The effect of acetate on the regulation of the branched chain α-keto acid and the pyruvate dehydrogenase complexes in the perfused rat liver

The regulatory consequences of acetate infusion on the pyruvate and the branched chain α-keto acid dehydrogenase reactions in the isolated, perfused rat liver were investigated. Metabolic flux through these two decarboxylation reactions was monitored by measuring the rate of ¹⁴CO₂ production from infused 1-¹⁴C-labeled substrates. When acetate was presented to the liver as the sole substrate the rate of ketogenesis which resulted was maximal at concentrations of acetate in excess of 10 mm. The increase in hepatic ketogenesis during acetate infusion was not accompanied by an alteration of the mitochondrial oxidation-reduction state as measured by the ratio of β-hydroxybutyrate/ acetoacetate in the effluent perfusate. While acetate infusion did not affect the rate of α-keto[1-¹⁴C]isocaproate decarboxylation, the rate of α-keto[1-¹⁴C]isovalerate decarboxylation was stimulated appreciably upon acetate addition. No change was observed in the amount of extractable branched chain α-keto acid dehydrogenase during acetate infusion. The rate of [1-¹⁴C]pyruvate decarboxylation was stimulated in the presence of acetate at low (<1 mm) but not at high (>1 mm) perfusate pyruvate concentrations. The stimulation of the metabolic flux through the pyruvate dehydrogenase reaction upon acetate infusion was accompanied by an increase in the activation state of the pyruvate dehydrogenase complex from 25.7 to 35.6% in the active form. In a liver perfused in the presence of the pyruvate dehydrogenase kinase inhibitor, dichloroacetate, at a low concentration of pyruvate (0.05 mm) the infusion of acetate did not affect the rate of pyruvate decarboxylation. As the rate of mitochondrial acetoacetate efflux is increased during acetate infusion the stimulation of pyruvate and α-ketoisovalerate decarboxylation is attributed to an accelerated rate of exchange of mitochondrial acetoacetate for cytosolic pyruvate or α-ketoisovalerate on the monocarboxylate transporter.     

Oxidation of branched-chain amino acids in skeletal muscle and liver of rat Effects of octanoate and energy state

The effect of octanoate on the oxidative decarboxylation of ¹⁴C-labeled amino acids has been studied in perfused hindquarter and liver of rat. Regulation of the branched-chain α-keto acid dehydrogenase has been further studied with α-[¹⁴C-1]ketoisovalerate in isolated rat muscle and liver mitochondria. (1) Octanoate has a stimulatory effect on the oxidation of branched-chain amino acids in perfused hindquarter. The oxidative decarboxylation of other amino acids are inhibited. Octanoate inhibits the oxidative decarboxylation of all amino acids in perfused liver. (2) The oxidation of valine is stimulated by octanoate and hexanoate also in isolated muscle mitochondria. The stimulatory effect is probably related to activation of the fatty acids since acyl-carnitines inhibit the oxidation. (3) The oxidation of α-ketoisovalerate in mitochondria is inhibited by competing substrates (pyruvate, α-ketoglutarate and succinate). This inhibition is counteracted by octanoate and ADP. (4) Low concentrations (1–5 μM) of 2,4-dinitrophenol (DNP) activates wheras higher concentrations inactivates the branched-chain α-keto acid dehydrogenase in intact but not in solubilized muscle mitochondria. The inactivation is counteracted by ATP, but is increased by octanoate. (5) The observations seem to suggest that the activation (like the inactivation) of branched-chain α-keto acid dehydrogenase in skeletal muscle is dependent on the mitochondrial energy state which therefore may regulate both activation and inactivation of the dehydrogenase.     

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.     

