Ethanol and leucine oxidation--II. Leucine oxidation by rat tissue in vitro

The effect of ethanol upon leucine oxidation by rat tissues in vitro is reported. The activities of branched chain amino acid aminotransferase and 2-oxo acid dehydrogenase were decreased by chronic administration of ethanol (20% v/v solution as drinking water for 35 d) in muscle and kidney but were increased, although not significantly, in liver. Acute administration of ethanol (8 g kg-1 body-weight 0.73) did not affect enzyme activities. Tissue NAD+:NADH ratios, calculated from lactate:pyruvate ratios, were significantly decreased in the liver and kidney of rats receiving ethanol acutely. These data are consistent with the view that ethanol decreases leucine oxidation by decreasing availability of NAD+ when given acutely and by decreasing enzyme activity when administered chronically.     

Effect of chronic ethanol ingestion on pancreatic protein synthesis

The effect of chronic ethanol feeding on pancreatic protein synthesis was assessed by studying the rate of incorporation of [3H]leucine into proteins in isolated rat pancreatic acini in vitro. Chronic ethanol feeding increased the rate of protein synthesis (2-3-fold) compared to controls fed an isocaloric diet. The onset of the increase in protein synthesis was detectable 2 days after the beginning of ethanol feeding, reached a maximum after 7 days and remained constant for up to 4 months. The increased incorporation of [3H]leucine was not due to an increased turnover of proteins as measured in pulse-chase experiments. After separation of individual digestive enzymes by SDS-polyacrylamide gel electrophoresis and determination of the distribution of radioactivity in different proteins, a general increase in the rate of incorporation of the label into all of the proteins was observed. In contrast to the observations made with isolated acini, there was no significant difference between the control and ethanol-fed groups when the rate of pancreatic protein synthesis was measured in vivo. However, overnight withdrawal of ethanol led to an increase of approx. 70% in protein synthesis in the ethanol-fed group. These results suggest that chronic ethanol ingestion modifies the control of pancreatic protein synthesis; the enhanced protein synthesis is expressed in isolated acini, i.e., in the absence of physiological factors present during chronic ethanol ingestion and in vivo after ethanol withdrawal.     

Ethanol-induced inhibition of leukotriene degradation by omega-oxidation

omega-Oxidation of leukotrienes is a major pathway in the degradation and inactivation of these proinflammatory mediators. Ethanol inhibited this process in vivo and in vitro. In rat liver in vivo the catabolism of LTE4 to omega-carboxylated leukotrienes was inhibited by 57% by an ethanol dose of 25 mmol/kg body mass administered intragastrically. The site of inhibition was the oxidation of omega-hydroxy-N-acetyl-LTE4 to omega-carboxy-N-acetyl-LTE4 resulting in an accumulation of omega-hydroxy-N-acetyl-LTE4 and of N-acetyl-LTE4. Analogous results were obtained for the oxidative degradation of LTB4 and omega-hydroxy-LTB4 in rat hepatocyte suspensions. Ethanol, at a concentration of 12.5 mmol/l (0.07%; by vol.), caused 68% inhibition of the oxidation of omega-hydroxy-LTB4 by 50% in hepatocyte suspensions. The conversion of omega-hydroxy-LTB4 to omega-carboxy-LTB4 by rat and human liver cytosol was inhibited by ethanol with half maximal concentrations of 100 mumols/l and 300 mumols/l, respectively. Our measurements indicate that direct interference by ethanol of the omega-oxidation of leukotrienes as well as an increased NADH/NAD+ ratio induced by ethanol led to the inhibition of leukotriene degradation. The impairment of leukotriene inactivation in the liver by ethanol may contribute to the development of the inflammatory reaction in acute alcoholic liver disease.     

Ethanol and leucine oxidation--III. Leucine oxidation by rat heart in vitro

The activities of leucine aminotransferase (BCAT) and 2-oxoisocaproate dehydrogenase (OADH) were measured in rat heart in vitro. The effect upon these enzyme activities of both ethanol and acetaldehyde, administered either acutely or chronically, was determined. Enzyme activities were not significantly altered by either acetaldehyde or ethanol when given chronically. Ethanol administered acutely to rats decreased OADH activity but BCAT was unaffected. Acetaldehyde administered acutely did not alter significantly BCAT activity but significantly increased OADH activity.     

