What stimulates protein synthesis

What stimulates protein synthesis DEFAULT

Nutrition and muscle protein synthesis: a descriptive review

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Stimulation of Muscle Protein Synthesis by Prolonged Parenteral Infusion of Leucine Is Dependent on Amino Acid Availability in Neonatal Pigs1,2

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Maximizing Post-exercise Anabolism: The Case for Relative Protein Intakes

Introduction

Lean body tissues, including skeletal muscle, are constantly being remodeled through the continuous and simultaneous processes of protein synthesis, and protein breakdown (collectively referred to as “turnover”). This constant turnover functions to breakdown old and/or damaged proteins and synthesize new proteins to help maintain protein mass and quality. Importantly, the algebraic difference between synthesis and breakdown determines net protein balance of a given tissue (e.g., muscle) and, ultimately, whether it is gaining or losing mass. To this end, resistance exercise increases muscle protein turnover for up to 48 h in the fasted state (1). Due to the greater stimulation of muscle protein synthesis compared to breakdown, muscle net balance is improved but, in the absence of exogenous amino acids, remains in a net negative balance (1, 2). It is only until a source of exogenous amino acids that net balance becomes positive due primarily the enhancement of muscle protein synthesis (3). Ultimately, the synergistic effects of resistance exercise and amino acid ingestion provides the requisite anabolic environment to support net tissue growth (i.e., muscle hypertrophy) characteristic of resistance training.

Notwithstanding the technical and logistical challenges associated with measuring rates of muscle protein breakdown in humans (especially in the postprandial state) (4), muscle protein synthesis is generally regarded as the prime-regulated variable in healthy humans in response to exercise and/or nutrition (5, 6). For example, the characteristic increase in muscle protein breakdown that occurs after resistance exercise in the fasted state is negated by the provision of exogenous amino acids, which subsequently supports greater rates of muscle protein synthesis, and an increased (and positive) net protein balance (3). The post-exercise increase in muscle protein synthesis that occurs with the ingestion of different dietary proteins (e.g., milk vs. soy) has also been shown to qualitatively predict training-induced increases in muscle hypertrophy and lean mass gains in young individuals (7–9). Importantly, measurement of the contractile myofibrillar protein subfraction, which is preferentially enhanced by resistance exercise and protein/amino acid ingestion (10–12), enhances the predictive ability of long-term (i.e., 24–48 h) rates of synthesis for muscle hypertrophy (13). Thus, identifying nutritional factors that may augment the exercise-induced increase in myofibrillar protein synthesis during this prolonged (i.e., >24 h) recovery period would ostensibly be an effective strategy to promote muscle hypertrophy. Therefore, the present review will focus on how dietary protein ingestion enhances post-exercise rates of muscle protein synthesis with a focus on the contractile myofibrillar protein fraction as a means to enhance recovery from, and adaptation to resistance exercise. The overall aim of this review will be to objectively determine the “optimal” relative bolus protein ingestion during the post-exercise recovery period as defined by one that maximizes myofibrillar protein synthesis while concomitantly minimizing estimated rates of amino acid oxidation. Potential biological (e.g., sex, age, body composition, active muscle mass), and nutritional (e.g., macronutrient co-ingestion, habitual protein intake, food matrix) confounders will be discussed to explore potential translational issues with recommending a per meal relative protein intake based on a preponderance of studies in young adults utilizing an isolated protein source (i.e., whey).

Regulation of Muscle Protein Synthesis After Exercise by Dietary Amino Acids

Since the first observations that skeletal muscle protein turnover is elevated in response to resistance exercise and that exogenous amino acids augment the increase in net protein balance of this tissue (2, 3), studies have investigated the nutritional factors that contribute to the optimal enhancement of post-exercise anabolism. This line of research has revealed that the most critical factor to enhance post-exercise muscle protein synthesis is the provision of dietary amino acids with the essential amino acids (EAA) primarily driving the response (14–17). A series of seminal studies from the Wolfe laboratory were the first to suggest a potential amino acid dose-response existed during recovery from resistance exercise in humans (15, 17, 18). These parallel studies demonstrated that lower EAA intakes (6–12 g) were associated with an apparent graded increase in muscle net balance (17, 18). When amino acid intakes were greater (i.e., 15 vs. 40 g EAA) there was a similar increase in post-exercise anabolism (15), suggestive of a potential ceiling effect. These seminal studies performed with crystalline amino acids provided the framework for future research into the nutritional regulation of post-exercise muscle protein synthesis. Importantly, as dietary amino acids are generally consumed as complete proteins, the next wave of muscle protein metabolism research investigated the ability of dietary protein to enhance post-exercise muscle remodeling.

Absolute Protein Intake to Maximize Post-exercise MPS

The first study to address the post-exercise ingested protein dose-response required healthy young resistance trained subjects with an average body mass of ~86 kg to perform a bout of heavy bilateral leg-based resistance exercise (i.e., leg press, knee extension, leg curl) before ingesting a variable amount of egg protein to enhance mixed muscle protein synthesis (19). Consistent with earlier results using crystalline amino acids (17, 18, 20), it was observed that even small amounts of protein (i.e., 5 and 10 g) were sufficient to enhance post-exercise mixed muscle protein synthesis (19). Importantly, mixed muscle protein synthesis was further enhanced by 20 g of protein but revealed an apparent plateau as a doubling of ingested protein to 40 g had no additive effect on the post-exercise protein synthetic response. These data ultimately conformed to a one-phase exponential decay relationship (Figure 1) that is characteristic of many allosterically regulated enzymes of the body, such as those within the mTOR pathway that control mRNA translation and muscle protein synthesis (21, 22), and is consistent with a ingested protein dose-response curve. It was subsequently demonstrated that the myofibrillar protein fraction displays a similar ingested protein dose-response relationship with 20 g of whey protein eliciting a maximal synthetic response (23). A unique feature of the study by Witard et al. (23) was that the post-exercise whey protein dose-response occurred ~4 h after participants consumed a high protein (~30% energy) breakfast, highlighting that the pre-exercise nutritional state (i.e., fasted vs. fed) does not appear to have a substantial impact on the post-exercise protein requirement to maximize muscle protein synthesis. This may be particular relevant for many athletes who have reported to consume ~5 daily meals and would therefore be in a postprandial state for the majority of their waking hours (24). Therefore, similar to rested skeletal muscle (23), 20 g of high quality dietary protein appears to be sufficient to support maximal post-exercise rates of muscle protein synthesis in average weight (i.e., 80–85 kg) young adult males.

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Figure 1. Percent-change from fasted (i.e., 0 g protein ingestion) of mixed muscle protein synthesis (A) and whole body leucine oxidation (B) after resistance exercise in response to graded intakes of egg white protein, as adapted from Moore et al. (19). Data conform to one phase-exponential decay and linear correlation, respectively (Graphpad Prism V.6). Hashed line represents 95% CI.

In contrast to the plateau observed with muscle protein synthesis, whole body leucine oxidation (a surrogate measure of protein oxidation) increases in a linear fashion with graded protein intakes (Figure 1) (19). This linear relationship may be related to the combination of a relatively low Km for the rate-controlling enzyme for leucine oxidation (i.e., branched-chain ketoacid dehydrogenase) (25), and a greater overall substrate supply (i.e., leucine, valine, and isoleucine) with higher protein intakes. Importantly, this increase in leucine oxidation in conjunction with a concomitant increase in urea synthesis (23) highlights that dietary amino acids provided at levels that are in excess of their ability to be incorporated into new (muscle) proteins results in their deamination and, in the case of the branched chain amino acids, irreversible oxidation (Figure 1). This pattern of dietary amino acid oxidation is arguably an inefficient use of ingested protein if the specific goal is to maximize post-exercise muscle protein synthesis and anabolism. In fact, the marked increase in whole body leucine oxidation concomitant with a sustained elevation in blood amino acid concentration (19) is consistent with a metabolic pattern that has been suggested to be characteristic of an upper limit of intake for this macronutrient (26). Therefore, given the ability to induce a plateau in muscle protein synthesis yet minimize amino acid oxidation and urea synthesis (19, 23), 20 g of high quality protein (e.g., egg or whey protein) arguably represents an “optimal” or absolute protein intake to efficiently enhance muscle remodeling after resistance exercise in young adults.

Relative Protein Intake to Maximize Myofibrillar Protein Synthesis

Based on previous studies that provided absolute protein intakes, the ingestion of 20 g of protein that was shown to maximize both mixed muscle and myofibrillar protein synthesis yet minimize whole body leucine oxidation, and ureagenesis in ~85 kg males translates into a relative protein intake of ~0.24 g protein/kg body weight. However, the ability to extrapolate these relative intakes into an “optimal” one-size-fits-all recommendation is arguably limited by the small sample size (i.e. n = 54) of “average” body mass individuals. In addition, the qualitative (albeit not statistically significant) ~10% increase in muscle protein synthetic rates between 20 and 40 g of protein could be interpreted as reflecting the “true” maximal intake as being within these two doses. Therefore, logical questions such as “would intakes greater than 20 g of protein further enhance muscle protein synthesis?” and “would 20 g of protein be the target intake for both 55 and 120 kg athletes?” naturally flow from these acute, absolute protein intake studies. In addition, recommendation of absolute meal protein intakes is at odds with daily recommendations for this macronutrient, which are almost universally prescribed relative to body mass.

To address these types of generalizability concerns, an unsystematic review was performed in Pubmed from its inception to July 1, 2019 consisting of keywords related to this review topic such as “whey,” “myofibrillar protein synthesis,” and “exercise.” As maximizing post-exercise myofibrillar protein synthesis would be essential for those interested in enhancing muscle growth and potentially strength with training, studies investigating the synthesis of this muscle fraction were selected to increase homogeneity as well as reflect the greater contractile and nutrient sensitivity of this protein fraction (12). Moreover, studies that utilized a bolus protein feeding of whey protein after exercise and measured the synthesis of the myofibrillar protein fraction by traditional primed-constant stable isotope infusion during the subsequent 3–5 h postprandial period were included. Given that the preponderance of studies fitting these criteria have been performed in young adults, only this age group (i.e., <35 y) was included in the final dose-response analysis to minimize any confounding effects of age (see below for additional discussion). Finally, given the variability in fractional synthetic rates across different stable isotopes and precursor pools (27), post-exercise myofibrillar protein synthetic rates were expressed as a change from reported (when available) or estimated basal rates to better compare across studies. Details of the studies utilized for the subsequent analysis are presented in Table 1. Only articles in English were assessed with reference lists cross-checked for any additional relevant articles.

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Table 1. Overview of studies investigating the post-exercise stimulation of myofibrillar protein synthesis with bolus whey protein ingestion.

By utilizing a step-wise modeling comparison similar to our previous study at rest (39), it was observed that the increase in post-exercise myofibrillar protein synthesis in young adults with protein ingestion displayed a bi-phase linear response that is consistent with the previous observation of a dose-response relationship (Figure 2). Breakpoint analysis revealed that the bi-phase linear response plateaued at ~0.31 g protein/kg body weight (i.e., estimated average requirement), which when accounting for a typical ~25% individual response variance in young adults (39) that would not be reflected in mean study responses could result in a safe intake of ~0.39 g/kg as an upper limit. This protein intake of ~0.31 g/kg is slightly higher than the relative intake calculated from the estimated plateau in protein synthesis and average group body weight previously determined in the mixed [~0.23 g/kg; (19)], and myofibrillar [~0.24 g/kg; (23)] protein fractions after the ingestion of 20 g of protein. This could explain in part the ~10% non-significant increases in protein synthesis from the 20 to 40 g doses (19, 23), which could suggest that the 20 g dose was not sufficient to maximize protein synthesis in all subjects whereas 40 g was clearly surfeit. In fact, the apparent lack of a true plateau in previous dose-response studies had led some to suggest that the protein intake to maximize muscle protein synthesis were within this range (i.e., >20 g) and that the upper level (i.e., 40 g) was necessary to obtain a maximal anabolic response (41). However, in contrast to the suggestion that 0.4–0.5 g protein/kg lean body mass (~0.34–0.43 g protein/kg body mass, assuming an average 15% body fat) should be ingested both before and after exercise (41), the data presented herein would suggest that only a moderately higher level of protein [i.e., ~0.31 vs. ~0.24 g/kg; (23)] should be ingested to reach a plateau in post-exercise myofibrillar protein synthesis.

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Figure 2. Increase in post-exercise myofibrillar protein synthesis above control relative to ingested protein normalized to total body weight (for study details, see Table 1). Bi-phase linear regression was performed with the slope of the second line segment constrained to zero and the average protein intake to maximize myofibrillar protein synthesis determined by breakpoint analysis (indicated by hashed arrow; 0.31 ± 0.08 g protein/kg body weight; mean ± SE; N = 23 protein intakes; analysis performed by Graphpad Prism 6.0). Applying a typical ~25% variance when analyzing individual myofibrillar protein synthetic rates (39) as compared to a collapsed mean study response, a safe intake could represent ~0.38 g/kg. There was a strong trend for a bi-phasic linear regression model to explain a greater proportion of variance vs. a simple linear regression model (r2 = 0.27 vs. 0.129, respectively; P = 0.056), suggesting the data conformed to a saturable dose-response relationship. First line segment described by: y = 254x + 63. Estimated maximal increase in myofibrillar protein synthesis above control is ~142% (as determined from equation above at 0.31 g protein/kg).

Would Sex Affect the Relative Protein Requirement?

Currently, research that evaluates the nutritional factors that enhance muscle protein synthesis after resistance exercise is primarily performed in young males with that of female athletes being unfortunately under-represented. For example, the only studies to evaluate the ingested protein dose-response, either at rest or after resistance exercise, have performed these investigations in males only (19, 23, 39, 42). The reason(s) for the unfortunate disparity in sex-based research is unclear but may include, in part, the potential influence of menstrual phase on protein kinetics, which has been reported to alter the basic requirements for some EAA (e.g., lysine) at rest (43) as well as influence whole body protein metabolism during endurance exercise (44). However, the stimulation of myofibrillar protein synthesis after resistance exercise is uninfluenced by the menstrual phase (45). Moreover, both the rested (46, 47), and the exercise-induced stimulation (48) of muscle protein synthesis are similar between young men and women in the fasted state, suggesting sex per se has little influence on the regulation of muscle protein remodeling in the absence of any nutritional manipulation.

With respect to the nutrient sensitivity of muscle protein synthesis, seminal work that investigated the nutritional factors that enhance post-exercise muscle anabolism reported no differences between males and females in their mixed study populations (15–18); this could suggest there are no overt differences in post-exercise nutrient sensitivity of muscle protein metabolism between sexes. It has also been demonstrated that the stimulation of myofibrillar protein synthesis with resistance exercise and a 25-g bolus of dietary protein ingestion is similar between young men and women (38). This study (38) provided an absolute amount of protein (25 g) to all participants that would likely translate into a saturating dose for both the men (~0.32 g/kg) and, especially, women (~37 g/kg), which makes it difficult to determine if potential sex differences exist at lower protein intakes. Nevertheless, the ability of whey protein to enhance post-exercise rates of myofibrillar protein synthesis during energy restriction is essentially identical between females and males when normalized to fat free mass (FFM) over a range of intakes (i.e., 0–0.8 g/kg FFM) (49). Therefore, despite a relative dearth of research studying the nutritional requirements of females after resistance exercise, it is difficult to envision, based on the current literature, a scenario in which acute protein requirements would be markedly disparate between the sexes.

Carbohydrate Co-ingestion

Carbohydrate ingestion during the recovery from resistance exercise is important for glycogen resynthesis (50, 51) and can contribute to the daily positive energy balance that is a general requisite to support muscle mass growth with training. Aside from providing additional energy during post-exercise recovery, it was first demonstrated that the co-ingestion of carbohydrate with crystalline amino acids improved post-exercise muscle net balance to a greater degree than amino acids alone (18). Subsequent studies revealed that this greater net anabolism was due primarily to an insulin-induced suppression of muscle protein breakdown rather than an augmentation of muscle protein synthesis (52, 53). In fact, as little as ~30 g of carbohydrate (and the associated insulin response) is sufficient to suppress post-exercise muscle protein catabolism (52). Provided dietary protein is provided at a level that would optimize muscle protein synthesis (i.e., ≥20 g), carbohydrate co-ingestion from 30–270 g has no additive effect on post-exercise muscle protein synthetic rates (52, 54, 55). Therefore, although it is unclear if carbohydrate co-ingestion may improve the synthetic effect of smaller (i.e., <20 g or <0.31 g/kg) amounts of dietary protein, it is clear that optimal protein ingestion is of paramount importance to maximize muscle protein synthesis after resistance exercise with mixed protein-carbohydrate beverages.

Does the Amount of Active Muscle Mass Influence Post-exercise Protein Requirements?

