Increased Amino Acid Availability Research Articles

Initiation of muscle protein synthesis was unrelated to simultaneously upregulated local production of IGF-1 by amino acids in non-proliferating L6 muscle cells.

BackgroundIGF-1 is considered an important regulator of muscle protein synthesis. However, its role in stimulation of muscle protein synthesis by amino acids (AA) is not clear, despite pronounced alterations in IGF-1 mRNA expression and signaling in muscle tissues by feeding. This study evaluates the role of locally produced IGF-1 and IGF-1 signaling when skeletal muscle protein synthesis is activated by increased amino acid availability in confluent, non-proliferating cells.MethodsL6 skeletal muscle cells were subjected to amino acid starvation (24 h, 0.14 mM) followed by 18 h amino acid refeeding in Low AA (0.28 mM) or High AA concentrations (9 mM). Protein synthesis rates were estimated by L-[U-14C]-phenylalanine incorporation into cellular proteins. IGF-1 and IGF-1 receptor mRNA expression were quantified by real time PCR. SiRNA knockdown, antibodies and chemical inhibitors were used to attenuate muscle IGF-1 production and signaling.ResultsHigh AA concentrations (9mM) increased IGF-1 mRNA expression (+ 30%, p<0.05) and increased L-[U-14C]-phenylalanine incorporation compared to Low AA in confluent, non-proliferating muscle cells. Blocking IGF-1 signaling by chemical inhibitors reduced IGF-1 mRNA upregulation (~50%, p< 0.01), without decrease of protein synthesis. SiRNA knockdown of IGF-1 reduced protein synthesis, mainly explained by reduced cell proliferation. High AA or IGF-1 inhibitors did not change IGF-1 receptor mRNA expressions.ConclusionAmino acids increased IGF-1 mRNA expression and stimulated muscle protein synthesis. However, simultaneous upregulation of IGF-1 mRNA did not relate to increased protein synthesis by amino acids. The results indicate that increased IGF-1 mRNA expression is rather a covariate to amino acid initiation of protein synthesis in non-proliferating muscle cells; effects that may be related to unrecognized metabolic activities, such as transport of amino acids.

Improved recovery from skeletal muscle damage is largely unexplained by myofibrillar protein synthesis or inflammatory and regenerative gene expression pathways.

The contribution of myofibrillar protein synthesis (MyoPS) to recovery from skeletal muscle damage in humans is unknown. Recreationally active men and women consumed a daily protein-polyphenol beverage targeted at increasing amino acid availability and reducing inflammation (PPB; n = 9), both known to affect MyoPS, or an isocaloric placebo (PLA; n = 9) during 168 h of recovery from 300 maximal unilateral eccentric contractions (EE). Muscle function was assessed daily. Muscle biopsies were collected for 24, 27, 36, 72, and 168 h for MyoPS measurements using 2H2O and expression of 224 genes using RT-qPCR and pathway analysis. PPB improved recovery of muscle function, which was impaired for 5 days after EE in PLA (interaction P < 0.05). Acute postprandial MyoPS rates were unaffected by nutritional intervention (24-27 h). EE increased overnight (27-36 h) MyoPS versus the control leg (PLA: 33 ± 19%; PPB: 79 ± 25%; leg P < 0.01), and PPB tended to increase this further (interaction P = 0.06). Daily MyoPS rates were greater with PPB between 72 and 168 h after EE, albeit after function had recovered. Inflammatory and regenerative signaling pathways were dramatically upregulated and clustered after EE but were unaffected by nutritional intervention. These results suggest that accelerated recovery from EE is not explained by elevated MyoPS or suppression of inflammation.NEW & NOTEWORTHY The present study investigated the contribution of myofibrillar protein synthesis (MyoPS) and associated gene signaling to recovery from 300 muscle-damaging, eccentric contractions. Measured with 2H2O, MyoPS rates were elevated during recovery and observed alongside expression of inflammatory and regenerative signaling pathways. A nutritional intervention accelerated recovery; however, MyoPS and gene signaling were unchanged compared with placebo. These data indicate that MyoPS and associated signaling do not explain accelerated recovery from muscle damage.

