.

     

Utilization in vivo of glucose and volatile fatty acids by sheep brain for the synthesis of acidic amino acids

1. Free glutamic acid, aspartic acid, glutamic acid from glutamine and, in some instances, the glutamic acid from glutathione and the aspartic acid from N-acetyl-aspartic acid were isolated from the brains of sheep and assayed for radioactivity after intravenous injection of [2-¹⁴C]glucose, [1-¹⁴C]acetate, [1-¹⁴C]butyrate or [2-¹⁴C]propionate. These brain components were also isolated and analysed from rats that had been given [2-¹⁴C]propionate. The results indicate that, as in rat brain, glucose is by far the best precursor of the free amino acids of sheep brain. 2. Degradation of the glutamate of brain yielded labelling patterns consistent with the proposal that the major route of pyruvate metabolism in brain is via acetyl-CoA, and that the short-chain fatty acids enter the brain without prior metabolism by other tissue and are metabolized in brain via the tricarboxylic acid cycle. 3. When labelled glucose was used as a precursor, glutamate always had a higher specific activity than glutamine; when labelled fatty acids were used, the reverse was true. These findings add support and complexity to the concept of the metabolic `compartmentation' of the free amino acids of brain. 4. The results from experiments with labelled propionate strongly suggest that brain metabolizes propionate via succinate and that this metabolic route may be a limited but important source of dicarboxylic acids in the brain.     

Glycolytic origin of alanine formed in rat diaphragm muscle in vitro.

In quarter-diaphragms from 40 h-starved rats the rate of glycogen mobilization is sufficient to account for the rate of lactate+pyruvate+alanine production. It is concluded, therefore, that alanine derives its carbon skeleton predominantly via glycolysis and not via synthesis de novo from tricarboxylic acid-cycle intermediates and related amino acids.     

Aldehyde dehydrogenase induction by glutamate in Escherichia coli. Role of 2-oxoglutarate.

In Escherichia coli, an aldehyde dehydrogenase that catalyzes the oxidation of L-lactaldehyde to L-lactate is induced not only by L-fucose, L-rhamnose or D-arabinose, but also by growth in the presence of glutamate or amino acids yielding glutamate, with the exception of proline. Induction by these amino acids requires glutamate accumulation. 4-Aminobutyric acid also induces this aldehyde dehydrogenase through its transamination to glutamate. Growth on 2-oxoglutarate, the tricarboxylic acid cycle intermediate with which glutamate is in equilibrium, also induces this aldehyde dehydrogenase. Conditions in which the conversion of 2-oxoglutarate into glutamate is highly restricted displayed unchanged rates of induction by 2-oxoglutarate, indicating that glutamate induces the aldehyde dehydrogenase through 2-oxoglutarate formation. Evidence is presented showing that L-fucose- and 2-oxoglutarate-inducing systems share the same regulatory protein. Induction by growth on either of these two compounds is repressed both by glucose and by glycerol. Addition of cAMP to these cultures partially recovers the glucose-repressed aldehyde dehydrogenase activity, while this nucleotide has no effect on the glycerol-mediated repression. These results indicate that ald is under carbon regulation mediated by at least two different mechanisms.     

Utilization of acetate by Methanomonas emthanooxidans.

Methanomonas methanooxidans incorporates both carbon atoms of acetate into the glutamate and aspartate families of amino acids during growth on methane; carbon dioxide is also evolved from both carbon atoms of acetate. The distribution of carboxyl-labeled acetate incorporated into convalently bound glutamate is consistent with the operation of the tricarboxylic acid cycle in this species, and the presence of alpha-ketoglutarate dehydrogenase was demonstrated in cell-free extracts.     

The incorporation of carbon dioxide into milk citrate in the isolated perfused goat udder

1. Isolated perfused goat udders supplied with glucose, acetate and amino acids were infused for several hours with NaH¹⁴CO₃. 2. Lactose, milk-fat fatty acids and glycerol had very little radioactivity. The specific radioactivity (counts./min./mg. of C) of milk citrate was 9–16% that of the carbon dioxide in the perfusion fluid and 19% that estimated for tissue carbon dioxide. The specific radioactivity of tissue citrate resembled that of milk citrate. 3. The radioactivity in citrate was predominantly in C-6, suggesting some carboxylation of α-oxoglutarate in addition to carboxylation of C₃ compounds. 4. [1-¹⁴C]Glutamate was infused in a similar experiment, and milk citrate radioactivity was predominantly in C-1+C-5. 5. The results are discussed in relation to the contribution of glucose and acetate carbon to citrate. The implications of the carboxylation of α-oxoglutarate are considered.     