It is customary for individuals engaged in resistance training for the goal of gaining muscle mass to perform whole body resistance exercise, which is in contrast to many acute studies aimed at understanding the local (i.e., muscle-specific) nutrient requirements to maximize muscle protein synthesis. This led MacNaughton et al. (31) to design an elegant study whereby groups of participants with markedly different body compositions were provided with moderate (20 g) and higher (40 g) doses of protein after a strenuous bout of whole body resistance exercise. The authors hypothesized that total lean body mass (LBM), and thus active lean (i.e., muscle) mass, would modify the acute requirement for dietary protein to maximize muscle protein synthesis during recovery. In contrast to their hypothesis and arguably the most compelling case against any impact of active muscle mass on acute protein requirements was the observation that participants with ~20 kg difference in LBM (i.e., ~59 vs. 79 kg LBM) had identical rates of myofibrillar protein synthesis after consumption of a moderate 20 g dose of whey protein. This finding is not without precedence as it has been shown previously that performing an intense bout of lower body resistance exercise (i.e., leg press, knee extension, leg curl), which would increase total body active muscle mass, does not impact blood flow during recovery to the arm nor post-exercise rates of myofibrillar protein synthesis with a moderate 25 g protein dose in the small biceps brachii (36, 56). Macnaughton et al. argued that the lower rates of myofibrillar protein synthesis in their whole body exercise protocol relative to a previous study utilizing unilateral leg resistance exercise (23) concomitant with statistically greater rates of synthesis with the larger (i.e., 40 g) dose were nevertheless indicative of greater post-exercise protein requirement with a greater active muscle mass (23). However, the study and cohort differences in myofibrillar synthesis rates are within the general inter-study variability (i.e., ±25%) for tracer-derived rates of human muscle protein synthesis (27). Arguably the most plausible reason for the greater myofibrillar synthetic rates with 40 as compare to 20 g of protein would be a greater statistical power to detect the relatively small ~20% difference between conditions, which the authors allude to in their discussion (31). For example, post hoc power analysis of previous absolute protein dose-response studies (19, 23) suggest that ~35 participants would be required to achieve statistical significance for the ~10% greater muscle protein synthetic rates with 40 g as compared to 20 g protein ingestion. This is markedly similar to the results in MacNaughton et al. (31) given that statistical significance between 20 and 40 g of protein was only achieved when the low and high LBM cohorts were collapsed (i.e., n = 30 total).

In order to more objectively estimate the impact of the amount of active muscle mass on post-exercise protein requirements, the increase in myofibrillar protein synthesis was compared to the amount of dietary protein ingested relative to the estimated active muscle mass (Table 1; Figure 3). If one were to expect the amount of active muscle mass influenced the ability of dietary protein to stimulate post-exercise muscle remodeling, then it would be likely that a greater protein intake per active muscle mass would also result in a greater increase in myofibrillar protein synthesis. Despite a greater than ~10-fold difference in relative protein intakes there was no observable relationship with the stimulation of myofibrillar protein synthesis, which suggests active muscle mass has little bearing on post-exercise protein requirement. The observation that the stimulation of muscle protein synthesis is apparently unrelated to the amount of protein ingested per unit of active muscle is not surprising given that resistance exercise is inherently anabolic and has been shown to improve intracellular amino acid recycling (1, 2). This enhanced intracellular amino acid reutilization would ultimately lessen the requirement for exogenous amino acids to support the exercise-induced stimulation of muscle protein synthesis, although protein/amino acid ingestion is still required to induce a net positive muscle protein balance. Therefore, presently available data suggest that the amount of active muscle mass has little bearing on the ability of or requirement for post-exercise protein ingestion to enhance muscle protein remodeling.

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Figure 3. Increase in myofibrillar protein synthesis above control after resistance exercise compared to ingested protein normalized to the estimated active muscle mass (for details, see Table 1). Data were analyzed using a linear correlation (Graphpad Prism V6). Non-significant slope defined by: y = 3.91x + 116 (r = 0.30; P = 0.18; N = 21 as only conditions with protein ingestion were included).

What About Maximizing Whole Body Anabolism?

During the post-exercise recovery period muscle protein synthesis is maximized with the ingestion ~0.31 g/kg of protein whereas muscle protein breakdown has been demonstrated to be maximally suppressed with a moderate insulin response (e.g., from ~30 g of carbohydrate) (52). Collectively, this provides compelling evidence that muscle protein net balance is saturable and primarily dictated by the nutritional enhancement of rates of muscle protein synthesis, as highlighted previously (6). In contrast, it has recently been suggested that there is no practical maximal anabolic response to dietary protein at the whole body level given the hypothesized role of an inexhaustible ability to suppress protein breakdown at high protein intakes (57, 58). For example, ingesting 70 g (~0.82 g/kg) as compared to 40 g (~0.48 g/kg) of dietary protein has been shown to enhance whole body net protein balance, in the absence of any further increase in rested or post-exercise rates of mixed muscle protein synthesis, through a proportional reduction in estimates of whole body protein breakdown (59). Based on these findings as well as those from older adults (60), the authors recently collapsed their data across ages and reanalyzed using a linear model to support their suggestion of there being no practical limit (58). This appeared to confirm their previous hypothesis on this topic (57) and potentially influenced previous suggestions of a target meal protein intake for resistance-trained athletes of ~0.4–0.55 g/kg (61). However, we recently demonstrated that whole body net balance plateaus with dietary protein ingestion after resistance exercise in females (62) and variable intensity stop-and-go exercise in both sexes (63) despite a linear increase in estimates of amino acid deamination (i.e., increased urinary urea:creatinine ratio), and presumably oxidation. The apparent discrepancy may be related in part to the choice of statistical model in our research (62, 63) as compared to others (58) (i.e., biphase vs. linear regression, respectively). In potential support, extraction and reanalysis of whole body net protein balance data from just their young adults relative to body weight-normalized protein ingestion from Kim et al. (58) revealed that the data is better fit by a segmental bi-phase linear regression as compared to standard linear model (i.e., r2 = 0.62 vs. 0.53, respectively; P < 0.05; Graphpad Prism V6). This analysis revealed a breakpoint in whole body net balance at ~0.71 g/kg, which is slightly greater than our recent estimates of ~0.5–0.6 g/kg (62, 63), and suggests that the capacity to assimilate dietary protein at the whole body level is substantially greater than at the muscle. While it has been suggested that these amino acids sequestered at the whole body level (e.g., within splanchnic tissues and/or circulating proteins) may be made available for muscle protein synthesis during the post-absorptive period (58), this possibility has yet to be empirically demonstrated. Some may also view these relatively higher per meal protein estimates as being unrealistic, although many Western populations with a skewed daily protein distribution routinely consume on average ~0.55 g/kg in their evening meal (64). Therefore, in contrast to prior suggested meal protein intakes of up to ~0.5 g/kg that are based on the supposition of no maximal whole body anabolism (61), it is argued that a more prudent “muscle-centric” target that maximizes muscle protein synthesis yet minimizes excess amino acid oxidative losses would place a more efficient intake at no more than ~0.39 g/kg.

Potential Caveats to Acute Relative Protein Requirements

The reanalysis of the relative protein intake to maximize post-exercise myofibrillar protein synthesis performed herein incorporates studies performed in healthy young individuals consuming a single, high-quality protein source (i.e., whey). While this approach increases homogeneity and allows for greater ease of comparison between studies, the results could be viewed as representing relative protein requirements under “ideal” conditions, notwithstanding the increased appreciation for the anabolic potential of whole foods (discussed in more detail below) (65–67). The following sections will briefly discuss conditions under which relative protein intakes may not be transferable and/or require further study.

Exercise Modality

Dietary protein is important for the remodeling of skeletal muscle after not only resistance exercise but also after high-intensity sprint exercise (68), steady-state endurance exercise (69), and combinations thereof (i.e., concurrent training) (70, 71). Unlike resistance exercise, which provides a predominantly muscle-specific stimulus (72), endurance exercise can increase whole body oxidative disposal of amino acids that must ultimately be replaced via dietary sources (73). This may contribute to the increased protein requirements of endurance athletes (74, 75). Studies from the same laboratory utilizing identical tracer methodology have demonstrated that the ingestion of 0 g (~0.057 vs. ~0.051%/h, respectively), and 20 g (~0.087 vs. ~0.070%/h, respectively) of whey protein elicit broadly similar rates of myofibrillar protein synthesis after 90 min of endurance exercise (~77% maximal aerobic capacity), and traditional resistance exercise (23, 69), which could be interpreted as reflecting a similar post-exercise protein requirement after these dichotomous exercise stimuli. However, it has recently been demonstrated in a group design that post-exercise rates of myofibrillar protein synthesis were ~16% greater after the ingestion of 20 g (~0.27 g/kg) of milk protein (whey, casein, and milk protein concentrate) compared to a protein-free control after an acute bout of concurrent exercise (71), which is slightly lower than the reported ~32% difference in myofibrillar protein synthetic rates between 25 g of whey (~0.32 g/kg) protein and a protein-free placebo after concurrent exercise in a crossover study (70). Although the relative differences in myofibrillar protein synthetic rates between 0 g protein and a moderate relative intake (i.e., ~0.26–0.32 g/kg) in these concurrent exercise studies seem muted compared to the present post-resistance exercise analysis (i.e., ~16–32 vs. ~79%), the estimated increase from basal may be moderately more comparable (i.e., ~78–147 vs. ~152%; Figure 3). Therefore, while the consumption of ~ 0.31 g/kg of protein would enhance myofibrillar remodeling after all forms of exercise, additional research may be warranted to confirm that this represents a saturable dose and/or is sufficient to fully replace any endurance exercise-induced oxidative amino acid losses. This is notwithstanding the other potential benefits of increased protein ingestion in endurance athletes during periods of intensified training that may be dissociated from myofibrillar remodeling, such as enhanced immune function and/or exercise performance (76, 77).

Population Age

Both young and old adults are capable of mounting an enhanced muscle protein synthetic response after resistance exercise in the fasted state (78, 79), which is consistent with the ability to increase muscle mass with this type of training across the lifespan (80). However, it has been observed that the combined effects of resistance exercise and amino acid ingestion on the enhancement of muscle protein synthesis may be delayed (81), and/or blunted in older adults (82, 83), suggesting nutrient sensitivities may be compromised with advancing age. In potential support, it has been shown that the ingestion of 40 g (~0.49 g/kg) of whey protein enhanced rates of post-exercise myofibrillar protein synthesis over and above that observed with 20 g (~0.25 g/kg) in older (~70 y) adults (82). However, the relative dose may not be substantially greater than younger adults as 30 g (~0.37 g/kg) of milk protein concentrate was recently demonstrated to enhance post-exercise myofibrillar protein synthetic rates in healthy older adults with no further benefit at 45 g (~0.56 g/kg) (84). Given that the anabolic potential of exercise and/or nutrition may be intimately tied to the “biological” age of a muscle as dictated by its habitual activity (85, 86), additional research is needed to confirm whether greater relative intakes are required to maximize post-exercise anabolism in older age and, if so, what lifestyle and/or biological factors may need to be considered (e.g., daily step count, presence/absence of sub-clinical chronic inflammation, excess body fat, etc.).

Protein Type

The studies examining the post-exercise ingested protein dose-response utilized high quality (i.e., enriched in EAA), rapidly digested protein sources (i.e., egg and whey) (19, 23). Moreover, the estimates for the relative protein requirements derived herein were obtained with studies utilizing whey protein, which due to its rapid digestion (37), and/or greater leucine content (87, 88) elicits an early (i.e., within 3 h) and robust post-exercise stimulation of muscle protein synthesis. In contrast, proteins that contain lower quantities of the branched-chain amino acids (e.g., plant-based, caseinate), and/or are slowly digested (e.g., micellar casein) generally result in a suboptimal muscle protein synthetic response compared to an equal amount of whey protein (88), although recent research with dairy proteins may not support this “rapid rate of leucinemia” requirement for post-exercise myofibrillar remodeling (89). Nevertheless, studies have suggested that proteins with suboptimal essential amino acid and/or leucine content may ultimately be compensated for by ingesting a greater absolute protein amount. For example, it has been reported that the post-exercise stimulation of mixed muscle protein synthesis over 5 h (90), and myofibrillar protein synthesis over 3–5 h (34) of recovery is similar with the ingestion of ~20 g of a mixed protein (i.e., whey, casein, soy blend) and ~17 g of whey. Therefore, the optimal intake of proteins that may be relatively deficient in EAA and/or leucine and/or slowly digested may need to be addressed in future studies. Alternatively, individuals who prefer to ingest lower quality proteins (insofar as the stimulation of muscle protein synthesis is concerned) may consider consuming intakes at the upper “safe” intake of ~0.39 g/kg.

Food Matrix

Early studies investigating the nutritional regulation of muscle protein synthesis have primarily provided dietary protein in beverage form. However, recent focus has been placed on the importance of studying whole foods (e.g., egg, beef) given these are typically nutrient-dense and arguably more representative of “normal” habitual dietary patterns (66, 67). Inasmuch as the peak and/or the rate of change in blood amino acid concentration regulates post-exercise muscle protein synthesis (37), the typically delayed digestion and absorption of solid foods may result in an attenuated muscle protein synthetic response (91). In this event, it is unclear if consuming a greater protein intake to account for any attenuated hyperaminoacidemia from solid food ingestion may be required to maximize post-exercise muscle protein synthesis. However, digestion rate may not be the only (or even primary) variable that influences the anabolic potential of whole food as minced beef has been demonstrated to induce a more rapid postprandial aminoacademia than skim milk but a lower early (i.e., <2 h), and potentially cumulative (i.e., 0–5 h) post-exercise myofibrillar protein synthetic response (92). Other studies have also demonstrated whole milk as more anabolic than skim milk (93) and skim milk more anabolic than soy juice (8) during post-exercise recovery. Finally, we recently demonstrated that whole egg supports a greater post-exercise myofibrillar protein synthetic response than an isonitrogenous quantity of egg white protein, which was supported by a greater lysosomal targeting of the mechanistic target of rapamycin (mTOR) as the potential underlying physiological mechanism (94, 95). This could suggest there may be circumstances whereby whole, nutrient-dense foods may require a lower relative intake to maximize post-exercise anabolism than other isolated protein sources. Although additional research is warranted to define the anabolic potential of whole food and its associated dose-response relationship to post-exercise anabolism, a target of ~0.31 g/kg protein could arguably represent a reasonable starting point for individuals aiming to enhance myofibrillar protein synthetic rates in the interim.

Habitual Protein Intake

Although it is generally accepted that daily protein requirements are elevated in strength athletes (96), habitual intakes of populations engaged in chronic resistance training generally far exceed most recommendations (i.e., >2 g/kg/d) (97). Habitually high protein diets increase the capacity for protein catabolism and amino acid oxidation as a means to manage this excess macronutrient load (98). From an acute feeding standpoint, rodent models have demonstrated that adaptation to a high protein intake is accompanied by a greater splanchnic extraction of dietary nitrogen, which results in an attenuated post-prandial delivery to and deposition of dietary nitrogen in peripheral tissues (99). In this way, the gut may act as a buffer to ensure amino acid delivery to peripheral tissues (including muscle) is relatively constant regardless of habitual dietary protein intake. This has some support in humans as there is reduced dietary amino acid availability after consumption of 25 g of milk protein when adapted to a moderate (1.5 g/kg/d) as compared to low (0.7 g/kg/d) protein diet (100), suggesting a potentially greater splanchnic amino acid sequestration. Although rested postprandial rates of myofibrillar protein synthesis were non-statistically attenuated by ~50% in the moderate compared to the low protein group in the study by Gorissen et al. (100), Pasiakos et al. (101) demonstrated that the postprandial stimulation of mixed muscle protein synthesis by 20 g of protein was attenuated when consuming 1.6 vs. 0.8 g/kg/d and was not enhanced with a 2.4 g/kg/d controlled diet. Collectively these data could suggest that individuals habituated to lower protein diet approximating the recommended dietary allowance (RDA; 0.83 g/kg/d) may be able to support maximal rates of muscle protein synthesis after exercise with intakes lower than ~0.31 g/kg. In contrast, those adapted to higher habitual intakes, as is common in many strength athletes, may require a greater relative intake to account for an attenuated peripheral dietary amino acid appearance and/or enhanced amino acid oxidative capacity. However, the threshold at which this greater acute requirement may manifest could be relatively high (e.g., ~3x the RDA) given that previous post-exercise dose-response studies recruited participants with relatively high self-reported habitual intakes (i.e., 1.4–2.3 g/kg/d) yet still demonstrated approximate plateaus in muscle protein synthetic rates with 20 g (~0.24 g/kg) protein ingestion (19, 23).

