Leucine Decarboxylation Research Articles

Branched-chain amino acid metabolism in rat muscle: abnormal regulation in acidosis.

Branched-chain amino acid (BCAA) metabolism is frequently abnormal in pathological conditions accompanied by chronic metabolic acidosis. To study how metabolic acidosis affects BCAA metabolism in muscle, rats were gavage fed a 14% protein diet with or without 4 mmol NH4Cl X 100 g body wt-1 X day-1. Epitrochlearis muscles were incubated with L-[1-14C]-valine and L-[1-14C]leucine, and rates of decarboxylation, net transamination, and incorporation into muscle protein were measured. Plasma and muscle BCAA levels were lower (P less than 0.05) in acidotic rats. Rates of valine and leucine decarboxylation and net transamination were higher (P less than 0.05) in muscles from acidotic rats; these differences were associated with a 79% increase in the total activity of branched-chain alpha-keto acid dehydrogenase and a 146% increase in the activated form of the enzyme. We conclude that acidosis affects the regulation of BCAA metabolism by enhancing flux through the transaminase and by directly stimulating oxidative catabolism through activation of branched-chain alpha-keto acid dehydrogenase.

The movement of organic solutes between the retina and pigment epithelium

The transport of several membrane precursors across the bullfrog retinal pigment epithelium (RPE)-choroid was studied in a modified Ussing chamber. The substrates choline, ethanolamine, and glucose did not exhibit a net flux across the tissue, while all amino acids tested showed a net flux in the retina to choroid direction. The 'sink' for a particular amino acid, leucine, was measured in bullfrog retinas in vitro. Decarboxylation of leucine was the major fate of the incorporated amino acid, while much less was directed toward protein synthesis. The subretinal fluid concentration of leucine was estimated to be in the order of 10 microM, approximately eight-fold less than in plasma, and consistent with levels in cerebrospinal fluid. The sink for leucine in the retina at 10 microM leucine is smaller than the fluxes across the RPE-choroid under conditions of an eight-fold gradient. This suggests that the RPE, not the retina, is responsible for controlling the concentrations of solutes in the subretinal fluid.

Ammonia detoxification by accelerated oxidation of branched chain amino acids in brains of acute hepatic failure rats.

BCAA aminotransferase and BCKA dehydrogenase activities are increased in the mitochondrial fractions from the brains of hepatic failure rats treated with two-thirds removal of CCl4-injured liver. Cerebral leucine decarboxylation was accelerated, and it well correlated with arterial blood ammonia levels. Elevation of brain ammonia content following an intraperitoneal injection of ammonium acetate to hepatic failure rats could be prevented by intravenous infusion of BCAA. Significantly increased brain glutamic acid, glutamine, and alanine contents were noted. These results suggested that accelerated brain BCAA catabolism in acute hepatic failure rats reduce the neurotoxicity of ammonia by promoting the synthesis of glutamic acid and glutamine from BCAA.

LEUCINE METABOLISM IN PERFUSED RAT SKELETAL MUSCLE DURING CONTRACTIONS

An isolated single rat hindlimb muscle preparation was used to examine leucine metabolism during steady-state conditions as a function of metabolic rate (VO2) and leucine concentration. The rates of muscle leucine uptake and leucine oxidation (measured as alpha-decarboxylation) were dependent on leucine delivery. At a physiological leucine concentration (0.1 mM), leucine uptake and alpha-ketoisocaproic acid (KIC) release during rest was 12.8 +/- 0.4 and 1.86 +/- 0.06 nmol.min-1.g-1 g, respectively. Leucine oxidation was 2.35 +/- 0.11 nmol.min-1.g-1 (n = 24) and if fully oxidized could account for only 3-4% of the resting VO2. This fraction was reduced to approximately 1% during contractions. The rate of leucine oxidation progressively increased, up to two to three times above rest (6-7 nmol.min-1.g-1), during contractions of graded frequency (7.5, 15, 30, 45, and 60 tetani/min) in a manner related to the eightfold increase in VO2 of the mixed fiber muscle. The fraction of muscle leucine uptake that was transaminated (i.e., leucine decarboxylation + KIC release) increased from 33% at rest to approximately 60% during contractions. The increase in leucine oxidation during contractions was probably primarily due to the high oxidative fast-twitch, red muscle mass, whose VO2 was estimated to increase up to 24-fold above rest. On the basis of our observed rates of muscle leucine alpha-decarboxylation, it is reasonable to attribute the rates of whole-body leucine oxidation of nontrained individuals during exercise to leucine oxidation by the working muscle.

