Rate Of Leucine Oxidation Research Articles

Underpinning the Food Matrix Regulation of Postexercise Myofibrillar Protein Synthesis by Comparing Salmon Ingestion With the Sum of Its Isolated Nutrients in Healthy Young Adults

BackgroundProtein is most commonly consumed as whole foods as opposed to single nutrients. However, the food matrix regulation of the postprandial muscle protein synthetic response has received little attention. ObjectivesThe purpose of this study was to assess the effects of eating salmon (SAL) and of ingesting the same nutrients as an isolated mixture of crystalline amino acids and fish oil (ISO) on the stimulation of postexercise myofibrillar protein synthesis (MPS) and whole-body leucine oxidation rates in healthy young adults. MethodsTen recreationally active adults (24 ± 4 y; 5 men, 5 women) performed an acute bout of resistance exercise, followed by the ingestion of SAL or ISO in a crossover fashion. Blood, breath, and muscle biopsies were collected at rest and after exercise during primed continuous infusions of L-[ring-2H5]phenylalanine and L-[1-13C]leucine. All data are presented as means ± SD and/or mean differences (95% CIs). ResultsPostprandial essential amino acid (EAA) concentrations peaked earlier (P = 0.024) in the ISO group than those in the SAL group. Postprandial leucine oxidation rates increased over time (P < 0.001) and peaked earlier in the ISO group (1.239 ± 0.321 nmol/kg/min; 63 ± 25 min) than those in the SAL group (1.230 ± 0.561 nmol/kg/min; 105 ± 20 min; P = 0.003). MPS rates for SAL (0.056 ± 0.022 %/h; P = 0.001) and ISO (0.046 ± 0.025 %/h; P = 0.025) were greater than the basal rates (0.020 ± 0.011 %/h) during the 0- to 5-h recovery period, with no differences between conditions (P = 0.308). ConclusionWe showed that the postexercise ingestion of SAL or ISO stimulate postexercise MPS rates with no differences between the conditions. Thus, our results indicate that ingesting protein from SAL as a whole-food matrix is similarly anabolic to ISO in healthy young adults. This trial was registered at www.clinicaltrials.gov as NCT03870165.

Chronic Fetal Leucine Infusion Increases Rate of Leucine Oxidation but Not of Protein Synthesis in Late Gestation Fetal Sheep

BackgroundLeucine increases protein synthesis rates in postnatal animals and adults. Whether supplemental leucine has similar effects in the fetus has not been determined. ObjectiveTo determine the effect of a chronic leucine infusion on whole-body leucine oxidation and protein metabolic rates, muscle mass, and regulators of muscle protein synthesis in late gestation fetal sheep. MethodsCatheterized fetal sheep at ∼126 d of gestation (term = 147 d) received infusions of saline (CON, n = 11) or leucine (LEU; n = 9) adjusted to increase fetal plasma leucine concentrations by 50%–100% for 9 d. Umbilical substrate net uptake rates and protein metabolic rates were determined using a 1-13C leucine tracer. Myofiber myosin heavy chain (MHC) type and area, expression of amino acid transporters, and abundance of protein synthesis regulators were measured in fetal skeletal muscle. Groups were compared using unpaired t tests. ResultsPlasma leucine concentrations were 75% higher in LEU fetuses compared with CON by the end of the infusion period (P < 0.0001). Umbilical blood flow and uptake rates of most amino acids, lactate, and oxygen were similar between groups. Fetal whole-body leucine oxidation was 90% higher in LEU (P < 0.0005) but protein synthesis and breakdown rates were similar. Fetal and muscle weights and myofiber areas were similar between groups, however, there were fewer MHC type IIa fibers (P < 0.05), greater mRNA expression levels of amino acid transporters (P < 0.01), and a higher abundance of signaling proteins that regulate protein synthesis (P < 0.05) in muscle from LEU fetuses. ConclusionsA direct leucine infusion for 9 d in late gestation fetal sheep does not increase protein synthesis rates but results in higher leucine oxidation rates and fewer glycolytic myofibers. Increasing leucine concentrations in the fetus stimulates its own oxidation but also increases amino acid transporter expression and primes protein synthetic pathways in skeletal muscle.

