Age and sex-specific changes in muscle protein synthesis

The natural biological ageing process is associated with a gradual decline in skeletal muscle mass, strength and functional capacity in later life. Accordingly, although muscle mass usually peaks in early adulthood (between 20 and 30 years of age), the onset of muscle mass loss can begin as early as the fourth decade of life^{(Reference Janssen, Heymsfield and Wang14)}. Muscle atrophy typically proceeds at a rate of about 1 % of total muscle mass per year until the age of 70 years^{(Reference Mitchell, Williams and Atherton15)}, increasing to about 1⋅5 % of total muscle mass per year beyond 80 years of age^{(Reference Delmonico, Harris and Visser16)}. At the ultrastructural level, muscle atrophy is primarily underpinned by a decline in cross-sectional area (CSA) of the larger force-generating type II muscle fibres rather than oxidative type I fibres^{(Reference Verdijk, Koopman and Schaart17,Reference Lexell, Taylor and Sjöström18)} . In comparison, the age-associated decline in muscle strength proceeds at an even faster rate (2–3 % per year)^{(Reference Goodpaster, Park and Harris19)} due primarily to additional age-associated changes in muscle architecture, innervation and deposition of non-contractile tissue (i.e. fat and connective tissue). Perhaps under-appreciated is the notion that periods of muscle disuse, ranging in severity from injury-induced immobilisation to illness-inflicted bedrest (i.e. cancer cachexia), exacerbate the trajectory of muscle atrophy with advancing age^{(Reference Wall, Dirks and Snijders20)}. Hence, once muscle mass and strength fall below a critical level – often referred to as the disability threshold – the functional capacity of older adults is likely impaired, manifesting in a reduction in mobility, loss of independence and increased risk of falls and bone fractures in later life.

The primary metabolic mechanism that underpins the decline in skeletal muscle mass with advancing age in healthy older adults is an impaired response of MPS to key anabolic stimuli, namely nutrition and/or physical activity/exercise^{(Reference Guillet, Prod'homme and Balage21)}. This phenomenon is termed anabolic resistance. In theory, two metabolic drivers of muscle atrophy exist. First, an increase in the rate of muscle protein degradation into amino acid precursors (termed muscle protein breakdown, MPB) and secondly, a decrease in the rate by which amino acids are synthesised into muscle protein (termed MPS). While some^{(Reference Wilkes, Selby and Atherton22)}, but not all, trials^{(Reference Groen, Horstman and Hamer23)} have reported the suppressive action of insulin on MPB to be diminished in older adults, studies have shown no clear difference in basal post-absorptive rates of MPS between healthy young and older adults^{(Reference Cuthbertson, Smith and Babraj24,Reference Volpi, Sheffield-Moore and Rasmussen25)} . In contrast, the response of MPS to ingesting meal-like quantities of protein^{(Reference Guillet, Prod'homme and Balage21,Reference Cuthbertson, Babraj and Smith26)} and/or other anabolic stimuli such as muscle loading^{(Reference Durham, Casperson and Dillon27–Reference Kumar, Selby and Rankin29)} is impaired in older adults compared to their younger counterparts^{(Reference Wall, Gorissen and Pennings30,Reference Shad, Thompson and Breen31)} . In theory, over time, anabolic resistance would contribute to the gradual loss of skeletal muscle mass with advancing age^{(Reference Phillips, Glover and Rennie32)}. Hence, nutritional interventions focused on combatting the age-related decline in skeletal muscle mass in otherwise healthy older adults are primarily targeted at restoring a youthful response of MPS to anabolic stimuli. Regarding compromised populations, such as cancer cachexic patients, the role of MPB in mediating muscle atrophy appears to be more pronounced. However, the metabolic action of n-3 PUFA in this specific clinical sub-population is beyond the scope of this review and interested readers are directed to the following excellent reviews^{(Reference Ferguson, Seigel and McGlory3,Reference McGlory4)} .

