Leucine Infusion Research Articles

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.

Muscle Protein Synthesis after Protein Administration in Critical Illness.

Rationale: Dietary protein may attenuate the muscle atrophy experienced by patients in the ICU, yet protein handling is poorly understood. Objectives: To quantify protein digestion and amino acid absorption and fasting and postprandial myofibrillar protein synthesis during critical illness. Methods: Fifteen mechanically ventilated adults (12 male; aged 50 ± 17 yr; body mass index, 27 ± 5 kg⋅m-2) and 10 healthy control subjects (6 male; 54 ± 23 yr; body mass index, 27 ± 4 kg⋅m-2) received a primed intravenous L-[ring-2H5]-phenylalanine, L-[3,5-2H2]-tyrosine, and L-[1-13C]-leucine infusion over 9.5 hours and a duodenal bolus of intrinsically labeled (L-[1-13C]-phenylalanine and L-[1-13C]-leucine) intact milk protein (20 g protein) over 60 minutes. Arterial blood and muscle samples were taken at baseline (fasting) and for 6 hours following duodenal protein administration. Data are mean ± SD, analyzed with two-way repeated measures ANOVA and independent samples t test. Measurements and Main Results: Fasting myofibrillar protein synthesis rates did not differ between ICU patients and healthy control subjects (0.023 ± 0.013% h-1 vs. 0.034 ± 0.016% h-1; P = 0.077). After protein administration, plasma amino acid availability did not differ between groups (ICU patients, 54.2 ± 9.1%, vs. healthy control subjects, 61.8 ± 13.1%; P = 0.12), and myofibrillar protein synthesis rates increased in both groups (0.028 ± 0.010% h-1 vs. 0.043 ± 0.018% h-1; main time effect P = 0.046; P-interaction = 0.584) with lower rates in ICU patients than in healthy control subjects (main group effect P = 0.001). Incorporation of protein-derived phenylalanine into myofibrillar protein was ∼60% lower in ICU patients (0.007 ± 0.007 mol percent excess vs. 0.017 ± 0.009 mol percent excess; P = 0.007). Conclusions: The capacity for critically ill patients to use ingested protein for muscle protein synthesis is markedly blunted despite relatively normal protein digestion and amino acid absorption.

Insects are a viable protein source for human consumption: from insect protein digestion to postprandial muscle protein synthesis in vivo in humans: a double-blind randomized trial

ABSTRACTBackgroundInsects have recently been identified as a more sustainable protein-dense food source and may represent a viable alternative to conventional animal-derived proteins.ObjectivesWe aimed to compare the impacts of ingesting lesser mealworm– and milk-derived protein on protein digestion and amino acid absorption kinetics, postprandial skeletal muscle protein synthesis rates, and the incorporation of dietary protein–derived amino acids into de novo muscle protein at rest and during recovery from exercise in vivo in humans.MethodsIn this double-blind randomized controlled trial, 24 healthy, young men ingested 30 g specifically produced, intrinsically l-[1-13C]-phenylalanine and l-[1-13C]-leucine labeled lesser mealworm– or milk-derived protein after a unilateral bout of resistance-type exercise. Primed continuous l-[ring-2H5]-phenylalanine, l-[ring-3,5-2H2]-tyrosine, and l-[1-13C]-leucine infusions were applied, with frequent collection of blood and muscle tissue samples.ResultsA total of 73% ± 7% and 77% ± 7% of the lesser mealworm and milk protein–derived phenylalanine was released into the circulation during the 5 h postprandial period, respectively, with no significant differences between groups (P < 0.05). Muscle protein synthesis rates increased after both lesser mealworm and milk protein concentrate ingestion from 0.025 ± 0.008%/h to 0.045 ± 0.017%/h and 0.028 ± 0.010%/h to 0.056 ± 0.012%/h at rest and from 0.025 ± 0.012%/h to 0.059 ± 0.015%/h and 0.026 ± 0.009%/h to 0.073 ± 0.020%/h after exercise, respectively (all P < 0.05), with no differences between groups (both P > 0.05). Incorporation of mealworm and milk protein-derived l-[1-13C]-phenylalanine into de novo muscle protein was greater after exercise than at rest (P < 0.05), with no differences between groups (P > 0.05).ConclusionsIngestion of a meal-like amount of lesser mealworm–derived protein is followed by rapid protein digestion and amino acid absorption and increases muscle protein synthesis rates both at rest and during recovery from exercise. The postprandial protein handling of lesser mealworm does not differ from ingesting an equivalent amount of milk protein concentrate in vivo in humans.This trial was registered at www.trialregister.nl as NL6897.