Leucine Is More Readily Oxidized When Ingested as an Isolated Nutrient versus Incorporated in Its Whole-Food Matrix

Abstract Objectives The ingestion of free amino acids, or isolated sources of protein, results in faster postprandial release of dietary amino acids into circulation, which stimulates muscle protein synthesis rates. However, indirect evidence suggests that this rapid release of dietary amino acids after the ingestion of free amino acids is coupled with higher amino acid oxidation rates when compared to a whole-food source. Whole food protein results in a reduced peak amplitude and prolonged postprandial aminoacidemia. This study aimed to assess the effects of eating a whole food source of protein on the stimulation of whole-body leucine oxidation rates versus eating these same nutrients in isolated form in healthy young adults. Methods In a crossover design, 10 recreationally active adults (24 ± 4 y; 5 M, 5 F) performed an acute bout of resistance exercise followed by the ingestion of salmon (SAL) (20.5 g protein and 7.5 g fat) or its matched constituents as crystalline amino acids and fish oil (ISO). Participants received priming doses of NaH13CO2 and L-[1–13C]leucine before initiating a constant L-[1–13C]leucine infusion. Blood and breath samples were collected at rest and after resistance exercise at regular intervals for the measurement of whole-body leucine oxidation rates and plasma leucine profiles. Data were tested using linear fixed effects models with time and group as fixed factors with Bonferroni's post hoc test. Results Postprandial plasma leucine concentrations did not differ between the SAL and ISO conditions (P &amp;gt; 0.05). Time to peak plasma leucine concentrations was faster in ISO (50 ± 27 min) vs. SAL (114 ± 64 min, P = 0.022) condition. Postprandial leucine oxidation rates were elevated from baseline at t = 30 min to t = 120 min in ISO and t = 60 min to t = 180 min in SAL (P &amp;lt; 0.001), with no total differences between group (P = 0.129). Time to peak leucine oxidation occurred sooner in ISO (66 ± 22 min; 1.358 ± 0.699 nmol · kg−1 · min−1) when compared to the SAL condition (105 ± 20 min; 1.067 ± 0.3076 nmol · kg−1 · min−1; P = 0.002). Conclusions We show that the ingestion of a whole-food source of protein resulted in a delayed stimulation of leucine oxidation when compared to free amino acid ingestion, but a similar net increase in oxidation during the 5 h postprandial period in healthy young adults. Funding Sources USDA National Institute of Food and Agriculture Hatch project 1017928

Elevated Plasma Branched-Chain Amino Acid Levels Correlate With Type 2 Diabetes-Related Metabolic Disturbances.

Patients with type 2 diabetes mellitus (T2DM) have elevated plasma branched-chain amino acid (BCAA) levels. The underlying cause, however, is not known. Low mitochondrial oxidation of BCAA levels could contribute to higher plasma BCAA levels. We aimed to investigate ex vivo muscle mitochondrial oxidative capacity and in vivo BCAA oxidation measured by whole-body leucine oxidation rates in patients with T2DM, first-degree relatives (FDRs), and control participants (CONs) with overweight or obesity. An observational, community-based study was conducted. Fifteen patients with T2DM, 13 FDR, and 17 CONs were included (age, 40-70 years; body mass index, 27-35 kg/m2). High-resolution respirometry was used to examine ex vivo mitochondrial oxidative capacity in permeabilized muscle fibers. A subgroup of 5 T2DM patients and 5 CONs underwent hyperinsulinemic-euglycemic clamps combined with 1-13C leucine-infusion to determine whole-body leucine oxidation. Total BCAA levels were higher in patients with T2DM compared to CONs, but not in FDRs, and correlated negatively with muscle mitochondrial oxidative capacity (r = -0.44, P < .001). Consistently, whole-body leucine oxidation rate was lower in patients with T2DM vs CON under basal conditions (0.202 ± 0.049 vs 0.275 ± 0.043 μmol kg-1 min-1, P < .05) and tended to be lower during high insulin infusion (0.326 ± 0.024 vs 0.382 ± 0.013 μmol kg-1 min-1, P = .075). In patients with T2DM, a compromised whole-body leucine oxidation rate supports our hypothesis that higher plasma BCAA levels may originate at least partly from a low mitochondrial oxidative capacity.

