Regulation of oxidative decarboxylation of branched-chain 2-oxo acids in rat liver mitochondria

At 0.1 mM 2-oxo[1-14C]isocaproate or 2-oxo[1-14C]isovalerate plots of the reciprocal of the rate of 14CO2 formation by branched-chain 2-oxo acid dehydrogenase complex in mitochondria vs alpha-cyanocinamate concentration were linear up to high inhibitor concentrations, indicating that the monocarboxylate carrier-mediated transport was the rate-limiting step. At low (0.025 mM) concentration of 2-oxo[1-14C]isocaproate or 2-oxo[1-14C]isovalerate the 1/v vs I plots became nonlinear indicating that the branched-chain 2-oxo acid dehydrogenase activity determined the rate of 14CO2 formation. Inhibition of branched-chain 2-oxo acid dehydrogenase complex by clofibric acid or arsenite showed that at 0.1 mM 2-oxoisovalerate the activity of the complex became the rate-limiting step of the pathway. The availability of the 2-oxoisocaproate or 2-oxoisovalerate seems to affect the phosphorylation and the activity of the branched-chain 2-oxo acid dehydrogenase complex only at low, physiological concentrations of these substrates (less than 0.025 mM).     

Interaction of octanoate with branched-chain 2-oxo acid oxidation in rat and human muscle in vitro

Acetate and butanoate inhibited and hexanoate and octanoate increased the 14CO2 production from 0.1 mM [1-14C]-labelled 2-oxoisocaproate (KIC) and 2-oxoisovalerate (KIV) in rat hemidiaphragms. Octanoate increased KIC and KIV oxidation in rat soleus muscle, too, inhibited it in human skeletal muscle and had a divergent effect in rat and human heart slices. In rat hemidiaphragms octanoate primarily affected the process of oxidative decarboxylation. No effect was found on transamination rates of branched-chain amino acids and on the CO2 production beyond alpha-decarboxylation. The reverse transamination of branched-chain 2-oxo acids and their incorporation into protein decreased in the presence of octanoate. Octanoate had no effect on KIC and KIV oxidation at higher 2-oxo acid concentrations and in hemidiaphragms from 3-day-starved rats. The observed interactions are discussed and related to regulatory mechanisms, which are known to affect the branched-chain 2-oxo acid dehydrogenase complex.     

Transamination and oxidation of leucine and valine in rat adipose tissue

Leucine was oxidized by rat adipose tissue at a rate which was not limited by the activity of branched chain amino acid transaminase since high concentrations (10 mM) of [1-14C]leucine and its transamination product, alpha-keto[1-14C]isocaproate, were oxidized at similar rates. Despite the apparent abundance of transaminase activity, however, [1-14C]valine was oxidized at only 10 to 25% of the rate of its transamination product, alpha-keto[1-14C]isovalerate. The net rate at which [1-14C] valine was transaminated by intact tissues was estimated as the sum of the rates of 14CO2 production and alpha-ketoiso[1-14C]valerate release into the medium. Transamination did not limit the rate of valine oxidation since valine was transaminated 3 times as fast as it was oxidized. The rate of valine transamination increased 18-fold when its concentration was raised 100-fold, but the fraction of [1-14C]valine oxidized to 14CO2 remained constant over the range of incubation conditions studied. The oxidation/transamination ratio for leucine was also constant and exceeded the oxidation/transamination ratio for valine unless valine oxidation was stimulated, either by the addition of glucose or leucine. Stimulation of valine oxidation did not increase its transamination but reduced the rate at which alpha-ketoisovalerate was released from the tissue. The faster oxidation of alpha-ketoisocaproate than of alpha-ketoisovalerate may be due to the activation of branched chain alpha-keto acid dehydrogenase by alpha-ketoisocaproate, but the alpha-keto acid oxidation rates do not fully account for the faster transamination of leucine than of valine.     

