Biochemical effects of the hypoglycaemic compound pent-4-enoic acid and related non-hypoglycaemic fatty acids. Effects of the free acids and their carnitine esters on coenzyme A-dependent oxidations in rat liver mitochondria

1. The synthesis of pent-4-enoyl-l-carnitine, cyclopropanecarbonyl-l-carnitine and cyclobutanecarbonyl-l-carnitine is described. 2. Pent-4-enoate strongly inhibits palmitoyl-l-carnitine oxidation in coupled but not in uncoupled mitochondria. Pent-4-enoyl-l-carnitine strongly inhibits palmitoyl-l-carnitine oxidation in uncoupled mitochondria. Prior intramitochondrial formation of pent-4-enoyl-CoA is therefore necessary for inhibition. 3. There was a small self-limiting pulse of oxidation of pent-4-enoyl-l-carnitine during which the ability to inhibit the oxidation of subsequently added palmitoyl-l-carnitine developed. 4. Pent-4-enoate and pent-4-enoyl-l-carnitine are equally effective inhibitors of the oxidation of all even-chain acylcarnitines of chain length C(4)-C(16). Pent-4-enoyl-l-carnitine also inhibits the oxidation of pyruvate and of 2-oxoglutarate. 5. Pent-4-enoate strongly inhibits the oxidation of palmitate but not that of octanoate. This is presumably due to competition between octanoate and pent-4-enoate for medium-chain acyl-CoA ligase. 6. There was less inhibition of the oxidation of pyruvate by pent-4-enoyl-l-carnitine, and of palmitoyl-l-carnitine by cyclopropanecarbonyl-l-carnitine, after pre-incubation with 10mm-arsenate. This suggests that these inhibitions were caused either by depletion of free CoA or by increase of acyl-CoA concentrations, since arsenate deacylates intramitochondrial acyl-CoA. There was little effect on the inhibition of palmitoyl-l-carnitine oxidation by pent-4-enoyl-l-carnitine. 7. Penta-2,4-dienoate strongly inhibited palmitoyl-l-carnitine oxidation in coupled mitochondria; acrylate only inhibited slightly. 8. Pent-4-enoate (0.1mm) caused a rapid and almost complete decrease in free CoA and a large increase in acid-soluble acyl-CoA when incubated with coupled mitochondria. Cyclopropanecarboxylate caused a similar decrease in CoA, with an equivalent rise in acid-soluble acyl-CoA concentrations. n-Pentanoate caused extensive lowering of CoA and a large increase in acid-soluble acyl-CoA and acetyl-CoA concentrations. Octanoate caused a 50% lowering of CoA and an increase in acid-soluble acyl-CoA and acetyl-CoA concentrations. 9. Cyclopropanecarboxylate and n-pentanoate were less potent inhibitors of palmitate oxidation than was pent-4-enoate. 10. It is concluded that pent-4-enoate causes a specific inhibition of beta-oxidation after the formation intramitochondrially of its metabolites.     

Potentiation by ammonia of the metabolic effects of pent-4-enoate in isolated rat hepatocytes.

The metabolic effects of pent-4-enoate were studied in isolated rat hepatocytes; 1 mM-pent-4-enoate did not significantly inhibit gluconeogenesis from lactate, alanine and glycerol, but significantly decreased glucose synthesis from pyruvate. The addition of 1 mM-NH4Cl led to a drastic inhibition of glucose synthesis from all these substrates. In hepatocytes incubated with 10 mM-alanine and 1 mM-oleate, pent-4-enoate at 0.05-1 mM slightly inhibited glucose synthesis and ketogenesis. The addition of ammonia resulted in a dramatic potentiation of the metabolic effects of pent-4-enoate. Half-maximum effect of ammonia was observed at 0.2 mM concentration. Concomitant cellular concentrations of ATP and acetyl-CoA were also decreased by the addition of ammonia, as were lactate/pyruvate ratio and beta-hydroxybutyrate/acetoacetate ratio. These data suggest that ammonia seriously interferes with the cellular metabolism of pent-4-enoate and leads to a dramatic potentiation of its effects.     