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.

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.

Accelerated leucine decarboxylation in the rat brain in relation to increased blood ammonia levels during acute hepatic failure.

Leucine decarboxylation in rat brain was investigated during acute hepatic failure, induced by partial hepatectomy after carbon tetrachloride (CCl4) pretreatment of rats. These rats presented metabolic alkalosis, and had significantly higher levels of arterial blood and brain ammonia than control and CCl4-treated rats. Brain leucine decarboxylation was elevated in rats with hepatic failure. This alteration correlated with arterial blood ammonia concentrations, and probably with elevated brain ammonia levels, as brain ammonia levels were directly related to arterial blood ammonia.

Isotopic determination of amino acid-urea interactions in exercise in humans.

We recently reported that in light exercise (30% VO2max) the oxidation of [1-13C]leucine was significantly increased but the rate of urea production was unchanged (J. Appl. Physiol: Respirat. Environ. Exercise Physiol. 52: 458-466, 1982). We have therefore tested three possible explanations for this apparent incongruity. 1) We infused NaH13CO3 throughout rest and exercise and found that, although altered bicarbonate kinetics in exercise resulted in greater recovery of 13CO2, the difference between rest and recovery was small compared with the increase in the rate of 13CO2 excretion during exercise when [1-13C]leucine was infused. 2) We infused [15N]leucine and isolated plasma urea N to determine directly the rate of incorporation of the 15N. During exercise there was no increase in the rate of 15N incorporation. Simultaneously, we infused [2,3-13C]alanine and quantified the rate of incorporation of 15N in alanine. We found that [15N]alanine production from [15N] leucine more than doubled in exercise, and by deduction, alanine production from other amino acids also doubled. 3) We tested our previous assumption that [1-13C]leucine metabolism in exercise was representative of the metabolism of other essential amino acids by infusing [1-13C] and [alpha-15N]lysine throughout rest and exercise. We found that the rate of breakdown of lysine during exercise was not increased in a manner comparable to that of leucine. Thus, these data confirm our original findings that leucine decarboxylation is enhanced in light exercise but urea production is unchanged.(ABSTRACT TRUNCATED AT 250 WORDS)

Skeletal muscle metabolism in neonatal lean and obese pigs.

Nine lean and nine obese male pigs were examined at 14 d of age. The biceps femoris muscle of the obese pigs had a greater (P less than .05) percentage dry matter, protein and triglycerides than the muscle of lean pigs. The rate of oxidation of glucose to CO2 by the biceps femoris muscle was not influenced (P greater than .05) by phenotype but the rate was greater (P less than .05) when incubations were conducted in either the presence of leucine or palmitate. Glycolytic flux was lower (P less than .05) in the muscle of the obese pigs than in the muscle of lean pigs. Glycolytic flux was enhanced (P less than .05) by addition of leucine or palmitate to the incubation media. The rate of release of lactate, pyruvate, alanine, glutamine and glutamate into the media was similar between phenotypes and was not influenced by the presence of leucine or palmitate (P greater than .05). The total amount of leucine transaminated was greater (P less than .05) in the muscle of lean pigs than in the muscle of obese pigs. This was because of greater (P less than .05) rates of decarboxylation of leucine and release of alpha-ketoisocaproic acid into the media by the muscle of lean pigs when compared with the muscle of obese pigs. However, the ratio of leucine decarboxylated to alpha-ketoisocaproic acid released was similar (P greater than .05) between the two phenotypes. The ratio of palmitate oxidized (to CO2) to palmitate esterified was greater (P less than .01) in the muscle of the lean pigs than in the muscle of obese pigs. This latter finding may partially explain the greater triglyceride content of obese pig muscle. The generally lower rates of oxidation of substrates and of glycolytic flux in the biceps femoris muscle of obese pigs, when compared with lean pigs, may be associated with differences in body composition that develop during growth of lean and obese pigs.