Soil aminopeptidase induction is unaffected by inorganic nitrogen availability

Soil microbes release a variety of aminopeptidase enzymes to degrade proteins into their constituent amino acid monomers, thereby increasing amino acid availability in the near-cell environment. We investigated how inorganic N availability controls the substrate induction response of aminopeptidase enzymes by conducting protein amendment experiments via gelatin addition to fertilized and unfertilized soil from a long-term N addition study. Protein addition triggered a measurable increase in enzyme activity, which we interpreted as an induction response, in all five of the aminopeptidase enzymes we investigated. The magnitude of these induction responses did not differ by fertilization regime even when unamended aminopeptidase activity differed between long-term fertilization treatments. Though soil microbes are thought to release aminopeptidase enzymes as a means of cellular N acquisition at steady-state concentrations, our results suggest that they use the same enzymes to access protein-derived C as an energetic resource at non-steady-state conditions, such as those triggered by the substrate induction response. The substrate induction response of aminopeptidase enzymes is therefore best viewed as a means of microbial competition for the substantial energetic resources available in protein subsidies.

Prolactin regulates LAT1 expression via STAT5 (signal transducer and activator of transcription 5) signaling in mammary epithelial cells of dairy cows

The l-type amino acid transporter 1 (LAT1; also known as SLC7A5) is a transporter that allows the uptake of large neutral amino acids into mammalian cells. In dairy cows, LAT1 is highly expressed in lactating mammary tissues and involved in milk protein synthesis. Prolactin (PRL) has a lactogenic role and is capable of inducing milk production in ruminants. However, the relationship between PRL stimulation and LAT1 expression in dairy cow mammary gland has not been well understood. In this study, we showed that PRL stimulation increased expression of LAT1 and β-casein in mammary epithelial cells of dairy cows. The stimulatory effect of PRL on milk protein production was inhibited by LAT1-specific inhibitor or LAT1 knockdown, suggesting that PRL-induced milk protein production is involved in LAT1 expression. To determine whether the PRL signaling pathway participates in regulation of LAT1 expression, PRLR (PRL receptor) or STAT5 (signal transducer and activator of transcription 5) was knocked down by short interfering (si)RNA in mammary epithelial cells of dairy cows. Western blot results showed that knockdown of PRLR or STAT5 with siRNA markedly decreased PRL-stimulated LAT1 expression. In addition, we observed a marked increase in plasma membrane expression of LAT1 in PRL-stimulated cells compared with control cells. These observations indicated that PRL signaling can regulate LAT1 expression and activity in mammary epithelial cells of dairy cows, contributing to increased amino acid availability and milk protein synthesis in mammary gland of dairy cow.

The Effects of Dietary Omega-3s on Muscle Composition and Quality in Older Adults.

This review will focus on findings from the few studies performed to date in humans to examine changes in muscle protein turnover, lean or muscle mass and physical function following fish oil-derived omega-3 fatty acid treatment. Although considerable gaps in our current knowledge exist, hypertrophic responses (e.g., improvements in the rate of muscle protein synthesis and mTOR signaling during increased amino acid availability and an increase in muscle volume) have been reported in older adults following prolonged (8 to 24 weeks) of omega-3 fatty acid supplementation. There is also accumulating evidence that increased omega-3 fatty acid levels in red blood cells are positively related to strength and measures of physical function. As a result, increased omega-3 fatty acid consumption may prove to be a promising low-cost dietary approach to attenuate or prevent aging associated declines in muscle mass and function.

Adaptation of in vivo amino acid kinetics facilitates increased amino acid availability for fetal growth in adolescent and adult pregnancies alike.