In vivo induction of 4-enoyl-CoA reductase by clofibrate in liver mitochondria and its effect on pent-4-enoate metabolism

Feeding of clofibrate to male rats leads to a 4–7 fold increase in the activity of the 4-enoyl-CoA reductase in the liver. Concomitantly the inhibition of fatty acid oxidation by pent-4-enoate is abolished, and an increased glucose formation in the presence of pent-4-enoate is observed. It is suggested that pent-4-enoate is converted to propionyl-CoA via the reaction sequence pent-4-enoyl-CoA→pent-2,4-dienoyl-CoA→pent-2-enoyl-CoA→propionyl-CoA + acetyl-CoA.     

Effects of pent-4-enoate on cellular redox state, glycolysis and fatty acid oxidation in isolated perfused rat heart

The metabolic effects of pent-4-enoate were studied in beating and potassium-arrested perfused rat hearts. The addition of 0.8mm-pent-4-enoate to the fluid used to perfuse a potassium-arrested heart resulted in a 70% increase in the O(2) consumption and a 66% decrease in the glycolytic flux as measured in terms of the de-tritiation of [3-(3)H]glucose, although the proportion of the O(2) consumption attributable to glucose oxidation decreased from an initial 30% to 10%. The pent-4-enoate-induced increase in O(2) consumption was only 15% in the beating heart. In the potassium-arrested heart, pent-4-enoate stimulated palmitate oxidation by more than 100% when measured in terms of the production of (14)CO(2) from [1-(14)C]palmitate, but in the beating heart palmitate oxidation was inhibited. Perfusion of the heart with pent-4-enoate had no effect on the proportion of pyruvate dehydrogenase found in the active form, in spite of large changes in the CoASH and acetyl-CoA concentrations and changes in their concentration ratios. The effects of pent-4-enoate on the cellular redox state were dependent on the ATP consumption of the heart. In the beating heart, pent-4-enoate caused a rapid mitochondrial NAD(+) reduction that subsequently faded out, so that the final state was more oxidized than the initial state. The arrested heart, however, remained in a more reduced state than initially, even after the partial re-oxidation that followed the initial rapid NAD(+) reduction. The ability of pent-4-enoate to increase or decrease fatty acid oxidation can be explained on the basis of the differential effects of pent-4-enoate on the concentration of citric acid-cycle intermediates under conditions of high or low ATP consumption of the myocardial cell. The proportion of the fatty acids in the fuel consumed by the heart is probably primarily determined by the regulatory mechanisms of glycolysis. When pent-4-enoate causes an increase in the citric acid-cycle intermediates, feedback inhibition of glycolysis results in an increase in the oxidation of fatty acids.     

Inhibition of urea synthesis by pent-4-enoate associated with decrease in N-acetyl-L-glutamate concentration in isolated rat hepatocytes

Pent-4-enoate at 0.1 to 1.0 mM strongly inhibited urea synthesis in isolated rat hepatocytes. Pent-4-enoate at the same concentrations markedly decreased concentrations of $\text{N-}\text{acetyl-}\text{L}\text{-glutamate}$, an essential activator of carbamoyl-phosphate synthase-I (EC 2.7.2.5), and the decrease was well parallel with the inhibition of urea synthesis by pent-4-enoate. This compound also lowered cellular concentrations of acetyl-CoA, a substrate of acetylglutamate synthase (EC 2.3.1.1). Pent-4-enoate in a dose of 1 mM did not significantly affect cellular concentrations of ATP, and had no direct effect on acetylglutamate synthase activity. These results suggest that the inhibition of urea synthesis by pent-4-enoate is due to decrease in $\text{N-}\text{acetyl-}\text{L}\text{-glutamate}$ concentration and that the decrease is probably brought about by decreased rate of its synthesis due to the lowered concentration of cellular acetyl-CoA.     

