Activation of rat liver branched-chain 2-oxo acid dehydrogenase in vivo by glucagon and adrenaline.

The activity of liver branched-chain 2-oxo acid dehydrogenase complex was measured in rats fed on low-protein diets and given adrenaline, glucagon, insulin or dibutyryl cyclic AMP in vivo. Administration of glucagon or adrenaline (200 micrograms/100 g body wt.) resulted in a 4-fold increase in the percentage of active complex. As with glucagon and adrenaline, treatment of rats with cyclic AMP (5 mg/100 g body wt.) resulted in marked activation of branched-chain 2-oxo acid dehydrogenase. Insulin administration (1 unit/100 g body wt.) also resulted in activation of enzyme; however, these effects were less than those observed with glucagon and adrenaline. In contrast with the results obtained with low-protein-fed rats, administration of adrenaline (200 micrograms/100 g body wt.) to rats fed with an adequate amount of protein resulted in only a modest (14%) increase in the activity of the complex. The extent to which these hormones activate branched-chain 2-oxo acid dehydrogenase appears to be correlated with their ability to stimulate amino acid uptake into liver.     

Microbial metabolism of amino alcohols. Formation of coenzyme B12-dependent ethanolamine ammonia-lyase and its concerted induction in Escherichia coli.

1. A branched-chain 2-oxo acid dehydrogenase was partially purified from ox liver mitochondria. 2. The preparation oxidized 4-methyl-2-oxopentanoate, 3-methyl-2-oxobutyrate and D- and L-3-methyl-2-oxopentanoate. The apparent Km values for the oxo acids and for thiamin pyrophosphate, CoA, NAD+ and Mg2+ were determined. 3. The oxidation of each oxo acid was inhibited by isovaleryl (3-methylbutyryl)-CoA (competitive with CoA) and by NADH (competitive with NAD+); Ki values were determined. 4. The preparation showed substrate inhibition with each 2-oxo acid. The oxidative decarboxylation of 4-methyl-2-oxo[1-14C]pentanoate was inhibited by 3-methyl-2-oxobutyrate and DL-3-methyl-2-oxopentanoate, but not by pyruvate. The Vmax. with 3-methyl-2-oxobutyrate as variable substrate was not increased by the presence of each of the other 2-oxo acids. 5. Ox heart pyruvate dehydrogenase did not oxidize these branched-chain 2-oxo acids and it was not inhibited by isovaleryl-CoA. The branched-chain 2-oxo acid dehydrogenase activity (unlike that of pyruvate dehydrogenase) was not inhibited by acetyl-CoA. 6. It is concluded that the branched-chain 2-oxo acid dehydrogenase activity is distinct from that of pyruvate dehydrogenase, and that a single complex may oxidize all three branched-chain 2-oxo acids.     

Functional differences in the catabolism of branched-chain L-amino acids in cultured normal and maple syrup urine disease fibroblasts

Possible functional differences in the catabolism of the four branched-chain L-amino acids in maple syrup urine disease were assessed using cultured human skin fibroblast stains. Transamination and oxidative decarboxylation were comparatively studied in 90-min incubations with 1 mmole/liter of 1-14C-labeled substrates. In normal cell strains (n = 5), apparent transamination rates (sum of branched-chain 2-oxo[14C]acid and 14CO2 release; means expressed in nmole/90 min/mg of cell protein) were in the order L-leucine (32) greater than L-valine (17) greater than or equal to L-isoleucine (14) greater than L-allo-isoleucine (8); 14CO2 production was in the order L-valine (9) greater than L-isoleucine (6) greater than or equal to L-leucine (5) greater than L-allo-isoleucine (2). In variant (n = 5) as well as classical (n = 2) MSUD cell lines, branched-chain 2-oxo-[14C]acid release rates were generally comparable to the control values. As compared to the 14CO2 release in controls (= 100%), branched-chain 2-oxo acid dehydrogenase activity in MSUD fibroblasts was individually reduced and varied considerably between strains (residual activity 2-38%). Within individual strains, only small differences in the residual decarboxylation activity were observed in incubations with L-valine, L-leucine, and L-isoleucine. It was remarkably high, however, when L-allo-isoleucine was applied as a substrate. With the exception of L-allo-isoleucine, apparent total transamination rates of branched-chain L-amino acids were therefore distinctly lower in MSUD cells than in normal cells.     

