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)     

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.     

Alanine and inter-organ relationships in branched-chain amino and 2-oxo acid metabolism

Branched-chain amino acid metabolism in skeletal muscte promotes the production of alanine, an important precursor in hepatic gluconeogenesis. There is controversy concerning the origin of the carbon skeleton of alanine produced in muscle, specifically whether it is derived from carbohydrate via glycolysis (the glucose-alanine cycle) or from amino acid precursors (viz. glutamate, valine, isoleucine, methionine, aspartate, asparagine) via a pathway involving phosphoenolpyruvate (PEP) carboxykinase and pyruvate kinase, or NADP-malate dehydrogenase (malic enzyme). The relevant literature is reviewed and it is concluded that neogenic flux from amino acids is unlikely to be of major quantitative importance for provision of the carbon skeleton of alanine either in vitro or in vivo. Evidence is presented that branched-chain amino acid oxidation in muscle is incomplete and that the branched-chain 2-oxo acids and the products of their partial oxidation (including glutamine) are released. The role of these metabolites is discussed in the context of fuel homeostasis in starvation.     

Specificity of the effects of leucine and its metabolites on protein degradation in skeletal muscle.

The effects of leucine, its metabolites, and the 2-oxo acids of valine and isoleucine on protein synthesis and degradation in incubated limb muscles of immature and adult rats were tested. Leucine stimulated protein synthesis but did not reduce proteolysis when leucine transamination was inhibited. 4-Methyl-2-oxopentanoate at concentrations as low as 0.25 mM inhibited protein degradation but did not change protein synthesis. The 2-oxo acids of valine and isoleucine did not change protein synthesis or degradation even at concentrations as high as 5 mM. 3-Methylvalerate, the irreversibly decarboxylated product of 4-methyl-2-oxopentanoate, decreased protein degradation at concentrations greater than or equal to 1 mM. This was not due to inhibition of 4-methyl-2-oxopentanoate catabolism, because 0.5 mM-3-methylvalerate did not suppress proteolysis, even though it inhibited leucine decarboxylation by 30%; higher concentrations of 3-methylvalerate decreased proteolysis progressively without inhibiting leucine decarboxylation further. During incubation with [1-14C]- and [U-14C]-leucine, it was found that products of leucine catabolism formed subsequent to the decarboxylation of 4-methyl-2-oxopentanoate accumulated intracellularly. This pattern was not seen during incubation with radiolabelled valine. Thus, the effect of leucine on muscle proteolysis requires transamination to 4-methyl-2-oxopentanoate. The inhibition of muscle protein degradation by leucine is most sensitive to, but not specific for, its 2-oxo acid, 4-methyl-2-oxopentanoate.     

Increase of the activity state and loss of total activity of the branched-chain 2-oxo acid dehydrogenase in rat diaphragm during incubation.

Actual and total branched-chain 2-oxo acid dehydrogenase activities were determined in homogenates of incubated diaphragms from fed and starved rats. Incubation in Krebs-Ringer buffer increased the activity state, but caused considerable loss of total activity. Palmitate oxidation rates and citrate synthase activities did not significantly change on incubation. Starved muscles showed a higher extent of activation after 15 min of incubation (not after 30 and 60 min) and a smaller loss of total activity. Experiments with the transaminase inhibitor amino-oxyacetate confirm that the contribution of endogenous amino acids to the oxidation precursor pool is also smaller in diaphragms from starved rats on incubation in vitro. These phenomena together cause the higher 14CO2 production from 14C-labelled branched-chain amino acids and 2-oxo acids in muscles from starved than from fed rats. High concentrations of branched-chain 2-oxo acids, and the presence of 2-chloro-4-methyl-pentanoate, octanoate or ketone bodies, increase the extent of activation of the dehydrogenase complex; glucose and pyruvate had no effect. The observed changes of the activity state by these metabolites are discussed in relation to their interaction with branched-chain 2-oxo acid oxidation in incubated hemidiaphragms.     

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.     