Biochemical effects of the hypoglycaemic compound pent-4-enoic acid and related non-hypoglycaemic fatty acids. Effects of their coenzyme A esters on enzymes of fatty acid oxidation

1. Pent-4-enoyl-CoA and its metabolites penta-2,4-dienoyl-CoA and acryloyl-CoA, as well as n-pentanoyl-CoA, cyclopropanecarbonyl-CoA and cyclobutanecarbonyl-CoA, were examined as substrates or inhibitors of purified enzymes of beta-oxidation in an investigation to locate the site of inhibition of fatty acid oxidation by pent-4-enoate. 2. The reactions of various acyl-CoA derivatives with l-carnitine and of various acyl-l-carnitine derivatives with CoA, catalysed by carnitine acetyltransferase, were investigated and V(max.) and K(m) values were determined. Pent-4-enoyl-CoA and n-pentanoyl-CoA were good substrates, whereas cyclobutanecarbonyl-CoA, cyclopropanecarbonyl-CoA and acryloyl-CoA reacted more slowly. A very slow rate with penta-2,4-dienoyl-CoA was detected. Pent-4-enoyl-l-carnitine, n-pentanoyl-l-carnitine and cyclobutanecarbonyl-l-carnitine were good substrates and cyclopropanecarbonyl-l-carnitine reacted more slowly. 3. Pent-4-enoyl-CoA and n-pentanoyl-CoA were substrates for butyryl-CoA dehydrogenase and for octanoyl-CoA dehydrogenase, and both compounds were equally effective competitive inhibitors of these enzymes with butyryl-CoA or palmitoyl-CoA respectively as substrates. V(max.), K(m) and K(i) values were determined. 4. None of the acyl-CoA derivatives inhibited enoyl-CoA hydratase or 3-hydroxybutyryl-CoA dehydrogenase. Penta-2,4-dienoyl-CoA was a substrate for enoyl-CoA hydratase when the reaction was coupled to that catalysed by 3-hydroxybutyryl-CoA dehydrogenase. 5. In a reconstituted sequence with purified enzymes crotonoyl-CoA was largely converted into acetyl-CoA, and pent-2-enoyl-CoA into acetyl-CoA and propionyl-CoA. Penta-2,4-dienoyl-CoA was slowly converted into acetyl-CoA and acryloyl-CoA. 6. Penta-2,4-dienoyl-CoA, a unique metabolite of pent-4-enoate, was the only compound that specifically inhibited an enzyme of the beta-oxidation sequence, 3-oxoacyl-CoA thiolase. The formation of penta-2,4-dienoyl-CoA could explain the strong inhibition of fatty acid oxidation in intact mitochondria by pent-4-enoate.     

Enhanced isoamyl acetate production upon manipulation of the acetyl-CoA node in Escherichia coli

Coenzyme A (CoA) and its thioester derivative acetyl-Coenzyme A (acetyl-CoA) participate in over 100 different reactions in intermediary metabolism of microorganisms. Earlier results indicated that overexpression of upstream rate-limiting enzyme pantothenate kinase with simultaneous supplementation of precursor pantothenic acid to the culture media increased intracellular CoA levels significantly ( approximately 10-fold). The acetyl-CoA levels also increased ( approximately 5-fold) but not as much as that of CoA, showing that the carbon flux from the pyruvate node is rate-limiting upon an increase in CoA levels. In this study, pyruvate dehydrogenase was overexpressed under elevated CoA levels to increase carbon flux from pyruvate to acetyl-CoA. This coexpression did not increase intracellular acetyl-CoA levels but increased the accumulation of extracellular acetate. The production of isoamyl acetate, an industrially useful compound derived from acetyl-CoA, was used as a model reporter system to signify the beneficial effects of this metabolic engineering strategy. In addition, a strain was created in which the acetate production pathway was inactivated to relieve competition at the acetyl-CoA node and to efficiently channel the enhanced carbon flux to the ester production pathway. The synergistic effect of cofactor CoA manipulation and pyruvate dehydrogenase overexpression in the acetate pathway deletion mutant led to a 5-fold increase in isoamyl acetate production. Under normal growth conditions the acetate pathway deletion mutant strains accumulate intracellular pyruvate, leading to excretion of pyruvate. However, upon enhancing the carbon flux from pyruvate to acetyl-CoA, the excretion of pyruvate was significantly reduced.     