The metabolic fate of branched-chain amino acids and 2-oxo acids in rat muscle homogenates and diaphragms

After incubation of muscle preparations with [U-14C]branched-chain amino acids or 2-oxo acids, radioactive metabolites were separated, identified and quantified. Homogenates of rat heart and skeletal muscle incubated with 4-methyl-2-oxopentanoate accumulated isovalerate, 3-hydroxyisovalerate and the corresponding carnitine esters. Incubation with 3-methyl-2-oxobutanoate resulted in the production of isobutyrate, 3-hydroxyisobutyrate and their carnitine esters. Addition of L-carnitine increased the production of the esters. The enzymes 3-methylcrotonyl-CoA carboxylase and 3-hydroxyisobutyric acid dehydrogenase apparently are inactive during incubation of muscle homogenates. With liver homogenates the degradation of both 2-oxo acids was more complete. Rat hemidiaphragms incubated with leucine, valine and isoleucine accumulated the corresponding branched-chain 2-oxo acids, fatty acids and hydroxylated fatty acids. The degradation of valine was markedly limited by the release of these metabolites. Considerable amounts (relatively smaller for valine) of radioactivity were also recovered in CO2 and glutamine and glutamate. Incubations with branched-chain 2-oxo acids gave the same radioactive products, except for glutamine and glutamate. Radioactivity was never found in lactate, pyruvate or alanine. These data indicate that the carbon-chains of amino acids entering the citric acid cycle in muscle, are not used for oxidation or for alanine synthesis, but are converted exclusively to glutamine.     

Interaction of various metabolites and agents with branched-chain 2-oxo acid oxidation in rat and human muscle in vitro

The interaction of various metabolites and agents with the 14CO2 production from 0.1 mM [1-14C]-labelled 2-oxoisocaproate (KIC) and 2-oxoisovalerate (KIV) was studied in rat and human heart and skeletal muscle preparations. Glucose and carnitine had no effect in any of the studied systems; palmitate gave a small increase of KIC oxidation only in soleus muscle. With rat hemidiaphragms a considerable decrease was found in the presence of high concentrations of a competitive branched-chain 2-oxo acid and of pyruvate, and in the presence of ketone bodies. A considerable increase was found in the presence of the branched-chain 2-oxo acid dehydrogenase kinase inhibitor 2-chloroisocaproate and the transminase inhibitor amino-oxyacetate. 2-Oxoglutarate increased and clofibric acid decreased only KIC oxidation. Divergent effects were given by intermediates of the degradation route of KIC and KIV and by monocarboxylate translocator inhibitors. The observed interactions are discussed and related to regulatory mechanisms which are known to affect the branched-chain 2-oxo acid dehydrogenase complex.     

Metabolism of valine and 3-methyl-2-oxobutanoate by the isolated perfused rat kidney.

Metabolism of branched-chain amino and 2-oxo acids was studied in the isolated perfused kidney. Significant amounts of 2-oxo acids were released by perfused kidney with all concentrations of amino acids tested (0.1-1.0 mM each), despite the high activity of branched-chain 2-oxo acid dehydrogenase in kidney. As perfusate valine concentration was increased from 0.2 to 1.0 mM, [1-14C]valine transamination (2-oxo acid oxidized + released) increased roughly linearly; [1-14C]valine oxidation, however, increased exponentially. Increasing perfusate concentration of 3-methyl-2-oxo[1-14C]butanoate from 0 to 1.0 mM resulted in a linear increase in the rate of its oxidation and a rise in perfusate valine concentration; at the same time significant decreases occurred in perfusate isoleucine and leucine concentrations, with corresponding increases in rates of release of their respective 2-oxo acids. Comparison of rates of oxidation of [1-14C]valine and 3-methyl-2-oxo[1-14C]butanoate suggests that 2-oxo acid arising from [1-14C]valine transamination has freer access to the 2-oxo acid dehydrogenase than has the 2-oxo acid from the perfusate. The observations indicate that, when branched-chain amino and 2-oxo acids are present in perfusate at near-physiological concentrations, rates of transamination of the amino and 2-oxo acids by isolated perfused kidney are greater than rates of oxidation.     

Adrenergic inhibition of branched-chain 2-oxo acid dehydrogenase in rat diaphragm muscle in vitro.

