Dietary protein induces transient muscle anabolism until ‘muscle full’

The anabolic effects of feeding, which are driven by the transfer and incorporation of AA obtained from dietary sources into skeletal muscle proteins^{(Reference Atherton and Smith16)}, were realised >30 years ago when the provision of a mixed-macronutrient meal (containing protein, carbohydrate and fat) was shown to stimulate acute MPS in human subjects^{(Reference Rennie, Edwards and Halliday29)}. It later transpired this robust MPS stimulation is highly dependent on the AA content of the meal^{(Reference Bennet, Connacher and Scrimgeour30)}, which was uniquely attributed to the essential amino acids (EAA)^{(Reference Smith, Barua and Watt31,Reference Smith, Reynolds and Downie32)} . This was eloquently demonstrated where bolus feeding of EAA (e.g. leucine, phenylalanine, threonine) stimulated MPS, but non-essential amino acids (NEAA; e.g. arginine, glycine, serine) did not^{(Reference Smith, Barua and Watt31,Reference Smith, Reynolds and Downie32)} . This AA-induced stimulation of MPS is purportedly dose-dependent with maximal anabolic stimulation being achieved with about 10–20 g EAA^{(Reference Cuthbertson, Smith and Babraj33)} or about 20–40 g high-quality protein^{(Reference Moore, Churchward-Venne and Witard34–Reference Macnaughton, Wardle and Witard38)}. However, of all EAA, the branched-chain amino acid (BCAA), leucine, can robustly stimulate MPS in isolation^{(Reference Wilkinson, Hossain and Hill39)}, and so it is likely that the dose–response of MPS to protein/EAA is not driven by AA quantity per se but instead by the leucine content^{(Reference Bukhari, Phillips and Wilkinson40)}. Reflecting this, it was shown that 3 g EAA containing 40 % leucine elicited comparable MPS to that of 20 g whey in older women^{(Reference Bukhari, Phillips and Wilkinson40)}. An important implication of this is that low doses of leucine-enriched EAA may provide less satiating yet anabolically robust nutritional protein sources (which is particularly relevant for older adults where the satiety of a feed is a key consideration^{(Reference Bukhari, Phillips and Wilkinson40,Reference Benelam41)} ). The digestion rate of the protein source is also purported to determine the subsequent anabolic impact, whereby ‘fast’ (e.g. whey) proteins result in rapid aminoacidemia and greater protein accretion compared to ‘slow’ (e.g. casein) proteins. Whilst there is data to show that 20 g whey elicits a greater MPS response v. a matched dose of casein, it is important to note that these protein sources differed in AA constituency, particularly in leucine content^{(Reference Burd, Yang and Moore42,Reference Pennings, Boirie and Senden43)} . Consequently, to address the importance of the EAA delivery profile upon muscle anabolism, Mitchell et al. provided older males with EAA consumed as either a single 15 g bolus (eliciting rapid aminoacidemia) or in four smaller pulse doses of 3⋅75 g received every 45 min (eliciting slower aminoacidemia), and demonstrated no differences in MPS^{(Reference Mitchell, Phillips and Williams44)}. As such, it is plausible to suggest that the content of leucine is a major determinant of the ensuing anabolic response. However, it should be noted that although leucine stimulates MPS in the absence of other AA via depletion of endogenous intra-myocellular pools, MPS would eventually become limited by the availability of other EAA^{(Reference Stokes, Hector and Morton20)} i.e. as an extreme example, if one were to replace protein in meals to leucine alone. In addition to acting as a signal and substrate for MPS, as a BCAA, and along with other BCAA (isoleucine and valine)^{(Reference Wagenmakers45)}, leucine may also be metabolised within skeletal muscle. This occurs via BCAA deamination and decarboxylation with ensuing metabolic steps leading to the formation of derivative molecules which feed into myriad metabolic pathways, such as the tricarboxylic acid cycle. On this basis, it has been suggested that BCAA metabolites may possess anabolic properties beyond the intact BCAA^{(Reference Mitchell, Wilkinson and Phillips46)}. Indeed, α-ketoisocaproate (KIC), which is the resultant keto-acid from leucine transamination, stimulates comparable MPS rates to leucine, albeit not in human muscle^{(Reference Escobar, Frank and Suryawan47)}. However, plasma leucine concentrations increase with KIC infusion and the reversible transamination of KIC back to leucine may mean that leucine was accountable for the stimulation of MPS^{(Reference Mitchell, Wilkinson and Phillips46)}. Whilst the KIC-branched-chain α-keto acid dehydrogenase pathway is the fate of most leucine metabolism, a minority (5 %) is converted to the metabolite, β-hydroxy-β-methylbutyrate (HMB)^{(Reference Van Koevering and Nissen48)} via KIC dioxygenase. Although primarily involved in cholesterol synthesis, HMB possesses robust anabolic and anti-catabolic properties^{(Reference Wilkinson, Hossain and Hill39,Reference Mitchell, Wilkinson and Phillips46,Reference Wilkinson, Hossain and Limb49)} , demonstrated by the provision of 3 g free acid-HMB stimulating MPS, similar to that of leucine, while also suppressing MPB in young human subjects^{(Reference Wilkinson, Hossain and Hill39,Reference Wilkinson, Hossain and Limb49)} . Importantly, despite proposed differences in bioavailability, the pro-anabolic and anti-catabolic properties of HMB also extend to the calcium salt form of HMB^{(Reference Wilkinson, Hossain and Limb49)}.