During pregnancy, adult women with a normal BMI synthesise extra amino acids after an overnight fast by increasing body protein breakdown and decreasing amino acid oxidation. It is not known whether adolescent girls can make these adaptations during pregnancy. The present study aimed to measure and compare the protein, glutamine and alanine kinetics of adult women and adolescent girls at early-, mid- and late-pregnancy. Kinetics were measured in the overnight fasted state using intravenous infusions of 13C-leucine, 15N-glutamine and 15N-alanine in ten adults and twenty adolescents aged 14-17 years in the first and second trimesters (phase 1 study) and infusions of 13C-leucine and 15N2-urea in ten adults and eleven adolescents aged 16-17 years in the first and third trimesters (phase 2 study). In phase 1 study, there were no significant differences between the groups with regard to any of the kinetic parameters measured. In both groups, leucine flux increased (P< 0.05), the percentage of leucine flux oxidised decreased (P< 0.05) and non-oxidative leucine disposal to protein synthesis increased (P< 0.05) from the first to the second trimester. In phase2 study, leucine flux was significantly slower (P< 0.05) in the adult group than in the adolescent group during both trimesters, and whole-body leucine flux and non-oxidative leucine disposal increased significantly in the adolescent group (P< 0.05, respectively) and were higher in the adult group from the first to the third trimester. These results suggest that similar to their adult counterparts after an overnight fast, adolescent girls with a normal BMI provide extra amino acids required for net protein deposition during pregnancy by increasing protein breakdown and decreasing amino acid oxidation.

Soy-dairy protein blend and whey protein ingestion after resistance exercise increases amino acid transport and transporter expression in human skeletal muscle

Increasing amino acid availability (via infusion or ingestion) at rest or postexercise enhances amino acid transport into human skeletal muscle. It is unknown whether alterations in amino acid availability, from ingesting different dietary proteins, can enhance amino acid transport rates and amino acid transporter (AAT) mRNA expression. We hypothesized that the prolonged hyperaminoacidemia from ingesting a blend of proteins with different digestion rates postexercise would enhance amino acid transport into muscle and AAT expression compared with the ingestion of a rapidly digested protein. In a double-blind, randomized clinical trial, we studied 16 young adults at rest and after acute resistance exercise coupled with postexercise (1 h) ingestion of either a (soy-dairy) protein blend or whey protein. Phenylalanine net balance and transport rate into skeletal muscle were measured using stable isotopic methods in combination with femoral arteriovenous blood sampling and muscle biopsies obtained at rest and 3 and 5 h postexercise. Phenylalanine transport into muscle and mRNA expression of select AATs [system L amino acid transporter 1/solute-linked carrier (SLC) 7A5, CD98/SLC3A2, system A amino acid transporter 2/SLC38A2, proton-assisted amino acid transporter 1/SLC36A1, cationic amino acid transporter 1/SLC7A1] increased to a similar extent in both groups (P < 0.05). However, the ingestion of the protein blend resulted in a prolonged and positive net phenylalanine balance during postexercise recovery compared with whey protein (P < 0.05). Postexercise myofibrillar protein synthesis increased similarly between groups. We conclude that, while both protein sources enhanced postexercise AAT expression, transport into muscle, and myofibrillar protein synthesis, postexercise ingestion of a protein blend results in a slightly prolonged net amino acid balance across the leg compared with whey protein.

Minced beef is more rapidly digested and absorbed than beef steak, resulting in greater postprandial protein retention in older men