In theory, the complete oxidation to CO2 of amino acids that are metabolized by conversion into tricarboxylic acid-cycle intermediates may proceed via their conversion into acetyl-CoA. The possible adrenergic modulation of this oxidative pathway was investigated in isolated hemidiaphragms from 40 h-starved rats. Adrenaline (5.5 microM), phenylephrine (0.49 mM) and dibutyryl cyclic AMP (10 microM) inhibited 14CO2 production from 3 mM-[U-14C]valine by 35%, 28% and 19% respectively. At the same time, these agents stimulated glycogen mobilization (measured as a decrease in glycogen content) and glycolysis (measured as lactate release). Adrenaline, phenylephrine and dibutyryl cyclic AMP did not inhibit 14CO2 production from 3 mM-[U-14C]aspartate or 3 mM-[U-14C]glutamate, although, as in the presence of valine, the agents stimulated glycogen mobilization and glycolysis. The rate of proteolysis (measured as tyrosine release in the presence of cycloheximide) was not changed by adrenaline. The data indicate that the adrenergic inhibition of 14CO2 production from [U-14C]valine was not a consequence of radiolabel dilution. Inhibition was apparently specific for branched-chain amino acid metabolism in that the adrenergic agonists also inhibited 14CO2 production from [1-14C]valine, [1-14C]leucine and [U-14C]isoleucine. Since 14CO2 production from the 1-14C-labelled substrates is a specific measure of decarboxylation in the reaction catalysed by the branched-chain 2-oxo acid dehydrogenase complex, it is at this site that the adrenergic agents are concluded to act.     

Transport of 2-oxoisocaproate in isolated hepatocytes and liver mitochondria

The transport of 2-oxoisocaproate into isolated hepatocytes and liver mitochondria of rat was studied using [U-14C]2-oxoisocaproate and the silicone oil filtration procedure. 2-Oxoisocaproate uptake by hepatocytes was composed of: rapid adsorption, unmediated diffusion and carrier-mediated transport. The carrier-mediated transport was strongly inhibited by 4,4'-diisothiocyano-2,2'-stilbenedisulphonic acid and p-chloromercuribenzoate, was less sensitive to alpha-cyano-4-hydroxycinnamate and insensitive to p-chloromercuriphenylsulphonate. Other 2-oxo acids: pyruvate, 2-oxoisovalerate and 2-oxo-3-methylvalerate, were also inhibitory. The kinetic parameters of the carrier-mediated transport were Km 30.6 mM and Vmax 23.4 nmol/min per mg wet wt, at 37 degrees C. It is concluded that at its low, physiological, concentration, 2-oxoisocaproate penetrates the hepatocyte membrane mainly by unmediated diffusion. The uptake of 2-oxoisocaproate by isolated liver mitochondria was partly inhibited by alpha-cyano-4-hydroxycinnamate, the inhibitor of mitochondrial monocarboxylate carrier. The remaining uptake was linearly dependent on 2-oxoisocaproate concentration and represented unmediated diffusion. The carrier-mediated transport exhibited the following kinetic parameters: Km 0.47 mM, Vmax 1.0 nmol/min per mg protein at 6 degrees C; and Km 0.075 mM and Vmax about 8 nmol/min per mg protein at 37 degrees C.     

Interaction of short-chain and branched-chain fatty acids and their carnitine and CoA esters and of various metabolites and agents with branched-chain 2-oxo acid oxidation in rat muscle and liver mitochondria