Regulation of leucine catabolism by metabolic fuels in mammary epithelial cells

Lactation is associated with elevated catabolism of branched-chain amino acids (BCAA) in mammary glands to produce glutamate, glutamine, alanine, aspartate, and asparagine. This study determined effects of metabolic fuels on the catabolism of leucine (a representative BCAA) in bovine mammary epithelial cells. Cells were incubated at 37 °C for 2 h in Krebs buffer containing 0.5 mM l-leucine and either l-[1-¹⁴C]leucine or l-[U-¹⁴C]leucine. The medium also contained 0–5 mM d-glucose, 0–2 mM l-glutamine, 0–4 mM dl-β-hydroxybutyrate, or 0–2 mM oleic acid. Rates of leucine decarboxylation were 60 % lower, but rates of α-ketoisocaproate production were 34 % higher, in the presence of 2 mM glucose than in its absence. All variables of leucine catabolism did not differ between 2 and 5 mM glucose or between 0 and 4 mM dl-β-hydroxybutyrate. Compared with 0–0.25 mM glutamine, 0.5 and 2 mM l-glutamine reduced leucine transport, transamination, and decarboxylation. In contrast, increasing the concentration of oleic acid from 0 to 2 mM dose-dependently stimulated leucine transamination, decarboxylation, and oxidation of carbons 2–6. Oleic acid also enhanced the abundance of cytosolic BCAA transaminase, while reducing the phosphorylated level (inactive state) of the E1α subunit of the mitochondrial branched-chain α-ketoacid dehydrogenase complex. Thus, hypoglycemia or ketosis in early lactation does not likely affect BCAA metabolism in mammary epithelial cells. Increasing circulating levels of BCAA and oleic acid may have great potential to increase the syntheses of glutamate, glutamine, aspartate, alanine, and asparagine by lactating mammary glands, thereby leading to enhanced production of milk for suckling neonates.     

Inhibition of glycine synthase by branched-chain α-keto acids: A possible mechanism for abnormal glycine metabolism in ketotic hyperglycinemia

The effect of various amino acid metabolites on glycine oxidation by rat liver homogenate was investigated. Three compounds, α-ketoisovaleric acid, α-ketoisocaproic acid, and α-keto-β-methylvaleric acid, were found to inhibit glycine oxidation by 40–60%. In addition, these compounds also inhibited the glycine-CO₂ exchange reaction, a partial reaction of glycine synthase. The reverse reaction, glycine synthesis, was stimulated 4-fold by these α-keto acids. Pyruvate and α-ketoglutarate had no effect on any of these reactions. The parent amino acids, valine, isoleucine, and leucine, also had no effect on the reactions nor did any of their other metabolites with the exception of the branched-chain α-keto acids. The concentration dependence of the inhibition of glycine oxidation and stimulation of glycine synthesis by these branched-chain α-keto acids suggested that the inhibition of glycine oxidation by these compounds was the result of their further oxidation by branched-chain α-keto acid dehydrogenase. However, the products of the branched-chain α-keto acid dehydrogenase, isobutyryl CoA, isovaleryl CoA, or α-methylbutyryl CoA had no effect on glycine oxidation. Thus, it appeared that either the branched-chain α-keto acids altered glycine oxidation by direct binding to glycine synthase or that electrons derived from the oxidation of branched-chain α-keto acids were transferred to the glycine synthase system. It is proposed that glycine synthase and branched-chain α-keto acid dehydrogenase either share a common subunit, possibly lipoamide dehydrogenase, or are so arranged on the mitochondrial membrane that electron transfer between these two enzymes occurs.     

Hormonal regulation of leucine catabolism in mammary epithelial cells

Branched-chain amino acids (BCAA) are actively taken up and catabolized by the mammary gland during lactation for syntheses of glutamate, glutamine and aspartate. Available evidence shows that the onset of lactation is associated with increases in circulating levels of cortisol, prolactin and glucagon, but decreases in insulin and growth hormone. This study determined the effects of physiological concentrations of these hormones on the catabolism of leucine (a representative BCAA) in bovine mammary epithelial cells. Cells were incubated at 37 °C for 2 h in Krebs buffer containing 3 mM d-glucose, 0.5 mM l-leucine, l-[1-¹⁴C]leucine or l-[U-¹⁴C]leucine, and 0–50 μU/mL insulin, 0–20 ng/mL growth hormone 0–200 ng/mL prolactin, 0–150 nM cortisol or 0–300 pg/mL glucagon. Increasing extracellular concentrations of insulin did not affect leucine transamination or oxidative decarboxylation, but decreased the rate of oxidation of leucine carbons 2–6. Elevated levels of growth hormone dose dependently inhibited leucine catabolism, α-ketoisocaproate (KIC) production and the syntheses of glutamate plus glutamine. In contrast, cortisol and glucagon increased leucine transamination, leucine oxidative decarboxylation, KIC production, the oxidation of leucine 2–6 carbons and the syntheses of glutamate plus glutamine. Prolactin did not affect leucine catabolism in the cells. The changes in leucine degradation were consistent with alterations in abundances of BCAA transaminase and phosphorylated levels of branched-chain α-ketoacid dehydrogenase. Reductions in insulin and growth hormone but increases in cortisol and glucagon with lactation act in concert to stimulate BCAA catabolism for glutamate and glutamine syntheses. These coordinated changes in hormones may facilitate milk production in lactating mammals.     

