Muscle Amino Acid Research Articles

Observations of Branched-Chain Amino Acid Administration in Humans

Since the in vitro study of Buse and Reid in 1975 showing a stimulatory effect of leucine upon rat muscle protein synthesis and reduction in proteolysis, a similar effect has been sought in humans. In 1978, Sherwin demonstrated in humans an improvement in N balance with infusion of leucine in obese subjects fasting to lose weight. A variety of subsequent studies have been performed in humans where leucine alone or the BCAAs have been administered in varying amounts and durations, and the effect upon protein metabolism has been measured. Measurements of changes in muscle amino acid metabolism were made by arteriovenous difference measurements and by biopsies. An anabolic effect of leucine and the branched-chain amino acids (BCAAs) on reduction of muscle protein breakdown was found in these studies, with no measured effect upon muscle protein synthesis. Later studies using stable isotope tracers to define both whole-body protein turnover and leg or arm protein metabolism have similarly concluded that leucine administration specifically induces a reduction in protein breakdown without increasing protein synthesis. This anabolic effect, produced through a reduction of protein breakdown in vivo in humans by leucine is contrary to in vitro studies of rat muscle where stimulation of protein synthesis, has been demonstrated by leucine. Likewise an increase in protein synthesis has also been demonstrated by insulin in rat muscle that is not seen in humans. Of the various studies administering BCAAs or leucine to humans for varying periods of time and amount, the results have been consistent. In addition, no untoward effects have been reported in any of these studies from infusion of the BCAAs at upward 3 times basal flux or 6 times normal dietary intake during the fed portion of the day.

Dietary Protein to Support Anabolism with Resistance Exercise in Young Men

Resistance exercise is fundamentally anabolic and as such stimulates the process of skeletal muscle protein synthesis (MPS) in an absolute sense and relative to skeletal muscle protein breakdown (MPB). However, the net effect of resistance exercise is to shift net protein balance (NPB = MPS − MPB) to a more positive value; however, in the absence of feeding NPB remains negative. Feeding stimulates MPS to an extent where NPB becomes positive, for a transient time. When combined, resistance exercise and feeding synergistically interact to result in NPB being greater than with feeding alone. This feeding- and exercise-induced stimulation of NPB is what, albeit slowly, results in muscle hypertrophy. With this rudimentary knowledge we are now at the point where we can manipulate variables within the system to see what impact these interventions have on the processes of MPS, MPB, and NPB and ultimately and perhaps most importantly, muscle hypertrophy and strength. We used established models of skeletal muscle amino acid turnover to examine how protein source (milk versus soy) acutely affects the processes of MPS and MPB after resistance exercise. Our findings revealed that even when balanced quantities of total protein and energy are consumed that milk proteins are more effective in stimulating amino acid uptake and net protein deposition in skeletal muscle after resistance exercise than are hydrolyzed soy proteins. Importantly, the finding of increased amino acid uptake would be independent of the differences in amino acid composition of the two proteins. We propose that the improved net protein deposition with milk protein consumption is also not due to differences in amino acid composition, but is due to a different pattern of amino acid delivery associated with milk versus hydrolyzed soy proteins. If our acute findings are accurate then we hypothesized that chronically the greater net protein deposition associated with milk protein consumption post-resistance exercise would eventually lead to greater net protein accretion (i.e., muscle fiber hypertrophy), over a longer time period. In young men completing 12 weeks of resistance training (5d/wk) we observed a tendency (P = 0.11) for greater gains in whole body lean mass and whole as greater muscle fiber hypertrophy with consumption of milk. While strength gains were not different between the soy and milk-supplemented groups we would argue that the true significance of a greater increase in lean mass that we observed with milk consumption may be more important in groups of persons with lower initial lean mass and strength such as the elderly.