Notably, skeletal muscles are receptive to the anabolic effects of AA for only a short period in the rested state; thus exhibiting temporal regulation. Following feeding, there is an initial lag of about 45–90 min post AA intake due to the time taken for digestion, absorption, arrival at the target tissue (i.e. muscle capillary perfusion and crossing the interstitium) and activation of intracellular anabolic signalling pathways^{(Reference Atherton and Smith16)}. Subsequently, MPS increases about 2–3-fold, peaking between about 1⋅5–2 h, prior to returning to baseline a total of about 2–3 h later^{(Reference Atherton and Smith16)}. Thereafter, MPS displays tachyphylaxis despite continued EAA availability and elevated intracellular anabolic signalling^{(Reference Deane, Wilkinson and Phillips50)}, a phenomenon termed muscle full^{(Reference Atherton, Etheridge and Watt51)}, whereby muscle cells intrinsically sense excess EAA and divert them away from incorporation into muscle proteins, instead towards oxidation^{(Reference Brook and Wilkinson23,Reference Witard, Jackman and Breen37)} . The notion that MPS may not be continually stimulated by dietary protein intake is reflected by the fact that dietary protein alone, even in excess, cannot induce muscle hypertrophy^{(Reference Mitchell, Wilkinson and Phillips46)}. Despite this muscle full phenomenon, the mechanisms regulating MPS-related tachyphylaxis despite adequate EAA provision and appropriate intracellular signalling remain elusive, although may reflect attenuated translation elongation^{(Reference Wilson, Layman and Moulton52,Reference Wilson, Moulton and Garlick53)} or endoplasmic reticulum stress^{(Reference Mitchell, Wilkinson and Phillips46)}. Another notable characteristic of the muscle full state is the fact that it cannot be overcome by proximal intake of additional nutrient signals/substrates regulating MPS (i.e. leucine)^{(Reference Mitchell, Phillips and Hill54)}. Therefore, a refractory period must exist before a further stimulation of MPS is possible, although the precise kinetics (i.e. duration, etc.) of this period remain ill-defined. Finally, muscle full is also refractory to pharmacological and nutraceutical enhancement of nutritive muscle blood flow, demonstrated by vasodilatory methacholine^{(Reference Phillips, Atherton and Varadhan55)} and cocoa flavanols^{(Reference Phillips, Atherton and Varadhan56)} having no added effects on MPS despite enhancing leg blood flow and microvascular blood volume; as such muscle full is not a state induced by muscle hypo-perfusion. Finally, muscle full manifests independently of whether protein intake occurs in a bolus or intermittent feeding pattern^{(Reference Mitchell, Phillips and Williams44)}, thus suggesting differing rates of aminoacidemia (and dose-dependence) do not dictate muscle full.

Whilst being substrates for MPS, certain EAA also act as key signalling molecules regulating MPS responses^{(Reference Mitchell, Wilkinson and Phillips46)}. Following trans-sarcolemmal EAA transport, leucine (and perhaps other EAA) activates the mechanistic target of rapamycin complex-1 (mTORC1), independent of proximal insulin (e.g. PI3 K/AKT) signalling^{(Reference Atherton and Smith16)}. mTORC1 activation induces phosphorylation of 4E-binding protein and ribosomal protein S6 kinase, stimulating the binding of the eukaryotic initiation factor (eIF) 4A and eIF4E to eIF4G, forming the eIF4F complex^{(Reference Kimball57)}. Thereafter, the eIF4F complex mediates mRNA binding to the 43S preinitiation complex, ultimately resulting in the formation of the 48S preinitiation complex^{(Reference Kimball57)}. This relay system triggered by intracellular EAA accumulation results in an upregulation in rates of mRNA translation and thus, MPS. Whilst the EAA-induced upstream regulation of mTORC1 is incompletely defined, recent evidence suggests leucine binding to Sestrin-2 (an intracellular leucine sensor of the mTOR pathway) and subsequent dissociation of Sestrin-2 and the GTPase-activating protein, GATOR2, leads to mTORC1 activation^{(Reference Wolfson, Chantranupong and Saxton58)}. For more in-depth discussions on nutrient-led signalling to MPS, readers are encouraged towards the following reviews^{(Reference Kimball57,Reference Proud59,Reference Proud60)} .