Interaction between leucine and palmitate catabolism in 3T3-L1 adipocytes and primary adipocytes from control and obese rats

Metabolic profiling studies have highlighted increases in the plasma free fatty acid (FFA) and branched-chain amino acid (BCAA) concentrations, which are hallmarks of the obese and insulin-resistant phenotype. However, little is known about how the increase of the BCAA concentration modifies the metabolic fate of FFA, and vice versa, in adipocytes. Therefore, we incubated differentiated 3T3-L1 adipocytes or primary adipocytes from rats fed a control or a high-fat diet with: (1) 0, 250, 500 and 1000 μM of leucine and determined the oxidation and incorporation of [1-¹⁴C]-palmitate into lipids or proteins or (2) 0, 250, 500 or 1000 μM of palmitate and evaluated the oxidation and incorporation of [U-¹⁴C]-leucine into lipids or proteins. Leucine decreased palmitate oxidation and increased its incorporation into the lipid fraction in adipocytes; the latter was reduced in adipocytes from obese rats. However, palmitate increased leucine oxidation in adipocytes as well as reduced leucine incorporation into the protein and lipid fractions in adipocytes from obese rats. These results demonstrate that leucine modifies the metabolic fate of palmitate, and vice versa, in adipocytes and that the metabolic interaction between leucine and palmitate catabolism is altered in adipocytes from obese rats.     

Studies on the regulation of leucine catabolism in the liver. Stimulation by pyruvate and dichloroacetate

The rate of oxidation of L-[1-14C]leucine to 14CO2 by isolated rat hepatocytes is increased by pyruvate and dichloroacetate. This effect is specific for L-leucine, not being observed for L-valine, L-isoleucine, or D-leucine. Transamination, the rate-limiting step of L-leucine catabolism in the liver, is the site of stimulation, because uptake of L-leucine by the cells and the oxidation of its transamination product, alpha-ketoisocaproate, are not increased. Measurement of steady state levels of alpha-ketoisocaproate indicate that both pyruvate and dichloroacetate promote the transamination of L-leucine, thereby increasing the availability of substrate for decarboxylation by the alpha-ketoisocaproate dehydrogenase complex (EC 1.2.4.3). Pyruvate stimulation of transamination is secondary to the provision of keto acid acceptors for the amino group of L-leucine. The mechanism of the effect of dichloroacetate remains unknown.     

Interrelationships of Leucine and Glutamate Metabolism in Cultured Astrocytes

Abstract: The aim was to study the extent to which leu-cine furnishes α-NH₂ groups for glutamate synthesis via branched-chain amino acid aminotransferase. The transfer of N from leucine to glutamate was determined by incubating astrocytes in a medium containing [¹⁵N]leucine and 15 unlabeled amino acids; isotopic abundance was measured with gas chromatography-mass spectrometry. The ratio of labeling in both [¹⁵N]glutamate/[¹⁵N]leucine and [2-¹⁵N]glutamine/[¹⁵N]leucine suggested that at least one-fifth of all glutamate N had been derived from leucine nitrogen. At the same time, enrichment in [¹⁵N]leucine declined, reflecting dilution of the ¹⁶N label by the unlabeled amino acids that were in the medium. Isotopic abundance in [¹⁶N]-isoleucine increased very quickly, suggesting the rapidity of transamination between these amino acids. The appearance of ¹⁵N in valine was more gradual. Measurement of branched-chain amino acid transaminase showed that the reaction from leucine to glutamate was approximately six times more active than from glutamate to leucine (8.72 vs. 1.46 nmol/min/mg of protein). However, when the medium was supplemented with α-ketoisocaproate (1 mM), the ketoacid of leucine, the reaction readily ran in the “reverse” direction and intraastrocytic [glutamate] was reduced by ～50% in only 5 min. Extracellular concentrations of α-ketoisocaproate as low as 0.05 mM significantly lowered intracellular [glutamate]. The relative efficiency of branched-chain amino acid transamination was studied by incubating astrocytes with 15 unlabeled amino acids (0.1 mM each) and [¹⁵N]glutamate. After 45 min, the most highly labeled amino acid was [¹⁵N]alanine, which was closely followed by [¹⁵N]leucine and [¹⁵N]isoleucine. Relatively little ¹⁵N was detected in any other amino acids, except for [¹⁵N]serine. The transamination of leucine was ～17 times greater than the rate of [1-¹⁴C]leucine oxidation. These data indicate that leucine is a major source of glutamate nitrogen. Conversely, reamination of a-ketoisocaproate, the ketoacid of leucine, affords a mechanism for the temporary “buffering” of intracellular glutamate.     

