Leucine Oxidation Research Articles

Iridium(III) Catalyzed Oxidation of Leucine by Chloramine-T in Acidic Medium: A Kinetic and Mechanistic Study

The present paper deals with the kinetics and mechanism of homogeneously Ir(III) chloride catalyzed oxidation of Leucine by chloramine-T (CAT) in perchloric acidic medium in the temperature range 30–45 °C. The reaction is carried out in the presence of mercuric acetate as a scavenger for chloride ion. The experimental results show first order kinetics with respect to the oxidant (CAT) and catalyst [Ir(III)] while positive effect with respect to substrate, i.e., Leucine was observed. The reaction shows negligible effect of [Hg(OAc)2], [H+] and ionic strength of the medium. Chloride ion positively influenced the rate of reaction. The various activation parameters were calculated from the rate measurments at different temperatures (30–45 °C). The reaction between chloramine-T and leucine in acid medium shows 1:1 stoichiometry. On the basis of kinetic studies, reaction stoichiometry and product analysis, a suitable mechanism has been proposed and rate law has been derived.

The dietary protein paradox and threonine 15 N-depletion: Pyridoxal-5'-phosphate enzyme activity as a mechanism for the δ15 N trophic level effect.

Nitrogen stable isotope ratios (δ15 N values) are used to reconstruct dietary patterns, but the biochemical mechanism(s) responsible for the diet to tissue trophic level effect and its variability are not fully understood. Here δ15 N amino acid (AA) values and physiological measurements (nitrogen intake, plasma albumin concentrations, liver-reduced glutathione concentrations and leucine oxidation rates) are used to investigate increased dietary protein consumption and oxidative stress (vitamin E deficiency) in rat total plasma protein. Using gas chromatography/combustion/isotope ratio mass spectrometry, the δ15 N values from N-pivaloyl-i-propyl esters of 15 AAs are reported for rats (n=40) fed casein-based diets with: adequate protein (AP, 13.8%; n=10), medium protein (MP, 25.7%; n=10), high protein (HP, 51.3%; n=10) or HP without vitamin E (HP-E; n=10) for 18weeks. Between the HP and AP groups, the δ15 NAA values of threonine (-4.0‰), serine (+1.4‰) and glycine (+1.2‰) display the largest differences and show significant correlations with: nitrogen intake, plasma albumin concentrations, liver-reduced glutathione concentrations and leucine oxidation rates. This indicates increased AA catabolism by the dietary induction of shared common metabolic pathways involving the enzymes threonine ammonia-lyase (EC 4.3.1.19), serine hydroxymethyltransferase (EC 2.1.2.1) and the glycine cleavage system (EC 2.1.2.10). The δ15 NAA values of the HP-E and HP groups were not found to be significantly different. The 15 N-depleted results of threonine are linked to increased activity of threonine ammonia-lyase, and show potential as a possible biomarker for protein intake and/or gluconeogenesis. We hypothesize that the inverse nitrogen equilibrium isotope effects of Schiff base formation, between AAs and pyridoxal-5'-phosphate cofactor enzymes, play a key role in the bioaccumulation and depletion of 15 N in the biomolecules of living organisms and contributes to the variability in the nitrogen trophic level effect. Copyright © 2017 John Wiley & Sons, Ltd.

Dehydration Causes Increased Reliance on Protein Oxidation in Mice: A Test of the Protein-for-Water Hypothesis in a Mammal.