Negative Energy Balance

Muscle protein synthesis is an energetically expensive process and is down-regulated during periods of cellular energy stress, such as during a diet-induced negative energy balance (49, 102). The post-exercise stimulation of myofibrillar protein synthesis with dietary protein ingestion is not affected by low levels of muscle glycogen (103), highlighting that acute energy restriction does not constrain post-exercise muscle remodeling with exogenous amino acid ingestion. In contrast, more chronic periods of negative energy balance (i.e., 5–10 d) suppress resting mixed and myofibrillar protein synthesis (49, 102, 104). In addition, after a 5-day moderate protein (i.e., 1.4 g/kg/d) low energy (30 kcal/kg fat-free mass/d) diet, post-exercise myofibrillar protein synthesis is increased in a linear dose-dependent fashion with 15 and 30 g of dietary protein (49). Although the maximal absolute protein intake was lower than previous dose-response studies during energy balance (i.e., 30 vs. 40 g) (19, 23), there was no apparent plateau in post-exercise myofibrillar protein synthesis within the range of relative protein intakes studied (i.e., up to ~0.5 g/kg body weight) (49). Additionally, the estimated maximal myofibrillar protein synthesis with 30 g protein ingestion (determined by the group mean response) was ~82% above the rested fasted rate during energy deficit (49), which is less than the estimated plateau of ~142% during energy balance in the present review and could suggest a saturable protein intake was not provided during this negative energy balance. While it is possible that maximal rates of myofibrillar protein synthesis may generally be constrained during chronic diet-induced negative energy balance, the lack of a plateau and the relatively modest increase in myofibrillar protein synthesis with 30 g of protein could also suggest that the protein intake required to maximize post-exercise myofibrillar protein synthesis is slightly greater during a period of energy restriction. This would generally be in line with the observations that high daily dietary protein intakes (i.e., at least ~2 times the RDA) are required to maintain lean body mass and muscle protein synthesis during a negative energy diet with (104, 105) or without resistance exercise (101). Additional benefits for higher protein intakes during negative energy balance could be increased satiety and post-prandial thermogenesis (106), both of which would help support weight loss goals. Therefore, although it has been suggested that 0.25–0.3 g protein/kg body weight should be targeted after exercise in athletes aiming to maintain lean body mass during weight loss (107), the ~0.31 g protein/kg body mass determined herein could be viewed as a minimum intake with a safe intake closer to ~0.4 g/kg for individuals consuming a sub-optimal energy intake.

Obesity

Beyond traditional derangements in glucose metabolism, it is becoming appreciated that excess body fat may also be an independent factor contributing to the dysregulation of muscle protein synthesis in obese populations (108). For example, obesity has been associated with a blunted myofibrillar protein synthetic response to dietary protein ingestion (i.e., 36 g or ~0.35 g/kg) (109), and resistance exercise (110). In addition, this anabolic resistance, which is not reported in relatively active obese individuals (i.e., ~7,400 steps/day) (111), may be exacerbated by inactivity (112), which suggests this anabolic resistance of obesity, similar to older adults (85, 86), has a strong lifestyle component to its manifestation and severity. Thus, inasmuch as this anabolic resistance extends to the post-exercise sensitivity to dietary amino acids, it could be argued that obese individuals may require a greater relative protein intake than their lean counterparts when normalized to the metabolically active lean body mass. However, studies used in the present analysis that yielded a relative protein intake of ~0.31 g/kg included participants of average body fat (~15%). Therefore, providing recommendations relative to total body mass would result in a greater dose per kg lean body mass in obese individuals (i.e., ~0.34 vs. ~0.41 g/kg lean body mass, respectively, assuming 30% body fat), which subsequently may be sufficient to overcome any obesity-related anabolic resistance.

Practical Application of Acute Relative Protein Intakes

A single bout of resistance exercise can increase muscle protein synthesis for up to 24–48 h with the duration for which it is elevated influenced by training history of the athlete (13, 113) and the specific exercise stimulus (11), which ultimately factor into the general inability of single acute (i.e., <6 h) “snapshots” of myofibrillar protein synthesis to predict training-induced muscle hypertrophy (114). However, individuals who are able to support greater rates of myofibrillar protein synthesis over this 24–48 h post-exercise recovery period have been shown to experience greater training-induced gains in muscle hypertrophy (13). Given that individuals who engage in resistance training for the goal of enhancing muscle mass and/or muscle strength typically train 3–5 times per week (115), athletes are generally in some state of post-exercise recovery. Dietary protein consumed at any point during this prolonged 24–48 h recovery period would ultimately contribute to the remodeling of skeletal muscle. Outside of the response after a single meal, the pattern and distribution of dietary protein ingestion has been shown to influence muscle protein synthesis over 12–24 h both at rest (116) and after resistance exercise (28, 117, 118). For example, the repeated ingestion of 20 g of whey protein (~0.25 g/kg) at 3 h intervals has been shown to support the greatest rates of myofibrillar protein synthesis and whole body net protein balance over the 12 h after an acute bout of resistance exercise (28, 117). This has led to the suggesting that 4–5 meal occasions, which is the typical feeding frequency already adopted by many elite athletes (24), would be the most favorable and metabolically efficient means to consume one's daily protein intake if the goal is to maximize skeletal muscle remodeling while simultaneously minimizing irreversible amino acid oxidative catabolism (28, 117). Therefore, if one were to take a “muscle-centric” view for the daily protein requirement then the optimal amount and pattern of protein intake would translate into ~1.24–1.55 g/kg/d for a resistance-trained individual aiming to maximize skeletal muscle remodeling and/or net protein accretion. Even if one were to apply a conservative ~20% correction-factor (i.e., ~0.37 g/kg) to account for less anabolic proteins [e.g., plant-based; (88)], then this pattern of protein intake would provide ~1.48–1.85 g/kg/d. Both of these estimates are within the range of intakes suggested to maximize lean mass growth with training (119) and are in line with current sports science consensus recommendations for daily protein intake (96).

Conclusion

The present review puts forth the argument that protein recommendations should be normalized to the body weight of an individual for a greater ease of translation of the dose that maximizes muscle protein synthesis and minimizes amino acid oxidation during the recovery from resistance exercise. Based on re-analysis of previously published literature, an intake of ~0.31 g/kg of high quality protein represents a suitable target to maximize myofibrillar protein synthesis during recovery from resistance exercise, regardless of sex, and quantity of active muscle mass. Though additional research is warranted to confirm whether acute protein requirements to maximize post-exercise rates of muscle protein synthesis are influenced by age, chronic energy status, and/or food matrix, a moderate intake of ~0.31 g/kg of high quality protein represents a good approximation for individuals of all body sizes aiming to efficiently enhance the repair, remodeling, and net synthesis of skeletal muscle tissue after resistance exercise.

Author Contributions

DM wrote and approved the final version the manuscript.

Conflict of Interest Statement

The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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Sours: https://www.frontiersin.org/articles/10.3389/fnut.2019.00147/full
Protein Synthesis and Muscle Growth, Meal Timing Matters

Abstract

Studies in vitro as well as in vivo in rodents have suggested that amino acids (AA) not only serve as substrates for protein synthesis, but also as nutrient signals to enhance mRNA translation and protein synthesis in skeletal muscle. However, the physiological relevance of these findings to normal humans is uncertain. To examine whether AA regulate the protein synthetic apparatus in human skeletal muscle, we infused an AA mixture (10% Travesol) systemically into 10 young healthy male volunteers for 6 h. Forearm muscle protein synthesis and degradation (phenylalanine tracer method) and the phosphorylation of protein kinase B (or Akt), eukaryotic initiation factor 4E-binding protein 1, and ribosomal protein S6 kinase (p70S6K) in vastus lateralis muscle were measured before and after AA infusion. We also examined whether AA affect urinary nitrogen excretion and whole body protein turnover.

Postabsorptively all subjects had negative forearm phenylalanine balances. AA infusion significantly improved the net phenylalanine balance at both 3 h (P < 0.002) and 6 h (P < 0.02). This improvement in phenylalanine balance was solely from increased protein synthesis (P = 0.02 at 3 h and P < 0.003 at 6 h), as protein degradation was not changed. AA also significantly decreased whole body phenylalanine flux (P < 0.004). AA did not activate Akt phosphorylation at Ser473, but significantly increased the phosphorylation of both eukaryotic initiation factor 4E-binding protein 1 (P < 0.04) and p70S6K (P < 0.001). We conclude that AA act directly as nutrient signals to stimulate protein synthesis through Akt-independent activation of the protein synthetic apparatus in human skeletal muscle.

SKELETAL MUSCLE PROTEIN synthesis is closely regulated in vivo, and the phosphatidylinositol 3-kinase (PI3-knase)/mammalian target of rapamycin (mTOR) pathway has been implicated as having a pivotal role in this process. Within this pathway, protein kinase B (or Akt) (1), eukaryotic initiation factor 4E (eIF4E)-binding protein 1 (4E-BP1) (2), and ribosomal protein S6 kinase (p70S6K) (3) are several key intermediates involved in the regulation of translation initiation and protein synthesis (4, 5). Activation of Akt, a Thr/Ser kinase downstream of PI3-kinase, promotes the phosphorylation and activation of mTOR (1, 6–8), which, in turn, phosphorylates 4E-BP1 and p70S6K. Dephosphoryl-ated 4E-BP1 represses mRNA translation initiation by binding to eIF4E. Phosphorylation of 4E-BP1 frees eIF4E, which can then associate with eIF4G to form the preinitiation complex and initiate protein synthesis. Phosphorylation of p70S6K increases the phosphorylation of ribosomal protein S6 (9) and facilitates the synthesis of some ribosomal proteins, initiation factors, and elongation factors that play important roles in protein synthesis (5, 10).

Amino acids (AA) have been shown to stimulate skeletal muscle protein synthesis in humans (11, 12). Recent evidence suggests that AA not only function as substrates for protein synthesis, but they also provide nutritional signals to activate translation initiation and protein synthesis (13–15). Many studies have demonstrated that AA, especially branched chain AA (BCAA) stimulate the phosphorylation of 4E-BP1 and p70S6K in various cell preparations and animal studies (15–28). It appears that AA signal to 4E-BP1 and p70S6K via mTOR activation, since the AA-stimulated translation initiation is abrogated by rapamycin, a specific inhibitor of mTOR (15–18, 29).

Most studies examining AA- or BCAA-stimulated translation initiation and protein synthesis, whether conducted in vivo in animals or in vitro in cells, used high concentrations of AA or BCAA, leaving uncertain the physiological significance of the above findings in human skeletal muscle. We previously reported that physiological concentrations of BCAA stimulate 4E-BP1 and p70S6K phosphorylation without increasing protein synthesis in human skeletal muscles (30). The major purpose of the current study was to examine whether mixed AA containing all essential AA at physiological concentrations regulate muscle protein synthesis and degradation and whether AA provide nutrient signals to regulate translation initiation in human skeletal muscle in vivo. We quantitated the effect of mixed AA on protein synthesis and degradation using the forearm phenylalanine tracer kinetic method (31) and their effects on Akt, 4E-BP1, and p70S6K phosphorylation in biopsied vastus lateralis muscle samples. The results showed that AA significantly stimulated forearm protein synthesis and improved forearm protein net balance, without affecting muscle protein degradation. The phosphorylation of both 4E-BP1 and p70S6K, but not of Akt, was increased significantly after AA infusion. These findings suggest that AA stimulate protein synthesis through an Akt-independent activation of translation initiation in human skeletal muscle.

Subjects and Methods

Subjects

Ten healthy young male volunteers were studied. Subjects ranged in age from 19–26 yr (22.6 ± 0.9 yr), with an average body mass index of 23.4 ± 0.8 kg/m2. They had no history of major organ system disease and were not taking any medication. Informed written consent was obtained from each volunteer before the study. The study protocol was approved by the human investigation committee and the general clinical research center advisory committee at University of Virginia before subject recruitment.

Study protocol

Subjects were kept on a meat-free diet for 3 d and then were admitted to the University of Virginia General Clinical Research Center the evening before the study. After a 12-h overnight fast, a brachial artery and an ipsilateral, retrograde, median deep antecubital vein in the study arm were catheterized percutaneously. The patency of the catheters was maintained by a slow infusion of normal saline. Another catheter was placed into a contralateral arm vein, and a primed (45 μCi) continuous (0.5 μCi/min) infusion of l-[ring-2,6-3H]phenylalanine was given for 8 h. After a 2-h tracer equilibration period, a mixed AA solution was infused systemically for the next 6 h. Figure 1 shows the overall study protocol. The mixed AA solution (Travesol, 10% in water; Travenol Laboratories, Deerfield, IL) was a mixture of various essential and nonessential AA (see Fig. 1 for detailed composition of this mixture) and was infused at a rate of 0.015 ml/min·kg body weight. Quadruplicate paired arterial and deep venous blood samples were obtained at 10-min intervals at the end of the tracer equilibration period (basal period at −30, −20, −10, and 0 min) and at the end of 3 h (150, 160, 170, and 180 min) and 6 h (330, 340, 350, and 360 min) of AA infusion for measurements of AA, insulin, glucose, lactate, oxygen balance, phenylalanine balance, and phenylalanine kinetics. For 2 min before and during the withdrawal of each deep venous blood sample, a pediatric sphygmomanometer cuff was inflated about the wrist to 200 mm Hg to exclude blood flow to the hand. Forearm blood flow was measured after each pair of blood samples using capacitance plethysmography. Urine samples were collected during the 12-h period before the infusion and during the 6-h period during the AA infusion for measurement of nitrogen and creatinine excretion. Just before beginning the systemic infusion of AA, the subject underwent a biopsy of vastus lateralis muscle, using a Bergstrom biopsy needle. Muscle biopsy was repeated in the opposite leg at the end of the study. Muscle tissues were immediately frozen and stored in liquid nitrogen for later analysis of Akt, 4E-BP1, and p70S6K. The detailed description of muscle biopsy procedure was reported previously (30).

Figure 1.

Study protocol. AA were infused systemically for 6 h at a rate of 0.015 ml/kg·min, and each 100 ml infusate contained 480 mg histidine, 600 mg isoleucine, 730 mg leucine, 580 mg lysine, 400 mg methionine, 560 mg phenylalanine, 420 mg threonine, 180 mg tryptophan, 580 mg valine, 2070 mg alanine, 1150 mg arginine, 1030 mg glycine, 680 mg proline, 500 mg serine, and 40 mg tyrosine.

Figure 1.

Study protocol. AA were infused systemically for 6 h at a rate of 0.015 ml/kg·min, and each 100 ml infusate contained 480 mg histidine, 600 mg isoleucine, 730 mg leucine, 580 mg lysine, 400 mg methionine, 560 mg phenylalanine, 420 mg threonine, 180 mg tryptophan, 580 mg valine, 2070 mg alanine, 1150 mg arginine, 1030 mg glycine, 680 mg proline, 500 mg serine, and 40 mg tyrosine.

Calculations of forearm phenylalanine kinetics

Net forearm balances for glucose, lactate, oxygen, and phenylalanine were calculated using the Fick principle: net balance = ([A] − [V]) × F, where [A] and [V] are arterial and venous substrate concentrations, and F is forearm blood flow in milliliters per minute per 100 ml forearm volume.

The forearm phenylalanine kinetics, determined using steady state isotope dilution equations, were calculated as previously described (30). In brief, phenylalanine is neither synthesized nor metabolized in muscle, and the balance of phenylalanine reflects the difference between its uptake for protein synthesis and its release from protein degradation. Simultaneous and paired sampling of forearm arterial and venous blood enables us to measure the concentrations and specific activities of phenylalanine and to calculate the uptake [rate of disappearance (Rd)] and the dilution [rate of appearance (Ra)] of [3H]phenylalanine during steady state infusion of [3H]phenylalanine, which can be used to estimate protein synthesis and degradation, respectively. Three formulas were used for calculations: 1) net balance = ([A] − [V]) × flow, 2) protein synthesis (Rd) = ([dpmartery − dpmvein] × flow)/SAvein, and 3) muscle protein breakdown = synthesis − net balance.

Calculation of whole body phenylalanine fluxes

Whole body phenylalanine fluxes were estimated from the ratio of the tracer infusion rate to the arterial specific activity at steady state, both basally (−30 to 0 min) and at the end of AA infusion (330–360 min), as described previously (32).