Likely individuality of the enzymes catalyzing the phosphorylation of choline and ethanolamine.

Dichloroacetate has effects upon hepatic metabolism which are profoundly different from its effects on heart, skeletal muscle, and adipose tissue metabolism. With hepatocytes prepared from meal-fed rats, dichloroacetate was found to activate pyruvate dehydrogenase, to increase the utilization of lactate and pyruvate without effecting an increase in the net utilization of glucose, to increase the rate of fatty acid synthesis, and to decrease slightly [1-¹⁴C]oleate oxidation to ¹⁴CO₂ without decreasing ketone body formation. With hepatocytes isolated from 48-h-starved rats, dichloroacetate was found to activate pyruvate dehydrogenase, to have no influence on net glucose utilization, to inhibit gluconeogenesis slightly with lactate as substrate, and to stimulate gluconeogenesis significantly with alanine as substrate. The stimulation of fatty acid synthesis by dichloroacetate suggests that the activity of pyruvate dehydrogenase can be rate determining for fatty acid synthesis in isolated liver cells. The minor effects of dichloroacetate on gluconeogenesis suggest that the regulation of pyruvate dehydrogenase is only of marginal importance in the control of gluconeogenesis.     

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.     

Glycine,Serine, and Leucine Metabolism in Different Regions of Rat Central Nervous System

We have investigated the glycine, serine and leucine metabolism in slices of various rat brain regions of 14-day-old or adult rats, using [1-¹⁴C]glycine, [2-¹⁴C]glycine, L-[3-¹⁴C]serine and L-[U-¹⁴C]leucine. We showed that the [1-¹⁴C]glycine oxidation to CO₂ in all regions studied occurs almost exclusively through its cleavage system (GCS) in brains of both 14-day-old and adults rats. In 14-day-old rats, the highest oxidation of [1-¹⁴C]glycine was in cerebellum and the lowest in medulla oblongata. In these animals, the L-[U-¹⁴C]leucine oxidation was lower than the [1-¹⁴C]glycine oxidation, except in medulla oblongata where both oxidations were the same. Serine was the amino acid that showed lowest oxidation to CO₂ in all structure studied. In adult rats brains, the highest oxidation of [1-¹⁴C]glycine was in cerebral cortex and the lowest in medulla oblongata. We have not seen difference in the lipid synthesis from both glycine labeled, neither in 14-day-old rats nor in adult ones, indicating that the lipids formed from glycine were not neutral. Lipid synthesis from serine was significantly high than lipid synthesis and from all other amino acids studied in all studied structures. Protein synthesis from L-[U-¹⁴C]leucine was significantly higher than that from glycine in all regions and ages studied.     

Studies on the role of β-cell metabolism in the insulinotropic effect of α-ketoisocaproic acid

α-Ketoisocaproic acid has been shown to be apotent insulin secretagogue but the mechanism has not been elucidated. To define the role of β-cell metabolism in the insulinotropic activity of α-ketoisocaproic acid the utilization of glucose and the oxidation of α-ketoisocaproic and isovaleric acid by incubated islets of obese hyperglycemic mice were measured.Glucose metabolism was never enhanced by α-ketoisocaproic acid. The same ¹⁴CO₂ amounts were released from the non-secretagogue [1-¹⁴C]isovaleric acid (10 mM) or from α-keto [2-¹⁴C]isocaproic acid (5–20 mM). Pyruvate (20 mM) did not inhibit α-ketoisocaproic acid-induced insulin secretion in spite of reduction of decarboxylation of α-ketoisocaproic acid by more than 40%.The results indicate that stimulated insulin release in response to α-ketoisocaproic acid is not mediated by an indirect increase in glucose metabolism and further suggest that isovaleryl-CoA and following CoA-esters in α-ketoisocaproic acid degradation are not likely recognized as signals. The possibility, however, remains that enhanced intramitochondrial production of reducing equivalents elicits insulin secretion.     