Evidence for the exchangeability of acidic ribosomal proteins on cytoplasmic ribosomes in regenerating rat liver

We purified acidic ribosomal proteins (P1 and P2) in good yield from rat liver ribosomes by precipitation of ribosomes with MgCl2 prior to ethanol extraction and chromatography of the extract on a column of CM-cellulose at pH 4.8. The newly-synthesized acidic ribosomal proteins in regenerating rat liver, labeled in vivo with [3H]leucine, were rapidly incorporated into cytoplasmic ribosomes without any detectable time lag and, after reaching a maximum at 30 min, they gradually disappeared from the ribosomes, suggesting a short metabolic-life. However, it was found later that they were re-incorporated slowly when newly-labeled proteins were "chased" by an injection of a large amount of cold leucine intraperitoneally at 15 min after the injection of [3H]leucine. Furthermore, in a long-term experiment, acidic ribosomal proteins were found to disappear with a half-life of 100 h from the ribosomes. Thus, these results suggest that acidic ribosomal proteins have a long metabolic life and are exchangeable on cytoplasmic ribosomes in regenerating rat liver.     

Effect of chronic ethanol, catalase inhibitor 3-amino-1,2,4-triazole and clofibrate treatment on lipid peroxidation in rat myocardium

1. The effect of chronic alcohol consumption, catalase inhibitor 3-amino-1,2,4-triazole (amino-triazole) and peroxisome proliferator clofibrate on the level of Fe/ADP-ascorbate-induced lipid peroxidation has been studied in the rat myocardium. The intensity of lipid peroxidation was measured using chemiluminescence technique and malondialdehyde formation. 2. Combined us well as separate treatment with ethanol (36% of dietary calories) and aminotriazole caused elevation of the rate of lipid peroxidation in the nuclear-free homogenate or total particulate fraction of the rat heart. The most pronounced effect was noted during combined application of ethanol and aminotriazole. 3. Prolonged clofibrate treatment significantly increased the level of nonenzymatic lipid peroxidation in the rat myocardium. 4. Peroxidative alteration of the myocardial lipids in vivo was evaluated by measurement of conjugated dienes (absorbance at 233 nm). Separate ethanol, aminotriazole or clofibrate treatment did not affect the level of u.v. absorption of lipids from the total particulate fraction. However, when ethanol and aminotriazole were administered simultaneously an increase of conjugated diene formation was observed. 5. The data obtained confirm the hypothesis that ethanol or clofibrate-induced activation of the myocardial lipid peroxidation may be due to the increase of hydrogen peroxide-generating capacity of the heart microperoxisomes.     

Hyperosmotic oxidation of glucose and decarboxylation of histidine in the gastric mucosa.

Paired slices of rat gastric mucosa were incubated with labeled glucose or histidine in isosomotic solution of 3-fold hyperosmotic solutions concentrated in NaCl, KCl, or ethanol. The rate of (1-14C)glucose oxidation to 14CO2 in isosmotic solution was reduced by 74% in hyperosmotic NaCl and by 28% in hyperosmotic KCl. The rate of (6-14C)glucose oxidation to 14CO2 in isosmotic solution was reduced by 64% in hyperosmotic NaCl and by 53% in hyperosmotic KCl. Reductions of glucose oxidation in hyperosmotic ethanol were not significant. The ratio of 14CO2 formed from (1-14C)glucose to that formed from (6-14C)glucose was not significantly changes by hyperosmotic NaCl or ethanol, but was significantly raised by hyperosmotic KCl. The rate of (carboxyl-14C)histidine decarboxylation in isosomotic solution was reduced significantly by 48% in hyperosmotic NaCl, by 30% in hyperosmotic KCl, and by 27% in hyperosmotic ethanol. We conclude that hyperosmotic solutions reduce glucose oxidation and histidine decarboxylation by rat gastric mucosa in the order of potency: NaCl greater than Kcl greater than or equal to ethanol. Thus hyperosmotic solutions inhibit the source of metabolic energy for stimulated acid secretion, the citric acid cycle, and the formation of the secretagogue histamine.     