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).     

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.     

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.     

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.     

Branched-chain 2-oxo acid dehydrogenase interferes with the measurement of the activity and activity state of hepatic pyruvate dehydrogenase.

Oxidative decarboxylation of pyruvate by branched-chain 2-oxo acid dehydrogenase can result in overestimation of the expressed and total activity of hepatic pyruvate dehydrogenase. Pyruvate is a poor substrate for branched-chain 2-oxo acid dehydrogenase relative to the branched-chain oxo acids; however, the comparable total activities of the two complexes in liver, the much greater activity state of branched-chain 2-oxo acid dehydrogenase compared with pyruvate dehydrogenase in most physiological states, and the use of high pyruvate concentrations, explain the interference that can occur in conventional radiochemical or indicator-enzyme linked assays of pyruvate dehydrogenase. Goat antibody that specifically inhibited branched-chain 2-oxo acid dehydrogenase was used in this study to provide a more specific assay for pyruvate dehydrogenase.     

Oxidation of 2-oxoisocaproate and 2-oxoisovalerate by the perfused rat heart. Interactions with fatty acid oxidation

The interactions between fatty acid oxidation and the oxidation of the 2-oxo acids of the branched chain amino acids were studied in the isolated Langendorff-perfused heart. 2-Oxoisocaproate inhibited the oxidation of oleate, but 2-oxoisovalerate and 2-oxo-3-methylvalerate did not. This difference was not attributable to the magnitude of the flux through the branched chain 2-oxo acid dehydrogenase, which was slightly higher with 2-oxoisovalerate than with 2-oxoisocaproate. Oxidation of 2-oxoisocaproate in the perfused heart was virtually complete, since more than 80% of the isovaleryl-CoA formed from 2-oxo[1-14C]isocaproate was further metabolized to CO2, as determined by comparing 14CO2 production from 2-oxo[14C(U)]isocaproate with that from the 1-14C-labelled compound. Only twice as much 14CO2 was produced from 2-oxo[14C(U)]isovalerate as from the 1-14C-labelled compound, indicating incomplete oxidation. This was confirmed by the accumulation in the perfusion medium of substantial quantities of labelled 3-hydroxyisobutyrate (an intermediate in the pathway of valine catabolism), when hearts were perfused with 2-oxo[14C(U)]isovalerate. The failure of 2-oxoisovalerate to inhibit fatty acid oxidation, then, can be attributed to the fact that its partial metabolism in the heart produces little ATP. We have previously shown that 3-hydroxyisobutyrate is a good gluconeogenic substrate in liver and kidney, and postulate that 3-hydroxyisobutyrate serves as an interorgan metabolite such that valine can serve as a glucogenic amino acid, even when its catabolism proceeds beyond the irreversible 2-oxo acid dehydrogenase in muscle.     

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.     

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.     

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.     

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.     

Regulation of the branched-chain 2-oxo acid dehydrogenase complex in hepatocytes isolated from rats fed on a low-protein diet.

Hepatocytes isolated from rats fed on a chow diet or a low-protein (8%) diet were used to study the effects of various factors on flux through the branched-chain 2-oxo acid dehydrogenase complex. The activity of this complex was also determined in cell-free extracts of the hepatocytes. Hepatocytes isolated from chow-fed rats had greater flux rates (decarboxylation rates of 3-methyl-2-oxobutanoate and 4-methyl-2-oxopentanoate) than did hepatocytes isolated from rats fed on the low-protein diet. Oxidizable substrates tended to inhibit flux through the branched-chain 2-oxo acid dehydrogenase, but inhibition was greater with hepatocytes isolated from rats fed on the low-protein diet. 2-Chloro-4-methylpentanoate (inhibitor of branched-chain 2-oxo acid dehydrogenase kinase), dichloroacetate (inhibitor of both pyruvate dehydrogenase kinase and branched-chain 2-oxo acid dehydrogenase kinase) and dibutyryl cyclic AMP (inhibitor of glycolysis) were effective stimulators of branched-chain oxo acid decarboxylation with hepatocytes from rats fed on a low-protein diet, but had little effect with hepatocytes from rats fed on chow diet. Activity measurements indicated that the branched-chain 2-oxo acid dehydrogenase complex was mainly (96%) in the active (dephosphorylated) state in hepatocytes from chow-fed rats, but only partially (50%) in the active state in hepatocytes from rats fed on a low-protein diet. Oxidizable substrates markedly decreased the activity state of the enzyme in hepatocytes from rats fed on a low-protein diet, but had much less effect in hepatocytes from chow-fed rats. 2-Chloro-4-methylpentanoate and dichloroacetate increased the activity state of the enzyme in hepatocytes from rats fed on a low-protein diet, but had no effect on the activity state of the enzyme in hepatocytes from chow-fed rats. The results indicate that protein starvation greatly increases the sensitivity of the hepatic branched-chain 2-oxo acid dehydrogenase complex to regulation by covalent modification.     

