Free Leucine Research Articles

Comparison of the effects of rumen-protected and unprotected L-leucine on fermentation parameters, bacterial composition, and amino acids metabolism in in vitro rumen batch cultures

This study was conducted to compare the effects of rumen-protected (RP-Leu) and unprotected L-leucine (RU-Leu) on the fermentation parameters, bacterial composition, and amino acid metabolism in vitro rumen batch incubation. The 5.00 g RP-Leu or RU-Leu products were incubated in situ in the rumen of four beef cattle (Bos taurus) and removed after 0, 2, 4, 6, 12, 16, and 24 h to determine the rumen protection rate. In in vitro incubation, both RP-Leu and RU-Leu were supplemented 1.5 mmol/bottle (L-leucine HCl), and incubated after 0, 2, 4, 6, 8, 12, and 16 h to measure gas production (GP), nutrient degradability, fermentation parameters, bacterial composition, and amino acids metabolism. Results from both in vitro and in situ experiments confirmed that the rumen protection rate was greater (p < 0.01) in RP-Leu than in RU-Leu, whereas the latter was slow (p < 0.05) degraded within incubation 8 h. Free leucine from RP-Leu and RU-Leu reached a peak at incubation 6 h (p < 0.01). RU-Leu supplementation increased (p < 0.05) gas production, microbial crude protein, branched-chain AAs, propionate and branched-chain VFAs concentrations, and Shannon and Sobs index in comparison to the control and RP-Leu supplementation. RU-Leu and RP-Leu supplementation decreased (p < 0.05) the relative abundance of Bacteroidota, which Firmicutes increased (p < 0.05). Correlation analysis indicated that there are 5 bacteria at the genus level that may be positively correlated with MCP and propionate (p < 0.05). Based on the result, we found that RP-Leu was more stable than RU-Leu in rumen fluid, but RU-Leu also does not exhibit rapid degradation by ruminal microbes for a short time. The RU-Leu was more beneficial in terms of regulating rumen fermentation pattern, microbial crude protein synthesis, and branched-chain VFAs production than RP-Leu in vitro rumen conditions.

In vitro digestion of two protein-rich dairy products in the ageing gastrointestinal tract.

It is still unclear if changes in protein digestibility and absorption kinetics in old age may affect the anabolic effect of high-protein foods. The objective of this study was to investigate the digestion of two high-protein (10% w/w) dairy products in vitro: a fermented dairy product formulated with a ratio of whey proteins to caseins of 80 to 20% (WBD) and a Skyr containing mainly caseins. The new static in vitro digestion model adapted to the general older adult population (≥65 years) proposed by the INFOGEST international consortium was implemented to investigate the digestion of these products and compared with the standard version of the protocol. Kinetics of proteolysis was compared between both models for each product, in the gastric and intestinal phases of digestion. Protein hydrolysis was studied by the OPA method, SDS-PAGE, and LC-MS/MS, and amino acids were quantified by HPLC. Protein hydrolysis by pepsin was slower with the older adult model than with the young adult model, and consequently, in spite of a longer gastric phase duration, the degree of proteolysis (DH) at the end of the gastric phase was lower. Two different scenarios were observed depending on the type of dairy product studied: -10 and -40% DH for Skyr and WBD, respectively. In the intestinal phase, lower concentrations of free leucine were observed in older adult conditions (approx. -10%), but no significant differences in proteolysis were observed overall between the models. Therefore, the digestion conditions used influenced significantly the rate and extent of proteolysis in the gastric phase but not in the intestinal phase.

Leucine ingestion promotes mTOR translocation to the periphery and enhances total and peripheral RPS6 phosphorylation in human skeletal muscle.

The activation of the mechanistic target of rapamycin complex 1 (mTORC1), a master regulator of protein synthesis, by anabolic stimuli (such as muscle contraction or essential amino acids) involves its translocation to the cell periphery. Leucine is generally considered the most anabolic of amino acids for its ability to independently modulate muscle protein synthesis. However, it is currently unknown if free leucine impacts region-specific mTORC1-mediated phosphorylation events and protein-protein interactions. In this clinical trial (NCT03952884; registered May 16, 2019), we used immunofluorescence methods to investigate the role of dietary leucine on the postprandial regulation of mTORC1 and ribosomal protein S6 (RPS6), an important downstream readout of mTORC1 activity. Eight young, healthy, recreationally active males (n = 8; 23 ± 3 yrs) ingested 2g of leucine with vastus lateralis biopsies collected at baseline, 30, 60, and 180min postprandial. Leucine promoted mTOR translocation to the periphery (~ 18-29%; p ≤ 0.012) and enhanced mTOR localization with the lysosome (~ 16%; both p = 0.049) at 30 and 60min post-feeding. p-RPS6Ser240/244 staining intensity, a readout of mTORC1 activity, was significantly elevated at all postprandial timepoints in both the total fiber (~ 14-30%; p ≤ 0.032) and peripheral regions (~ 16-33%; p ≤ 0.014). Additionally, total and peripheral p-RPS6Ser240/244 staining intensity at 60min was positively correlated (r = 0.74, p = 0.036; r = 0.80, p = 0.016, respectively) with rates of myofibrillar protein synthesis over 180min. The ability of leucine to activate mTORC1 in peripheral regions favors an enhanced rate of MPS, as this is the intracellular space thought to be replete with the cellular machinery that facilitates this anabolic process.