The control of tricarboxylate-cycle oxidations in blowfly flight muscle. The steady-state concentrations of coenzyme A, acetyl-coenzyme A and succinyl-coenzyme A in flight muscle and isolated mitochondria

(1) A ;cycling' method involving citrate synthase (EC 4.1.3.7) and malate dehydrogenase (EC 1.1.1.37) was modified by the inclusion of succinyl-CoA synthetase (EC 6.2.1.5) and hexokinase (EC 2.7.1.1) to permit the determination of very small amounts of succinyl-CoA in addition to CoA and acetyl-CoA. (2) Application of this technique to blowfly (Phormia regina) flight-muscle extracts reveals no change in acetyl-CoA concentration, a slight fall in CoA concentration and a rise in succinyl-CoA concentration during flight. (3) Extraction of isolated mitochondria during controlled (state 4) pyruvate oxidation reveals essentially only acetyl-CoA. Activation of respiration by ADP (state 3) or uncoupling agents leads to a fall in acetyl-CoA and a rise in CoA and succinyl-CoA content. (4) The presence of glycerol phosphate in addition to pyruvate results in a lower acetyl-CoA content in state 4. (5) It is contended that these results are consistent with a primary control of one of the reactions of the tricarboxylate cycle, rather than of pyruvate dehydrogenase, during the state 4 oxidation of pyruvate by isolated mitochondria, and that the modulation of citrate synthase activity by the ratio of acetyl-CoA/succinyl-CoA is unimportant under these conditions.     

Metabolic effects of propionate in normal and vitamin B12-deficient rats

1. Administration of propionate caused a twofold increase in the concentrations of lactate and pyruvate in the blood of vitamin B(12)-deficient rats, whereas there was a slight decrease in lactate and a 50% increase in pyruvate in normal rats. 2. Concentrations of total ketone bodies in the blood of normal rats were not significantly altered by propionate administration but the [3-hydroxybutyrate]/[acetoacetate] ratio decreased from 3.0 to 2.0. In the vitamin B(12)-deficient rats there was a 40% decrease in total ketone bodies and a change in the ratio from 3.4 to 1.2. 3. The changes in the concentration of ketone bodies in freeze-clamped liver preparations were similar in pattern to those observed in blood. 4. Propionate administration caused a decrease in the concentration of acetyl-CoA in the livers of both groups of animals, but the absolute decrease was greater in the vitamin B(12)-deficient group. The decrease in the concentration of CoA was similar in both groups. 5. As in blood, there were threefold increases in the concentrations of lactate and pyruvate in the livers of the vitamin B(12)-deficient rats after propionate administration, whereas there was no significant change in the concentrations of these metabolites in the normal rats. 6. There was a 50% inhibition of glucose synthesis in perfused livers from vitamin B(12)-deficient rats when lactate and propionate were substrates as compared with lactate alone. 7. It is concluded that the conversion of lactate into glucose is inhibited in vitamin B(12)-deficient rats after propionate administration, and that this effect is due to inhibition of the pyruvate carboxylase step resulting from a decrease in acetyl-CoA concentration and a postulated increase in methylmalonyl-CoA concentration.     