Biosynthesis of the macrolide antibiotic chlorothricin: basic building blocks

The biosynthesis of chlorothricin (I), a macrolide antibiotic isolated from Streptomyces antibioticus Tü 99, has been studied by feeding experiments with 14C- and 3H-labeled precursors. Acetate and propionate, but not methionine and mevalonate, were incorporated into the macrocylic aglycone of the antibiotic. Glucose and the various carbon atoms of tyrosine, except the carboxyl carbon, also contributed label to the aglycone. Glucose also seems to be a specific precursor of the 2-deoxyrhamnose moiety, probably via a process involving a hydrogen shift from C-4 to C-6 of the hexose. The substituted 6-methylsalicylic acid moiety seems to be derived from acetate and one O-methyl group provided by methionine; shikimic acid is not incorporated.     

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.     

Carbohydrate catabolism in the plerocercoids of Schistocephalus solidus (Cestoda: Pseudophyllidea)

The plerocercoids of S. solidus possess a complete sequence of glycolytic and tricarboxylic acid cycle enzymes. The presence of phosphoenolpyruvate carboxykinase and fumarate reductase activity and the relatively low activities of aconitase and isocitrate dehydrogenase suggest that carbon dioxide fixation is an important pathway in this parasite. Carbon balances show that glycogen is the main energy source under both aerobic and anaerobic conditions and there is only a slight Pasteur effect. Aerobically 22·5% of the glycogen catabolized is excreted as acetate and propionate (4:1), anaerobically 70% of the glycogen utilized can be accounted for as acetate and propionate (1:3). The results indicate that anaerobically the plerocercoids fix carbon dioxide and have a partial reversed tricarboxylic acid cycle, whilst under aerobic conditions at least part of the carbohydrate may be oxidized via a functional tricarboxylic acid cycle.     

Autotrophic CO2 fixation in Chlorobium limicola. Evidence for the operation of a reductive tricarboxylic acid cycle in growing cells

Chlorobium limicola was grown on a mineral salts medium with CO₂ as the main carbon source supplemented with specifically labeled ¹⁴C propionate and the incorporation of ¹⁴C into alanine ( intracellular pyruvate), aspartate ( oxaloacetate), and glutamate ( -ketoglutarate) was studied in long term labeling experiments. During growth in presence of propionate 30% of the cell carbon were derived from propionate and 70% from CO₂. Propionate was not oxidized to CO₂.All three amino acids were found to be labeled. The labeling patterns indicate that propionate was assimilated via propionyl CoA, methylmalonyl CoA and succinyl CoA. When 1-¹⁴C propionate was the labeled precursor no radioactivity was found in the carboxyl group(s) of alanine, aspartate and glutamate, excluding the incorporation of propionate into the amino acids via succinate oxidation to fumarate. With 1-¹⁴C propionate preferentially aspartate (C-3) and glutamate (C-2) became labeled, with 2-¹⁴C propionate alanine (C-3) and glutamate (C-4). These findings indicate that propionate was incorporated into the amino acids via succinyl CoA, -ketoglutarate, isocitrate, and citrate, followed by a si-type cleavage of citrate to oxaloacetate and acetyl CoA (or acetate). Similar experiments with U-¹⁴C acetate confirm these conclusions. Thus, all reactions of the proposed reductive tricarboxylic acid cycle could be demonstrated in autotrophically growing cells.     

On the role of the mitochondrial 2-oxoglutarate dehydrogenase complex in amino acid metabolism