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

Chronic Fetal Leucine Infusion Does Not Potentiate Glucose-Stimulated Insulin Secretion or Affect Pancreatic Islet Development in Late-Gestation Growth-Restricted Fetal Sheep

Growth-restricted fetuses have attenuated glucose-stimulated insulin secretion (GSIS), smaller pancreatic islets, less pancreatic β-cells, and less pancreatic vascularization compared with normally growing fetuses. Infusion of leucine into normal late-gestation fetal sheep potentiates GSIS, as well as increases pancreatic islet size, the proportion of the pancreas and islet comprising β-cells, and pancreatic and islet vascularity. In addition, leucine stimulates hepatocyte growth factor (HGF ) mRNA expression in islet endothelial cells isolated from normal fetal sheep. We hypothesized that a 9-d leucine infusion would potentiate GSIS and increase pancreatic islet size, β-cells, and vascularity in intrauterine fetal growth restriction (IUGR) fetal sheep. We also hypothesized that leucine would stimulate HGF mRNA in islet endothelial cells isolated from IUGR fetal sheep. Late-gestation Columbia-Rambouillet IUGR fetal sheep (singleton or twin) underwent surgeries to place vascular sampling and infusion catheters. Fetuses were randomly allocated to receive a 9-d leucine infusion to achieve a 50-100% increase in leucine concentrations or a control saline infusion. GSIS was measured and pancreas tissue was processed for histologic analysis. Pancreatic islet endothelial cells were isolated from IUGR fetal sheep and incubated with supplemental leucine. Data were analyzed by mixed-models ANOVA; Student, Mann-Whitney, or a paired t test; or a test of equality of proportions. Chronic leucine infusion in IUGR fetuses did not affect GSIS, islet size, the proportion of the pancreas comprising β-cells, or pancreatic or pancreatic islet vascularity. In isolated islet endothelial cells from IUGR fetuses, HGF mRNA expression was not affected by supplemental leucine. IUGR fetal sheep islets are not responsive to a 9-d leucine infusion with respect to insulin secretion or any histologic features measured. This is in contrast to the response in normally growing fetuses. These results are important when considering nutritional strategies to prevent the adverse islet and β-cell consequences in IUGR fetuses.

Leucine acutely potentiates glucose-stimulated insulin secretion in fetal sheep.

A 9-day infusion of leucine into fetal sheep potentiates fetal glucose-stimulated insulin secretion (GSIS). However, there were accompanying pancreatic structural changes that included a larger proportion of β-cells and increased vascularity. Whether leucine can acutely potentiate fetal GSIS in vivo before these structural changes develop is unknown. The mechanisms by which leucine acutely potentiates GSIS in adult islets and insulin-secreting cell lines are well known. These mechanisms involve leucine metabolism, including leucine oxidation. However, it is not clear if leucine-stimulated metabolic pathways are active in fetal islets. We hypothesized that leucine would acutely potentiate GSIS in fetal sheep and that isolated fetal islets are capable of oxidizing leucine. We also hypothesized that leucine would stimulate other metabolic pathways associated with insulin secretion. In pregnant sheep we tested in vivo GSIS with and without an acute leucine infusion. In isolated fetal sheep islets, we measured leucine oxidation with a [1-14C] l-leucine tracer. We also measured concentrations of other amino acids, glucose, and analytes associated with cellular metabolism following incubation of fetal islets with leucine. In vivo, a leucine infusion resulted in glucose-stimulated insulin concentrations that were over 50% higher than controls (P < 0.05). Isolated fetal islets oxidized leucine. Leucine supplementation of isolated fetal islets also resulted in significant activation of metabolic pathways involving leucine and other amino acids. In summary, acute leucine supplementation potentiates fetal GSIS in vivo, likely through pathways related to the oxidation of leucine and catabolism of other amino acids.

Intermittent leucine pulses during continuous feeding alters novel components involved in skeletal muscle growth of neonatal pigs.

When neonatal pigs continuously fed formula are supplemented with leucine pulses, muscle protein synthesis and body weight gain are enhanced. To identify the responsible mechanisms, we combined plasma metabolomic analysis with transcriptome expression of the transcriptome and protein catabolic pathways in skeletal muscle. Piglets (n = 23, 7-day-old) were fed continuously a milk replacement formula via orogastric tube for 21days with an additional parenteral infusion (800μmolkg-1h-1) of either leucine (LEU) or alanine (CON) for 1h every 4h. Plasma metabolites were measured by liquid chromatography-mass spectrometry. Gene and protein expression analyses of longissimus dorsi muscle were performed by RNA-seq and Western blot, respectively. Compared with CON, LEU pigs had increased plasma levels of leucine-derived metabolites, including 4-methyl-2-oxopentanoate, beta-hydroxyisovalerate, β-hydroxyisovalerylcarnitine, and 3-methylglutaconate (P ≤ 0.05). Leucine pulses downregulated transcripts enriched in the Kyoto Encyclopedia of Genes and Genomes terms "spliceosome," "GAP junction," "endocytosis," "ECM-receptor interaction," and "DNA replication". Significant correlations were identified between metabolites derived from leucine catabolism and muscle genes involved in protein degradation, transcription and translation, and muscle maintenance and development (P ≤ 0.05). Further, leucine pulses decreased protein expression of autophagic markers and serine/threonine kinase 4, involved in muscle atrophy (P ≤ 0.01). In conclusion, results from our studies support the notion that leucine pulses during continuous enteral feeding enhance muscle mass gain in neonatal pigs by increasing protein synthetic activity and downregulating protein catabolic pathways through concerted responses in the transcriptome and metabolome.

