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

Open AccessPublished:March 01, 2021DOI:https://doi.org/10.1016/j.nutos.2021.01.005

      Summary

      Skeletal muscle is the largest organ of the human body and plays a pivotal role in whole-body homeostasis through the maintenance of physical and metabolic health. Establishing strategies aimed at increasing the amount, and minimising loss, of muscle mass are of upmost importance. Muscle mass is primarily dictated by the meal-to-meal fluctuations in muscle protein synthesis (MPS) and muscle protein breakdown (MPB), each of which can be quantified through the use of stable isotopically labelled tracers. Importantly, both MPS and MPB can be influenced by external factors such as nutritional manipulation, specifically protein ingestion, and changes in loading via exercise. To date, research involving stable isotopic tracers has focused on determining the optimal dose, timing surrounding bouts of exercise, distribution and composition of protein to maximally stimulate MPS and inhibit MPB, both at rest and following exercise. In this review we focus on the use of these stable isotopically-labeled tracers to unravel the intricacies of skeletal muscle protein turnover in response to specific nutritional interventions.

      Keywords

      1. Introduction

      Skeletal muscle is a remarkably plastic tissue that can change its phenotype in response to changes in loading demands. Skeletal muscle also plays an integral role in whole-body metabolism and homeostasis. The rate of muscle protein turnover is dependent on the balance between two opposing, ongoing, but interrelated kinetic processes: muscle protein synthesis (MPS) and muscle protein breakdown (MPB). This continuous turnover of muscle proteins results in efficient repair and renewal of damaged (whether mechanically, via oxidation, misfolding, nitrosylation, or otherwise) proteins and underpins the plasticity of skeletal muscle in response to contractile and nutritional perturbations [
      • Bell R.A.
      • Al-Khalaf M.
      • Megeney L.A.
      The beneficial role of proteolysis in skeletal muscle growth and stress adaptation.
      ]. In the postabsorptive state, MPB exceeds MPS and muscle proteins are catabolized to supply amino acids (AA) back into the free pool, but most of which are recycled and reused. However, some AA are lost from muscle, mostly as alanine and glutamine (nitrogen carriers) for glucose production via gluconeogenesis, or as a fuel for enterocytes. When MPB exceeds MPS, the net catabolism of skeletal muscle, or a negative net protein balance (NPBAL) is transient, however [
      • Biolo G.
      • Fleming R.Y.D.
      • Maggi S.P.
      • Wolfe R.R.
      Transmembrane transport and intracellular of amino acids in human skeletal muscle kinetics.
      ]. Ingestion of a mixed-meal, and the ensuing rise in plasma AA and insulin, stimulates MPS and suppresses MPB leading to net accretion of protein, and a positive muscle NPBAL. In healthy, active adults, assuming adequate intakes of protein, periods of postabsorptive catabolism remain in dynamic equilibrium with periods of postprandial anabolism over a 24hr period and muscle mass is maintained. This is likely true in fully grown adults in their third decade of life and onward into their fourth and possibly fifth decade; however, at a certain point NPBAL begins to shift toward a net negative state and muscle is slowly lost. This slow loss of muscle with aging is termed sarcopenia [
      • Cao L.
      • Morley J.E.
      Sarcopenia is recognized as an independent condition by an international classification of disease, tenth revision, clinical modification (ICD-10-CM) code.
      ].
      Exercise increases muscle protein turnover. Specifically exercise, independent of nutrition, results in an increase in both MPS and MPB, but the increase in MPB outweighs that of MPS and thus resulting in a negative NPBAL. However, the consumption of protein is able to increase the MPS response and drive a positive NPBAL. Resistance exercise (RE) leads to the sensitization of the muscle protein translational machinery to the presence of AA for at least 24–48 h [
      • Burd N.A.
      • West D.W.
      • Moore D.R.
      • Atherton P.J.
      • Staples A.W.
      • Prior T.
      • et al.
      Enhanced amino acid sensitivity of myofibrillar protein synthesis persists for up to 24 h after resistance exercise in young men.
      ], resulting in an additive stimulation of MPS over that due to hyperaminoacidemia alone [
      • Biolo G.
      • Tipton K.D.
      • Klein S.
      • Wolfe R.R.
      An abundant supply of amino acids enhances the metabolic effect of exercise on muscle protein.
      ]. The increase in MPS following exercise is dependent on the type of exercise completed. For example, RE is commonly associated with increases in muscle size, whereas endurance exercise (EE) is characterized by remodelling of the muscle towards a more oxidative phenotype. Initially, the stress of RE and EE in untrained adults upregulates myofibrillar and mitochondrial protein synthesis [
      • Di Donato D.M.
      • West D.W.
      • Churchward-Venne T.A.
      • Breen L.
      • Baker S.K.
      • Phillips S.M.
      Influence of aerobic exercise intensity on myofibrillar and mitochondrial protein synthesis in young men during early and late postexercise recovery.
      ]; however, as training progresses the response is refined to be more specific to form, resistance or endurance, of exercise. An acute bout of RE after 10 weeks of resistance training (RT) increased myofibrillar but not mitochondrial protein synthesis [
      • Wilkinson S.B.
      • Phillips S.M.
      • Atherton P.J.
      • Patel R.
      • Yarasheski K.E.
      • Tarnopolsky M.A.
      • et al.
      Differential effects of resistance and endurance exercise in the fed state on signalling molecule phosphorylation and protein synthesis in human muscle.
      ]. In contrast, acute EE increased mitochondrial protein synthesis after 10 weeks of endurance training, with no detectable effect on the myofibrillar sub-fraction [
      • Wilkinson S.B.
      • Phillips S.M.
      • Atherton P.J.
      • Patel R.
      • Yarasheski K.E.
      • Tarnopolsky M.A.
      • et al.
      Differential effects of resistance and endurance exercise in the fed state on signalling molecule phosphorylation and protein synthesis in human muscle.
      ]. Post-exercise protein intake supports the synthesis of proteins in these exercise-responsive protein sub-fractions [
      • Wilkinson S.B.
      • Phillips S.M.
      • Atherton P.J.
      • Patel R.
      • Yarasheski K.E.
      • Tarnopolsky M.A.
      • et al.
      Differential effects of resistance and endurance exercise in the fed state on signalling molecule phosphorylation and protein synthesis in human muscle.
      ]. These data underscore the importance of measuring fraction-specific protein turnover to understand the specificity of skeletal muscle adaptation.
      It is now possible to combine stable isotopes with liquid chromatography and mass-spectrometry to investigate the fractional synthesis rate and abundance of hundreds of individual proteins within a given muscle sub-fraction [
      • Camera D.M.
      • Burniston J.G.
      • Pogson M.A.
      • Smiles W.J.
      • Hawley J.A.
      Dynamic proteome profiling of individual proteins in human skeletal muscle after a high-fat diet and resistance exercise.
      ,
      • Murphy C.H.
      • Shankaran M.
      • Churchward-Venne T.A.
      • Mitchell C.J.
      • Kolar N.M.
      • Burke L.M.
      • et al.
      Effect of resistance training and protein intake pattern on myofibrillar protein synthesis and proteome kinetics in older men in energy restriction.
      ]. Determining the abundance and synthesis rate of individual proteins also permits the calculation of protein breakdown rates. Once changes in individual protein abundance and FSR are obtained by D2O ingestion and alanine labelling, the absolute rate of individual protein breakdown can be calculated by difference. This allows researchers to circumvent issues associated with bulk MPB measurements using the tracer dilution technique (i.e. the need for a physiological steady-state) and multiple biopsies during the dilution of the tracer [
      • Phillips S.
      • Tipton K.
      • Ferrando A.
      • Wolfe R.
      Resistance training reduces the acute exercise-induced increase in muscle protein turnover.
      ,
      • Phillips S.
      • Tipton K.
      • Aarsland A.
      • Wolf S.
      • Wolfe R.
      Mixed muscle protein synthesis and breakdown after resistance exercise in humans.
      ].
      In this review, we focus on the application of stable isotope tracers to elucidate the impact of protein ingestion and exercise on skeletal muscle protein turnover in humans (Fig. 1). Specifically, we consider the influence of total protein intake, protein source and daily protein distribution on muscle protein synthesis and, where data are available, muscle protein breakdown. Given the breadth of information on this topic, and the consideration of distinct clinical populations in accompanying reviews in this special issue, we limit our discussion primarily to healthy young and older adults without existing clinical comorbidities.
      Fig. 1
      Fig. 1Schematic representation of the use of AA stable isotope tracers and D2O for the measurement of muscle protein turnover. (A) To measure acute (typically 0–6 h post-exercise and/or feeding) MPS, stable AA isotope tracers are infused intravenously which results in an increased plasma enrichment of the isotope. The AA isotope is taken up by the muscle (rate of disappearance) into the intracellular AA pool. The labeled AA is incorporated into new proteins (MPS). As infusion time progresses the change in incorporated AA stable isotope is proportionate to the rate of total MPS. Alternatively, MPB can be measured through the release of labelled AA stable isotope into the intracellular AA pool or inferred from the rate of appearance of the labelled AA into the venous blood samples. (B) To measure integrated (free-living) MPS D2O is ingested and results in an increase in the enrichment of the body water pool. Once entering the cell the deuterium becomes incorporated into alanine during the transamination process, these deuterium labelled alanine AA are then incorporated into new muscle proteins and are indicative of MPS. (C) For both methods, muscle protein/plasma/saliva enrichment is determined using mass spectrometry following derivatization of AA. To calculate the rate of MPS, the enrichment of one muscle sample (T1) is subtracted from the enrichment of a later muscle sample (T2) and divided by the mean enrichment of the precursor (plasma enrichment when using stable AA isotope; body water enrichment when using D2O) multiplied by the time.

      2. Protein dose

      In young healthy individuals skeletal muscle accounts for ~40% of total body mass and serves as an important hub for dietary protein uptake and utilization, a protein dose normalized for total body mass may seem practical. As such, the current guidelines (or recommended daily allowance (RDA)) suggest the amount of protein required for a healthy adult is equivalent to 0.8 g/kg/d (RDA) [
      Food, Nutrition Board IOM
      Dietary reference intakes for energy, carbohydrate, fiber, fat, fatty acids, cholesterol, protein, and amino acids (macronutrients).
      ]. However, these recommendations were established from early nitrogen balance studies, within which the primary focus was the achievement of nitrogen balance and the avoidance of protein deficiency [
      • Wolfe R.R.
      • Miller S.L.
      The recommended dietary allowance of protein: a misunderstood concept.
      ]. Thus, current protein intake recommendations are inadequate, particularly for those looking to increase skeletal muscle mass with exercise training, mitigate situations of muscle loss (i.e., limb immobilization or bed rest) or prevent age-related sarcopenic muscle loss. The utilization of stable isotopes, and the and continuous development of the associated analytical techniques, has paved the way for a plethora of investigations within human metabolic research. At the forefront of protein metabolism research, the late Professor Mike Rennie and colleagues pioneered the first investigations, in humans, to demonstrate that the incorporation of a stable isotope tracer (13C-Leucine) into skeletal muscle was increased following protein feeding [
      • Rennie M.J.
      • Edwards R.H.
      • Halliday D.
      • Matthews D.E.
      • Wolman S.L.
      • Millward D.J.
      Muscle protein synthesis measured by stable isotope techniques in man: the effects of feeding and fasting.
      ] indicating a higher rate of MPS. Subsequently, since the inception of stable isotopic tracers within metabolic research, the influence that nutritional intricacies exert on skeletal muscle protein metabolism has become a niche field that has flourished in recent years.