During fasting, animals rely on a mixture of fats, carbohydrates, and proteins that are derived solely from endogenous sources. The relative contributions of these metabolic fuels chiefly depend on the duration of the fast, but other factors including previous diet, environmental temperature, and activity level can modulate the fuel mixture. It has long been held that endogenous proteins are spared from catabolism until the final stages of prolonged fasting and contribute a significant proportion of energy once the other metabolic fuels have been depleted. However, evidence is mounting that protein is catabolized supplemental to fat metabolism under some circumstances. This has been shown in migratory birds that exhibit dramatic reductions in lean mass during flights. One hypothesis to explain this seemingly maladaptive metabolic strategy is that the catabolism and oxidation of protein in situ yields five times more metabolic water than that generated through fat oxidation alone. Recent support for this hypothesis is that birds benefit from such a strategy due to their uricotelic nature. However, it remains unclear whether ureotelic mammals would also employ this strategy. Here we test the protein-for-water hypothesis in resting laboratory mice subjected to water deprivation during fasting while we tracked rates of protein and lipid catabolism using endogenously incorporated 13C-leucine and 13C-palmitic acid. We found no differences in instantaneous leucine oxidation; however, cumulative differences in instantaneous leucine oxidation ultimately resulted in a higher total leucine oxidation after 72 h of fasting in water-deprived animals. We also found that lipid oxidation was 8% higher in the hydrated mice, but the difference was not significant presumably because of a concomitant reduction in metabolic rates of the water-deprived mice. Our results indicate that mammals do increase rates of protein catabolism during dehydration but to a lesser degree than birds. The ability of mammals to produce highly concentrated urine and their lower inherent rates of protein turnover apparently preclude mammals from taking full advantage of the protein-for-water strategy during fasting under dehydrating conditions.

Testosterone prevents protein loss via the hepatic urea cycle in human.

The urea cycle is a rate-limiting step for amino acid nitrogen elimination. The rate of urea synthesis is a true indicator of whole-body protein catabolism. Testosterone reduces protein and nitrogen loss. The effect of testosterone on hepatic urea synthesis in humans has not been studied. To determine whether testosterone reduces hepatic urea production. An open-label study. Eight hypogonadal men were studied at baseline, and after two weeks of transdermal testosterone replacement (Testogel, 100 mg/day). The rate of hepatic urea synthesis was measured by the urea turnover technique using stable isotope methodology, with 15N2-urea as tracer. Whole-body leucine turnover was measured, from which leucine rate of appearance (LRa), an index of protein breakdown and leucine oxidation (Lox), a measure of irreversible protein loss, were calculated. Testosterone administration significantly reduced the rate of hepatic urea production (from 544.4 ± 71.8 to 431.7 ± 68.3 µmol/min; P < 0.01), which was paralleled by a significant reduction in serum urea concentration. Testosterone treatment significantly reduced net protein loss, as measured by percent Lox/LRa, by 19.3 ± 5.8% (P < 0.05). There was a positive association between Lox and hepatic urea production at baseline (r2 = 0.60, P < 0.05) and after testosterone administration (r2 = 0.59, P < 0.05). Testosterone replacement reduces protein loss and hepatic urea synthesis. We conclude that testosterone regulates whole-body protein metabolism by suppressing the urea cycle.

Safety and Tolerability of Leucine Supplementation in Elderly Men

Leucine, a branched-chain amino acid, has been shown to stimulate muscle protein synthesis and has been suggested to play a role in the prevention of age-related muscle atrophy (sarcopenia). Although leucine supplementation may be beneficial, the efficacious dose of leucine is unknown. Before conducting studies with increased doses of leucine, the Tolerable Upper Intake Level (UL) for leucine needs to be determined. The objective of this review is to describe 2 current studies to determine the UL for leucine in young and elderly men. Initially, in young men we tested the conceptual model of determining the maximum oxidative capacity of an amino acid to be an ideal marker for identifying the UL. Leucine oxidation, measured with the use of L-[1-13C]leucine, increased with increasing leucine intakes and reached a plateau at higher intakes. Two-phase linear regression analysis identified a breakpoint of 550 mg ⋅ kg−1 ⋅ d−1 (95% CI: 454, 646 mg ⋅ kg−1 ⋅ d−1), with a simultaneous increase in blood ammonia concentrations above normal values (35 μmol/L). Recently, a similar study was conducted in elderly men (∼72 y old). A breakpoint in leucine oxidation was observed at 431 mg ⋅ kg−1 ⋅ d−1 (95% CI: 351, 511 mg ⋅ kg−1 ⋅ d−1), with blood ammonia concentrations above normal (35 μmol/L) at leucine intakes >550 mg ⋅ kg−1 ⋅ d−1. Taking the data together, the UL for leucine intake in healthy elderly men could be set at a value similar to young men, at 500 mg ⋅ kg−1 ⋅ d–1, or ∼35 g/d for an individual weighing 70 kg; or, as a cautious estimate, the leucine UL could also be considered as 351 mg ⋅ kg−1 ⋅ d−1 (the lower 95% CI), which would be ∼24.5 g/d for an elderly individual weighing 70 kg. These studies to determine the UL for leucine in humans are acute diet studies, and future studies with additional biomarkers and long-term supplementation of leucine will be necessary.

