Understanding the effects of nutrition and post-exercise nutrition on skeletal muscle protein turnover: Insights from stable isotope studies

MPS is a modifiable process, the magnitude and duration of which can be influenced by a variety of feeding strategies. An early line of enquiry for researchers was to decipher the maximal capacity of the human body to digest, absorb and subsequently utilize the constituent AA for anabolism of contractile and metabolically functional proteins. Fittingly, Mike Rennie's group were amongst the first to demonstrate that MPS was elevated in a dose–response manner to increasing concentrations of circulating (extracellular) AA in resting skeletal muscle []. Organ tissues utilize AA derived from dietary protein intake to replace damaged proteins and to synthesize an array of molecules required for normal bodily function. Acute infusion studies using labelled AA ¹³C₆-Phenylalanine, have demonstrated that muscle protein synthesis is maximally stimulated by protein intakes of ~0.24 g/kg and ~0.4 g/kg body mass in healthy young and older adults at rest in a post-prandial state, respectively []. Beyond these amounts, oxidation of AA – measured in this case as an increase in breath ¹³CO₂ enrichment – is accelerated as is ureagenesis. However, the meal-induced rise in MPS, without the influence of exercise, is transient and returns to basal rates after 2–3 h despite a persistent hyperaminoacidemia and intramuscular anabolic signaling []. Together these findings provide a rationale for evenly distributing protein throughout the day such that each meal maximally stimulates MPS, while simultaneously minimizing catabolism. In support of this approach, 20 g protein beverages consumed every 3 h stimulated myofibrillar protein synthesis to a greater degree than a pulsed (8 × 10 g protein every 1.5 h) or bolus dosing strategy (2 × 40 g protein every 6 h) when measured over 12 h in resistance-trained young men [].

Most adults in Western society consume their daily protein intakes in a skewed manner. A disproportionate amount (~40–50%) of daily protein is consumed at the late-day dinner meal, with the remainder divided amongst breakfast and lunchtime meals. Because of this unequal distribution, <50% of young adults and only ~7.5% of older adults consume the amount of protein at breakfast and lunch that would be required to maximally stimulate MPS []. From a muscle-centric perspective, increasing the amount of protein consumed at breakfast and lunch, while simultaneously reducing the amount consumed at dinner should foster an enhanced anabolic environment throughout the day without increasing daily intake. Indeed, evenly distributing protein intake over breakfast, lunch and dinner meals (30 g at each meal) led to a ~25% greater stimulation of 24 h mixed-muscle protein synthesis compared to a skewed intake of the same total amount of protein (10 g, 15 g, and 65 g at breakfast, lunch and dinner) in middle-aged adults []. Consuming a protein supplement at breakfast also augmented lean mass accretion after 12 RT in young men compared to when the same supplement was consumed at dinner []. Similarly, older adults who increased their protein intake at breakfast and lunch above 0.4 g/kg via milk protein supplementation demonstrated a significantly greater increase in lean body mass over a 24-week dietary intervention period compared to those given a maltodextrin control []. However, the need to evenly distribute protein ingestion has been challenged. When older adults consumed either 0.8 or 1.5 g of protein/kg/day distributed either evenly or skewed (15% breakfast, 20% lunch and 65% dinner) between 3 meals for 4 days, both groups consuming higher protein had greater MPS than the groups consuming less protein, suggesting that protein quantity and not protein distribution affected MPS (Kim et al., 2015). The concept of even protein distribution was further challenged by the same research group when they demonstrated that when older adults consumed 1.1 g of protein/kg/day either distributed evenly or skewed (15% breakfast, 20% lunch and 65% dinner) over 3 meals for 8 weeks no differences in MPS, lean body mass or strength were observed between groups (Kim et al., 2018). Although the protein consumed in this study was of high-quality total protein consumption per day (1.1 g/kg/day) may not be sufficient in maximally stimulating MPS in older individuals and therefore may mask any differences that could exists between protein distribution. Specifically, consuming 1.1.g of protein/kg/day in a balanced manner would result in consuming approximately 0.36 g of protein/kg at each meal which may not be able to maximally stimulate MPS in older adults.