Prolonged amino acid infusion into intrauterine growth-restricted fetal sheep increases leucine oxidation rates.

Overcoming impaired growth in an intrauterine growth-restricted (IUGR) fetus has potential to improve neonatal morbidity, long-term growth, and metabolic health outcomes. The extent to which fetal anabolic capacity persists as the IUGR condition progresses is not known. We subjected fetal sheep to chronic placental insufficiency and tested whether prolonged amino acid infusion would increase protein accretion in these IUGR fetuses. IUGR fetal sheep were infused for 10 days with either mixed amino acids providing ~2 g·kg-1·day-1 (IUGR-AA) or saline (IUGR-Sal) during late gestation. At the end of the infusion, fetal plasma leucine, isoleucine, lysine, methionine, and arginine concentrations were higher in the IUGR-AA than IUGR-Sal group ( P < 0.05). Fetal plasma glucose, oxygen, insulin, IGF-1, cortisol, and norepinephrine concentrations were similar between IUGR groups, but glucagon concentrations were fourfold higher in the IUGR-AA group ( P < 0.05). Net umbilical amino acid uptake rate did not differ between IUGR groups; thus the total amino acid delivery rate (net umbilical amino acid uptake + infusion rate) was higher in the IUGR-AA than IUGR-Sal group (30 ± 4 vs. 19 ± 1 μmol·kg-1·min-1, P < 0.05). Net umbilical glucose, lactate, and oxygen uptake rates were similar between IUGR groups. Fetal leucine oxidation rate, measured using a leucine tracer, was higher in the IUGR-AA than IUGR-Sal group (2.5 ± 0.3 vs. 1.7 ± 0.3 μmol·kg-1·min-1, P < 0.05). Fetal protein accretion rate was not statistically different between the IUGR groups (1.6 ± 0.4 and 0.8 ± 0.3 μmol·kg-1·min-1 in IUGR-AA and IUGR-Sal, respectively) due to variability in response to amino acids. Prolonged amino acid infusion into IUGR fetal sheep increased leucine oxidation rates with variable anabolic response.

Growth and Clinical Variables in Nitrogen-Restricted Piglets Fed an Adjusted Essential Amino Acid Mix: Effects of Free Amino Acid–Based Diets

Excess protein intake in early life has been linked to obesity and metabolic syndrome in later life. Yet protein, and in particular the essential amino acids (EAAs), need to be present in adequate quantity to support growth. With the use of a piglet model restricted in dietary amino acids (AAs), we compared the efficacy and safety of a standard formula with a low-AA formula containing an adjusted AA composition. Female piglets (3-7 d old; Landrace × Yorkshire × Duroc) were fed 1 of 4 isoenergetic AA-based formulas for 14 d (700 kJ·kg body weight-1·d-1). The formulas contained a set control amount (44 g/L) and AA compositions referred to as the experimental standard (ST-100, n=22), or 20% or 50% lower total AAs (respectively, ST-80, n=19 and ST-50, n=13), or 20% lower total AAs with an optimally adjusted EAA composition (O-80, n=17). A series of clinical and paraclinical endpoints were measured. Growth rates were similar for ST-100, O-80 and ST-80 piglets (all ∼15 g·kg-1·d-1), whereas ST-50 had a markedly lower weight gain relative to all groups (all P<0.05). Relative to ST-100, all groups with reduced AA intake showed ∼16% reduction in plasma albumin and ∼30% reduction in plasma urea (both P<0.05). The absolute leucine oxidation rate was ∼30% lower for O-80 than for ST-100 piglets (P<0.05). These data show that a 20% reduction in total AA intake for both the control (ST-80) and the adjusted AA (O-80) formula did not have any short-term adverse effects on growth in artificially reared, AA-restricted piglets. The lower absolute leucine oxidation rate observed in O-80 supports the development of an infant formula with an improved AA composition and a moderate reduction in total protein to support adequate growth in healthy infants.