Sex differences in skeletal muscle mass and MPS rates appear to be evident in older adults^{(Reference Hansen33)}. At the ultrastructural level, when stratified by sex, the magnitude of type II muscle fibre atrophy was shown to be more pronounced in older women than men relative to their younger counterparts^{(Reference Martel, Roth and Ivey34)}. The age-related decline in muscle mass in women coincides with the onset of the menopause is accelerated during the transition into menopause^{(Reference Forbes and Reina35)}, and then proceeds at a slower rate than in men^{(Reference Kyle, Genton and Hans36)}. Although the magnitude of muscle hypertrophy in response to resistance exercise training is reduced in older women compared with older men^{(Reference Bamman, Hill and Adams37)}, the degree of muscle atrophy in response to muscle disuse is reduced in older women than men^{(Reference Yasuda, Glover and Phillips38,Reference Callahan, Tourville and Miller39)} . Moreover, based on a limited number of relevant studies, sex differences in the response of MPS to anabolic stimuli are evident in healthy older adults. Although basal rates of MPS appear to be greater in older women than older men^{(Reference Smith, Atherton and Villareal40)}, the MPS response to the intravenous infusion of amino acids and insulin^{(Reference Smith, Atherton and Villareal40)}, the ingestion of a mixed macronutrient meal^{(Reference Smith, Atherton and Villareal40)} and an acute bout of resistance exercise^{(Reference Smith, Reeds and Hall41)} has been shown to be impaired to a greater extent in older women than age-matched men. As such, older women appear more susceptible to anabolic resistance than older men^{(Reference Smith, Atherton and Villareal40)} which appears to be mediated by lower oestrogen levels in post-menopausal women^{(Reference Hansen, Skovgaard and Reitelseder42)}. Sex comparisons in the response of MPB are yet to be elucidated in older adults. Nonetheless, taken together these combined data underscore the potential need for sex-specific nutritional recommendations to mitigate the age-related decline in muscle mass. Specifically, given recent evidence regarding the anabolic properties of n-3 PUFA in stimulating MPS, the role of dietary n-3 PUFA ingestion in mitigating muscle loss in later life has received considerable attention, especially in older women.

Incorporation of ո-3 PUFA into skeletal muscle membranes

The most plausible mechanism linking n-3 PUFA ingestion with the modulation of muscle protein metabolism relates to the incorporation of EPA and DHA into the skeletal muscle phospholipid membrane, and subsequent modulation of intracellular anabolic cell signalling pathways. This notion is supported by two key lines of evidence. First, using an animal model of tumour-bearing mice, we recently demonstrated that the incorporation and subsequent uptake of EPA and DHA into both plasma and skeletal muscle membranes was directly proportional to the relative EPA and DHA content of a controlled diet following a 3-week experimental period^{(Reference Dijk, van Dijk and Dorresteijn43)}. Similar findings were reported in human studies whereby the provision of a fish oil containing medical food formulation over 7 d resulted in an increased percentage incorporation of EPA into the phospholipid membrane of leucocytes in healthy Caucasian adults^{(Reference Faber, Berkhout and Vos44)}. Hence, a direct relationship appears to exist between dietary n-3 PUFA intake and the propensity for tissue EPA/DHA incorporation.

Secondly, the uptake and incorporation of n-3 PUFA into human skeletal muscle cells is evidently congruent with the kinase activity of cell signalling proteins known to trigger MPS. The time course of changes in n-3 PUFA incorporation appears to differ between blood and muscle tissue. Consistent with the slower turnover rates of skeletal muscle tissue compared with blood, McGlory et al. demonstrated that ≥2-weeks of n-3 PUFA intake is required to increase the EPA/DHA content of skeletal muscle, whereas the incorporation of EPA and DHA into whole blood was increased after only 1 week of fish oil supplementation in healthy young adults^{(Reference McGlory, Galloway and Hamilton7)}. In this study^{(Reference McGlory, Galloway and Hamilton7)}, about 2-fold change in the proportion of n-3 PUFA incorporated into the skeletal muscle cell was observed after 4 weeks of fish oil supplementation, although no plateau in EPA/DHA incorporation was reached. Importantly, this incorporation of EPA/DHA into the skeletal muscle membrane was accompanied by an increased kinase activity of candidate cell signalling proteins known to regulate MPS, namely focal adhesion kinase and mTORC1. Interestingly, a recent study in healthy females that administered 5 g/d of fish oil (3⋅50 g/d EPA, 0⋅90 g/d DHA) over an extended 8-week time course reported a plateau in muscle phospholipid n-3 PUFA incorporation after 6 weeks of fish oil supplementation^{(Reference McGlory, Gorissen and Kamal45)}. Moreover, other studies that provided lower doses of n-3 PUFA over an extended timeframe reported similar proportional increases in n-3 PUFA at the end of an 8-week^{(Reference Smith, Atherton and Reeds46)} and 12-week^{(Reference Gerling, Mukai and Chabowski6)} supplementation period. In contrast, only 6 d of high-dose n-3 PUFA ingestion resulted in increased muscle tissue n-3 PUFA content^{(Reference Wardle, Macnaughton and McGlory47)}. Taken together, these studies suggest that the duration and dose of n-3 PUFA supplementation are cogent factors that modulate the magnitude of n-3 PUFA incorporation into skeletal muscle, and thus provide the primary biological rationale that underpins the regulation of MPS with n-3 PUFA ingestion (Table 1).