Effect of 4-Pentenoic Acid on Intermediate Metabolism of Tetrahymena*

SYNOPSIS. The growth of Tetrahymena pyriformis strain HSM was strongly inhibited by 4-pentenoic acid. Supplementing the medium with acetate reversed the growth inhibition, but pyruvate was ineffective. Glycogen content was much lower in cells grown with 4-pentenoic acid than in controls; this effect was not reversed by acetate or by pyruvate. There was little effect of 4-pentenoic acid on the incorporation of label from [1-¹⁴C]acetate, [2-¹⁴C]glycerol, [1-³⁴]ribose, [U-¹⁴C]fructose, or [1-¹⁴C]glucose into CO₂, but incorporation of label into glycogen was inhibited, the strongest inhibition being on acetate and the weakest (～ 20%) on ribose, fructose, and glucose. A 3-compartment model for quantitation of labeled acetyl CoA fluxes was shown to be applicable to Tetrahymena grown in the presence of 4-pentenoic acid, and experiments were performed to establish the flux of [1-¹⁴C]acetyl CoA into glycogen, lipids, CO₂, glutamate, and alanine. It was evident from the results of these experiments that 4-pentenoic acid did not appreciably inhibit β-oxidation or lipogenesis, but markedly decreased the glyconeogenic flux of labeled acetyl-CoA from the peroxismal and outer mitochondrial compartments. At least 2 mechanisms have been proposed for the action of 4-pentenoic acid: (a) reduction of the levels of acetyl CoA or free CoA and (b) direct inhibition of enzymes by 4-pentenoyl CoA or its metabolites. Although 4-pentenoic acid has little effect on acetyl-CoA metabolism in the inner mitochondrial compartment, the present data suggest that the flux through the outer mitochondrial compartment of acetyl-CoA derived from pyruvate is inhibited largely by the first, and that the glyconeogenic flux of acetyl-CoA is inhibited largely by the 2nd mechanism.     

Dark metabolism of acetate in Rhodospirillum rubrum cells, grown under photoheterotropic conditions

The mechanism of the dark assimilation of acetate in the photoheterotrophically grown nonsulfur bacterium Rhodospirillum rubrum was studied. Both in the light and in the dark, acetate assimilation in Rsp. rubrum cells, which lack the glyoxylate pathway, was accompanied by the excretion of glyoxylate into the growth medium. The assimilation of propionate was accompanied by the excretion of pyruvate. Acetate assimilation was found to be stimulated by bicarbonate, pyruvate, the C4-dicarboxylic acids of the Krebs cycle, and glyoxylate, but not by propionate. These data implied that the citramalate (CM) cycle in Rsp. rubrum cells grown aerobically in the dark can function as an anaplerotic pathway. This supposition was confirmed by respiration measurements. The respiration of cells oxidizing acetate depended on the presence of CO2 in the medium. The fact that the intermediates of the CM cycle (citramalate and mesaconate) markedly inhibited acetate assimilation but had almost no effect on cell respiration indicative that citramalate and mesaconate are intermediates of the acetate assimilation pathway. The inhibition of acetate assimilation and cell respiration by itaconate was due to its inhibitory effect on propionyl-CoA carboxylase, an enzyme of the CM cycle. The addition of 5 mM itaconate to extracts of Rsp. rubrum cells inhibited the activity of this enzyme by 85%. The data obtained suggest that the CM cycle continues to function in Rsp. rubrum cells that have been grown anaerobically in the light and then transferred to the dark and incubated aerobically.     

Pathways of acetate and propionate production in adult Fasciola hepatica

In adult F. hepatica pyruvate is decarboxylated via pyruvate dehydrogenase to acetyl-CoA; acetyl-CoA is then cleaved to acetate via three possible mechanisms (1) carnitine dependent hydrolysis, (2) CoA transferase, (3) reversal of a GTP dependent acyl-CoA synthetase. Of these three systems, CoA transferase has by far the greatest activity. Propionate production by F. hepatica is similar to the mammalian system, succinate being metabolized via succinic thiokinase, methylmalonyl-CoA isomerase, methyl-malonyl-CoA racemase and propionyl-CoA carboxylase to propionyl-CoA. Propionyl-CoA is then cleaved to propionate by the same three pathways as acetyl-CoA. No ATP or GTP production could be demonstrated when acetyl- or propionyl-CoA were incubated with homogenates of F. hepatica. This indicates that carnitine dependent hydrolysis or CoA transferase are the major pathways of acetyl- or propionyl-CoA breakdown. The CoA transferase reaction would result in the conservation of the bond energy although there is no net ATP synthesis.     