Metabolic Fate of Branched-Chain Amino Acids During Adipogenesis, in Adipocytes From Obese Mice and C2C12 Myotubes.

Branched-chain amino acid (BCAA) catabolism is regulated by the branched-chain aminotransferase (BCAT2) and the branched-chain α-keto acid dehydrogenase complex (BCKDH). BCAT2 and BCKDH expression and activity are modified during adipogenesis and altered in adipose tissues of mice with genetic or diet-induced obesity. However, little is known about how these modifications and alterations affect the intracellular metabolic fate of BCAAs during adipogenesis, in adipocytes from mice fed a control or high-fat diet or in C2C12 myotubes. Here, we demonstrate that BCAAs are mainly incorporated into proteins during the early stages of adipocyte differentiation. However, they are oxidized and incorporated into lipids during the late days of differentiation. Conversely, 92% and 97% of BCAA were oxidized, 1.6% and 6% were used for protein synthesis and 1.2% and 1.5% were incorporated into lipids in adipocytes from epididymal and subcutaneous adipose tissue, respectively. All three pathways were decreased in adipocytes from mice fed a high-fat diet. In C2C12 myotubes, leucine is mainly used for protein synthesis and palmitate is incorporated into lipids. Interestingly, leucine decreased both palmitate oxidation and its incorporation to lipids and proteins; and palmitate increased leucine oxidation and decreased its incorporation to lipids and proteins in a dose-dependent manner. These results demonstrate that BCAA metabolic fate differs between the early and late stages of adipocyte differentiation and in adipocytes from mice fed a control or high-fat diet; and that leucine affects the metabolic fate of palmitate and vice versa in C2C12 myotubes. J. Cell. Biochem. 118: 808-818, 2017. © 2016 Wiley Periodicals, Inc.

Insulin resistance of protein anabolism accompanies that of glucose metabolism in lean, glucose-tolerant offspring of persons with type 2 diabetes

ObjectiveTo test whether protein anabolic resistance is an early defect in type 2 diabetes (T2D).Research design and methodsSeven lean, normoglycemic T2D offspring (T2D-O) and eight matched participants without family history...

Pattern of Amino Acid Oxidation in Low BMI South Indian Pregnant Women

It is not known whether Indian pregnant women with low Body Mass Index (BMI) have adequate protein stores to provide sufficient amino acids for the growing fetus and how this interacts with the maternal body fat (energy) stores in early pregnancy. This study aimed to measure amino acid oxidation in pregnant women with low BMI and to evaluate the influence of maternal body fat on amino acid oxidation in early pregnancy. In two experimental studies of low BMI women, leucine oxidation rates significantly reduced from 1&lt;sup&gt;st&lt;/sup&gt; to 3&lt;sup&gt;rd&lt;/sup&gt; trimester (post absorptive state, p&amp;lt;0.002 and fed state, p&amp;lt;0.003, Wilcoxon sign rank test). In the post absorptive state in the 1st trimester, leucine oxidation negatively correlated with maternal fat percentage (r=-0.32, p=0.04). Hence, it would appear that the availability of body fat as an energy store in early pregnancy could reduce the utilization of amino acid as a substrate for the energy demands of the growing feto-placental unit.