Whey Protein Supplementation Is Superior to Leucine-Matched Collagen Peptides to Increase Muscle Thickness During a 10-Week Resistance Training Program in Untrained Young Adults.

The purpose of this study was to investigate the effects of supplementation of whey protein (WP) versus leucine-matched collagen peptides (CP) on muscle thickness MT and performance after a resistance training (RT) program in young adults. Twenty-two healthy untrained participants were randomly assigned to either a WP (n = 11) or leucine-matched CP (n = 11) group and then submitted to a supervised 10-week RT program (3days/week). The groups were supplemented with an equivalent amount of WP (35g, containing 3.0g of leucine) and CP (35g, containing 1.0g of leucine and 2.0g of free leucine) during the intervention period (after each workout and in the evening on nontraining days). MT of the vastus lateralis and biceps brachii, isokinetic peak torque and mean power output of the elbow flexors, and peak power output of the lower body were assessed before and after the RT program. The WP group experienced a greater (interaction, p < .05) increase in the vastus lateralis (effect size, WP = 0.68 vs. CP = 0.38; % Δ, WP = 8.4 ± 2.5 vs. CP = 5.6 ± 2.6%) and biceps brachii muscle thickness (effect size, WP = 0.61 vs. CP = 0.35; % , WP = 10.1 ± 3.8 vs. CP = 6.0 ± 3.2%), with a similar increase in muscle performance (peak torque, mean power output, and peak power output) between groups (time p < .05). Supplementation with WP was superior to leucine content-matched CP supplementation in increasing muscle size, but not strength and power, after a 10-week RT program in young adults.

Effects of rumen-protected leucine on production performance and starch digestion in the small intestine of lactating goats

This study was conducted to evaluate the effects of rumen-protected leucine (RP-Leu) on production performance and starch digestion in the small intestine of lactating goats. Thirty lactating goats (body weight: 43.07 ± 5.06 kg; first parity, day in milk: 135 ± 15 days; milk yield: 2.06 ± 0.44 kg/d) were randomly assigned into one of the following dietary treatments: (1) CON: basal diet and (2) RPL: basal diet + 10.6 g/d RP-Leu. The experiment lasted 65 days, 15 days for adaption, 45 days for treatment, and 5 days for measurement. Goats fed the diet supplemented with RP-Leu had higher (P < 0.05) average daily gain than those fed the CON diet, but no significant difference (P > 0.05) was observed in milk yield and milk composition. The addition of RP-Leu did not affect dry matter intake, apparent total tract digestibility of starch, and rumen fermentation parameters in lactating goats (P > 0.05). The feces of goats fed the diet supplemented with RP-Leu had reduced molar proportion of butyrate and increased pH and molar proportion of acetate compared with that of goats fed the basal diet (P < 0.05). Dietary supplementation of RP-Leu increased the relative abundance of Christensenellaceae, Christensenellaceae_R-7, Ruminococcaceae, and Ruminococcus in feces, while reducing the relative abundance of Lachnospiraceae and Anaerostipes (P < 0.05). Serum low-density lipoprotein and free leucine were greater (P < 0.05) for goats fed the RPL diet than those of goats fed the CON diet. In addition, the concentrations of total cholesterol and high-density lipoprotein in goats fed the RPL diet tended to be higher (0.05 ≤ P < 0.10) than those of goats fed the CON diet. In conclusion, dietary supplementation of RP-Leu probably improved starch digestibility in the small intestine of lactating goats showing as reduced hindgut starch fermentation and unchanged starch intake, apparent total tract digestibility of starch and rumen fermentation parameters.