Effects of lactation on L-leucine metabolism in the rat. Studies in vivo and in vitro.

1. The turnover rate of L-[1-14C]leucine was increased by 35% in lactating rats compared with virgin rats. Starvation or removal of pups (24 h) returned the value to that of the virgin rat. 2. Incorporation of L-[U-14C]leucine into lipid and protein of mammary glands of lactating rats in vivo increased 7-fold and 6-fold respectively compared with glands of virgin rats. Lactation caused no change in the incorporation of L-[U-14C]leucine into hepatic lipid and protein. 3. The production of 14CO2 from L[l-14C]leucine (in the presence of glucose) was similar in isolated acini from glands of fed (chow) and starved lactating rats. Feeding with a 'cafeteria' diet caused a slight decrease, and removal of pups a large decrease, in the oxidative decarboxylation of leucine. 4. Oxidation of L-[2-14C]leucine to 14CO2 was increased about 3-fold in acini from starved lactating rats or lactating rats fed on a 'cafeteria' diet compared with rats fed on a chow diet. Insulin decreased the formation of 14CO2 in all three situations. 5. Incorporation of L-[U-14C]- and [2-14C]-leucine into lipid was decreased in acini from starved lactating rats and lactating rats fed on a 'cafeteria' diet. Insulin tended to increase the conversion of [2-14C]leucine into lipid, but this was significant only in the case of the acini from 'cafeteria'-fed rats. 6. Experiments with (-)-hydroxycitrate indicate that the major route for conversion of leucine carbon into lipid in acini is via citrate translocation from the mitochondria. 7. The physiological implications of these findings are discussed.

Leucine degradation and release of glutamine and alanine by adipose tissue.

Adipose tissue from fed rats rapidly degrades [l14C]leucine to 14C02. This process is stimulated by glucose (but not by pyruvate) and by insulin, which also enhances the degradation of isoleucine and valine. Fasting and protein deficiency markedly reduced the catabolism of [l-‘4C]leucine in epididymal fat pads by decreasing the decarboxylation of a-ketoisocaproic acid and the transamination of leucine. In tissue homogenates, the rates of these reactions decreased by 63 and 47%, respectively, after 2 days of food deprivation. Within only 1 day of food deprivation, total decarboxylation of leucine in tissue homogenates was 73% slower. In adipose tissue from the fasted animals, leutine oxidation was also stimulated by glucose but not by insulin. In fat pads from fed (but not fasted) rats, leucine decreased the oxidation of pyruvate and thus may spare use of glucose-derived acetyl coenzyme A for lipogenesis. When fed and fasted rats were injected with [1-14CJleucine, their rates of expiration of 14C02 did not differ, but when they were injected with p-‘4C]leucine, 14C02 expiration was greater in the fasted rats. Therefore, food deprivation did not affect the total amount of leucine degradation by the organism but seemed to increase the oxidation of leucine by muscle, while decreasing its conversion to triglycerides. The incubated fat pads appear to synthesize and release large amounts of glutamine and smaller quantities of alanine and glutamate. Addition of leucine increased release into the medium of glutamine and, to a lesser extent, that of alanine and glutamate (without affecting tissue protein balance or the release of other amino acids). In fasting, when degradation of leucine fell, the accumulation of glutamine and alanine in the medium decreased. Isoleucine and valine were degraded more slowly than leucine and stimulated glutamine and alanine release to a lesser extent. In adipose tissue, as in muscle, production of glutamine, as well as alanine and glutamate, seems to be the route for disposal of amino groups released in the transamination of branched-chain amino acids.

The stimulus-secretion coupling of amino acid-induced insulin release: metabolism and cationic effects of leucine.