Biotinylation: a novel posttranslational modification linking cell autonomous circadian clocks with metabolism.

Circadian clocks are critical modulators of metabolism. However, mechanistic links between cell autonomous clocks and metabolic processes remain largely unknown. Here, we report that expression of the biotin transporter slc5a6 gene is decreased in hearts of two distinct genetic mouse models of cardiomyocyte-specific circadian clock disruption [i.e., cardiomyocyte-specific CLOCK mutant (CCM) and cardiomyocyte-specific BMAL1 knockout (CBK) mice]. Biotinylation is an obligate posttranslational modification for five mammalian carboxylases: acetyl-CoA carboxylase α (ACCα), ACCβ, pyruvate carboxylase (PC), methylcrotonyl-CoA carboxylase (MCC), and propionyl-CoA carboxylase (PCC). We therefore hypothesized that the cardiomyocyte circadian clock impacts metabolism through biotinylation. Consistent with decreased slc5a6 expression, biotinylation of all carboxylases is significantly decreased (10-46%) in CCM and CBK hearts. In association with decreased biotinylated ACC, oleate oxidation rates are increased in both CCM and CBK hearts. Consistent with decreased biotinylated MCC, leucine oxidation rates are significantly decreased in both CCM and CBK hearts, whereas rates of protein synthesis are increased. Importantly, feeding CBK mice with a biotin-enriched diet for 6 wk normalized myocardial 1) ACC biotinylation and oleate oxidation rates; 2) PCC/MCC biotinylation (and partially restored leucine oxidation rates); and 3) net protein synthesis rates. Furthermore, data suggest that the RRAGD/mTOR/4E-BP1 signaling axis is chronically activated in CBK and CCM hearts. Finally we report that the hepatocyte circadian clock also regulates both slc5a6 expression and protein biotinylation in the liver. Collectively, these findings suggest that biotinylation is a novel mechanism by which cell autonomous circadian clocks influence metabolic pathways.

MON-PP218: Increased Whole Body Transamination and Oxidation of Leucine in Chronic Heart Failure

MON-PP218: Increased Whole Body Transamination and Oxidation of Leucine in Chronic Heart Failure

Abstract 384: Leucine, Serine and Methionine Differentially Affect Protein Synthesis in Rat Heart

Alternating flux through metabolic pathways enables the heart to rapidly adapt to stress. As a consequence, compensatory mechanisms are often associated with changes in mitochondrial function and cellular redox state. The collective importance of these metabolic changes to energy provision and protein turnover remains unknown. We therefore integrated radioactive tracer studies with a mathematical model of cardiac metabolism - CardioNet. We assessed through flux rate analysis the impact of altered amino acid supply on mitochondrial metabolism. In combination with leucine, both serine and methionine resulted in a two- to threefold increased variance of mitochondrial flux rates. Next we used the isolated working rat heart to determine whether supply of glucose and leucine, in combination with either serine or methionine at different concentrations, affects cardiac protein turnover and energy provision. While glucose remained the major energy source, oxidation of leucine contributed to ATP production. Methionine, but not serine, markedly increased both myocardial oxygen consumption and cardiac power at normal physiologic concentrations. Protein synthesis rates (units given in μmol/min/g dry wt) did not significantly differ from leucine alone (0.037±0.001) or leucine in combination with serine (0.038±0.003) or methione (0.045±0.007). However, with increased serine supply, protein synthesis significantly increased (0.0907±0.009) compared to methionine (0.059±0.006, p=0.024) or leucine only (0.015±0.002, p=0.031). The results suggest that myocardial protein synthesis is differentially regulated by specific amino acids.