FEATURES OF LEUCINE TRANSAMINATION IN THE SKELETAL MUSCLES OF RATS UNDER CONDITIONS OF NUTRIENT IMBALANCE IN FOOD RATION

The paper presents studies of the features of transamination of leucine in skeletal muscles of rats under conditions of nutrient imbalance in the diet. The distribution of the content of free leucine in muscles and blood plasma, the activity of leucine aminotransferase in muscles under conditions of consumption of excessive sucrose content, lack of dietary protein and the combined effect of two dietary factors were studied. During the experiment, experimental animals consumed a semi-synthetic AIN-93 diet in accordance with the recommendations of the American Institute of Nutrition. For the purpose of simulating alimentary protein deprivation, the animals received a semi-synthetic low-protein diet containing 1/3 of the generally accepted daily protein requirement daily for 28 days. A high-sucrose diet was simulated by increasing the amount of sucrose in the diet by 4 times. The content of leucine in skeletal muscles and blood plasma was determined by the method of chromatographic analysis on an automatic amino acid analyzer T 339. Leucine aminotransferase activity in the mitochondrial fraction of skeletal muscles of rats was assessed by the amount of α-ketoisocaproate formed. A significant decrease in leucine content in skeletal muscle of protein-deficient rats was found by 42% and by approximately 70% in groups of animals that received excessive amounts of sucrose compared to the values of control animals. Since the excessive consumption of sucrose leads to a maximum decrease in the level of this amino acid in the muscles of animals, this can be considered as one of the mechanisms of disruption of the supply of glucose to insulin-dependent tissues. At the same time, a 40% decrease in leucine aminotransferase activity was recorded in the skeletal muscles of protein-deficient rats; when consuming a high-sucrose diet, the activity of the studied enzyme is reduced by 50% compared to control indicators. The research results show a decrease in the concentration of leucine in the blood plasma of all experimental groups of animals compared to control indicators. The lowest values of the level of this amino acid were recorded under conditions of consumption of an excess amount of sucrose, regardless of the protein content in the diet. Nutrient imbalance in the diet is accompanied by a violation of leucine transamination processes in the skeletal muscles of rats, which is characterized by a decrease in leucine aminotransferase activity in the mitochondrial fraction, and is obviously associated with a decrease in the content of leucine, the substrate of this reaction. Excessive consumption of sucrose is a key factor in reducing the content of leucine in the blood plasma of rats, which can be considered as a characteristic of the intracellular deficiency of this amino acid in muscles.

Leucine did not stimulate growth and accretion in either stressed or unstressed Atlantic salmon

The aim of the current trial was to test whether leucine affected growth and accretion including test any effects on leucine upon stress. Quadruplicate tanks each containing 50 Atlantic salmon (Salmo salar) (mean start body weight of 524 ± 28 g) were fed diets containing 27.3, 30.0, 35.0 and 41.0 g leucine/kg diet for 74 days. Two tanks per dietary group were exposed to a stressor (5 min chasing) three days a week to test whether enriched leucine diet aid coping with chronic stress, while two tanks per dietary groups were left unstressed. The stressed fish consumed less feed and grew less than the unstressed fish, irrespective of diet. Leucine inclusion did not affect protein accretion, but leucine retention declined with increasing dietary leucine in both stressed and unstressed fish. No difference between the stressed or unstressed fish was present. Leucine did not affect relative liver size, but unstressed fish had slightly higher relative liver size compared with stressed fish (p = 0.05). Free leucine in the muscle and liver was not affected by dietary leucine, but unstressed fish had higher concentration of valine and isoleucine in the muscle compared with the stressed fish. Muscle of fish fed elevated leucine had lower mRNA expression of murf1 (p = 0.037) and higher expression of ppara (p = 0.012). Muscle of stressed fish had higher expression of the oxydative genes mnsod (p = 0.049) and catalase (p = 0.037) compared with the fish left unstressed, while in liver, there were no differences of expression of any of the genes tested. In conclusion, diets enriched in leucine had minor effects and neither protein accretion nor growth was affected in either stressed or unstressed fish.

Ameliorative effect of enhanced Fischer ratio flaxseed protein hydrolysate in combination with antioxidant micronutrients on ethanol-induced hepatic damage in a rat model

Alcohol abuse causes severe metabolic abnormalities inducing hepatic damage and malnutrition. Since higher Fischer ratio proteins have therapeutic value in liver diseases, an investigation was undertaken to study the ameliorative effect of the enhanced Fischer ratio flaxseed protein hydrolysate (EFR-FPH) alone and in combination with antioxidant micronutrients on ethanol-induced hepatotoxicity in a rat model. The EFR-FPH was prepared by dual enzymatic hydrolysis and charcoal treatment of flaxseed protein. The ratio of the branched-chain:aromatic amino acids (Fischer ratio) was found to be 7·08. The EFR-FPH, characterised using LC-MS/MS, showed the abundance of free leucine and isoleucine compared with phenylalanine and tyrosine. The matrix-assisted laser desorption/ionisation-time of flight MS analysis revealed the larger peptides present in EFR-FPH with mass 2·3 kDa. The EFR-FPH improved the nutritional status, liver function and antioxidant defense in the ethanol hepatotoxicity-induced rat model. The hepatoprotective effect of EFR-FPH was significantly enhanced when combined with selenium or vitamin E. Ethanol-induced changes in the liver tissue were effectively suppressed in the groups receiving EFR-FPH. Flaxseed-based hepatoprotective dietary supplement was formulated incorporating an optimum level of EFR-FPH (10 %) based on sensory acceptability and was fortified with selenium and vitamin E. The hepatoprotective formulation significantly lowered aspartate transaminase, alanine transaminase, alkaline phosphatase and bilirubin by 47, 61, 55 and 78 %, respectively, and improved the antioxidant defense in the ethanol hepatotoxicity-induced rat model. The current investigation suggests that EFR-FPH in synergy with antioxidant micronutrients is potent in ameliorating ethanol-induced hepatotoxicity and has a potential to form a hepatoprotective dietary supplement.