Western immunoblotting technique

Pieces (∼40 mg) of frozen vastus lateralis muscle tissue were weighed and powdered in frozen 25 mm Tris-HCl buffer [26 mm potassium fluoride and 5 mm EDTA (pH 7.5)], then disrupted by sonication using a microtip probe (0.5 sec on/0.5 sec off for 45 sec total) at a 3.0 power setting on the Fisher XL2020 sonicator (Fisher Scientific, Pittsburgh, PA). The homogenate was centrifuged at 2000 rpm for 2 min, and the protein concentration was measured in the supernatant using the Bradford method (33). For Akt, one aliquot of the supernatant containing approximately 60 μg protein was diluted with an equal volume of sodium dodecyl sulfate sample buffer and electrophoresed on an 8% polyacrylamide gel. For 4E-BP1, one aliquot of the supernatant containing approximately 60 μg protein was diluted with an equal volume of sodium dodecyl sulfate sample buffer and electrophoresed on a 15% polyacrylamide gel. For p70S6K, another aliquot of supernatant containing about 50 μg protein was diluted with an equal volume of sodium dodecyl sulfate sample buffer and electrophoresed on an 8% polyacrylamide gel. Proteins were then electrophoretically transferred to nitrocellulose membranes. After being blocked with 5% low fat milk in Tris-buffered saline plus Tween 20, membranes were incubated with either rabbit polyclonal Akt antibody or phospho-Akt (Ser473) antibody (New England Biolabs, Inc., Beverly, MA) overnight at 4–8 C or rabbit anti-4E-BP1 or p70S6K (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) for 1 h at room temperature. This was followed by a donkey antirabbit IgG coupled to horseradish peroxidase, and the blots were developed using an enhanced chemiluminescence Western blotting kit (Amersham Life Sciences, Piscataway, NJ).

Quantitation of Akt, 4E-BP1, and p70S6K phosphorylation state

Autoradiographic films were scanned densitometrically (Molecular Dynamics, Inc., Sunnyvale, CA) and quantitated using ImageQuant 3.3. Figure 2 illustrates the Akt, 4E-BP1, and p70S6K phosphorylation status on Western blots of biopsied muscle samples obtained during the basal period and at the end of AA infusion. For Akt, we intended to quantitate both the total and phospho-Akt (Ser473) densities and calculate the ratios of phospho-specific Akt density to total Akt density if there was any phosphorylation of Akt at Ser473. For both 4E-BP1 and p70S6K, we exploited the different electrophoretic mobility properties of variously phosphorylated proteins to quantitate the phosphorylation status. When subjected to SDS-PAGE, the least phosphorylated portion (the α-form) migrates most rapidly, whereas the most phosphorylated portion (the γ-form) moves the slowest, with the modestly phosphorylated portion (the β-form) in between. The densities of all bands (α + β + γ) were measured, and the fraction of protein migrating more slowly (β + γ) was determined as the appropriate ratios (β + γ/total). The β- and γ-forms represent the more highly phosphorylated proteins, and the available data support a good correlation between electrophoretic mobility and biological activity for both 4E-BP1 (3, 34) and p70S6K (35). We also demonstrated a good correlation between the ratios of β + γ/total for both 4E-BP1 and p70S6K and the bulk protein synthesis rates in vivo in animal studies (data not shown).

Figure 2.

Gel patterns of human skeletal muscle phospho-Akt (A), total Akt (B), 4E-BP1 (C), and p70S6K (D) on SDS-PAGE. For 4E-BP1 and p70S6K, the α-band is the least phosphorylated portion and moves most rapidly. The β- and γ-bands are more phosphorylated and have slower electrophoretic mobility. Basal, Basal muscle biopsy sample; AA, muscle sample obtained after 6 h of AA infusion. The two left lanes are Akt gel patterns using muscle samples obtained from a young healthy human subject before and after systemic insulin infusion (20 mU/kg·min euglycemic clamp) for 2 h. As our positive control, insulin significantly stimulated the phosphorylation of Akt at Ser473.

Figure 2.

Gel patterns of human skeletal muscle phospho-Akt (A), total Akt (B), 4E-BP1 (C), and p70S6K (D) on SDS-PAGE. For 4E-BP1 and p70S6K, the α-band is the least phosphorylated portion and moves most rapidly. The β- and γ-bands are more phosphorylated and have slower electrophoretic mobility. Basal, Basal muscle biopsy sample; AA, muscle sample obtained after 6 h of AA infusion. The two left lanes are Akt gel patterns using muscle samples obtained from a young healthy human subject before and after systemic insulin infusion (20 mU/kg·min euglycemic clamp) for 2 h. As our positive control, insulin significantly stimulated the phosphorylation of Akt at Ser473.

Analytic methods

Whole blood glucose and lactate concentrations were measured in duplicate using a combined glucose/lactate analyzer (YSI, Inc., Yellow Springs, OH). Plasma insulin concentrations were determined using an insulin ELISA (Diagnostic Systems Laboratories, Inc., Webster, TX). Blood oxygen content was measured spectrophotometrically using an OSM2 hemoximeter (Radiometer, Copenhagen, Denmark). Plasma AA concentrations were measured using an automated ion exchange chromatographic technique (D-500, Dionex, Sunnyvale, CA). Phenylalanine concentration and specific activity in arterial and venous blood were determined using an HPLC procedure as described previously (36).

Statistical analysis

All data are presented as the mean ± sem. Data for glucose, lactate, oxygen, and phenylalanine were averaged over the four time points in the basal (−30 to 0 min) and AA infusion (150–180 min and 330–360 min) periods for each subject. Statistical comparisons between the basal and AA infusion periods were made using two-tailed paired t test.

Results

Effects of AA infusion on forearm blood flow, and insulin and substrate levels

The postabsorptive forearm blood flow, and blood glucose, lactate, and insulin concentrations are shown in Table 1. AA infusion did not significantly alter arterial insulin concentrations, forearm blood flow, glucose balance, or oxygen balance, but increased forearm lactate release from −0.20 ± 0.11 to −0.51 ± 0.15 μmol/min·100 ml (P < 0.02). The basal venous phenylalanine concentrations were significantly higher than arterial phenylalanine concentrations, reflecting a net protein breakdown after an overnight fast (Table 1). This negative venous-arterial phenylalanine differential was either reversed (3 h) or abrogated (6 h) after AA infusion. Table 2 lists the concentrations of all AA measured at the basal period and at the end of 6-h infusion. Total AA concentrations were increased by 57 ± 3.5%, and total BCAA concentrations were increased by 109 ± 8.4% (P < 0.00001 for both) after 6-h AA infusion. The concentrations of asparagine, glutamic acid, and tyrosine were decreased by 13–21%, whereas the concentrations of all other AA were increased (by 9–227%) after AA infusion. The increment in AA concentrations was statistically significant for all AA except glutamine (9 ± 4.5%; P < 0.07).

Table 1.

Effects of AA infusion on forearm blood flow and substrate balances

Basal . 3 h . 6 h . 
Blood flow (ml/min·100 ml) 4.31 ± 0.74 4.97 ± 0.86 5.17 ± 1.15 
Glucose balance (μmol/min·100 ml) 0.60 ± 0.14 0.68 ± 0.17 1.05 ± 0.25 
Lactate balance (μmol/min·100 ml) −0.20 ± 0.11 −0.51 ± 0.12a−0.51 ± 0.15a
Oxygen balance (μmol/min·100 ml) 7.3 ± 1.27 8.23 ± 1.73 8.85 ± 2.01 
Arterial insulin (pmol/liter) 36 ± 3.6 41 ± 3.5 40 ± 4.7 
Arterial phenylalanine (μmol/liter) 52.4 ± 3.6 106.0 ± 5.1b118.5 ± 5.1b
Venous phenylalanine (μmol/liter) 58.8 ± 4.2c101.9 ± 5.0bd116.7 ± 5.2b
Basal . 3 h . 6 h . 
Blood flow (ml/min·100 ml) 4.31 ± 0.74 4.97 ± 0.86 5.17 ± 1.15 
Glucose balance (μmol/min·100 ml) 0.60 ± 0.14 0.68 ± 0.17 1.05 ± 0.25 
Lactate balance (μmol/min·100 ml) −0.20 ± 0.11 −0.51 ± 0.12a−0.51 ± 0.15a
Oxygen balance (μmol/min·100 ml) 7.3 ± 1.27 8.23 ± 1.73 8.85 ± 2.01 
Arterial insulin (pmol/liter) 36 ± 3.6 41 ± 3.5 40 ± 4.7 
Arterial phenylalanine (μmol/liter) 52.4 ± 3.6 106.0 ± 5.1b118.5 ± 5.1b
Venous phenylalanine (μmol/liter) 58.8 ± 4.2c101.9 ± 5.0bd116.7 ± 5.2b

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Table 1.

Effects of AA infusion on forearm blood flow and substrate balances

Basal . 3 h . 6 h . 
Blood flow (ml/min·100 ml) 4.31 ± 0.74 4.97 ± 0.86 5.17 ± 1.15 
Glucose balance (μmol/min·100 ml) 0.60 ± 0.14 0.68 ± 0.17 1.05 ± 0.25 
Lactate balance (μmol/min·100 ml) −0.20 ± 0.11 −0.51 ± 0.12a−0.51 ± 0.15a
Oxygen balance (μmol/min·100 ml) 7.3 ± 1.27 8.23 ± 1.73 8.85 ± 2.01 
Arterial insulin (pmol/liter) 36 ± 3.6 41 ± 3.5 40 ± 4.7 
Arterial phenylalanine (μmol/liter) 52.4 ± 3.6 106.0 ± 5.1b118.5 ± 5.1b
Venous phenylalanine (μmol/liter) 58.8 ± 4.2c101.9 ± 5.0bd116.7 ± 5.2b
Basal . 3 h . 6 h . 
Blood flow (ml/min·100 ml) 4.31 ± 0.74 4.97 ± 0.86 5.17 ± 1.15 
Glucose balance (μmol/min·100 ml) 0.60 ± 0.14 0.68 ± 0.17 1.05 ± 0.25 
Lactate balance (μmol/min·100 ml) −0.20 ± 0.11 −0.51 ± 0.12a−0.51 ± 0.15a
Oxygen balance (μmol/min·100 ml) 7.3 ± 1.27 8.23 ± 1.73 8.85 ± 2.01 
Arterial insulin (pmol/liter) 36 ± 3.6 41 ± 3.5 40 ± 4.7 
Arterial phenylalanine (μmol/liter) 52.4 ± 3.6 106.0 ± 5.1b118.5 ± 5.1b
Venous phenylalanine (μmol/liter) 58.8 ± 4.2c101.9 ± 5.0bd116.7 ± 5.2b

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Table 2.

Serum amino acid concentrations (μmol/liter) before and after amino acid infusion

Basal . 6 h . % Increase . P
α-Aminobutyrate 18 ± 0.9 32 ± 1.7 81 ± 11.1 <0.0002 
Alanine 354 ± 38.7 669 ± 42.2 100 ± 16.3 <0.00001 
Arginine 100 ± 3.4 237 ± 9.0 137 ± 9.6 <0.00001 
Asparagine 39 ± 2.5 31 ± 3.0 −21 ± 4.5 <0.001 
Aspartic acid 14 ± 1.1 16 ± 1.7 16 ± 4.8 <0.02 
Citrulline 33 ± 1.0 44 ± 1.5 34 ± 4.6 <0.0002 
Glutamic acid 199 ± 20 161 ± 14.3 −18 ± 1.8 <0.001 
Glutamine 726 ± 57.8 788 ± 59.1 9 ± 4.5 <0.07 
Glycine 225 ± 6.2 449 ± 18.2 99 ± 4.7 <0.00001 
Histidine 80 ± 3.1 138 ± 5.2 73 ± 7.2 <0.00001 
Isoleucine 60 ± 3.0 175 ± 5.6 199 ± 15.6 <0.00001 
Leucine 109 ± 6.1 223 ± 8.9 108 ± 9.4 <0.00001 
Lysine 134 ± 6.8 198 ± 8.5 49 ± 7.2 <0.00009 
Methionine 26 ± 0.9 83 ± 3.8 227 ± 14.7 <0.00001 
Ornithine 50 ± 2.5 80 ± 4.9 61 ± 8.4 <0.0002 
Phenylalanine 50 ± 2.8 116 ± 5.1 134 ± 7.9 <0.00001 
Serine 108 ± 4.7 190 ± 6.3 77 ± 6.8 <0.00001 
Taurine 41 ± 1.7 45 ± 1.3 10 ± 3.8 <0.04 
Threonine 106 ± 5.6 178 ± 8.6 70 ± 6.2 <0.00001 
Tryptophan 52 ± 2.9 79 ± 4.5 54 ± 11.3 <0.0008 
Tyrosine 49 ± 3.3 43 ± 3.2 −13 ± 2.8 <0.003 
Valine 203 ± 8.7 372 ± 13.8 85 ± 6.9 <0.00001 
Total BCAA 371 ± 17.0 770 ± 27.6 109 ± 8.4 <0.00001 
Total AA 2777 ± 74.2 4348 ± 151.3 57 ± 3.5 <0.00001 
Basal . 6 h . % Increase . P
α-Aminobutyrate 18 ± 0.9 32 ± 1.7 81 ± 11.1 <0.0002 
Alanine 354 ± 38.7 669 ± 42.2 100 ± 16.3 <0.00001 
Arginine 100 ± 3.4 237 ± 9.0 137 ± 9.6 <0.00001 
Asparagine 39 ± 2.5 31 ± 3.0 −21 ± 4.5 <0.001 
Aspartic acid 14 ± 1.1 16 ± 1.7 16 ± 4.8 <0.02 
Citrulline 33 ± 1.0 44 ± 1.5 34 ± 4.6 <0.0002 
Glutamic acid 199 ± 20 161 ± 14.3 −18 ± 1.8 <0.001 
Glutamine 726 ± 57.8 788 ± 59.1 9 ± 4.5 <0.07 
Glycine 225 ± 6.2 449 ± 18.2 99 ± 4.7 <0.00001 
Histidine 80 ± 3.1 138 ± 5.2 73 ± 7.2 <0.00001 
Isoleucine 60 ± 3.0 175 ± 5.6 199 ± 15.6 <0.00001 
Leucine 109 ± 6.1 223 ± 8.9 108 ± 9.4 <0.00001 
Lysine 134 ± 6.8 198 ± 8.5 49 ± 7.2 <0.00009 
Methionine 26 ± 0.9 83 ± 3.8 227 ± 14.7 <0.00001 
Ornithine 50 ± 2.5 80 ± 4.9 61 ± 8.4 <0.0002 
Phenylalanine 50 ± 2.8 116 ± 5.1 134 ± 7.9 <0.00001 
Serine 108 ± 4.7 190 ± 6.3 77 ± 6.8 <0.00001 
Taurine 41 ± 1.7 45 ± 1.3 10 ± 3.8 <0.04 
Threonine 106 ± 5.6 178 ± 8.6 70 ± 6.2 <0.00001 
Tryptophan 52 ± 2.9 79 ± 4.5 54 ± 11.3 <0.0008 
Tyrosine 49 ± 3.3 43 ± 3.2 −13 ± 2.8 <0.003 
Valine 203 ± 8.7 372 ± 13.8 85 ± 6.9 <0.00001 
Total BCAA 371 ± 17.0 770 ± 27.6 109 ± 8.4 <0.00001 
Total AA 2777 ± 74.2 4348 ± 151.3 57 ± 3.5 <0.00001 

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Table 2.