      2.1 Protein distribution

      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 [
      • Bohe J.
      • Low A.
      • Wolfe R.R.
      • Rennie M.J.
      Human muscle protein synthesis is modulated by extracellular, not intramuscular amino acid availability: a dose-response study.
      ]. 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 [
      • Moore D.R.
      • Churchward-Venne T.A.
      • Witard O.
      • Breen L.
      • Burd N.A.
      • Tipton K.D.
      • et al.
      Protein ingestion to stimulate myofibrillar protein synthesis requires greater relative protein intakes in healthy older versus younger men.
      ]. 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 [
      • Atherton P.J.
      • Etheridge T.
      • Watt P.W.
      • Wilkinson D.
      • Selby A.
      • Rankin D.
      • et al.
      Muscle full effect after oral protein: time-dependent concordance and discordance between human muscle protein synthesis and mTORC1 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 [
      • Areta J.L.
      • Burke L.M.
      • Ross M.L.
      • Camera D.M.
      • West D.W.
      • Broad E.M.
      • et al.
      Timing and distribution of protein ingestion during prolonged recovery from resistance exercise alters myofibrillar protein synthesis.
      ].
      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 [
      • Smeuninx B.
      • Greig C.A.
      • Breen L.
      Amount, source and pattern of dietary protein intake across the adult lifespan: a cross-sectional study.
      ]. 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 [
      • Mamerow M.M.
      • Mettler J.A.
      • English K.L.
      • Casperson S.L.
      • Arentson-Lantz E.
      • Sheffield-Moore M.
      • et al.
      Dietary protein distribution positively influences 24-h muscle protein synthesis in healthy 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 [
      • Yasuda J.
      • Tomita T.
      • Arimitsu T.
      • Fujita S.
      Evenly distributed protein intake over 3 meals augments resistance exercise-induced muscle hypertrophy in healthy young men.
      ]. 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 [
      • Norton C.
      • Toomey C.
      • McCormack W.G.
      • Francis P.
      • Saunders J.
      • Kerin E.
      • et al.
      Protein supplementation at breakfast and lunch for 24 Weeks beyond habitual intakes increases whole-body lean tissue mass in healthy older adults.
      ]. 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 [
      • Hudson J.L.
      • Iii R.E.B.
      • Campbell W.W.
      Protein distribution and muscle-related outcomes: does the evidence support the concept?.
      ]. 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 [
      • Soop M.
      • Nehra V.
      • Henderson G.C.
      • Boirie Y.
      • Ford G.C.
      • Nair K.S.
      Coingestion of whey protein and casein in a mixed meal: demonstration of a more sustained anabolic effect of casein.
      ]. 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 [
      • Soop M.
      • Nehra V.
      • Henderson G.C.
      • Boirie Y.
      • Ford G.C.
      • Nair K.S.
      Coingestion of whey protein and casein in a mixed meal: demonstration of a more sustained anabolic effect of casein.
      ]. 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 [
      • van Vliet S.
      • Beals J.W.
      • Holwerda A.M.
      • Emmons R.S.
      • Goessens J.P.
      • Paluska S.A.
      • et al.
      Time-dependent regulation of postprandial muscle protein synthesis rates after milk protein ingestion in young men.
      ]. 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 [
      • Atherton P.J.
      • Etheridge T.
      • Watt P.W.
      • Wilkinson D.
      • Selby A.
      • Rankin D.
      • et al.
      Muscle full effect after oral protein: time-dependent concordance and discordance between human muscle protein synthesis and mTORC1 signaling.
      ,
      • Bohe J.
      • Low J.F.
      • Wolfe R.R.
      • Rennie M.J.
      Latency and duration of stimulation of human muscle protein synthesis during continuous infusion of amino acids.
      ]. 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 [
      • Areta J.L.
      • Burke L.M.
      • Camera D.M.
      • West D.W.
      • Crawshay S.
      • Moore D.R.
      • et al.
      Reduced resting skeletal muscle protein synthesis is rescued by resistance exercise and protein ingestion following short-term energy deficit.
      ]. Our laboratory has also shown a reduction in fed-state myofibrillar protein synthesis following a 4-week hypocaloric dietary intervention in older men [
      • Murphy C.H.
      • Churchward-Venne T.A.
      • Mitchell C.J.
      • Kolar N.M.
      • Kassis A.
      • Karagounis L.G.
      • et al.
      Hypoenergetic diet-induced reductions in myofibrillar protein synthesis are restored with resistance training and balanced daily protein ingestion 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) [
      • Murphy C.H.
      • Churchward-Venne T.A.
      • Mitchell C.J.
      • Kolar N.M.
      • Kassis A.
      • Karagounis L.G.
      • et al.
      Hypoenergetic diet-induced reductions in myofibrillar protein synthesis are restored with resistance training and balanced daily protein ingestion in older men.
      ]. 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 [
      • Murphy C.H.
      • Shankaran M.
      • Churchward-Venne T.A.
      • Mitchell C.J.
      • Kolar N.M.
      • Burke L.M.
      • et al.
      Effect of resistance training and protein intake pattern on myofibrillar protein synthesis and proteome kinetics in older men in energy restriction.
      ]. 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 [
      • Murphy C.H.
      • Shankaran M.
      • Churchward-Venne T.A.
      • Mitchell C.J.
      • Kolar N.M.
      • Burke L.M.
      • et al.
      Effect of resistance training and protein intake pattern on myofibrillar protein synthesis and proteome kinetics in older men in energy restriction.
      ]. 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 [
      • Groen B.B.
      • Res P.T.
      • Pennings B.
      • Hertle E.
      • Senden J.M.
      • Saris W.H.
      • et al.
      Intragastric protein administration stimulates overnight muscle protein synthesis in elderly men.
      ]. 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 [
      • Trommelen J.
      • Holwerda A.M.
      • Kouw I.W.
      • Langer H.
      • Halson S.L.
      • Rollo I.
      • et al.
      Resistance exercise augments postprandial overnight muscle protein synthesis rates.
      ] and older adults [
      • Holwerda A.M.
      • Kouw I.W.
      • Trommelen J.
      • Halson S.L.
      • Wodzig W.K.
      • Verdijk L.B.
      • et al.
      Physical activity performed in the evening increases the overnight muscle protein synthetic response to presleep protein ingestion in older men.
      ] and augmented RT-induced gains in skeletal muscle mass and strength after a 12 week intervention [
      • Snijders T.
      • Res P.T.
      • Smeets J.S.
      • van Vliet S.
      • van Kranenburg J.
      • Maase K.
      • et al.
      Protein ingestion before sleep increases muscle mass and strength gains during prolonged resistance-type exercise training in healthy young men.
      ]. 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.

      2.2 Individual/per-feed protein dose

      To extend upon the early work from Bob Wolfe's group, which elegantly demonstrated the sensitization of skeletal muscle to AA provision following RE [
      • Biolo G.
      • Tipton K.D.
      • Klein S.
      • Wolfe R.R.
      An abundant supply of amino acids enhances the metabolic effect of exercise on muscle protein.
      ]. 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 [
      • Moore D.R.
      • Robinson M.J.
      • Fry J.L.
      • Tang J.E.
      • Glover E.I.
      • Wilkinson S.B.
      • et al.
      Ingested protein dose response of muscle and albumin protein synthesis after resistance exercise in young men.
      ]. 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 [
      • Moore D.R.
      • Robinson M.J.
      • Fry J.L.
      • Tang J.E.
      • Glover E.I.
      • Wilkinson S.B.
      • et al.
      Ingested protein dose response of muscle and albumin protein synthesis after resistance exercise in young men.
      ]. 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 [
      • Witard O.C.
      • Jackman S.R.
      • Breen L.
      • Smith K.
      • Selby A.
      • Tipton K.D.
      Myofibrillar muscle protein synthesis rates subsequent to a meal in response to increasing doses of whey protein at rest and after resistance exercise.
      ]. 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).
      Fig. 2
      Fig. 2Dose–response of muscle protein synthesis, to protein ingestion following exercise. Protein ingestion after (A) resistance exercise and (B) endurance exercise presented as a percent change from the ingestion of no protein (0 g) results in an increases in myofibrillar FSR. Following RE the consumption of 40 g versus 20 g of whey protein increased myofibrillar FSR only slightly and the increase was statistically significant indicating that 20 g of protein maximally stimulated MPS following RE (A). Following endurance exercise 45 g of carbohydrates was consumed with 0 g, 15 g, 30 g, and 45 g of intrinsically labelled milk protein. Myofibrillar FSR was maximally stimulated with the ingestion of 45 g of carbohydrates and 30 g of protein, the consumption of 15 g of protein did not significantly increase MPS rates above that of the consumption of 0 g of protein. Figure was altered from Stokes and colleagues 2018 [
      • Stokes T.
      • Hector A.J.
      • Morton R.W.
      • McGlory C.
      • Phillips S.M.
      Recent perspectives regarding the role of dietary protein for the promotion of muscle hypertrophy with resistance exercise training.
      ], data re-drawn from Witard and colleagues 2014 [
      • Witard O.C.
      • Jackman S.R.
      • Breen L.
      • Smith K.
      • Selby A.
      • Tipton K.D.
      Myofibrillar muscle protein synthesis rates subsequent to a meal in response to increasing doses of whey protein at rest and after resistance exercise.
      ] and Churchward-Venne and colleagues 2020 [
      • Churchward-Venne T.A.
      • Pinckaers P.J.
      • Smeets J.S.
      • Betz M.W.
      • Senden J.M.
      • Goessens J.P.
      • et al.
      Dose-response effects of dietary protein on muscle protein synthesis during recovery from endurance exercise in young men: a double-blind randomized trial.
      ].
      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 [
      • Macnaughton L.S.
      • Wardle S.L.
      • Witard O.C.
      • McGlory C.
      • Hamilton D.L.
      • Jeromson S.
      • et al.
      The response of muscle protein synthesis following whole-body resistance exercise is greater following 40 g than 20 g of ingested whey protein.
      ]. 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% [
      • Witard O.C.
      • Jackman S.R.
      • Breen L.
      • Smith K.
      • Selby A.
      • Tipton K.D.
      Myofibrillar muscle protein synthesis rates subsequent to a meal in response to increasing doses of whey protein at rest and after resistance exercise.
      ] and 8% [
      • Moore D.R.
      • Robinson M.J.
      • Fry J.L.
      • Tang J.E.
      • Glover E.I.
      • Wilkinson S.B.
      • et al.
      Ingested protein dose response of muscle and albumin protein synthesis after resistance exercise in young men.
      ]; 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 [
      • Macnaughton L.S.
      • Wardle S.L.
      • Witard O.C.
      • McGlory C.
      • Hamilton D.L.
      • Jeromson S.
      • et al.
      The response of muscle protein synthesis following whole-body resistance exercise is greater following 40 g than 20 g of ingested whey protein.
      ]. 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) [
      • Churchward-Venne T.A.
      • Pinckaers P.J.
      • Smeets J.S.
      • Betz M.W.
      • Senden J.M.
      • Goessens J.P.
      • et al.
      Dose-response effects of dietary protein on muscle protein synthesis during recovery from endurance exercise in young men: a double-blind randomized trial.
      ] (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 [
      • Boirie Y.
      • Fauquant J.
      • Rulquin H.
      • Maubois J.-L.
      • Beaufrère B.
      Production of large amounts of [13C] leucine-enriched milk proteins by lactating cows.
      ], in 2009, Professor Luc van Loon, and his research group, extended upon a novel tool for use within muscle protein metabolism research [
      • Van Loon L.
      • Boirie Y.
      • Gijsen A.
      • Fauquant J.
      • De Roos A.
      • Kies A.
      • et al.
      The production of intrinsically labeled milk protein provides a functional tool for human nutrition 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 [
      • Groen B.B.
      • Horstman A.M.
      • Hamer H.M.
      • De Haan M.
      • Van Kranenburg J.
      • Bierau J.
      • et al.
      Post-prandial protein handling: you are what you just ate.
      ]. 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 [
      • Groen B.B.
      • Horstman A.M.
      • Hamer H.M.
      • De Haan M.
      • Van Kranenburg J.
      • Bierau J.
      • et al.
      Post-prandial protein handling: you are what you just ate.
      ]. 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) [
      • Churchward-Venne T.A.
      • Pinckaers P.J.
      • Smeets J.S.
      • Betz M.W.
      • Senden J.M.
      • Goessens J.P.
      • et al.
      Dose-response effects of dietary protein on muscle protein synthesis during recovery from endurance exercise in young men: a double-blind randomized trial.
      ]. Thus, by utilizing the intrinsically labelled protein method a number of the intricacies associated with individual protein doses have been teased out.

      2.3 Total protein dose

      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 [
      • Bauer J.
      • Biolo G.
      • Cederholm T.
      • Cesari M.
      • Cruz-Jentoft A.J.
      • Morley J.E.
      • et al.
      Evidence-based recommendations for optimal dietary protein intake in older people: a position paper from the PROT-AGE Study Group.
      ,
      • Deutz N.E.
      • Bauer J.M.
      • Barazzoni R.
      • Biolo G.
      • Boirie Y.
      • Bosy-Westphal A.
      • et al.
      Protein intake and exercise for optimal muscle function with aging: recommendations from the ESPEN Expert Group.
      ,
      • Wolfe R.R.
      • Cifelli A.M.
      • Kostas G.
      • Kim I.Y.
      Optimizing protein intake in adults: interpretation and application of the recommended dietary allowance compared with the acceptable macronutrient distribution range.
      ], 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 [
      • Morton R.W.
      • Murphy K.T.
      • McKellar S.R.
      • Schoenfeld B.J.
      • Henselmans M.
      • Helms E.
      • et al.
      A systematic review, meta-analysis and meta-regression of the effect of protein supplementation on resistance training-induced gains in muscle mass and strength in healthy adults.
      ]. 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 [
      • Mazzulla M.
      • Sawan S.A.
      • Williamson E.
      • Hannaian S.J.
      • Volterman K.A.
      • West D.W.
      • et al.
      Protein intake to maximize whole-body anabolism during postexercise recovery in resistance-trained men with high habitual intakes is severalfold greater than the current recommended dietary allowance.
      ]. 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 [
      • Ussing H.H.
      The rate of protein renewal in mice and rats studied by means of heavy hydrogen.
      ], there has been a resurgence of interest in the use of D2O for measuring muscle protein turnover [
      • Wilkinson D.J.
      • Franchi M.V.
      • Brook M.S.
      • Narici M.V.
      • Williams J.P.
      • Mitchell W.K.
      • et al.
      A validation of the application of D(2)O stable isotope tracer techniques for monitoring day-to-day changes in muscle protein subfraction synthesis in humans.
      ]. 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 [
      • Hector A.J.
      • McGlory C.
      • Damas F.
      • Mazara N.
      • Baker S.K.
      • Phillips S.M.
      Pronounced energy restriction with elevated protein intake results in no change in proteolysis and reductions in skeletal muscle protein synthesis that are mitigated by resistance exercise.
      ]; 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 [
      • Oikawa S.Y.
      • Bahniwal R.
      • Holloway T.M.
      • Lim C.
      • McLeod J.C.
      • McGlory C.
      • et al.
      Potato protein isolate stimulates muscle protein synthesis at rest and with resistance exercise in young women.
      ]. 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.