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.

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.

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.

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.

Oral low-dose testosterone administration induces whole-body protein anabolism in postmenopausal women: a novel liver-targeted therapy

In hypopituitary men, oral delivery of unesterified testosterone in doses that result in a solely hepatic androgen effect enhances protein anabolism during GH treatment. In this study, we aimed to determine whether liver-targeted androgen supplementation induces protein anabolism in GH-replete normal women. Eight healthy postmenopausal women received 2-week treatment with oral testosterone at a dose of 40 mg/day (crystalline testosterone USP). This dose increases portal concentrations of testosterone, exerting androgenic effects on the liver without a spillover into the systemic circulation. The outcome measures were whole-body leucine turnover, from which leucine rate of appearance (LRa, an index of protein breakdown) and leucine oxidation (Lox, a measure of irreversible protein loss) were estimated, energy expenditure and substrate utilization. We measured the concentration of liver transaminases as well as of testosterone, SHBG and IGF1. Testosterone treatment significantly reduced LRa by 7.1 ± 2.5% and Lox by 14.6 ± 4.5% (P<0.05). The concentration of liver transaminases did not change significantly, while that of serum SHBG fell within the normal range by 16.8 ± 4.0% and that of IGF1 increased by 18.4 ± 7.7% (P<0.05). The concentration of peripheral testosterone increased from 0.4 ± 0.1 to 1.1 ± 0.2 nmol/l (P<0.05), without exceeding the upper normal limit. There was no change in energy expenditure and fat and carbohydrate utilization. Hepatic exposure to unesterified testosterone by oral delivery stimulates protein anabolism by reducing protein breakdown and oxidation without inducing systemic androgen excess in women. We conclude that a small oral dose of unesterified testosterone holds promise as a simple novel treatment of protein catabolism and muscle wasting.

Propylthiouracil-induced hypothyroidism during lactation alters leucine and mannose metabolism in rat cerebellar slices

Thyroid hormone deficiency during perinatal development results in significant alterations in neurological functions. The relationship between such events and brain metabolism is not completely understood. The aim of this study was to investigate the effects of hypothyroidism on leucine, mannose, glucose and lactate metabolism in rat cerebellar slices. Experimental hypothyroidism was induced by exposing mothers and pups to propylthiouracil (PTU) until weaning - postnatal day 21. Metabolic analyses were performed in postnatal day 10 (PND10) and 21 (PND21) animals. A matching group of animals received the same oral treatment also after weaning until adulthood PND60 with T3 supplement during lactation (P1-P21). In PND21 animals, PTU treatment significantly increased the rate of leucine oxidation to CO2, although glucose and lactate oxidations were not affected. PTU treatment also increased the oxidation of leucine to CO2 at PND60 (adult animals). PND10 hypothyroidism animals showed a decrease in conversion of mannose to glycolipids and glycoprotein compared with the control group. However, PTU treatment increased the conversion of mannose to glycolipids and glycoprotein in PND21 animals. The replacement of T3 normalized mannose and leucine metabolism in adult rats. These results indicate that deficits in thyroid hormones during lactation could delay or alter brain development and metabolism.

Application of Key Events Dose Response Framework to Defining the Upper Intake Level of Leucine in Young Men1–4