Interaction of various compounds with the 14CO2 production from [1-14C]-labelled branched-chain 2-oxo acids was studied in intact rat quadriceps muscle and liver mitochrondria. In the absence of carnitine, CoA esters of short-chain and branched-chain fatty acids, CoA and acetyl-L-carnitine stimulated oxidation of 4-methyl-2-oxopentanoate and 3-methyl-2-oxobutanoate in muscle mitochondria. Octanoyl-L-carnitine inhibited oxidation of the latter, but stimulated that of the former substrate. Isovaleryl-L-carnitine was inhibitory with both substrates. Carnitine stimulates markedly 3-methyl-2-oxobutanoate oxidation in liver mitochondria at substrate concentrations higher than 0.1 mM, in contrast to 4-methyl-2-oxopentanoate oxidation. In the presence of carnitine, 3-methyl-2-oxobutanoate oxidation was inhibited in muscle and liver mitochondria by octanoate, octanoyl-L-carnitine and isovaleryl-L-carnitine. The latter ester and octanoyl-D-carnitine inhibited also 4-methyl-2-oxopentanoate oxidation in muscle mitochondria. Branched-chain 2-oxo acids inhibited mutaly their oxidation, except that 3-methyl-2-oxobutanoate did not inhibit 4-methyl-2-oxopentanoate oxidation in liver mitochondria. Their degradation products, isovalerate, 3-methylcrotonate, isobutyrate and 3-hydroxyisobutyrate inhibited to a different extent 2-oxo acid oxidation in liver mitochondria. The effect of CoA esters was studied in permeabilized and with cofactors reinforced mitochondria. Acetyl-CoA and isovaleryl-CoA inhibited only 3-methyl-2-oxobutanoate oxidation in muscle mitochondria. Octanoyl-CoA inhibited oxidation of both 2-oxo acids in muscle and 4-methyl-2-oxopentanoate oxidation in liver mitochondria.(ABSTRACT TRUNCATED AT 250 WORDS)     

The metabolism of 4-methyl-2-oxopentanoate in rat pancreatic islets.

1. Radioactively labelled 4-methyl-2-oxopentanoate was taken up by isolated pancreatic islets in a concentration- and pH-dependent manner and led to the intracellular accumulation of labelled amino acid and to a decrease in the intracellular pH. Uptake of 4-methyl-2-oxopentanoate did not appear to be either electrogenic or Na+-dependent. The islet content of 2-oxo acid radioactivity was not affected by either 2-cyano-3-hydroxy-cinnamate (10mM) or pyruvate (10mM), although both these substances inhibited the oxidation of [U-14C]4-methyl-2-oxopentanoate by islet tissue. 2. 4-Methyl-2-oxopentanoate markedly stimulated islet-cell respiration, ketone-body formation and biosynthetic activity. The metabolism of endogenous nutrients by islets appeared to be little affected by the compound. 3. Studies with the 3H- and 14C-labelled substrate revealed that 4-methyl-2-oxopentanoate was incorporated by islets into CO2, water, acetoacetate, L-leucine and to a lesser extent into islet protein and lipid. Carbon atoms C-2, C-3 and C-4 of the acetoacetate produced were derived from the carbon skeleton of the 4-methyl-2-oxopentanoate, but the acetoacetate carboxy group was derived from the incorporation of CO2. These results, and consideration of the relative rates of 14CO2 and acetoacetate formation from 1-14C-labelled as opposed to U-14C-labelled 4-methyl-2-oxopentanoate, led to the conclusion that the pathway of catabolism of this 2-oxo acid in pancreatic islets is identical with that described in other tissues. The amination of 4-methyl-2-oxopentanoate by islets was attributed to the presence of a branched-chain amino acid aminotransferase (EC 2.6.1.42) activity in the tissue. Although glutamate dehydrogenase activity was demonstrated in islet tissue, the reductive amination of 2-oxoacids did not seem to be of importance in the formation of leucine from 4-methyl-2-oxopentanoate. 4. The results of experiments with respiratory inhibitors and uncouplers, and the finding that 14CO2 production and islet respiration were linked in a 1:1 stoicheiometry suggested that 4-methyl-2-oxopentanoate catabolism was coupled to mitochondrial oxidative phosphorylation. The catabolism of 4-methyl-2-oxopentanoate in islet tissue appeared to be regulated at the level of the initial 2-oxo acid dehydrogenase (EC 1.2.1.25) reaction.     