      2.4 Protein timing surrounding resistance exercise

      Protein ingestion and RE have a synergistic influence on MPS and muscle growth over time [
      • Morton R.W.
      • Murphy K.T.
      • McKellar S.R.
      • Schoenfeld B.J.
      • Henselmans M.
      • Helms E.
      • et al.
      A systematic review, meta-analysis and meta-regression of the effect of protein supplementation on resistance training-induced gains in muscle mass and strength in healthy adults.
      ,
      • Cermak N.M.
      • Res P.T.
      • de Groot L.C.
      • Saris W.H.
      • van Loon L.J.
      Protein supplementation augments the adaptive response of skeletal muscle to resistance-type exercise training: a meta-analysis.
      ]. 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) [
      • Tipton K.D.
      • Rasmussen B.B.
      • Miller S.L.
      • Wolf S.E.
      • Owens-Stovall S.K.
      • Petrini B.E.
      • et al.
      Timing of amino acid-carbohydrate ingestion alters anabolic response of muscle to resistance exercise.
      ]. 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 [
      • Tipton K.D.
      • Rasmussen B.B.
      • Miller S.L.
      • Wolf S.E.
      • Owens-Stovall S.K.
      • Petrini B.E.
      • et al.
      Timing of amino acid-carbohydrate ingestion alters anabolic response of muscle to resistance exercise.
      ]. However, when measured directly (tracer incorporation into muscle proteins [
      • Fujita S.
      • Dreyer H.C.
      • Drummond M.J.
      • Glynn E.L.
      • Volpi E.
      • Rasmussen B.B.
      Essential amino acid and carbohydrate ingestion before resistance exercise does not enhance postexercise muscle protein synthesis.
      ]) as opposed to indirectly (i.e. rate of tracer disappearance from plasma [
      • Tipton K.D.
      • Rasmussen B.B.
      • Miller S.L.
      • Wolf S.E.
      • Owens-Stovall S.K.
      • Petrini B.E.
      • et al.
      Timing of amino acid-carbohydrate ingestion alters anabolic response of muscle to resistance exercise.
      ]), Fujita and colleagues failed to demonstrate a superior effect of pre-exercise EAA + CHO consumption compared to fasted-state RE on MPS [
      • Fujita S.
      • Dreyer H.C.
      • Drummond M.J.
      • Glynn E.L.
      • Volpi E.
      • Rasmussen B.B.
      Essential amino acid and carbohydrate ingestion before resistance exercise does not enhance postexercise muscle protein synthesis.
      ]. 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 [
      • Beelen M.
      • Koopman R.
      • Gijsen A.P.
      • Vandereyt H.
      • Kies A.K.
      • Kuipers H.
      • et al.
      Protein coingestion stimulates muscle protein synthesis during resistance-type exercise.
      ], which is likely due to enhanced intramuscular anabolic signaling activation [
      • Burke L.M.
      • Hawley J.A.
      • Ross M.L.
      • Moore D.R.
      • Phillips S.M.
      • Slater G.R.
      • et al.
      Preexercise aminoacidemia and muscle protein synthesis after resistance exercise.
      ]. 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 [
      • Schoenfeld B.J.
      • Aragon A.A.
      • Krieger J.W.
      The effect of protein timing on muscle strength and hypertrophy: a meta-analysis.
      ]. Thus, total protein intake more so than when it was ingested had the largest effect on muscle hypertrophy [
      • Schoenfeld B.J.
      • Aragon A.A.
      • Krieger J.W.
      The effect of protein timing on muscle strength and hypertrophy: a meta-analysis.
      ], which is consistent with a recent meta-analysis from our laboratory [
      • Morton R.W.
      • Murphy K.T.
      • McKellar S.R.
      • Schoenfeld B.J.
      • Henselmans M.
      • Helms E.
      • et al.
      A systematic review, meta-analysis and meta-regression of the effect of protein supplementation on resistance training-induced gains in muscle mass and strength in healthy adults.
      ]. Additionally, skeletal muscle remains sensitized to AA for at least 24 h post-exercise [
      • Burd N.A.
      • West D.W.
      • Moore D.R.
      • Atherton P.J.
      • Staples A.W.
      • Prior T.
      • et al.
      Enhanced amino acid sensitivity of myofibrillar protein synthesis persists for up to 24 h after resistance exercise in young men.
      ] 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.
      Fig. 3
      Fig. 3Muscle protein synthesis is increased following a bout of resistance exercise. Skeletal muscle becomes more sensitized to the ingestion of protein for at least 24 h following resistance exercise. Thus, total protein intake rather than the exact time at which protein is consumed surrounding resistance exercise has the greatest impact on muscle protein synthesis. Figure originally from and has been re-drawn from Churchward-Venne and colleagues [
      • Churchward-Venne T.A.
      • Burd N.A.
      • Phillips S.M.
      Nutritional regulation of muscle protein synthesis with resistance exercise: strategies to enhance anabolism.
      ].

      3. Protein quality

      In addition to protein dose and timing, protein quality has been the subject of intense scientific focus and is considered to be an important variable that should be incorporated in dietary protein recommendations. The quantity of a protein is determined based on the nitrogen content in a sample of isolated protein or food, whereas protein quality is dictated by the capacity of the protein to fulfil key metabolic requirements. Therefore, protein quality is organism and physiology-dependent, rather than being limited to a function of chemical or biochemical properties of the protein (i.e. available nitrogen content). It is the EAA content and the proteins inherent digestibility within a particular protein source that are key determinants of the metabolic effects induced by protein ingestion [
      • Wolfe R.R.
      • Rutherfurd S.M.
      • Kim I.Y.
      • Moughan P.J.
      Protein quality as determined by the Digestible Indispensable Amino Acid Score: evaluation of factors underlying the calculation.
      ,
      • Phillips S.M.
      The impact of protein quality on the promotion of resistance exercise-induced changes in muscle mass.
      ,
      • Phillips S.M.
      Current concepts and unresolved questions in dietary protein requirements and supplements in adults.
      ].
      In 1991, the Food and Agriculture Organization (FAO) of the United Nations recommended the use of the protein digestibility-corrected amino acid score (PDCAAS) for the evaluation of protein quality in food products - Protein Quality Evaluation: Report of Joint FAO/WHO Expert Consultation [
      FAO/WHO
      Dietary protein quality evaluation in human nutrition. Report of an FAQ Expert Consultation.
      ]. Briefly, PDCAAS is determined by the AA score of one given crude dietary protein (CP) multiplied by its true faecal nitrogen digestibility. However, PDCAAS had several limitations requiring it to be updated by new, more accurate approaches [
      • Phillips S.M.
      Current concepts and unresolved questions in dietary protein requirements and supplements in adults.
      ,
      FAO/WHO
      Dietary protein quality evaluation in human nutrition. Report of an FAQ Expert Consultation.
      ,
      • Rutherfurd S.M.
      • Fanning A.C.
      • Miller B.J.
      • Moughan P.J.
      Protein digestibility-corrected amino acid scores and digestible indispensable amino acid scores differentially describe protein quality in growing male rats.
      ]. Since then, an expert Consultation on Protein Evaluation in Human Nutrition report, published in 2013, contained a recommendation to use the digestible indispensable amino acid score (DIAAS); as this method addresses some limitations of the PDCAAS. Specifically, the DIAAS indicates a percentage of the daily requirement for the most limiting EAA in a given protein when an amount equals to the estimated average requirement (EAR) is ingested and it also uses ileal rather than faecal digestibility. Because EAR for different AA varies according to age ranges, the same protein or food protein source can have different DIAAS values based on the requirements applied during the calculation. As opposed to PDCAAS, DIAAS is also not truncated at 1.0 and instead recognized that EAA at levels above those required for growth also needs to be recognized. Interested readers can find a deeper description and discussion on the limitations and advantages of DIAAS or further comparison between DIAAS over PDCAAS elsewhere [
      • Wolfe R.R.
      • Rutherfurd S.M.
      • Kim I.Y.
      • Moughan P.J.
      Protein quality as determined by the Digestible Indispensable Amino Acid Score: evaluation of factors underlying the calculation.
      ,
      • Phillips S.M.
      The impact of protein quality on the promotion of resistance exercise-induced changes in muscle mass.
      ,
      • Marinangeli C.P.F.
      • House J.D.
      Potential impact of the digestible indispensable amino acid score as a measure of protein quality on dietary regulations and health.
      ].
      Protein quality has been a known independent variable dictating MPS response. Still, most studies have tested high-quality proteins (i.e. milk-derived proteins, whole-egg protein, etc.) to stimulate MPS in humans [
      • Moore D.R.
      • Robinson M.J.
      • Fry J.L.
      • Tang J.E.
      • Glover E.I.
      • Wilkinson S.B.
      • et al.
      Ingested protein dose response of muscle and albumin protein synthesis after resistance exercise in young men.
      ,
      • Churchward-Venne T.A.
      • Breen L.
      • Di Donato D.M.
      • Hector A.J.
      • Mitchell C.J.
      • Moore D.R.
      • et al.
      Leucine supplementation of a low-protein mixed macronutrient beverage enhances myofibrillar protein synthesis in young men: a double-blind, randomized trial.
      ,
      • Mitchell C.J.
      • McGregor R.A.
      • D'Souza R.F.
      • Thorstensen E.B.
      • Markworth J.F.
      • Fanning A.C.
      • et al.
      Consumption of milk protein or whey protein results in a similar increase in muscle protein synthesis in middle aged men.
      ,
      • Kramer I.F.
      • Verdijk L.B.
      • Hamer H.M.
      • Verlaan S.
      • Luiking Y.C.
      • Kouw I.W.K.
      • et al.
      Both basal and post-prandial muscle protein synthesis rates, following the ingestion of a leucine-enriched whey protein supplement, are not impaired in sarcopenic older males.
      ]. Therefore, the vast majority of the knowledge in this field is restricted to considered high-quality proteins. A robust protein synthetic response can be stimulated by proteins like whey protein [
      • Churchward-Venne T.A.
      • Breen L.
      • Di Donato D.M.
      • Hector A.J.
      • Mitchell C.J.
      • Moore D.R.
      • et al.
      Leucine supplementation of a low-protein mixed macronutrient beverage enhances myofibrillar protein synthesis in young men: a double-blind, randomized trial.
      ]; however, particular characteristics of whey protein as rapid digestibility and high leucine content [
      • Soop M.
      • Nehra V.
      • Henderson G.C.
      • Boirie Y.
      • Ford G.C.
      • Nair K.S.
      Coingestion of whey protein and casein in a mixed meal: demonstration of a more sustained anabolic effect of casein.
      ,
      • Gorissen S.H.
      • Horstman A.M.
      • Franssen R.
      • Crombag J.J.
      • Langer H.
      • Bierau J.
      • et al.
      Ingestion of wheat protein increases in vivo muscle protein synthesis rates in healthy older men in a randomized trial.
      ]. were fundamental to discovering the role of Leucine in MPS activation [
      • Soop M.
      • Nehra V.
      • Henderson G.C.
      • Boirie Y.
      • Ford G.C.
      • Nair K.S.
      Coingestion of whey protein and casein in a mixed meal: demonstration of a more sustained anabolic effect of casein.
      ,
      • Churchward-Venne T.A.
      • Breen L.
      • Di Donato D.M.
      • Hector A.J.
      • Mitchell C.J.
      • Moore D.R.
      • et al.
      Leucine supplementation of a low-protein mixed macronutrient beverage enhances myofibrillar protein synthesis in young men: a double-blind, randomized trial.
      ,
      • Gorissen S.H.
      • Horstman A.M.
      • Franssen R.
      • Crombag J.J.
      • Langer H.
      • Bierau J.
      • et al.
      Ingestion of wheat protein increases in vivo muscle protein synthesis rates in healthy older men in a randomized trial.
      ], as discussed in the following section.