Free amino acid and glutathione concentrations in muscle during short-term starvation and refeeding

The effects of short-term starvation and refeeding on the free amino acids and glutathione in skeletal muscle in healthy man are not known. This is necessary baseline knowledge when studying the effects of nutrition, trauma and sepsis on protein, amino acid and glutathione metabolism. Concentrations of free amino acids and glutathione in muscle and plasma from young healthy male volunteers (n = 8) were measured before and after a 3-day fast and then again after 2 days refeeding. Nitrogen balance was determined during the study period. The cumulated nitrogen loss was 36.9+/-5.4 g during the fasting period indicating a condition of protein catabolism. During the fasting period decreases were seen in muscle glutamate by 48 +/- 20% and in glutamine by 38 +/- 12%. These changes were returned back to normal levels during the refeeding period. The changes seen in other muscle amino acids during the study period were reflected by similar changes in plasma amino acids, again with normalisation after the refeeding period. Muscle glutathione concentration and the redox status of glutathione remained unaffected of short-term starvation and refeeding. A short-term fasting followed by a refeeding period induced changes in the concentrations of concentrations of glutamate, glutamine, branched chained and basic amino acids in muscle and plasma. Despite this, no changes were seen regarding the glutathione levels in muscle and plasma or its redox status, indicating that the glutathione system is of priority.

Whole body and forearm substrate metabolism in hyperthyroidism: evidence of increased basal muscle protein breakdown

Thyroid hormones have significant metabolic effects, and muscle wasting and weakness are prominent clinical features of chronic hyperthyroidism. To assess the underlying mechanisms, we examined seven hyperthyroid women with Graves' disease before (Ht) and after (Eut) medical treatment and seven control subjects (Ctr). All subjects underwent a 3-h study in the postabsorptive state. After regional catheterization, protein dynamics of the whole body and of the forearm muscles were measured by amino acid tracer dilution technique using [15N]phenylalanine and [2H4]tyrosine. Before treatment, triiodothyronine was elevated (6.6 nmol/l) and whole body protein breakdown was increased 40%. The net forearm release of phenylalanine was increased in hyperthyroidism (microg.100 ml(-1).min(-1)): -7.0 +/- 1.2 Ht vs. -3.8 +/- 0.8 Eut (P = 0.04), -4.2 +/- 0.3 Ctr (P = 0.048). Muscle protein breakdown, assessed by phenylalanine rate of appearance, was increased (microg.100 ml(-1).min(-1)): 15.5 +/- 2.0 Ht vs. 9.6 +/- 1.4 Eut (P = 0.03), 9.9 +/- 0.6 Ctr (P = 0.02). Muscle protein synthesis rate did not differ significantly. Muscle mass and muscle function were decreased 10-20% before treatment. All abnormalities were normalized after therapy. In conclusion, our results show that hyperthyroidism is associated with increased muscle amino acid release resulting from increased muscle protein breakdown. These abnormalities can explain the clinical manifestations of sarcopenia and myopathy.

NOS3 is involved in the increased protein and arginine metabolic response in muscle during early endotoxemia in mice

Sepsis is a severe catabolic condition. The loss of skeletal muscle protein mass is characterized by enhanced release of the amino acids glutamine and arginine, which (in)directly affects interorgan arginine and the related nitric oxide (NO) synthesis. To establish whether changes in muscle amino acid and protein kinetics are regulated by NO synthesized by nitric oxide synthase-2 or -3 (NOS2 or NOS3), we studied C57BL6/J wild-type (WT), NOS2-deficient (NOS2-/-), and NOS3-deficient (NOS3-/-) mice under control (unstimulated) and lipopolysaccharide (LPS)-treated conditions. Muscle amino acid metabolism was studied across the hindquarter by infusing the stable isotopes L-[ring-2H5]phenylalanine, L-[ring-2H2]tyrosine, L-[guanidino-15N2]arginine, and L-[ureido-13C,2H2]citrulline. Muscle blood flow was measured using radioactive p-aminohippuric acid dilution. Under baseline conditions, muscle blood flow was halved in NOS2-/- mice (P < 0.1), with simultaneous reductions in muscle glutamine, glycine, alanine, arginine release and glutamic acid, citrulline, valine, and leucine uptake (P < 0.1). After LPS treatment, (net) muscle protein synthesis increased in WT and NOS2-/- mice [LPS vs. control: 13 +/- 3 vs. 8 +/- 1 (SE) nmol.10 g(-1).min(-1) (WT), 18 +/- 5 vs. 7 +/- 2 nmol.10 g(-1).min(-1) (NOS2-/-); P < 0.05 for LPS vs. control]. This response was absent in NOS3-/- mice (LPS vs. control: 11 +/- 4 vs. 10 +/- 2 nmol.10 g(-1).min(-1)). In agreement, the increase in muscle arginine turnover after LPS was also absent in NOS3-/- mice. In conclusion, disruption of the NOS2 gene compromises muscle glutamine release and muscle blood flow in control mice, but had only minor effects after LPS. NOS3 activity is crucial for the increase in muscle arginine and protein turnover during early endotoxemia.

