Leucine Decarboxylation Research Articles

Functional assay for analysis of variants of uncertain significance in maple syrup urine disease

Introduction: Enhanced molecular characterisation using high-throughput sequencing has significantly improved patient diagnosis and management. However, the identification of variants of uncertain significance (VUS) limits its use for clinical decision making, such as prenatal diagnosis in subsequent pregnancies. Case report: We report a female infant who presented with mild gross motor delay at 4 months and developed seizures with hypoglycaemia at 5 months. Plasma amino acid and urine organic acid findings were consistent with Maple syrup urine disease (MSUD), despite a normal newborn screening test for this condition. MSUD results in deficiency of branched-chain alpha-ketoacid dehydrogenase, responsible for leucine, isoleucine, and valine degradation, caused by biallelic pathogenic variants in BCKDHA, BCKDHB, or DBT genes. Results: Molecular analysis in this patient identified two VUS, c.434-15_434-4del and c.365A>G (p. Tyr122Cys) in the DBT gene. Functional testing, in the form of leucine decarboxylation studies in fibroblasts revealed deficient function and mRNA splicing studies provided additional data that permitted variant reclassification and subsequent prenatal testing. Conclusion: This case reinforces the need for specialist biochemical genetic laboratories to retain their ability to perform functional assays to aid the resolution of VUS.

Thermal decomposition of model compound of algal biomass

AbstractThis contribution investigates thermal decomposition of leucine, as a representative model compound for amino acids in algal biomass. We map out potential energy surface for a wide array of unimolecular and self‐condensation reactions operating in the decomposition of leucine. Decarboxylation and dehydration of leucine ensues by eliminating CO2 and –OH, respectively, from the –COOH group attached to the α‐carbon. The molecular channel for deamination involves cleavage of NH2 from α‐carbon of leucine. The activation energies for direct elimination of CO2, NH3, and H2O from a leucine molecule lie within 20.7 kJ/mol of each other. Activation energies for these decomposition pathways reside below the bond dissociation enthalpy of H–C(α) of 323.1 kJ/mol. The decarboxylation, deamination, and dehydration pathways, via radical‐prompted pathways, systematically require lower energy barriers, in reference to closed‐shell reaction corridors. Detailed computations at the CBS‐QB3 level provide the Arrhenius rate parameters for the unimolecular and bimolecular reactions, and standard enthalpies of formation, standard entropies, and heat capacities for all the products and intermediates. A kinetic analysis of gas‐phase reactions, within the context of a plug‐flow reactor model, accounts qualitatively for the formation of major products observed experimentally in the thermal degradation of the condensed‐phase leucine. Among notable N‐containing species, the model predicts the prevailing of NH3 over HCN and HNCO, in addition to corresponding appreciable concentrations of amines, imines, and nitriles. Our detailed kinetic investigation illustrates a negligible contribution of the self‐condensation reactions of leucine in the gas phase.

Kinetics and Mechanistic Approach to Palladium (II)-Catalyzed Oxidative Deamination and Decarboxylation of Leucine and Isoleucine by Anticancer Platinum (IV) Complex in Perchlorate Solutions

Oxidations of two aliphatic α-amino acids (AA), namely, leucine and isoleucine by hexachloroplatinate (IV) as an anticancer platinum (IV) complex has been studied using a spectrophotometric technique in perchlorate solutions in the presence of palladium (II) catalyst at a constant ionic strength of 1.0 mol dm-3 and at 25°C. The reactions did not proceed in the absence of the catalyst. The reactions of both amino acids showed a first order dependence on both [PtIV] and [PdII], and less than unit order dependences with respect to both [AA] and [H+]. Increasing ionic strength and dielectric constant of the reactions medium increased the rates of the reactions. A probable oxidations mechanism has been suggested and the rate law expression has been derived. Both spectral and kinetic evidences revealed formation of 1:1 intermediate complexes between AA and PdII before the rate-controlling step. The oxidation products of the investigated amino acids were identified as the corresponding aldehyde, ammonium ion and carbon dioxide. The activation parameters of the second order rate constants were evaluated and discussed

Diverse functional evolution of serine decarboxylases: identification of two novel acetaldehyde synthases that uses hydrophobic amino acids as substrates.