      3.1 Leucine: the trigger amino acid

      More recently, an emphasis has been placed on identifying specific EAA that stimulate MPS, specifically leucine [
      • Phillips S.M.
      Current concepts and unresolved questions in dietary protein requirements and supplements in adults.
      ]. Altogether, EAA are the main drivers of a sustained MPS response [
      • Volpi E.
      • Kobayashi H.
      • Sheffield-Moore M.
      • Mittendorfer B.
      • Wolfe R.R.
      Essential amino acids are primarily responsible for the amino acid stimulation of muscle protein anabolism in healthy elderly adults.
      ], with leucine being the AA that in muscle, acts to activate the process of MPS [
      • Aguiar A.F.
      • Grala A.P.
      • da Silva R.A.
      • Soares-Caldeira L.F.
      • Pacagnelli F.L.
      • Ribeiro A.S.
      • et al.
      Free leucine supplementation during an 8-week resistance training program does not increase muscle mass and strength in untrained young adult subjects.
      ]. Several studies have shown that leucine content, within a protein-containing meal, is the main driver of MPS activation [
      • Churchward-Venne T.A.
      • Breen L.
      • Di Donato D.M.
      • Hector A.J.
      • Mitchell C.J.
      • Moore D.R.
      • et al.
      Leucine supplementation of a low-protein mixed macronutrient beverage enhances myofibrillar protein synthesis in young men: a double-blind, randomized trial.
      ,
      • Devries M.C.
      • McGlory C.
      • Bolster D.R.
      • Kamil A.
      • Rahn M.
      • Harkness L.
      • et al.
      Protein leucine content is a determinant of shorter- and longer-term muscle protein synthetic responses at rest and following resistance exercise in healthy older women: a randomized, controlled trial.
      ,
      • Devries M.C.
      • McGlory C.
      • Bolster D.R.
      • Kamil A.
      • Rahn M.
      • Harkness L.
      • et al.
      Leucine, not total protein, content of a supplement is the primary determinant of muscle protein anabolic responses in healthy older women.
      ]. Because of this property, a leucine trigger hypothesis has been advocated to better understand how protein ingestion activates MPS [
      • Phillips S.M.
      The impact of protein quality on the promotion of resistance exercise-induced changes in muscle mass.
      ,
      • Garlick P.J.
      The role of leucine in the regulation of protein metabolism.
      ,
      • Tipton K.D.
      • Elliott T.A.
      • Ferrando A.A.
      • Aarsland A.A.
      • Wolfe R.R.
      Stimulation of muscle anabolism by resistance exercise and ingestion of leucine plus protein.
      ]. The hypothesis stipulates that MPS activation is a function of leucine concentration within the intracellular AA pool [
      • Phillips S.M.
      The impact of protein quality on the promotion of resistance exercise-induced changes in muscle mass.
      ,
      • Churchward-Venne T.A.
      • Breen L.
      • Di Donato D.M.
      • Hector A.J.
      • Mitchell C.J.
      • Moore D.R.
      • et al.
      Leucine supplementation of a low-protein mixed macronutrient beverage enhances myofibrillar protein synthesis in young men: a double-blind, randomized trial.
      ]. 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) [
      • Kimball S.R.
      • Gordon B.S.
      • Moyer J.E.
      • Dennis M.D.
      • Jefferson L.S.
      Leucine induced dephosphorylation of Sestrin2 promotes mTORC1 activation.
      ]. The exact mechanism by which leucine activates mTORC1 pathway has been identified, and it involves proteins of the Sestrin family [
      • Kimball S.R.
      • Gordon B.S.
      • Moyer J.E.
      • Dennis M.D.
      • Jefferson L.S.
      Leucine induced dephosphorylation of Sestrin2 promotes mTORC1 activation.
      ,
      • Wolfson R.L.
      • Chantranupong L.
      • Saxton R.A.
      • Shen K.
      • Scaria S.M.
      • Cantor J.R.
      • et al.
      Sestrin2 is a leucine sensor for the mTORC1 pathway.
      ]. Specifically, leucine binds to Sestrin 2 changing its phosphorylation state and decreasing its interaction with GAP activity toward the Rag GTPases 2 (GATOR2) [
      • Kimball S.R.
      • Gordon B.S.
      • Moyer J.E.
      • Dennis M.D.
      • Jefferson L.S.
      Leucine induced dephosphorylation of Sestrin2 promotes mTORC1 activation.
      ]. 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) [
      • Kimball S.R.
      • Gordon B.S.
      • Moyer J.E.
      • Dennis M.D.
      • Jefferson L.S.
      Leucine induced dephosphorylation of Sestrin2 promotes mTORC1 activation.
      ]. 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 [
      • Devries M.C.
      • McGlory C.
      • Bolster D.R.
      • Kamil A.
      • Rahn M.
      • Harkness L.
      • et al.
      Protein leucine content is a determinant of shorter- and longer-term muscle protein synthetic responses at rest and following resistance exercise in healthy older women: a randomized, controlled trial.
      ,
      • Devries M.C.
      • McGlory C.
      • Bolster D.R.
      • Kamil A.
      • Rahn M.
      • Harkness L.
      • et al.
      Leucine, not total protein, content of a supplement is the primary determinant of muscle protein anabolic responses in healthy older women.
      ,
      • Murphy C.H.
      • Saddler N.I.
      • Devries M.C.
      • McGlory C.
      • Baker S.K.
      • Phillips S.M.
      Leucine supplementation enhances integrative myofibrillar protein synthesis in free-living older men consuming lower- and higher-protein diets: a parallel-group crossover study.
      ]. Katsanos and colleagues [
      • Katsanos C.S.
      • Kobayashi H.
      • Sheffield-Moore M.
      • Aarsland A.
      • Wolfe R.R.
      A high proportion of leucine is required for optimal stimulation of the rate of muscle protein synthesis by essential amino acids in the elderly.
      ] 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 [
      • Katsanos C.S.
      • Kobayashi H.
      • Sheffield-Moore M.
      • Aarsland A.
      • Wolfe R.R.
      A high proportion of leucine is required for optimal stimulation of the rate of muscle protein synthesis by essential amino acids in the elderly.
      ]. Thus, it seems total leucine content rather than total protein quantity may be rate limiting in stimulating MPS [
      • Churchward-Venne T.A.
      • Breen L.
      • Di Donato D.M.
      • Hector A.J.
      • Mitchell C.J.
      • Moore D.R.
      • et al.
      Leucine supplementation of a low-protein mixed macronutrient beverage enhances myofibrillar protein synthesis in young men: a double-blind, randomized trial.
      ,
      • Devries M.C.
      • McGlory C.
      • Bolster D.R.
      • Kamil A.
      • Rahn M.
      • Harkness L.
      • et al.
      Protein leucine content is a determinant of shorter- and longer-term muscle protein synthetic responses at rest and following resistance exercise in healthy older women: a randomized, controlled trial.
      ]. 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 [
      • Traylor D.A.
      • Gorissen S.H.M.
      • Phillips S.M.
      Perspective: protein requirements and optimal intakes in aging: are we ready to recommend more than the recommended daily allowance?.
      ]. 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 [
      • Churchward-Venne T.A.
      • Breen L.
      • Di Donato D.M.
      • Hector A.J.
      • Mitchell C.J.
      • Moore D.R.
      • et al.
      Leucine supplementation of a low-protein mixed macronutrient beverage enhances myofibrillar protein synthesis in young men: a double-blind, randomized trial.
      ]. Nevertheless, older subjects require a higher protein and leucine content per meal, to achieve an equivalent MPS response compared with young subjects [
      • Moore D.R.
      • Churchward-Venne T.A.
      • Witard O.
      • Breen L.
      • Burd N.A.
      • Tipton K.D.
      • et al.
      Protein ingestion to stimulate myofibrillar protein synthesis requires greater relative protein intakes in healthy older versus younger men.
      ].
      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 [
      • Murphy C.H.
      • Saddler N.I.
      • Devries M.C.
      • McGlory C.
      • Baker S.K.
      • Phillips S.M.
      Leucine supplementation enhances integrative myofibrillar protein synthesis in free-living older men consuming lower- and higher-protein diets: a parallel-group crossover study.
      ]. 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 [
      • Murphy C.H.
      • Saddler N.I.
      • Devries M.C.
      • McGlory C.
      • Baker S.K.
      • Phillips S.M.
      Leucine supplementation enhances integrative myofibrillar protein synthesis in free-living older men consuming lower- and higher-protein diets: a parallel-group crossover study.
      ]. 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) [
      • Devries M.C.
      • McGlory C.
      • Bolster D.R.
      • Kamil A.
      • Rahn M.
      • Harkness L.
      • et al.
      Leucine, not total protein, content of a supplement is the primary determinant of muscle protein anabolic responses in healthy older women.
      ]. 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 [
      • Katsanos C.S.
      • Kobayashi H.
      • Sheffield-Moore M.
      • Aarsland A.
      • Wolfe R.R.
      A high proportion of leucine is required for optimal stimulation of the rate of muscle protein synthesis by essential amino acids in the elderly.
      ].