Maternal post-absorptive leucine kinetics during late pregnancy in US women with HIV taking antiretroviral therapy: A cross-sectional pilot study

Despite the success of combination antiretroviral therapy (cART) for the prevention of mother to child transmission of HIV, infants exposed to cART in utero frequently are born smaller and have mild cardiac abnormalities. The mechanisms responsible for lower birth weight and cardiac abnormalities in children exposed to cART are unclear but could be related to dysregulation of maternal amino acid metabolism during pregnancy. Previous data in HIV(-) women have shown a relationship between abnormal maternal protein metabolism during pregnancy and low infant birth weight and animal data demonstrate a relationship between altered maternal protein metabolism and increased risk for offspring cardiovascular abnormalities. The objectives of this study were to: characterize post-absorptive maternal leucine kinetics during late pregnancy andexamine the relationships between maternal leucine kinetics and offspring birth weight and cardiac function. Post-absorptive maternal leucine kinetics (evaluated by using stable isotope tracer methodology) in 16 HIV(+) women receiving cART and 14 HIV(-) US women during the 3rd trimester of pregnancy were compared. Relationships between post-absorptive maternal leucine kinetics, cardiac function (echocardiography) and birth weight were statistically examined. Maternal plasma leucine concentration (HIV(-): 82.8 ± 10.7 vs. HIV(+): 72.3 ± 13.5 μM, p=0.06) and leucine oxidation rate (HIV(-): 6.1 ± 1.6 vs. HIV(+): 4.9 ± 1.8 μmol/kgBW/min, p=0.03) were lower in HIV+ women compared to controls. Total leucine turnover rate, non-oxidative leucine disposal rate and post-absorptive maternal glucose and palmitate kinetics did not differ between groups. Left ventricular fractional shortening tended to be lower in children born to HIV(+) compared to controls (HIV(-): 42 ± 1 vs. HIV+: 36 ± 5 %, p=0.08) and associated with lower maternal plasma leucine concentration (r= 0.43, p=0.08). Preliminary results indicate that post-absorptive maternal leucine metabolism during late pregnancy is mildly altered in HIV+ US women taking cART. The clinical significance of maternal leucine metabolism on adverse infant outcomes is unclear and should be further explored in more expansive studies.

Octanoic acid promotes branched-chain amino acid catabolisms via the inhibition of hepatic branched-chain alpha-keto acid dehydrogenase kinase in rats

ObjectiveIt has been reported that administration of octanoic acid, one of medium-chain fatty acids (MCFAs), promoted leucine oxidation in vitro and in vivo, but it remained unclear how octanoic acid stimulated leucine oxidation. Therefore, the aim of this study was to elucidate the mechanism that octanoic acid facilitates branched-chain amino acid (BCAA) catabolism. Materials/MethodsIn in vivo experiments, male rats were orally administered MCFAs as free fatty acids or triacylglycerol (trioctanoin), and then activities of hepatic branched-chain α-ketoacid dehydrogenase (BCKDH) complex (BCKDC) and BCKDH kinase (BDK) and alterations in the concentration of blood components were analyzed. In in vitro experiments, purified BCKDC associated with BDK (BCKDH–BDK complex) was reacted with various concentrations of hexanoic, octanoic, and decanoic acids. ResultsOral administration of trioctanoin in rats activated hepatic BCKDC via down-regulation of BDK activity in association with a decrease in plasma BCAA concentration and an increase in serum ketone body concentration. In vitro experiments using purified BCKDH–BDK complex showed that MCFAs (hexanoic, octanoic, and decanoic acids) inhibited BDK activity and that this inhibition was higher in hexanoic and octanoic acids than in decanoic acid. Oral administration of octanoic acid, but not decanoic acid, in rats activated hepatic BCKDC via down-regulation of BDK activity by decreasing the amount of BDK bound to the complex. The serum ketone body level was elevated by both administration of octanoic acid and decanoic acid. ConclusionThese results suggest that octanoic acid promotes BCAA catabolism in vivo by activation of BCKDC via decreasing the bound form of BDK.