Nociceptin/orphanin FQ prevents ethanol-induced gastric lesions in the rat

Nociceptin/orphanin FQ (N/OFQ), the endogenous ligand of the NOP receptor, exerts a variety of effects on the gastrointestinal tract. The present study was aimed at evaluating the possible implication of N/OFQ in the maintenance of gastric mucosal integrity. N/OFQ was given either centrally or peripherally 30 min prior to intragastric administration (i.g.) of 1 ml/rat of ethanol (either 25% or 50%, v/v), which produces macroscopically visible gastric lesions. Intracerebroventricular (i.c.v.) injection of 2 microg/rat of N/OFQ significantly reduced lesions caused by 50% ethanol, while 1 microg/rat was enough to significantly reduce lesions caused by 25% ethanol. Intracerebroventricular injection of 5 microg/rat of the selective NOP receptor antagonist, UFP-101, completely reversed the protective effect of N/OFQ, 1 or 4 microg/rat against 25% or 50% ethanol, respectively. The intraperitoneal (i.p.) injection of N/OFQ produced a significant reduction of lesions induced by 50% ethanol, the peak effect being observed at 10 microg/kg. Intraperitoneal pretreatment with UFP-101, 120 microg/kg, completely abolished the protective effect of peripherally injected N/OFQ. Therefore, N/OFQ acts both centrally and peripherally as a protective agent against ethanol-induced gastric lesions, and its effect is mediated by NOP receptors.     

Evidence for the generation of transaminase inhibitor(s) during ethanol metabolism by rat liver homogenates: a potential mechanism for alcohol toxicity

Since ethanol consumption decreases hepatic aminotransferase activities in vivo, mechanisms of ethanol-mediated transaminase inhibition were explored in vitro using mitochondria-depleted rat liver homogenates. When homogenates were incubated at 37 degrees with 50 mM ethanol for 1 hr, alanine aminotransferase decreased by 20%, while aspartate aminotransferase was unchanged. After 2 hr, aspartate aminotransferase decreased by 20% and by 3 hr, alanine and aspartate aminotransferases were decreased by 31 and 23%, respectively. Levels of acetaldehyde generated during ethanol oxidation were 525 +/- 47 microM at 1 hr, 855 +/- 14 microM at 2 hr, and 1293 +/- 140 microM at 3 hr. Although inhibition of alcohol oxidation with methylpyrazole or cyanide markedly decreased ethanol-mediated transaminase inhibition, neither incubation with acetate nor generation of reducing equivalents by oxidation of lactate, malate, xylitol, or sorbitol altered the activity of either enzyme. However, semicarbazide, an aldehyde scavenger, prevented inhibition of both aminotransferases by ethanol. Moreover, incubation with 5 mM acetaldehyde for 1 hr inhibited alanine and aspartate aminotransferases by 36 and 26%, respectively. Cyanamide, an aldehyde dehydrogenase inhibitor, had little effect on ethanol-mediated transaminase inhibition. Thus, metabolism of ethanol by rat liver homogenates produces transaminase inhibition similar to that described in vivo and this effect requires acetaldehyde generation but not acetaldehyde oxidation. Since addition of pyridoxal 5'-phosphate to assay mixes did not reverse ethanol effects, aminotransferase inhibition does not result from displacement of vitamin B6 coenzymes.     