      3.2 Plant-derived sources

      Protein quality relies mostly on AA composition and bioavailability [
      • Phillips S.M.
      Current concepts and unresolved questions in dietary protein requirements and supplements in adults.
      ]. 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 [
      • Phillips S.M.
      Current concepts and unresolved questions in dietary protein requirements and supplements in adults.
      ,
      • Mathai J.K.
      • Liu Y.
      • Stein H.H.
      Values for digestible indispensable amino acid scores (DIAAS) for some dairy and plant proteins may better describe protein quality than values calculated using the concept for protein digestibility-corrected amino acid scores (PDCAAS).
      ,
      • Hodgkinson S.M.
      • Montoya C.A.
      • Scholten P.T.
      • Rutherfurd S.M.
      • Moughan P.J.
      Cooking conditions affect the true ileal digestible amino acid content and digestible indispensable amino acid score (DIAAS) of bovine meat as determined in pigs.
      ,
      • Bailey H.M.
      • Mathai J.K.
      • Berg E.P.
      • Stein H.H.
      Pork products have digestible indispensable amino acid scores (DIAAS) that are greater than 100 when determined in pigs, but processing does not always increase DIAAS.
      ]. 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 [
      • Rutherfurd S.M.
      • Fanning A.C.
      • Miller B.J.
      • Moughan P.J.
      Protein digestibility-corrected amino acid scores and digestible indispensable amino acid scores differentially describe protein quality in growing male rats.
      ,
      • Cervantes-Pahm S.K.
      • Liu Y.
      • Stein H.H.
      Digestible indispensable amino acid score and digestible amino acids in eight cereal grains.
      ,
      • Han F.
      • Han F.
      • Wang Y.
      • Fan L.
      • Song G.
      • Chen X.
      • et al.
      Digestible indispensable amino acid scores of nine cooked cereal grains.
      ]. 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 [
      • Rutherfurd S.M.
      • Fanning A.C.
      • Miller B.J.
      • Moughan P.J.
      Protein digestibility-corrected amino acid scores and digestible indispensable amino acid scores differentially describe protein quality in growing male rats.
      ], 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 [
      • Bindari Y.R.
      • Laerke H.N.
      • Norgaard J.V.
      Standardized ileal digestibility and digestible indispensable amino acid score of porcine and bovine hydrolyzates in pigs.
      ]. 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 [
      • Wilkinson S.B.
      • Tarnopolsky M.A.
      • Macdonald M.J.
      • Macdonald J.R.
      • Armstrong D.
      • Phillips S.M.
      Consumption of fluid skim milk promotes greater muscle protein accretion after resistance exercise than does consumption of an isonitrogenous and isoenergetic soy-protein beverage.
      ]. 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 [
      • Wilkinson S.B.
      • Tarnopolsky M.A.
      • Macdonald M.J.
      • Macdonald J.R.
      • Armstrong D.
      • Phillips S.M.
      Consumption of fluid skim milk promotes greater muscle protein accretion after resistance exercise than does consumption of an isonitrogenous and isoenergetic soy-protein beverage.
      ]. However, this acute difference in MPS seems to be present only when comparing soy-based protein beverages with skim milk [
      • Wilkinson S.B.
      • Tarnopolsky M.A.
      • Macdonald M.J.
      • Macdonald J.R.
      • Armstrong D.
      • Phillips S.M.
      Consumption of fluid skim milk promotes greater muscle protein accretion after resistance exercise than does consumption of an isonitrogenous and isoenergetic soy-protein beverage.
      ] or whey protein [
      • Tang J.E.
      • Moore D.R.
      • Kujbida G.W.
      • Tarnopolsky M.A.
      • Phillips S.M.
      Ingestion of whey hydrolysate, casein, or soy protein isolate: effects on mixed muscle protein synthesis at rest and following resistance exercise in young men.
      ], but not with the ingestion of casein [
      • Tang J.E.
      • Moore D.R.
      • Kujbida G.W.
      • Tarnopolsky M.A.
      • Phillips S.M.
      Ingestion of whey hydrolysate, casein, or soy protein isolate: effects on mixed muscle protein synthesis at rest and following resistance exercise in young men.
      ].
      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 [
      • van Vliet S.
      • Burd N.A.
      • van Loon L.J.
      The skeletal muscle anabolic response to plant- versus animal-based protein consumption.
      ]. 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 [
      • Reidy P.T.
      • Walker D.K.
      • Dickinson J.M.
      • Gundermann D.M.
      • Drummond M.J.
      • Timmerman K.L.
      • et al.
      Protein blend ingestion following resistance exercise promotes human muscle protein synthesis.
      ]. Similar findings were observed in middle-aged and older men, despite the use of a greater protein dose [
      • Borack M.S.
      • Reidy P.T.
      • Husaini S.H.
      • Markofski M.M.
      • Deer R.R.
      • Richison A.B.
      • et al.
      Soy-dairy protein blend or whey protein isolate ingestion induces similar postexercise muscle mechanistic target of rapamycin complex 1 signaling and protein synthesis responses in older men.
      ]. 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) [
      • Kim I.Y.
      • Shin Y.A.
      • Schutzler S.E.
      • Azhar G.
      • Wolfe R.R.
      • Ferrando A.A.
      Quality of meal protein determines anabolic response in older adults.
      ]. 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 [
      • van Vliet S.
      • Burd N.A.
      • van Loon L.J.
      The skeletal muscle anabolic response to plant- versus animal-based protein consumption.
      ,
      • Gorissen S.H.M.
      • Witard O.C.
      Characterising the muscle anabolic potential of dairy, meat and plant-based protein sources in older adults.
      ,
      • Gorissen S.H.M.
      • Crombag J.J.R.
      • Senden J.M.G.
      • Waterval W.A.H.
      • Bierau J.
      • Verdijk L.B.
      • et al.
      Protein content and amino acid composition of commercially available plant-based protein isolates.
      ]. 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 [
      • Churchward-Venne T.A.
      • Pinckaers P.J.M.
      • Smeets J.S.J.
      • Peeters W.M.
      • Zorenc A.H.
      • Schierbeek H.
      • et al.
      Myofibrillar and mitochondrial protein synthesis rates do not differ in young men following the ingestion of carbohydrate with milk protein, whey, or micellar casein after concurrent resistance- and endurance-type exercise.
      ]. Fractional synthetic rates in myofibrillar and mitochondrial protein fractions were equivalent in the 6 h following exercise and ingestion of the protein beverages [
      • Churchward-Venne T.A.
      • Pinckaers P.J.M.
      • Smeets J.S.J.
      • Peeters W.M.
      • Zorenc A.H.
      • Schierbeek H.
      • et al.
      Myofibrillar and mitochondrial protein synthesis rates do not differ in young men following the ingestion of carbohydrate with milk protein, whey, or micellar casein after concurrent resistance- and endurance-type exercise.
      ]. 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) [
      • Gorissen S.H.
      • Horstman A.M.
      • Franssen R.
      • Crombag J.J.
      • Langer H.
      • Bierau J.
      • et al.
      Ingestion of wheat protein increases in vivo muscle protein synthesis rates in healthy older men in a randomized trial.
      ]. 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 [
      • Gorissen S.H.
      • Horstman A.M.
      • Franssen R.
      • Crombag J.J.
      • Langer H.
      • Bierau J.
      • et al.
      Ingestion of wheat protein increases in vivo muscle protein synthesis rates in healthy older men in a randomized trial.
      ].
      Table 1Essential amino acids (EAA) and leucine content in different dietary protein sources.
      Protein sourceEAA g/100
      Values derived from Ref. [92].
      EAA g/25 g
      Values derived from Ref. [92].
      Leucine g/100 g
      Values derived from Ref. [92].
      Leucine g/25 g of protein
      Values derived from Ref. [92].
      Protein dose (g) to ingest 3 g of Leucine
      Values derived from Ref. [92].
      Animal-derived proteins
       Egg16.54.16.91.743
       Whey34.18.510.82.728
       Milk30.37.58.72.234
       Caseinate32.88.29.02.333
       Casein24.86.27.92.038
      Vegetal-derived proteins
       Soy19.94.96.81.744
       Wheat18.02.46.01.550
       Pea23.65.97.11.842
       Potato29.37.38.22.037
       Corn21.05.213.53.422
       Oat13.73.45.71.452
      EAA: essential amino acids.
      a Values derived from Ref. [
      • Gorissen S.H.M.
      • Crombag J.J.R.
      • Senden J.M.G.
      • Waterval W.A.H.
      • Bierau J.
      • Verdijk L.B.
      • et al.
      Protein content and amino acid composition of commercially available plant-based protein isolates.
      ].
      Ingestion of supplemental plant-based protein isolates can support significant differences in daily MPS [
      • Oikawa S.Y.
      • Bahniwal R.
      • Holloway T.M.
      • Lim C.
      • McLeod J.C.
      • McGlory C.
      • et al.
      Potato protein isolate stimulates muscle protein synthesis at rest and with resistance exercise in young women.
      ]. 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 [
      • Oikawa S.Y.
      • Bahniwal R.
      • Holloway T.M.
      • Lim C.
      • McLeod J.C.
      • McGlory C.
      • et al.
      Potato protein isolate stimulates muscle protein synthesis at rest and with resistance exercise in young women.
      ]. 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 [
      • Babault N.
      • Paizis C.
      • Deley G.
      • Guerin-Deremaux L.
      • Saniez M.H.
      • Lefranc-Millot C.
      • et al.
      Pea proteins oral supplementation promotes muscle thickness gains during resistance training: a double-blind, randomized, Placebo-controlled clinical trial vs. Whey protein.
      ]. 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 [
      • Katsanos C.S.
      • Kobayashi H.
      • Sheffield-Moore M.
      • Aarsland A.
      • Wolfe R.R.
      A high proportion of leucine is required for optimal stimulation of the rate of muscle protein synthesis by essential amino acids in the elderly.
      ].

      4. Muscle protein breakdown

      Although much of the current research centred on the influence of dietary intake on muscle protein turnover has focussed on unravelling the mechanisms of MPS, MPB is also a critical process of skeletal muscle remodelling. Importantly, protein accretion occurs only when an individual is in a state of positive NPBAL, which occurs when MPS exceeds that of MPB achieved through an increased rate of MPS or by the suppression of MPB or a combination of both. The breakdown of skeletal muscle protein is regulated by three inter-connected systems: the ubiquitin-proteasomal pathway (UPP), lysosomal autophagy, and the calpain Ca2+-dependent cysteine proteases [
      • Tipton K.D.
      • Hamilton D.L.
      • Gallagher I.J.
      Assessing the role of muscle protein breakdown in response to nutrition and exercise in humans.
      ]. The assessment of skeletal MPB is complex, and it can be estimated from static or measured directly by dynamic techniques. Static measurement of muscle protein breakdown is commonly assessed at the gene and protein level predominantly through the determination of molecular, mRNA or protein abundance, responses within each of the three protein breakdown pathways. In addition, the appearance of 3-methylhistidine (3-MH), a post-translationally methylated histidine present in myofibrillar proteins that cannot be metabolized or reutilized within skeletal muscle, within circulation has been used to provide a crude estimate of MPB [
      • Ballard F.J.
      • Tomas F.M.
      3-Methylhistidine as a measure of skeletal muscle protein breakdown in human subjects: the case for its continued use.
      ].
      As previously mentioned, the assessment of MPB is complex. Specifically, the use of stable isotopically labelled tracers is an invasive process and requires the infusion of a labelled amino acid combined with multiple arterial and venous blood samples throughout an experiment [
      • Tipton K.D.
      • Hamilton D.L.
      • Gallagher I.J.
      Assessing the role of muscle protein breakdown in response to nutrition and exercise in humans.
      ,
      • Holm L.
      • O'Rourke B.
      • Ebenstein D.
      • Toth M.J.
      • Bechshoeft R.
      • Holstein-Rathlou N.H.
      • et al.
      Determination of steady-state protein breakdown rate in vivo by the disappearance of protein-bound tracer-labeled amino acids: a method applicable in humans.
      ]. Briefly, the two-pool arteriovenous (AV) method allows for the determination of release of AA (assumed to be from muscle) into circulation, and by subtraction uptake, of a labelled AA that is not metabolized by the muscle [
      • Tipton K.D.
      • Hamilton D.L.
      • Gallagher I.J.
      Assessing the role of muscle protein breakdown in response to nutrition and exercise in humans.
      ]. The three-pool method is very similar to the two-pool method, with the critical distinction that the addition of intramuscular determination of labelled AA enrichment and concentration allows for the calculation of true intramuscular protein breakdown and synthesis and not blood proxies per the two-pool model [
      • Tipton K.D.
      • Hamilton D.L.
      • Gallagher I.J.
      Assessing the role of muscle protein breakdown in response to nutrition and exercise in humans.
      ,
      • Zhang X.J.
      • Chinkes D.L.
      • Sakurai Y.
      • Wolfe R.R.
      An isotopic method for measurement of muscle protein fractional breakdown rate in vivo.
      ]. One fundamental limitation is that the two- and three-pool methods require physiological and isotope steady state, and therefore cannot be conducted following feeding. In addition, in an experimental setting, arterial catherization is not always possible, therefore the incorporation of the two- and three-pool model may not be feasible. Breakdown can also be determined by the infusion of two different isotopes and the sampling of arterialized venous blood and muscle tissue. Although, this method does not require arterial catherization, physiological steady state must still be achieved and additional muscle biopsies are required; potentially negating the reduced invasiveness [
      • Tipton K.D.
      • Hamilton D.L.
      • Gallagher I.J.
      Assessing the role of muscle protein breakdown in response to nutrition and exercise in humans.
      ,
      • Zhang X.J.
      • Chinkes D.L.
      • Sakurai Y.
      • Wolfe R.R.
      An isotopic method for measurement of muscle protein fractional breakdown rate in vivo.
      ]. The complexity associated with the assessment of MPB, makes it clear that less information is available that examines the influence of exercise and nutrition on MPB specifically, when compared with the extensive literature that pertains to MPS. Nevertheless, stable isotopes provide a valuable tool in the quest to understand muscle protein turnover, and the associated difficulties of MPB assessments have not hampered the curiosity of researchers to explore the impact of various nutritional interventions of MPB.

      4.1 Insulin

      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) [
      • Witard O.C.
      • Jackman S.R.
      • Breen L.
      • Smith K.
      • Selby A.
      • Tipton K.D.
      Myofibrillar muscle protein synthesis rates subsequent to a meal in response to increasing doses of whey protein at rest and after resistance exercise.
      ]. Although the systemic rise in insulin has been shown to be permissive for increasing MPS, hyperinsulinemia plays an essential role in the inhibition MPB [
      • Koopman R.
      • Beelen M.
      • Stellingwerff T.
      • Pennings B.
      • Saris W.H.
      • Kies A.K.
      • et al.
      Coingestion of carbohydrate with protein does not further augment postexercise muscle protein synthesis.
      ,
      • Staples A.W.
      • Burd N.A.
      • West D.W.
      • Currie K.D.
      • Atherton P.J.
      • Moore D.R.
      • et al.
      Carbohydrate does not augment exercise-induced protein accretion versus protein alone.
      ]. 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 [
      • Möller-Loswick A.C.
      • Zachrisson H.
      • Hyltander A.
      • Körner U.
      • Matthews D.E.
      • Lundholm K.
      Insulin selectively attenuates breakdown of nonmyofibrillar proteins in peripheral tissues of normal men.
      ]. 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 [
      • Biolo G.
      • Maggi S.P.
      • Williams B.D.
      • Tipton K.D.
      • Wolfe R.R.
      Increased rates of muscle protein turnover and amino acid transport after resistance exercise in humans.
      ]. However, the rise in MPB was abolished when RE was immediately followed by the infusion of AA [
      • Biolo G.
      • Tipton K.D.
      • Klein S.
      • Wolfe R.R.
      An abundant supply of amino acids enhances the metabolic effect of exercise on muscle protein.
      ].
      In a hyperlipidemic, hyperglycaemic and hyperinsulinemic state MPB is reduced compared to the post absorptive state [
      • Bell J.A.
      • Volpi E.
      • Fujita S.
      • Cadenas J.G.
      • Sheffield-Moore M.
      • Rasmussen B.B.
      Skeletal muscle protein anabolic response to increased energy and insulin is preserved in poorly controlled type 2 diabetes.
      ]. 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 [
      • Greenhaff P.L.
      • Karagounis L.
      • Peirce N.
      • Simpson E.J.
      • Hazell M.
      • Layfield R.
      • et al.
      Disassociation between the effects of amino acids and insulin on signalling, ubiquitin-ligases and protein turnover in human muscle.
      ]. 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 [
      • Greenhaff P.L.
      • Karagounis L.
      • Peirce N.
      • Simpson E.J.
      • Hazell M.
      • Layfield R.
      • et al.
      Disassociation between the effects of amino acids and insulin on signalling, ubiquitin-ligases and protein turnover in human muscle.
      ]. 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 [
      • Glynn E.L.
      • Fry C.S.
      • Drummond M.J.
      • Dreyer H.C.
      • Dhanani S.
      • Volpi E.
      • et al.
      Muscle protein breakdown has a minor role in the protein anabolic response to essential amino acid and carbohydrate intake following resistance exercise.
      ]. This, and a recent meta-analysis [
      • Abdulla H.
      • Smith K.
      • Atherton P.J.
      • Idris I.
      Role of insulin in the regulation of human skeletal muscle protein synthesis and breakdown: a systematic review and 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 [
      • Koopman R.
      • Beelen M.
      • Stellingwerff T.
      • Pennings B.
      • Saris W.H.
      • Kies A.K.
      • et al.
      Coingestion of carbohydrate with protein does not further augment postexercise muscle protein synthesis.
      ,
      • Staples A.W.
      • Burd N.A.
      • West D.W.
      • Currie K.D.
      • Atherton P.J.
      • Moore D.R.
      • et al.
      Carbohydrate does not augment exercise-induced protein accretion versus protein alone.
      ]. 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).
      Fig. 4
      Fig. 4Chronic (free-living) MPS and acute MPS/MPB can be quantified by the infusion of isotope tracers such as D2O and labelled AA, respectively. In a fasted state MPB is greater than MPS and thus the muscle is in a catabolic state of negative NPBAL. However, following exercise and an increase in dietary intake, specifically protein, MPS increases, MPB is inhibited and the muscle is in an anabolic state of positive NPBAL. Specifically manipulating dose, timing, distribution and composition of protein intake can maximize the muscle's anabolic response.