In addition to stimulating MPS, protein/EAA intake (with or without carbohydrate) provides a second route for anabolism via suppression of MPB. This is a facet of nutrient-mediated protein accretion which is entirely attributable to insulin action^{(Reference Atherton and Smith16)}. Illustrating this, insulin at 3× postabsorptive concentrations (i.e. about 15 μU/ml) was sufficient to inhibit MPB by about 50 %^{(Reference Wilkes, Selby and Atherton61)}; an observation which could not be recapitulated by the provision of EAA infusions during postabsorptive insulin clamps (i.e. at 5 μU/ml)^{(Reference Greenhaff, Karagounis and Peirce62)}. Thus, since the provision of protein/AA only suppresses MPB when insulin is allowed to rise above post-absorptive levels^{(Reference Greenhaff, Karagounis and Peirce62)}, it is insulin and not EAA that is responsible for the anti-catabolic effects of feeding^{(Reference Wilkes, Selby and Atherton61)}. Supporting this, a recent systematic review and meta-analysis of >40 human studies concluded insulin has a permissive role in MPS but does have a prominent anti-catabolic role in attenuating MPB^{(Reference Abdulla, Smith and Atherton63)}. Nonetheless, changes in MPS are likely to be greater than those of MPB (at least in healthy muscle), and so MPS remains the key driver of protein feeding-induced anabolism^{(Reference Atherton and Smith16)}.

Dietary protein and exercise interactions delay the onset of muscle full

In addition to being a prerequisite for skeletal muscle maintenance, dietary protein is critical for the ensuing muscle adaptation in response to RE. This is evidenced by the fact that exercise-induced increases in MPS in the absence of protein/EAA result in a prolonged elevation in MPB, therein contributing to an overall negative muscle protein balance^{(Reference Biolo, Maggi and Williams64)}. As such, exercise must be coupled with sufficient protein/EAA in order to achieve positive net protein balance, yielding small amounts of muscle protein accrual after each individual bout of RE, that ultimately culminate over a period of resistance exercise training (RET) resulting in muscle hypertrophy^{(Reference Stokes, Hector and Morton20)}.

A prominent feature of exercise is that it increases the anabolic sensitivity of the muscle therein delaying the onset of muscle full. Specifically, increasing the availability of dietary EAA post-exercise enhances the magnitude and duration of the MPS response^{(Reference Pennings, Koopman and Beelen65)}, with no further anabolic effects (i.e. increase in MPS and/or decrease in MPB) evidenced with the addition of carbohydrates^{(Reference Staples, Burd and West66)}. To further evidence this, older women exhibited comparable increases in MPS for 2 h following whey or low-dose leucine-enriched EAA feeding in the absence of exercise, whereas exercise (i.e. RE) combined with protein feeding (regardless of composition) prolonged this response, stimulating MPS for 4 h^{(Reference Bukhari, Phillips and Wilkinson40)}. In some cases, these exercise-enhanced MPS responses to protein intake can last for 24 h^{(Reference Burd, West and Moore67)}. The maximisation of the post-exercise MPS response also relates to studies investigating the optimal timing of protein intake in and around exercise. However, considering an acute bout of RE delays muscle full considerably (i.e. up to 24 h^{(Reference Burd, West and Moore67)}), nutrient consumption pre-, during or post-exercise is perhaps not as critical as presumed; and rather, protein sufficiency (that being quantity and quality) is the most fundamental consideration^{(Reference Atherton and Smith16)}. Despite exercise sensitising the muscle to protein feeding, limits still exist whereby muscle full is still reached. Specifically, in response to unilateral leg-based RE, MPS displays a dose–response relationship to dietary protein ingestion until maximal stimulation with 20 g intact protein, where doses above which (i.e. 40 g) lead to irreversible oxidation and thus excess protein is catabolised^{(Reference Atherton and Smith16,Reference Moore, Robinson and Fry35)} . However, in situations of whole-body RE the protein requirements tend to be greater where 40 g whey protein stimulated MPS to a greater extent than 20 g, although the dose needed to reach muscle full was not determined^{(Reference Macnaughton, Wardle and Witard38)}. Taken together, protein-intake coupled with RE delays, but does not fully overcome an eventual muscle full, to permit additional use of available EAA for MPS to promote appropriate muscle remodelling and/or growth.