Determination of the Tolerable Upper Intake Level of Leucine in Healthy Elderly (70‐75y)

Leucine, a branched‐chain amino acid, has been shown to stimulate muscle protein synthesis, and thus it has been proposed to prevent age related muscle atrophy (sarcopenia). Therefore, leucine supplementation may have potential benefits in elderly populations. The UL (tolerable upper intake level) for leucine intake in young men has been recently determined to be 500 mg/kg/day, and increases in ammonia concentrations were seen at intake levels above 500/mg/kg/day. The objective of the current study was to determine the UL for leucine in healthy elderly men, as this is currently unknown. Five healthy elderly men (70 – 76 yrs) received graded stepwise increases in leucine intakes ranging from 50 ‐ 650 mg/kg/day, provided on eight separate study days. The UL of leucine was identified by measurements of plasma and urinary biochemical variables, including blood ammonia, glucose and insulin concentrations. Further, the participants received an oral primed‐continuous protocol of 1‐13C‐leucine on all study days to measure changes in leucine oxidation. None of the participants reported any adverse physical symptoms on study days. Leucine oxidation appears to plateau with increasing leucine intakes. Blood ammonia concentrations above normal values (35 µmol/L) were observed at leucine intake of 350mg/kg/day and above. Plasma glucose stayed within normal ranges. These preliminary results suggest that the UL for leucine in healthy elderly men may be lower than the current UL of 500 mg/kg/d for healthy young men.(Supported by ICAAS)

Formoterol, a Highly β2-Selective Agonist, Induces Gender-Dimorphic Whole Body Leucine Metabolism in Humans

ObjectiveFormoterol is a β2-selective agonist that enhances protein anabolism in rodents. Whether formoterol imparts anabolic benefits in humans is unknown. The objective of the study was to investigate the effects of formoterol on whole body protein rates of turnover, oxidative loss and synthesis. DesignOpen label intervention study. PatientsFifteen healthy adults (8 men). MeasurementsVolunteers were treated with oral formoterol 160μg/day for one week. Changes in leucine turnover (LRa; index of protein breakdown), oxidation (Lox; irreversible protein loss) and incorporation into protein (LIP; index of protein synthesis) were assessed using the whole body 1-[13C]leucine turnover technique before/after treatment. ResultsLRa, Lox and LIP correlated significantly with lean body mass (LBM). LRa, adjusted for LBM was significantly higher (P<0.05, 160±6 vs 109±3μmol/min) in men but not fractional Lox and LIP (expressed as a proportion of LRa). Formoterol reduced LRa (−9±4%) in men but stimulated LRa (9±3%) in women. Formoterol significantly reduced (P<0.05) fractional Lox, an effect greater in women (−4±1 vs −1±1 %). It stimulated fractional LIP in women (∆4±1%, P<0.05) but not in men (∆1±1%). Formoterol induced an absolute anabolic effect that was greater in women (30 vs 8%). Heart rate, systolic and diastolic blood pressures were unaffected. ConclusionIn a therapeutic dose, formoterol stimulates protein anabolism in humans. It induced gender-dimorphic effects on protein turnover and on the partitioning of amino acids from oxidative loss toward protein synthesis, effects that are greater in women than in men. Formoterol holds promise as a treatment for sarcopenia.

Enhanced Glutamine Availability Exerts Different Effects on Protein and Amino Acid Metabolism in Skeletal Muscle From Healthy and Septic Rats.

Enhanced glutamine (GLN) intake may affect the catabolism of branched-chain amino acids (BCAAs; valine, leucine, and isoleucine), which play a regulatory role in protein turnover. We examined the effects of enhanced GLN availability on leucine oxidation, amino acid concentrations, and protein metabolism in muscles from healthy and septic rats. Cecal ligation and puncture were used as a model of sepsis. Twenty-four hours after surgery, the soleus (SOL, red muscle) and the extensor digitorum longus (EDL, white muscle) were incubated in medium containing 0.5 or 2.0 mM GLN. Protein breakdown, protein synthesis, and leucine oxidation were determined via 3-methylhistidine release, muscle L-[1-(14)C]leucine radioactivity, and the radioactivity of released (14)CO2, respectively. In muscles from septic animals, increased proteolysis and leucine oxidation and decreased protein synthesis were detected. These effects were more pronounced in the EDL. In septic muscles, the addition of GLN decreased leucine oxidation in both muscles and increased protein synthesis in the EDL. In muscles from untreated animals, decreased leucine oxidation after the addition of GLN to the medium was associated with decreased protein synthesis in the SOL and decreased concentrations of serine, glycine, histidine, alanine, arginine, proline, and lysine in both muscles. White muscle fibers are more sensitive to septic stimuli than red fibers are. In sepsis, enhanced GLN intake may ameliorate GLN deficiency, inhibit BCAA catabolism, and stimulate protein synthesis. In the healthy state, surplus of GLN may lead to severe alterations in the intramuscular concentration of several amino acids and impair protein synthesis.