Effect of chronic ethanol treatment on lipid peroxidation in rat liver homogenate and subcellular fractions

1. The effect of chronic ethanol treatment on the level of lipid peroxidation in rat liver homogenate and subcellular fractions was measured using chemiluminescence technique and malondialdehyde formation. 2. It was shown that after chronic ethanol treatment the level of Fe/ADP-ascorbate-induced lipid peroxidation was decreased in the whole and "postnuclear" liver homogenates. Dilution of the homogenates prevented depressive effect of ethanol on lipid peroxidation. 3. Chronic ethanol treatment did not affect the intensity of the Fe/ADP-ascorbate-induced process in rat liver mitochondria and microsomes. 4. Peroxidative alteration of the liver lipids in vivo was evaluated by measurement of conjugated dienes (absorbance at 233 nm). It was shown that ethanol did not increase the level of u.v. absorption of lipids from mitochondria and microsomes. Chronic alcohol treatment did not influence the steady-state concentration of malonic dialdehyde in the whole liver homogenate. 5. The data obtained indicate that cytosol from the ethanol treated rat liver contains a factor(s) which prevents Fe/ADP-ascorbate-dependent lipid peroxidation in biological membranes.     

Potential role of leucine metabolism in the leucine-signaling pathway involving mTOR

Leucine has been shown to stimulate adipose tissue protein synthesis in vivo as well as leptin secretion, protein synthesis, hyper-plastic growth, and tissue morphogenesis in in vitro experiments using freshly isolated adipocytes. Recently, others have proposed that leucine oxidation in the mitochondria may be required to activate the mammalian target of rapamycin (mTOR), the cytosolic Ser/Thr protein kinase that appears to mediate some of these effects. The first irreversible and rate-limiting step in leucine oxidation is catalyzed by the branched-chain alpha-keto acid dehydrogenase (BCKD) complex. The activity of this complex is regulated acutely by phosphorylation of the E1alpha-subunit at Ser293 (S293), which inactivates the complex. Because the alpha-keto acid of leucine regulates the activity of BCKD kinase, it has been suggested as a potential target for leucine regulation of mTOR. To study the regulation of BCKD phosphorylation and its potential link to mTOR activation, a phosphopeptide-specific antibody recognizing this site was developed and characterized. Phospho-S293 (pS293) immunoreactivity in liver corresponded closely to diet-induced changes in BCKD activity state. Immunoreactivity was also increased in TREMK-4 cells after the induction of BCKD kinase by a drug-inducible promoter. BCKD S293 phosphorylations in adipose tissue and gastrocnemius (which is mostly inactive in vivo) were similar. This suggests that BCKD complex in epididymal adipose tissue from food-deprived rats is mostly inactive (unable to oxidize leucine), as is the case in muscle. To begin to test the leucine oxidation hypothesis of mTOR activation, the dose-dependent effects of orally administered leucine on acute activation of S6K1 (an mTOR substrate) and BCKD were compared using the pS293 antibodies. Increasing doses of leucine directly correlated with increases in plasma leucine concentration. Phosphorylation of S6K1 (Thr389, the phosphorylation site leading to activation) in adipose tissue was maximal at a dose of leucine that increased plasma leucine approximately threefold. Changes in BCKD phosphorylation state required higher plasma leucine concentrations. The results seem more consistent with a role for BCKD and BCKD kinase in the activation of leucine metabolism/oxidation than in the activation of the leucine signal to mTOR.     

Experimental alcoholism in rats. Effect of acute ethanol intoxication on the in vitro incorporation of ( 3 H)leucine into neuronal and glial proteins

Abstract— Ethanol administered in vivo or in vitro during incubation of brain slices was studied with respect to its effect on brain protein synthesis. In the in vivo series the rats were given a single intraperitoneal injection of ethanol 3 h before death. Slices of cerebral cortex and liver were incubated in isotonic saline media containing [³H]leucine. Amounts of free and protein-bound radioactivity were determined. Subcellular fractions and fractions enriched in neuronal perikarya and in glial cells were prepared from cortical slices subsequent to incubation, and the specific radioactivity determined for each cell type. The incorporation of [³H]leucine into brain proteins was inhibited while incorporation into liver proteins was stimulated in ethanol-treated rats. The levels of TCA-soluble radio-activity, however, did not differ between the ethanol group and the controls. In the fractionated material from cerebral cortex, the specific radioactivity in the neuronal fraction was unaffected by ethanol, while the radioactivity in the glial fraction was significantly depressed. In vitro administration of ethanol induced a non-linear response in both brain and liver, with depression of leucine incorporation into proteins of cerebral cortex at all concentrations used. When brain slices were exposed to ethanol in vitro, in concentrations corresponding to the in vivo experiments, a similar reduction of the leucine incorporation into the glial fraction was obtained. Incorporation of leucine into subcellular fractions from whole brain cortex was also investigated. The specific sensitivity of the glial fraction to ethanol is discussed in relation to the involvement of the different cell types with transport processes in the brain.     