Metabolism of valine and the exchange of amino acids across the hind-limb muscles of fed and starved sheep

A combination of the isotope-dilution and arterio-venous (AV) difference techniques was used to study simultaneously the metabolism of valine in the whole body and in the hind-limb muscles of fed and starved (40 h) sheep. The net exchange of gluconeogenic amino acids across hind-limb muscles was also studied. Valine entry rate was unaffected by nutritional status. There was significant extraction of valine by hind-limb muscles in both fed and starved sheep. The percentage of valine uptake decarboxylated was higher (P less than 0.05) in fed sheep but the amount of valine decarboxylated was not significantly different. The proportion of valine uptake that was transaminated was about 30 times higher in starved sheep. About 54% of valine taken up by hind-limb muscle of starved sheep was metabolized. The corresponding value for fed sheep was 21%. The contribution of CO2 from valine decarboxylation to total hind-limb muscle CO2 output was about 0.2%. The output of alanine in both fed and starved sheep was low but the output of glutamine was relatively high and roughly equivalent to the amounts of aspartate, glutamate and branched-chain amino acids that were catabolized. This study has confirmed that valine is catabolized in sheep skeletal muscle, and shown that glutamine is a major carrier of amino nitrogen out of muscle.     

Leucine degradation in cell-free extracts of skeletal muscle.

Since skeletal muscle is the major site in the body for oxidation of leucine, isoleucine and valine, the pathway and control of leucine oxidation were investigated in cell-free preparations of rat muscle. Leucine was found to be transaminated to 4-methyl-2-oxopentanoate, which was then oxidatively decarboxylated. On differential centrifugation 70--80% of the transaminase activity was recovered in the soluble fraction of the cell, and the remaining amount in the mitochondrial fraction. The transaminase, from both fractions had similar pH optima and both were markedly inhibited by Ca2+. Thus changes in cellular Ca2+ concentration may regulate transaminase activity. Both transaminases had a much higher affinity for 2-oxoglutarate than for pyruvate. Therefore the utilization of amino groups from leucine for the biosynthesis of alanine in muscle [Odessey, Khairallah & Goldberg (1974) J. Biol. Chem. 249, 7623--7629] in vivo involves transamination with 2-oxoglutarate to produce glutamate, which is then transaminated with pyruvate to produce alanine. The dehydrogenase activity assayed by the decarboxylation of methyl-2-oxo[1-14C]pentanoate was localized exclusively in the fraction containing mitochondria and required NAD+, CoA and thiamin pyrophosphate for optimal activity. Measurements of competitive inhibition suggested that the oxo acids of leucine, isoleucine and valine are all decarboxylated by the same enzyme. The enzyme activity was decreased by 90% upon freezing or sonication and was stimulated severalfold by Mg2+, K+ and phosphate ions. In addition, it was markedly inhibited by ATP, but not by non-metabolizable analogues. This observation suggests that splitting of ATP is required for inhibition. The oxidative decarboxylation of 4-methyl-2-oxopentanoate by the dehydrogenase appears to be the rate-limiting step for leucine oxidation in muscle homogenates and also in intact tissues. In fact, rat muscles incubated with [1-14C]leucine release 1-14C-labelled oxo acid into the medium at rates comparable with the rate of decarboxylation. Intact muscles also released the oxo acids of [1-14C]valine or [1-14C]isoleucine, but not of other amino acids. These findings suggest that muscle is the primary source of the branched-chain oxo acids found in the blood.     

The effect of acylcarnitines on the oxidation of branched chain alpha-keto acids in mitochondria

The oxidation of 14C-labelled branched-chain alpha-keto acids corresponding to the branched-chain amino acids valine, isoleucine and leucine has been studied in isolated mitochondria from heart, liver and skeletal muscle. 1. Heart and liver mitochondria have similar capacities to oxidize these alpha-keto acids based on protein content. Skeletal muscle mitochondria also show significant activity. 2. Half maximum rates are obtained with approximately 0.1 mM of the alpha-keto acids under optimal conditions. Added NAD and CoA had no effect on the oxidation rate, showing that endogenous mitochondrial NAD and CoA are required for the oxidation. 3. Addition of carnitine esters of fatty acids (C6--C16), succinate, pyruvate, or alpha-ketoglutarate inhibited the oxidation of the branched chain alpha-keto acids, especially in a high-energy state (no ADP added). In heart mitochondria the addition of AD (low-energy state) decreased the inhibitory effects of acylcarnitines of medium chain length or of pyruvate, and abolished the inhibitory effect of succinate. It is suggested that the oxidation rate is regulated mainly by the redox state of the mitochondria under the conditions used. 4. The results are discussed in relation to the regulation of branched-chain amino acid metabolism in the body.     