Control of the tricarboxylate cycle and its interactions with glycolysis during acetate utilization in rat heart

1. Transient and steady-state changes caused by acetate utilization were studied in perfused rat heart. The transient period occupied 6min and steady-state changes were followed in a further 6min of perfusion. 2. In control perfusions glucose oxidation accounted for 75% of oxygen utilization; the remaining 25% was assumed to represent oxidation of glyceride fatty acids. With acetate in the steady state, acetate oxidation accounted for 80% of oxygen utilization, which increased by 20%; glucose oxidation was almost totally suppressed. The rate of tricarboxylate-cycle turnover increased by 67% with acetate perfusion. The net yield of ATP in the steady state was not altered by acetate. 3. Acetate oxidation increased muscle concentrations of acetyl-CoA, citrate, isocitrate, 2-oxoglutarate, glutamate, alanine, AMP and glucose 6-phosphate, and lowered those of CoA and aspartate; the concentrations of pyruvate, ATP and ADP showed no detectable change. The times for maximum changes were 1min, acetyl-CoA, CoA, alanine and AMP; 6min, citrate, isocitrate, glutamate and aspartate; 2-4min, 2-oxoglutarate. Malate concentration fell in the first minute and rose to a value somewhat greater than in the control by 6min. There was a transient and rapid rise in glucose 6-phosphate concentration in the first minute superimposed on the slower rise over 6min. 4. Acetate perfusion decreased the output of lactate, the muscle concentration of lactate and the [lactate]/[pyruvate] ratio in perfusion medium and muscle in the first minute; these returned to control values by 6min. 5. During the first minute acetate decreased oxygen consumption and lowered the net yield of ATP by 30% without any significant change in muscle ATP or ADP concentrations. 6. The specific radioactivities of cycle metabolites were measured during and after a 1min pulse of [1-(14)C]acetate delivered in the first and twelfth minutes of acetate perfusion. A model based on the known flow rates and concentrations of cycle metabolites was analysed by computer simulation. The model, which assumed single pools of cycle metabolites, fitted the data well with the inclusion of an isotope-exchange reaction between isocitrate and 2-oxoglutarate+bicarbonate. The exchange was verified by perfusions with [(14)C]bicarbonate. There was no evidence for isotope exchange between citrate and acetyl-CoA or between 2-oxoglutarate and malate. There was rapid isotope equilibration between 2-oxoglutarate and glutamate, but relatively poor isotope equilibration between malate and aspartate. 7. It is concluded that the citrate synthase reaction is displaced from equilibrium in rat heart, that isocitrate dehydrogenase and aconitate hydratase may approximate to equilibrium, that alanine aminotransferase is close to equilibrium, but that aspartate transamination is slow for reasons that have yet to be investigated. 8. The slow rise in citrate concentration as compared with the rapid rise in that of acetyl-CoA is attributed to the slow generation of oxaloacetate by aspartate aminotransferase. 9. It is proposed that the tricarboxylate cycle may operate as two spans: acetyl-CoA-->2-oxoglutarate, controlled by citrate synthase, and 2-oxoglutarate-->oxaloacetate, controlled by 2-oxoglutarate dehydrogenase; a scheme for cycle control during acetate oxidation is outlined. The initiating factors are considered to be changes in acetyl-CoA, CoA and AMP concentrations brought about by acetyl-CoA synthetase. 10. Evidence is presented for a transient inhibition of phosphofructokinase during the first minute of acetate perfusion that was not due to a rise in whole-tissue citrate concentration. The probable importance of metabolite compartmentation is stressed.     