The effect of ethanol on erythrocyte carbonic anhydrase isoenzymes activity: an in vitro and in vivo study

The ethanol is a widely consumed as sedative-hypnotic drug throughout the world. In this study, the effects of ethanol were investigated on carbonic anhydrase (CA) enzyme activities both in vitro in human erythrocyte and in vivo in Sprague-Dawley rat erythrocyte. For in vitro study, the human carbonic anhydrase-I (HCA-I) and -II (HCA-II) are purified by Sepharose 4B-L-tyrosine-sulphanilamide affinity chromatography. In vivo CA enzyme activity was determined colorimetrically by using CO(2)-hydration method of Wilbur and Anderson. Rat blood samples were taken from each rat before and after the ethanol administration at different times (1 h, 3 h, and 5 h). Rat erythrocyte CA activity was significantly inhibited by pharmacological dosage of the ethanol (2 mL.kg(- 1)) for up to 3 h (p < 0.001) following intraperitoneally administration. The ethanol showed in vitro inhibitory effects on HCA-I and HCA-II hydratase activity, determined by colorimetrically using the CO(2)-hydratase method. The inhibitor concentrations causing up to 50% inhibition (IC(50)) were 2.09 M for HCA-I (r(2):0.9273) and 1.83 M for HCA-II (r(2):9749). In conclusion, it was demonstrated that carbonic anhydrase enzyme in erythrocytes was significantly inhibited by the ethanol both in in vitro and in vivo.     

The effect of insulin on amino acid incorporation into exocrine pancreatic cells of the rat.

The rate of incorporation of radioactive leucine per cell in the acinar pancreatic cells of the rat increases by 50 per cent within one hour after subcutaneous administration of insulin, an effect that lasts for at least one more hour. The rate of incorporation has been measured by quantitative radioautography and by determination of the radioactivity per mug DNA in TCA-precipitable material from tissue homogenates. The capacity for amino acid (leucine and lysine) incorporation as measured by incubating pancreatic fragments in vitro is not enhanced by insulin treatment of the rat in vivo during one or more hours. Insulin was found to lower the serum concentration of most amino acids significantly, leucine by 50 per cent. The apparent effect of insulin on the incorporation of radioactive leucine in vivo can be explained by the difference in the specific radioactivity of the circulating amino acid in the treated rats as compared to the untreated ones. A change in amino acid concentration in the serum may likewise be the explanation of the decrease in amino acid incorporation rate in alloxan diabetic rats. The absence of a short term effect of insulin on the rate of protein synthesis does not exclude a long term effect as suggested by the higher rate of incorporation in the cells of peri-insular acini.     

Amino Acid Metabolism in Rat Hippocampus During the Period of Brain Growth Spurt

We studied protein synthesis, lipid synthesis and CO₂ production by oxidation of glycine, alanine and leucine by slices of rat hippocampus during the period of brain growth spurt. The metabolism of the three amino acids decreased with the age of the animals, A major reduction was observed in protein synthesis, which was 4 times higher at 7 days of age than at 21 days of age for all amino acids studied. Glycine oxidation to CO₂ was twice as high as alanine oxidation and ten times higher than leucine oxidation. The major pathway of leucine utilization was incorporation into proteins. Glycine was the amino acid that had the highest metabolic rate.     

Effect of maternal ethanol consumption on foetal and neonatal rat hepatic protein synthesis.