BackgroundType II pyridoxal 5′-phosphate decarboxylases are an important group of phylogenetically diverse enzymes involved in amino acid metabolism. Within plants, this group of enzymes is represented by aromatic amino acid decarboxylases, glutamate decarboxylases and serine decarboxylases. Additional evolutionary divergence of plant aromatic amino acid decarboxylases has resulted in further subcategories with distinct substrate specificities and enzymatic activities. Despite shared homology, no such evolutionary divergence has been characterized within glutamate decarboxylases or serine decarboxylases (SDC).ResultsComparative analysis of two previously characterized serine decarboxylase-like (SDC-like) enzymes demonstrates distinct substrate specificities despite their highly conserved primary sequence. The alternate substrate preference of these homologous SDC-like proteins indicated that functional divergence might have occurred with in SDC-like proteins. In an effort to identify additional SDC-like functional divergence, two uncharacterized SDC-like enzymes were recombinantly expressed and characterized.ConclusionsAn extensive biochemical analysis of two serine decarboxylases-like recombinant proteins led to an interesting discovery; both proteins catalyze the formation of acetaldehyde derivatives from select hydrophobic amino acids substrates. Specifically, Medicago truncatula [GenBank: XP_003592128] and Cicer arietinum [GenBank: XP_004496485] catalyze the decarboxylation and oxidative deamination of phenylalanine, methionine, leucine and tryptophan to generate their corresponding acetaldehydes. The promiscuous aldehyde synthase activity of these proteins yields novel products of 4-(methylthio) butanal, 3-methylbutanal (isovaleraldehyde) and indole-3-acetaldehyde from methionine, leucine and tryptophan respectively. A comparative biochemical analysis of the Medicago truncatula and Cicer arietinum enzymes against two previously characterized SDC-like enzymes further emphasizes the unusual substrate specificity and activity of these novel aldehyde synthases. Due to the strong substrate preference towards phenylalanine, it is likely that both enzymes function as phenylacetaldehyde synthesis in vivo. However, due to their significant sequence divergence and unusual substrate promiscuity these enzymes are functionally and evolutionary divergent from canonical phenylacetaldehyde synthesis enzymes. This work further elaborates on the functional complexity of plant type II PLP decarboxylases and their roles in secondary metabolite biosynthesis.Electronic supplementary materialThe online version of this article (doi:10.1186/s12870-014-0247-x) contains supplementary material, which is available to authorized users.

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-(14)C]leucine or L-[U-(14)C]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.

Rate of metabolic decarboxylation of leucine as assessed by a l[1‐13C1]leucine breath test combined with indirect calorimetry of broiler chickens fed isocaloric diets with different protein:fat ratio

Research has shown that broiler chickens reared on a low-protein diet have a more efficient protein digestion. However, information on the fate of absorbed amino acids in relation to the dietary crude protein level in poultry is sparse. Therefore, this study aimed at developing a methodology for a 1-(13)C(1)-leucine breath test combined with indirect calorimetry, and to apply this technique using broiler diets known to induce differences in protein retention. From 14 days of age onwards, broiler chickens were reared on one of two isocaloric diets with substitutions between fat and protein [low-protein (LP) vs. high-protein (HP) diet: 130.4 vs. 269 g protein/kg; and 101.8 vs. 27.9 g fat/kg]. Every 4 or 5 days, three chickens per diet were placed in the respiratory cells for 48 h. The broilers were intubated with 40 mg 1-(13)C(1)-leucine/kg body weight, followed by breath sampling for 4 h at 15-min intervals and mass spectrometric analysis of the (13)C:(12)C ratio in the samples. The CO(2) level in the respiratory cell air was monitored and excreta samples were collected. The methodology to study l[1-(13)C(1)]leucine decarboxyation in chickens using a breath test combined with indirect calorimetry was accomplished. Results of the nitrogen balance test indicated that the LP broilers had an improved dietary protein retention compared with the HP animals. Moreover, LP chickens decarboxylated a significantly lower percentage of l[1-(13)C(1)]leucine, demonstrating several 'protein- or amino acid-sparing' mechanisms in animals reared on a diet with lower protein level, both at the digestive and at the postabsorptive level.