Serum amino acid concentrations (μmol/liter) before and after amino acid infusion

Basal . 6 h . % Increase . P
α-Aminobutyrate 18 ± 0.9 32 ± 1.7 81 ± 11.1 <0.0002 
Alanine 354 ± 38.7 669 ± 42.2 100 ± 16.3 <0.00001 
Arginine 100 ± 3.4 237 ± 9.0 137 ± 9.6 <0.00001 
Asparagine 39 ± 2.5 31 ± 3.0 −21 ± 4.5 <0.001 
Aspartic acid 14 ± 1.1 16 ± 1.7 16 ± 4.8 <0.02 
Citrulline 33 ± 1.0 44 ± 1.5 34 ± 4.6 <0.0002 
Glutamic acid 199 ± 20 161 ± 14.3 −18 ± 1.8 <0.001 
Glutamine 726 ± 57.8 788 ± 59.1 9 ± 4.5 <0.07 
Glycine 225 ± 6.2 449 ± 18.2 99 ± 4.7 <0.00001 
Histidine 80 ± 3.1 138 ± 5.2 73 ± 7.2 <0.00001 
Isoleucine 60 ± 3.0 175 ± 5.6 199 ± 15.6 <0.00001 
Leucine 109 ± 6.1 223 ± 8.9 108 ± 9.4 <0.00001 
Lysine 134 ± 6.8 198 ± 8.5 49 ± 7.2 <0.00009 
Methionine 26 ± 0.9 83 ± 3.8 227 ± 14.7 <0.00001 
Ornithine 50 ± 2.5 80 ± 4.9 61 ± 8.4 <0.0002 
Phenylalanine 50 ± 2.8 116 ± 5.1 134 ± 7.9 <0.00001 
Serine 108 ± 4.7 190 ± 6.3 77 ± 6.8 <0.00001 
Taurine 41 ± 1.7 45 ± 1.3 10 ± 3.8 <0.04 
Threonine 106 ± 5.6 178 ± 8.6 70 ± 6.2 <0.00001 
Tryptophan 52 ± 2.9 79 ± 4.5 54 ± 11.3 <0.0008 
Tyrosine 49 ± 3.3 43 ± 3.2 −13 ± 2.8 <0.003 
Valine 203 ± 8.7 372 ± 13.8 85 ± 6.9 <0.00001 
Total BCAA 371 ± 17.0 770 ± 27.6 109 ± 8.4 <0.00001 
Total AA 2777 ± 74.2 4348 ± 151.3 57 ± 3.5 <0.00001 
Basal . 6 h . % Increase . P
α-Aminobutyrate 18 ± 0.9 32 ± 1.7 81 ± 11.1 <0.0002 
Alanine 354 ± 38.7 669 ± 42.2 100 ± 16.3 <0.00001 
Arginine 100 ± 3.4 237 ± 9.0 137 ± 9.6 <0.00001 
Asparagine 39 ± 2.5 31 ± 3.0 −21 ± 4.5 <0.001 
Aspartic acid 14 ± 1.1 16 ± 1.7 16 ± 4.8 <0.02 
Citrulline 33 ± 1.0 44 ± 1.5 34 ± 4.6 <0.0002 
Glutamic acid 199 ± 20 161 ± 14.3 −18 ± 1.8 <0.001 
Glutamine 726 ± 57.8 788 ± 59.1 9 ± 4.5 <0.07 
Glycine 225 ± 6.2 449 ± 18.2 99 ± 4.7 <0.00001 
Histidine 80 ± 3.1 138 ± 5.2 73 ± 7.2 <0.00001 
Isoleucine 60 ± 3.0 175 ± 5.6 199 ± 15.6 <0.00001 
Leucine 109 ± 6.1 223 ± 8.9 108 ± 9.4 <0.00001 
Lysine 134 ± 6.8 198 ± 8.5 49 ± 7.2 <0.00009 
Methionine 26 ± 0.9 83 ± 3.8 227 ± 14.7 <0.00001 
Ornithine 50 ± 2.5 80 ± 4.9 61 ± 8.4 <0.0002 
Phenylalanine 50 ± 2.8 116 ± 5.1 134 ± 7.9 <0.00001 
Serine 108 ± 4.7 190 ± 6.3 77 ± 6.8 <0.00001 
Taurine 41 ± 1.7 45 ± 1.3 10 ± 3.8 <0.04 
Threonine 106 ± 5.6 178 ± 8.6 70 ± 6.2 <0.00001 
Tryptophan 52 ± 2.9 79 ± 4.5 54 ± 11.3 <0.0008 
Tyrosine 49 ± 3.3 43 ± 3.2 −13 ± 2.8 <0.003 
Valine 203 ± 8.7 372 ± 13.8 85 ± 6.9 <0.00001 
Total BCAA 371 ± 17.0 770 ± 27.6 109 ± 8.4 <0.00001 
Total AA 2777 ± 74.2 4348 ± 151.3 57 ± 3.5 <0.00001 

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Effects of AA infusion on whole body and forearm muscle protein metabolism

AA infusion slightly decreased urinary nitrogen excretion, although the difference was not statistically significant (6.72 ± 0.41 vs. 5.79 ± 0.35 g nitrogen/g creatinine, basal vs. AA infusion; P = 0.09; Fig. 3). However, whole body phenylalanine flux was dramatically decreased by AA infusion, from 0.54 ± 0.03 to 0.36 ± 0.04 μmol/min·kg (P < 0.004; Fig. 3).

Figure 3.

Effects of AA infusion on urinary nitrogen excretion and whole body phenylalanine flux. AA infusion slightly decreased urinary nitrogen excretion, but this did not achieve statistical significance (*, P = 0.09), and significantly decreased whole body phenylalanine flux (#, P < 0.004).

Figure 3.

Effects of AA infusion on urinary nitrogen excretion and whole body phenylalanine flux. AA infusion slightly decreased urinary nitrogen excretion, but this did not achieve statistical significance (*, P = 0.09), and significantly decreased whole body phenylalanine flux (#, P < 0.004).

Consistent with our previous reports (30, 37), all subjects had negative forearm protein balance after an overnight fast. AA infusion significantly improved forearm phenylalanine balance at 3 h, and the effect persisted at 6 h (Fig. 4). Protein synthesis was increased significantly at both 3 and 6 h. It appears that the improvement in protein balance at both 3 and 6 h can be accounted for solely by the increment in protein synthesis, as the rate of protein degradation (Ra) was not changed by AA infusion.

Figure 4.

Effects of AA infusion on forearm muscle protein metabolism. Postabsorptively all subjects had net release of phenylalanine from the forearm, resulting in negative protein balance. AA infusion significantly improved phenylalanine balance at both 3 h (*, P < 0.002) and 6 h (**, P < 0.02). This improvement is solely from increased forearm phenylalanine Rd [#, P = 0.02; ##, P < 0.003 (vs. basal)]. Forearm phenylalanine Ra was not changed by AA infusion.

Figure 4.

Effects of AA infusion on forearm muscle protein metabolism. Postabsorptively all subjects had net release of phenylalanine from the forearm, resulting in negative protein balance. AA infusion significantly improved phenylalanine balance at both 3 h (*, P < 0.002) and 6 h (**, P < 0.02). This improvement is solely from increased forearm phenylalanine Rd [#, P = 0.02; ##, P < 0.003 (vs. basal)]. Forearm phenylalanine Ra was not changed by AA infusion.

Effects of AA infusion on Akt, 4E-BP1, and p70S6K phosphorylation

Phosphorylation of Akt at Thr308 and Ser473 is required for Akt kinase activity, and phosphorylation of Thr308 leads to the phosphorylation of Ser473 (8). AA infusion did not enhance Akt phosphorylation at Ser473 in our study subjects (Fig. 1, two right lanes in A and B).

To quantify the extent of phosphorylation of 4E-BP1 and p70S6K, we measured the ratio of the intensity of the more slowly migrating species (β + γ) to that of the total integrated intensity (α + β + γ). AA infusion significantly increased the (β + γ)/(α + β + γ) ratio of 4E-BP1 by decreasing the amount of rapidly migrating species (α) and increasing the density of the more slowly migrating forms (β + γ; 0.26 ± 0.04 vs. 0.32 ± 0.03; P < 0.04; Fig. 5), suggesting increases in 4E-BP1 phosphorylation and the amount of eIF4E available to initiate translation. For p70S6K, the overall p70S6K kinase activity is dependent on the phosphorylation of at least seven Ser/Thr residues at three separate domains (9, 35). The uppermost bands (β and γ) represent the more highly phosphorylated forms of p70S6K and generally correspond to species with greater kinase activity. Similar to the effect on 4E-BP1, AA infusion significantly increased the ratios of (β + γ)/(α + β + γ) of p70S6K (0.161 ± 0.014 vs. 0.271 ± 0.019; P < 0.001; Fig. 5).

Figure 5.

Effects of AA infusion on 4E-BP1 and p70S6K phosphorylation. AA infusion significantly increased the phosphorylation of both 4E-BP1 and p70S6K, evidenced by the increased ratios of (β + γ)/(α + β + γ) for both proteins.

Figure 5.

Effects of AA infusion on 4E-BP1 and p70S6K phosphorylation. AA infusion significantly increased the phosphorylation of both 4E-BP1 and p70S6K, evidenced by the increased ratios of (β + γ)/(α + β + γ) for both proteins.

Discussion

Results from the current study demonstrated that moderate increments (within their physiological ranges) of circulating AA, similar to those seen postprandially (38), stimulated human skeletal muscle protein synthesis, improved protein balance, and enhanced the phosphorylation of both 4E-BP1 and p70S6K. These findings indicate that changes in AA concentrations within the physiological ranges were sufficient to stimulate protein synthesis in human skeletal muscle by providing a significant anabolic signal to activate mRNA translation initiation.

Inasmuch as AA have been shown to phosphorylate 4E-BP1 and p70S6K via an mTOR-dependent manner in a variety of in vitro cell studies and in vivo animal studies (15–18, 29), and mTOR is phosphorylated and thereby activated by Akt (1, 6–8), we must consider the possibility that AA activate mRNA translation initiation through the PI3-kinase/Akt signaling pathway. However, we believe that this is highly unlikely, and that AA must act downstream of Akt to signal the mRNA translation initiation. AA have been shown to provide positive signals for the maintenance of protein stores while inhibiting other actions of insulin at multiple levels, including insulin-mediated tyrosine phosphorylation of insulin receptor substate-1 and -2 and activation of PI3-kinase (15, 16, 39). In the current study AA infusion increased 4E-BP1 and p70S6K phosphorylation and protein synthesis, but did not increase Akt phosphorylation and did not affect glucose uptake by muscle. This is consistent with a recent report that leucine activates p70S6K through an Akt-independent mechanism (40).

The time course of AA action on protein synthesis remains unclear. A recent study (41) reported that human muscle protein synthesis responds rapidly (within 30–120 min) to increased availability of AA, but is then inhibited (after 120 min) despite continued AA availability. However, our results clearly indicate that protein synthesis was stimulated after 3 h, and this effect lasted for the entire 6 h of continuous AA infusion. There are several significant differences between these two studies, making direct comparisons difficult. Firstly, their study population was 10 yr older (33 ± 1 vs. 23.4 ± 0.8 yr), and their subjects’ body weights were 6 kg heavier (80 ± 5 vs. 74 ± 4 kg). Younger and leaner subjects tend to engage in more physical activity and may respond better to anabolic stimuli, including AA. Secondly, in the study by Bohe and colleagues (41) the serum insulin levels increased approximately 3-fold within the first 30 min and remained elevated for more than 3 h. This insulinotropic effect was probably due to the large AA prime (54 mg/kg) and higher AA infusion rate (2.7 mg/kg·min) used in that study. We did not use a primed infusion, and the concentrations of blood glucose and insulin remained steady in our study population. We chose the infusion rate of 0.015 ml/kg·min (1.5 mg/kg·min) in the present study to avoid the potential confounding effect of AA-induced insulin secretion on the interpretation of results. Previous work has shown that iv infusion of mixed AA at 0.5, 1, and 2 mg/kg·min does not increase plasma insulin concentrations at 1, 2, and 3 h (42). Thirdly, different radiotracers and analytic techniques were used to assess the rate of protein synthesis in these two studies. In the current study, consistent with protein synthesis data, 4E-BP1 and p70S6K remained hyperphosphoryl-ated after 6 h of AA infusion. Studies performed by others and us suggest that the effects of AA on protein synthesis and the phosphorylation of 4E-BP1 and/or p70S6K remain as long as the provocative stimulus exists. Infusion of mixed AA was able to improve protein balance and increase protein synthesis within 3 h (12), and infusion of leucine alone for 2 h caused a 4-fold increase in the phosphorylation of p70S6K (40) in postabsorptive humans. No in vivo human study has examined the acute effect (<2 h) of AA on the phosphorylation of 4E-BP1 and p70S6K. In rats, oral administration of leucine stimulated phosphorylation of both proteins within 1 h (27).

We have previously reported that moderate elevation of plasma BCAA concentrations stimulates the phosphorylation of 4E-BP1 and p70S6K and decreases whole body phenylalanine flux and skeletal muscle protein degradation; however, skeletal muscle protein synthesis was not affected (30, 36, 43). These data suggest that BCAA activate mRNA translation initiation, but without the anticipated increase in protein synthesis. One possible explanation for this apparent discrepancy is that BCAA inhibit proteolysis and thereby decrease the arterial concentrations of other AA (36). The availability of AA from plasma has been shown to affect the rate of protein synthesis in muscle (11, 12). In the current study, mixed AA infusion doubled the total BCAA concentrations, whereas the phosphorylation of 4E-BP1 and p70S6K increased dramatically, and muscle protein synthetic rates were significantly stimulated, consistent with our data in laboratory rats reported previously (19). Taken together, we believe that mixed AA infusion not only provides substrates for protein synthesis, but it also directly provides nutrient signals to activate the protein synthetic apparatus.

It is of interest to note that the serum concentrations of BCAA were much lower in the current study than those we observed during the infusion of BCAA alone (36) (372 ± 13.8 vs. 662 ± 21, 223 ± 8.9 vs. 441 ± 14, and 175 ± 5.6 vs. 395 ± 16 μmol/liter for valine, leucine, and isoleucine, respectively). This occurred despite a slightly higher BCAA infusion rate in the current study (2.27 vs. 1.66 μmol/min·kg). Thus, in the current study clearance of infused BCAA was more than doubled compared with that seen during the infusion of BCAA alone. This probably arose from increased utilization of BCAA for protein synthesis in the current study. Despite this, we observed a similar increase in the extent of 4E-BP1 and p70S6K phosphorylation in the current study compared with that in our previous study using the 1.66 μmol BCAA/min·kg infusion rate (30). This suggests that either BCAA can stimulate mRNA translation initiation at much lower concentrations and/or other AA may have also contributed in providing nutrient signals to activate this process in the current study.

In conclusion, mixed AA infusion stimulates protein synthesis, promotes positive protein balance, and enhances the phosphorylation of 4E-BP1 and p70S6K through an Akt-independent mechanism in human skeletal muscle. Our findings suggest that AA not only function as substrates for protein synthesis, but also play a major signaling role in mRNA translation in human skeletal muscle.

Acknowledgements

This work was supported by NIH Grants RR-15540 (to Z.L.), DK-38578 and DK-54058 (to E.J.B.), and RR00847 (to University of Virginia General Clinical Research Center).

Abbreviations:

  • AA,

  • BCAA,

    branched chain amino acids;

  • 4E-BP1,

    eukaryotic initiation factor 4E-binding protein 1;

  • eIF4E,

    eukaryotic initiation factor 4E;

  • mTOR,

    mammalian target of rapamycin;

  • PI3-kinase,

    phosphatidylinositol 3-kinase;

  • Ra,

  • Rd,

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Protein synthesis stimulates what

Abstract

Oral administration of a single bolus of leucine in an amount equivalent to the daily intake (1.35 g/kg body wt) enhances skeletal muscle protein synthesis in food-deprived rats. To elucidate whether smaller amounts of leucine can also stimulate protein synthesis, rats were administered the amino acid at concentrations ranging from 0.068 to 1.35 g/kg body wt by oral gavage. Thirty minutes following the administration of doses of leucine as low as 0.135 g/kg body wt, skeletal muscle protein synthesis was significantly greater than control values. The increase in protein synthesis was associated with changes in the regulation of biomarkers of mRNA translation initiation as evidenced by upregulated phosphorylation of the translational repressor, eukaryotic initiation factor (eIF)4E-binding protein 1 (4E-BP1), the association of eIF4G with the mRNA cap binding protein eIF4E, and the phosphorylation of the 70-kDa ribosomal protein S6 kinase. Alterations in the phosphorylation of eIF4G, as well as the association of 4E-BP1 with eIF4E, were observed following leucine administration; however, these changes appeared to be biphasic with maximal changes occurring when circulating insulin concentrations were elevated. Thus it appears that leucine administration affects mRNA translation and skeletal muscle protein synthesis through modulation of multiple biomarkers of mRNA translation. The ability of small doses of leucine to stimulate skeletal muscle protein synthesis suggests that future research on the regulation of skeletal muscle protein synthesis by orally administered leucine will be feasible in humans.

translation initiation, gastrocnemius muscle, mTOR

Ingestion of a mixed meal typically stimulates skeletal muscle protein synthetic rates in food-deprived animals (1,2). However, consumption of a protein-deficient meal does not elicit this response (3,4) and it is now clear that an adequate supply of amino acids is essential for feeding-induced changes in skeletal muscle protein synthesis. Moreover, recent studies suggest that it is the supply of branched-chain amino acids, and leucine in particular, that modulates the protein synthetic response in skeletal muscle to meal feeding (5,6).

Both in vitro and in vivo experiments have demonstrated that the mechanism(s) whereby leucine ingestion stimulates skeletal muscle protein synthesis involves the enhancement of mRNA translation initiation rates (7–9). However, leucine stimulates insulin secretion (10–12) and when administered in large doses, as is the case with the majority of studies performed to date, it causes a transient but significant increase in serum insulin concentrations (13). The stimulatory effects of insulin on mRNA translation initiation in skeletal muscle have been well documented (14–21) and as such, it has been difficult to characterize the direct contribution of leucine to the stimulation of skeletal muscle protein synthesis in vivo.