Effect of dichloroacetate and glucagon on the incorporation of labeled substrates into glucose and on pyruvate dehydrogenase in hepatocytes from fed and starved rats

Dichloroacetate (2 mm) stimulated the conversion of [1-¹⁴C]lactate to glucose in hepatocytes from fed rats. In hepatocytes from rats starved for 24 h, where the mitochondrial $\text{NADH}\text{NAD}{}^{\mathrm{+}}$ ratio is elevated, dichloroacetate inhibited the conversion of [1-¹⁴C]lactate to glucose. Dichloroacetate stimulated ¹⁴CO₂ production from [1-¹⁴C]lactate in both cases. It also completely activated pyruvate dehydrogenase and increased flux through the enzyme. The addition of β-hydroxybutyrate, which elevates the intramitochondrial $\text{NADH}\text{NAD}{}^{\mathrm{+}}$ ratio, changed the metabolism of [1-¹⁴C]lactate in hepatocytes from fed rats to a pattern similar to that seen in hepatocytes from starved rats. Thus, the effect of dichloroacetate on labeled glucose synthesis from lactate appears to depend on the mitochondrial oxidation-reduction state of the hepatocytes. Glucagon (10 nm) stimulated labeled glucose synthesis from lactate or alanine in hepatocytes from both fed and starved rats and in the absence or presence of dichloroacetate. The hormone had no effect on pyruvate dehydrogenase activity whether or not the enzyme had been activated by dichloroacetate. Thus, it appears that pyruvate dehydrogenase is not involved in the hormonal regulation of gluconeogenesis. Glucagon inhibited the incorporation of 10 mm [1-¹⁴C]pyruvate into glucose in hepatocytes from starved rats. This inhibition has been attributed to an inhibition of pyruvate dehydrogenase by the hormone (Zahlten et al., 1973, Proc. Nat. Acad. Sci. USA70, 3213–3218). However, dichloroacetate did not prevent the inhibition of glucose synthesis. Nor did glucagon alter the activity of pyruvate dehydrogenase in homogenates of cells that had been incubated with 10 mm pyruvate in the absence or presence of dichloroacetate. Thus, the inhibition by glucagon of pyruvate gluconeogenesis does not appear to be due to an inhibition of pyruvate dehydrogenase.     

Effect of alanyl-glutamine on leucine and protein metabolism in irradiated rats

Summary. The mechanism by which glutamine produces a favorable effect in the treatment of sepsis, injury, burns and abdominal irradiation is not completely understood. The main aim of this study was to evaluate the effect of alanyl-glutamine (AlaGln) administration on the metabolism of proteins in irradiated rats. The rats were exposed to whole-body irradiation (8 Gy) and then fed intragastrically with a mixture of glucose and amino acids either with AlaGln or without AlaGln. At 48 hours after irradiation, parameters of whole-body protein metabolism and DNA synthesis in intestinal mucosa were investigated using a primed, continuous infusion of [1-¹⁴C]leucine and [³H]thymidine. In addition, we evaluated the effect of irradiation and AlaGln on gut morphology, blood count and amino acid concentrations in blood plasma and skeletal muscle. Control rats were not irradiated but were given identical treatment. An increase in whole-body leucine oxidation, and insignificant changes in whole-body proteolysis and in protein synthesis were observed after irradiation. In irradiated rats we observed a decrease in muscle glutamine concentration, a decrease in protein synthesis in jejunum, colon and heart, and an increase in synthesis of proteins of blood plasma and spleen. Morphological examination and measurement of DNA synthesis failed to demonstrate any favorable effect of AlaGln supplementation on irradiated gut. However, administration of AlaGln resulted in a decrease in whole-body proteolysis and leucine oxidation which caused an increase in the fraction of leucine incorporated into the pool of body proteins. We conclude that the data obtained demonstrate that irradiation induces metabolic derangement associated with increased oxidation of essential branched-chain amino acids (valine, leucine and isoleucine) and that these disturbances can be ameliorated by administration of AlaGln. Received February 14, 2000 Accepted July 12, 2000     

Indole alkaloid biosynthesis : An investigation into leucine as a possible precursor of the monoterpene portion of vindoline