Microdialysis to investigate tissue amino acid kinetics

This review discusses recent developments in the utilization of microdialysis to assess tissue amino acid kinetics. Three questions regarding the use of microdialysis to assess amino acid kinetics have recently been addressed. Does glutamate measured in the interstitial fluid of the brain represent neurotransmission activity? Can local nitric oxide production be determined with the use of microdialysis? Finally, how can skeletal muscle amino acid metabolism be studied in more detail utilizing microdialysis? The most appropriate way to assess tissue kinetics is by the use of tracers. The number of studies actually utilizing microdialysis in combination with tracers to assess amino acid kinetics is rather limited. However, these studies show exiting new insides into in-vivo amino acid kinetics.

Ceramide down‐regulates System A amino acid transport and protein synthesis in rat skeletal muscle cells

Skeletal muscle is a major insulin target tissue and has a prominent role in the control of body amino acid economy, being the principal store of free and protein-bound amino acids and a dominant locus for amino acid metabolism. Interplay between diverse stimuli (e.g., hormonal/nutritional/mechanical) modulates muscle insulin action to serve physiological need through the action of factors such as intramuscular signaling molecules. Ceramide, a product of sphingolipid metabolism and cytokine signaling, has a potent contra-insulin action with respect to the transport and deposition of glucose in skeletal muscle, although ceramide effects on muscle amino acid turnover have not previously been documented. Here, membrane permeant C2-ceramide is shown to attenuate the basal and insulin-stimulated activity of the Na+-dependent System A amino acid transporter in rat muscle cells (L6 myotubes) by depletion of the plasma membrane abundance of SNAT2 (a System A isoform). Concomitant with transporter down-regulation, ceramide diminished both intramyocellular amino acid abundance and the phosphorylation of translation regulators lying downstream of mTOR. The physiological outcome of ceramide signaling in this instance is a marked reduction in cellular protein synthesis, a result that is likely to represent an important component of the processes leading to muscle wasting in catabolic conditions.

Exposure to air, but not seawater, increases the glutamine content and the glutamine synthetase activity in the marsh clamPolymesoda expansa

Polymesoda expansa spends a considerable portion of its life exposed to air in mangrove swamps where salinity fluctuates greatly. Thus, the aim of this study was to evaluate the effects of aerial exposure (transfer from 10 per thousand brackish water directly to air) or salinity changes (transfer from 10 per thousand brackish water directly to 30 per thousand seawater) on nitrogen metabolism in P. expansa. We concluded that P. expansa is non-ureogenic because carbamoyl phosphate (CPS) III activity was undetectable in the adductor muscle, foot muscle, hepatopancreas and mantle when exposed to brackish water (control), seawater or air for 17 days. It is ammonotelic as it excretes nitrogenous wastes mainly as ammonia in brackish water or seawater. After transfer to seawater for 17 days, the contents of total free amino acids (TFAA) in the adductor muscle, foot muscle, hepatopancreas and mantle increased significantly. This could be related to an increase in protein degradation because exposure to seawater led to a greater rate of ammonia excretion on days 15 and 17, despite unchanged tissue ammonia contents. Alanine was the major free amino acid (FAA) in P. expansa. The contribution of alanine to the TFAA pool in various tissues increased from 43-48% in brackish water to 62-73% in seawater. In contrast, in clams exposed to air for 17 days there were no changes in alanine content in any of the tissues studied. Thus, the functional role of alanine in P. expansa is mainly connected with intracellular osmoregulation. Although 8.5-16.1% of the TFAA pool of P. expansa was attributable to glutamine, the glutamine contents in the adductor muscle, foot muscle, hepatopancreas and mantle were unaffected by 17 days of exposure to seawater. However, after exposure to air for 17 days, there were significant increases in ammonia content in all these tissues in P. expansa, accompanied by significant increases in glutamine content (2.9-, 2.5-, 4.5- and 3.4-fold, respectively). Simultaneously, there were significant increases in glutamine synthetase activities in the adductor muscle (1.56-fold) and hepatopancreas (3.8-fold). This is the first report on the accumulation of glutamine associated with an upregulation of glutamine synthetase in a bivalve species in response to aerial exposure, and these results reveal that the evolution of glutamine synthesis as a means for detoxification of ammonia first occurred among invertebrates.