A Chronic Fetal Leucine Infusion Potentiates Fetal Insulin Secretion and Increases Pancreatic Islet Size, Vascularity, and β Cells in Late-Gestation Sheep

Background:Infusion of a complete amino acid mixture into normal late-gestation fetal sheep potentiates glucose-stimulated insulin secretion (GSIS). Leucine acutely stimulates insulin secretion in late-gestation fetal sheep and isolated fetal sheep islets in vitro. Objectives:We hypothesized that a 9-d leucine infusion would potentiate GSIS in fetal sheep. Methods:Columbia-Rambouillet fetal sheep at 126 days of gestation received a 9-d leucine infusion to achieve a 50%–100% increase in leucine concentrations or a control infusion. At the end of the infusion we measured GSIS, pancreatic morphology, and expression of pancreatic mRNAs. Pancreatic islet endothelial cells (ECs) were isolated from fetal sheep and incubated with supplemental leucine or vascular endothelial growth factor A (VEGFA) followed by collection of mRNA. Data measured at multiple time points were compared with a repeated-measures 2-factor ANOVA. Data measured at 1 time point were compared using Student's t test or the Mann–Whitney test. Results:Glucose-stimulated insulin concentrations were 80% higher in leucine-infused (LEU) fetuses than in controls (P < 0.05). In the pancreas, LEU fetuses had a higher proportion of islets >5000 μm2 than controls (75% more islets >5000 μm2; P < 0.05) and a larger proportion of the pancreas that stained for β cells (12% greater; P < 0.05). Pancreatic and pancreatic islet vascularity were both 25% greater in LEU fetuses (P < 0.05). PancreaticVEGFAand hepatocyte growth factor (HGF) mRNA expressions were 38% and 200% greater in LEU fetuses than in controls (P < 0.05), respectively. In isolated islet ECs,HGFmRNA was 20% and 50% higher after incubation in ce:supplemental leucine (P < 0.05) or VEGFA (P < 0.01), respectively. Conclusions:A 9-d leucine infusion potentiates fetal GSIS, demonstrating that glucose and leucine act synergistically to stimulate insulin secretion in fetal sheep. A greater proportion of the pancreas being comprised of β cells and higher pancreatic vascularity contributed to the higher GSIS.

Hot-water Immersion Does Not Increase Post-exercise Muscle Protein Synthesis Rates

Protein ingestion and heating are strategies employed by athletes to improve post-exercise recovery and, as such, to facilitate muscle reconditioning following exercise. However, whether post-exercise heating affects post-prandial protein handling and subsequent muscle protein synthesis rates during recovery from exercise has not been assessed. PURPOSE: To assess the impact of post-exercise heating on post-prandial myofibrillar protein synthesis rates during recovery from a single bout of resistance-type exercise in healthy, young males. METHODS: Twelve healthy, male adults (age: 23±1 y) performed a single bout of resistance-type exercise followed by 20 min water immersion of both legs. One leg was immersed in hot water (46°C: HWI) while the other leg was immersed in thermoneutral water (30°C: CON). After water immersion, a beverage was ingested containing 20 g intrinsically L-[1-13C]-phenylalanine and L-[1-13C]-leucine labelled milk protein with 45 g of carbohydrates. In addition, primed continuous L-[ring-2H5]-phenylalanine and L-[1-13C]-leucine infusions were applied, with frequent collection of blood and muscle samples to assess myofibrillar protein synthesis rates in vivo over a 5 h recovery period. RESULTS: Muscle temperature immediately after water immersion was higher in the HWI compared to the CON leg (37.5±0.1 vs 35.2±0.2 °C; P<0.001). Incorporation of dietary protein-derived L-[1-13C]-phenylalanine into myofibrillar protein did not differ significantly between the HWI and CON leg during the 5 h recovery period (0.025±0.003 vs 0.024±0.002 MPE; P=0.953). Post-exercise myofibrillar protein synthesis rates did not differ between the HWI and the CON leg based upon L-[1-13C]-leucine (0.050±0.005 vs 0.049±0.002%·h-1, respectively; P=0.815) as well as L-[ring-2H5]-phenylalanine (0.048±0.002 vs 0.047±0.003%·h-1, respectively; P=0.877). CONCLUSIONS: Hot-water immersion during recovery from resistance-type exercise does not increase the post-prandial rise in myofibrillar protein synthesis rates. In addition, post-exercise heating does not increase the capacity of the muscle to use dietary protein derived amino acids for de novo muscle protein accretion during subsequent recovery.