Formation of 3-Methylbutanal and 3-Methylbutan-1-ol Recognized as Malty during Fermentation in Swiss Raclette-Type Cheese, Reconstituted Milk, and de Man, Rogosa, and Sharpe Broth.

This work aimed to determine the formation over time of 3-methylbutanal and 3-methylbutan-1-ol recognized as malty during the manufacture of Raclette-type cheese and the fermention of reconstituted skim milk, and filter-sterilized MRS broth. Using dynamic headspace-vacuum transfer in trap extraction followed by gas chromatography coupled with mass spectrometry-olfactometry (DHS-VTT-GC-MS-O) as a screening method for the malty compounds, five compounds (2-methylpropanal, 2- and 3-methylbutanal, and 2- and 3-methylbutan-1-ol) were identified as potential compounds causing the malty aroma in starter culture development and Raclette-type cheeses. Focus on compounds having a predominant sensorial effect (3-methylbutanal and 3-methylbutan-1-ol), spikings of leucine, 13C-labeled leucine, α-ketoisocaproic acid, and α-ketoglutaric acid provided a better understanding of their formation pathway. This study highlighted the discrepancies in the formation of 3-methylbutanal and 3-methylbutan-1-ol between the growth media; namely, despite the presence of free leucine available in MRS and the addition of an excess, no increase of the target compounds was observed. The concentration of these compounds in MRS increased only when α-ketoglutaric acid or α-ketoisocaproic acid was added, and a preference for the pathway to α-hydroxyisocaproic acid instead of 3-methylbutanal was shown. In addition, a formation of 3-methylbutanal when the bacteria were not yet active was observed when spiking α-ketoisocaproic acid, which potentially indicates that this part of the metabolism could take place extracellularly. These results could potentially unveil other, not-yet-identified reactants, directly influencing the production of compounds responsible for the malty aroma in Raclette cheese.

Leucine Supplementation Has No Further Effect on Training-induced Muscle Adaptations.

Several acute studies have suggested that leucine is a key amino acid to drive muscle protein synthesis. However, there are very few studies on the long-term effects of leucine supplementation on resistance training (RT)-induced gains in muscle mass and strength. We sought to determine the impact of 10 g of leucine on muscle mass and strength in response to RT in healthy young men. Twenty-five, resistance-trained men (27 ± 5 yr; 78.4 ± 11.6 kg; 24.8 ± 3.0 kg·m) consuming 1.8 ± 0.4 g protein·kg·d, were randomly assigned to receive 2 × 5 g·d supplementation of either free leucine (LEU n = 12) or alanine (PLA n = 13) while undergoing a supervised 12-wk, twice-weekly lower-limb RT program. One-repetition maximum (leg-press 1RM) and muscle cross-sectional area (mCSA) of the vastus lateralis were determined before (PRE) and after (POST) the intervention. Additionally, three 24-h dietary recalls were also performed at PRE and POST. Protein intake was roughly double that of the RDA in both groups and remained unchanged across time with no differences detected between groups. Similar increases were observed between groups in leg-press 1RM (LEU, 19.0% ± 9.4% and PLA, 21.0% ± 10.4%, P = 0.31) and mCSA (LEU, 8.0% ± 5.6% and PLA, 8.4% ± 5.1%, P = 0.77). High-dose leucine supplementation did not enhance gains in muscle strength and mass after a 12-wk RT program in young resistance-trained males consuming adequate amounts of dietary protein.