Reducing plasma HIV RNA improves muscle amino acid metabolism.

We reported (Yarasheski KE, Zachwieja JJ, Gischler J, Crowley J, Horgan MM, and Powderly WG. Am J Physiol Endocrinol Metab 275: E577-E583, 1998) that AIDS muscle wasting was associated with an inappropriately low rate of muscle protein synthesis and an elevated glutamine rate of appearance (Ra Gln). We hypothesized that high plasma HIV RNA caused dysregulation of muscle amino acid metabolism. We determined whether a reduction in HIV RNA (> or =1 log) increased muscle protein synthesis rate and reduced R(a) Gln and muscle proteasome activity in 10 men and 1 woman (22-57 yr, 60-108 kg, 17-33 kg muscle) with advanced HIV (CD4 = 0-311 cells/microl; HIV RNA = 10-375 x 10(3) copies/ml). We utilized stable isotope tracer methodologies ([13C]Leu and [15N]Gln) to measure the fractional rate of mixed muscle protein synthesis and plasma Ra Gln in these subjects before and 4 mo after initiating their first or a salvage antiretroviral therapy regimen. After treatment, median CD4 increased (98 vs. 139 cells/microl, P = 0.009) and median HIV RNA was reduced (155,828 vs. 100 copies/ml, P = 0.003). Mixed muscle protein synthesis rate increased (0.062 +/- 0.005 vs. 0.078 +/- 0.006%/h, P = 0.01), Ra Gln decreased (387 +/- 33 vs. 323 +/- 15 micromol.kg fat-free mass(-1).h(-1), P = 0.04), and muscle proteasome chymotrypsin-like catalytic activity was reduced 14% (P = 0.03). Muscle mass was only modestly increased (1 kg, P = not significant). We estimated that, for each 10,000 copies/ml reduction in HIV RNA, approximately 3 g of additional muscle protein are synthesized per day. These findings suggest that reducing HIV RNA increases muscle protein synthesis and reduces muscle proteolysis, but muscle protein synthesis relative to whole body protein synthesis rate is not restored to normal, so muscle mass is not substantially increased.

An integrative approach to in-vivo protein synthesis measurement: from whole tissue to specific proteins.

In-vivo estimation of protein turnover by stable isotopes in animals and humans has provided much relevant information on metabolic regulation and alterations for decades. While it was first appreciated at the whole body level in the 1970s and 1980s, new approaches have allowed inter-organ or tissue protein turnover rates to be measured, notably the incorporation rate of a labelled amino acid in muscle. These technical improvements have recently been completed by new isolation methods for the study of protein synthesis rates in various muscle and hepatic protein fractions in different blood cells or tissues such as bone and skin. This new insight into tissue protein synthesis opens the door for exploration of single proteins, which may be fully achievable in the near future through the combination of proteomics analysis and technical progress in mass spectrometry. This is, therefore, a new area in which not only quantitative but also qualitative changes in specific proteins will be considered for a fully integrative approach to assessing protein metabolism in physiology and disease. To understand the mechanisms by which protein metabolism is altered during physiopathological situations, it is of importance to measure the effect on specific proteins rather than on the body as a whole. Procedures are currently under development with the aim of isolating individuals proteins and to measure their synthesis rates by isotopic methods. Such technical progress is needed to gain a better understanding of the regulation of protein metabolism in situations in which loss of body protein mass occurs.

Proteinous amino acids in muscle cytosol of rats' heart, after their treatment with propranolol, pentylenetetrazol or reserpine.

Tissue levels of nineteen amino acids and total free amino acids, were assayed by gas-liquid chromatography in cytosols of rat atrial and ventricular muscle cardiomyocytes. The tissues were assayed after the rats had been administered IP with the three cardioactive drugs, exerting a significant effect on their heart action: propranolol, pentylenetetrazol and reserpine. It was demonstrated that while in the atrial and ventricular cardiac muscle cytosols of control rats, arginine, glutamine and cysteine were detected in high levels (35.1% and 17.6%; 14.8% and 51.6%; 9.9% and 0.25% of the total free amino acids, respectively), all three drugs significantly reduced the total amounts of cytosolic free amino acids in both atrial and ventricular heart muscles. All three drugs (with reserpine in particular) modified the levels of arginine, cysteine, phenylalanine, tryptophan, isoleucine and tyrosine. The role of these amino acids in the heart muscle cytosol, and their involvement in the mechanism of action of these three cardioactive drugs, is discussed.