Ingestion of Free Amino Acids as Opposed to Intact Protein Increases Amino Acid Absorption but Does Not Further Augment Postprandial Muscle Protein Synthesis Rates

Abstract Objectives The rate of protein digestion and amino acid absorption determines the postprandial rise in circulating amino acids and, as such, modulates postprandial muscle protein synthesis rates. This study compares protein digestion and amino acid absorption kinetics and the subsequent muscle protein synthetic response following ingestion of intact protein versus an equivalent amount of free, crystalline amino acids. Methods Twenty-four healthy, young subjects (age: 22 ± 3 y, BMI: 23 ± 2 kg·m−2, sex: 12 M/12F) ingested 30 g intrinsically L-[1–13C]-phenylalanine and L-[1–13C]-leucine labeled milk protein (PROT; n = 12) or an equivalent amount of free amino acids (AA; n = 12). In addition, subjects received primed continuous L-[ring-2H5]-phenylalanine, L-[ring-3,5–2H2]-tyrosine, and L-[1–13C]-leucine infusions. Blood samples and muscle biopsies were obtained frequently to assess protein digestion and amino acid absorption kinetics and subsequent muscle protein synthesis rates over a 6 h postprandial period. An unpaired t-test was used to compare overall exogenous phenylalanine release in plasma. For other parameters repeated measures ANOVA were applied to determine differences between groups over time (time as within, and group as between-subjects factor). Data are expressed as mean ± SD. Results Postprandial plasma amino acid concentrations and exogenous phenylalanine appearance rates increased after ingestion of PROT and AA (both, P &amp;lt; 0.001), with a greater increase following ingestion of AA when compared to PROT (time*group interaction P &amp;lt; 0.001). Exogenous phenylalanine release in plasma assessed over the 6 h postprandial period, was greater in AA (76 ± 9%) compared with PROT (59 ± 10%; P &amp;lt; 0.001). Ingestion of AA and PROT strongly increased muscle protein synthesis rates based upon L-[ring-2H5]-phenylalanine (time effect P &amp;lt; 0.001), with no differences between groups (from 0.037 ± 0.015 to 0.053 ± 0.014%·h−1 and from 0.039 ± 0.016 to 0.051 ± 0.010%·h−1, respectively; time*group interaction P = 0.629). Conclusions Ingestion of free amino acids as opposed to intact milk protein is followed by more rapid amino acid absorption and greater postprandial plasma amino acid availability, but this does not further augment postprandial muscle protein synthesis rates. Funding Sources This research did not receive external funding.

Hot-water immersion does not increase postprandial muscle protein synthesis rates during recovery from resistance-type exercise in healthy, young males

The purpose of this study was to assess the impact of postexercise hot-water immersion on postprandial myofibrillar protein synthesis rates during recovery from a single bout of resistance-type exercise in healthy, young men. Twelve healthy, adult men (age: 23 ± 1 y) performed a single bout of resistance-type exercise followed by 20 min of water immersion of both legs. One leg was immersed in hot water [46°C: hot-water immersion (HWI)], while the other leg was immersed in thermoneutral water (30°C: CON). After water immersion, a beverage was ingested containing 20 g intrinsically L-[1-13C]-phenylalanine and L-[1-13C]-leucine labeled milk protein with 45 g of carbohydrates. In addition, primed continuous L-[ring-2H5]-phenylalanine and L-[1-13C]-leucine infusions were applied, with frequent collection of blood and muscle samples to assess myofibrillar protein synthesis rates in vivo over a 5-h recovery period. Muscle temperature immediately after water immersion was higher in the HWI compared with the CON leg (37.5 ± 0.1 vs. 35.2 ± 0.2°C; P < 0.001). Incorporation of dietary protein-derived L-[1-13C]-phenylalanine into myofibrillar protein did not differ between the HWI and CON leg during the 5-h recovery period (0.025 ± 0.003 vs. 0.024 ± 0.002 MPE; P = 0.953). Postexercise myofibrillar protein synthesis rates did not differ between the HWI and CON leg based upon L-[1-13C]-leucine (0.050 ± 0.005 vs. 0.049 ± 0.002%/h; P = 0.815) and L-[ring-2H5]-phenylalanine (0.048 ± 0.002 vs. 0.047 ± 0.003%/h; P = 0.877), respectively. Hot-water immersion during recovery from resistance-type exercise does not increase the postprandial rise in myofibrillar protein synthesis rates. In addition, postexercise hot-water immersion does not increase the capacity of the muscle to incorporate dietary protein-derived amino acids in muscle tissue protein during subsequent recovery.NEW & NOTEWORTHY This is the first study to assess the effect of postexercise hot-water immersion on postprandial myofibrillar protein synthesis rates and the incorporation of dietary protein-derived amino acids into muscle protein. We observed that hot-water immersion during recovery from a single bout of resistance-type exercise does not further increase myofibrillar protein synthesis rates or augment the postprandial incorporation of dietary protein-derived amino acids in muscle throughout 5 h of postexercise recovery.