Growth, biochemical composition and photosynthetic performance of Scenedesmus acuminatus under different initial sulfur supplies

The effects of sulfur availability on growth, biochemical composition and photosynthetic capacity of the freshwater green microalga Scenedesmus acuminatus were investigated. Modified BG-11 media with Na2SO4 concentrations of 0.076 mM (0.25S), 0.153 mM (0.5S), 0.306 mM (control group), 0.459 mM (1.5S) and 0.612 mM (2.0S) were used to culture S. acuminatus. Sulfur repletion promoted the growth and reproduction of this species, while low sulfur supply markedly decreased the reproduction, nitrogen uptake, soluble protein and chlorophyll content, and photosynthetic activity. Elemental analysis showed that nitrogen, sulfur and carbon content per cell was significantly higher in the sulfur-limitation groups than in the control group on day 18. The metabolic response to sulfur limitation was divided into two stages: first, carbon equivalents were diverted from soluble protein to carbohydrates; then, carbohydrates were converted into neutral lipids. The total lipid content in the 0.25S and 0.5S groups reached 55.2% dry weight (DW) and 52.5% DW, respectively; neutral lipids accounted for 92.1% and 90.3% of the total lipids. Low-sulfur (0.5S) and sulfur-replete conditions both enhanced the lipid productivity. The initial sulfur level caused significant changes in the fatty acid composition of S. acuminatus. The sulfur-limited groups accumulated C16:0 and C18:1, while the sulfur-replete groups were mainly enriched in C16:0. Levels of free leucine, isoleucine, valine, lysine, glycine, alanine, aspartic acid and proline were closely related to the initial sulfur concentration. Changes in free phenylalanine, tryptophan, threonine and serine were due more to the duration of culturing. The results of this study will be useful as a reference for regulation of the initial sulfur supply to enhance lipid productivity of S. acuminatus for use in applications such as biodiesel production.

Leucine coingestion augments the muscle protein synthetic response to the ingestion of 15 g of protein following resistance exercise in older men.

Older adults have shown an attenuated postexercise increase in muscle protein synthesis rates following ingestion of smaller amounts of protein compared with younger adults. Consequently, it has been suggested that older adults require the ingestion of more protein to increase postexercise muscle protein synthesis rates compared with younger adults. We investigated whether coingestion of 1.5 g of free leucine with a single 15-g bolus of protein further augments the postprandial muscle protein synthetic response during recovery from resistance-type exercise in older men. Twenty-four healthy older men (67 ± 1 yr) were randomly assigned to ingest 15 g of milk protein concentrate (MPC80) with (15G+LEU; n = 12) or without (15G; n = 12) 1.5 g of free leucine after performing a single bout of resistance-type exercise. Postprandial protein digestion and amino acid absorption kinetics, whole body protein metabolism, and postprandial myofibrillar protein synthesis rates were assessed using primed, continuous infusions with l-[ring-2H5]phenylalanine, l-[ring-2H2]tyrosine, and l-[1-13C]leucine combined with ingestion of intrinsically l-[1-13C]phenylalanine-labeled milk protein. A total of 70 ± 1% (10.5 ±0.2 g) and 75 ± 2% (11.2 ± 0.3 g) of the protein-derived amino acids were released in the circulation during the 6-h postexercise recovery phase in 15G+LEU and 15G, respectively (P < 0.05). Postexercise myofibrillar protein synthesis rates were 16% (0.058 ± 0.003 vs. 0.049 ± 0.002%/h, P < 0.05; based on l-[ring-2H5]phenylalanine) and 19% (0.071 ± 0.003 vs. 0.060 ± 0.003%/h, P < 0.05; based on l-[1-13C]leucine) greater in 15G+LEU compared with 15G. Leucine coingestion further augments the postexercise muscle protein synthetic response to the ingestion of a single 15-g bolus of protein in older men.

Human Muscle Protein Synthesis Rates after Intake of Hydrolyzed Porcine-Derived and Cows' Milk Whey Proteins-A Randomized Controlled Trial.

Background: Whey protein has been shown to be one of the best proteins to stimulate muscle protein synthesis rate (MPS), but other high quality proteins, e.g., animal/porcine-derived, could have similar effects. Objective: To investigate the effects of hydrolyzed porcine proteins from blood (HPB) and muscle (HPM), in comparison to hydrolyzed whey protein (HW), on MPS after intake of 15 g alone or 30 g protein as part of a mixed meal. We hypothesized that the postprandial MPS would be similar for porcine proteins and whey protein. Design: Eighteen men (mean ± SD age: 24 ± 1 year; BMI: 21.7 ± 0.4 kg/m2) participated in the randomized, double-blind, three-way cross-over study. Subjects consumed the three test products (HPB, HPM and HW) in a random order in two servings at each test day. Serving 1 consisted of a drink with 15 g protein and serving 2 of a drink with 30 g protein together with a mixed meal. A flood-primed continuous infusion of (ring-13C6) phenylalanine was performed and muscle biopsies, blood and urine samples were collected for determination of MPS, muscle free leucine, plasma amino acid concentrations and urea excretion. Results: There were no statistical differences between the MPS measured after consuming 15 g protein alone or 30 g with a mixed meal (p = 0.53) of HPB (0.048 ± 0.007 vs. 0.049 ± 0.008%/h, resp.), HPM (0.063 ± 0.011 vs. 0.062 ± 0.011 %/h, resp.) and HW (0.058 ± 0.007 vs. 0.071 ± 0.013%/h, resp.). However, the impact of protein type on MPS reached statistical tendency (HPB vs. HPM (p = 0.093) and HPB vs. HW (p = 0.067)) with no difference between HPM and HW (p = 0.88). Plasma leucine, branched-chain, essential and total amino acids were generally higher for HPB and HW than HPM (p < 0.01), which reflected their content in the proteins. Muscle-free leucine was higher for HPB than HW and HPM (p < 0.05). Conclusion: Hydrolyzed porcine proteins from blood and muscle resulted in an MPS similar to that of HW, although with a trend for porcine blood proteins to be inferior to muscle proteins and whey. Consequently, these porcine-derived muscle proteins can be used similarly to whey protein to support maintenance of skeletal muscle as part of supplements and ingredients in foods.