Ammonia toxicity to the brain and creatine

Symptoms of hyperammonemia are age-dependent and some are reversible. Multiple mechanisms are involved. Hyperammonemia increases the uptake of tryptophan into the brain by activation of the L-system carrier while brain glutamine plays a still undefined role. The uptake of tryptophan by the brain is enhanced when the plasma levels of branched-chain amino acids competing with the other large neutral amino acids are low. Hyperammonemia increases the utilization of branched-chain amino acids in muscle when ketoglutarate is low, and this is further enhanced by glutamine depletion (as a result of therapy with ammonia scavengers like phenylbutyrate). Anorexia, most likely a serotoninergic symptom, might further aggravate the deficiency of indispensable amino acids (e.g., branched-chain and arginine). The role of increased glutamine production in astrocytes and the excitotoxic and metabotropic effects of increased extracellular glutamate have been extensively investigated and found to differ between models of acute and chronic hyperammonemia. Using an in vitro model of cultured embryonic rat brain cell aggregates, we studied the role of creatine in ammonia toxicity. Cultures exposed to ammonia before maturation showed impaired cholinergic axonal growth accompanied by a decrease of creatine and phosphocreatine, a finding not observed in mature cultures. By using different antibodies, we have shown that the phosphorylated form of the intermediate neurofilament protein is affected. Adding creatine to the culture medium partially prevents impairment of axonal growth and the presence of glia in the culture is a precondition for this protective effect. Adequate arginine substitution is essential in the treatment of urea cycle defects as creatine is inefficiently transported into the brain.

Biochemical changes in the abdominal muscle of mud crab Scylla serrata during storage

: Changes in levels of pH, volatile basic nitrogen (VBN), K-value, glycogen, adenosine triphosphate (ATP) and its related compounds (ARC), and free amino acids (FAA) in the abdominal muscle of live mud crabs stored at 10°C and 25°C were investigated. Levels of pH, VBN, and K-value did not increase until 2 days of storage, whereas the energy charge and glycogen levels declined rapidly during the early period of storage. The onset of initial decomposition of the mud crab muscle was observed after storage at 10°C for 6 days and 25°C for 3 days. The initial ATP concentration was high but decreased by 76–80% on day 2 of storage. The total nucleotide contents in the 2-day-stored samples accounted for 82–83% of the total ARC. The value decreased to 50% after storage at 10°C for 4 days. Glycine, arginine, glutamine, alanine and proline were the major FAA. Total amounts of FAA and taste-active amino acids including glycine, arginine, alanine and glutamic acid had no apparent change during storage at 10°C, while an increase of 14–38% was found prior to the initial decomposition stage at 25°C. In both storages, ornithine, citrulline and ammonia increased markedly in the stage of initial decomposition.

Proteinous Amino Acids in Muscle Cytosol of Rats' Heart, After Their Treatment with Propranolol, Pentylenetetrazol or Reserpine

Tissue levels of nineteen amino acids and total free amino acids, were assayed by gas-liquid chromatography in cytosols of rat atrial and ventricular muscle cardiomiocytes. The tissues were assayed after the rats had been administered IP with the three cardioactive drugs, exerting a significant effect on their heart action: propranolol, pentylenetetrazol and reserpine. It was demonstrated that while in the atrial and ventricular cardiac muscle cytosols of control rats, arginine, glutamine and cysteine were detected in high levels (35.1% and 17.6%; 14.8% and 51.6%; 9.9% and 0.25% of the total free amino acids, respectively), all three drugs significantly reduced the total amounts of cytosolic free amino acids in both atrial and ventricular heart muscles. All three drugs (with reserpine in particular) modified the levels of arginine, cysteine, phenylalanine, tryptophan, isoleucine and tyrosine. The role of these amino acids in the heart muscle cytosol, and their involvement in the mechanism of action of these three cardioactive drugs, is discussed.+ Deceased.* Affiliated with the David R. Bloom Center for Pharmacy at the Hebrew University.