A recent study demonstrated that protein synthetic rates are significantly elevated in the gastrocnemius and plantaris muscles of diabetic rats 1 h following oral administration of a large dose of leucine (1.35 g/kg body wt) in comparison with diabetic controls (22). The change in protein synthesis was associated with alterations in phosphorylation or function of proteins associated with the regulation of mRNA translation initiation; however, the magnitude of the change in both protein synthetic rate and initiation factors was smaller than that observed in nondiabetic rats administered leucine. In an additional study, alterations in mRNA translation initiation factors were observed 30 min following oral leucine administration (1.35 g/kg body wt) when food-deprived rats were infused with somatostatin to inhibit insulin release and maintain serum insulin concentrations at fasting levels (13). There was not, however, a significant increase in protein synthetic rates in the gastrocnemius and plantaris muscles in rats administered somatostatin. Thus it appears that the oral administration of large doses of leucine can stimulate mRNA translation initiation in skeletal muscle of food-deprived rats independently of increased serum insulin concentrations. However, leucine-induced increases in circulating insulin appear to be necessary to elevate synthetic rates above values observed under food-deprived conditions.

The dose of leucine employed in the aforementioned studies is quite large, equivalent to that consumed in a 24-h period by age- and strain-matched rats when allowed free access to standard lab chow (9). Due to the relative insolubility of leucine, such a dose is unlikely to be compatible with human studies. Thus, the aim of the present study was to define the minimal dose of leucine required to stimulate protein synthesis in skeletal muscle and to identify the biomarkers of mRNA translation that mediate the response.

MATERIALS AND METHODS

Animal care.

The animal facilities and the experimental protocol used in the studies reported herein were reviewed and approved by the Institutional Animal Care and Use Committee of the Pennsylvania State University College of Medicine. Male Sprague-Dawley rats (∼200 g) were maintained on a 12-h light:dark cycle with a standard diet (Harlan-Teklad Rodent Chow 8604) and water provided ad libitum.

Experimental design.

Rats were food deprived for 18 h prior to experimentation. A suspension of 54.0 g L-leucine/L water was prepared and rats were administered measured volumes corresponding to 0.068 (5%, n = 12), 0.135 (10%, n = 12), 0.338 (25%, n = 8), 0.675 (50%, n = 10), and 1.35 g L-leucine/kg body wt (100%, n = 12) by oral gavage. Because the administered leucine was in the form of a suspension, rats were divided into their respective groups based on serum leucine concentrations. Rats in which serum leucine values differed significantly from the mean were removed from the study. The highest concentration of L-leucine employed is equivalent to that consumed in a 24-h period by age- and strain-matched rats when allowed free access to standard lab chow (9). Control rats (n = 12) were administered 0.155 mol/L NaCl at a volume of 2.5 mL/100 g body wt. This volume of saline is equivalent to the volume of leucine suspension administered to rats in the 100% leucine group and was chosen to control for any possible volume-induced effects of oral gavage, i.e., gastric expansion-induced signaling. There were fewer rats in the 25 and 50% groups because this study represents 2 separate experiments, wherein the first experiment did not include these groups.

Administration of metabolic tracer and sample collection.

Twenty minutes following oral gavage, a flooding dose (1.0 mL/100 g body wt) of L-[2,3,4,5,6-3H]phenylalanine (150 mmol/L, containing 3.70 GBq/L) was administered via tail vein injection for the measurement of protein synthesis (23). Rats were killed by decapitation 10 min later. Serum was obtained from trunk blood by centrifugation at 1800 × g for 10 min at 4°C. The right gastrocnemius and plantaris muscles were quickly excised as 1 unit (hereafter referred to as gastrocnemius) and homogenized in 7 vol of buffer consisting of 20 mmol/L N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid (pH 7.4), 100 mmol/L KCl, 0.2 mmol/L EDTA, 2 mmol/L ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid, 1 mmol/L dithiothreitol, 50 mmol/L sodium fluoride, 50 mmol/L β-glycerophosphate, 0.1 mmol/L phenylmethylsulfonyl fluoride, 1 mmol/L benzamidine, and 0.5 mmol/L sodium vanadate. An aliquot (0.5 mL) of the homogenate was used for the measurement of muscle protein synthesis as described under Measurement of muscle protein synthesis. The remainder of the homogenate was immediately centrifuged at 10,000 × g for 10 min at 4°C. The supernatant was used for analysis of mRNA translation initiation factors as described under Analysis of initiation factor phosphorylation state and Analysis of eIF4E complexes. The remaining tissue was used to assess eIF2B activity as described under Analysis of eIF2B activity.

Serum measurements.

Serum insulin concentrations were analyzed using a commercial RIA kit for rat insulin (Linco Research). Serum leucine concentrations were analyzed by derivatization with phenylisothiocyanate and HPLC analysis as described previously (24).

Measurement of muscle protein synthesis.

Fractional rates of protein synthesis were assessed from the rate of incorporation of radioactive phenylalanine into total mixed muscle protein as described previously (25). The time from injection of the metabolic tracer until homogenization of the muscle was recorded as the actual time for radiolabeled phenylalanine incorporation.

Analysis of initiation factor phosphorylation state.

Phosphorylation of eukaryotic initiation factor (eIF)3 4G, 4E-binding protein 1 (4E-BP1), and ribosomal protein S6 kinase (S6K1) at Thr389 was evaluated in 10,000 × g supernatants of muscle homogenates by protein immunoblot analysis as described previously (26–28).

Analysis of eIF4E complexes.

eIF4E was immunoprecipitated from 10,000 × g supernatants using a monoclonal eIF4E antibody (28). Samples were subjected to immunoblot analysis using polyclonal antibodies to either 4E-BP1 or eIF4G to determine the association of 4E-BP1 and eIF4G with eIF4E, respectively (28).

Analysis of eIF2B activity.

The guanine nucleotide exchange activity of eIF2B was assessed as the rate of exchange of [3H]GDP bound to eIF2 for nonradioactively labeled GDP as described previously (15).

Statistical analysis.

Data are means ± SEM. Statistical outliers within each treatment group were identified using a Grubbs test (GraphPad Software) and removed. All remaining data were analyzed by the InStat Version 3 statistical software package (GraphPad Software). Correlation coefficients were determined by Pearson correlation test. Means were compared using a one-way ANOVA. When ANOVA indicated a significant overall effect, differences among individual means were assessed using the Sidak test for multiple comparisons as described previously (29). Differences with P values < 0.05 were considered significant.

RESULTS

Serum leucine concentrations rose in proportion to the amount of the amino acid administered and by 30 min were significantly greater than controls at all doses (Fig. 1A). In accordance with what has been demonstrated previously, serum insulin concentrations were also elevated 30 min following oral administration of leucine at the 100% dose (13) and also at the 50% dose (Fig. 1B). However, administration of leucine at lower doses did not affect serum insulin concentrations at this time point.

FIGURE 1

Changes in (A) serum leucine and (B) insulin concentrations following oral leucine administration in rats. Serum leucine and insulin concentrations were measured 30 min following administration of saline or leucine at doses ranging from 0.068 to 1.35 g leucine/kg body wt. Values are means ± SE, n = 8–12. *Different from saline-treated controls, P < 0.05.

FIGURE 1

Changes in (A) serum leucine and (B) insulin concentrations following oral leucine administration in rats. Serum leucine and insulin concentrations were measured 30 min following administration of saline or leucine at doses ranging from 0.068 to 1.35 g leucine/kg body wt. Values are means ± SE, n = 8–12. *Different from saline-treated controls, P < 0.05.

The synthetic rate of total mixed protein in the gastrocnemius was increased by 31, 30, 37, and 43% of control values 30 min following oral administration of leucine at the 10, 25, 50, and 100% doses, respectively (Fig. 2). Protein synthetic rates were not statistically different from control values in the 5 or 25% groups [probably due to the small sample size in the 25% group (n = 6 vs. 10–12)], but rates of protein synthesis did correlate with serum leucine concentrations (r = 0.33, P = 0.0057). In contrast, there was no correlation between synthesis rates and serum insulin concentrations.

FIGURE 2

Rates of protein synthesis in gastrocnemius muscle of rats following oral leucine administration. Protein synthesis was measured by the incorporation of [3H]phenylanlanine into protein 30 min following administration of saline or leucine at doses ranging from 0.068 to 1.35 g leucine/kg body wt. Values are means ± SE, n = 6–12. *Different from saline-treated controls, P < 0.05.

FIGURE 2

Rates of protein synthesis in gastrocnemius muscle of rats following oral leucine administration. Protein synthesis was measured by the incorporation of [3H]phenylanlanine into protein 30 min following administration of saline or leucine at doses ranging from 0.068 to 1.35 g leucine/kg body wt. Values are means ± SE, n = 6–12. *Different from saline-treated controls, P < 0.05.

The synthesis of most proteins is regulated acutely at the stage of translation initiation (30). For initiation to occur in most eukaryotic systems, a complex including eIF2-bound GTP delivers an initiator methionyl-tRNA (met-tRNAiMet) to the 40S ribosomal subunit where GTP is hydrolyzed to GDP. For subsequent rounds of initiation to occur the guanine nucleotide exchange protein, eIF2B, must catalyze the exchange of GDP for GTP on eIF2 (31). Therefore, to examine whether eIF2B activity was affected 30 min following oral leucine administration, extracts from the gastrocnemius were assayed, using eIF2 · [3H]GDP as substrate, for guanine nucleotide exchange activity. However, eIF2B activity in the gastrocnemius of food-deprived controls and rats administered leucine at the 10, 50, and 100% doses did not differ (data not shown; measurements were not made with the remaining doses). Thus an alteration in eIF2B activity does not appear to contribute to the aforementioned changes in protein synthetic rates.

Translation initiation may also be regulated through the binding of mRNA to a protein complex, composed of eIF4A, eIF4E, and eIF4G and referred to as eIF4F, which facilitates transport of mRNA to the 40S ribosomal subunit (32). The formation of eIF4F can be limited by sequestration of one of the component proteins of the eIF4F complex, i.e., eIF4E, by the eIF4E binding proteins (4E-BPs) (33). To evaluate the effect of oral leucine administration on eIF4F assembly in the gastrocnemius, coimmunoprecipitation experiments were performed. As demonstrated in Fig. 3A, the disassociation of 4E-BP1 from eIF4E 30 min following leucine administration preceded the observed changes in protein synthetic rates, with significant differences existing between controls and rats administered doses of leucine as low as 5%. The ability of 4E-BPs to sequester eIF4E is demarcated by their phosphorylation state, because hyperphosphorylation of the 4E-BPs results in a decreased binding affinity for eIF4E (34). Accordingly, SDS-PAGE analysis revealed that 4E-BP1 phosphorylation was increased in the gastrocnemius of rats fed leucine compared to controls (Fig. 3B), with significant differences from controls observed at all doses of leucine aside from the 5% dose.

FIGURE 3

Changes in the association of (A) eIF4E-BP1 with eIF4E and (B) 4E-BP1 phosphorylation in gastrocnemius muscle of rats following oral leucine administration. The amount of 4E-BP1 bound to eIF4E was assessed by coimmunoprecipitation of the 2 proteins 30 min following administration of saline or leucine at doses ranging from 0.068 to 1.35 g leucine/kg body wt. When subjected to SDS-PAGE, 4E-BP1 is resolved into multiple electrophoretic forms whereby the most highly phosphorylated form, i.e., the γ-form, exhibits the slowest mobility. Phosphorylation of 4E-BP1 was assessed by Western blot analysis 30 min following administration of saline or leucine at doses ranging from 0.068 to 1.35 g leucine/kg body wt using a polycolonal antibody that recognizes phosphorylated and unphosphorylated forms of 4E-BP1 and expressed as the percentage of protein in the γ-form. Results of typical blots are shown in the inset. 4E-BP1, coimmunoprecipitated 4E-BP1; eIF4E, total immunoprecipitated eIF4E; α, α-form of 4E-BP1; β, β-form of 4E-BP1; γ, γ-form of 4E-BP. Values are means ± SE, n = 8–12. *Different from saline-treated controls, P < 0.05.

FIGURE 3

Changes in the association of (A) eIF4E-BP1 with eIF4E and (B) 4E-BP1 phosphorylation in gastrocnemius muscle of rats following oral leucine administration. The amount of 4E-BP1 bound to eIF4E was assessed by coimmunoprecipitation of the 2 proteins 30 min following administration of saline or leucine at doses ranging from 0.068 to 1.35 g leucine/kg body wt. When subjected to SDS-PAGE, 4E-BP1 is resolved into multiple electrophoretic forms whereby the most highly phosphorylated form, i.e., the γ-form, exhibits the slowest mobility. Phosphorylation of 4E-BP1 was assessed by Western blot analysis 30 min following administration of saline or leucine at doses ranging from 0.068 to 1.35 g leucine/kg body wt using a polycolonal antibody that recognizes phosphorylated and unphosphorylated forms of 4E-BP1 and expressed as the percentage of protein in the γ-form. Results of typical blots are shown in the inset. 4E-BP1, coimmunoprecipitated 4E-BP1; eIF4E, total immunoprecipitated eIF4E; α, α-form of 4E-BP1; β, β-form of 4E-BP1; γ, γ-form of 4E-BP. Values are means ± SE, n = 8–12. *Different from saline-treated controls, P < 0.05.

Assembly of the eIF4F complex was evaluated by measuring the association of eIF4G with eIF4E and, whereas the disassociation of 4E-BP1 from eIF4E following leucine administration was significant at the 5% dose, coimmunoprecipitation experiments revealed that significant increases in eIF4G/eIF4E association only occurred following the administration of leucine at the 10% dose or greater (Fig. 4A). eIF4F assembly may also be regulated through phosphorylation of eIF4G, because phosphorylation of eIF4G on Ser1108 facilitates its association with eIF4E (35). Western blot analysis with a phospho-specific eIF4G antibody demonstrated (Fig. 4B) that changes in Ser1108 phosphorylation precede the observed changes in protein synthetic rates, with significant differences existing between controls and rats administered doses of leucine as low as 5%. These results indicate that modulation of eIF4F complex formation is a mechanism whereby low physiological doses of orally administered leucine may affect muscle protein synthesis in the gastrocnemius of food-deprived rats.

FIGURE 4

Changes in (A) the phosphorylation state of eIF4G and (B) its association with eIF4E in gastrocnemius muscle of rats following oral leucine administration. The amount of eIF4G bound to eIF4E was assessed by coimmunoprecipitation of the 2 proteins 30 min following administration of saline or leucine at doses ranging from 0.068 to 1.35 g leucine/kg body wt. Phosphorylation of eIF4G was assessed by Western blot analysis 30 min following administration of saline or leucine at doses ranging from 0.068 to 1.35 g leucine/kg body wt using an anti-phospho-eIF4G antibody that specifically recognizes the protein when it is phosphorylated on Ser1108. Results of typical blots are shown in the inset. eIF4G(P), eIF4G phosphorylated on Ser1108; eIF4G, total eIF4G content; eIF4E, total immunoprecipitated eIF4E. Values are means ± SE, n = 8–12. *Different from saline-treated controls, P < 0.05.

FIGURE 4

Changes in (A) the phosphorylation state of eIF4G and (B) its association with eIF4E in gastrocnemius muscle of rats following oral leucine administration. The amount of eIF4G bound to eIF4E was assessed by coimmunoprecipitation of the 2 proteins 30 min following administration of saline or leucine at doses ranging from 0.068 to 1.35 g leucine/kg body wt. Phosphorylation of eIF4G was assessed by Western blot analysis 30 min following administration of saline or leucine at doses ranging from 0.068 to 1.35 g leucine/kg body wt using an anti-phospho-eIF4G antibody that specifically recognizes the protein when it is phosphorylated on Ser1108. Results of typical blots are shown in the inset. eIF4G(P), eIF4G phosphorylated on Ser1108; eIF4G, total eIF4G content; eIF4E, total immunoprecipitated eIF4E. Values are means ± SE, n = 8–12. *Different from saline-treated controls, P < 0.05.

Phosphorylation of the ribosomal protein S6 kinase, S6K1, represents another mechanism whereby translation initiation may be regulated (36–38). Hyperphosphorylated S6K1 represents one mechanism for enhancing the translation of mRNAs that contain a terminal oligopyrimidine sequence (TOP mRNAs) (39). The pertinence of these TOP mRNAs stems from the fact that they often encode components of the translational apparatus itself. Hyperphosphorylation of S6K1 may thereby facilitate the immediate upregulation of a small subset of proteins necessary for any subsequent increase in global rates of protein synthesis. Only when S6K1 is already highly phosphorylated does it become phosphorylated at Thr389 (37). Thus by employing a phospho-specific antibody against Thr389, it was possible to assess S6K1 hyperphosphorylation. The phosphorylation of Thr389 in the gastrocnemius of rats administered leucine (Fig. 5) was significantly greater than control values at all doses investigated. Therefore, the activation of S6K1 may represent an additional mechanism whereby low doses of leucine stimulate skeletal muscle protein synthesis.