The regulation of hepatic gluconeogenesis using alpha-ketoisovalerate and propionate as precursors

The regulation of gluconeogenesis from alpha-ketoisovalerate and propionate was investigated in perfused livers from fasted rats. With alpha-ketoisovalerate as the gluconeogenic precursor, infusion of beta-hydroxybutyrate and acetate stimulated the rate of alpha-ketoisovalerate decarboxylation, but inhibited the rate of glucose production. Oleate, on the other hand, inhibited both alpha-ketoisovalerate decarboxylation and glucose production. When propionate was the primary gluconeogenic substrate, oleate, beta-hydroxybutyrate, and acetate infusion did not significantly alter hepatic glucose production. The present studies suggest that gluconeogenesis from alpha-ketoisovalerate is regulated at the level of various dehydrogenases prior to formation of propionyl-CoA, but subsequent to the branched-chain alpha-keto acid dehydrogenase reaction.     

Studies on the regulation of leucine catabolism: Mechanism responsible for oxidizable substrate inhibition and dichloroacetate stimulation of leucine oxidation by the heart

In the absence of any other oxidizable substrate, the perfused rat heart oxidizes [1-¹⁴C]leucine to ¹⁴CO₂ at a rapid rate and releases only small amounts of α-[1-¹⁴C]ketoisocaproate into the perfusion medium. The branched-chain α-keto acid dehydrogenase complex, assayed in extracts of mitochondria prepared from such perfused hearts, is very active. Under such perfusion conditions, dichloroacetate has almost no effect on [1-¹⁴C]leucine oxidation, α-[1-¹⁴C]ketoisocaproate release, or branched-chain α-keto acid dehydrogenase activity. Perfusion of the heart with some other oxidizable substrate, e.g., glucose, pyruvate, ketone bodies, or palmitate, results in an inhibition of [1-¹⁴C]leucine oxidation to ¹⁴CO₂ and the release of large amounts of α-[1-¹⁴C]ketoisocaproate into the perfusion medium. The branched-chain α-keto acid dehydrogenase complex, assayed in extracts of mitochondria prepared from such hearts, is almost completely inactivated. The enzyme can be reactivated, however, by incubating the mitochondria at 30 °C without an oxidizable substrate. With hearts perfused with glucose or ketone bodies, dichloroacetate greatly increases [1-¹⁴C]leucine oxidation, decreases α-[1-¹⁴C]ketoisocaproate release into the perfusion medium, and activates the branched-chain α-keto acid dehydrogenase complex. Pyruvate may block dichloroacetate uptake because dichloroacetate neither stimulates [1-¹⁴C]leucine oxidation nor activates the branched-chain α-keto acid dehydrogenase complex of pyruvate-perfused hearts. It is suggested that leucine oxidation by heart is regulated by the activity of the branched-chain α-keto acid dehydrogenase complex which is subject to interconversion between active and inactive forms. Oxidizable substrates establish conditions which inactivate the enzyme. Dichloroacetate, known to activate the pyruvate dehydrogenase complex by inhibition of pyruvate dehydrogenase kinase, causes activation of the branched-chain α-keto acid dehydrogenase complex, suggesting the existence of a kinase for this complex.     

Decreased flux through pyruvate dehydrogenase during calcium ion movements induced by vasopressin, alpha-adrenergic agonists and the ionophore A23187 in perfused rat liver.