Free amino acids in muscle of Norway lobster ( Nephrops novergicus ( L.)) in controlled and modified atmospheres during chilled storage

Changes in free amino acids (FAAs) in relation to freshness were examined in the muscle of Norway lobster during bulk storage in controlled and modified atmospheres containing two different gas mixes (1: 60/15/25 and 2: 40/40/20, CO 2%/O 2%/N 2%). The essential free amino acids in the highest concentrations were threonine, leucine, valine, lysine and arginine, all over 40 mg/100 g. Threonine, valine, lysine and arginine concentrations decreased significantly ( p<0.05) during storage. These decreases were more pronounced in the lots kept in the CO 2-rich mix (1) than the lots kept in the O 2-rich mix (2) and this was independent of whether the storage was under controlled (C) or modified (M) atmosphere. The dipeptide anserine showed decreases with the same trend. The most abundant non-essential FAAs were glycine, alanine and glutamic acid (57.9, 57.2 and 31.2 mg/100 g, respectively), along with the dipeptide anserine (52.9 mg/100 g). The non-essential FAAs presented differences attributable to the gas mixes used. No differences were detected in any of the FAA groups studied with respect to the different types of atmospheres (C or M). Only ornithine and tryptophan levels increased significantly ( p<0.05) in the course of storage. These increases were significantly ( p<0.05) different only in the control lot. These FAAs would appear to be suitable as freshness indices for Norway lobster stored in ice. Moreover, the significant ( p<0.05) decrease of the dipeptide anserine could be a suitable freshness index for Norway lobster stored in either ice or in atmospheres (C or M).

Effect of different matrices on physiological amino acids analysis by liquid chromatography: evaluation and correction of the matrix effect

Free amino acids in plasma, muscle and liver tissues were determined using the Pico-Tag method which involves HPLC and pre-column derivatization. A study of the accuracy in the determination is carried out by means of external calibration, standard-added calibration, Youden calibration and analyte recovery. It is conclusively shown that the method is affected by a proportional systematic error due to matrix effects, but not by a constant one. A new function named matrix-corrected calibration is proposed to correct for proportional bias when free analyte matrices for matrix-matched addition is unavailable.

A leucine-supplemented diet improved protein content of skeletal muscle in young tumor-bearing rats.

Cancer cachexia induces host protein wastage but the mechanisms are poorly understood. Branched-chain amino acids play a regulatory role in the modulation of both protein synthesis and degradation in host tissues. Leucine, an important amino acid in skeletal muscle, is higher oxidized in tumor-bearing animals. A leucine-supplemented diet was used to analyze the effects of Walker 256 tumor growth on body composition in young weanling Wistar rats divided into two main dietary groups: normal diet (N, 18% protein) and leucine-rich diet (L, 15% protein plus 3% leucine), which were further subdivided into control (N or L) or tumor-bearing (W or LW) subgroups. After 12 days, the animals were sacrificed and their carcass analyzed. The tumor-bearing groups showed a decrease in body weight and fat content. Lean carcass mass was lower in the W and LW groups (W = 19.9 0.6, LW = 23.1 1.0 g vs N = 29.4 1.3, L = 28.1 1.9 g, P < 0.05). Tumor weight was similar in both tumor-bearing groups fed either diet. Western blot analysis showed that myosin protein content in gastrocnemius muscle was reduced in tumor-bearing animals (W = 0.234 0.033 vs LW = 0.598 0.036, N = 0.623 0.062, L = 0.697 0.065 arbitrary intensity, P < 0.05). Despite accelerated tumor growth, LW animals exhibited a smaller reduction in lean carcass mass and muscle myosin maintenance, suggesting that excess leucine in the diet could counteract, at least in part, the high host protein wasting in weanling tumor-bearing rats.

White muscle free amino acid concentrations following feeding a maize gluten dietary protein in Atlantic salmon ( Salmo salar L.)