Postexercise cooling impairs muscle protein synthesis rates in recreational athletes.

Key points Protein ingestion and cooling are strategies employed by athletes to improve postexercise recovery and, as such, to facilitate muscle conditioning. However, whether cooling affects postprandial protein handling and subsequent muscle protein synthesis rates during recovery from exercise has not been assessed.We investigated the effect of postexercise cooling on the incorporation of dietary protein‐derived amino acids into muscle protein and acute postprandial (hourly) as well as prolonged (daily) myofibrillar protein synthesis rates during recovery from resistance‐type exercise over 2 weeks.Cold‐water immersion during recovery from resistance‐type exercise lowers the capacity of the muscle to take up and/or direct dietary protein‐derived amino acids towards de novo myofibrillar protein accretion. In addition, cold‐water immersion during recovery from resistance‐type exercise lowers myofibrillar protein synthesis rates during prolonged resistance‐type exercise training.Individuals aiming to improve skeletal muscle conditioning should reconsider applying cooling as a part of their postexercise recovery strategy. We measured the impact of postexercise cooling on acute postprandial (hourly) as well as prolonged (daily) myofibrillar protein synthesis rates during adaptation to resistance‐type exercise over 2 weeks. Twelve healthy males (aged 21 ± 2 years) performed a single resistance‐type exercise session followed by water immersion of both legs for 20 min. One leg was immersed in cold water (8°C: CWI), whereas the other leg was immersed in thermoneutral water (30°C: CON). After water immersion, a beverage was ingested containing 20 g of intrinsically (l‐[1‐13C]‐phenylalanine and l‐[1‐13C]‐leucine) labelled milk protein with 45 g of carbohydrates. In addition, primed continuous l‐[ring‐2H5]‐phenylalanine and l‐[1‐13C]‐leucine infusions were applied, with frequent collection of blood and muscle samples to assess myofibrillar protein synthesis rates in vivo over a 5 h recovery period. In addition, deuterated water (2H2O) was applied with the collection of saliva, blood and muscle biopsies over 2 weeks to assess the effects of postexercise cooling with protein intake on myofibrillar protein synthesis rates during more prolonged resistance‐type exercise training (thereby reflecting short‐term training adaptation). Incorporation of dietary protein‐derived l‐[1‐13C]‐phenylalanine into myofibrillar protein was significantly lower in CWI compared to CON (0.016 ± 0.006 vs. 0.021 ± 0.007 MPE; P = 0.016). Postexercise myofibrillar protein synthesis rates were lower in CWI compared to CON based upon l‐[1‐13C]‐leucine (0.058 ± 0.011 vs. 0.072 ± 0.017% h−1, respectively; P = 0.024) and l‐[ring‐2H5]‐phenylalanine (0.042 ± 0.009 vs. 0.053 ± 0.013% h−1, respectively; P = 0.025). Daily myofibrillar protein synthesis rates assessed over 2 weeks were significantly lower in CWI compared to CON (1.48 ± 0.17 vs. 1.67 ± 0.36% day−1, respectively; P = 0.042). Cold‐water immersion during recovery from resistance‐type exercise reduces myofibrillar protein synthesis rates and, as such, probably impairs muscle conditioning.