When isolated rat pancreatic islets are exposed to L-leucine (20 mM), the rate of NH4 production is close to the summed rates of L-[1-14C] leucine decarboxylation and alpha-ketoisocarproate production, whereas the rates of acetoacetate production and L-[U-14C]-leucine oxidation are compatible with conversion of each mole of the amino acid to one mole of acetoacetate and three moles of CO2. ATP content, ATP/ADP ratio, and adenylate charge are maintained at normal values by L-leucine, whereas the NADH/NAD+ ratio (but not the NADPH/NADP+ ratio) is significantly increased. The release of insulin evoked by L-leucine is potentiated by 2-ketoisovalerate, unaffected by L-valine, and inhibited by menadione. L-leucine mimicks the effect of D-glucose on 86Rb+ and 45Ca2+ handling by the islets. However, relative to its rate of oxidation, the insulinotropic effect of L-leucine is less marked than that of D-glucose. This may be due, in part at least, to a decrease in the oxidation of endogenous nutrients. It is concluded that the metabolic, cationic, and secretory effects of L-leucine in isolated islets are not incompatible with the fuel hypothesis for insulin release.

Rhythmic variations of valine and leucine decarboxylation in rat diaphragm

Daily rhythmic variations of valine and leucine decarboxylation in the rat diaphragm were measured. Both valine and leucine decarboxylation increased during the hours of darkness and decreased during hours of light. Hypophysectomy eliminated the daily variation of decarboxylation. When food is available ad lib. to normal rats, the time of most active feeding coincides with the hours of darkness. Therefore, the period of darkness and maximum feeding coincides with the maximum oxidation of these two essential amino acids, valine and leucine, by diaphragm, and an active pituitary appears to be necessary to maintain this relationship. This model can be used to study interrelationships of behavioral, neurohumoral, and metabolic rhythms.

Carrier-mediated Uptake of L-Leucine by the Brown Alga Giffordia mitchellae

Summary The uptake of 17 different amino acids by the marine f ilamentous brown alga Gi f f ordia mitchellae was measured; L-Leucine revealed the highest uptake rate. This uptake is a light independent process which is not simple diffusion. The pH- and temperature optimum is pH 6 and 20 to 21 °C. Under nearly natural conditions of pH 8.2 and 17 °C, KM is 0.3 to 1.4 - 10-4 M, and VMa~ is 3000 to 3800 nmol (100 mg dry weight - h)-1. An activation energy of 43.9 k J - mol-1 was calculated. Leucine uptake can be inhibited competitively and by CCCP. The amino acid is accumulated up to 27 fold within the cells; a decarboxylation of Leucine to 1-amino-3-methyl-butane was not detected. The uptake cannot be induced by Leucine or enhanced by a sea water medium with a low level of nitrogen. The results indicate a carrier mediated uptake system with a rather low affinity for L-Leucine and a weak substrate specifity.

Differential alterations in branched-chain amino acid decarboxylation in liver of hypophysectomized rats.

Valine decarboxylation was significantly increased and leucine decarboxylation was significantly decreased in rat liver slices following hypophysectomy. In both normal and hypophysectomized rats decarboxylation of leucine exceeded that of valine in slices whereas the reverse was observed with the respective keto acids and mitochondria.

Leucine oxidation in diabetes and starvation: Effects of ketone bodies on branched-chain amino acid oxidation in vitro

Both starvation and diabetes markedly increase the rate of α-decarboxylation of leucine by gastrocnemius muscle homogenate of rat. Since hyperketonemia is common to both these conditions, the effect of acetoacetate and β-hydroxybutyrate on the rate of α-decarboxylation of leucine by tissue homogenates of fed rats was investigated. The rate of leucine decarboxylation by muscle homogenate increased markedly when 1–20 m M acetoacetate was added to the incubation medium (109% increase at 20 m M). In contrast, β-hydroxybutyrate was without significant effect until its concentration in the medium was increased to 30 m M (24% increase). Acetoacetate also increased α-decarboxylation of valine, but had no effect on other amino acids, namely, alanine and glutamate. There was a significant correlation between the rates of leucine decarboxylation and the endogenous concentrations of acetoacetate in the gastrocnemius muscle of fed, starved, and diabetic rats ( r = 0.83, p < 0.01). The stimulatory effect of acetoacetate on leucine decarboxylation was also observed when homogenates of other skeletal muscles, namely, soleus, vastus lateralis, and rectus abdominis were used. The rate of α-decarboxylation of leucine by the other tissue homogenates was not altered (heart and diaphragm), was decreased (liver), or was slightly increased (kidney) by the addition of acetoacetate. The energy-yielding potential of acetoacetate did not appear responsible for enhancing the leucine decarboxylation rate by the muscle. Acetoacetate had no significant effect on the rate of leucine transamination, nor did it stimulate the uptake of cycloleucine by muscle mitochondria. Acetoacetate, however, markedly increased (over 100%) the rate of α-decarboxylation of α-ketoisocaproate by the muscle mitochondria. These results suggest that acetoacetate may have a metabolic role in regulation of the skeletal muscle decarboxylation of branched-chain amino acids in rats.