Leucine is sold in large doses in health food stores and is ingested by weight-training athletes. The safety of ingestion of large doses of leucine is unknown. Before designing chronic high-dose leucine supplementation experiments, we decided to determine the effect of graded doses of leucine in healthy participants. The Key Events Dose Response Framework is an organizational and analytical framework that dissects the various biologic steps (key events) that occur between exposure to a substance and an eventual adverse effect. Each biologic event is looked at for its unique dose-response characteristics. For nutrients, there are a number of biologic homeostatic mechanisms that work to keep circulating/tissue levels in a safe, nontoxic range. If a response mechanism at a particular key event is especially vulnerable and easily overwhelmed, this is known as a determining event, because this event drives the overall slope or shape of the dose-response relationship. In this paper, the Key Events Dose Framework has been applied to the problem of leucine toxicity and leucine’s tolerable upper level. After analyzing the experimental data vis a vis key events for leucine leading to toxicity, it became evident that the rate of leucine oxidation was the determining event. A dose-response study has been conducted to graded intakes of leucine in healthy human adult male volunteers. All participants were started at the mean requirement level of leucine [50 mg/(kg · d)] and the highest leucine intake was 1250 mg/( kg · d), which is 25 times the mean requirement. No gut intolerance was seen. Blood glucose fell progressively but remained within normal values without any changes in plasma insulin. Maximal leucine oxidation levels occurred at an intake of 550 mg leucine/( kg · d), after which plasma leucine progressively increased and plasma ammonia also increased in response to leucine intakes >500 mg/( kg · d). Thus, the “key determining event” appears to be when the participants reach their maximal leucine oxidation level, after which the risk of metabolic adverse effects progressively increased.

Cellular signaling of amino acids towards mTORC1 activation in impaired human leucine catabolism

The regulation of cell growth and protein biosynthesis is triggered by the mammalian target of rapamycin complex 1 (mTORC1) responding to amino acids, especially leucine. The molecular mechanisms linking leucine to mTORC1 activation are not well understood. We analyzed whether the free intracellular leucine availability, a metabolite of leucine catabolism or the process of leucine oxidation activates mTORC1 signaling. We further investigated whether mTORC1 signaling is subject to altered regulation in disturbed leucine metabolism. Human fibroblasts with deficiencies in leucine catabolic steps and those from healthy control subjects were utilized. In all cells, leucine-induced mTORC1 signaling was significantly related to leucine pool size and leucine repletion capacity. The leucine/glutamine antiporter SLC7A5/SLC3A2 and the amino acid sensor MAP4K3 were identified as crucial determinants of signaling leucine availability to downstream targets. In cells with defective leucine catabolism, mTORC1 signaling towards phosphorylation of ribosomal protein S6 kinase 1 (S6K1) was significantly increased, whereas transcriptional down-regulation of MAP4K3 upon reduced amino acid supply was abrogated. Remarkably, these effects were observed irrespective of the localization of the enzymatic blockage. Our data provide evidence that mechanisms determining intracellular leucine availability and the amino acid sensor MAP4K3 are key upstream modulators of nutrient-sensitive mTORC1 signaling, whereas specific leucine metabolites or leucine oxidation rates do not play a role. In human fibroblasts deficient in leucine catabolic steps, we observed regulation consistent with sustaining a more efficient MAP4K3 and mTOR-S6K1 signaling. Such regulatory circuit might serve to protect cells against detrimental consequences of reduced nutrient utilization in human conditions associated with disturbed leucine metabolism.

Acute supplementation of amino acids increases net protein accretion in IUGR fetal sheep

Placental insufficiency decreases fetal amino acid uptake from the placenta, plasma insulin concentrations, and protein accretion, thus compromising normal fetal growth trajectory. We tested whether acute supplementation of amino acids or insulin into the fetus with intrauterine growth restriction (IUGR) would increase net fetal protein accretion rates. Late-gestation IUGR and control (CON) fetal sheep received acute, 3-h infusions of amino acids (with euinsulinemia), insulin (with euglycemia and euaminoacidemia), or saline. Fetal leucine metabolism was measured under steady-state conditions followed by a fetal muscle biopsy to quantify insulin signaling. In CON, increasing amino acid delivery rates to the fetus by 100% increased leucine oxidation rates by 100%. In IUGR, amino acid infusion completely suppressed fetal protein breakdown rates but increased leucine oxidation rate by only 25%, resulting in increased protein accretion rates by 150%. Acute insulin infusion, however, had very little effect on amino acid delivery rates, fetal leucine disposal rates, or fetal protein accretion rates in CON or IUGR fetuses despite robust signaling of the fetal skeletal muscle insulin-signaling cascade. These results indicate that, when amino acids are given directly into the fetal circulation independently of changes in insulin concentrations, IUGR fetal sheep have suppressed protein breakdown rates, thus increasing net fetal protein accretion.