Date supporting the importance of even protein distribution throughout the day is equivocal, therefore there is no concordance on the efficacy of evenly distributed protein the potential relevance and generalizability of the distribution hypothesis to daily nutritional practices []. Most notable is the reality that protein is often consumed as part of a mixed-meal containing carbohydrate and lipid rather than as an isolated nutrient. This ‘whole-food matrix’ alters the digestion and absorption kinetics of protein and can influence the subsequent anabolic response. Indeed, the consumption of an intrinsically-labelled whey protein beverage induces a rapid and transient rise in the rate of phenylalanine appearance, which peaks at ~30–60 min and returns to basal levels by ~180–240 min []. In contrast, the rate of phenylalanine appearance into plasma is sustained at an elevated level for a longer period of time following the ingestion of labelled casein protein []. These distinct absorption profiles probably explain the recent finding that, when whey and casein are co-ingested (i.e. as milk protein), MPS remains elevated throughout the 5hr postprandial period []. This observation is in contrast to rapid return of MPS to basal levels 2–3 h after the ingestion of a whey protein isolate and suggest that ingestion of a mixed-meal may protract the anabolic response. These data argue against a strict 3 h spacing window between meals as implied by earlier work that formed the foundation for the muscle-full hypothesis [,]. More research is now needed to investigate the dose–response of MPS to whole-food protein sources and the daily eating strategies that maximize anabolism over the course of the day to place these findings into a broader nutritional context.

An even rather than a skewed intake of dietary protein may be beneficial during periods of energy restriction (ER). This thesis rests on the observation that ER leads to a reduction in basal and postprandial MPS in young adults despite protein intakes of ~2x RDA []. Our laboratory has also shown a reduction in fed-state myofibrillar protein synthesis following a 4-week hypocaloric dietary intervention in older men []. However, participants who consumed their daily protein evenly distributed across four daily meals (25% daily protein per meal) demonstrated an attenuated decline in acute myofibrillar protein synthesis relative to participants consuming protein in a skewed manner (7:17:72:4% at breakfast, lunch, dinner and pre-bed snack, respectively) []. The effects were subtle, however, and follow up analysis (using biopsy tissue from the same participants) showed that the difference in MPS between groups is no longer significant when measured using D₂O to capture integrated changes over a 2-week period []. Nonetheless, when viewed at the level of individual muscle proteins, balanced and skewed protein intakes differentially regulated the synthesis of proteins belonging to distinct biological pathways. For instance, a balanced intake of dietary protein increased the abundance of proteins involved in myofibril assembly to a greater extent compared to skewed protein intake during ER and RT []. Taken together, these data suggest that balanced protein consumption may be an ideal strategy for distributing daily protein during periods of energy deficit, particularly when combined with RT, and also underscore the importance of using complimentary tracer techniques to capture nuanced but potentially important changes in the muscle proteome that are diluted when quantified as ‘bulk’ sub-fractional averages.

Finally, protein ingestion before sleep should also be considered when determining ideal daily protein distribution patterns. For most individuals sleep represents the longest period spent in the fasted state, and thus a negative NPBAL. In one of the first proof of principle investigations to assess the effectiveness of pre-sleep protein ingestion on muscle anabolism, Groen and colleagues provided participants with 40 g of casein protein via a nasogastric tube during sleep []. The AA contained in the beverage were effectively digested and absorbed, thus increasing plasma AA availability throughout sleep and augmenting MPS. The combination of RE in the evening and pre-sleep protein ingestion had an even greater stimulatory effect on MPS in healthy young [] and older adults [] and augmented RT-induced gains in skeletal muscle mass and strength after a 12 week intervention []. Therefore, the ingestion of pre-sleep protein may be an effective way to further extend an individual's time spent in a positive NPBAL.

Evenly distributing protein intake throughout the day is a pragmatic strategy to enhance skeletal muscle anabolism at each meal – especially when combined with RT. However, with increasing daily protein intakes, the benefits of an even vs. skewed protein consumption pattern ostensibly become less important. Thus, the benefits observed when balancing protein intake across meals may be secondary to an increase in total daily protein intake and therefore reflect the inadequacy of current protein intake guidelines rather than an effect of protein distribution per se.