Alanine and glutamine synthesis and release from skeletal muscle. II. The precursor role of amino acids in alanine and glutamine synthesis.

The synthesis and release of alanine and glutamine have been studied in the intact rat epitrochlaris skeletal muscle preparation. Aspartate, cysteine, leucine, valine, methionine, isoleucine, serine, theronine, and glycine increased significantly the formation and release of alanine from muscle. Cysteine, leucine, valine, methionine, isoleucine, tyrosine, lysine, and phenylalanine increased the rate of glutamine synthesis. Only ornithine, arginine, and tryptophan were without effect on the synthesis of either alanine or glutamine. Half-maximal stimulation of alanine and glutamine formation by added amino acids was observed with concentrations ranging between 0.5 and 1.0 mM. Increases in alanine and glutamine formation were not accompanied by changes in pyruvate production or glucose uptake. The progressive decline in alanine and glutamine synthesis noted on prolonged incubation was prevented by the addition of amino acids to the incubation medium. Stimulation of alanine synthesis by added amino acids was unaffected by inhibition of glycolysis with iodoacetate. Inhibition of alanine aminotransferase with aminooxyacetate significantly decreased alanine formation. Pyruvate and ammonium chloride did not increase further the rate of either alanine or glutamine formation above that produced by added amino acids. These data indicate that most amino acids are precursors for alanine and glutamine synthesis in skeletal muscle. A general mechanism is presented for the de novo formation of alanine from amino acids in skeletal muscle, and the importance of proteolysis for the supply of amino acid precursors for alanine and glutamine synthesis is discussed.     

The differential transport of amino acids into the phloem of Ricinus communis L. seedlings as shown by the analysis of sieve-tube sap

The cotyledons of castor bean (Ricinus communis L.) act as absorption organs for amino acids, which are supplied to the medium. The analysis of the sieve-tube sap, which exudes from the cut hypocotyl, demonstrated the ability of the cotyledons to load particular amino acids into the phloem and to reject the loading of others. The sieve-tube sap of cotyledons, which were embedded in the endosperm, contained 150 mM amino acids, with 50 mM glutamine as the major amino acid, and 10–15 mM each of valine, isoleucine, lysine and arginine. Removal of the endosperm led to a drastic decline in the amino-acid content of sieve-tube sap down to 16 mM. Addition of single amino acid species to the medium increased the amino acid concentration in the sieve-tube sap in specific manner: glutamine caused the largest increase (up to 140 mM in exudate), glutamate and alanine smaller increases (up to 60 mM), and arginine the smallest. In addition, the amino acid composition of the sieve-tube sap changed, for instance, glutamine or alanine readily appeared in the sieve-tube sap upon incubation in glutamine or alanine, respectively, whereas glutamate was hardly discernible even in the case of incubation with glutamate; arginine was loaded into the sieve tubes only reluctantly. In general, glutamine and alanine accumulated four- to tenfold in the sieve tubes. The uptake of amino acids and of sucrose into the sieve tubes was interdependent: the loading of sucrose strongly reduced the amino acid concentration in the sieve-tube exudate and loading of amino acids decreased the sucrose concentration. Comparison of the concentrations of various amino acids on their way from the endosperm via the cotyledon-endosperm interface, through the cotyledons and into the sieve tubes showed that glutamine, valine, isoleucine and lysine are accumulated on this pathway, whereas glutamate and arginine are more concentrated in the cotyledons than in the sieve tubes. Obviously the phloem-loading system has a transport specificity different from that of the amino acid uptake system of the cotyledon in general and it strongly discriminates between amino acids within the cotyledons.     