Decreased pancreatic islet response to L-leucine in the spontaneously diabetic GK rat: enzymatic, metabolic and secretory data.

Pancreatic islets from hereditarily non-insulin-dependent diabetic Goto-Kakizaki (GK) rats have a deficient insulin response not only to D-glucose but also to L-leucine. Our aim was to explain the cellular mechanism(s) underlying the beta-cell unresponsiveness to this amino acid. Freshly collagenase isolated islets from GK rats and healthy Wistar control rats matched with them for sex and age were compared. Leucine uptake, metabolic fluxes and insulin secretory capacity were investigated on batch incubated-islets. Enzymatic activities were measured on sonicated islets. In GK rat islets, neither leucine transport nor leucine transaminase activity was disturbed. By contrast, 14CO2 production from either L-[U-14C]leucine or L-[1-14C]leucine was decreased. The L-[U-14C]leucine oxidation: L-[1-14C]leucine decarboxylation ratio was unaffected, indicating that the acetyl-CoA generated from leucine undergoes normal oxidation in the Krebs cycle. The leucine non-metabolizable analogue 2-amino-bicyclo[2,2,1]heptane-2-carboxylic acid induced insulin release and enhanced the secretory response to leucine as in controls, whereas leucine failed to amplify the response to the leucine analogue. Moreover, the potentiating action of L-glutamine on leucine-mediated insulin release was preserved. This coincided with normal glutamate dehydrogenase activity and L-[U-14C]glutamine oxidation. Finally, the secretory response to the leucine deamination product 2-ketoisocaproate was decreased, as was the 2-keto[1-14C]isocaproate oxidation. In islet beta cells from GK rats, the defective secretory response to leucine cannot be ascribed to a deteriorated leucine-stimulated glutamate metabolism but rather to an impaired leucine catabolism. A reduced generation of acetyl-CoA from 2-ketoisocaproate, due to the defective oxidative decarboxylation of this keto-acid by the mitochondrial branched-chain 2-ketoacid dehydrogenase, is incriminated.

Interphotoreceptor Retinoid-Binding Protein (IRBP) is Rapidly Cleared From theXenopusInterphotoreceptor Matrix

The interphotoreceptor matrix (IPM) is a highly-organized extracellular matrix critical to retinal development and function. Although the concentrations of its components are carefully regulated, little is known about the mechanisms of this regulation. Interphotoreceptor retinoid-binding protein (IRBP) is the most abundant soluble protein component of the IPM. Although its rate of clearance is thought to be an important factor regulating the concentration of IRBP within the IPM, no study has measured the rate of its extracellular turnover. Here we determine the rate of turnover of matrix IRBP inXenopus.The rate of IRBP turnover was estimated by measuring the loss of radioactivity from protein labeled by a single injection of a radiolabeled protein precursor. To provide an estimate of the rate of IRBP turnover, we have examined the following issues: (1) Quantitative extraction of IRBP from the IPM for biochemical analysis. (2) Routes of delivery of radiolabeled precursor to achieve a pulse label in vivo. (3) Selection of labeled precursor in order to minimize reutilization of radiolabel. Using Western blot analysis, immunoprecipitation and immuno-electron microscopy, we found that IRBP can be quantitatively extracted from the IPM by a simple saline wash. IRBP was radiolabeled by systemic or intravitreal injection of either [35S]methionine or carboxyl-terminal labeled [1-14C]leucine. The specific activity of matrix IRBP was determined by either phosphorimaging or fluorography of Coomassie blue-stained SDS-polyacrylamide gels. Intravitreal injection of tracer was more effective than systemic delivery in achieving a pulse of radiolabel to the retina. This may be due to intravitreal injection allowing the body to act as a ‘sink’ for radiolabeled amino acid. When radiolabeled precursor was delivered by intravitreal injection, the calculated half-life of matrix IRBP using [35S]methionine was 25.6±0.82 hr; in contrast, it was 10.7±2.9 hr using [1-14C]leucine. The faster apparent IRBP turnover using [1-14C]leucine is interpreted in context of the early decarboxylation of leucine during its degradation. Our results demonstrate rapid turnover of IRBP in theXenopusIPM in vivo and suggest that the IPM is a dynamic structure undergoing continuous renewal.