Assessment of Effect of Starvation, Glucose, Fatty Acids and Hormones on α-Decarboxylation of Leucine in Skeletal Muscle of Rat

The present investigations of rates of oxidation of [U-14C] or [1-14C]leucine by homogenates of gastrocnemius muscle of fed and starved rats have indicated that 14CO2 production is mainly the result of alpha-decarboxylation of leucine in this tissue. This incomplete oxidation was not the result of imparied tricarboxylic acid cycle since the oxidation of palmitate proceeded to completion within the experimental conditions. In the subsequent studies, the effect of altered nutrition and metabolic factors on alpha-decarboxylation of leucine by gastrocnemius muscle homogenates was investigated. Starvation increased the rate of alpha-decarboxylation of leucine. Glucose or palmitate (C16) added in physiological concentrations to the incubation medium were without effect on decarboxylation of leucine, but this reaction was stimulated by addition of 1 mM hexanoate (C6) or octanoate (C8) to the incubation medium. However, when fatty acid chain length was elongated to C10 (decanoate), the stimulatory effect was not only abolished, but this fatty acid significantly inhibited the rate of leucine decarboxylation. Addition of insulin, epinephrine, glucagon and cyclic AMP within a wide range of concentrations to the incubation medium did not significantly affect the rate of decarboxylation of leucine. These studies indicate a complex interrelationship between the metabolism of leucine and that of fatty acids.

Metabolic effects of hypoglycemic sulfonylureas—I: In vitro effect of sulfonylureas on leucine incorporation and metabolism and on respiration of rat tissues

The hypoglycemic sulfonylureas: chlorpropamide, tolbutamide, carbutamide and glycodiazine were found to inhibit leucine incorporation into the protein of rat diaphragm, liver, kidney and adipose tissue. This inhibition was not the result of a reduced amino acid transport, an alteration in the metabolism of leucine or an impaired endogenous respiration of these tissues. A stimulation of oxygen uptake was observed in muscle and liver early during the incubation. Glucose and insulin relieve to some extent the inhibition of leucine incorporation by sulfonylureas and this could indicate that the inhibition of protein synthesis is related to an impaired energy metabolism. The oxidative decarboxylation of leucine, in the four tissues studied, is strongly inhibited by chlorpropamide and is mainly responsible for the increased cellular levels of leucine found in diaphragm after incubation with chlorpropamide and tolbutamide. The possibility of a link between sulfonylurea- and leucine hypoglycemia is discussed.

Decarboxylation of neutral amino acids in Proteus vulgaris.

SUMMARY: Washed suspensions and cell-free extracts of Proteus vulgaris decarboxylate leucine, valine, norvaline, isoleucine, and α-amino-n-butyric acid. The system differs from most bacterial decarboxylases in being optimally active near pH 7 and in not requiring acid conditions for its formation. The system is adaptive (inducible); the presence of either leucine, valine or isoleucine will simultaneously induce decarboxylase activity against each of the five amino acids listed above. No additive effects were found when two amino acids were offered to the system simultaneously. Pyridoxal phosphate is required as coenzyme at least for valine and leucine decarboxylation; the affinity between apo- and co-enzyme is greater during decarboxylation of valine than leucine.