Older individuals generally experience a reduced food-chewing efficiency. As a consequence, food texture may represent an important factor that modulates dietary protein digestion and absorption kinetics and the subsequent postprandial protein balance. We assessed the effect of meat texture on the dietary protein digestion rate, amino acid availability, and subsequent postprandial protein balance in vivo in older men. Ten older men (mean ± SEM age: 74 ± 2 y) were randomly assigned to a crossover experiment that involved 2 treatments in which they consumed 135 g of specifically produced intrinsically L-[1-(13)C]phenylalanine-labeled beef, which was provided as beef steak or minced beef. Meat consumption was combined with continuous intravenous L-[ring-(2)H5]phenylalanine and L-[ring-(2)H2]tyrosine infusion to assess beef protein digestion and absorption kinetics as well as whole-body protein balance and skeletal muscle protein synthesis rates. Meat protein-derived phenylalanine appeared more rapidly in the circulation after minced beef than after beef steak consumption (P < 0.05). Also, its availability in the circulation during the 6-h postprandial period was greater after minced beef than after beef steak consumption (61 ± 3% compared with 49 ± 3%, respectively; P < 0.01). The whole-body protein balance was more positive after minced beef than after beef steak consumption (29 ± 2 compared with 19 ± 3 μmol phenylalanine/kg, respectively; P < 0.01). Skeletal muscle protein synthesis rates did not differ between treatments when assessed over a 6-h postprandial period. Minced beef is more rapidly digested and absorbed than beef steak, which results in increased amino acid availability and greater postprandial protein retention. However, this does not result in greater postprandial muscle protein synthesis rates. This trial was registered at clinicaltrials.gov as NCT01145131.

Amino Acid Infusion Alters the Expression of Growth-Related Genes in Multiple Skeletal Muscles

Exercise and nutritional interventions have been examined independently as countermeasures to offset the loss of skeletal muscle mass with unloading, yet a protocol to completely preserve the soleus has not been identified. Little is known regarding the combined effect of exercise and nutrition on factors regulating skeletal muscle growth. The purpose of this investigation was to evaluate the influence of amino acid (AA) infusion on myogenic (MRF-4, MyoD, and Myogenin), proteolytic (MuRF-1, Atrogin-1, FOXO3A, Calpain-1, Calpain-2, Caspase-3, Cathepsin L1), and cytokine (IL-6, IL-8, and IL-15) mRNA transcripts in two skeletal muscles that respond distinctly to microgravity unloading. Muscle biopsies were obtained from the vastus lateralis (VL) and soleus of eight male subjects prior to and after 4 h of AA infusion for analysis of mRNA expression. All subjects performed a standardized exercise bout (45-min treadmill run) 24 h prior to the AA infusion. In the VL, proteolytic factors MuRF-1 and FOXO3A were reduced (44 +/- 9 and 28 +/- 6%, respectively) in response toAA infusion. In the soleus, mRNA transcripts of myogenic factor MRF-4 (91 +/- 36%) and cytokines IL-6, IL-8, and IL-15 were elevated while the proteolytic marker FOXO3A mRNA was reduced by 19 +/- 9%. These data suggest that the expression of genes related to skeletal muscle remodeling is altered during acute AA infusion 24 h post-exercise. It appears that increased amino acid availability in concert with exercise may create an intramuscular environment favorable for the prevention of muscle atrophy associated with unloading, which may be particularly beneficial for the soleus.

Root carbon inputs to the rhizosphere stimulate extracellular enzyme activity and increase nitrogen availability in temperate forest soils

The exudation of carbon (C) by tree roots stimulates microbial activity and the production of extracellular enzymes in the rhizosphere. Here, we investigated whether the strength of rhizosphere processes differed between temperate forest trees that vary in soil organic matter (SOM) chemistry and associate with either ectomycorrhizal (ECM) or arbuscular mycorrhizal (AM) fungi. We measured rates of root exudation, microbial and extracellular enzyme activity, and nitrogen (N) availability in samples of rhizosphere and bulk soil influenced by four temperate forest tree species (i.e., to estimate a rhizosphere effect). Although not significantly different between species, root exudation ranged from 0.36 to 1.10 g C m−2 day−1, representing a small but important transfer of C to rhizosphere microbes. The magnitude of the rhizosphere effects could not be easily characterized by mycorrhizal associations or SOM chemistry. Ash had the lowest rhizosphere effects and beech had the highest rhizosphere effects, representing one AM and one ECM species, respectively. Hemlock and sugar maple had equivalent rhizosphere effects on enzyme activity. However, the form of N produced in the rhizosphere varied with mycorrhizal association. Enhanced enzyme activity primarily increased amino acid availability in ECM rhizospheres and increased inorganic N availability in AM rhizospheres. These results show that the exudation of C by roots can enhance extracellular enzyme activity and soil-N cycling. This work suggests that global changes that alter belowground C allocation have the potential to impact the form and amount of N to support primary production in ECM and AM stands.