Older skeletal muscle displays ‘anabolic resistance’ to dietary protein intake and exercise (i.e. resistance exercise)

For more than a decade, work developing the concept of ‘anabolic resistance’ has greatly aided explanation of age-related declines in skeletal muscle mass^{(Reference Wilkinson, Piasecki and Atherton68)}. Although some early, small cohort studies reported depressed basal (post-absorptive, rested) MPS rates in older compared to younger age^{(Reference Welle, Thornton and Jozefowicz69,Reference Welle, Thornton and Statt70)} , it is now largely accepted that post-absorptive rates of MPS and MPB are unchanged in healthy ageing^{(Reference Breen and Phillips22,Reference Moore, Churchward-Venne and Witard34,Reference Phillips, Hill and Atherton71,Reference Wall, Gorissen and Pennings72)} . Anabolic resistance, therefore, centres upon the blunting of increases in MPS and/or suppression of MPB to key anabolic drivers, namely exercise and nutrition^{(Reference Moore, Churchward-Venne and Witard34,Reference Phillips, Hill and Atherton71–Reference Marshall, Smeuninx and Morgan73)} . Reflecting this, a plethora of accumulating evidence (i.e.^{(Reference Moore, Churchward-Venne and Witard34,Reference Phillips, Hill and Atherton71–Reference Mitchell, Williams and Atherton75)} ) suggests that anabolic resistance is likely a major driver of age-related muscle atrophy (sarcopenia); though that is not to say mixed results engender this contentious^{(Reference Symons, Sheffield-Moore and Wolfe36,Reference Chevalier, Goulet and Burgos76)} . Seminal work by Cuthbertson et al. demonstrated a maximal MPS response in young adults to 10 g EAA, with older adults having suppressed rates of MPS above 5 g EAA^{(Reference Cuthbertson, Smith and Babraj33)}. Furthermore, a 40 g bolus of EAA failed to elicit a similar MPS response in older adults to that seen in young adults at 10 g EAA^{(Reference Cuthbertson, Smith and Babraj33)}. Two recent meta-analyses further support the existence of anabolic resistance in aged muscle, with Wall et al. reporting depressed post-prandial MPS rates (−16 %) in older adults^{(Reference Wall, Gorissen and Pennings72)} and Moore et al. reporting a ‘rightward shift’ whereby myofibrillar fractional synthesis rate (i.e. MPS) plateaus with respect to relative protein intake in older (about 0⋅4 g/kg) compared to young (about 0⋅24 g/kg) men^{(Reference Moore, Churchward-Venne and Witard34)}.

Fortifying protein with leucine may provide another nutraceutical avenue for combating anabolic resistance in ageing muscle. Older adults typically exhibit increased satiety after consuming a meal^{(Reference Cuthbertson, Smith and Babraj33)}, which likely contributes to the inadequate daily consumption of protein (RDA: 1⋅0–1⋅2 g/kg BW/d^{(Reference Bauer, Biolo and Cederholm77)}) commonly seen in the older population^{(Reference Cuthbertson, Smith and Babraj33,Reference Marshall, Smeuninx and Morgan73)} . As such, specific EAA, such as leucine, may be considered as an adjuvant nutritional strategy to maximise MPS that can also alleviate satiating effects^{(Reference Marshall, Smeuninx and Morgan73)}. Through the consumption of a submaximal protein drink (10 g) enriched with 4⋅5 g leucine, older adults exhibited elevated MPS (0⋅14 (sem 0⋅01) %/4 h), compared to enrichment with 4⋅5 g alanine (0⋅11 (sem 0⋅01) %/4 h), with fractional synthesis rate remaining elevated above baseline beyond the measured 4 h^{(Reference Atherton, Kumar and Selby78)}. Moreover, anabolic signalling was robustly triggered when administering about 6 g BCAA (about 2⋅6 g leucine), hence leading to an increase in acute MPS response in both young muscle post-RE^{(Reference Jackman, Witard and Philp79)} and in older adults^{(Reference Fuchs, Hermans and Holwerda80)}. Conversely, it was reported that ingestion of leucine alone failed to stimulate MPS in the absence of a full EAA profile in postmenopausal (aged 50–65 years) women^{(Reference van Vliet, Smith and Porter81)}. Bell et al. reported that with 12 weeks of combined RET (about 80 % 1-repetition maximum (1-RM)) and high-intensity interval training (about 90 % maximal heart-rate), alongside consumption of a multi-ingredient supplement (consisting of 30 g whey protein, 2⋅5 g creatine, 500 IU vitamin D, 400 mg calcium and 1500 mg n-3 PUFA), myofibrillar fractional synthesis rate post-training was elevated by about 30 % and 7 % at 0–24 and 24–48 h, respectively; however, the control group demonstrated about 20 % increase in MPS compared to resting levels 24–48 h post-training^{(Reference Bell, Brook and Snijders82)}. Therefore, it is inconclusive whether standalone or adjuvant supplementation of leucine is effective to sufficiently stimulate MPS across populations, and it remains to be investigated whether submaximal doses of complete protein enriched with leucine (at least 3 g^{(Reference Wilkinson, Hossain and Hill39)}) or multi-ingredient supplementation may lead to enhanced muscle anabolism in older adults^{(Reference Marshall, Smeuninx and Morgan73)}.