Vasopressin or alpha-adrenergic agents such as phenylephrine or adrenaline, but not glucagon, elicited an initial decrease in flux through pyruvate dehydrogenase assayed by 14CO2 production from [1-14C]pyruvate in perfused rat liver. This rapid decrease in 14CO2 production was maximal within 1-2 min of exposure, concomitant with a rise in effluent pyruvate concentration: a subsequent return towards initial values in both parameters was completed well before 5 min. This time course was superposed with Ca2+ efflux from perfused liver, maximal (at 116 nmol/min per g wet wt. of liver) at 1-2 min of exposure. The percentage of the active (dephospho) form of pyruvate dehydrogenase was not decreased at 2 min of exposure. The effect on flux through pyruvate dehydrogenase by phenylephrine was abolished by prazosine, phentolamine or phenoxybenzamine. Ionophore A23187 also caused a depression in 14CO2 production from [1-14C]pyruvate and a rise in effluent pyruvate concentration, but this effect was stable for longer times, and it was delayed when Ca2+ was omitted from the perfusion medium. Responses of phenylephrine and A23187 were not additive. The results demonstrate that under the experimental conditions employed in intact perfused liver, the mitochondrial multienzyme system of pyruvate dehydrogenase is sensitive to vasopressin, alpha-adrenergic agents and A23187. The similar time course in Ca2+ efflux may be indicative of the involvement of Ca2+ in mediating this effect.     

Regulation of branched-chain amino acid oxidation in isolated muscles, nerves and aortas of rats.

1. The oxidation of the three branched-chain amino acids was regulated in parallel fashion in rat tissues studied in vitro. 2. With 0.1 mM-[1-14C]isoleucine as substrate in the presence of 5.5 mM-glucose, 14CO2 production was 0.6 mumol/2 h per g in the aorta, 0.3 in peripheral nerve, 0.2 in muscle and 0.13 in spinal cord. 3. The ratio 14C oxidized/14C incorporated into proteins with 0.1 mM-[1-14C]leucine was 1.3 in hemidiaphragms, 3.3 in sciatic nerve and 1.0 in nerves undergoing Wallerian degeneration. Leucine oxidation decreased only slightly during degeneration, but protein synthesis doubled. 4. Hemidiaphragms incubated with [1-14C]leucine or 4-methyl-2-oxo[1-14C]pentanoate increased 14CO2 production 7-9-fold as substrate concentration was increased from 0.1 to 0.5 mM; under the same conditions 14CO2 production by nerves increased only 2-3-fold. 5. 2-Oxoglutarate stimulated the oxidation of the branched-chain amino acids by muscles and peripheral nerves and the oxidation of 4-methyl-2-oxopentanoate by hemidiaphragms but not by nerves. 6. Octanoate (0.1-1.0 mM) markedly stimulated the oxidation of branched-chain amino acids and of 4-methyl-2-oxopentanoate in hemidiaphragms, but inhibited oxidation of both by peripheral nerves and spinal cord. In aortas, oxidation of isoleucine (the only substance tested) was inhibited by octanoate. 7. The effects of octanoate and 2-oxoglutarate on leucine oxidation by hemidiaphragms were additive at low concentrations. When maximally stimulating concentrations of either agent were used, addition of the other was ineffective. 8. Pyruvate inhibited the oxidation of branched-chain amino acids and 4-methyl-2-oxopentanoate in all tissues tested. 9. Insulin did not affect the oxidation of 4-methyl-2-oxopentanoate by muscles or nerves. 10. The oxidative decarboxylation of the branched-chain alpha-oxo acids is suggested as a regulatory site of branched-chain amino acid oxidation. Differences in regulation between muscle on the one hand, and nerve and aorta on the other, are discussed.     

The stimulation of tricarboxylic acid-cycle flux by alpha-adrenergic agonists in perfused rat liver.

Output of 14CO2 from 1-14C-labelled glutamate, 2-oxoglutarate or octanoate and from 4-methyl-2-oxo[2-14C]pentanoate was increased by more than 100% after infusion of phenylephrine into perfused livers of fed rats. Infusion of ethanol or sorbitol raised 3-hydroxybutyrate/acetoacetate ratios and decreased the output of 14CO2. Increases in 14CO2 output induced by phenylephrine were observed in the presence or absence of ethanol or sorbitol and were accompanied by elevated 3-hydroxybutyrate/acetoacetate ratios under all conditions examined. Phenylephrine had no effect on total tissue ATP/ADP ratios in livers from fed or starved rats. The data suggest that phenylephrine-induced increases in tricarboxylic acid-cycle flux do not arise from lowered matrix NADH/NAD+ or ATP/ADP ratios.     

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.