To extend upon the early work from Bob Wolfe's group, which elegantly demonstrated the sensitization of skeletal muscle to AA provision following RE []. Moore and colleagues provided the first evidence that, following an intense acute bout of RE of the leg in healthy previously trained young men, MPS was saturated at a relatively moderate dose – 20 g – of protein []. Specifically, ingestion of isolated egg protein stimulated MPS in a dose-dependent manner up to 20 g (equivalent to 8.6 g essential amino acids [EAA]), and when the protein dose was doubled (40 g) the rate of MPS was not further augmented []. This dose–response phenomenon was replicated by Witard and colleagues, who showed that RE-induced MPS was maximally stimulated following the provision of 20 g whey protein in young, resistance-trained, men []. Taken together, this supports the notion that MPS, in response to resistance exercise and protein feeding, is saturated at a ‘moderate’ dose (~20 g or ~0.25 g protein/kg) of high quality, protein (Fig. 2).

Although a moderate dose of protein appears sufficient to saturate the RE-induced increase in MPS, others report that following whole-body RE the consumption of 40 g (~0.5 g/kg), compared with 20 g, led to a small but significantly higher (~18%) acute MPS response []. The authors propose that the small benefit observed, with double the dose of protein, may be explained by the amount of muscle mass recruited for the whole-body RE, which would likely increase the demand for AA; though this is yet to be confirmed. Importantly, however, two other studies found that when ingesting 40 g of protein the stimulation of MPS was numerically but not statistically greater than that seen with ingestion of 20 g of protein by 10% [] and 8% []; thus, it seems that the statistically significant increase seen by Macnaughton and colleagues is small and likely of little consequence in affecting long-term muscle NPBAL []. Following endurance exercise, MPS was observed to be saturated with the ingestion of 30 g (~0.49 g/kg) milk protein and the authors also report that whole-body NPBAL displayed a clear dose–response relationship (i.e., 45 g PRO > 30 g PRO > 15 g PRO) [] (Fig. 2).

In recent years, a number of developments within isotopic tracer methodologies, has significantly advanced the field and enabled a deeper, yet more wholistic, understanding of skeletal muscle protein metabolism. Following initial attempts [], in 2009, Professor Luc van Loon, and his research group, extended upon a novel tool for use within muscle protein metabolism research []. Specifically, Holstein cows were infused with 1-[1-¹³C]-phenylalanine at the beginning of lactation, which enabled them to produce milk that yielded intrinsically l-[1-¹³C]phenylalanine-labeled milk proteins when processed. This enabled precise determination of how much of the ingested milk protein was first-pass cleared by splanchnic AA extraction, AA uptake into the muscle, and subsequent MPS within a single in vivo experiment []. The ingestion of intrinsically labelled proteins allows for the assessment of protein digestion and absorption kinetics, whereas the ingestion of labelled free amino acids does not. Measuring the muscle protein synthetic response to intrinsically labelled foods is less likely to disturb the tracer equilibrium and accounts for the “true” physiology of food more closely relate to the consumption of a ‘regular’ meal, instead of infusing individuals with labelled amino acids. Accordingly, from a single 20 g dose of l-[1-¹³C]phenylalanine-labelled milk protein ~50% of the constituent AA are extracted by splanchnic tissues (i.e., gut and liver) and the remaining ~50% becomes available within the circulation. It is important to note that digestion and extraction values reported are specific to the use of phenylalanine as an isotopically labeled tracer and vary between amino acids. Perhaps surprisingly, only ~10% of ingested protein (~2.2 g) was utilized for de novo myofibrillar protein synthesis; whereas, the remainder of the protein underwent catabolism and ureagenesis or used for production of other proteins []. Subsequently, Churchward-Venne and colleagues were the first to demonstrate a protein dose–response of incorporation of ingested AA into de novo mitochondrial proteins in response to endurance exercise (i.e., 45 g PRO > 30 g PRO > 15 g PRO) []. Thus, by utilizing the intrinsically labelled protein method a number of the intricacies associated with individual protein doses have been teased out.