Ovine fetal leucine kinetics and protein metabolism during acute metabolic acidosis.

Fetal acidosis is associated with poor fetal growth. Because protein accretion is an important component of fetal growth, we used seven chronically prepared fetal lambs (10-16 days postoperation) to find whether fetal metabolic acidosis affected fetal protein accretion, and, if so, whether such effects were due to decreased synthesis or increased breakdown of proteins. Fetal leucine kinetics were measured during infusion of [1-(14)C]leucine by the reciprocal pool method. After control measurements, metabolic acidosis was induced by fetal infusion of 0.5 N HCl, and the measurements were repeated. Although fetal leucine concentration rose (164 +/- 11 vs. 216 +/- 15 microM; P < 0.001), fetal leucine uptake fell during acidosis (3.33 +/- 0.30 vs. 1.43 +/- 0.35 micromol x kg(-1) x min(-1); P < 0.05). However, the influx of leucine from protein breakdown increased (12.6 +/- 2.6 vs. 14.7 +/- 2.6 micromol x kg(-1) x min(-1); P < 0.02). The incorporation of leucine into fetal protein was unaffected by acidosis, so that fetal protein accretion fell (0.48 +/- 1.04 vs. -2.32 +/- 1.53 micromol x kg(-1) x min(-1); P < 0.001). Fetal leucine decarboxylation increased during acidosis (2.85 +/- 0.33 vs. 3.75 +/- 0.61 micromol x kg(-1) x min(-1); P < 0.05). We conclude that fetal metabolic acidosis stimulates pathways to degrade both protein and at least one of the subsequently derived amino acids, leucine. The consequence of such changes induced by acidosis is decreased protein accretion, a finding incompatible with normal fetal growth.

Effects of increased cortisol concentration on ovine fetal leucine kinetics and protein metabolism

Fetal protein accretion decreases as gestation progresses, primarily because protein synthesis decreases. Also, glucocorticoid primarily because protein synthesis decreases. Also, glucocorticoid concentrations increase late in gestation, and restraint of growth is one of the most consistently noted effects of glucocorticoids. Therefore, this study was done to determine whether fetal protein accretion is decreased by increased cortisol concentration and whether such an effect might be due to decreased protein synthesis or increased proteolysis. Six days after surgery, fetal leucine and protein metabolism was measured in eight pregnant ewes (114-119 days of gestation) at normal and elevated cortisol concentrations. Arterial blood leucine concentration, fetal leucine disposal rate, and exogenous leucine uptake were unaffected by cortisol infusion. Fetal leucine decarboxylation, however, increased by 19% (P < 0.001). Increased fetal cortisol concentration increased fetal proteolysis by 11% (P < 0.001) but did not affect the use of leucine for protein synthesis. Consequently, fetal protein accretion fell by 34% (P < 0.002). We conclude that increased fetal plasma cortisol concentration increases fetal proteolysis, thereby decreasing the rate of fetal protein accretion, an effect different from the decreased protein synthesis reported in late gestation.