Effects of ketone bodies on amino acid metabolism in isolated rat diaphragm.

1. Diaphragms from 48h-starved rats were incubated in Krebs-Ringer bicarbonate medium at 37degreesC for 30min and then transferred into new medium and incubated for 1, 2 and 3 h. 2. The amount of free amino acids found at the end of each time of incubation was larger than the amount at the beginning of incubation, indicating that in this system proteolysis is prevailing. 3. The diaphragms was releasing mainly alanine and glutamine into the incubation medium. 4. Within the periods of incubation the release and metabolism of free amino acids was proceeding at a constant rate. 5. Addition of sodium DL-3-hydroxybutyrate decreased the tissue content of several amino acids, among which were tyrosine and phenylalanine, suggesting that proteolysis was decreased by ketone bodies. 6. In the presence of glucose (10mM) and branched-chain amino acids (0.5mM), sodium DL-3-hydroxybutyrate at concentrations of 4 or 6 mM resulted in 30% decrease in tissue alanine content and a 20% decline in alanine release. Release of taurine and glutamine was decreased by 19 and 16% respectively with 6 mM-sodium DL-3-hydroxybutyrate. Addition of sodium acetoacetate (1-3mM) also resulted in a 20-35% decrease in tissue content of alanine, glutamine and taurine and in a 15-24% decrease of alanine and glutamine release. Smaller decreases (less than 15%) in the release of glycine, threonine, proline, serine and aspartate were also observed in the presence of sodium DL-3-hydroxybutyrate or sodium acetoacetate. 7. Substitution of pyruvate (1.0mM) for glucose in the presence of acetoacetate restored alanine and glutamine production to control values. In the presence of acetoacetate, pyruvate also increased the tissue content of aspartate by 77% and decreased the tissue content of glutamate by 30%. 8. It is suggested that in diaphragms from starved rats, ketone bodies (a) in the absence of other substrates inhibit protein catabolism and (b) in the presence of glucose and branched-chain amino acids decrease alanine and glutamine production, by inhibiting glycolysis.     

Formation of alanine and glutamine in chick (Gallus domesticus) skeletal muscle

1. Incubation of extensor digitorum communis muscles from fed chicks in the presence of plasma concentrations of branched-chain amino acids (BCAA) increased the formation of glutamate, glutamine and alanine. This effect was inhibited by 1.5 mM L-cycloserine. 2. 2-Oxoisocaproate (0.1 and 0.5 mM) increased the formation of leucine but decreased that of glutamate, glutamine and alanine. 3. NH4Cl (0.3 mM) increased the formation of glutamine, and decreased the release and intracellular concentrations of glutamate and alanine. 4. Our results demonstrate that alanine and glutamine are synthesized de novo in chick skeletal muscle and demonstrate the similarity in alanine and glutamine synthesis in skeletal muscle between the domestic fowl and mammals.     

Regulatory effects of fatty acids on decarboxylation of leucine and 4-methyl-2-oxopentanoate in the perfused rat heart.

The regulatory effects of fatty acids on the oxidative decarboxylation of leucine and 4-methyl-2-oxopentanoate were investigated in the isolated rat heart. Infusion of the long-chain fatty acid palmitate resulted in both an inactivation of the branched-chain 2-oxo acid dehydrogenase and an inhibition of the measured metabolic flux through this enzyme complex. Pyruvate addition also caused both an inactivation and an inhibition of the flux through the complex. On the other hand, the medium-chain fatty acid octanoate caused an activation of and a stimulation of flux through the branched-chain 2-oxo acid dehydrogenase when the perfusion conditions before octanoate addition maintained the enzyme complex in its inactive state. When the enzyme complex was activated before octanoate infusion, this fatty acid caused a significant inhibition of the flux through the branched-chain 2-oxo acid dehydrogenase reaction. Inclusion of glucose in the perfusion medium prevented the octanoate-mediated activation of the branched-chain 2-oxo acid dehydrogenase.     