Because of the nonspecific incorporation of acetate into the monoterpenoid portion of the indole alkaloids, the amino acid leucine has been investigated as an alternative precursor. Incorporations of leucine into the alkaloids vindoline and catharanthine in Vinca rosea are found to be 0.07 and 0.02%, respectively, levels comparable with those of acetate. An equation is developed to calculate the quantity of [¹³C]leucine needed to be fed in order to obtain alkaloids with sufficient ¹³C enrichment to be analysed by ¹³C nmr spectroscopy. This equation assumes that percentage incorporations do not decrease with increasing quantities of leucine fed, an assumption found to be true. [2-¹³C]Leucine was synthesized from [2-¹³C]acetic acid in an eight-step procedure in 11.5% overall yield, and fed to Vinca rosea plants. Incorporations into vindoline obtained using both ¹⁴C and ¹³C were in reasonable agreement, and the activity was found to be spread over seven carbon atoms, none of which corresponded to the two atoms at which activity was expected. It is concluded that the leucine → mevalonate → monoterpene route does not operate in indole alkaloid biosynthesis, leaving open the question of the origin of the monoterpene portion of the alkaloids. An attempt to confirm these results by degradation of ¹⁴C-labelled alkaloids was unsuccessful, vindoline proving to be unexpectedly resistant to oxidation.     

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.     

Desilicification from the High Silica Containing Kraft Black Liquor by the Improved Carbon Dioxide Method

Addition of NADH to crude but not to pure branched-chain α-keto acid decarboxylase decreased the CO₂ production from α-keto-β-methylvalerate (KMV) suggesting the presence of an NADH dependent inhibitor in the crude enzyme from Bacillus subtilis. This NADH-dependent decarboxylase inhibitor was purified to homogeneity by a fast protein liquid chromatography system.

The purified inhibitor was identical with leucine dehydrogenase as to N-terminal amino acid squence (35 residues) and molecular weight, and catalyzed the oxidative deamination of three branched chain amino acids (BCAAs), valine, leucine, and isoleucine. The decarboxylase inhibitor was therefore identified as leucine dehydrogenase. A decreased substrate availability caused by leucine dehydrogenase thus reasonably accounted for the NADH dependent inhibition of the decarboxylation. In turn, the observation that leucine dehydrogenase competes with the decarboxylase for branched-chain α-keto acid (BCKA) suggested an involvement of this enzyme in the branched chain fatty acid (BCFA) biosynthesis. This view was supported by the observation that addition of NAD to crude fatty acid synthetase increased the incorporation of isoleucine into BCFAs. Pyridoxal-5′-phosphate and α-ketoglutarate, cofactors for BCAA transaminase, modulated BCFA biosynthesis from isoleucine in vitro, suggesting also the involvement of transaminase reaction in BCFA biosynthesis.     

THE INFLUENCE OF PORTOCAVAL ANASTOMOSIS ON THE METABOLISM OF LABELLED OCTANOATE, BUTYRATE AND LEUCINE IN RAT BRAIN

Rats were given a portocaval anastomosis and 3 weeks later, when the only ultrastructural change in the CNS is watery swelling of astrocytes, several aspects of brain metabolism were studied. The uptake of leucine by the brain, its incorporation into protein and its oxidation were followed after the simultaneous injection of a mixture of L-[1¹⁴C]leucine and L-[4,5-³H]leucine. The concentration of leucine in blood was lowered in the operated animals whereas in brain it was increased. The specific radioactivity of leucine in the brain was comparable to values in control animals and there was no evidence of a decrease in incorporation of [1-¹⁴C]leucine into brain proteins over the short experimental time period studied. The only difference from the controls in the oxidation of [4,5-³H]leucine was a greater accumulation in glutamine. The amount of glutamine in the brains of the operated animals had increased 4-fold at the time of the metabolic studies. From dual-labelled experiments in which a mixture containing [1-¹⁴C]butyrate and L-[4,5-³H]leucine was injected intravenously, it was shown that, in both control and operated animals, the pools of brain glutamate and glutamine labelled from butyrate were metabolically distinct from those labelled from leucine. The total radioactivity appearing in brain from [1-¹⁴C]butyrate was markedly reduced in operated animals, but the radioactivity from L-[4,5-³H]leucine was not. The metabolism of [1-¹⁴C]octanoate was compared with that of [1-¹⁴C]butyrate. In control animals the labelling of metabolites was almost identical with either precursor. In operated animals there was no reduction in the uptake of [1-¹⁴C]octanoate into the brain. There was evidence that the size of the glutamine pool labelled, relative to glutamate, was increased but that it had a slower fractional turnover coefficient. A link between astroglial changes and an impairment to the carrier mechanism for transport of short chain monocarboxylic acids across the blood-brain barrier is suggested.