Effect of palmitate on the metabolic fate of leucine and vice versa in primary adipocytes of a rat model of diet‐induced obsesity (575.7)

Metabolic profiling studies have spotlighted altered fatty acid (FA) or branched chain amino acid (BCAA) oxidation that marks the obese and insulin‐resistant phenotype. Interestingly, mRNA expression of Carnitine palmitoyltransferase I (CPT1) ‐the rate‐limiting enzyme of FA oxidation‐ and Isovaleryl‐CoA dehydrogenase (IVDH) ‐a mitochondrial enzyme involved in BCAA oxidation‐ are inversely correlated in the livers of 41 recombinant inbred mouse strains of the BxD genetic reference population (http://www.genenetwork.org/). The inverse correlation between CPT1 and IVDH suggest that a high rate of FA oxidation could decrease BCAA oxidation and vice versa. To test this hypothesis, we have evaluated the effect of increasing concentrations of palmitate or leucine in the metabolic fate of leucine or palmitate in primary adipocytes of a rat model of diet‐induced obsesity. We incubated primary adipocytes with: 1) 0, 250, 500 or 1000 µM of palmitate and evaluated the oxidation, incorporation to lipids or proteins of [U‐14C]‐leucine; or 2) 0, 250, 500 and 1000 µM of leucine and determined the oxidation, incorporation to lipids or proteins of [1‐14C]‐palmitic acid. Interstingly, palmitate increased leucine oxidation and decreased its incorporation to lipids and proteins in a dose‐dependent manner. Furthermore, leucine decreased both palmitate oxidation and its incorporation to lipids and proteins in a dose‐dependent manner. Overall, our results demonstrate that palmitate affects the metabolic fate of leucine and that leucine modifies the metabolic fate of palmitate in primary adipocytes of a rat model of diet‐induced obsesity.Grant Funding Source: CONACYT 155949

Enrichment of amino acid-oxidizing, acetate-reducing bacteria

In anaerobic condition, amino acids are oxidatively deaminated, and decarboxylated, resulting in the production of volatile fatty acids. In this process, excess electrons are produced and their consumption is necessary for the accomplishment of amino acid degradation. In this study, we anaerobically constructed leucine-degrading enrichment cultures from three different environmental samples (compost, excess sludge, and rice field soil) in order to investigate the diversity of electron-consuming reaction coupled to amino acid oxidation. Constructed enrichment cultures oxidized leucine to isovalerate and their activities were strongly dependent on acetate. Analysis of volatile fatty acids (VFAs) profiles and community structure analysis during batch culture of each enrichment indicated that Clostridium cluster I coupled leucine oxidation to acetate reduction in the enrichment from the compost and the rice field soil. In these cases, acetate was reduced to butyrate. On the other hand, Clostridium cluster XIVb coupled leucine oxidation to acetate reduction in the enrichment from the excess sludge. In this case, acetate was reduced to propionate. To our surprise, the enrichment from rice field soil oxidized leucine even in the absence of acetate and produced butyrate. The enrichment would couple leucine oxidation to reductive butyrate synthesis from CO2. The coupling reaction would be achieved based on trophic link between hydrogenotrophic acetogenic bacteria and acetate-reducing bacteria by sequential reduction of CO2 and acetate. Our study suggests anaerobic degradation of amino acids is achieved yet-to-be described reactions.