      4.2 ‘Excess’ protein consumption

      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 [
      • Moore D.R.
      • Robinson M.J.
      • Fry J.L.
      • Tang J.E.
      • Glover E.I.
      • Wilkinson S.B.
      • et al.
      Ingested protein dose response of muscle and albumin protein synthesis after resistance exercise in young men.
      ,
      • Witard O.C.
      • Jackman S.R.
      • Breen L.
      • Smith K.
      • Selby A.
      • Tipton K.D.
      Myofibrillar muscle protein synthesis rates subsequent to a meal in response to increasing doses of whey protein at rest and after resistance exercise.
      ], whereas approximately 10 g of EAA maximally stimulates MPS [
      • Cuthbertson D.
      • Smith K.
      • Babraj J.
      • Leese G.
      • Waddell T.
      • Atherton P.
      • et al.
      Anabolic signaling deficits underlie amino acid resistance of wasting, aging muscle.
      ]. 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 [
      • Zello G.A.
      • Wykes L.J.
      • Ball R.O.
      • Pencharz P.B.
      Recent advances in methods of assessing dietary amino acid requirements for adult humans.
      ].
      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 [
      • Witard O.C.
      • Jackman S.R.
      • Breen L.
      • Smith K.
      • Selby A.
      • Tipton K.D.
      Myofibrillar muscle protein synthesis rates subsequent to a meal in response to increasing doses of whey protein at rest and after resistance exercise.
      ]. 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) [
      • Moore D.R.
      • Robinson M.J.
      • Fry J.L.
      • Tang J.E.
      • Glover E.I.
      • Wilkinson S.B.
      • et al.
      Ingested protein dose response of muscle and albumin protein synthesis after resistance exercise in young men.
      ]. 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 [
      • Moore D.R.
      • Robinson M.J.
      • Fry J.L.
      • Tang J.E.
      • Glover E.I.
      • Wilkinson S.B.
      • et al.
      Ingested protein dose response of muscle and albumin protein synthesis after resistance exercise in young men.
      ,
      • Witard O.C.
      • Jackman S.R.
      • Breen L.
      • Smith K.
      • Selby A.
      • Tipton K.D.
      Myofibrillar muscle protein synthesis rates subsequent to a meal in response to increasing doses of whey protein at rest and after resistance exercise.
      ]. 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 [
      • Jahoor F.
      • Wolfe R.R.
      Reassessment of primed constant-infusion tracer method to measure urea kinetics.
      ]. 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 [
      • Witard O.C.
      • Jackman S.R.
      • Breen L.
      • Smith K.
      • Selby A.
      • Tipton K.D.
      Myofibrillar muscle protein synthesis rates subsequent to a meal in response to increasing doses of whey protein at rest and after resistance exercise.
      ]. 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 [
      • Trommelen J.
      • Betz M.W.
      • van Loon L.J.
      The muscle protein synthetic response to meal ingestion following resistance-type exercise.
      ], therefore researchers must consider practical inferences that can be made based on these results.

      5. The role of adjunctive nutritional compounds to stimulate MPS

      The combination of RE and protein ingestion is well accepted as an effective way to increase muscle mass. Maximizing muscle mass and function is important in various populations such as athletes, older individuals and in several clinical conditions. As such, researchers have focused on other nutritional compounds/supplements which, in addition to protein, may stimulate MPS, the effects of adjunctive nutritional compounds on MPS is summarized in Table 2.
      Table 2The effect of various nutritional compounds on MPS.
      SupplementStateMPS measureResult
      LeucineFedacuteincrease [
      • Devries M.C.
      • McGlory C.
      • Bolster D.R.
      • Kamil A.
      • Rahn M.
      • Harkness L.
      • et al.
      Protein leucine content is a determinant of shorter- and longer-term muscle protein synthetic responses at rest and following resistance exercise in healthy older women: a randomized, controlled trial.
      ,
      • Cuthbertson D.
      • Smith K.
      • Babraj J.
      • Leese G.
      • Waddell T.
      • Atherton P.
      • et al.
      Anabolic signaling deficits underlie amino acid resistance of wasting, aging muscle.
      ,
      • Katsanos C.S.
      • Kobayashi H.
      • Sheffield-Moore M.
      • Aarsland A.
      • Wolfe R.R.
      A high proportion of leucine is required for optimal stimulation of the rate of muscle protein synthesis by essential amino acids in the elderly.
      ]
      Fed + REacuteincrease [
      • Devries M.C.
      • McGlory C.
      • Bolster D.R.
      • Kamil A.
      • Rahn M.
      • Harkness L.
      • et al.
      Protein leucine content is a determinant of shorter- and longer-term muscle protein synthetic responses at rest and following resistance exercise in healthy older women: a randomized, controlled trial.
      ]
      Free-livingintegratedincrease [
      • Devries M.C.
      • McGlory C.
      • Bolster D.R.
      • Kamil A.
      • Rahn M.
      • Harkness L.
      • et al.
      Protein leucine content is a determinant of shorter- and longer-term muscle protein synthetic responses at rest and following resistance exercise in healthy older women: a randomized, controlled trial.
      ,
      • Murphy C.H.
      • Saddler N.I.
      • Devries M.C.
      • McGlory C.
      • Baker S.K.
      • Phillips S.M.
      Leucine supplementation enhances integrative myofibrillar protein synthesis in free-living older men consuming lower- and higher-protein diets: a parallel-group crossover study.
      ]
      Free-living + REintegratedincrease [
      • Devries M.C.
      • McGlory C.
      • Bolster D.R.
      • Kamil A.
      • Rahn M.
      • Harkness L.
      • et al.
      Protein leucine content is a determinant of shorter- and longer-term muscle protein synthetic responses at rest and following resistance exercise in healthy older women: a randomized, controlled trial.
      ,
      • Murphy C.H.
      • Saddler N.I.
      • Devries M.C.
      • McGlory C.
      • Baker S.K.
      • Phillips S.M.
      Leucine supplementation enhances integrative myofibrillar protein synthesis in free-living older men consuming lower- and higher-protein diets: a parallel-group crossover study.
      ]
      n-3 PUFAFastedacuteno change [
      • Smith G.I.
      • Atherton P.
      • Reeds D.N.
      • Mohammed B.S.
      • Rankin D.
      • Rennie M.J.
      • et al.
      Dietary omega-3 fatty acid supplementation increases the rate of muscle protein synthesis in older adults: a randomized controlled trial.
      ,
      • Smith G.I.
      • Atherton P.
      • Reeds D.N.
      • Mohammed B.S.
      • Rankin D.
      • Rennie M.J.
      • et al.
      Omega-3 polyunsaturated fatty acids augment the muscle protein anabolic response to hyperinsulinaemia-hyperaminoacidaemia in healthy young and middle-aged men and women.
      ]
      Fedacuteincrease [
      • Smith G.I.
      • Atherton P.
      • Reeds D.N.
      • Mohammed B.S.
      • Rankin D.
      • Rennie M.J.
      • et al.
      Dietary omega-3 fatty acid supplementation increases the rate of muscle protein synthesis in older adults: a randomized controlled trial.
      ,
      • Smith G.I.
      • Atherton P.
      • Reeds D.N.
      • Mohammed B.S.
      • Rankin D.
      • Rennie M.J.
      • et al.
      Omega-3 polyunsaturated fatty acids augment the muscle protein anabolic response to hyperinsulinaemia-hyperaminoacidaemia in healthy young and middle-aged men and women.
      ]
      Fastedacuteno change [
      • McGlory C.
      • Wardle S.L.
      • Macnaughton L.S.
      • Witard O.C.
      • Scott F.
      • Dick J.
      • et al.
      Fish oil supplementation suppresses resistance exercise and feeding-induced increases in anabolic signaling without affecting myofibrillar protein synthesis in young men.
      ]
      Fedacuteno change [
      • McGlory C.
      • Wardle S.L.
      • Macnaughton L.S.
      • Witard O.C.
      • Scott F.
      • Dick J.
      • et al.
      Fish oil supplementation suppresses resistance exercise and feeding-induced increases in anabolic signaling without affecting myofibrillar protein synthesis in young men.
      ]
      Fed + REacuteno change [
      • McGlory C.
      • Wardle S.L.
      • Macnaughton L.S.
      • Witard O.C.
      • Scott F.
      • Dick J.
      • et al.
      Fish oil supplementation suppresses resistance exercise and feeding-induced increases in anabolic signaling without affecting myofibrillar protein synthesis in young men.
      ]
      Free-living (+RE)integratedno change [
      • Da Boit M.
      • Sibson R.
      • Sivasubramaniam S.
      • Meakin J.R.
      • Greig C.A.
      • Aspden R.M.
      • et al.
      Sex differences in the effect of fish-oil supplementation on the adaptive response to resistance exercise training in older people: a randomized controlled trial.
      ]
      Free-living (+immobilization)integratedincrease [
      • McGlory C.
      • Calder P.C.
      • Nunes E.A.
      The influence of omega-3 fatty acids on skeletal muscle protein turnover in health, disuse, and disease.
      ]
      CollagenFree-living (+aerobic exercise)integratedno change [
      • Oikawa S.Y.
      • MacInnis M.J.
      • Tripp T.R.
      • McGlory C.
      • Baker S.K.
      • Phillips S.M.
      Lactalbumin, not collagen, augments muscle protein synthesis with aerobic exercise.
      ]
      Free living (+inactivity and energy restriction)integratedno change [
      • Oikawa S.Y.
      • McGlory C.
      • D'Souza L.K.
      • Morgan A.K.
      • Saddler N.I.
      • Baker S.K.
      • et al.
      A randomized controlled trial of the impact of protein supplementation on leg lean mass and integrated muscle protein synthesis during inactivity and energy restriction in older persons.
      ]
      Free living (+RT)integratedno change [
      • Oikawa S.Y.
      • Kamal M.J.
      • Webb E.K.
      • McGlory C.
      • Baker S.K.
      • Phillips S.M.
      Whey protein but not collagen peptides stimulate acute and longer-term muscle protein synthesis with and without resistance exercise in healthy older women: a randomized controlled trial.
      ]
      Fastedacuteno change [
      • Oikawa S.Y.
      • Kamal M.J.
      • Webb E.K.
      • McGlory C.
      • Baker S.K.
      • Phillips S.M.
      Whey protein but not collagen peptides stimulate acute and longer-term muscle protein synthesis with and without resistance exercise in healthy older women: a randomized controlled trial.
      ]
      Fedacuteno change [
      • Oikawa S.Y.
      • Kamal M.J.
      • Webb E.K.
      • McGlory C.
      • Baker S.K.
      • Phillips S.M.
      Whey protein but not collagen peptides stimulate acute and longer-term muscle protein synthesis with and without resistance exercise in healthy older women: a randomized controlled trial.
      ]
      Fed + REacuteno change [
      • Oikawa S.Y.
      • Kamal M.J.
      • Webb E.K.
      • McGlory C.
      • Baker S.K.
      • Phillips S.M.
      Whey protein but not collagen peptides stimulate acute and longer-term muscle protein synthesis with and without resistance exercise in healthy older women: a randomized controlled trial.
      ]
      CreatineFastedacuteno change [
      • Louis M.
      • Poortmans J.R.
      • Francaux M.
      • Hultman E.
      • Berre J.
      • Boisseau N.
      • et al.
      Creatine supplementation has no effect on human muscle protein turnover at rest in the postabsorptive or fed states.
      ,
      • Parise G.
      • Mihic S.
      • MacLennan D.
      • Yarasheski K.E.
      • Tarnopolsky M.A.
      Effects of acute creatine monohydrate supplementation on leucine kinetics and mixed-muscle protein synthesis.
      ]
      Fedacuteno change [
      • Louis M.
      • Poortmans J.R.
      • Francaux M.
      • Hultman E.
      • Berre J.
      • Boisseau N.
      • et al.
      Creatine supplementation has no effect on human muscle protein turnover at rest in the postabsorptive or fed states.
      ]
      Red + REacuteno change [
      • Louis M.
      • Poortmans J.R.
      • Francaux M.
      • Hultman E.
      • Berre J.
      • Boisseau N.
      • et al.
      Creatine supplementation has no effect on human muscle protein turnover at rest in the postabsorptive or fed states.
      ]
      ArginineFedacuteno change [
      • Tang J.E.
      • Lysecki P.J.
      • Manolakos J.J.
      • MacDonald M.J.
      • Tarnopolsky M.A.
      • Phillips S.M.
      Bolus arginine supplementation affects neither muscle blood flow nor muscle protein synthesis in young men at rest or after resistance exercise.
      ]
      Fed + REacuteno change [
      • Tang J.E.
      • Lysecki P.J.
      • Manolakos J.J.
      • MacDonald M.J.
      • Tarnopolsky M.A.
      • Phillips S.M.
      Bolus arginine supplementation affects neither muscle blood flow nor muscle protein synthesis in young men at rest or after resistance exercise.
      ]
      CitrullineFastedacuteno change [
      • Churchward-Venne T.A.
      • Cotie L.M.
      • MacDonald M.J.
      • Mitchell C.J.
      • Prior T.
      • Baker S.K.
      • et al.
      Citrulline does not enhance blood flow, microvascular circulation, or myofibrillar protein synthesis in elderly men at rest or following exercise.
      ]
      Fedacuteno change [
      • Churchward-Venne T.A.
      • Cotie L.M.
      • MacDonald M.J.
      • Mitchell C.J.
      • Prior T.
      • Baker S.K.
      • et al.
      Citrulline does not enhance blood flow, microvascular circulation, or myofibrillar protein synthesis in elderly men at rest or following exercise.
      ]
      Fed + REacuteno change [
      • Churchward-Venne T.A.
      • Cotie L.M.
      • MacDonald M.J.
      • Mitchell C.J.
      • Prior T.
      • Baker S.K.
      • et al.
      Citrulline does not enhance blood flow, microvascular circulation, or myofibrillar protein synthesis in elderly men at rest or following exercise.
      ]
      Phosphatidic acidFastedacuteno change [
      • Smeuninx B.
      • Nishimura Y.
      • McKendry J.
      • Limb M.
      • Smith K.
      • Atherton P.J.
      • et al.
      The effect of acute oral phosphatidic acid ingestion on myofibrillar protein synthesis and intracellular signaling in older males.
      ]
      REacutereduced [
      • Smeuninx B.
      • Nishimura Y.
      • McKendry J.
      • Limb M.
      • Smith K.
      • Atherton P.J.
      • et al.
      The effect of acute oral phosphatidic acid ingestion on myofibrillar protein synthesis and intracellular signaling in older males.
      ]