Rapamycin does not affect post-absorptive protein metabolism in human skeletal muscle

Administration of the mTORC1 inhibitor, rapamycin, to humans blocks the increase in skeletal muscle protein synthesis in response to resistance exercise or amino acid ingestion. ObjectiveTo determine whether rapamycin administration influences basal post-absorptive protein synthesis or breakdown in human skeletal muscle. Materials/MethodsSix young (26±2 years) subjects were studied during two separate trials, in which each trial was divided into two consecutive 2h basal periods. The trials were identical except during one trial a single oral dose (16mg) of rapamycin was administered immediately prior to the second basal period. Muscle biopsies were obtained from the vastus lateralis at 0, 2, and 4h to examine protein synthesis, mTORC1 signaling, and markers of autophagy (LC3B-I and LC3B-II protein) associated with each 2h basal period. ResultsDuring the Control trial, muscle protein synthesis, whole body protein breakdown (phenylalanine Ra), mTORC1 signaling, and markers of autophagy were similar between both basal periods (p>0.05). During the Rapamycin trial, these variables were similar to the Control trial (p>0.05) and were unaltered by rapamycin administration (p>0.05). Thus, post-absorptive muscle protein metabolism and mTORC1 signaling were not affected by rapamycin administration. ConclusionsShort-term rapamycin administration may only impair protein synthesis in human skeletal muscle when combined with a stimulus such as resistance exercise or increased amino acid availability.

Anabolic resistance: the effects of aging, sexual dimorphism, and immobilization on human muscle protein turnoverThis paper is one of a selection of papers published in this Special Issue, entitled 14th International Biochemistry of Exercise Conference – Muscles as Molecular and Metabolic Machines, and has undergone the Journal’s usual peer review process.

In healthy active older persons, there is no derangement of muscle protein metabolism. However, there is a major deficit in the ability of older muscles to regulate their maintenance during feeding and exercise. The dose-response relationship between myofibrillar protein synthesis and the availability of essential amino acids (EAA) is shifted down and to the right, and giving extra amino acids is unable to overcome this. There is no sex difference in basal or fed muscle protein metabolism in the young, but postmenopausal women have a greater anabolic resistance than older men. Anabolic resistance is also shown by the decreased phosphorylation in the PKB-mTOR-eIF4BP1 pathway in response to increased EAA. The muscle synthetic system is refractory to EAA provision, irrespective of the availability of insulin, insulin-like growth factor 1, and growth hormone. However, insulin is a major regulator of muscle protein breakdown, and there is a blunting of the ability of older muscle to decrease proteolysis in response to low concentrations of insulin, such as those observed after a light breakfast. Providing more EAA seems not to be useful, and modern N-balance data confirm that the dietary protein requirements of older persons are not increased. The sigmoidal dose-response relationship between muscle protein synthesis and resistance exercise intensity is shifted downward and to the right in older men. Decreased physical activity itself, even in young subjects, can produce anabolic resistance of muscle protein synthesis, which cannot be overcome by increasing amino acid availability. Exercise may retune the amino acid and (or) insulin sensitivity of muscle in older people.