Individual protein dose aside, the chief determinant of skeletal muscle growth, and/or maintenance, is total daily protein intake. As previously mentioned, the current guidelines are far from optimal if one were looking to maximize MPS in response to RE. A host of researchers and expert groups have since advocated for increased daily protein intakes particularly for older persons [, , ], many of which rely on important information obtained from stable isotope tracer studies. A recent meta-analysis reported, using a bi-phasic regression analysis of 49 studies and 1863 participants, that total protein intakes beyond ~1.6 g/kg/day (95% confidence interval: 1.0–2.2 g/kg/d) resulted in no further increases in RE-induced gains in fat-free mass []. However, following advances in the indicator amino acid oxidation (IAAO) method, whereby one EAA is limiting and the oxidation of the indicator AA is provided in excess to determine a breakpoint where whole-body protein synthesis is maximal have yielded different answers. In one study it was reported that post-exercise whole body anabolism, or NPBAL, was maximized with a protein intake of ~2.0 g/kg/d []. It is however important to note that these inferences were made based on whole-body protein metabolism measures and these are not specific to the skeletal muscle. Importantly, the individuals recruited for this particular study, were resistance trained and habitually consumed a higher protein diet. Nevertheless, this study, and the previous meta-analysis, suggests that a protein intake (~1.6 to as high as 2.2 g/kg/d) far in excess of the current recommendations (0.8 g/kg/d) may be optimal to maximize the daily body protein balance and muscle protein accrual.

Many years following the development and application of deuterated water (D₂O) for use in protein metabolism research [], there has been a resurgence of interest in the use of D₂O for measuring muscle protein turnover []. As a far more convenient and arguably ecologically valid measure of MPS, versus an infusion of labeled AA, D₂O has provided researchers with a vital tool capable of determining MPS in a free-living setting (Fig. 1). Thus, D₂O enables the assessment of daily integrated MPS; which may be much more reflective of the anabolic potential of exercise and nutritional interventions. For example, integrated rates of MPS were shown to be greater in young men consuming a higher (~2.35 g/k/d) compared with a lower (~1.2 g/kg/d) protein diet []; during pronounced ER. Importantly, a recent study that employed the D₂O methodology has also demonstrated that the benefits of a higher protein diet combined with RE extend to a young female population []. The utility of D₂O combined with the recent expansion of proteomics has enabled an increasingly detailed examination of entire protein networks and their regulation during altered muscle protein turnover. Thus, the use of stable isotopic tracers, regardless of methodology (i.e., infusion, IAAO, intrinsically labelled, D₂O), offer an invaluable tool to the field of muscle protein metabolism.

Protein ingestion and RE have a synergistic influence on MPS and muscle growth over time [,]. Therefore, peri-workout nutrition has garnered significant research attention, both in athletic populations attempting to maximize their adaptive potential to training and in older individuals with the goal of preserving muscle mass over time. Tipton and colleagues were the first to assess the anabolic effects of pre-vs. post-RE consumption of EAA and carbohydrates (CHO) []. The authors demonstrated a superior NPBAL, measured by the rate of appearance and disappearance of labelled L-[ring-²H₅]-Phenylalanine in the blood, in participants who consumed 6 g of EAA + 35 g of CHO prior to RE as opposed to 1hr post-RE []. However, when measured directly (tracer incorporation into muscle proteins []) as opposed to indirectly (i.e. rate of tracer disappearance from plasma []), Fujita and colleagues failed to demonstrate a superior effect of pre-exercise EAA + CHO consumption compared to fasted-state RE on MPS []. In contrast, pre-exercise protein and CHO co-ingestion followed by periodic pulses every 15 min during a 2 h RE session augmented MPS compared to a CHO placebo [], which is likely due to enhanced intramuscular anabolic signaling activation []. While most of the data regarding pre- and intra-RE protein supplementation demonstrate positive effects on MPS and thus NPBAL, the most popular time to ingest protein is arguably post-RE, when signaling proteins responsive to muscle contraction are activated and the muscle is essentially primed to synthesize new proteins. A meta-analysis showed that consumption of protein in close temporal proximity to RE appeared to positively influence accretion of lean mass, however, no effect of protein timing on muscle hypertrophy was observed when the confounding influence of total protein intake was considered []. Thus, total protein intake more so than when it was ingested had the largest effect on muscle hypertrophy [], which is consistent with a recent meta-analysis from our laboratory []. Additionally, skeletal muscle remains sensitized to AA for at least 24 h post-exercise [] and thus remains highly responsive to post-RE nutrition (Fig. 3). Taken together, peri-workout nutrition plays a minimal role in muscle hypertrophy when total protein intake is sufficient to support muscle hypertrophy.