      5.1 Omega-3 polyunsaturated fatty acids (n3-PUFA)

      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 [
      • Arterburn L.M.
      • Hall E.B.
      • Oken H.
      Distribution, interconversion, and dose response of n-3 fatty acids 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 [
      • Witard O.C.
      • Combet E.
      • Gray S.R.
      Long-chain n-3 fatty acids as an essential link between musculoskeletal and cardio-metabolic health in older adults.
      ]. 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 [
      • Witard O.C.
      • Combet E.
      • Gray S.R.
      Long-chain n-3 fatty acids as an essential link between musculoskeletal and cardio-metabolic health in older adults.
      ,
      • McGlory C.
      • Calder P.C.
      • Nunes E.A.
      The influence of omega-3 fatty acids on skeletal muscle protein turnover in health, disuse, and disease.
      ].
      Seminal work from Smith and colleagues [
      • Smith G.I.
      • Atherton P.
      • Reeds D.N.
      • Mohammed B.S.
      • Rankin D.
      • Rennie M.J.
      • et al.
      Dietary omega-3 fatty acid supplementation increases the rate of muscle protein synthesis in older adults: a randomized controlled trial.
      ,
      • Smith G.I.
      • Atherton P.
      • Reeds D.N.
      • Mohammed B.S.
      • Rankin D.
      • Rennie M.J.
      • et al.
      Omega-3 polyunsaturated fatty acids augment the muscle protein anabolic response to hyperinsulinaemia-hyperaminoacidaemia in healthy young and middle-aged men and women.
      ] demonstrated that in healthy older adults [
      • Smith G.I.
      • Atherton P.
      • Reeds D.N.
      • Mohammed B.S.
      • Rankin D.
      • Rennie M.J.
      • et al.
      Dietary omega-3 fatty acid supplementation increases the rate of muscle protein synthesis in older adults: a randomized controlled trial.
      ], and young-to-middle-aged adults [
      • Smith G.I.
      • Atherton P.
      • Reeds D.N.
      • Mohammed B.S.
      • Rankin D.
      • Rennie M.J.
      • et al.
      Omega-3 polyunsaturated fatty acids augment the muscle protein anabolic response to hyperinsulinaemia-hyperaminoacidaemia in healthy young and middle-aged men and women.
      ] 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 [
      • McGlory C.
      • Wardle S.L.
      • Macnaughton L.S.
      • Witard O.C.
      • Scott F.
      • Dick J.
      • et al.
      Fish oil supplementation suppresses resistance exercise and feeding-induced increases in anabolic signaling without affecting myofibrillar protein synthesis in young men.
      ] 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 [
      • McGlory C.
      • Wardle S.L.
      • Macnaughton L.S.
      • Witard O.C.
      • Scott F.
      • Dick J.
      • et al.
      Fish oil supplementation suppresses resistance exercise and feeding-induced increases in anabolic signaling without affecting myofibrillar protein synthesis in young men.
      ] 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 [
      • Moore D.R.
      • Churchward-Venne T.A.
      • Witard O.
      • Breen L.
      • Burd N.A.
      • Tipton K.D.
      • et al.
      Protein ingestion to stimulate myofibrillar protein synthesis requires greater relative protein intakes in healthy older versus younger men.
      ], however, the recommended dose may be difficult to achieve [
      • Volpi E.
      • Campbell W.W.
      • Dwyer J.T.
      • Johnson M.A.
      • Jensen G.L.
      • Morley J.E.
      • et al.
      Is the optimal level of protein intake for older adults greater than the recommended dietary allowance?.
      ]. With this in mind, n3-PUFA feeding may provide more of an anabolic benefit to older adults. Indeed, compared to younger adults [
      • Smith G.I.
      • Atherton P.
      • Reeds D.N.
      • Mohammed B.S.
      • Rankin D.
      • Rennie M.J.
      • et al.
      Omega-3 polyunsaturated fatty acids augment the muscle protein anabolic response to hyperinsulinaemia-hyperaminoacidaemia in healthy young and middle-aged men and women.
      ], older adults [
      • Smith G.I.
      • Atherton P.
      • Villareal D.T.
      • Frimel T.N.
      • Rankin D.
      • Rennie M.J.
      • et al.
      Differences in muscle protein synthesis and anabolic signaling in the postabsorptive state and in response to food in 65-80 year old men and women.
      ] demonstrated greater relative increases in rates of mixed MPS following n3-PUFA feeding. However, Da Boit and colleagues [
      • Da Boit M.
      • Sibson R.
      • Sivasubramaniam S.
      • Meakin J.R.
      • Greig C.A.
      • Aspden R.M.
      • et al.
      Sex differences in the effect of fish-oil supplementation on the adaptive response to resistance exercise training in older people: a randomized controlled trial.
      ] 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 [
      • Da Boit M.
      • Sibson R.
      • Sivasubramaniam S.
      • Meakin J.R.
      • Greig C.A.
      • Aspden R.M.
      • et al.
      Sex differences in the effect of fish-oil supplementation on the adaptive response to resistance exercise training in older people: a randomized controlled trial.
      ] 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 [
      • Da Boit M.
      • Sibson R.
      • Sivasubramaniam S.
      • Meakin J.R.
      • Greig C.A.
      • Aspden R.M.
      • et al.
      Sex differences in the effect of fish-oil supplementation on the adaptive response to resistance exercise training in older people: a randomized controlled trial.
      ] 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 [
      • Wall B.T.
      • Dirks M.L.
      • Snijders T.
      • van Dijk J.W.
      • Fritsch M.
      • Verdijk L.B.
      • et al.
      Short-term muscle disuse lowers myofibrillar protein synthesis rates and induces anabolic resistance to protein ingestion.
      ]. 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 [
      • McGlory C.
      • Gorissen S.H.M.
      • Kamal M.
      • Bahniwal R.
      • Hector A.J.
      • Baker S.K.
      • et al.
      Omega-3 fatty acid supplementation attenuates skeletal muscle disuse atrophy during two weeks of unilateral leg immobilization in healthy young women.
      ,
      • McGlory C.
      • von Allmen M.T.
      • Stokes T.
      • Morton R.W.
      • Hector A.J.
      • Lago B.A.
      • et al.
      Failed recovery of glycemic control and myofibrillar protein synthesis with 2 wk of physical inactivity in overweight, prediabetic older adults.
      ,
      • Oikawa S.Y.
      • McGlory C.
      • D'Souza L.K.
      • Morgan A.K.
      • Saddler N.I.
      • Baker S.K.
      • et al.
      A randomized controlled trial of the impact of protein supplementation on leg lean mass and integrated muscle protein synthesis during inactivity and energy restriction in older persons.
      ]. Strategies such as RT [
      • Moore D.R.
      • Kelly R.P.
      • Devries M.C.
      • Churchward-Venne T.A.
      • Phillips S.M.
      • Parise G.
      • et al.
      Low-load resistance exercise during inactivity is associated with greater fibre area and satellite cell expression in older skeletal muscle.
      ] and neuromuscular electrical stimulation [
      • Dirks M.L.
      • Wall B.T.
      • Snijders T.
      • Ottenbros C.L.
      • Verdijk L.B.
      • van Loon L.J.
      Neuromuscular electrical stimulation prevents muscle disuse atrophy during leg immobilization in humans.
      ] 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 [
      • Smith G.I.
      • Atherton P.
      • Reeds D.N.
      • Mohammed B.S.
      • Rankin D.
      • Rennie M.J.
      • et al.
      Dietary omega-3 fatty acid supplementation increases the rate of muscle protein synthesis in older adults: a randomized controlled trial.
      ,
      • Smith G.I.
      • Atherton P.
      • Reeds D.N.
      • Mohammed B.S.
      • Rankin D.
      • Rennie M.J.
      • et al.
      Omega-3 polyunsaturated fatty acids augment the muscle protein anabolic response to hyperinsulinaemia-hyperaminoacidaemia in healthy young and middle-aged men and women.
      ] 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 [
      • McGlory C.
      • Gorissen S.H.M.
      • Kamal M.
      • Bahniwal R.
      • Hector A.J.
      • Baker S.K.
      • et al.
      Omega-3 fatty acid supplementation attenuates skeletal muscle disuse atrophy during two weeks of unilateral leg immobilization in healthy young women.
      ]. 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 [
      • McGlory C.
      • Gorissen S.H.M.
      • Kamal M.
      • Bahniwal R.
      • Hector A.J.
      • Baker S.K.
      • et al.
      Omega-3 fatty acid supplementation attenuates skeletal muscle disuse atrophy during two weeks of unilateral leg immobilization in healthy young women.
      ]. 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 [
      • McGlory C.
      • Calder P.C.
      • Nunes E.A.
      The influence of omega-3 fatty acids on skeletal muscle protein turnover in health, disuse, and disease.
      ].