Specificity of Leucine Effect on Protein Degradation in Perfused Rat Heart

The primary objective of the present study was to assess whether the inhibitory effect of leucine in rat hearts on protein degration is mediated by leucine itself, α-ketoisocaproate (α-KIC) or the decarboxylated products of leucine. Protein degradation, as measured by the release of phenylalanine, was inhibited by 1 mM leucine in hearts supplied with glucose, despite low intracellular α-KIC concentration (6.3 ± 0.5 μM). The inhibition of protein degradation by leucine occurred in hearts supplied with pyruvate, a substrate that completely abolished leucine decarboxylation. Under this condition, leucine was transaminated to α-KIC. Since the transamination could not be inhibited more than 38% by 10 mM l-cycloserine, it was difficult to exclude that leucine exerted its inhibitory effect via transamination to α-KIC. A clear correlation between protein degradation and intracellular leucine or α-KIC concentration could not be established. However, a high concentration of leucine (1 mM) or α-KIC (0.3 mM) in the perfusate inhibited protein degradation by 30 and 20%, respectively. This suggested that both leucine and α-KIC act on the plasma membrane regulatory sites. Of the various structural analogues of leucine that were tested, only l-leucinol inhibited protein degradation. This inhibition could be attributed to a direct lysosomotropic effect of this amino alcohol. The results indicate that high concentrations of extracellular leucine and α-KIC appear to inhibit heart protein degradation in vitro.

Ovine fetal protein metabolism during decreased glucose delivery

During decreased fetal glucose delivery, endogenous substrates increasingly support metabolic rate. If oxidation of fetal amino acid stores (i.e., protein) is to provide such substrate, either protein synthesis must decrease or protein breakdown must increase. Both of these changes would diminish fetal protein accretion, an important component of fetal growth. This study was performed, therefore, to find if decreased glucose delivery alters fetal leucine metabolism. Catheters were placed in six sheep fetuses under maternal general anesthesia, and, after 6 days recovery, fetal leucine metabolism was measured by infusion of [1-14C]leucine before and after fetal glucose delivery was decreased by infusing insulin to the ewe. Later (2 days) the experiment was repeated in reverse order. Decreased fetal glucose delivery was associated with a 19% decrease in the rate of fetal leucine disposal (P < 0.001), a 42% decrease in the rate of exogenous leucine uptake (P < 0.01), and no change in the rate of fetal leucine decarboxylation. The use of leucine for protein synthesis by the fetus fell by 23% (P < 0.001), whereas the rate of protein breakdown did not change. Consequently, during decreased fetal glucose delivery, leucine accretion into fetal proteins was 28% (P < 0.02) of the control rate. In summary, fetal oxidation of amino acids derived from increased protein breakdown is not the source of endogenous substrate needed by the fetus with restricted glucose availability. Fetal protein synthesis did decrease, however, diminishing the accretion of leucine into protein.

149 MITOCHONDRIAL MYOPATHY: DEFICIENCY OF HEAT SHOCK PROTEIN 60 (HSP60)

Mitochondrial (mt) myopathies are neuromuscular disorders caused by defects in mt metabolism. Hsp70 and 60 stabilize structures of mt polypeptides to assure membrane transport resp. assembly into functional enzymes. In one of 30 patient fibroblast cultures hsp60 was hardly detectable. The patient, from consanguineous parents, suffered from lactic acidosis and died 2 days after birth. Heat shock of the fibroblasts caused a minor increase of hsp60 and normal hsp70 induction. Increase of hsp60 mRNA was normal, suggesting defective translation or unstable hsp60 protein. Hsp60 deficiency is likely to cause defects in mt enzymes: activities of cyt. c oxidase, succinate cyt. c reductase and propionyl CoA carboxylase and leucine decarboxylation were deficient. Experiments to show the relation between hsp60 deficiency and the enzyme deficiencies will be discussed.