More recently, an emphasis has been placed on identifying specific EAA that stimulate MPS, specifically leucine []. Altogether, EAA are the main drivers of a sustained MPS response [], with leucine being the AA that in muscle, acts to activate the process of MPS []. Several studies have shown that leucine content, within a protein-containing meal, is the main driver of MPS activation [,,]. Because of this property, a leucine trigger hypothesis has been advocated to better understand how protein ingestion activates MPS [,,]. The hypothesis stipulates that MPS activation is a function of leucine concentration within the intracellular AA pool [,]. Currently, it is known that the increase in intercellular concentration of leucine signals to activate MPS through a mechanism involving the regulation of the mechanistic target of rapamycin (mTOR) []. The exact mechanism by which leucine activates mTORC1 pathway has been identified, and it involves proteins of the Sestrin family [,]. Specifically, leucine binds to Sestrin 2 changing its phosphorylation state and decreasing its interaction with GAP activity toward the Rag GTPases 2 (GATOR2) []. Accordingly, GATOR2 interacts with GATOR1, decreasing its inhibition on mTOR, which in turn leads to an increased protein-synthetic response by the activation of downstream proteins (i.e. ribosomal protein S6 kinase beta-1 – p706SK1) []. Therefore, different protein sources will produce distinct protein synthetic responses; reflective of the leucine and other EAA content.

Due to leucine being the primary AA that activates MPS, it has been used to enhance the anabolic capacity of a given protein meal [,,]. Katsanos and colleagues [] showed that a mixture of EAA, containing leucine at 1.7 g, stimulated MPS in younger adults, but not in the older adults. However, increased rates of MPS were observed in older subjects when the leucine content was increased to 2.8 g []. Thus, it seems total leucine content rather than total protein quantity may be rate limiting in stimulating MPS [,]. As a result, modifying the leucine content of a meal/protein source may be an effective strategy to augment the MPS response to a dietary protein-containing meal in situations when a high-quality protein source is unavailable. However, the exact amount of leucine necessary to maximize MPS is not known []. For instance, studies have shown that 6 g of whey protein supplemented to contain ~5 g of leucine (4 g as crystalline leucine plus the leucine in 6 g of whey protein) can produce a similar MPS response when compared with 25 g of whey protein []. Nevertheless, older subjects require a higher protein and leucine content per meal, to achieve an equivalent MPS response compared with young subjects [].

Leucine content seems to be an essential variable determining the MPS response in older as well as younger subjects and independent of total daily protein ingestion []. For example, the ingestion of 5 g crystalline leucine added to each main meal increased rates of MPS over 3 days, and this effect was independent of whether older subjects consumed 0.8 or 1.2 g protein/kg/d of protein []. In addition, Devries and colleagues showed that the ingestion of 10 g of leucine enriched-milk protein (3 g leucine) produced a similar myofibrillar protein synthetic response in comparison to 25 g of whey protein isolate (3 g leucine) []. Therefore, MPS is stimulated by leucine in younger and older subjects, and the manipulation of leucine content per meal seems to be a useful strategy to maximally stimulate MPS. However, adjustments in the leucine content per meal based on age may be necessary (at least to ~3 g of available leucine), since older subject require a greater leucine ingestion to stimulate the same anabolic response observed in young subjects [].

Protein quality relies mostly on AA composition and bioavailability []. Most animal-derived proteins (i.e. beef, dairy, eggs, milk) are considered high quality and provide all of the EAA to activate (i.e. leucine trigger effect) and sustain a robust MPS response [,, , ]. When comparing plant-derived proteins with animal-derived proteins, vegetal-source proteins are usually lower quality due to lower quantities of an EAA. For instance, the first limiting AA in cereals and grains is most commonly lysine, whereas legumes have methionine as their limiting AA [,,]. Even high-quality vegetal proteins like soy isolate (DIAAS = 90–91) are limited in the EAA methionine and cysteine. In contrast, most animal-derived proteins have a DIAAS closer to or greater than 100 [], meaning that there is no real limiting AA when ingesting such proteins and daily protein recommendations are covered. One relevant exception to this pattern is the animal-derived protein collagen, which has a DIAAS of 0 based on the total absence of tryptophan in its composition []. The result of differences in EAA abundance in plant-derived proteins on acute MPS was shown by Wilkinson and colleagues comparing a soy protein-based beverage (~18.2 g protein) versus skim milk after RE []. Skim milk ingestion promoted a ~25–30% higher fractional synthetic rate in muscle proteins, in the 3 h post-exercise, when compared to the soy-based beverage []. However, this acute difference in MPS seems to be present only when comparing soy-based protein beverages with skim milk [] or whey protein [], but not with the ingestion of casein [].