Glial Metabolism of Valine

The three essential amino acids, valine, leucine and isoleucine, constitute the group of branched-chain amino acids (BCAAs). BCAAs are rapidly taken up into the brain parenchyma, where they serve several distinct functions including that as fuel material in brain energy metabolism. As one function of astrocytes is considered the production of fuel molecules that support the energy metabolism of adjacent neural cells in brain. Astroglia-rich primary cultures (APC) were shown to rapidly dispose of the BCAAs, including valine, contained in the culture medium. While the metabolisms of leucine and isoleucine by APC have already been studied in detail, some aspects of valine metabolism remained to be determined. Therefore, in the present study an NMR analysis was performed to identify the ¹³C-labelled metabolites that are generated by APC during catabolism of [U-¹³C]valine and that are subsequently released into the incubation medium. The results presented show that APC (1) are potently disposing of the valine contained in the incubation medium; (2) are capable of degrading valine to the tricarboxylic acid (TCA) cycle member succinyl-CoA; and (3) release into the extracellular milieu valine catabolites and compounds generated from them such as [U-¹³C]2-oxoisovalerate, [U-¹³C]3-hydroxyisobutyrate, [U-¹³C]2-methylmalonate, [U-¹³C]isobutyrate, and [U-¹³C]propionate as well as several TCA cycle-dependent metabolites including lactate. This article is dedicated to Dr. George DeVries.     

GDP binding to brown-adipose-tissue mitochondria of mice treated chronically with corticosterone.

A procedure is described to convert rates of (14)CO(2) production into rates of mitochondrial acetyl-CoA production from a (14)C-labelled substrate. The principle is illustrated in perfused rat liver and kidney by the differential yield of (14)CO(2) from 4-methyl-2-oxo[1-(14)C]valerate and 4-methyl-2-oxo[2-(14)C]valerate.     

Modulation of branched-chain amino acid oxidation in rat hemidiaphragms in vitro by glucose and ketone bodies

Branched-chain amino acid metabolism in hemidiaphragms from 40 h-starved rats is influenced by the provision of glucose as co-substrate. Glucose inhibits 14CO2 production from [l-14C]valine and [U-14C]valine but stimulates 14CO2 production from [l-14C]leucine, [U-14C]leucine and [U-14C]isoleucine. In the presence of glucose, ketone bodies inhibit alanine release and 14CO2 production from [l-14C]valine, [l-14C]leucine and [U-14C]isoleucine, but inhibition is not observed in the absence of glucose as cosubstrate. Glucose-dependent inhibition by ketone bodies of branched-chain amino acid oxidation via inhibition of the branched-chain 2-oxo acid dehydrogenase complex or branched-chain amino acid aminotransferase may account in part for the reported hypoalanaemic action of ketone bodies in vivo.     

Metabolism of branched-chain amino acids in starved rats: the role of hepatic tissue

Parameters of branched-chain amino acids (BCAA; leucine, isoleucine and valine) and protein metabolism were evaluated using L-[1-(14)C]leucine and alpha-keto[1-(14)C]isocaproate (KIC) in the whole body and in isolated perfused liver (IPL) of rats fed ad libitum or starved for 3 days. Starvation caused a significant increase in plasma BCAA levels and a decrease in leucine appearance from proteolysis, leucine incorporation into body proteins, leucine oxidation, leucine-oxidized fraction, and leucine clearance. Protein synthesis decreased significantly in skeletal muscle and the liver. There were no significant differences in leucine and KIC oxidation by IPL. In starved animals, a significant increase in net release of BCAA and tyrosine by IPL was observed, while the effect on other amino acids was non-significant. We conclude that the protein-sparing phase of uncomplicated starvation is associated with decreased whole-body proteolysis, protein synthesis, branched-chain amino acid (BCAA) oxidation, and BCAA clearance. The increase in plasma BCAA levels in starved animals results in part from decreased BCAA catabolism, particularly in heart and skeletal muscles, and from a net release of BCAA by the hepatic tissue.     

Acute regulation of the branched-chain 2-oxo acid dehydrogenase complex by adrenaline and glucagon in the perfused rat heart.