Prolonged infusion of amino acids increases leucine oxidation in fetal sheep

Maternal high-protein supplements designed to increase birth weight have not been successful. We recently showed that maternal amino acid infusion into pregnant sheep resulted in competitive inhibition of amino acid transport across the placenta and did not increase fetal protein accretion rates. To bypass placental transport, singleton fetal sheep were intravenously infused with an amino acid mixture (AA, n = 8) or saline [control (Con), n = 10] for ∼12 days during late gestation. Fetal leucine oxidation rate increased in the AA group (3.1 ± 0.5 vs. 1.4 ± 0.6 μmol·min(-1)·kg(-1), P < 0.05). Fetal protein accretion (2.6 ± 0.5 and 2.2 ± 0.6 μmol·min(-1)·kg(-1) in AA and Con, respectively), synthesis (6.2 ± 0.8 and 7.0 ± 0.9 μmol·min(-1)·kg(-1) in AA and Con, respectively), and degradation (3.6 ± 0.6 and 4.5 ± 1.0 μmol·min(-1)·kg(-1) in AA and Con, respectively) rates were similar between groups. Net fetal glucose uptake decreased in the AA group (2.8 ± 0.4 vs. 3.9 ± 0.1 mg·kg(-1)·min(-1), P < 0.05). The glucose-O(2) quotient also decreased over time in the AA group (P < 0.05). Fetal insulin and IGF-I concentrations did not change. Fetal glucagon increased in the AA group (119 ± 24 vs. 59 ± 9 pg/ml, P < 0.05), and norepinephrine (NE) also tended to increase in the AA group (785 ± 181 vs. 419 ± 76 pg/ml, P = 0.06). Net fetal glucose uptake rates were inversely proportional to fetal glucagon (r(2) = 0.38, P < 0.05), cortisol (r(2) = 0.31, P < 0.05), and NE (r(2) = 0.59, P < 0.05) concentrations. Expressions of components in the mammalian target of rapamycin signaling pathway in fetal skeletal muscle were similar between groups. In summary, prolonged infusion of amino acids directly into normally growing fetal sheep increased leucine oxidation. Amino acid-stimulated increases in fetal glucagon, cortisol, and NE may contribute to a shift in substrate oxidation by the fetus from glucose to amino acids.

Examination of Chinese habitual dietary protein requirements of Chinese young female adults by indicator amino acid method.

To determine protein requirement of Chinese young female adults on habitual Chinese diet through indicator amino acid oxidation (IAAO) technique. Twenty women with a mean (SD) age of 21.6 (0.9) years were healthy based on questionnaire, physical examinations and screening tests. There were three consecutive periods of 7 days each with six different intakes of protein (0.70, 0.78, 0.86, 0.94, 1.02 and 1.10 g/kg·d) within Chinese habitual diets (proportions of good-quality protein were 40 to 45%). Subjects were randomly allocated equally into two groups (1.10, 0.86, 0.78 g/kg·d for group 1 and 1.02, 0.94, 0.70 g/kg·d for group 2 from period 1 to period 3 in turn). Adaptation days were from day 1 to day 6 and the isotope study day was day 7 in each period. Amino acid kinetics was measured in non-menstrual periods, based on the IAAO technique. Two indicators (rate of release of 13CO2 and rate of leucine oxidation) were used to estimate protein requirement by breakpoint analysis with a two-phase linear regression crossover model. Mean and population safe protein requirements of Chinese habitual diets in non-menstrual periods from the rate of release of 13CO2 were 0.91 and 1.09 g/kg·d, respectively. And from the rate of leucine oxidation were 0.92 and 1.10 g/kg·d, respectively. The protein requirement of young women on Chinese habitual diets in non-menstrual period was lower than the current protein reference intake for Chinese females. Further studies are necessary to explore female protein requirements during the whole menstrual cycle.