Mitochondria are tightly linked to cellular nutrient sensing, and provide not only energy, but also intermediates for the de novo synthesis of cellular compounds including amino acids. Mitochondrial metabolic enzymes as generators and/or targets of signals are therefore important players in the distribution of intermediates between catabolic and anabolic pathways. The highly regulated 2-oxoglutarate dehydrogenase complex (OGDHC) participates in glucose oxidation via the tricarboxylic acid cycle. It occupies an amphibolic branch point in the cycle, where the energy-producing reaction of the 2-oxoglutarate degradation competes with glutamate (Glu) synthesis via nitrogen incorporation into 2-oxoglutarate. To characterize the specific impact of the OGDHC inhibition on amino acid metabolism in both plant and animal mitochondria, a synthetic analog of 2-oxoglutarate, namely succinyl phosphonate (SP), was applied to living systems from different kingdoms, both in situ and in vivo. Using a high-throughput mass spectrometry-based approach, we showed that organisms possessing OGDHC respond to SP by significantly changing their amino acid pools. By contrast, cyanobacteria which lack OGDHC do not show perturbations in amino acids following SP treatment. Increases in Glu, 4-aminobutyrate and alanine represent the most universal change accompanying the 2-oxoglutarate accumulation upon OGDHC inhibition. Other amino acids were affected in a species-specific manner, suggesting specific metabolic rearrangements and substrate availability mediating secondary changes. Strong perturbation in the relative abundance of amino acids due to the OGDHC inhibition was accompanied by decreased protein content. Our results provide specific evidence of a considerable role of OGDHC in amino acid metabolism.     

Regulation of the acuF Gene, Encoding Phosphoenolpyruvate Carboxykinase in the Filamentous Fungus Aspergillus nidulans

Phosphoenolpyruvate carboxykinase (PEPCK) is a key enzyme required for gluconeogenesis when microorganisms grow on carbon sources metabolized via the tricarboxylic acid (TCA) cycle. Aspergillus nidulans acuF mutants isolated by their inability to use acetate as a carbon source specifically lack PEPCK. The acuF gene has been cloned and shown to encode a protein with high similarity to PEPCK from bacteria, plants, and fungi. The regulation of acuF expression has been studied by Northern blotting and by the construction of lacZ fusion reporters. Induction by acetate is abolished in mutants unable to metabolize acetate via the TCA cycle, and induction by amino acids metabolized via 2-oxoglutarate is lost in mutants unable to form 2-oxoglutarate. Induction by acetate and proline is not additive, consistent with a single mechanism of induction. Malate and succinate result in induction, and it is proposed that PEPCK is controlled by a novel mechanism of induction by a TCA cycle intermediate or derivative, thereby allowing gluconeogenesis to occur during growth on any carbon source metabolized via the TCA cycle. It has been shown that the facB gene, which mediates acetate induction of enzymes specifically required for acetate utilization, is not directly involved in PEPCK induction. This is in contrast to Saccharomyces cerevisiae, where Cat8p and Sip4p, homologs of FacB, regulate PEPCK as well as the expression of other genes necessary for growth on nonfermentable carbon sources in response to the carbon source present. This difference in the control of gluconeogenesis reflects the ability of A. nidulans and other filamentous fungi to use a wide variety of carbon sources in comparison with S. cerevisiae. The acuF gene was also found to be subject to activation by the CCAAT binding protein AnCF, a protein homologous to the S. cerevisiae Hap complex and the mammalian NFY complex.     

Pathway of propionate oxidation by a syntrophic culture of Smithella propionica and Methanospirillum hungatei

The pathway of propionate conversion in a syntrophic coculture of Smithella propionica and Methanospirillum hungatei JF1 was investigated by (13)C-NMR spectroscopy. Cocultures produced acetate and butyrate from propionate. [3-(13)C]propionate was converted to [2-(13)C]acetate, with no [1-(13)C]acetate formed. Butyrate from [3-(13)C]propionate was labeled at the C2 and C4 positions in a ratio of about 1:1.5. Double-labeled propionate (2,3-(13)C) yielded not only double-labeled acetate but also single-labeled acetate at the C1 or C2 position. Most butyrate formed from [2,3-(13)C]propionate was also double labeled in either the C1 and C2 atoms or the C3 and C4 atoms in a ratio of about 1:1.5. Smaller amounts of single-labeled butyrate and other combinations were also produced. 1-(13)C-labeled propionate yielded both [1-(13)C]acetate and [2-(13)C]acetate. When (13)C-labeled bicarbonate was present, label was not incorporated into acetate, propionate, or butyrate. In each of the incubations described above, (13)C was never recovered in bicarbonate or methane. These results indicate that S. propionica does not degrade propionate via the methyl-malonyl-coenzyme A (CoA) pathway or any other of the known pathways, such as the acryloyl-CoA pathway or the reductive carboxylation pathway. Our results strongly suggest that propionate is dismutated to acetate and butyrate via a six-carbon intermediate.