Similar to protein feeding, there is evidence of anabolic resistance in response to exercise in older age. Kumar et al. demonstrated at 1–2 h post-RET (20–90 % 1-RM), that MPS responses were blunted in older compared to younger adults^{(Reference Kumar, Selby and Rankin83)}, and with Fry et al. highlighting impaired skeletal muscle mTORC1 signalling (a possible mechanism underlying age-related blunting of MPS) and MPS up to 24 h post-exercise^{(Reference Fry, Glynn and Drummond84)}. Brook et al. also highlighted the presence of anabolic resistance following 6 weeks of unilateral leg RET (75 % 1-RM), with MPS becoming elevated after 3 weeks in young (1⋅6 (sem 0⋅01) % daily) but not older (1⋅49 (sem 0⋅08) % daily) adults, which was coupled with an absence of thigh fat-free mass gains in older muscle^{(Reference Brook, Wilkinson and Mitchell74)}. Similarly, Durham et al. reported a reduced sensitivity to AA following endurance type (i.e. walking) exercise at about 40 % VO_2peak in older compared to younger adults^{(Reference Durham, Casperson and Dillon85)}. In contrast, Symons et al. reported a 50 % increase in MPS in young and older muscle when exercise was coupled to a 30 g protein meal^{(Reference Symons, Sheffield-Moore and Wolfe36)}, suggesting aged muscle simply exhibits delays in the anabolic response to exercise coupled to nutrition^{(Reference Drummond, Glynn and Fry86)}. In terms of exercise intensity, it has been suggested that low-intensity high-volume RET may have comparative effects to high-intensity low-volume RET, a modality which would be of particular practicable benefit to older individuals^{(Reference Breen and Phillips22)}. Indeed, results from Fry et al. highlight, in older compared to young adults, low-intensity RET (20 % 1-RM) coupled with blood flow occlusion heightened mTORC1 signalling and MPS by 56 % above basal levels 3 h post-exercise^{(Reference Fry, Glynn and Drummond84)}. However, in the absence of blood flow occlusion, MPS remained unchanged post-exercise^{(Reference Fry, Glynn and Drummond84)}. Interestingly, although in younger adults, it was demonstrated that unilateral leg extension exercise at 30 % v. 90 % 1-RM to failure was more effective at increasing and maintaining elevated MPS (199 %) 24 h post-exercise^{(Reference Burd, West and Staples87)}. If RET volume rather than intensity is key for stimulating MPS, a greater portion of older adults may be able to perform RET due to associated sarcopenic comorbidities (e.g. arthritis) which may limit the performance of high-intensity exercise^{(Reference Breen and Phillips22)}; thus offering a potential effective intervention to offset the impact of anabolic resistance and age-related muscle functional declines, although this requires further investigation.