Protein consumption following aerobic exercise increases whole-body protein turnover in older adults

Research measuring whole-body protein turnover (WBPT) after both exercise and nutrition has generally focused on resistance exercise; however, there is a paucity of data regarding the effect of postaerobic exercise nutrition, especially in older adults. It is not known if postexercise protein feeding has a beneficial effect on protein turnover after low- to moderate-intensity exercise. We investigated whether consuming protein plus carbohydrate (PRO) immediately after an acute bout of aerobic exercise has an additive effect over carbohydrate alone (CHO) on WBPT in older individuals. Twelve healthy older adults (age, 59 ± 4 years) were studied on 2 separate occasions after 1 h of exercise at approximately 50% of maximal rate of oxygen uptake, followed by 4 h of recovery. Immediately following exercise, subjects ingested a CHO (60 g) or an isocaloric PRO beverage (40 g carbohydrate, 20 g whey protein). Whole-body protein metabolism was determined using [1-13C]leucine infusion (60 mg prime; 75 mgh(-1) continuous), and sampling blood and expired breath. Rates of whole-body leucine appearance and oxidation, and nonoxidative leucine disposal during the third and fourth hours of postexercise recovery were higher in the PRO group (2.51 ± 0.55, 0.78 ± 0.37, and 1.71 ± 0.44 micromol kg(-1)·min(-1), respectively) than in the CHO group (1.81 ± 0.27, 0.33 ± 0.14, and 1.47 ± 0.25 micromol kg(-1)·min-1, respectively; p = 0.001). Our results indicate that consumption of a PRO beverage after aerobic exercise increased WBPT to a greater extent than a CHO beverage.

Prolonged maternal amino acid infusion in late-gestation pregnant sheep increases fetal amino acid oxidation

Protein supplementation during human pregnancy does not improve fetal growth and may increase small-for-gestational-age birth rates and mortality. To define possible mechanisms, sheep with twin pregnancies were infused with amino acids (AA group, n = 7) or saline (C group, n = 4) for 4 days during late gestation. In the AA group, fetal plasma leucine, isoleucine, valine, and lysine concentrations were increased (P < 0.05), and threonine was decreased (P < 0.05). In the AA group, fetal arterial pH (7.365 +/- 0.007 day 0 vs. 7.336 +/- 0.012 day 4, P < 0.005), hemoglobin-oxygen saturation (46.2 +/- 2.6 vs. 37.8 +/- 3.6%, P < 0.005), and total oxygen content (3.17 +/- 0.17 vs. 2.49 +/- 0.20 mmol/l, P < 0.0001) were decreased on day 4 compared with day 0. Fetal leucine disposal did not change (9.22 +/- 0.73 vs. 8.09 +/- 0.63 micromol x min(-1) x kg(-1), AA vs. C), but the rate of leucine oxidation increased 43% in the AA group (2.63 +/- 0.16 vs. 1.84 +/- 0.24 micromol x min(-1) x kg(-1), P < 0.05). Fetal oxygen utilization tended to be increased in the AA group (327 +/- 23 vs. 250 +/- 29 micromol x min(-1) x kg(-1), P = 0.06). Rates of leucine incorporation into fetal protein (5.19 +/- 0.97 vs. 5.47 +/- 0.89 micromol x min(-1) x kg(-1), AA vs. C), release from protein breakdown (4.20 +/- 0.95 vs. 4.62 +/- 0.74 micromol x min(-1) x kg(-1)), and protein accretion (1.00 +/- 0.30 vs. 0.85 +/- 0.25 micromol x min(-1) x kg(-1)) did not change. Consistent with these data, there was no change in the fetal skeletal muscle ubiquitin ligases MaFBx1 or MuRF1 or in the protein synthesis regulators 4E-BP1, eEF2, eIF2alpha, and p70(S6K). Decreased concentrations of certain essential amino acids, increased amino acid oxidation, fetal acidosis, and fetal hypoxia are possible mechanisms to explain fetal toxicity during maternal amino acid supplementation.