Effects of maternal ethanol consumption were investigated on the rates of protein synthehsis by livers of foetal and neonatal rats both in vivo and in vitro, and on the activities of enzymes involved in protein synthesis and degradation. The rates of general protein synthesis by ribosomes in vitro studied by measuring the incorporation of [14C]leucine into ribosomal protein showed that maternal ethanol consumption resulted in an inhibition of the rates of protein synthesis by both foetal and neonatal livers from the ethanol-fed group. The rates of incorporation of intravenously injected [14C]leucine into hepatic proteins were also significantly lower in the foetal, neonatal and adult livers from the ethanol-fed group. Incubation of adult-rat liver slices with ethanol resulted in an inhibition of the incorporation of [14C]leucine into hepatic proteins; however, this effect was not observed in the foetal liver slices. This effect of externally added ethanol was at least partially prevented by the addition of pyrazole to the adult liver slices. Pyrazole addition to foetal liver slices was without significant effect on the rates of protein synthesis. Cross-mixing experiments showed that the capacity of both hepatic ribosomes and pH5 enzyme fractions to synthesize proteins was decreased in the foetal liver from the ethanol-fed group. Maternal ethanol consumption resulted in a decrease in hepatic total RNA content, RNA/DNA ratio and ribosomal protein content in the foetal liver. Foetal hepatic DNA content was not significantly affected. Ethanol consumption resulted in a significant decrease in proteolytic activity and the activity of tryptophan oxygenase in the foetal, neonatal and adult livers. It is possible that the mechanisms of inhibition of protein synthesis observed here in the foetal liver after maternal ethanol consumption may be responsible for at least some of the changes observed in 'foetal alcohol syndrome'.     

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.     

Effects of chronic administration of either ethanol or pentanol on rat duodenum morphology

The morphology of the rat duodenum after chronic treatment with 15% (v/v) ethanol and 4% (v/v) pentanol was studied. Male Wistar rats of experimental groups were given ethanol and pentanol for 15 weeks with food and fluid freely available. Ethanol-15% and 4% pentanol-fed rats showed a significantly reduced fluid and food intake as compared with control rats. The study of the mucosa indicated that the number of chronic inflammatory infiltrating (mononuclear cells) and goblet cells was higher in the groups of the ethanol- and pentanol-fed rats than in the control group. There was an increase in the thickness of the brush border in pentanol-fed rats. Intervillus adhesion was concurrently observed in the pentanol-fed rats but not in the control or ethanol-fed rats. After ethanol feeding many of the villi developed blebs at the apex of the villus or laterally on its upper half. These blebs generally remained intact. In contrast, after pentanol feeding no bleb formation was appreciated. The intake of ethanol and other short chain alcohols present in alcoholic beverages leads to mainfold disturbances on the rat duodenum. These findings suggest that the chronic ingestion of pentanol seems to promote cellular changes but less important than those observed after chronic ethanol ingestion.     

Activation of Glutamate Dehydrogenase by Leucine and Its Nonmetabolizable Analogue in Rat Brain Synaptosomes

Leucine and beta-(+/-)-2-aminobicyclo[2.2.1]heptane-2-carboxylic acid (BCH) stimulated, in a dose-dependent manner, reductive amination of 2-oxoglutarate in rat brain synaptosomes treated with Triton X-100. The concentration dependence curves were sigmoid, with 10-15-fold stimulations at 15 mM leucine (or BCH); oxidative deamination of glutamate also was enhanced, albeit less. In intact synaptosomes, leucine and BCH elevated oxygen uptake and increased ammonia formation, consistent with stimulation of glutamate dehydrogenase (GDH). Enhancement of oxidative deamination was seen with endogenous as well as exogenous glutamate and with glutamate generated inside synaptosomes from added glutamine. With endogenous glutamate, the stimulation of oxidative deamination was accompanied by a decrease in aspartate formation, which suggests a concomitant reduction in flux through aspartate aminotransferase. Activation of reductive amination of 2-oxoglutarate by BCH or leucine could not be demonstrated even in synaptosomes depleted of internal glutamate. It is suggested that GDH in synaptosomes functions in the direction of glutamate oxidation, and that leucine may act as an endogenous activator of GDH in brain in vivo.     

Transamination and oxidation of leucine and valine in rat adipose tissue

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