Physical inactivity induces a premature muscle full state in response to dietary protein

Physical inactivity, such as sedentarism or immobilisation due to bed-rest, induces a premature muscle full state characterised by blunted fasted and fed-state MPS (i.e. anabolic resistance)^{(Reference de Boer, Selby and Atherton88–Reference Wall, Snijders and Senden93)}, prompting insulin resistance^{(Reference Dirks, Wall and van de Valk94)} and muscle atrophy^{(Reference Kilroe, Fulford and Holwerda6)}, which over time may play a key role in the aetiology of sarcopenia^{(Reference Wall, Dirks and van Loon95)}. In certain cases, inactivity-induced atrophy is not fully recovered despite resumption of normal ambulation^{(Reference McGlory, von Allmen and Stokes96)} or even following RET^{(Reference Suetta, Hvid and Justesen97)}. As such, protein intervention, as a means to stimulate inactivity-induced declines in MPS, is a primary research avenue for offsetting inactivity-induced atrophy, particularly since modulating the type^{(Reference Pennings, Groen and van Dijk98)} and amount^{(Reference Pennings, Groen and de Lange99)} of dietary protein can somewhat overcome anabolic resistance in ageing^{(Reference Pennings, Groen and van Dijk98,Reference Pennings, Groen and de Lange99)} .

To date, results surrounding the anabolic efficacy of protein feeding during inactivity have been mixed. For example, provision of 20 g protein daily (on top of a partially controlled diet 1 g/kg/d) during 14 d of unilateral leg immobilisation did not attenuate muscle loss in middle-aged adults^{(Reference Mitchell, D'Souza and Mitchell100)}. More recently, a high protein diet (1⋅6 g protein/kg/d) provided at evenly spaced intervals throughout the day (strategically done so in order to avoid the refractory period), was shown to have no positive influence on MPS or muscle volume observed with 3 d of unilateral limb immobilisation in young males^{(Reference Kilroe, Fulford and Jackman101)}. This led the authors to suggest that dietary protein consumption of ≤1⋅6 g/kg/d during inactivity does not modulate muscle loss^{(Reference Kilroe, Fulford and Jackman101)}, perhaps calling for future studies to investigate whether larger protein doses can overcome the anabolic resistance that manifests during inactivity. On the contrary, higher dose EAA/leucine supplementation has shown more promising results. For example, high-dose EAA combined with carbohydrate during 28 d of bed-rest preserved MPS rates and ameliorated the loss of lean muscle mass^{(Reference Paddon-Jones, Sheffield-Moore and Urban102,Reference Paddon-Jones, Sheffield-Moore and Urban103)} . Further, middle-aged adults consuming leucine supplementation (0⋅06 g/kg/meal) during 14 d of bed rest illustrated attenuated reductions in post-absorptive MPS and reduced whole-body lean mass loss after 7 d (compared to an alanine control group)^{(Reference English, Mettler and Ellison104)}. However, not all studies are in agreement; 7⋅5 g/d of free leucine did not attenuate muscle mass declines in young adults undergoing 7 d of unilateral limb immobilisation^{(Reference Backx, Horstman and Marzuca-Nassr105)}. Whilst the precise cause(s) of discrepancies between study findings are unknown, the model of inactivity used (i.e. bed rest, unilateral limb immobilisation), volunteer characteristics, dietary control within studies, and importantly, the differing amount and quality of protein provided are likely factors. In line with the general consensus of the literature thus far, it is plausible that higher dose EAA/leucine, as opposed to high protein diets, may be a more effective intervention to overcome the premature onset of muscle full during inactivity. If so, it has been suggested that it is not the availability of AA per se that is limiting muscle anabolism during inactivity, but rather inactivity induces a significant increase in the threshold required for EAA/leucine to stimulate anabolism (e.g. intracellular signalling pathways)^{(Reference Kilroe, Fulford and Jackman101)}. To test this, future research should thus focus on protein strategies that maximise intracellular transport and delivery of AA, therein maximising anabolic signals during inactivity^{(Reference Kilroe, Fulford and Jackman101)}. In the search for alternative/effective protein therapeutics, NEAA (which seemingly play a trivial role in healthy muscle^{(Reference Davies, Bass and Carson106)}), may be important in ameliorating inactivity-induced muscle decline^{(Reference Holloway, McGlory and McKellar107)}. For example, glutamine is purported to harbour anti-catabolic properties^{(Reference Nishizaki, Ikegami and Tanaka108)} while arginine is implicated in the maintenance of muscle perfusion^{(Reference Mitchell, Phillips and Wilkinson109)} contributing to substrate delivery or MPS stimulation^{(Reference Holloway, McGlory and McKellar107,Reference Chantranupong, Scaria and Saxton110)} . As such, dietary formulations combining EAA and NEAA may confer unique mechanistic advantages over either AA profile in isolation due to the simultaneous targeting and therapizing of multiple inactivity-activated molecular processes contributing to muscle decline. Indeed, a novel AA formulation (AXA2678) containing select anabolic EAA (e.g. leucine) and NEAA (glutamine, arginine and n-acetylcysteine) consumed thrice daily, preserved muscle volume and cross-sectional area during 7 d of immobilisation in young men^{(Reference Holloway, McGlory and McKellar107)}. However, a confounder within this study was the use of a carbohydrate placebo, meaning the treatment group (AXA2678) received an additional about 66⋅8 g AA daily, maybe explaining the positive findings^{(Reference Holloway, McGlory and McKellar107)}. Future studies are warranted that compare supplements such as AXA2678 to appropriate controls, and to test their efficacy in situations of anabolic resistance (i.e. ageing).