This study aimed to investigate the effect of high maize gluten protein diets on the white muscle free amino acid (FAA) concentrations of Atlantic salmon following feeding and to examine whether these tissue free amino acids profiles highlighted any dietary deficiencies in essential amino acids (EAA). Two isocaloric and isonitrogenous diets (52% protein, 20% oil) were formulated and mixed proportionately to give a total of five feeds in which maize gluten provided 0%, 16%, 32%, 48% and 64% of the total dietary protein (diets 0MG, 16MG, 32MG, 48MG, 64MG, respectively). Triplicate groups of 19 g Atlantic salmon were fed each of the experimental diets for a period of 40 days. At the end of the growth experiment, groups of four salmon from each diet were taken at 4, 8 and 12 h after feeding for the white muscle amino acids analysis. Although fish doubled their weights during the growth period, no significant differences were observed in specific growth rates, feed conversion ratios, protein digestibility and protein growth rates among fish fed the test diets. Total free amino acid (FAA) concentrations did not change at various times after a meal and this was also true for the majority of individual FAAs (except asparagine, which increased significantly 12 h after feeding). The most notable diet-induced changes in the white muscle were an overall reduction in essential FAA concentrations (threonine and lysine) as dietary maize gluten content increased and an increase in histidine and leucine. Correlations between dietary essential amino acids patterns and white muscle protein-bound amino acids were examined. The possibility of partial replacement of fish meal maize gluten up to 50% was demonstrated.

Free amino acid pool and muscle protein balance after resistance exercise.

The aim of this study was to assess the effects of a resistance exercise session (RES) on free amino acid concentrations and protein synthesis and breakdown of the vastus lateralis (VL) muscle during recovery in male subjects. Both the exercise group (EG) and the control group (CG) consisted of six healthy physically active men. On the experiment day in fasting conditions, a stable isotopic tracer of L-[ring-2H(5)] phenylalanine was infused and EG started a heavy 50-min hypertrophic RES for lower extremities after 55 min of infusion. At the same time, CG was at rest. During recovery of 195 min after RES, several blood samples were drawn from the femoral artery (FA) and the femoral vein (FV) and muscle samples from the VL muscle. The enrichment was analyzed by GC/MS and leg muscle amino acid kinetics determined by three-pool compartment model between FA, FV, and VL. During recovery at 60 min after RES, there was no difference in muscle protein synthesis or muscle protein breakdown between the groups, but at 195 min, both muscle protein synthesis (P < 0.05) and muscle protein breakdown (P < 0.05) were increased in EG compared with CG. The protein net balance was negative and similar in both groups. Simultaneously in serum concentrations, there was a decrease in leucine (P < 0.05) associated with an increase in aspartate (P < 0.05). Furthermore, the exercise-induced increase in alanine concentration decreased both in serum and muscle. In fasting conditions, protein net balance is negative and RES induces an increase in muscle protein synthesis and breakdown at 195 min but not yet at 60 min of recovery.

Regulation of translation initiation by insulin and amino acids in skeletal muscle of neonatal pigs.

Previous studies have shown that intravenous infusion of insulin and/or amino acids reproduces the feeding-induced stimulation of muscle protein synthesis in neonates and that insulin and amino acids act independently to produce this effect. The goal of the present study was to delineate the regulatory roles of insulin and amino acids on muscle protein synthesis in neonates by examining translational control mechanisms, specifically the eukaryotic translation initiation factors (eIFs), which enable coupling of initiator methionyl-tRNAi and mRNA to the 40S ribosomal subunit. Insulin secretion was blocked by somatostatin in fasted 7-day-old pigs (n = 8-12/group), insulin was infused to achieve plasma levels of approximately 0, 2, 6, and 30 microU/ml, and amino acids were clamped at fasting or fed levels or, at the high insulin dose, below fasting. Both insulin and amino acids increased the phosphorylation of ribosomal protein S6 kinase (S6K1) and the eIF4E-binding protein (4E-BP1), decreased the binding of 4E-BP1 to eIF4E, increased eIF4E binding to eIF4G, and increased fractional protein synthesis rates but did not affect eIF2B activity. In the absence of insulin, amino acids had no effect on these translation initiation factors but increased the protein synthesis rates. Raising insulin from below fasting to fasting levels generally did not alter translation initiation factor activity but raised protein synthesis rates. The phosphorylation of S6K1 and 4E-BP1 and the amount of 4E-BP1 bound to eIF4E and eIF4E bound to eIF4G were correlated with insulin level, amino acid level, and protein synthesis rate. Thus insulin and amino acids regulate muscle protein synthesis in skeletal muscle of neonates by modulating the availability of eIF4E for 48S ribosomal complex assembly, although other processes also must be involved.