Because plant-derived proteins are limited in some EAA, the use of different protein blends (animal + plant-derived or different plant-derived) is commonly encouraged []. Reidy and colleagues observed a similar MPS response to resistance exercise (following 4 h) after the consumption of either a 19 g protein blend containing a mixture of 50% sodium caseinate, 25% whey and 25% soy protein isolate, versus ~18 g whey protein in young subjects []. Similar findings were observed in middle-aged and older men, despite the use of a greater protein dose []. Nevertheless, we lack the knowledge to describe the ability of various mixtures of plant-derived proteins on the MPS response. In this context, Kim and colleagues measured MPS and whole-body protein kinetics, in the fasted state and in response to an egg-based (EGG) or cereal-based (CEREAL) isocaloric (~500 kcal) and isonitrogenous (~26 g protein) breakfast followed by lunch (4 h later) containing mainly beef protein (~25 g) []. No significant differences were observed in MPS after the ingestion of the different breakfasts.

In addition to the limited amount of methionine or lysine, the content or availability of leucine for some plant-based proteins is also generally lower when compared with animal-based proteins (Table 1), but not at a limiting level [,,]. Therefore, when ingesting plant-based proteins, leucine content, and protein dose are additional relevant variables that have been the focus of some research. Recently, Churchward-Venne and colleagues, determined the MPS response to the ingestion of whey, soy, and leucine-enriched-soy protein beverages (all three providing the equivalent of 20 g of protein) after concurrent resistance and endurance exercise in young subjects []. Fractional synthetic rates in myofibrillar and mitochondrial protein fractions were equivalent in the 6 h following exercise and ingestion of the protein beverages []. Thus, it seems that if enough leucine and EAA are present, adding additional leucine to plant-based proteins does not further promote increases in MPS, at least in young subjects. Gorissen and colleagues assessed the protein synthetic response to plant-based isolate proteins (i.e. wheat protein and wheat protein hydrolysates) using whey protein or micellar casein as positive controls in older subjects (71 ± 1 yrs) []. The authors showed that 35 g of wheat protein hydrolysates produced the same plasma EAA and leucine increases when compared to micellar casein. However, to stimulate myofibrillar MPS, a 60 g wheat protein hydrolysates, containing equivalent amounts of leucine as 35 g of whey protein, was necessary [].

Ingestion of supplemental plant-based protein isolates can support significant differences in daily MPS []. For instance, young women ingesting 50g/day (2 × 25 g) of potato protein isolate (2.5 g of leucine per dose), and increasing total daily protein ingestion up to 1.6 g/kg/day, showed an enhanced myofibrillar protein synthesis (measured by ingestion of D₂O) during rest and after RE []. In addition, research monitoring muscle mass in response to supplementation with plant-based protein has shown promising results when compared to the ingestion of high-quality animal-based protein. Ingestion of either 50g/day pea protein isolate (1.6 g of leucine per dose) or 50 g of whey protein (2.1 g leucine per dose) in young subjects in combination with RT for 12 weeks resulted in similar increases in biceps brachii thickness []. Taken together, results of studies testing plant-derived proteins have shown that ingestion of such protein sources can be used to sustain MPS and support exercise-induced gains in muscle mass equivalent to high-quality animal-based protein. Hence, for some plant-based proteins, adjustments of doses focusing on providing enough leucine and other EAA is a reasonable strategy to ensure a maximal MPS response. This is especially important for older subjects, who demonstrate, compared to younger persons, an anabolic resistance at the level of their muscle to an equivalent hyperaminoacidemia [].