A parallel anabolic intervention to protein feeding in order to negate the premature onset of muscle full during inactivity is the re-introduction of some form of muscle contraction during the period of inactivity^{(Reference Bamman, Clarke and Feeback111,Reference Oates, Glover and West112)} . For example, RE performed every other day during 14 d bed rest or immobilisation in young adults prevented declines in type I and II myofibres^{(Reference Bamman, Clarke and Feeback111)} and muscle cross-sectional area^{(Reference Oates, Glover and West112)}, respectively. However, the ability to perform exercise during inactivity, both in the context of physical capacity and having access to an appropriate environment (e.g. equipment and expertise) are key considerations for this type of intervention, particularly in a clinical setting. In light of this, some researchers have applied neuromuscular electrical stimulation which, from a practical standpoint, has many benefits such as its relatively low cost, ease for hospital staff to apply, tolerance of volunteers, and no reported contraindications or side effects, and importantly, it is feasible and applicable during situations of inactivity or incapacity^{(Reference Dirks, Groen and Franssen113)}. Indeed, neuromuscular electrical stimulation prevented declines in quadricep cross-sectional area in young healthy males following 5 d unilateral leg immobilisation, with corresponding attenuations in the atrophy-associated markers, myostatin and muscle atrophy F-box^{(Reference Dirks, Wall and Snijders114)}. However, the interactions of neuromuscular electrical stimulation and protein feeding in the context of inactivity remain to be investigated, and across populations. Thus, whilst inactivity–activity-protein interactions show tremendous potential for mitigating inactivity-associated muscle decline, studies are still very much in the infancy stage, even more so in the context of a young clinical setting (e.g. hospitalisation).

Dietary protein to mitigate the premature muscle full state during physical inactivity in ageing

From a clinical perspective, older adults are more likely to experience bouts of inactivity (e.g. hospitalisation/physical debilitation with ensuing bed rest)^{(Reference Suetta, Hvid and Justesen97,Reference Arentson-Lantz, Fiebig and Anderson-Catania115)} , with most hospitalised patients over 65 years old and having longer lengths of hospital stays^{(Reference He, Sengupta and Velkoff116)}. This may induce acute sarcopenia. Indeed, the associated loss of muscle mass is significant and rapid, with older adults losing detectable muscle mass (about 1⋅5 %) within as few as 5 d unilateral limb immobilisation^{(Reference Dirks, Wall and Nilwik117)} and losing up to about 6 % (about 1 kg) of lean mass during 10 d bed rest^{(Reference Kortebein, Ferrando and Lombeida118)}; this is purportedly greater than the loss of muscle seen in younger subjects following 14 or 28 d bed rest^{(Reference Paddon-Jones, Sheffield-Moore and Urban103,Reference Ferrando, Lane and Stuart119)} . In older individuals, the premature onset of muscle full associated with inactivity may be further confounded by age-related anabolic resistance to nutrition. As such, ageing populations subjected to periods of physical inactivity stand to benefit considerably from optimised dietary protein–exercise intervention.