      5.2 Collagen

      Collagen is an extracellular protein that accounts for ~25% of total body protein mass [
      • Zdzieblik D.
      • Oesser S.
      • Baumstark M.W.
      • Gollhofer A.
      • Konig D.
      Collagen peptide supplementation in combination with resistance training improves body composition and increases muscle strength in elderly sarcopenic men: a randomised controlled trial.
      ]. As a nutritional supplement, collagen peptides are considered a low-quality protein source, due to the complete lack of the EAA, tryptophan, and a low leucine content. As described previously, the EAA content of a protein source, particularly leucine, is pertinent for increasing MPS. Work from our laboratory has demonstrated that, compared to high-quality protein sources, such as whey protein [
      • Oikawa S.Y.
      • McGlory C.
      • D'Souza L.K.
      • Morgan A.K.
      • Saddler N.I.
      • Baker S.K.
      • et al.
      A randomized controlled trial of the impact of protein supplementation on leg lean mass and integrated muscle protein synthesis during inactivity and energy restriction in older persons.
      ,
      • Oikawa S.Y.
      • Kamal M.J.
      • Webb E.K.
      • McGlory C.
      • Baker S.K.
      • Phillips S.M.
      Whey protein but not collagen peptides stimulate acute and longer-term muscle protein synthesis with and without resistance exercise in healthy older women: a randomized controlled trial.
      ] and alpha-lactalbumin (a leucine-enriched fraction of whey) [
      • Oikawa S.Y.
      • MacInnis M.J.
      • Tripp T.R.
      • McGlory C.
      • Baker S.K.
      • Phillips S.M.
      Lactalbumin, not collagen, augments muscle protein synthesis with aerobic exercise.
      ], collagen peptide ingestion was not effective in mounting comparable increases in MPS, despite all supplements being isonitrogenous [
      • Oikawa S.Y.
      • McGlory C.
      • D'Souza L.K.
      • Morgan A.K.
      • Saddler N.I.
      • Baker S.K.
      • et al.
      A randomized controlled trial of the impact of protein supplementation on leg lean mass and integrated muscle protein synthesis during inactivity and energy restriction in older persons.
      ,
      • Oikawa S.Y.
      • Kamal M.J.
      • Webb E.K.
      • McGlory C.
      • Baker S.K.
      • Phillips S.M.
      Whey protein but not collagen peptides stimulate acute and longer-term muscle protein synthesis with and without resistance exercise in healthy older women: a randomized controlled trial.
      ,
      • Oikawa S.Y.
      • MacInnis M.J.
      • Tripp T.R.
      • McGlory C.
      • Baker S.K.
      • Phillips S.M.
      Lactalbumin, not collagen, augments muscle protein synthesis with aerobic exercise.
      ]. In fact, collagen peptide ingestion did not elevate plasma leucine concentrations, nor did it increase acute and integrated rates of MPS above baseline [
      • Oikawa S.Y.
      • Kamal M.J.
      • Webb E.K.
      • McGlory C.
      • Baker S.K.
      • Phillips S.M.
      Whey protein but not collagen peptides stimulate acute and longer-term muscle protein synthesis with and without resistance exercise in healthy older women: a randomized controlled trial.
      ]. Despite this, collagen peptide supplements have garnered considerable attention [
      • Zdzieblik D.
      • Oesser S.
      • Baumstark M.W.
      • Gollhofer A.
      • Konig D.
      Collagen peptide supplementation in combination with resistance training improves body composition and increases muscle strength in elderly sarcopenic men: a randomised controlled trial.
      ,
      • Centner C.
      • Zdzieblik D.
      • Roberts L.
      • Gollhofer A.
      • Konig D.
      Effects of blood flow restriction training with protein supplementation on muscle mass and strength in older men.
      ,
      • Jendricke P.
      • Centner C.
      • Zdzieblik D.
      • Gollhofer A.
      • Konig D.
      Specific collagen peptides in combination with resistance training improve body composition and regional muscle strength in premenopausal women: a randomized controlled trial.
      ] for their ability to augment RT-induced increases in skeletal muscle mass that are so large as to position collagen as being as efficacious as testosterone administration [
      • Gharahdaghi N.
      • Rudrappa S.
      • Brook M.S.
      • Idris I.
      • Crossland H.
      • Hamrock C.
      • et al.
      Testosterone therapy induces molecular programming augmenting physiological adaptations to resistance exercise in older men.
      ]. However, in contrast to longer-term trials, our group has consistently demonstrated no benefit of collagen peptide supplementation on indices of skeletal muscle mass during periods of loading (i.e., physical activity [
      • Oikawa S.Y.
      • Kamal M.J.
      • Webb E.K.
      • McGlory C.
      • Baker S.K.
      • Phillips S.M.
      Whey protein but not collagen peptides stimulate acute and longer-term muscle protein synthesis with and without resistance exercise in healthy older women: a randomized controlled trial.
      ,
      • Oikawa S.Y.
      • MacInnis M.J.
      • Tripp T.R.
      • McGlory C.
      • Baker S.K.
      • Phillips S.M.
      Lactalbumin, not collagen, augments muscle protein synthesis with aerobic exercise.
      ] and unloading (i.e., physical inactivity [
      • Oikawa S.Y.
      • McGlory C.
      • D'Souza L.K.
      • Morgan A.K.
      • Saddler N.I.
      • Baker S.K.
      • et al.
      A randomized controlled trial of the impact of protein supplementation on leg lean mass and integrated muscle protein synthesis during inactivity and energy restriction in older persons.
      ]). Following a single bout of RE [
      • Oikawa S.Y.
      • Kamal M.J.
      • Webb E.K.
      • McGlory C.
      • Baker S.K.
      • Phillips S.M.
      Whey protein but not collagen peptides stimulate acute and longer-term muscle protein synthesis with and without resistance exercise in healthy older women: a randomized controlled trial.
      ], or several sessions of aerobic exercise training [
      • Oikawa S.Y.
      • MacInnis M.J.
      • Tripp T.R.
      • McGlory C.
      • Baker S.K.
      • Phillips S.M.
      Lactalbumin, not collagen, augments muscle protein synthesis with aerobic exercise.
      ], collagen peptide ingestion was not as effective as whey protein [
      • Oikawa S.Y.
      • Kamal M.J.
      • Webb E.K.
      • McGlory C.
      • Baker S.K.
      • Phillips S.M.
      Whey protein but not collagen peptides stimulate acute and longer-term muscle protein synthesis with and without resistance exercise in healthy older women: a randomized controlled trial.
      ] or lactalbumin ingestion [
      • Oikawa S.Y.
      • MacInnis M.J.
      • Tripp T.R.
      • McGlory C.
      • Baker S.K.
      • Phillips S.M.
      Lactalbumin, not collagen, augments muscle protein synthesis with aerobic exercise.
      ] to increase MPS in healthy older women [
      • Oikawa S.Y.
      • Kamal M.J.
      • Webb E.K.
      • McGlory C.
      • Baker S.K.
      • Phillips S.M.
      Whey protein but not collagen peptides stimulate acute and longer-term muscle protein synthesis with and without resistance exercise in healthy older women: a randomized controlled trial.
      ], or young, endurance trained individuals [
      • Oikawa S.Y.
      • MacInnis M.J.
      • Tripp T.R.
      • McGlory C.
      • Baker S.K.
      • Phillips S.M.
      Lactalbumin, not collagen, augments muscle protein synthesis with aerobic exercise.
      ]. In fact, collagen peptide ingestion did not act synergistically with RT to increase integrated rates of MPS above baseline [
      • Oikawa S.Y.
      • Kamal M.J.
      • Webb E.K.
      • McGlory C.
      • Baker S.K.
      • Phillips S.M.
      Whey protein but not collagen peptides stimulate acute and longer-term muscle protein synthesis with and without resistance exercise in healthy older women: a randomized controlled trial.
      ]. Furthermore, collagen peptide supplementation did not augment MPS or leg lean mass during convalescence from two weeks of physical inactivity, in healthy older adults; in contrast, whey protein supplementation was able to stimulate recovery of muscle protein synthesis and increase in leg lean mass [
      • Oikawa S.Y.
      • McGlory C.
      • D'Souza L.K.
      • Morgan A.K.
      • Saddler N.I.
      • Baker S.K.
      • et al.
      A randomized controlled trial of the impact of protein supplementation on leg lean mass and integrated muscle protein synthesis during inactivity and energy restriction in older persons.
      ]. Collectively, these findings further emphasize the importance of protein quality as determinant for increasing MPS.

      5.3 Creatine

      Creatine is essential in ATP re-synthesis and has been used as an ergogenic aid by athletes to improve performance specifically in tasks of repeated high-intensity short duration effort [
      • Branch J.D.
      Effect of creatine supplementation on body composition and performance: a meta-analysis.
      ]. Supplementation with creatine in combination with RT has been reported to increase lean body mass to a greater extent; when compared with placebo [
      • Branch J.D.
      Effect of creatine supplementation on body composition and performance: a meta-analysis.
      ]. However, the mechanism by which creatine affects muscle size is largely unknown. To our knowledge only two studies have examined the effects of creatine supplementation on muscle protein turnover. Creatine supplementation for 5 days raised muscle creatine levels [
      • Louis M.
      • Poortmans J.R.
      • Francaux M.
      • Hultman E.
      • Berre J.
      • Boisseau N.
      • et al.
      Creatine supplementation has no effect on human muscle protein turnover at rest in the postabsorptive or fed states.
      ,
      • Parise G.
      • Mihic S.
      • MacLennan D.
      • Yarasheski K.E.
      • Tarnopolsky M.A.
      Effects of acute creatine monohydrate supplementation on leucine kinetics and mixed-muscle protein synthesis.
      ] however basal MPS levels were not affected in either the fasted [
      • Louis M.
      • Poortmans J.R.
      • Francaux M.
      • Hultman E.
      • Berre J.
      • Boisseau N.
      • et al.
      Creatine supplementation has no effect on human muscle protein turnover at rest in the postabsorptive or fed states.
      ,
      • Parise G.
      • Mihic S.
      • MacLennan D.
      • Yarasheski K.E.
      • Tarnopolsky M.A.
      Effects of acute creatine monohydrate supplementation on leucine kinetics and mixed-muscle protein synthesis.
      ], or the fed state [
      • Louis M.
      • Poortmans J.R.
      • Francaux M.
      • Hultman E.
      • Berre J.
      • Boisseau N.
      • et al.
      Creatine supplementation has no effect on human muscle protein turnover at rest in the postabsorptive or fed states.
      ], nor following RE [
      • Louis M.
      • Poortmans J.R.
      • Francaux M.
      • Berre J.
      • Boisseau N.
      • Brassine E.
      • et al.
      No effect of creatine supplementation on human myofibrillar and sarcoplasmic protein synthesis after resistance exercise.
      ]. Although whole body protein oxidation, assessed via the rate of appearance plasma leucine appearance, was slightly reduced in the fasted state following creatine loading [
      • Parise G.
      • Mihic S.
      • MacLennan D.
      • Yarasheski K.E.
      • Tarnopolsky M.A.
      Effects of acute creatine monohydrate supplementation on leucine kinetics and mixed-muscle protein synthesis.
      ], Louis and colleagues observed no changes in MPB following creatine loading in the fasted or fed state [
      • Louis M.
      • Poortmans J.R.
      • Francaux M.
      • Hultman E.
      • Berre J.
      • Boisseau N.
      • et al.
      Creatine supplementation has no effect on human muscle protein turnover at rest in the postabsorptive or fed states.
      ]. These results support the notion that increased lean mass following creatine supplementation may be due factors other than increased MPS – specifically, creatine serves to increase PCr stores (delaying depletion), facilitate the rapid re-synthesis of PCr and ATP and provide an energy buffer [
      • Branch J.D.
      Effect of creatine supplementation on body composition and performance: a meta-analysis.
      ] which enables a greater training volume to be completed.
      MPS, muscle protein synthesis; RE, resistance exercise; PUFA, polyunsaturated fatty acid.

      6. Conclusion

      Skeletal muscle protein turnover has been, and remains, an important field of study. Muscle mass is determined by the difference between MPS and MPB, creating either a positive (anabolic state) or negative (catabolic state) NPBAL (summarised in Fig. 4). Through the use of labelled stable isotopes researchers have been able to determine the optimal dose, timing, distribution and composition of a protein source to maximally stimulate MPS, inhibit MPB and thus create a positive NPBAL, as summarised in Table 3. An exciting avenue of future research exists in exploring the ability of nutritional supplements, other than protein/AA ingestion, to stimulate MPS, such as the use of omega 3 fatty acids. Furthermore, it is now possible to combine stable isotopes (D2O) with proteomic mass-spectrometry to investigate the protein fractional synthesis rates and abundance of hundreds of individual proteins within a given muscle sub-fraction. This can also allow the calculation of the breakdown rate of these individual proteins, which would be extremely valuable information. The integration of stable isotopes and the emerging omics field will enable researchers to further elucidate the ability of nutritional interventions to influence biological networks that regulate muscle protein metabolism.
      Table 3Practical recommendations of protein intake to maximise MPS in young and old adults.
      Protein dose
      • ~1.6 g/kg body mass of protein per day
      • If total protein intake is not adequate, then consume ~0.4 g/kg body mass of protein at each meal and following RE
      Protein distribution
      • Evenly space each protein-containing meal
      • Consume protein 1–3 h before sleep
      Protein quality
      • Consume high quality (EAA rich) protein
      • Supplement low dose protein with leucine
      Protein ingestion surrounding RE
      • Not essential if sufficient AA/EAA are consumed
      MPS, muscle protein synthesis; RE, resistance exercise; EAA, essential amino acids; AA, amino acids.

      Statement of authorship

      SJ, JM, CL, EAN, TS, JCM, and SMP wrote the initial draft of the manuscript. All authors edited and approved the final version of the manuscript and agree to be accountable for all aspects of the work.

      Declaration of interests

      The authors declare no conflict of interest.

      Acknowledgments

      SMP thanks the Canada Research Chairs Program, the Canadian Institutes of Health Research , and the National Science and Engineering Research Council of Canada for their support during development of this work.

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