Glutamine deficiency in extracellular fluid exerts adverse effects on protein and amino acid metabolism in skeletal muscle of healthy, laparotomized, and septic rats

Characteristic feature of critical illness, such as trauma and sepsis, is muscle wasting associated with activated oxidation of branched-chain amino acids (valine, leucine, isoleucine) and enhanced release of glutamine (GLN) to the blood. GLN consumption in visceral tissues frequently exceeds its release from muscle resulting in GLN deficiency that may exert adverse effects on the course of the disease. In the present study, we investigated the effects of GLN depletion in extracellular fluid on GLN production and protein and amino acid metabolism in skeletal muscle of healthy, laparotomized, and septic rats. Cecal ligation and puncture (CLP) was used as a model of sepsis. After 24 h, soleus muscle (SOL, slow-twitch, red muscle) and extensor digitorum longus (EDL, fast-twitch, white muscle) were isolated and incubated in a medium containing 0.5 mM GLN or without GLN. L-[1-(14)C]leucine was used to estimate protein synthesis and leucine oxidation, 3-methylhistidine release was used to evaluate myofibrillar protein breakdown. CLP increased GLN release from muscle, protein breakdown and leucine oxidation, and decreased protein synthesis. The effects were more pronounced in EDL. Alterations induced by laparotomy were similar to those observed in sepsis, but of a lower extent. GLN deficiency in medium enhanced GLN release and decreased intramuscular GLN concentration, decreased protein synthesis in muscles of intact and laparotomized rats, and enhanced leucine oxidation in SOL of intact and protein breakdown in SOL of laparotomized rats. It is concluded that (1) fast-twitch fibers are more sensitive to septic stimuli than slow-twitch fibers, (2) extracellular GLN deficiency may exert adverse effects on protein and amino acid metabolism in skeletal muscle, and (3) muscles of healthy and laparotomized animals are more sensitive to GLN deficiency than muscles of septic animals.

Effects of Chinese herbal medicine on plasma glucose, protein and energy metabolism in sheep

BackgroundThe use of antibiotics in animal diets is facing negative feedback due to the hidden danger of drug residues to human health. Traditional Chinese herbal medicine has been used to replace antibiotics in the past two decades and played an increasingly important role in livestock production. The present study was carried out to assess the feeding effects of a traditional nourishing Chinese herbal medicine mixture on kinetics of plasma glucose, protein and energy metabolism in sheep. Ruminal fermentation characteristics were also determined.MethodsFour sheep were fed on either mixed hay (MH-diet) or MH-diet supplemented with 2% of Chinese herbal medicine (mixture of Astragalus root, Angelica root and Atractylodes rhizome; CHM-diet) over two 35-day periods using a crossover design. The turnover rate of plasma glucose was measured with an isotope dilution method using [U-13C]glucose. The rates of plasma leucine turnover and leucine oxidation, whole body protein synthesis (WBPS) and metabolic heat production were measured using the [1-13C]leucine dilution and open circuit calorimetry.ResultsBody weight gain of sheep was higher (P = 0.03) for CHM-diet than for MH-diet. Rumen pH was lower (P = 0.02), concentration of rumen total volatile fatty acid tended to be higher (P = 0.05) and acetate was higher (P = 0.04) for CHM-diet than for MH-diet. Turnover rates of plasma glucose and leucine did not differ between diets. Oxidation rate of leucine tended to be higher (P = 0.06) for CHM-diet than for MH-diet, but the WBPS did not differ between diets. Metabolic heat production tended to be greater (P = 0.05) for CHM-diet than for MH-diet.ConclusionsThe sheep fed on CHM-diet had a higher body weight gain and showed positive impacts on rumen fermentation and energy metabolism without resulting in any adverse response. Therefore, these results suggested that the Chinese herbal medicine mixture should be considered as a potential feed additive for sheep.