Effect of alpha-ketoacid dehydrogenase phosphorylation on branched-chain amino acid metabolism in muscle

The regulation of leucine and valine metabolism was evaluated in skeletal muscle of perfused rat hindlimb. Control of the branched-chain alpha-ketoacid dehydrogenase (BCKADH) via phosphorylation was removed with 0.4 mM alpha-chloroisocaproate (CIC). CIC activated the BCKADH complex 13- to 26-fold and led to increased rates of leucine and valine uptake into muscle, transamination to the corresponding alpha-ketoacid, and leucine (3- to 4-fold) and valine (6-fold) decarboxylation but led to decreased rates of alpha-ketoacid efflux from muscle. Although the increased rates of branched-chain amino acid (BCAA) decarboxylation were extensive, they were far below the extent of BCKADH activation as measured in vitro, suggesting that factors other than BCKADH activation become dominant in controlling the flux through alpha-ketoacid decarboxylation in skeletal muscle in situ. When the BCKADH capacity of muscle was increased 70-90% by a training-induced increase in mitochondrial content, the same 13- to 26-fold activation of the complex by CIC led to a rate of BCAA decarboxylation, which was only marginally greater (10-20%; P less than 0.05) than that of normal muscle. In addition, increasing the energy demand via muscle contractions led to a significant increase in leucine decarboxylation in the presence of complete activation of BCKADH by dephosphorylation. Thus BCKADH phosphorylation-dephosphorylation plays an important though not exclusive role in modulating the rates of BCAA metabolism in skeletal muscle. Differences in valine and leucine metabolism were apparent as valine catabolism bolstered citric acid cycle contents by increasing malate in red muscle with high mitochondrial content.(ABSTRACT TRUNCATED AT 250 WORDS)

Fetoplacental deamination and decarboxylation of leucine

Fetal and placental metabolism of leucine (Leu) and ketoisocaproic acid (KIC) were studied in seven fetal lambs at 132 +/- 1.3-days gestation. Fetal infusions of [1-13C]Leu, [1-14C]Leu, and antipyrine were carried out for 4 h. Uterine and umbilical blood flows were measured using the antipyrine steady-state diffusion technique. Leu and KIC concentrations, [14C]Leu-specific activities, 14CO2, [13C]Leu, and [13C]KIC enrichment (mole percent enrichment) were measured in the maternal artery, uterine vein, and umbilical artery and vein to calculate net fluxes of tracee and tracer molecules between fetus and placenta and between the uteroplacenta and the maternal circulation. There were net Leu and KIC fluxes into the fetus from the placenta with the KIC flux equal to approximately 19% of the combined Leu plus KIC flux. In addition, there was a net KIC flux into the uterine circulation. The fraction of infused tracer Leu escaping the placenta into the mother was small (approximately 6%). By contrast, there was a rapid exchange of tracer Leu carbon between placenta and fetus resulting in a significant flux of labeled KIC from placenta to fetus. Approximately 20% of the infused tracer carbon was converted to CO2 within the fetus. This rate of conversion was greater than 80% of the total fetoplacental conversion rate and significantly higher than the flux of KIC tracer carbon from placenta to fetus. Fetal KIC decarboxylation rate, calculated from the fetal KIC enrichment data, was 2.83 +/- 0.40 mumol.min-1.kg fetus-1 and approximately 60% of the combined net Leu and KIC flux into the fetus from the placenta.(ABSTRACT TRUNCATED AT 250 WORDS)

Differential sensitivity to beta-cell secretagogues in cultured rat pancreatic islets exposed to human interleukin-1 beta.