Table 1. Summary of studies characterising the incorporation on n-3 PUFA into blood cell fractions

n-3 PUFA, long-chain ո-3 PUFA.

Data are represented as % of total fatty acids, except *, where data are represented as nM/ml blood phospholipid content.

Alternative factors modulate the rate of n-3 PUFA incorporation into the skeletal muscle membrane including, (1) the cellular fraction (i.e. mitochondrial, sarcolemmal) measured, (2) the specific n-3 PUFA species (EPA v. DHA) of interest and (3) the sex of the individual. Regarding cellular fraction, Herbst et al. detected an increased incorporation of EPA and DHA into mitochondrial membranes of skeletal muscle after 12 weeks of fish oil supplementation^{(Reference Herbst, Paglialunga and Gerling48)}. Consistent with these findings, Gerling et al. demonstrated an increased enrichment of EPA and DHA into the skeletal muscle mitochondrial membrane, whereas only DHA incorporation was detected in the sarcolemmal fraction^{(Reference Gerling, Mukai and Chabowski6)}. Evidence from human studies also suggests that EPA exhibits a faster rate of tissue incorporation compared to DHA. Accordingly, several studies^{(Reference Browning, Walker and Mander49–Reference Katan, Deslypere and van Birgelen51)} have reported a more rapid uptake of EPA than DHA in multiple plasma and serum fractions and erythrocytes. This differential uptake and incorporation profile of EPA and DHA in blood appears to follow a similar pattern in skeletal muscle^{(Reference McGlory, Galloway and Hamilton7,Reference Herbst, Paglialunga and Gerling48,Reference Smith, Atherton and Reeds52)} which could be attributed to a preferential incorporation of distinct n-3 PUFA species into different phospholipid fractions. For instance, EPA has been shown to be preferentially incorporated into phosphatidylcholine species of erythrocytes that are primarily located on the outer membrane, whereas DHA is preferentially incorporated into phosphatidylethanolamine species that are located on the inner membrane and requires DHA to be transported through the cellular membrane for incorporation^{(Reference Cartwright, Pockley and Galloway53,Reference Pal, Metherel and Fiabane54)} . Although no data exist to confirm this thesis, this additional step in the biological fate of DHA has been proposed to underpin the consistent observation of slower incorporation rates of DHA v. EPA into the phospholipid membrane^{(Reference Pal, Metherel and Fiabane54)}.

The notion that the anabolic action of n-3 PUFA is primarily ascribed to EPA is supported by two lines of evidence from in vitro studies conducted in C₂C₁₂ myotubes but is not universally accepted. First, Kamolrat and Gray reported a 25 % increase in MPS rates in the EPA condition v. control cells, with no effect of DHA on MPS^{(Reference Kamolrat and Gray55)}. Furthermore, MPB rates were 22 % lower after incubation with EPA compared to control, with no changes in MPB in the DHA condition. Secondly, Jeromson et al. demonstrated that EPA treatment enhanced protein accretion via a suppression of MPB, while DHA elicited no impact on rates of MPS or MPB^{(Reference Jeromson, Mackenzie and Doherty56)}. However, Wang et al. reported a greater attenuation of protein degradation with DHA compared to EPA, as mediated via the upregulation of the PPARγ/NF-κB pathway. The authors attributed these distinct effects to the longer carbon chain of DHA compared to EPA^{(Reference Wang, Lin and Zheng57)}. Future human in vivo studies are warranted to fully elucidate the distinct physiological effects of EPA and DHA in terms of anabolic cell signalling, with application to mitigating age-related anabolic resistance.

The suggestion that sex differences exist with regards to tissue n-3 PUFA incorporation is supported by a series of preliminary observations. For example, Metherel et al. reported higher baseline DHA levels in erythrocytes in females than males^{(Reference Metherel, Armstrong and Patterson50)}. Moreover, a more pronounced increase in DHA incorporation has been observed in females following n-3 PUFA supplementation^{(Reference Metherel, Armstrong and Patterson50,Reference Patterson, Chalil and Aristizabal Henao58)} that may be linked to a higher conversion of ALA to EPA and DHA in the liver of females. A recent study also demonstrated a greater abundance of precursors necessary for DHA synthesis following EPA supplementation in females than males^{(Reference Rotarescu, Rezaei and Mutch59)} and thus may serve as an explanation for the apparent sex-based differences in baseline DHA levels. Conversely, Da Boit et al. reported no sex differences in the incorporation of EPA and DHA into blood and skeletal muscle following 12 weeks of n-3 PUFA supplementation, despite observing improvements in maximal isometric torque and muscle quality in older women only^{(Reference Da Boit, Sibson and Sivasubramaniam60)}. Acknowledging the relatively small sample size in human studies, a trend appears to exist regarding a greater propensity for EPA and DHA incorporation in females than males (Table 1). However, given that previous studies did not adjust the dose of ingested n-3 PUFA for individual body mass, the greater incorporation of n-3 PUFA in females may merely be a function of the smaller stature and subsequent higher relative dose of n-3 PUFA in women compared to men. Taken together, these data highlight the practical importance of establishing the optimum supplementation strategy to achieve a maximal, rapid and sustained incorporation of EPA/DHA into the muscle membrane, while accounting for the different biological roles of EPA and DHA and potential sex-specific differences in the biological fate of ingested n-3 PUFA.