Whole body protein anabolism in COPD patients and healthy older adults is not enhanced by adding either carbohydrates or leucine to a serving of protein

Carbohydrates (CHO) and leucine (LEU) both have insulinotropic properties, and could therefore enhance the protein anabolic capacity of dietary proteins, which are important nutrients in preventing muscle loss in patients with Chronic Obstructive Pulmonary Disease (COPD). LEU is also known to activate protein anabolic signaling pathways independent of insulin. Based on our previous findings in COPD, we hypothesized that whole body protein anabolism is enhanced to a comparable extent by the separate and combined co-ingestion of CHO and LEU with protein. To disentangle the protein anabolic effects of CHO and/or free LEU when co-ingested with a high-quality protein, we studied 10 patients with moderate to very severe COPD and dyspnea (GOLD: II-IV, mMRC dyspnea scale≥2), at risk for muscle loss, and 10 healthy age- and gender-matched controls. On four occasions, in a single-blind randomized crossover design, each subject ingested a drink containing 0.6g/kg fat-free mass (ffm) hydrolyzed casein protein with, a) no add-ons (protein), b) 0.3g/kg ffm CHO (protein+CHO), c) 0.095g/kg ffm leucine (protein+LEU), d) both add-ons (protein+CHO+LEU). Whole body protein breakdown (PB), protein synthesis (PS), and net protein balance (= PS - PB) were measured by IV primed and continuous infusion of L-[ring-2H5]-phenylalanine and L-[13C9,15N]-tyrosine. L-[15N]-phenylalanine was added to the protein drinks to measure splanchnic extraction. In both groups, whole body PS, PB and net protein balance responses were comparable between the four protein drinks, despite higher postprandial plasma LEU concentrations for the LEU supplemented drinks (P<0.05), and higher insulin concentrations for the CHO supplemented drinks as compared to the protein only drink (P<0.05). Adding CHO and/or LEU to a serving of high-quality protein does not further augment whole body protein anabolism in dyspneic COPD patients at risk for muscle loss or healthy older adults. ClinicalTrials.gov; No. NCT01734473; URL: www.clinicaltrials.gov.

Leucine Supplementation Does Not Attenuate Skeletal Muscle Loss during Leg Immobilization in Healthy, Young Men.

Background: Short successive periods of physical inactivity occur throughout life and contribute considerably to the age-related loss of skeletal muscle mass. The maintenance of muscle mass during brief periods of disuse is required to prevent functional decline and maintain metabolic health. Objective: To assess whether daily leucine supplementation during a short period of disuse can attenuate subsequent muscle loss in vivo in humans. Methods: Thirty healthy (22 ± 1 y) young males were exposed to a 7-day unilateral knee immobilization intervention by means of a full leg cast with (LEU, n = 15) or without (CON, n = 15) daily leucine supplementation (2.5 g leucine, three times daily). Prior to and directly after immobilization, quadriceps muscle cross-sectional area (computed tomography (CT) scan) and leg strength (one-repetition maximum (1-RM)) were assessed. Furthermore, muscle biopsies were taken in both groups before and after immobilization to assess changes in type I and type II muscle fiber CSA. Results: Quadriceps muscle cross-sectional area (CSA) declined in the CON and LEU groups (p < 0.01), with no differences between the two groups (from 7712 ± 324 to 7287 ± 305 mm2 and from 7643 ± 317 to 7164 ± 328 mm2; p = 0.61, respectively). Leg muscle strength decreased from 56 ± 4 to 53 ± 4 kg in the CON group and from 63 ± 3 to 55 ± 2 kg in the LEU group (main effect of time p < 0.01), with no differences between the groups (p = 0.052). Type I and II muscle fiber size did not change significantly over time, in both groups (p > 0.05). Conclusions: Free leucine supplementation with each of the three main meals (7.5 g/d) does not attenuate the decline of muscle mass and strength during a 7-day limb immobilization intervention.