FIGURE 5

Changes in the phosphorylation state of S6K1 in gastrocnemius muscle of rats following oral leucine administration. Phosphorylation of S6K1 was assessed 30 min following administration of saline or leucine at doses ranging from 0.068 to 1.35 g leucine/kg body wt by Western blot analysis using an anti-phospho-S6K1 antibody that specifically recognizes the protein when it is phosphorylated on Thr389. A typical blot is shown in the inset. S6K1(P), S6K1 phosphorylated on Thr389. Values are means ± SE, n = 8–12. *Different from saline-treated controls, P < 0.05.

FIGURE 5

Changes in the phosphorylation state of S6K1 in gastrocnemius muscle of rats following oral leucine administration. Phosphorylation of S6K1 was assessed 30 min following administration of saline or leucine at doses ranging from 0.068 to 1.35 g leucine/kg body wt by Western blot analysis using an anti-phospho-S6K1 antibody that specifically recognizes the protein when it is phosphorylated on Thr389. A typical blot is shown in the inset. S6K1(P), S6K1 phosphorylated on Thr389. Values are means ± SE, n = 8–12. *Different from saline-treated controls, P < 0.05.

DISCUSSION

Oral administration of a single bolus of leucine in an amount equivalent to the daily intake (1.35 g/kg body wt) stimulates skeletal muscle protein synthetic rates in food-deprived rats and this stimulation is mediated, at least in part, through the modulation of proteins that regulate the initiation stage of mRNA translation (9,13,22). It is currently unknown, however, whether lower doses of leucine may be as efficacious in stimulating protein synthesis and/or modulating translation initiation factors.

Similar to what has been reported previously (13), the synthesis rate of total mixed protein in the gastrocnemius of 18-h food-deprived rates was 43% greater than that of controls 30 min after oral administration of leucine at the 100% dose. Interestingly, protein synthesis rates were also enhanced in rats administered the 10% dose. It has been hypothesized (13) that a transient increase in serum insulin concentrations is necessary for the enhancement of protein synthetic rates in the gastrocnemius of food-deprived rats following oral administration of leucine. In this study, however, synthetic rates in the gastrocnemius were significantly greater than control values 30 min following leucine administration even with low doses of leucine that did not induce a significant insulin release. The results therefore suggest that leucine can stimulate skeletal muscle protein synthesis in food-deprived rats without concomitant increases in circulating insulin. Interestingly, similar results were attained in a recent study on the regulation of skeletal muscle protein synthesis in neonatal pigs infused with low doses of leucine (40).

A possible explanation for the leucine-induced insulin-independent changes in skeletal muscle protein synthesis observed in this study, but not in the study by Anthony et al. (13), is that the stimulation of skeletal muscle protein synthesis by leucine is transient (13). As such, significant changes in protein synthesis may be observable 30 min following leucine administration, as in the present study, but not at 1 h, as in the prior study. This suggestion is supported by the recent observation that 30 min following the administration of the leucine analogue norleucine, protein synthesis in the gastrocnemius of food-deprived rats is enhanced to the same extent as in rats administered leucine, but without an increase in serum insulin (41). A cautionary note should be made, however, because it is possible that insulin secretion induced by small doses of leucine may occur more rapidly than with large doses of leucine and, therefore, that a transient leucine-induced increase in circulating insulin may contribute to these results.

Independent of dose, leucine appears to mediate its effect on skeletal muscle protein synthesis, at least in part, via the regulation of mRNA translation initiation. Of the biomarkers of mRNA translation examined in the present study, the one that correlated best with the observed changes in protein synthesis was the amount of eIF4G associated with eIF4E. Both protein synthesis and eIF4G binding to eIF4E were slightly, but not significantly (P = 0.67 and 0.25), increased at the lowest leucine dose tested and both were maximally changed at 10% of the highest tested dose of the amino acid, i.e., when there is a relatively small increase in serum leucine concentrations, similar to that observed following intake of a complete meal. The other biomarkers examined were also affected by leucine concentrations in the 5–25% range, but were further altered in response to higher doses. Although changes in the other biomarkers did not correlate directly with altered rates of global protein synthesis or eIF4G association with eIF4E, a role for such changes in the regulation of mRNA translation cannot be eliminated. Indeed, it is likely that the observed changes in eIF4G phosphorylation and 4E-BP1 association with eIF4E contribute to the enhanced binding of eIF4G to eIF4E observed at lower leucine doses. Moreover, and as with changes in circulating insulin concentrations, it should be noted that the measurements in this study represent but a snapshot of possible effects induced by leucine administration. Thus it is possible that changes in some biomarkers occurred soon after leucine administration, but returned to control levels by 30 min. These changes would have also contributed to the observed changes in protein synthetic rate. A more thorough assessment of the contribution of translational control mechanisms to leucine-stimulated protein synthesis will therefore require earlier time course data.

Unlike protein synthesis and eIF4G binding to eIF4E, hyperphosphorylation of 4E-BP1 and phosphorylation of S6K1 on Thr389 were directly correlated with serum leucine concentration. However, the serum leucine concentration measured at the highest dose is several-fold greater than that observed after a complete meal and is thus supraphysiological. The finding that supraphysiological serum leucine concentrations directly correlate with enhanced phosphorylation of 4E-BP1 and S6K1 would argue against the effects being mediated through a receptor-based mechanism, which would be expected to exhibit maximal activation at leucine concentrations near, or slightly above, physiological levels. This is particularly relevant for 4E-BP1 phosphorylation because its only known function is to promote release of the protein from the 4E-BP1 · eIF4E complex to allow assembly of the eIF4G · eIF4E complex. In contrast, the finding that protein synthesis and eIF4G association with eIF4E are maximally stimulated at a physiological serum leucine concentration would be consistent with these events being regulated through a receptor-mediated process.

A potential explanation for the lack of correlation between leucine signaling to 4E-BP1 and changes in eIF4G association with eIF4E is that the amount of eIF4G available for formation of the eIF4G · eIF4E complex is limiting (42) and that release of a fraction of total eIF4E from the 4E-BP1 · eIF4E complex provides enough eIF4E to promote maximal formation of the eIF4G · eIF4E complex. Alternatively, eIF4G binding to eIF4E may be differentially regulated. For example, administration of the drug rapamycin, a highly specific inhibitor of the kinase mammalian target of rapamycin (mTOR), to rats in vivo completely abrogates the leucine-induced dissociation of 4E-BP1 from eIF4E in skeletal muscle, whereas the leucine-induced association of eIF4G with eIF4E is only partially inhibited (43). Thus whereas an mTOR-dependent pathway regulates the association of eIF4E with 4E-BP1, eIF4E's association with eIF4G is regulated by both mTOR-dependent and -independent pathways and a differential response in the association of eIF4G and 4E-BP1 with eIF4E in this study is not totally unexpected. Several biomarkers examined in the present study appear to exhibit a biphasic response to leucine administration, reaching a plateau at low to moderate doses of the amino acid and increasing further as the leucine dose is increased. For example, 4E-BP1 association with eIF4E is significantly decreased at the 5% leucine dose and then remains constant until 50%, when it decreases further. Phosphorylation of eIF4G on Ser1108 exhibits a similar pattern of change. Interestingly, for both biomarkers the dose of leucine that elicits the second change (i.e., 50%) also results in a significant increase in serum insulin concentration. Thus one explanation for the apparent biphasic response of eIF4G phosphorylation and 4E-BP1 association with eIF4E is that insulin released in response to provision of higher leucine concentrations mediates the changes observed at 50 and 100% doses. In support of this idea, leucine and insulin act synergistically to enhance S6 phosphorylation in skeletal muscle of neonatal pigs (44) and humans (45).

Although leucine-induced insulin secretion was not associated with significant increases in skeletal muscle protein synthetic rates in the current study, this rise in circulating insulin may still affect mRNA translation. For example, the translational control mechanisms initiated by leucine-induced insulin secretion may affect the translation of a subset of mRNAs, whose increased synthesis alone would not be detectable by the methods employed in this study. For example, mRNAs having highly structured 5′-untranslated regions appear to be preferentially translated under conditions of enhanced eIF4F assembly (49–51). Pertinently, several mRNAs encoding proteins that regulate cell growth and development have such untranslated structures.

In conclusion, the oral administration of low doses of leucine effectively stimulates skeletal muscle protein synthesis in food-deprived mature rats. Because proteins that facilitate both the transportation of mRNA to the 40S ribosomal subunit and the synthesis of the translational apparatus itself are affected by low-dose leucine administration, changes in the control of mRNA translation initiation likely contribute to this stimulation of protein synthesis. Although the study is limited in its ability to assess which pathways mediate the observed changes in translational control and protein synthesis, it does demonstrate that activation, or perhaps more correctly the degree of activation, of these pathways is dependent upon the dose of leucine administered. These results also suggest that the small increases in circulating insulin levels arising from the administration of high doses of leucine may further stimulate mRNA translation in the skeletal muscle of food-deprived mature rats. That the oral administration of small doses of leucine can stimulate skeletal muscle protein synthesis in food-deprived rats suggests that future research on the regulation of skeletal muscle protein synthesis by orally administered leucine will be feasible in humans. It has been suggested previously that leucine administration may prove to be an effective therapy for conditions such as type 2 diabetes, trauma, and infection that are characterized by both insulin-resistant and skeletal muscle wasting (45). The results of this study support this proposal, but further studies will be required to elucidate how long-term leucine administration affects protein turnover and skeletal muscle mass in humans.

The authors sincerely thank Lynne Hugendubler for assistance with eIF2B activity measurements and Courtney Bradley, Sharon Rannels, Jamie Crispino, Neil Kubica, and David Williamson for assistance with sample collection. We also recognize the Pennsylvania State University GCRC Microdialysis Core Lab for help with protein synthesis measurements.

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5 Ways to Increase Protein Synthesis without Eating More Protein

Protein timing and its effects on muscular hypertrophy and strength in individuals engaged in weight-training

Journal of the International Society of Sports Nutritionvolume 9, Article number: 54 (2012) Cite this article

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Abstract

The purpose of this review was to determine whether past research provides conclusive evidence about the effects of type and timing of ingestion of specific sources of protein by those engaged in resistance weight training. Two essential, nutrition-related, tenets need to be followed by weightlifters to maximize muscle hypertrophy: the consumption of 1.2-2.0 g protein.kg -1 of body weight, and ≥44-50 kcal.kg-1 of body weight. Researchers have tested the effects of timing of protein supplement ingestion on various physical changes in weightlifters. In general, protein supplementation pre- and post-workout increases physical performance, training session recovery, lean body mass, muscle hypertrophy, and strength. Specific gains, differ however based on protein type and amounts. Studies on timing of consumption of milk have indicated that fat-free milk post-workout was effective in promoting increases in lean body mass, strength, muscle hypertrophy and decreases in body fat. The leucine content of a protein source has an impact on protein synthesis, and affects muscle hypertrophy. Consumption of 3–4 g of leucine is needed to promote maximum protein synthesis. An ideal supplement following resistance exercise should contain whey protein that provides at least 3 g of leucine per serving. A combination of a fast-acting carbohydrate source such as maltodextrin or glucose should be consumed with the protein source, as leucine cannot modulate protein synthesis as effectively without the presence of insulin. Such a supplement post-workout would be most effective in increasing muscle protein synthesis, resulting in greater muscle hypertrophy and strength. In contrast, the consumption of essential amino acids and dextrose appears to be most effective at evoking protein synthesis prior to rather than following resistance exercise. To further enhance muscle hypertrophy and strength, a resistance weight- training program of at least 10–12 weeks with compound movements for both upper and lower body exercises should be followed.

Review

Purpose

Individuals who engage in resistance weight training, whether as competitive weightlifters or to promote optimal physical outcomes, would benefit by knowing the ideal nutritional intake protocol needed to maximize muscle hypertrophy and strength. The type, timing (pre/post workout) or amount of protein intake required to meet strength-training goals may not be clear to weightlifters or their trainers. The purpose of this review was to determine whether past research provides conclusive evidence about the effects of type and timing of ingesting specific protein sources by those engaged in resistance weight training. The review targets the effects of intake and timing of the following protein sources on physical outcomes: whey, casein, milk, soy and essential amino acids.

Protein and calorie intake

For maximal muscle hypertrophy to occur, weightlifters need to consume 1.2-2.0 grams (g). protein kilogram. (kg)-1 and > 44–50 kilocalories (kcal).kg-1 body weight daily[1–9]. This is considerably higher than the recommended dietary allowance (RDA) for protein (currently 0.8 g.kg-1) which meets the needs of 97.5% of all healthy adult Americans not engaged in weightlifting with the intent of gaining muscle mass[8]. Table1 summarizes ranges for protein intake for weightlifters based on previous literature reviews.

Full size table

Leucine and muscle protein synthesis

The leucine content of a protein source has an impact on protein synthesis, and affects muscle hypertrophy[10–15]. This section details the role of leucine in protein synthesis to illustrate its importance in the process.

Protein synthesis occurs when methionyl-transfer ribonucleic acid (methionyl-tRNA) binds to a eukaryotic small ribosomal subunit (40S ribosomal unit) resulting in the formation of a pre-initiation complex (43S pre-initiation complex)[16]. This initial step is mediated by eukaryotic initiation factor 2 (eIF2)[16]. The 43S complex subsequently binds to messenger ribonucleic acid (mRNA) near the cap structure. After successful engagement of the 43S pre-initiation complex to RNA, the molecule eukaryotic initiation factor 5 (eIF5) removes eIF2 while a molecule of guanosine triphospahte (GTP) is hydrolyzed so that eIF2 is recycled to its active form of eIF2-GTP[16]. This allows eIF2-GTP to continue with the initial step of protein synthesis. Once eIF2-GTP is released, the second step can occur. A ribosomal binding site/translation start site forms once eukaryotic initiation factor 4F (eIF4F) recognizes the molecule[16]. The eIF4F complex binds the eukaryotic initiation factor 4E (eIF4E) subunit of eIF4F to the m7GTP cap structure present in all eukaryotic mRNAs[16]. Replication of the mRNA strand occurs, thus indicating protein synthesis. The processes of protein synthesis appear to be highly regulated by the amino acid leucine[10–14].

Leucine plays a role in muscle protein synthesis mostly through stimulation of the mammalian target of rapamaycin (mTOR) signaling pathway[15, 17, 18]. Leucine interacts with two mTOR regulatory proteins, mTOR raptor (or raptor) and rashomolog enriched in the brain (or Rheb)[19, 20]. The importance of the regulation of mTOR is that when activated, it phosphorylates the proteins eIF4E binding protein 1 (4E-BP1) and ribosomal protein S6 kinase (S6K1) complex[21, 22]. When 4E-BP1 is phosphorylated, it becomes inactive, which allows the continuation of the second step initiation phase of translation by inhibiting its binding to eIF4F complex[10]. This allows additional translation to occur. When S6K1 is phosphorylated, it produces additional eIFs which increases the translation of mRNAs that encode components of the protein synthesis pathway[10, 12].

Leucine has been indicated as the sole stimulator of protein synthesis[10–15]. For example, Dreyer et al. conducted a study on 16 young, healthy untrained men to determine the effects of post-workout consumption of either no beverage or leucine-enhanced EAAs[15]. Those consuming the leucine-enhanced beverage one hour following a single bout of resistance exercise had greater rates of protein synthesis than did the control group. Another study conducted by Koopman et al.[23] concurs with the findings of Dreyer. Eight untrained men were randomly assigned to consume one of the three beverages: carbohydrates, carbohydrate and protein or carbohydrate, protein and free leucine following 45 minutes of resistance exercise. The results indicated that whole body net protein balance was significantly greater in the carbohydrate, protein and leucine group compared with values observed in the carbohydrate and protein and carbohydrate only groups, indicating the ability of leucine to augment protein synthesis[23].

Leucine alone appears to be nearly as effective in stimulating protein synthesis as when all branched chain amino acids (BCAAs) are consumed[24–26]. Leucine also seems to have both insulin-dependent and insulin-independent mechanisms for promoting protein synthesis[27, 28]. Approximately 3 to 4 g of leucine per serving is needed to promote maximal protein synthesis[29, 30]. See Table2 for the leucine content of protein sources for all protein ingestion timing studies referenced in this review.

Full size table

Types of protein

There are numerous protein sources available to the consumer. This review article focuses on studies that have used a variety of dairy- and soy-based protein sources. This section describes each of these protein sources and compares their quality on the two scales most relevant to this review: biological value and protein digestibility corrected amino acid score (PDCAAS)[44]. Biological value (BV), determines how efficiently exogenous protein leads to protein synthesis in body tissues once absorbed, and has a maximum score of 100[44]. PDCAAS numerically ranks protein sources based on the completeness of their essential amino acid content, and has a maximum score of 1.0[44]. The BV and PDCAAS are both important in understanding bioavailability and quality of different protein sources.