The consumption of AA, and ensuing aminoacidemia, stimulates MPS and is especially important in maintaining a positive muscle NPBAL. However, the ingestion of AA also leads to an increase in circulating insulin concentrations (hyperinsulinemia) []. Although the systemic rise in insulin has been shown to be permissive for increasing MPS, hyperinsulinemia plays an essential role in the inhibition MPB [,]. Using the two-pool method showed an decrease in MPB, as measured by the rate of appearance (or release) in response to the infusion of AA above postprandial levels and hyperinsulinemia []. These results were extended in a series of seminal studies, where Biolo and colleagues, using the three-pool AV method with the infusion of 2 different isotopes, demonstrating that RE increased MPB in the postabsorptive (i.e. fasted) state []. However, the rise in MPB was abolished when RE was immediately followed by the infusion of AA [].

In a hyperlipidemic, hyperglycaemic and hyperinsulinemic state MPB is reduced compared to the post absorptive state []. However, Greenhaff and colleagues demonstrated that muscle NPBAL was improved, in a dose-dependent manner (up to a plateau), following the infusion of a range of insulin concentrations (5–167 mU/l) compared to the postabsorptive state []. Importantly, increasing the insulin concentration did not augment MPS, highlighting a permissive (not stimulatory) role of insulin in MPS, while MPB was suppressed but not beyond insulin concentration of 30 mU/l []. Thus, the consumption of AA is essential to create a positive muscle NPBAL, both through the direct stimulation of MPS and insulin-mediated suppression of MPB. In further support of this, 20 g of EAA combined with high (90 g) or low (30 g) of CHO following RE led to significantly different systemic insulin concentrations, however, MPB, the associated protein signalling and gene expression were not different between conditions []. This, and a recent meta-analysis [], support the notion that the role of insulin to promote a positive muscle NPBAL is driven by the suppression of MPB, up to a point, and not by further stimulation of MPS [,]. Thus, the ingestion of a large enough bolus of protein/AA (~0.25 g/kg for younger adults, and 0.4 g/kg for older adults) is sufficient to maximize the acute MPS response, and the ensuing hyperinsulinemia is sufficient to maximally inhibit MPB, optimizing the acute anabolic response to exercise and nutrition; which, when repeated over time leads to protein accretion (this concept is summarised in Fig. 4).

Although it is widely accepted that sufficient AA consumption is essential to generate a positive NPBAL, what happens when an individual consumes protein in excess of what can be used? Several studies have examined the dose–response of protein provision on MPS, the ingestion of 20 g of protein (~0.25 g/kg) maximally stimulates MPS in young individuals [,], whereas approximately 10 g of EAA maximally stimulates MPS []. When AA are consumed in excess of the amount that can be utilized by protein-requiring processes including skeletal muscle for protein synthesis, and because they are not ‘stored’ in the traditional sense that fatty acids and carbohydrates are, they are deaminated and some carbon skeletal can then be oxidized. Therefore, the measurement of AA oxidation, of infused and appropriately labelled AA is an indicator of when AA supply exceeds the rates at which AA can be used for protein-requiring processes like protein synthesis [].

Labelled phenylalanine can be infused intravenously and the oxidation rates of the labelled amino acid can be determined from the conversion of L-[ring-¹³C₆]phenylalanine to L-[ring-¹³C₆]tyrosine in plasma enrichment []. Oxidation of AA can also be determined via the appearance of an appropriately labelled ¹³C-AA and the appearance of this label in expired air (i.e., ¹³CO₂) []. In MPS dose–response studies of ingested protein where MPS is maximally stimulated with the ingestion of 20 g of protein following exercise, consumption above 20 g of protein resulted in increased whole-body AA oxidation [,]. Additionally, the endogenous urea production rate is an indicator of protein catabolism, as the release of AA nitrogen requires the formation of urea for its safe disposal []. The infusion of ¹⁵N₂ urea allows for the determination of urea production rates by determining the ratio of urea enrichment in the plasma and the infusate. Following protein ingestion, similarly to what was observed with AA oxidation, urea production rates were greatest after the consumption of 40 g of whey []. These data demonstrate how the use of labelled isotopes can be used to determine whole body AA oxidation following ingestion of various protein doses to confirm that protein consumed in excess is irreversibly loss to oxidation. Important information is gained from studies in which whole-body protein turnover is measured, however it is not specific to the skeletal muscle itself and limitations exist surrounding these measurements [], therefore researchers must consider practical inferences that can be made based on these results.