Mass isotopomer study of anaplerosis from propionate in the perfused rat heart

Anaplerosis from propionate was investigated in rat hearts perfused with 0-2mM [(13)C(3)]propionate and physiological concentrations of glucose, lactate, and pyruvate. The data show that when the concentration of [(13)C(3)]propionate was raised from 0 to 2mM, total anaplerosis increased from 5% to 16% of the turnover of citric acid cycle intermediates. Then, [(13)C(3)]propionate abolished anaplerosis from endogenous substrates, glucose, lactate, and pyruvate. Also, while the contents of propionyl-CoA and methylmalonyl-CoA increased with [(13)C(3)]propionate concentration, the content of succinyl-CoA decreased, presumably via activation of succinyl-CoA hydrolysis by a decrease in free CoA. Under our conditions, [(13)C(3)]propionate was a purely anaplerotic substrate since there was no labeling of mitochondrial acetyl-CoA, reflected by the labeling of the acetyl moiety of citrate.     

Isolated rat heart mitochondria are able to metabolize pent-4-enoate to tricarboxylic acid-cycle intermediates.

The metabolism of four short-chain odd-number-carbon fatty acids, pentanoate, pent-4-enoate, propionate and acrylate, was studied in isolated rat heart mitochondria incubated in [14C]bicarbonate buffer. Under these conditions pentanoate was metabolized with a concomitant accumulation of malate and incorporation of 14CO2 into non-volatile compounds. The metabolism of propionate to tricarboxylic acid-cycle intermediates required the addition of ATP and oligomycin. After addition of a small amount of rotenone to the incubation medium, pent-4-enoate was metabolized with an increase in malate from less than 3 nmol/mg of protein to 34.0 +/- 1.5 nmol/mg in 40 min, during which time the amount of 14CO2 fixed in acid-stable compounds increased from 1.56 +/- 0.30 to 41.1 +/- 2.6 nmol/mg of protein. Acrylate was not metabolized under any of the conditions tested. The results show that cardiac mitochondria must have an enzyme system that is capable of reducing the double bond of either pent-4-enoate or its metabolities. That the metabolism of pent-4-enoate occurs through a reductive step and energy-dependent carboxylation is evident from the requirement for NAD+ reduction by partial inhibition of the mitochondrial respiratory chain and the presence of ATP and CO2. The results do not enable us to say whether the compound reduced is pent-4-enoyl-CoA or acryloyl-CoA.     

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].     

Investigation of the dark metabolism of acetate in photoheterotrophically grown cells ofRhodospirillum rubrum

The mechanism of the aerobic dark assimilation of acetate in the photoheterotrophically grown purple nonsulfur bacteriumRhodospirillum rubrum was studied. Both in the light and in the dark, acetate assimilation inRsp. rubrum cells, which lack the glyoxylate pathway, was accompanied by the excretion of glyoxylate into the growth medium. The assimilation of propionate was accompanied by the excretion of pyruvate. Acetate assimilation was found to be stimulated by bicarbonate, pyruvate, the C₄-dicarboxylic acids of the Krebs cycle, and glyoxylate, but not by propionate. These data implied that the citramalate (CM) cycle inRsp. rubrum cells can function as an anaplerotic pathway under aerobic dark conditions. This supposition was confirmed by respiration measurements. The respiration of cells oxidizing acetate depended on the presence of CO₂ in the medium. The fact that the intermediates of the CM cycle (citramalate and mesaconate) markedly inhibited acetate assimilation but had almost no effect on cell respiration indicated that citramalate and mesaconate were intermediates of the acetate assimilation pathway. The inhibition of acetate assimilation and cell respiration by itaconate was due to its inhibitory effect on propionyl-CoA carboxylase, an enzyme of the CM cycle. The addition of 5 mM itaconate to extracts ofRsp. rubrum cells inhibited the activity of this enzyme by 85%. The data obtained suggest that the CM cycle continues to function inRsp. rubrum cells that have been grown anaerobically in the light and then transferred to the dark and incubated aerobically.     