Factors regulating amino acid release from extrasplanchnic tissues in the rat. Interactions of alanine and glutamine.

1. Factors regulating the release of alanine and glutamine in vivo were investigated in starved rats by removing the liver from the circulation and monitoring blood metabolite changes for 30 min. 2. Alanine and glutamine were the predominant amino acids released into the circulation in this preparation. 3. Dichloroacetate, an activator of pyruvate dehydrogenase, inhibited net alanine release: it also interfered with the metabolism of the branched-chain amino acids valine, leucine and isoleucine. 4. L-Cycloserine, an inhibitor of alanine aminotransferase, decreased alanine accumulation by 80% after functional hepatectomy, whereas methionine sulphoximine, an inhibitor of glutamine synthetase, decreased glutamine accumulation by the same amount. 5. It was concluded that: (a) the alanine aminotransferase and the glutamine synthetase pathways respectively were responsible for 80% of the alanine and glutamine released into the circulation by the extrasplanchnic tissues, and extrahepatic proteolysis could account for a maximum of 20%; (b) alanine formation by the peripheral tissues was dependent on availability of pyruvate and not of glutamate; (c) glutamate availability could influence glutamine formation subject, possibly, to renal control.     

Phosphorylation of branched-chain 2-oxo acid dehydrogenase complex in isolated adipocytes. Effects of 2-oxo acids.

Isolated adipocytes from rat epididymal fat-pads were incubated with [32P]Pi, and intracellular phosphoproteins were then analysed by SDS/polyacrylamide-gel electrophoresis and autoradiography. A phosphorylated polypeptide of apparent Mr 46,000 was identified as the alpha-subunit of branched-chain 2-oxo acid dehydrogenase complex by immunoprecipitation using antiserum raised against the homogeneous E1 component of branched-chain 2-oxo acid dehydrogenase complex. Immunoprecipitation of this phosphoprotein is blocked in a competitive manner by purified branched-chain 2-oxo acid dehydrogenase complex. Peptide mapping of the isolated phosphoprotein indicates that two sites on the polypeptide are phosphorylated in the intact cells. Addition of branched-chain 2-oxo acids to the incubation medium causes diminution in the extent of labelling of both phosphorylation sites on the alpha-subunit, an effect presumably mediated via their known inhibitory action on branched-chain 2-oxo acid dehydrogenase kinase. These observations provide direct evidence for phosphorylation of branched-chain 2-oxo acid dehydrogenase complex in intact cells.     

Developmental changes in rat liver branched-chain 2-oxo acid dehydrogenase.

Branched-chain 2-oxo acid dehydrogenase catalyses the first irreversible step in the degradation of the branched-chain amino acids leucine, isoleucine and valine. With specifically labelled 4-methyl-2-oxo[1-14C]pentanoate as substrate, the enzyme's activity was measured in rat liver homogenates. Activity (per g wet wL of liver or per mg of protein) increased most rapidly during the perinatal period (2 days before to 1 day after birth), reaching approximately adult values by the time of weaning. The apparent Vmax, of the enzyme increased with age, but its Km appeared unchanged. The data suggest that hepatic branched-chain 2-oxo acid dehydrogenase is induced or activated during the perinatal period. The enzyme's activity at birth was unaffected by maternal diabetes, or by treating the mother with pharmacological doses of corticosterone or 3,3',5-tri-iodothyronine, during the last 5 days of pregnancy.     

Amino acid metabolism by perfused rat hindquarter. Effects of insulin, leucine and 2-chloro-4-methylvalerate.