Rates of transamination and decarboxylation of [1-14C]leucine at a physiological concentration (0.1 mM) were measured in the perfused rat heart. In hearts from fasted rats, metabolic flux through the branched-chain 2-oxo acid dehydrogenase reaction was low initially, but increased gradually during the perfusion period. The increase in 14CO2 production was accompanied by an increase in the amount of active branched-chain 2-oxo acid dehydrogenase complex present in the tissue. In hearts from rats fed ad libitum, extractable branched-chain dehydrogenase activity was low initially, but increased rapidly during perfusion, and high rates of decarboxylation were attained within the first 10 min. Infusion of glucagon, adrenaline, isoprenaline, or adrenaline in the presence of phentolamine all produced rapid, transient, inhibition (40-50%) of the formation of 4-methyl-2-oxo[1-14C]pentanoate and 14CO2 within 1-2 min, but the specific radioactivity of 4-methyl-2-oxo[14C]pentanoate released into the perfusate remained constant. Glucagon and adrenaline infusion also resulted in transient decreases (16-24%) in the amount of active branched-chain 2-oxo acid dehydrogenase. In hearts from fasted animals, infusion for 10 min of adrenaline, phenylephrine, or adrenaline in the presence of propranolol, but not infusion of glucagon or isoprenaline, stimulated the rate of 14CO2 production 3-fold, and increased 2-fold the extractable branched-chain 2-oxo acid dehydrogenase activity. These results demonstrate that stimulation of glucagon or beta-adrenergic receptors in the perfused rat heart causes a transient inhibition of branched-chain amino acid metabolism, whereas alpha-adrenergic stimulation causes a slower, more sustained, enhancement of branched-chain amino acid metabolism. Both effects reflect interconversion of the branched-chain 2-oxo acid dehydrogenase complex between active and inactive forms. Also, these studies suggest that the concentration of branched-chain 2-oxo acid available for decarboxylation can be regulated by adrenaline and glucagon.     

Amino acid catabolism by perfused rat hindquarter. The metabolic fates of valine.

Hindquarters from starved rats were perfused with plasma concentrations of amino acids, but without other added substrates. Release of amino acids was similar to that previously reported, but, if total amino acid changes were recorded, alanine and glutamine were not formed in excess of their occurrence in muscle proteins. In protein balance (excess insulin) there was no net formation of either alanine or glutamine, even though the branched-chain amino acids and methionine were consumed. If [U-14C]valine was present, radiolabelled 3-hydroxyisobutyrate and, to a lesser extent, 2-oxo-3-methylbutyrate accumulated and radiolabel was incorporated into citrate-cycle intermediates and metabolites closely associated with the citrate cycle (glutamine and glutamate, and, to a smaller extent, lactate and alanine). If a 2-chloro-4-methylvalerate was present to stimulate the branched-chain oxo acid dehydrogenase, flux through this step was accelerated, resulting in increased accumulation of 3-hydroxyisobutyrate, decreased accumulation of 2-oxo-3-methylbutyrate, and markedly increased incorporation of radiolabel (specific and total) into all measured metabolites formed after 3-hydroxyisobutyrate. It is concluded that: amino acid catabolism by skeletal muscle is confined to degradation of the branched-chain amino acids, methionine and those that are interconvertible with the citrate cycle; amino acid catabolism is relatively minor in supplying carbon for net synthesis of alanine and glutamine; and partial degradation products of the branched-chain amino acids are quantitatively significant substrates released from muscle for hepatic gluconeogenesis. For valine, 3-hydroxyisobutyrate appears to be quantitatively the most important intermediate released from muscle. A side path for inter-organ disposition of the branched-chain amino acids is proposed.     

Peroxisomal degradation of branched-chain 2-oxo acids

Branched-chain 2-oxo acids which are formed by transamination of leucine, isoleucine, and valine are metabolized by the peroxisomes from mung bean (Vigna radiata L.) hypocotyls. Acylcoenzyme A (CoA) thio ester intermediates of the pathways were separated by reversed-phase high performance liquid chromatography. Retention time and cochromatography of individual acyl-CoA reference standards were used for identification of the acyl-CoA esters separated from the assay mixtures. Based on the results of identification and those of kinetic experiments, pathways of the peroxisomal degradation of 2-oxoisocaproate, 2-oxoisovalerate, and 2-oxo-3-methylvalerate are suggested.     