The early stages of insulin-dependent diabetes mellitus are characterized by a selective inability to secrete insulin in response to glucose, coupled to a better response to nonnutrient secretagogues. The deficient glucose response may be a result of the autoimmune process directed toward the beta-cells. Interleukin-1 (IL-1) has been suggested to be one possible mediator of immunological damage of the beta-cells. In the present study we characterized the sensitivity of beta-cells to different secretagogues after human recombinant IL-1 beta (rIL-1 beta) exposure. Furthermore, experiments were performed to clarify the biochemical mechanisms behind the defective insulin response observed in these islets. Rat pancreatic islets were isolated and kept in tissue culture (medium RPMI-1640 plus 10% calf serum) for 5 days. The islets were subsequently exposed to 60 pM human recombinant IL-1 beta during 48 h in the same culture conditions as above and examined immediately after IL-1 exposure. The rIL-1 beta-treated islets showed a marked reduction of glucose-stimulated insulin release. Stimulation with arginine plus different glucose concentrations, and leucine plus glutamine partially counteracted the rIL-1 beta-induced reduction of insulin release. The activities of the glycolytic enzymes hexokinase, glucokinase, and glyceraldehyde 3-phosphate dehydrogenase, were similar in control and IL-1-exposed islets. Treatment with IL-1 also did not impair the activities of NADH+- and NADPH+-dependent glutamate dehydrogenase, glutamate-aspartate transaminase, glutamate-alanine transaminase, citrate synthase, and NAD+-linked isocitrate dehydrogenase. The oxidation of D-[6-14C]glucose and L-[U-14C]leucine were decreased by 50% in IL-1-treated islets. Furthermore, there was a significant decrease in the ratios of [2-14C]pyruvate oxidation/[1-14C]pyruvate decarboxylation and L-[U-14C]leucine oxidation/L-[1-14C]leucine decarboxylation, indicating that IL-1 decreases the proportion of generated acetyl-coenzyme-A residues undergoing oxidation. However, in the presence of IL-1 there was a significant increase in L-[U-14C]glutamate oxidation. These combined observations suggest that exposure to IL-1 induces a preferential decrease in glucose-mediated insulin release and mitochondrial glucose metabolism. This mitochondrial dysfunction seems to reflect an impairment in proximal steps of the Krebs cycle. It is conceivable that the IL-1-induced suppression and shift in islet metabolism can be an explanation for the beta-cell insensitivity to glucose observed in the early phases of human and experimental insulin-dependent diabetes mellitus.

Acute and Chronic‐Intermittent Isovaleric Acidemia: Diagnosis and Glycine Therapy

AbstractIsovaleric acidemia is caused by an inherited defect in isovaleryl‐CoA dehydrogenase (E.C. 1.3.99.10). L‐leucine restriction and glycine supplementation are used to decrease the toxic accumulation of isovaleric acid (IVA). Supplemental glycine augments conversion of IVA to isovalerylglycine gVG) through the alternate pathway glycine‐N‐acylase (E.C. 2.3.1.13). Two clinical phenotypes are described, acute and chronic‐intermittent, and are differentiated by enzyme activity in cultured fibroblasts. In this study we determined the optimum therapeutic glycine supplement for treating isovaleric acidemia under stable conditions of restricted leucine intake as compared to oral leucine loading. Under stable clinical conditions and on a restricted leucine intake of 55 mg/kg/day a nine year old with chronic intermittent IVA excreted 12.3 ± 5.8 of IVG which rose to 33.6 ± 14.2 mmoles IVG/gm creatinine as glycine supplements were increased from 0 to 50 mg/kg/day at weekly intervals. Excretion of IVC plateaued between glycine intakes of 50–150 mg/kg, but unexpectedly fell to 13.8 ± 0.5 and 16.3 ± 0.9 mmoles/gm creatinine when glycine supplements were further increased to 300 and 600 mglkglday. IVG excretion increased in a two year old with acute IVA while ingesting 45 mg leucine/kg/day from 5.5 ± 2.8 on no glycine supplement to 10.6 ± 0.8 mmoleslgm creatinine on 600 mg/kg glycine supplements. IVG production was compared in the older patient with chronic intermittent IVA. During acute leucine loads, glycine supplements of 190 or 600 mg/kg/day, resulted in urinary IVG of 194 and 419 mmoles/gm creatinine, respectively.These studies demonstrate that: acute and chronic‐intermittent isovaleric acidemia are differentiated with [1–14C] and [2–14C]‐leucine decarboxylation assays by intact fibroblasts; that glycine supplements of 150 to 250 mg/kg/day are optimum in both forms of the disease when patients are clinically stable but additional supplements will increase IVG production during acute leucine load conditions. However increases in glycine supplementation above 150 mg/kg/day may inhibit IVG production in patients with the chronic intermittent form of isovaleric acidemia, when intracellular IVA accumulation is minimized by diet, or if glycine‐N‐acylase is less competent.