Muscle-specific MicroRNAs Are Not Altered In Human Muscle Following Ingestion Of Leucine-enriched Essential Amino Acids

Introduction: Essential amino acid (EAA) ingestion results in a significant increase in mixed muscle protein synthesis in humans. The muscle-specific microRNAs (miR-1, -133a, and -206) have been postulated to participate in the regulation of skeletal muscle size by inhibiting translation of specific target mRNAs. Our laboratory has recently shown that miR-1 expression decreased immediately following the combined anabolic stimulus of resistance exercise and EAA ingestion. PURPOSE: We hypothesized that expression of miR-1, -133a, and -206 would be decreased immediately following ingestion of leucine-enriched EAA in young healthy humans. METHODS: Five young subjects (2 men, 3 women, 31±2 yr) ingested leucine-enriched (35%) EAA (10g) following an overnight fast. Muscle biopsies were sampled before and 1 and 2h following EAA ingestion. Standard laboratory procedures were utilized to isolate RNA. This was followed by real-time PCR using commercially available kits (mirVana) and primers in order to determine the expression of miR-1, -133a, and -206. RESULTS: The expression levels of miR-1, -133a, and -206 were unaltered following EAA ingestion as compared to baseline. CONCLUSION: We conclude that the muscle-specific microRNAs (miR-1, -133a, and -206) are not associated with the acute increase in human skeletal muscle protein synthesis during the first couple of hours following essential amino acid ingestion. Therefore, it appears that the changes we detected in microRNA expression following resistance exercise and EAA ingestion were likely due to muscle contraction rather than increased amino acid availability. However, future studies are needed to determine the individual effect of resistance exercise on microRNA expression and whether increased amino acid availability post-exercise is a contributing factor. Supported by ACSM Research Endowment Grant (MJD), P30 AG-024832 (MJD) and NIH/NIAMS grant RO1 AR049877 (BBR)

RalA Functions as an Indispensable Signal Mediator for the Nutrient-sensing System

Cells sense nutrients present in the extracellular environment and modulate the activities of intracellular signaling systems in response to nutrient availability. This study demonstrates that RalA and its activator RalGDS participate in nutrient sensing and are indispensable for activation of mammalian target of rapamycin complex 1 (mTORC1) induced by extracellular nutrients. Knockdown of RalA or RalGDS abolished amino acid- and glucose-induced mTORC1 activation, as judged by phosphorylation of S6 kinase and eukaryotic translation initiation factor 4E-binding protein 1. The amount of GTP-bound RalA increased in response to increased amino acid availability. In addition, RalA knockdown suppressed Rheb-induced S6 kinase phosphorylation, and the constitutively active form of RalA induced mTORC1 activation in the absence of Rheb. These results collectively suggest that RalGDS and RalA act downstream of Rheb and that RalA activation is a crucial step in nutrient-induced mTORC1 activation.

Amino acid supplementation prevents the loss of appetite for casein in old Lou/Cjall rats

A deleterious reduction of casein intake occurring earlier in males than in females had been previously observed in old Lou/Cjall rats. On the contrary, protein intake was observed to be maintained in old males when they were offered whey protein. Present studies were designed to investigate the effect of dietary casein modification on protein decrease. In two lifelong studies, male and female Lou/Cjall rats were tested every four months in order to study protein intake depending on the protein available: casein, whey protein or casein supplemented with an amino acid mixture (SC). In subsequent cross-sectional studies, young, adult, middle-aged and old rats were successively fed with casein, casein supplemented either with leucine or with alanine or with glycine. Supplementing casein with an amino acid mixture both globally increased protein intake and allowed old males to maintain a high rate of protein intake. In cross-sectional experiments, no effect of supplementation was seen in the young group. In older animals, the greatest effect was seen when casein was supplemented with alanine or glycine, independently of sex and age. We therefore, concluded that supplemented casein is more beneficial for old rats than casein alone, probably by increasing amino acid availability. We hypothesize that alanine could act through its effect on gluconeogenesis.