The biosynthesis of valine from isobutyrate by Peptostreptococcus elsdenii and Bacteroides ruminicola

1. Growing cultures of Peptostreptococcus elsdenii and Bacteroides ruminicola incorporate (14)C from [1-(14)C]isobutyrate into the valine of cell protein. With P. elsdenii some of the (14)C is also incorporated into leucine. 2. Crude cell-free extracts of both organisms in the presence of glutamine, carbon dioxide and suitable sources of energy and electrons incorporate (14)C from [1-(14)C]isobutyrate into valine but not into leucine. 3. With extracts of P. elsdenii treated with DEAE-cellulose the reaction is dependent on ATP, CoA, thiamin pyrophosphate, molecular hydrogen and a low-potential electron carrier (ferredoxin, flavodoxin or benzyl viologen). 4. The same extracts incorporate (14)C from NaH(14)CO(3) into valine in the presence of isobutyrate plus ATP, CoA, glutamine and ferredoxin; isobutyryl-CoA or isobutyryl phosphate plus CoA will replace the isobutyrate plus CoA and ATP. With acetyl phosphate in place of isobutyryl phosphate, (14)C is incorporated into alanine. With isovalerate or 2-methylbutyrate in place of isobutyrate, (14)C is incorporated into leucine and isoleucine respectively. 5. When carrier 2-oxoisovalerate is added to the carboxylating system (14)C from [1-(14)C]isobutyrate passes into the oxo acid fraction. 6. It is concluded that these two organisms form valine from isobutyrate by the sequence isobutyrate-->isobutyryl-CoA-->2-oxoisovalerate-->valine and that the reductive carboxylation of isobutyrate is catalysed by a system similar to the pyruvate synthetase of clostridia and photosynthetic bacteria.     

The effect of octanoate and palmitate on the metabolism of valine in perfused hindquarter of rat.

The effect of octanoate and palmitate on the oxidation of 14C-labelled valine has been studied in perfused hindquarter of rat. 1. The oxidation rate of valine increases 30 times when the concentration of valine is increased from 0.1 mM to 5 mM. 2. Octanoate at a 5 mM concentration effected a 10-fold increase in the flux through the alpha-ketoisovalerate dehydrogenase step and a 5-fold increase at 0.5 mM concentration. 3. Palmitate (1 mM) effects only a moderate increase in the valine oxidation. 4. With no octanoate there was a great accumulation of alpha-ketoisovalerate in both the muscle and the perfusion medium. 5. With octanoate little alpha-ketoisovalerate accumulated whereas 3-hydroxyisobutyrate was found in high concentration both in the muscle and in the medium. 6. Octanoate stimulated the production of citric-acid-cycle intermediates and lactate. 7. The results are discussed in relation to valine metabolism in the body.     

Regulation of hepatic glutamate metabolism. Role of 2-oxoacids in glutamate release from isolated perfused rat liver

In isolated perfused rat liver, addition of the oxoanalogues of leucine, isoleucine, methionine and phenylalanine is followed by a rapid and reversible stimulation of glutamate release. This is not observed with the corresponding amino acids or 2-oxoisovalerate, 2-oxoglutarate or oxaloacetate. The increased glutamate release by the liver is accompanied by a decrease in the tissue contents of 2-oxoglutarate and glutamate by about 25% and 50%, respectively. During the metabolism of glutamine, i.e. conditions with elevated tissue glutamate concentrations, 2-oxoacid-induced glutamate release is stimulated. In the presence of glutamine (5 mM), 2-oxoisocaproate, 2-oxo-4-methylvalerate and 2-oxo-4-methylthiobutyrate were found to be most effective and glutamate release by the liver increased linearly from about 80 nmol g-1 min-1 to 600 nmol g-1 min-1 at increasing 2-oxoacid concentrations up to 1 mM. When glutamate tissue levels were decreased by phenylephrine, stimulation of glutamate release by 2-oxoisocaproate was markedly diminished. 2-Oxoacid-stimulated glutamate release is independent of oxoacid metabolism, indicating that the effect is probably not explained by a 2-oxoacid/glutamate exchange across the liver plasma membrane. 2-Oxoacid-induced glutamate export predominantly occurs in a sodium-independent way. At low concentrations of 2-oxoisocaproate (below 0.2 mM), the increased glutamate release was accompanied by a slight inhibition of 14CO2 production from added [14C]glutamate, indicating a simultaneous glutamate uptake and release also under these conditions. Stimulation of glutamate release by 2-oxoisocaproate is followed by a decreased rate of urea and glutamine synthesis from portal ammonia, as a consequence of an increased glutamate release.