Hindquarters from starved rats were perfused without substrates but in the presence of an O2- and CO2-carrying perfluorocarbon emulsion to evaluate principally the metabolism of individual endogenous and protein-derived amino acids by this muscle preparation. This experimental model was shown, by a battery of metabolite measurements, to maintain cellular homoeostasis for at least 2h. The net appearance of most amino acids closely approximated their frequency of occurrence in muscle proteins, showing that they are not significantly metabolized. Exceptions were the branched-chain amino acids, methionine and those amino acids that are interconvertible with intermediates of the citrate cycle and pyruvate through coupled transaminations. The evidence indicates that only valine, isoleucine, aspartate and probably methionine can be catabolized by skeletal muscle to provide carbon precursors for glutamate/glutamine and alanine that are formed de novo by protein-catabolic muscle. The protein-sparing effects of insulin and leucine were confirmed. Although each decreased proteolysis and the net appearance of free amino acids, they were generally without effect on the ratios of amino acids formed. 2-Chloro-4-methylvalerate selectively stimulated the removal rate for the branched-chain amino acids, confirming the idea that the branched-chain oxo acid dehydrogenase normally limits the rate of their oxidation by muscle. It is also concluded that, since alanine was not formed in excess of that found in muscle proteins when no glucose was added as substrate, the excess of alanine (carbon) released from muscles in other studies is derived to a large extent, but not exclusively, from preformed carbohydrate.     

Effects of branched-chain amino acids on muscles under hyperammonemic conditions

The aim was to determine the effects of enhanced availability of branched-chain amino acids (BCAAs; leucine, isoleucine, and valine) on ammonia detoxification to glutamine (GLN) and protein metabolism in two types of skeletal muscle under hyperammonemic conditions. Isolated soleus (SOL, slow-twitch) and extensor digitorum longus (EDL, fast-twitch) muscles from the left leg of white rats were incubated in a medium with 1 mM ammonia (NH3 group), BCAAs at four times the concentration of the controls (BCAA group) or high levels of both ammonia and BCAA (NH3 + BCAA group). The muscles from the right leg were incubated in basal medium and served as paired controls. L-[1-¹⁴C]leucine was used to estimate protein synthesis and leucine oxidation, and 3-methylhistidine release was used to evaluate myofibrillar protein breakdown. We observed decreased protein synthesis and glutamate and α-ketoglutarate (α-KG) levels and increased leucine oxidation, GLN levels, and GLN release into medium in muscles in NH3 group. Increased leucine oxidation, release of branched-chain keto acids and GLN into incubation medium, and protein synthesis in EDL were observed in muscles in the BCAA group. The addition of BCAAs to medium eliminated the adverse effects of ammonia on protein synthesis and adjusted the decrease in α-KG found in the NH3 group. We conclude that (i) high levels of ammonia impair protein synthesis, activate BCAA catabolism, enhance GLN synthesis, and decrease glutamate and α-KG levels and (ii) increased BCAA availability enhances GLN release from muscles and attenuates the adverse effects of ammonia on protein synthesis and decrease in α-KG.     

Inhibition by the branched-chain 2-oxo acids of the 2-oxoglutarate dehydrogenase complex in developing rat and human brain

1. The effect of the branched-chain amino acids, namely leucine, isoleucine and valine and their corresponding 2-oxo acids on the metabolism of 2-oxoglutarate by developing rat and human brain preparations was investigated. 2. The decarboxylation of 2-oxo[1-(14)C]glutarate to (14)CO(2) by mitochondria from adult rat brain was inhibited by the branched-chain 2-oxo acids whereas the branched-chain amino acids had no inhibitory effect on this process. 3. The activity of 2-oxoglutarate dehydrogenase complex was about 0.2unit/g of brain from 2-day-old rats and increased by about fourfold reaching an adult value by the end of the third postnatal week. 4. The K(m) value for 2-oxoglutarate of the 2-oxoglutarate dehydrogenase complex in rat and human brain was 100 and 83mum respectively. 5. The branched-chain 2-oxo acids competitively inhibited this enzyme from suckling and adult rats brains as well as from foetal and adult human brains, whereas the branched-chain amino acids had no effect on this enzyme. 6. Approximate K(i) values for the branched-chain 2-oxo acids found for this enzyme were in the range found for these 2-oxo acids in plasma from patients with maple-syrup-urine disease. 7. The possible significance of the inhibition by the branched-chain 2-oxo acids of the 2-oxoglutarate dehydrogenase complex in brains of untreated patients with maple-syrup-urine disease is discussed in relation to the energy metabolism and the biosynthesis of lipids from ketone bodies.