Omega-3 polyunsaturated fatty acids (n3-PUFA) are fatty acids that contain two or more double bonds, with the first double bond located three carbons from the terminal methyl group. The most biologically potent n3-PUFA are eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), however the de novo conversion from its n3-PUFA precursor – alpha linoleic acid (ALA) – to EPA and DHA is fairly limited in humans []. With this in mind, EPA and DHA are considered conditionally essential fatty acids, and increasing dietary (i.e. oily fish) and/or supplemental (i.e. fish oil) intake is recommended []. N3-PUFA are traditionally known for their potent effects on cardiovascular and neurological function, and recently, n3-PUFA supplementation has garnered considerable attention for its effects on skeletal muscle health [,].

Seminal work from Smith and colleagues [,] demonstrated that in healthy older adults [], and young-to-middle-aged adults [] supplementation with EPA (1.86 g/d) and DHA (1.50 g/d) for 8 weeks, rates of MPS were potentiated during a constant infusion of insulin and AA, compared to a control group consuming corn oil at rest. However, McGlory and colleagues [] failed to show any benefit of 8 weeks of n3-PUFA supplementation on changes in MPS following either 30 g of whey protein ingestion, or when whey protein feeding was combined with RE in young men. It may be that the 30 g dose of whey protein used by McGlory and colleagues [] saturated the muscle protein synthetic response in younger persons, and that n3-PUFA supplementation conferred no anabolic benefit. In this respect, n3-PUFA supplementation may provide anabolic benefit during time periods where protein consumption is likely to be sub-optimal.

Older adults require a greater relative dose of protein to optimally stimulate rates of MPS [], however, the recommended dose may be difficult to achieve []. With this in mind, n3-PUFA feeding may provide more of an anabolic benefit to older adults. Indeed, compared to younger adults [], older adults [] demonstrated greater relative increases in rates of mixed MPS following n3-PUFA feeding. However, Da Boit and colleagues [] failed to show any measurable effect of n3-PUFA feeding on free-living (integrated) rates of MPS in healthy older adults undergoing RT. It may be that the lack of repeated measures design in the aforementioned study [] prevented the researchers from finding a discernible effect of n3-PUFA feeding. In addition, due to the lack of monitoring habitual protein intake, we cannot discount the possibility that the older adults recruited by Da Boit and colleagues [] were habitually consuming adequate dietary protein, effectively masking a discernible effect of n3-PUFA feeding. Nonetheless, future work investigating the effects of n3-PUFA feeding on integrated measures of muscle protein synthesis under conditions of sub-optimal protein intake are warranted.

Periods of physical inactivity (i.e. bed rest, muscle disuse, step reduction) result in decreased MPS in both the post-absorptive and post-prandial state []. Reduced rates of MPS during periods of physical inactivity results in a negative NPBAL, leading to a decline in muscle mass and strength over time [, , ]. Strategies such as RT [] and neuromuscular electrical stimulation [] are effective in attenuating skeletal muscle disuse atrophy. However, the previously mentioned strategies may not represent the most practical approaches, necessitating alternative strategies to combat disuse-induced skeletal muscle atrophy. Given that n3-PUFA feeding enhances post-prandial increases in MPS [,] it is possible that n3-PUFA may attenuate declines in MPS during periods of skeletal muscle disuse. Recently, we showed that young healthy women supplementing with EPA (2.97 g/d) and DHA (2.03 g/d) had higher integrated rates of MPS during 2 weeks of single-leg immobilization, and upon return to habitual physical activity levels, compared to a control group ingesting sunflower oil []. Moreover, EPA and DHA supplementation not only alleviated muscle atrophy during immobilization, but also facilitated full return of skeletal muscle volume after 2 weeks of recovery []. It has been proposed that accumulation of short periods of skeletal muscle disuse superimposed onto the natural biological decline in muscle mass with advancing age, may give rise to the development of sarcopenia [].