Regulation of pancreas development and enzymatic gene expression by duodenal infusion of leucine and phenylalanine in dairy goats

This study aimed to investigate the effect of a duodenal infusion of leucine and phenylalanine on pancreatic development and enzyme gene expression in dairy goats. Sixteen healthy yearling Guanzhong dairy goats of similar weights (29.7 ± 1.4 kg) had surgical duodenal intubation, and were used in a completely random design in this experimental. The animals were randomly divided into the 4 treatment groups, with 4 goats in each group: control, 3 g/d leucine (3 Leu), 9 g/d leucine (9 Leu) and 2 g/d phenylalanine (2 Phe), dissolved in and infused into the duodenum for 21 days. The goats were fed twice daily (0800 and 1800) with 350 g (dry matter) of total mixed rations, and were all slaughtered on the last day of the experimental period for pancreatic tissue sampling. The results showed that leucine and phenylalanine infusion did not affect pancreas weight or protein concentration (P > 0.05). However, 9 Leu treatment reduced pancreatic DNA concentration, and both 9 Leu and 2 Phe treatments increased the pancreatic protein:DNA ratio (P < 0.05). Leucine and phenylalanine infusions increased pancreatic amylase activity (P < 0.05), whereas had no effect on the total trypsin and lipase activities (P > 0.05). Treatment with 9 Leu and 2 Phe increased amylase mRNA levels (P < 0.05), and 2 Phe increased lipase mRNA levels (P < 0.05). Trypsin mRNA levels were unaffected by leucine and phenylalanine infusion (P > 0.05). The phosphorylation of eukaryotic translation initiation factor 4E-binding protein 1 was increased by leucine infusion (P < 0.05), but not by phenylalanine infusion (P > 0.05). Leucine infusion also increased the free leucine concentration in the pancreas (P < 0.05), and had no effect on glucose concentrations (P > 0.05). In conclusion, duodenal infusion of an appropriate amount of leucine and/or phenylalanine can effectively improve the expression and secretion of pancreatic amylase in goats, which is important for improving intestinal starch digestion in ruminant animals. The regulation of the pancreatic amylase expression by leucine and phenylalanine mainly occurs at the transcriptional and/or translational level in goats.

Differential effects of leucine and leucine-enriched whey protein on skeletal muscle protein synthesis in aged mice

It has been suggested that anabolic resistance, or a blunted protein synthetic response to anabolic stimuli, contributes to the failure of muscle mass maintenance in older adults. The amino acid leucine is one of the most prominent food-related anabolic stimuli. However, data on muscle protein synthesis (MPS) after administration of a single bolus of leucine in aged populations is lacking and long-term single leucine supplementation has not been shown to increase muscle mass. This study aimed to determine the MPS response to the administration of a single bolus of leucine or to leucine combined with whey protein, in aged mice. Overnight fasted C57/BL6RJ mice at 25-mo of age received an oral gavage with leucine or whey-protein enriched with leucine (0.75g/kg bodyweight total leucine in both) or 0.5mL water (fasted control). Subsequently, mice were s.c. injected with puromycin (0.04μmol/g bw at t=30, 45 or 60min) and were sacrificed 30min thereafter. Amino acid concentrations were determined in plasma and right muscle tibialis anterior (TA). Left TA was used to analyse MPS by SUnSET method and phosphorylation rate of Akt, 4E-BP1 and p70S6k by western blot. In aged mice, leucine administration failed to increase MPS, despite a 6-fold increase in plasma leucine and elevated muscle free leucine levels (P<0.05). In contrast, leucine-enriched whey protein significantly stimulated MPS in aged mice at 60min after gavage (P<0.05). Muscle free EAA, NEAA and the phosphorylation rate of Akt, 4E-BP1 and p70S6k increased significantly (P<0.05), only after administration of leucine-enriched whey protein. MPS is stimulated in aged mice by leucine-enriched whey protein but not by leucine administration only. Administration of other amino acids may be required for leucine administration to stimulate muscle protein synthesis in aged mice.

The Paradoxical Effect of Paraquat on Leucyl-tRNA Levels in &amp;lt;i&amp;gt;E. coli&amp;lt;/i&amp;gt; Provides New Insights for Amino Acid Therapy in Humans