Leucine degradation in sheep.

1. In vitro leucine catabolism in adipose tissue, skeletal muscle, kidney and liver homogenates was studied in sheep. 2. In Expt 1, Suffolk X Targhee ram lambs were slaughtered at 1, 28, 56, 84, 112, 140, 168, 196, 224 and 365 d of age. In Expt 2, 5-month-old crossbred wethers were fed on 80, 120 or 180 g crude protein (nitrogen X 6.25)/kg diets for 4 weeks or fed on 120 g crude protein/kg for 4 weeks and then fasted for 48 or 96 h before slaughter. Leucine catabolism was assayed in tissue homogenates for Expts 1 and 2. 3. Leucine deamination (per unit protein) was highest in skeletal muscle at day 1 and then declined; liver exhibited an activity pattern akin to muscle while kidney activity tended to rise over the duration of the study. Adipose tissue in vitro leucine deamination was higher at all ages studied and 9- to 50-fold higher than all other tissues at 365 d. Leucine decarboxylation (per unit protein) was highest at day 1 in muscle and declined to low levels (P less than 0.01) after 28 d; liver and kidney decarboxylation activities were higher than muscle at all ages with kidney showing the highest activity. Whilst adipose had high initial activity it declined significantly (P less than 0.01) by day 28 and remained low. 4. Dietary protein intake had no effect on leucine deamination in any tissue. Leucine decarboxylation tended to increase with protein intake for all tissues except kidney.(ABSTRACT TRUNCATED AT 250 WORDS)

Role of activation of myocardial branched-chain 2-oxo acid dehydrogenase complex in the regulation of leucine decarboxylation during cardiac work in vitro.

The oxidative decarboxylation of L-[1-14C]leucine was measured in the isolated perfused rat heart under both working and non-working conditions. Stimulation of decarboxylation of the labelled substrate was observed in working hearts as cardiac work was increased, and in Langendorff hearts upon increasing the coronary flow rate. The rate of L-[1-14C]leucine decarboxylation was significantly higher (P less than 0.05) in hearts working against moderate afterload pressure when compared to Langendorff hearts perfused at a matching coronary flow rate. The rate of release of 4-methyl-2-oxo[1-14C]pentanoate to the perfusate was high in Langendorff hearts, and was unaffected by changes in coronary flow. In contrast, perfusate levels of 14C-labelled 4-methyl-2-oxopentanoate decreased significantly upon the establishment of the working condition (P less than 0.05). These findings suggested an enhancement in the efficiency of the decarboxylation of the 2-oxo acid in response to cardiac work. The amount of branched-chain 2-oxo acid dehydrogenase complex present in the active form was measured in freeze-clamped hearts. Cardiac work resulted in a rapid activation of the complex (P less than 0.02) within 5 min of work when compared to control Langendorff hearts perfused at matching coronary flow rates. To a lesser extent, increasing the coronary flow rate in Langendorff-perfused hearts also led to activation of the enzyme complex. These studies suggest the following: a) L-leucine oxidation in myocardial tissue can be accelerated by exercise as it is in other tissues; b) this regulatory response can be evoked by the contractile activity of the heart itself, independent of contributions by circulating factors or nervous stimuli; and c) regulation of the activity state of the branched-chain 2-oxo acid dehydrogenase complex is involved in the mechanism by which metabolic flux through this pathway is controlled during cardiac work.

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.