Synergistic effects of caloric restriction together with protein intake on skeletal muscle metabolism in middle‐aged rats

Rationale Caloric restriction (CR) reverses some age-related alterations in muscle mitochondrial function. However, CR does not prevent the decline in ATP production necessary to sustain muscle protein synthesis rate (FSR) and contraction. We hypothesized that a limited ATP production is linked to a reduction in mitochondrial protein turnover, which could be counteracted by increasing amino acid availability during CR. Methods Wistar rats (16 mo, n=30) were fed either ad libitum (AL), 40% protein-energy restricted (PER) or 40% energy restricted (ER) for 5 months. Muscle mass and strength (grip force) were measured. Mitochondrial coupling, ATP production, and FSR (using 13C-valine as a tracer) were determined in tibialis anterior (TA) and soleus (SO) muscles mitochondria. Myosin and actin FSR were also investigated. Results ER diet improved mitochondrial ATP production and coupling in TA (p<0.05 vs AL) and in SO muscles (p=0.07). TA mitochondrial FSR was also enhanced by ER diet while PER decreased it (p<0.05 vs AL). ER diet was able to maintain TA mass and to improve myosin and actin FSR (p<0.05 vs PER and AL) and muscle strength (p<0.05 vs AL). Protein synthesis rates were not affected by the diets in SO muscle. Conclusions Deprivation of energy with maintenance of protein intake initiated in late middle age improves mitochondrial function, ATP production and protein turnover in skeletal muscles. The present study supports the hypothesis that sarcopenia is nutritionally preventable by using strategies associating limiting energy intake but favouring sufficient protein intake of various quality in the diet.

The response of intracellular signaling and muscle-protein metabolism to nutrition and exercise

In recent years, a body of literature examining the response of muscle-protein metabolism to exercise and nutrition has arisen. Developments in methods, especially stable isotopic tracer methodology, have allowed much information to be gathered in vivo in humans. The metabolic mechanism behind increased muscle mass requires that muscle-protein synthesis exceeds breakdown, i.e., net muscle-protein synthesis. Increased net muscle-protein balance may occur due to exercise, but net synthesis may occur only with the addition of nutrients, particularly a source of amino acids. The major impact of increased amino acid availability on net muscle-protein balance is due to stimulation of muscle-protein synthesis and less to inhibition of muscle-protein breakdown. Amino acids seem to stimulate muscle-protein synthesis, not only by mass action, i.e., providing substrate, but also as signals for initiation of protein synthesis. Stimulation of muscle-protein synthesis by amino acid ingestion may be linked to increased intracellular amino acid levels and/or to changing amino acid levels in the blood. Carbohydrate ingestion, most likely through the action of insulin, also may play a role in the response of muscle to exercise and nutrition. There is very little research in humans in vivo on the intracellular signaling that is linked to muscle-protein synthesis. It is clear that intracellular signaling responds to both insulin and amino acids, but the interactions with exercise are not well known; however, the details of the pathways have only just begun to be investigated, especially in humans. Delineation of these pathways is complicated, and there is little doubt that multiple intracellular signaling pathways with several levels of communication are involved in the hypertrophy process in response to nutrition and exercise. A systematic investigation of the relationship of the signaling to insulin and amino acids combined with exercise will provide important information, especially for populations vulnerable to muscle loss.

Overactivation of S6 Kinase 1 as a Cause of Human Insulin Resistance During Increased Amino Acid Availability