Leucine-enriched essential amino acid supplementation in mechanically ventilated trauma patients: a feasibility study

BackgroundCritically ill patients lose up to 2% of muscle mass per day. We assessed the feasibility of administering a leucine-enriched essential amino acid (L-EAA) supplement to mechanically ventilated trauma patients with the aim of assessing the effect on skeletal muscle mass and function.MethodsA randomised feasibility study was performed over six months in intensive care (ICU). Patients received 5 g L-EAA five times per day in addition to standard feed (L-EAA group) or standard feed only (control group) for up to 14 days. C-reactive protein, albumin, IL-6, IL-10, urinary 3-MH, nitrogen balance, protein turnover ([1-13C] leucine infusion), muscle depth change (ultrasound), functional change (Katz and Barthel indices) and muscle strength Medical Research Council (MRC) sum score to assess ICU Acquired Weakness were measured sequentially.ResultsEight patients (9.5% of screened patients) were recruited over six months. L-EAA doses were provided on 91/124 (73%) occasions. Inflammatory and urinary marker data were collected; serial muscle depth measurements were lacking due to short length of stay. Protein turnover studies were performed on five occasions. MRC sum score could not be performed as patients were not able to respond to the screening questions. The Katz and Barthel indices did not change. L-EAA delivery was achievable, but meaningful functional and muscle mass outcome measures require careful consideration in the design of a future randomised controlled trial.ConclusionL-EAA was practical to provide, but we found significant barriers to recruitment and measurement of the chosen outcomes which would need to be addressed in the design of a future, large randomised controlled trial.Trial registrationISRCTN Registry, ISRCTN79066838. Registered on 25 July 2012.

Effects of jugular infusions of isoleucine, leucine, methionine, threonine, and other amino acids on insulin and glucagon concentrations, mammalian target of rapamycin (mTOR) signaling, and lactational performance in goats

The supply and profile of absorbed AA may affect milk protein synthesis through hormonal changes and mammalian target of rapamycin (mTOR) signaling pathways; and Ile, Leu, Met, and Thr (ILMT) are the 4 AA that have been reported to have the greatest effect on mammary mTOR signaling. The extent to which ILMT and the other remaining AA (RAA) differ in their effects on milk protein synthesis needs to be systematically investigated. In this study, 5 lactating goats, averaging 120 ± 10 d in milk, fitted with jugular vein and carotid artery catheters, were fasted for 24 h, followed by intravenous infusions of a mixture containing AA and glucose for 8 h in a 5 × 5 Latin square design. The AA mixtures were formulated according to the profile of casein. The amounts of AA infused were calculated based on supplies of AA when metabolizable protein (MP) was at requirement (MR). Treatments were an infusate containing glucose without AA (NTAA); an infusate containing 3 × the MR of Ile, Leu, Met and Thr (3F0R); and infusates containing 3F0R plus 1, 2, or 3 × MR of RAA (3F1R, 3F2R, and 3F3R, respectively) according to amounts provided when fed to meet MP requirements for maintenance and lactation for each goat. Milk, arterial blood, and mammary tissue samples were collected immediately after halting the infusion. Relative to NTAA, supplementation of ILMT tended to increase milk protein production and plasma glucose concentrations, and increased milk and lactose production, but had no effects on production or content of milk fat. Graded supplementation of RAA tended to quadratically affect production of milk and lactose. Arterial glucose and glucagon concentrations decreased linearly, and plasma insulin concentrations decreased quadratically with increased RAA. Mammary p70-S6K1 phosphorylation was decreased by addition of ILMT compared with NTAA but increased linearly with increased RAA infusion. Furthermore, EIF4EBP1 gene expression was much lower for 3F-treated goats than for the NTAA treatment. Both MTOR and RPS6KB1 gene expressions were decreased quadratically with increased RAA supply. These results suggested that short-term milk protein yield tended to be increased by elevated ILMT availability, and this trend was not explained by variations in mammary mTOR signaling or pancreatic hormone secretions, whereas graded increase of RAA in combination with ILMT appeared to regulate the efficiency of conversion of glucose to lactose in a manner not involving milk protein production.

Post-exercise Cooling Impairs Muscle Protein Synthesis Rates In Healthy Young Males