Three sources of dairy protein typically used in studies of muscle hypertrophy and strength are bovine milk, casein and whey. Bovine milk is a highly bioavailable source of protein, comprising 80% casein and 20% whey[44]. Overall, bovine milk has a BV of 91 and a PDCAAS of 1.00 indicating that it is readily absorbed by the body, promoting protein synthesis and tissue repair, and provides all essential amino acids (EAAs). Casein, with a BV of 77 and a PDCAAS of 1.00, is the predominate protein in bovine milk and gives milk its white color[44]. It exists in micelle form, and within the stomach will gel or clot, thus resulting in a sustained release of amino acids[45]. Compared with milk, it is less bioavailable, but like milk, it provides all EAAs. Whey the other protein found in milk, is the liquid part of milk that remains after the process of cheese manufacturing[44]. With a BV of 104 and a PDCAAS of 1.00, whey is superior to both milk and casein. It contains all EAAs, and its excellent bioavailability leads to rapid protein synthesis[44, 45].

Soy is a vegetable-based protein source that is useful for vegetarians and individuals who are lactose- or casein-intolerant. Soy has a BV of 74 and PDCAAS of 1.00, indicating that it is not as bioavailable as milk based protein, but does contain all EAAs[44].

Whole-food protein intake studies: post workout only

The timing of protein intake has been an important condition in studies on muscle hypertrophy and strength in weight-trained individuals. In this section, studies using whole-food protein sources (i.e. bovine and soy milk) have been reviewed with respect to their intake following weight-resistance training.

Many studies on the effects of protein intake timing on physical changes have used protein supplements[31–36], but some studies have used milk and other fluid protein sources. In a study focused on protein intake following a single resistance training session, Elliot et al. examined milk consumption post-workout in 24 untrained men and women[37]. Subjects were randomly assigned to one of three groups: 237 g of fat-free milk, 237 g of whole milk, or 393 g of isocaloric fat-free milk. The findings indicated that in untrained individuals, threonine uptake was significantly higher for those consuming 237 g whole milk versus those consuming 237 g fat free milk. Threonine uptake is indicative of net muscle protein synthesis. The results of this study suggest that whole milk increased utilization of available amino acids for protein synthesis[37]. Tipton et al. conducted a study on 23 untrained men and women in which participants ingested 1) 20 g casein, 2) 20 g whey, or 3) artificially sweetened water one hour following heavy leg resistance exercise[46] Positive changes in net muscle protein balance resulted for both protein groups but not for the control group. This study indicated that milk proteins (both casein and whey) post-workout increased protein synthesis[46].

Various studies have compared whole-food protein sources to determine which is most effective in improving muscle mass and strength gains. Hartman et al. conducted a study comparing the use of milk, soy protein, or carbohydrate drinks by 56 young untrained males[38]. Subjects were assigned to one of three groups; each consumed 500-milliliter (mL) of a) fat-free milk, b) an isocaloric, isonitrogenous, and macronutrient- matched soy-protein beverage, or c) an isocaloric carbohydrate beverage immediately following and again one hour after resistance exercise. Body composition, muscle hypertrophy, and strength measurements were recorded at baseline and three days following 12 weeks of training 5 d.wk-1. The group using milk post-workout had significantly increased body weight and decreased body fat versus the other two groups, indicating an increase in lean body mass (LBM). Results indicated that consumption of fat-free milk post-workout was statistically more effective than soy protein in promoting increases in LBM (p<0.01), increases in type II muscle fiber area (p<0.05) and decreases in body fat (p<0.05)[38]. These results were similar to those found by Wilkinson et al.[39]. Researchers assigned eight weight-trained men to either 500 mL of skim milk or an isonitrogenous, isocaloric, and macronutrient-matched soy-protein beverage following resistance exercise[39]. A crossover design was used so that all participants consumed either milk or soy on their first trials and alternated to the other supplement on the second trials. Trials were separated by one week. Both protein drinks increased protein synthesis and promoted increases in muscle mass; however, the consumption of skim milk had a significantly greater impact on the development of muscle mass than did consumption of the soy protein[39]. Both Hartman et al.[38] and Wilkinson et al.[39] demonstrated the superiority of milk proteins over soy protein in building muscle mass. This may be due to the fact that soy has a lower BV than milk (74 versus 91 respectively), resulting in lower bioavailability, thus providing less protein synthesis in body tissues.

Rankin et al. studied the effects of milk versus carbohydrate consumption post-resistance exercise on body composition and strength[40]. Nineteen untrained men were randomly assigned to one of two groups that provided 5 kcal.kg-1 body weight of either chocolate milk, or a carbohydrate-electrolyte beverage. Subjects completed whole body dual-energy X-ray absorptiometry (DXA) scans and strength assessments prior to and after following a 3 d.wk-1 for 10-weeks weightlifting protocol. Results indicated that both groups had increases in LBM and strength, but there were no significant between-group differences[40]. The addition of a control group to this study would have helped determine whether increases in strength were due solely to the weightlifting program or to the combination of exercise and supplementation. The findings suggest that consumption of chocolate milk post-exercise may be effective in increasing LBM in weightlifters, but more studies using control groups are needed.

Milk consumption and resistance training also have been investigated in women. Josse et al. examined the effects of milk consumption post-workout on strength and body composition in 20 healthy untrained women[41]. Subjects were assigned to 500 mL of either fat-free milk or isocaloric maltodextrin. The women followed a weight training protocol 5 d.wk-1 for 12-weeks. Each participant completed strength assessments, DXA scans, and blood tests. The group consuming milk had statistically greater increases in LBM, greater fat mass losses and greater gains in strength, providing evidence that fat-free milk consumption post-workout was effective in promoting increased LBM and strength in women weightlifters[41]. The results of this study support those of previous studies completed in men showing that milk consumption post-workout has a favorable effect on MPS[37–40].

Protein supplement intake studies: a comparison of timing protocols

Protein and amino acid supplements have been used widely in studies showing their effectiveness on protein synthesis. Hoffman et al. compared protocols providing protein supplementation and subsequent effects on muscle strength and body composition in 33 strength-trained adult men[31]. Two protein-intake timing strategies were implemented over the course of 10-weeks of resistance weight-training[31]. One group consumed a protein supplement comprising enzymatically hydrolyzed collagen-, whey-, and casein-protein isolates pre/post-workout. A second group consumed the same supplement in the morning upon awakening and in the evening. A control group was not given the protein blend. The average caloric intake of the three groups was 29.1 ± 9.7 kcal.kg body mass-1.d-1.

Muscle strength was assessed through one-repetition maximum (1RM) on bench and leg press. Body composition was assessed using DXA[31]. There were no group differences in body composition based on timing of supplementation[31]. All groups increased the 1RM for squats, indicating increased muscle strength. Only the protein supplement groups also showed significant increases in the 1RM for bench press, indicating improved strength[31]. These findings indicated that supplementation was beneficial for increasing muscle strength in 1 RM bench press but timing of ingestion was not important. The results on body composition may have had different effects if participants had consumed adequate kcal.kg-1, as greater-than-maintenance-caloric needs are required for muscular hypertrophy to occur. Strength did increase, providing evidence to both the effectiveness of protein supplementation on strength and the effectiveness of the workout regimen used in this study. Future studies should ensure that participants are consuming greater than 44–50 kcal.kg-1 to maximize muscle hypertrophy[9].

Hoffman et al. conducted a double-blind study focusing on the use of protein supplements to hasten recovery from acute resistance weight training sessions[32]. Fifteen strength-trained men were matched for strength then randomly assigned to receive 42 g of either a) a proprietary protein blend (enzymatically hydrolyzed collagen-, whey-, or casein-protein isolates, plus 250 mg of additional BCAA pre and post workout), or b) a placebo of maltodextrin pre-and post-workout[32]. Participants initially performed a 1RM for squat, dead lift, and barbell lunge exercises. On the second visit, subjects performed four sets of at least 10 repetitions at 80% of their 1RM for the exercises with 90 seconds between sets. On visits three (24 hours from visit two) and four (48 hours from visit two), participants performed four sets of squats with the previous weight and performed as many repetitions per set as possible[32]. Hoffman et al.[32] found that the group receiving the proprietary protein blend performed significantly more repetitions at visits three and four than did subjects receiving the placebo. These findings provide evidence that protein supplementation pre- and post-workout is useful in maximizing weight-training performance, as well as in hastening exercise recovery 24 and 48 hours post-exercise.

Timing of supplementation in relation to the resistance workout also has been studied[33]. Cribb et al. assigned 23 male bodybuilders to one of two groups: those who received a supplement a) before and after a workout, or b) in the morning and evening. The supplement contained 40 g protein (from whey isolate), 43 g carbohydrate (glucose), and seven g creatine monohydrate per 100 g. Each participant was given the supplement in quantities of 1.0 g.kg-1 body weight. All participants followed a preliminary resistance weight-training program for 8–12 weeks before baseline measurements were taken. Participants then started the 10-week resistance weight-training session which was divided into three distinct stages: preparatory (70–75% 1RM), overload phase 1 (80–85%1RM), and overload phase 2 (90–95% 1RM)[33].

Results indicated significant differences in body composition in the group consuming the supplement pre- and post-workout[33]. This group experienced increased LBM and decreased body fat. Both groups demonstrated increases in strength, but the pre- and post-workout group demonstrated significantly greater gains[33], indicating that timing of the ingestion of the protein supplement was crucial. This is contradictory to the findings of Hoffman et al.[31] with respect to changes in body composition. This could be because Cribb et al.[33] used a supplement that was a combination of protein, carbohydrate and creatine whereas, Hoffman et al.[31] supplemented with protein only. The major finding of this study was that after 10 weeks of training, supplementation pre/post each workout resulted in greater improvements in 1RM strength and body composition (increased LBM and decreased body fat percentage) compared with a matched group who consumed supplement in the morning and evening, outside of the pre- and post-workout time frames.

The majority of studies of protein intake and resistance exercise have been conducted on younger adult males[31–33]. In contrast, Verdijk et al. investigated the impact of the protein, casein hydrolysate, on muscle hypertrophy in healthy untrained elderly men[34]. Researchers randomly assigned 28 elderly men to consume either a protein supplement or a placebo pre- and post-workout. Subjects performed a 12-week resistance weight-training program requiring weightlifting 3 d .wk-1. Baseline and ending measurements were obtained, including strength assessments, CT scans, DXA scans, blood samples, 24-hour urine samples, muscle biopsies, and immunohistochemistry tests. Results indicated no differences in ending measurements between the protein group and placebo group in muscle hypertrophy, strength, or body composition[34], suggesting that for elderly men, intake of 20 g casein hydrolysate before and after resistance training does not increase muscle hypertrophy or strength. In this study, however, only 20 g of casein was used, and it was divided into two servings. This protocol would not have provided participants with the required 3 g of leucine needed to maximize protein synthesis. Additionally, since casein is slow digesting[44, 45], it may not have been ideal for use in a study of elderly men. Future studies with this population should incorporate whey protein, which is highly bioavailable in an amount that would provide at least 3 g leucine[29, 30]. Studies comparing the effects of supplementation with adequate protein and those with creatine-enhanced protein pre-and post-workout also should be conducted to determine whether creatine is needed to produce the desired outcomes, as has been demonstrated in younger men[33] (See Table2).

The long-term use of whey protein pre- and post- resistance exercise was investigated by Hulmi et al.[35], by assigning participants to one of three groups:1) 15 g of whey protein before and after resistance exercise, 2) a placebo before and after resistance exercise, or 3) no supplement no participation in weightlifting but continued habitual exercise as they did prior to the study. Participants in the first two groups completed two resistance exercise sessions per week for 21 weeks consisting of both upper and lower body multi-joint lifts. All participants then had biopsies performed on their vastus lateralis. Results indicated that the whey protein group had significantly greater increases than the other groups in vastus lateralis hypertrophy, and greater overall muscle hypertrophy[35]. These findings provide evidence that whey protein supplementation pre- and post-workout is useful in increasing muscle hypertrophy.

Andersen et al. examined the effects of a mixed blend of proteins on muscle strength and muscle fiber size[36]. They studied the ingestion pre- and post-workout of 25 g of a protein blend (whey, casein, egg-white proteins, and l-glutamine), compared with a maltodextrin supplement, over the course of a 3 d.wk-1 14-week resistance-training program. Results of muscle biopsies from the vastus lateralis indicated that the protein supplementation group had greater increases in muscle hypertrophy and in squat jump height[36]. Results of this study provide evidence that supplementation with a blend of whey, casein, egg-white proteins, and l-glutamine pre- and post-workout helps promote muscle hypertrophy and improved physical performance.

Training effects

The effects of training protocols also are very important on increases in strength and muscle hypertrophy. All studies used in this review followed a resistance weight-lifting protocol[31–36, 38–41]. It appears from the studies referenced in this review that a training protocol tailored for muscle hypertrophy and strength should be at least 10–12 weeks in length and involve three to five training sessions weekly, consisting of compound lifts that include both the upper and lower body[31, 33, 35, 36, 38, 40, 41].

Conclusions

Researchers have tested the effects of types and timing of protein supplement ingestion on various physical changes in weightlifters. In general, protein supplementation pre- and/or post-workout increases physical performance[31–34, 38–41], training session recovery[32], lean body mass[33, 38–41], muscle hypertrophy[35, 38–41], and strength[31, 33, 38, 40, 41]. Specific gains, however, differ based on protein type and amounts[31–36]. For example, whey protein studies showed increases in strength[31, 33], whereas, supplementation with casein did not promote increases in strength[34]. Additional research is needed on the effects of a protein and creatine supplement consumed together, as one study has shown increases in strength and LBM[33].

Studies on timing of milk consumption have indicated that fat-free milk post-workout was effective in promoting increases in lean body mass, strength, muscle hypertrophy and decreases in body fat[38–41] Milk proteins have been shown to be superior to soy proteins in promoting lean body mass[38] and muscle mass development[39]. What is interesting about the milk studies[38–41] is that not one of them provided the 3–4 g of leucine needed to promote maximal MPS (See Table2), yet they all showed improvements in LBM and strength. This raises the question of whether other components in milk could have contributed to the changes observed. Future researchers should investigate whether other properties of milk help increase LBM when leucine intake is suboptimal to provide maximal MPS. Researchers should also investigate the effects of protein supplements when participants are consuming adequate kcal.kg-1 and g.kg-1 of protein to maximize muscle hypertrophy.

The effects of timing of ingestion of EAAs on physical changes following exercise also have been studied[47, 48]. Tipton et al.[47] found that the ingestion of EAAs prior to resistance exercise was more beneficial than post-ingestion in promoting protein synthesis[47], but these results did not hold true with respect to whey protein ingestion[48]. Once a protein has been consumed by an individual, anabolism is increased for about three hours postprandial with a peak at about 45–90 minutes[14]. After about three hours postprandial, MPS drops back to baseline even though serum amino acid levels remain elevated[14]. These data show that there is a limited time window within which to induce protein synthesis before a refractory period begins. With this in mind, an ideal protein supplement after resistance exercise should contain whey protein, as this will rapidly digest and initiate MPS, and provide 3–4 g of leucine per serving, which is instrumental in promoting maximal MPS[29, 30]. A combination of a fast-acting carbohydrate source such as maltodextrin or glucose should be consumed with the protein source, as leucine cannot modulate protein synthesis as effectively without the presence of insulin[27, 28] and studies using protein sources with a carbohydrate source tended to increase LBM more than did a protein source alone[33, 37–41]. Such a supplement would be ideal for increasing muscle protein synthesis, resulting in increased muscle hypertrophy and strength. In contrast, the consumption of essential amino acids and dextrose appears to be most effective at evoking protein synthesis prior to rather than following resistance exercise[47]. To further enhance muscle hypertrophy and strength, a resistance weight-training program of at least 10–12 weeks 3–5 d .wk-1 with compound movements for both upper and lower body exercises should be followed[31, 33, 35, 36, 38, 40, 41].

Abbreviations

Milliliter

Day

Gram

Kilogram

Week

Recommended dietary allowance

Lean body mass

Maximal protein synthesis

One-repetition maximum

Dual-energy X-ray absorptiometry

Computed tomography

Kilocalories per kilogram

Gram per kilogram

Branched chain amino acids

Essential amino acids

Hour

Methionyl-transfer ribonucleic acid

Eukaryotic small ribosomal subunit

Pre-initiation complex

Eukaryotic initiation factor 2

Messenger ribonucleic acid

Guanosine triphosphate

Eukaryotic initiation factor 4F

Eukaryotic initiation factor 4E

Mammalian target of rapamycin

mTOR raptor

Rashomolog enriched in the brain

eIF4E binding protein 1

Ribosomal protein S6 kinase.

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