To examine the molecular mechanisms by which plasma amino acid elevation impairs insulin action, we studied seven healthy men twice in random order during infusion of an amino acid mixture or saline (total plasma amino acid approximately 6 vs. approximately 2 mmol/l). Somatostatin-insulin-glucose clamps created conditions of low peripheral hyperinsulinemia ( approximately 100 pmol/l, 0-180 min) and prandial-like peripheral hyperinsulinemia ( approximately 430 pmol/l, 180-360 min). At low peripheral hyperinsulinemia, endogenous glucose production (EGP) did not change during amino acid infusion but decreased by approximately 70% during saline infusion (EGP(150-180 min) 11 +/- 1 vs. 3 +/- 1 mumol . kg(-1) . min(-1), P = 0.001). Prandial-like peripheral hyperinsulinemia completely suppressed EGP during both protocols, whereas whole-body rate of glucose disappearance (R(d)) was approximately 33% lower during amino acid infusion (R(d) (330-360 min) 50 +/- 4 vs. 75 +/- 6 mumol . kg(-1) . min(-1), P = 0.002) indicating insulin resistance. In skeletal muscle biopsies taken before and after prandial-like peripheral hyperinsulinemia, plasma amino acid elevation markedly increased the ability of insulin to activate S6 kinase 1 compared with saline infusion ( approximately 3.7- vs. approximately 1.9-fold over baseline). Furthermore, amino acid infusion increased the inhibitory insulin receptor substrate-1 phosphorylation at Ser312 and Ser636/639 and decreased insulin-induced phosphoinositide 3-kinase activity. However, plasma amino acid elevation failed to reduce insulin-induced Akt/protein kinase B and glycogen synthase kinase 3alpha phosphorylation. In conclusion, amino acids impair 1) insulin-mediated suppression of glucose production and 2) insulin-stimulated glucose disposal in skeletal muscle. Our results suggest that overactivation of the mammalian target of rapamycin/S6 kinase 1 pathway and inhibitory serine phosphorylation of insulin receptor substrate-1 underlie the impairment of insulin action in amino acid-infused humans.

Amino acid control of muscle protein turnover in renal disease

This review discusses the concept that skeletal muscle intracellular amino acids (ICAAs), in particular the essential amino acids, are regulated throughout a wide range of physiologic circumstances. Whether in critical illness, severe injury, or healthy states, ICAAs are closely regulated by a coordinated response in 1 or more of the processes of synthesis, breakdown, and tissue transport. For a given metabolic signal (hormonal, change in plasma amino acid concentrations), the regulation of ICAAs entails appropriate and corresponding changes in amino acid kinetics. These changes vary according to the strength of the metabolic signal and the existing requirement to maintain the ICAA pool. For the patient with end-stage renal disease (ESRD), frequent dialysis induces an abrupt removal of half of the circulating amino acids, which in turn results in a substantial efflux of amino acids from skeletal muscle. ICAAs are maintained through the increase in protein breakdown, and similar to other stress states, there is a concomitant increase in protein synthesis. Thus, the regulation of ICAAs often pushes subsequent adaptations in amino acid kinetics to maintain the existing homeostasis. This regulatory mechanism is evident in circumstances ranging from increased amino acid availability in healthy volunteers to a change in anabolic signal in severe injury. Despite the substantial evidence of ICAA regulation, its physiologic significance is not evident. However, the regulation of ICAAs represents a method by which skeletal muscle ensures its capacity for anabolism.

Metabolic effects of orally administered amino acid mixture in elderly subjects with poorly controlled type 2 diabetes mellitus*1

The reduction of muscle mass and increased protein catabolism in aging can determine the occurrence of metabolic alterations—such as hyperglycemia and reduced insulin sensitivity—in elderly subjects with diabetes mellitus. Therefore, the aim of the study was to evaluate the effect of nutritional supplementation with oral amino acid mixture (OAAM) in elderly subjects with type 2 diabetes. This approach was conducted in an attempt to antagonize muscle catabolism by means of increased endogenous protein synthesis and to improve glucose metabolism and insulin sensitivity. A randomized, open-label, crossover study was conducted in poorly controlled (glycosylated hemoglobin level [HbA1c] >7%) elderly subjects (age range, 65 to 85 years) with type 2 diabetes. OAAM significantly reduced fasting and postprandial blood glucose and HbA1c, whereas all parameters remained substantially unchanged in the group treated with placebo. Fasting insulin levels and insulin resistance increased at baseline in all subjects with diabetes and decreased during OAAM supplementation. These results persisted also after crossover from OAAM to placebo. No changes in blood lipid levels, creatinine, homocysteine, and urinary albumin excretion rate were observed throughout the study, whereas a mild but significant increase of high-density lipoprotein cholesterol was found after OAAM supplementation. We suggest that increased amino acid availability for skeletal muscle function and strength could ameliorate metabolic control and insulin sensitivity in elderly patients with poorly controlled type 2 diabetes.