As with younger adults, the ability of protein feeding in older adults to offset the premature muscle full state associated with inactivity has produced mixed results. For example, twice daily supplementation of protein (20⋅7 g), fat (3⋅0 g) and carbohydrate (9⋅3 g) on top of an ample protein diet (1⋅1 g/kg/d) during 5 d unilateral leg immobilisation did not attenuate muscle mass declines in older males^{(Reference Dirks, Wall and Nilwik117)}. This led the authors to speculate that maintaining dietary protein intake is necessary to prevent muscle loss during inactivity, but hyper-protein consumption would not further circumvent muscle loss^{(Reference Dirks, Wall and Nilwik117)}, although more recent data from the same group obtained in younger adults no longer supports this hypothesis^{(Reference Kilroe, Fulford and Jackman101)}. Further studies show that high-dose EAA supplementation (15 g thrice daily) on top of a diet containing 0⋅8 g/kg/d protein attenuated the decline in 24 h fractional synthesis rate during 10 d bed rest in older adults^{(Reference Ferrando, Paddon-Jones and Hays120)}. Similarly, healthy older men and women consuming daily meals supplemented with leucine (3–4 g/meal) during 7 d bed rest reduced the loss of leg lean mass (compared to alanine control)^{(Reference Arentson-Lantz, Fiebig and Anderson-Catania115)}. This demonstrates the anabolic potential of small amounts of supplemental leucine and highlights practical advantages such as the cost and ease of incorporating leucine into meal plans^{(Reference Arentson-Lantz, Fiebig and Anderson-Catania115)}. Although muscle loss is largely attributed to marked declines in MPS, potential undetectable changes in MPB cannot be disregarded; particularly in cohorts where the MPB response to inactivity is not well characterised (i.e. older adults), most likely due to difficulties surrounding MPB-related methodologies. As such, HMB has been tested as a potential therapeutic in inactive older adults due to its pro-anabolic and anti-catabolic properties^{(Reference Wilkinson, Hossain and Hill39)} and its efficacy for preserving older muscle health in the context of habitual living^{(Reference Flakoll, Sharp and Baier121)}. In these trials, older men and women consuming 3 g/d HMB during bed rest confinement for 10 d maintained MPS rates pre- to post-bed rest and preserved muscle mass (control volunteers lost about 2 kg lean mass)^{(Reference Deutz, Pereira and Hays122)}. As such, data thus far in older adults echo that of the young, where EAA/leucine, and possibly HMB, may demonstrate greater anabolic effects during inactivity, compared to high protein diets. As a collective, these studies demonstrate there is potential for refined nutritional interventions to offset inactivity-induced atrophy in ageing.

Considering protein feeding in isolation may not fully counteract the premature onset of muscle full during inactivity in older adults; adjunct interventions, such as clinically relevant, feasible (i.e. financially) and effective exercise interventions, are needed. Importantly, hospitalised older adults experience functional decline and are often limited to about 4 min walking every 3 h, which is even less so in women who typically engage in about 2 min walking every 3 h^{(Reference Mahoney123)}. As such, neuromuscular electrical stimulation is a logical adjunct intervention that is (as previously discussed) clinically feasible even for those who are totally incapable of weight bearing. Investigations thus far have shown that following 1 d best rest, older adults subjected to unilateral neuromuscular electrical stimulation followed by 40 g casein ingestion prior to sleep led to an about 18 % greater increase in MPS overnight (compared to the unstimulated leg)^{(Reference Dirks, Groen and Franssen113)}. In the context of longer periods of inactivity, 5 d bed rest combined with neuromuscular electrical stimulation (40 min, thrice daily) and supplemental protein (17 g/d) maintained thigh lean mass and attenuated increases in catabolic biomarkers, myostatin and muscle atrophy F-box, perhaps (at least partly) underlying the preservation of lean mass observed in this trial^{(Reference Reidy, McKenzie and Brunker124)}. Protein–exercise interventional approaches such as this may have translational ramifications for preserving muscle anabolic responses in older adults subjected to periods of inactivity in a clinical setting (i.e. hospitalisation). Future studies should aim to optimise such ‘EX-NUT’ interventions, particularly in women, considering apparent sexual dimorphism in ambulation in hospital settings.

It should be noted that whilst inactivity studies (i.e. bed rest, immobilisation, reduced physical activity) in healthy young and older volunteer populations allow for the metabolic and molecular investigations of inactivity-induced muscle decline, whilst controlling for lifestyle factors that can drastically influence metabolism (e.g. diet, activity, etc.), clinical translation must be treated with caution. For example, the onset of illness or occurrence of injury often results in hospital admittance where a hyper-metabolism (or other catabolic stressors such as inflammation) may result in excessive or accelerated muscle loss, particularly in those with comorbidities, which may have specific dietary challenges and requirements^{(Reference Phillips, Paddon-Jones and Layman125)} and, importantly, may not be recapitulated in healthy research volunteers^{(Reference Marshall, Smeuninx and Morgan73)}. As such, investigations into the effects of protein nutrition in both young and older clinical populations undergoing inactivity are imminently needed in order to optimise appropriate anabolic interventions. Furthermore, in an attempt to effectively and fully translate clinical trials into the clinical setting, it is imperative that day-to-day concerns, such as the quantity, taste and palatability of protein feeding^{(Reference Phillips, Paddon-Jones and Layman125)} and tolerability and compliance to exercise interventions are thoroughly trialled in appropriate clinical populations.