Protein ingestion and cooling are strategies employed by athletes to improve post-exercise recovery and, as such, to facilitate muscle reconditioning following exercise. However, whether post-exercise cooling affects postprandial protein handling and subsequent muscle protein synthesis rates during recovery from exercise has not been studied. PURPOSE: This study assessed the impact of post-exercise cooling on acute postprandial (hourly) and prolonged (daily) myofibrillar protein synthesis rates during recovery from resistance-type exercise over a 2-week period. METHODS: Twelve healthy, male adults (age: 21±1 y) performed a single session of resistance-type exercise followed by water immersion of both legs for 20 min. One leg was immersed in cold water (8°C: CWI) while the other leg was immersed in thermoneutral water (30°C: CON). After water immersion, a beverage was ingested containing 20 g intrinsically L-[1-13C]-phenylalanine and L-[1-13C]-leucine labelled milk protein with 45 g of carbohydrates. In addition, primed continuous L-[ring-2H5]-phenylalanine and L-[1-13C]-leucine infusions were applied, with frequent collection of blood samples and muscle biopsies to assess myofibrillar protein synthesis rates in vivo over a 5-h recovery period. In addition, deuterated water (2H2O) was ingested with the collection of saliva, blood and muscle biopsies over 2 weeks to assess the effects of post-exercise cooling with protein intake on myofibrillar protein synthesis rates during more prolonged resistance-type exercise training. RESULTS: Incorporation of dietary protein-derived L-[1-13C]-phenylalanine into myofibrillar protein was significantly lower in CWI compared to CON (0.016±0.002 vs 0.021±0.002 MPE; P=0.016). Post-exercise myofibrillar protein synthesis rates were lower in CWI compared to CON based upon L-[1-13C]-leucine (0.058±0.003 vs 0.072±0.005%·h-1, respectively; P=0.024) and L-[ring-2H5]-phenylalanine (0.042±0.003 vs 0.053±0.004%·h-1, respectively; P=0.025). Daily myofibrillar protein synthesis rates assessed over 2 weeks were significantly lower in CWI when compared to CON (1.48±0.05 vs 1.67±0.11%·d-1, respectively; P=0.042). CONCLUSION: Cold-water immersion during recovery from resistance-type exercise impairs myofibrillar protein synthesis rates.

In vivo assessment of increased oxidation of branched-chain amino acids in glioblastoma

Altered branched-chain amino acids (BCAAs) metabolism is a distinctive feature of various cancers and plays an important role in sustaining tumor proliferation and aggressiveness. Despite the therapeutic and diagnostic potentials, the role of BCAA metabolism in cancer and the activities of associated enzymes remain unclear. Due to its pivotal role in BCAA metabolism and rapid cellular transport, hyperpolarized 13C-labeled α-ketoisocaproate (KIC), the α-keto acid corresponding to leucine, can assess both BCAA aminotransferase (BCAT) and branched-chain α-keto acid dehydrogenase complex (BCKDC) activities via production of [1-13C]leucine or 13CO2 (and thus H13CO3−), respectively. Here, we investigated BCAA metabolism of F98 rat glioma model in vivo using hyperpolarized 13C-KIC. In tumor regions, we observed a decrease in 13C-leucine production from injected hyperpolarized 13C-KIC via BCAT compared to the contralateral normal-appearing brain, and an increase in H13CO3−, a catabolic product of KIC through the mitochondrial BCKDC. A parallel ex vivo13C NMR isotopomer analysis following steady-state infusion of [U-13C]leucine to glioma-bearing rats verified the increased oxidation of leucine in glioma tissue. Both the in vivo hyperpolarized KIC imaging and the leucine infusion study indicate that KIC catabolism is upregulated through BCAT/BCKDC and further oxidized via the citric acid cycle in F98 glioma.

Dysregulated Handling of Dietary Protein and Muscle Protein Synthesis After Mixed-Meal Ingestion in Maintenance Hemodialysis Patients

IntroductionSkeletal muscle loss is common in patients with renal failure who receive maintenance hemodialysis (MHD) therapy. Regular ingestion of protein-rich meals are recommended to help offset muscle protein loss in MHD patients, but little is known about the anabolic potential of this strategy.MethodsEight MHD patients (age: 56 ± 5 years; body mass index [BMI]: 32 ± 2 kg/m2) and 8 nonuremic control subjects (age: 50 ± 2 years: BMI: 31 ± 1 kg/m2) received primed continuous L-[ring-2H5]phenylalanine and L-[1-13C]leucine infusions with blood and muscle biopsy sampling on a nondialysis day. Participants consumed a mixed meal (546 kcal; 20-g protein, 59-g carbohydrates, 26-g fat) with protein provided as L-[5,5,5-2H3]leucine-labeled eggs.ResultsCirculating dietary amino acid availability was reduced in MHD patients (41 ± 5%) versus control subjects (61 ± 4%; P = 0.03). Basal muscle caspase-3 protein content was elevated (P = 0.03) and large neutral amino acid transporter 1 (LAT1) protein content was reduced (P = 0.02) in MHD patients versus control subjects. Basal muscle protein synthesis (MPS) was ∼2-fold higher in MHD patients (0.030 ± 0.005%/h) versus control subjects (0.014 ± 0.003%/h) (P = 0.01). Meal ingestion failed to increase MPS in MHD patients (absolute change from basal: 0.0003 ± 0.007%/h), but stimulated MPS in control subjects (0.009 ± 0.002%/h; P = 0.004).ConclusionsMHD patients demonstrated muscle anabolic resistance to meal ingestion. This blunted postprandial MPS response in MHD patients might be related to high basal MPS, which results in a stimulatory ceiling effect and/or reduced plasma dietary amino acid availability after mixed-meal ingestion.

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.