Secondary mitochondrial dysfunction in propionic aciduria: a pathogenic role for endogenous mitochondrial toxins

Mitochondrial dysfunction during acute metabolic crises is considered an important pathomechanism in inherited disorders of propionate metabolism, i.e. propionic and methylmalonic acidurias. Biochemically, these disorders are characterized by accumulation of propionyl-CoA and metabolites of alternative propionate oxidation. In the present study, we demonstrate uncompetitive inhibition of PDHc (pyruvate dehydrogenase complex) by propionyl-CoA in purified porcine enzyme and in submitochondrial particles from bovine heart being in the same range as the inhibition induced by acetyl-CoA, the physiological product and known inhibitor of PDHc. Evaluation of similar monocarboxylic CoA esters showed a chain-length specificity for PDHc inhibition. In contrast with CoA esters, non-esterified fatty acids did not inhibit PDHc activity. In addition to PDHc inhibition, analysis of respiratory chain and tricarboxylic acid cycle enzymes also revealed an inhibition by propionyl-CoA on respiratory chain complex III and alpha-ketoglutarate dehydrogenase complex. To test whether impairment of mitochondrial energy metabolism is involved in the pathogenesis of propionic aciduria, we performed a thorough bioenergetic analysis in muscle biopsy specimens of two patients. In line with the in vitro results, oxidative phosphorylation was severely compromised in both patients. Furthermore, expression of respiratory chain complexes I-IV and the amount of mitochondrial DNA were strongly decreased, and ultrastructural mitochondrial abnormalities were found, highlighting severe mitochondrial dysfunction. In conclusion, our results favour the hypothesis that toxic metabolites, in particular propionyl-CoA, are involved in the pathogenesis of inherited disorders of propionate metabolism, sharing mechanistic similarities with propionate toxicity in micro-organisms.     

Stoichiometric traps in the tricarboxylic acid cycle. I. Self-inhibition and triggering phenomena

The steady-state oxidation of 2 mM pyruvate in pigeon and rat heart mitochondria in the presence of ADP-glucose-hexokinase load can be strongly inhibited by excess (10-40 mM) of pyruvate or beta-hydroxybutyrate. This inhibition is accompanied by the accumulation of alpha-ketoglutarate and a decrease of malate. The mechanism of such substrate inhibition may be associated with the limitation of the tricarboxylic acid cycle flux by low levels of oxaloacetate and free CoA due to their being trapped as alpha-ketoglutarate and acetyl-CoA. Contrary to pyruvate, the ketone bodies in the absence of other substrates produce self-inhibition of their oxidation at as low concentrations as 0.5-1 mM. At 10-15 mM of acetoacetate, a complete suppression of respiration may develop. At a high load (preset by ADP or the uncoupler CCCP), the suppression is characterised by the accumulation of malate and a decrease of alpha-ketoglutarate. At low loads, the reverse distribution of the intermediates takes place. It is concluded that the system of ketone body oxidation in heart mitochondria is an example of biochemical triggers (systems with two alternative stable states).     

Mechanisms of growth inhibition by propionate and restoration of the growth by sodium bicarbonate or acetate in Rhodopseudomonas sphaeroides S

Mechanisms of growth inhibition by propionate on the growth of Rhodopseudomonas sphaeroides were studied. Partially purified pyruvate dehydrogenase complex (PDC) from R. sphaeroides was inhibited by propionyl-CoA, one of the metabolic intermediates of propionate, while propionate itself did not inhibit the enzyme. This suggests that the inhibitor of the growth in vivo is not propionate but propionyl-CoA. The inhibition by propionyl-CoA was competitive with respect to coenzyme A concentration. The K1 value for propionyl-CoA was 0.84 mM. Addition of NaHCO3, which restored the growth of this bacterium in the presence of propionate, increased the rate of propionate incorporation by 1.7-fold and decreased the intracellular level of propionyl-CoA by half. These findings suggest that HCO3-ion lowers the level of propionyl-CoA by accelerating its carboxylation reaction, which is catalyzed by propionyl-CoA carboxylase. Effects of NaHCO3 and acetate on the growth restoration were also studied by the use of propionyl-CoA carboxylase-deficient mutants. NaHCO3 did not restore the growth of the mutants, indicating an essential role of propionyl-CoA carboxylase on the restoration of growth by NaHCO3 as suggested above. Addition of acetate restores the growth of the mutants in the presence of propionate. Acetate probably restores the growth by supplying acetyl-CoA.