Dihydroxyacid dehydratase (DHAD), the rate-limiting enzyme in the synthesis of branched-chain (BCA) amino acids in bacteria and plants, is sensitive to oxyradical toxicity. Oxidant stress reversibly inactivates DHAD and causes starvation for BCA and reversible cessation of growth in Escherichia coli [1][2]. To better understand the underlying toxicity mechanisms, we have determined the cellular concentrations of charged-tRNAs for BCA, in E. coli treated with the redox-active chemical, paraquat. Contrary to expectation, in the paraquat-treated cells, the concentration of only charged leucyl-tRNA decreased dramatically; whereas, the concentrations of the other BCAs (valine and isoleucine) increased. This paradoxical result, the “paraquat effect” can be best explained if leucine is the most abundant amino acid in the E. coli proteins and therefore the rate-limiting building block in their synthesis. Based on this assumption, we investigated the concentration of free amino acids in E. coli and their relative abundances in E. coli proteins. Protein amino acid frequencies were determined by analyzing one-hundred gene bank protein sequences with software developed as described in Methods. Leucine is the most abundant amino acid in the E. coli proteins (10%) and consequently, the cellular free leucine concentration is smaller and the native charged-leucyl-tRNA levels are much higher than those of valine and isoleucine. This has relevance to humans because: leucine-deprivation was shown to be beneficial in tumor suppression [3], and leucine-supplementation was beneficial in the recovery from exercise-induced muscle loss [4][5], and leucine also occurs at a higher frequency in almost all human proteins. In three human protein categories, we examined it ranged from 9% to 17%. This predominance of leucine in proteins would make cells vulnerable to impairment of the leucine pools and could explain our results in E. coli and some of the biological effects of free leucine in humans.

The 15N-leucine single-injection method allows for determining endogenous losses and true digestibility of amino acids in cecectomized roosters.

This study was conducted to assess the influence of dietary protein content in poultry when using the 15N-leucine single-injection method to determine endogenous amino acid losses (EAALs) in poultry. Forty-eight cecectomized roosters (2.39 ± 0.23 kg) were randomly allocated to eight dietary treatments containing protein levels of 0, 3%, 6%, 9%, 12%, 15%, 18% and 21%. Each bird was precisely fed an experimental diet of 25 g/kg of body weight. After feeding, all roosters were subcutaneously injected with a 15N-leucine solution at a dose of 20 mg/kg of body weight. Blood was sampled 23 h after the injection, and excreta samples were continuously collected during the course of the 48-h experiment. The ratio of 15N-enrichment of leucine in crude mucin to free leucine in plasma ranged from 0.664 to 0.763 and remained relatively consistent (P > 0.05) across all treatments. The amino acid (AA) profiles of total endogenous AAs, except isoleucine, alanine, aspartic acid, cysteine, proline and serine, were not influenced (P > 0.05) by dietary protein contents. The predominant endogenous AAs in the excreta were glutamic acid, aspartic acid, threonine, serine and proline. The order of the relative proportions of these predominant AAs also remained relatively constant (P > 0.05). The endogenous losses of total AAs determined with the 15N-leucine single-injection method increased curvilinearly with the dietary protein contents. The true digestibility of most AAs and total AAs was independent of their respective dietary protein levels. Collectively, the 15N-leucine single-injection method is appropriate for determining EAALs and the true digestibility of AAs in poultry fed varying levels of protein-containing ingredients.

Presleep dietary protein-derived amino acids are incorporated in myofibrillar protein during postexercise overnight recovery.

The purpose of this study was to determine the impact of ingesting 30 g casein protein with and without 2 g free leucine before sleep on myofibrillar protein synthesis rates during postexercise overnight recovery. Thirty-six healthy young men performed a single bout of resistance-type exercise in the evening (1945) after a full day of dietary standardization. Thirty minutes before sleep (2330), subjects ingested 30 g intrinsically l-[1-13C]phenylalanine-labeled protein with (PRO+leu, n = 12) or without (PRO, n = 12) 2 g free leucine, or a noncaloric placebo (PLA, n = 12). Continuous intravenous l-[ ring-2H5]phenylalanine, l-[1-13C]leucine, and l-[ ring-2H2]tyrosine infusions were applied. Blood and muscle tissue samples were collected to assess whole body protein net balance, myofibrillar protein synthesis rates, and overnight incorporation of dietary protein-derived amino acids into myofibrillar protein. Protein ingestion before sleep improved overnight whole body protein net balance ( P < 0.001). Myofibrillar protein synthesis rates did not differ significantly between treatments as assessed by l-[ ring-2H5]phenylalanine (0.057 ± 0.002, 0.055 ± 0.002, and 0.055 ± 0.004%/h for PLA, PRO, and PRO+leu, respectively; means ± SE; P = 0.850) or l-[1-13C]leucine (0.080 ± 0.004, 0.073 ± 0.004, and 0.083 ± 0.006%/h, respectively; P = 0.328). Myofibrillar l-[1-13C]phenylalanine enrichments increased following protein ingestion but did not differ between the PRO and PRO+leu treatments. In conclusion, protein ingestion before sleep improves whole body protein net balance and provides amino acids that are incorporated into myofibrillar protein during sleep. However, the ingestion of 30 g casein protein with or without additional free leucine before sleep does not increase muscle protein synthesis rates during postexercise overnight recovery.