Protein Ingestion before Sleep Increases Overnight Muscle Protein Synthesis Rates in Healthy Older Men: A Randomized Controlled Trial

Background: The loss of skeletal muscle mass with aging has been attributed to the blunted anabolic response to protein intake. Presleep protein ingestion has been suggested as an effective strategy to compensate for such anabolic resistance.Objective: We assessed the efficacy of presleep protein ingestion on dietary protein digestion and absorption kinetics and overnight muscle protein synthesis rates in older men.Methods: In a randomized, double-blind, parallel design, 48 older men (mean ± SEM age: 72 ± 1 y) ingested 40 g casein (PRO40), 20 g casein (PRO20), 20 g casein plus 1.5 g leucine (PRO20+LEU), or a placebo before sleep. Ingestion of intrinsically l-[1-13C]-phenylalanine- and l-[1-13C]-leucine-labeled protein was combined with intravenous l-[ring-2H5]-phenylalanine and l-[1-13C]-leucine infusions during sleep. Muscle and blood samples were collected throughout overnight sleep.Results: Exogenous phenylalanine appearance rates increased after protein ingestion, but to a greater extent in PRO40 than in PRO20 and PRO20+LEU (P < 0.05). Overnight myofibrillar protein synthesis rates (based on l-[ring-2H5]-phenylalanine) were 0.033% ± 0.002%/h, 0.037% ± 0.003%/h, 0.039% ± 0.002%/h, and 0.044% ± 0.003%/h in placebo, PRO20, PRO20+LEU, and PRO40, respectively, and were higher in PRO40 than in placebo (P = 0.02). Observations were similar based on l-[1-13C]-leucine tracer (placebo: 0.047% ± 0.004%/h and PRO40: 0.058% ± 0.003%/h, P = 0.08). More protein-derived amino acids (l-[1-13C]-phenylalanine) were incorporated into myofibrillar protein in PRO40 than in PRO20 (0.033 ± 0.002 and 0.019 ± 0.002 MPE, respectively, P < 0.001) and tended to be higher than in PRO20+LEU (0.025 ± 0.002 MPE, P = 0.06).Conclusions: Protein ingested before sleep is properly digested and absorbed throughout the night, providing precursors for myofibrillar protein synthesis during sleep in healthy older men. Ingestion of 40 g protein before sleep increases myofibrillar protein synthesis rates during overnight sleep. These findings provide the scientific basis for a novel nutritional strategy to support muscle mass preservation in aging and disease. This trial was registered at www.trialregister.nl as NTR3885.

Consumption of whole eggs promotes greater stimulation of postexercise muscle protein synthesis than consumption of isonitrogenous amounts of egg whites in young men

Background: Protein in the diet is commonly ingested from whole foods that contain various macro- and micronutrients. However, the effect of consuming protein within its natural whole-food matrix on postprandial protein metabolism remains understudied in humans.Objective: We aimed to compare the whole-body and muscle protein metabolic responses after the consumption of whole eggs with egg whites during exercise recovery in young men.Design: In crossover trials, 10 resistance-trained men [aged 21 ± 1 y; 88 ± 3 kg; body fat: 16% ± 1% (means ± SEMs)] received primed continuous l-[ring-2H5]phenylalanine and l-[1-13C]leucine infusions and performed a single bout of resistance exercise. After exercise, participants consumed intrinsically l-[5,5,5-2H3]leucine-labeled whole eggs (18 g protein, 17 g fat) or egg whites (18 g protein, 0 g fat). Repeated blood and muscle biopsy samples were collected to assess whole-body leucine kinetics, intramuscular signaling, and myofibrillar protein synthesis.Results: Plasma appearance rates of protein-derived leucine were more rapid after the consumption of egg whites than after whole eggs (P = 0.01). Total plasma availability of leucine over the 300-min postprandial period was similar (P= 0.75) between the ingestion of whole eggs (68% ± 1%) and egg whites (66% ± 2%), with no difference in whole-body net leucine balance (P = 0.27). Both whole-egg and egg white conditions increased the phosphorylation of mammalian target of rapamycin complex 1, ribosomal protein S6 kinase 1, and eukaryotic translation initiation factor 4E-binding protein 1 during postexercise recovery (all P < 0.05). However, whole-egg ingestion increased the postexercise myofibrillar protein synthetic response to a greater extent than did the ingestion of egg whites (P= 0.04).Conclusions: We show that the ingestion of whole eggs immediately after resistance exercise resulted in greater stimulation of myofibrillar protein synthesis than did the ingestion of egg whites, despite being matched for protein content in young men. Our data indicate that the ingestion of nutrient- and protein-dense foods differentially stimulates muscle anabolism compared with protein-dense foods. This trial was registered at clinicaltrials.gov as NCT03117127.