Influence of ո-3 PUFA ingestion on muscle protein synthesis

The preponderance of evidence garnered from high-quality experiential studies conducted in human volunteers suggests a modulatory role of n-3 PUFA ingestion in stimulating MPS rates, particularly in healthy older adults (Table 2). Initial interest in the anabolic action of n-3 PUFA in older adults essentially stemmed from a seminal study conducted by Smith et al. in healthy older men and women^{(Reference Smith, Atherton and Reeds46)}. This tightly controlled repeated-measures clinical trial measured mixed (i.e. myofibrillar, mitochondrial and sarcoplasmic protein fractions combined) MPS rates under basal (fasted) and simulated fed conditions immediately before and after 8 weeks of fish oil-derived n-3 PUFA supplementation v. a placebo-controlled corn oil supplementation regimen. The simulated fed condition was represented by the intravenous infusion of amino acids and insulin (i.e. hyperaminoacidemic–hyperinsulinemic clamp) to mimic the nutrient profile and delivery kinetics of a protein-rich mixed macronutrient meal. Basal rates of MPS did not change following 8 weeks of n-3 PUFA supplementation. However, the main finding of this study was a potentiation in the simulated feeding-induced increase in MPS rates after 8 weeks of n-3 PUFA supplementation compared with before supplementation with no pre–post supplementation changes in the MPS response observed in the corn oil (placebo control) trial. Moreover, the increased stimulation of MPS with n-3 PUFA ingestion was accompanied by an upregulation in the phosphorylation status of intracellular cell signalling proteins (mTORC1–p70S6K1) known to trigger the rate-limiting translation initiation step of MPS. Interestingly, no changes in TNF-α or C-reactive protein concentration were observed with n-3 PUFA ingestion. Hence, from a mechanistic standpoint, the anti-inflammatory effects of n-3 PUFA did not appear to mediate the increased stimulation of MPS following 8 weeks of fish oil supplementation, at least in this cohort of healthy older adults. As such, this proof-of-concept study provided preliminary evidence regarding the metabolic action of chronic (8 weeks) n-3 PUFA ingestion in stimulating MPS, at least when the protein synthetic machinery of skeletal muscle is exposed to nutritional stimuli such as amino acids and insulin. In practical terms, these data provided the scientific platform to systematically investigate the efficacy of n-3 PUFA ingestion as an adjunct nutritional strategy to mitigate anabolic resistance in older adults.

Table 2. Summary of experimental studies investigating the impact of n-3 PUFA ingestion on muscle protein synthesis

ASR, absolute synthesis rates; BSA, body surface area; CHO, carbohydrate; CNO, coconut oil; CO, corn oil; d, day; FFM, fat-free mass; FO, fish oil; mo, months; PRO, protein; RET, resistance exercise training; SO, sunflower oil; T, trained; UT, untrained; wk, weeks; y, years.

*Data values not presented in paper.

To further interrogate the efficacy of chronic n-3 PUFA ingestion in modulating the stimulation of MPS, we and others conducted a series of arguably more physiologically relevant metabolic studies in young^{(Reference McGlory, Wardle and Macnaughton61)} and older^{(Reference Da Boit, Sibson and Sivasubramaniam60,Reference Murphy, Flanagan and De Vito62–Reference Kunz, Michie and Gries66)} adults, utilising protein feeding (rather than intravenous hyperaminoacidemic–hyperinsulinemic clamp)^{(Reference McGlory, Wardle and Macnaughton61,Reference Murphy, Flanagan and De Vito62)} and/or supervised bouts of exercise^{(Reference McGlory, Wardle and Macnaughton61,Reference Brook, Din and Tarum63–Reference Dalle, Van Roie and Hiroux65)} as primary anabolic stimuli. Two of these studies were conducted under tightly controlled laboratory conditions and utilised the intravenous tracer infusion of ¹³C₆ labelled phenylalanine to measure the acute 3–4-h response of MPS to a single bout of resistance exercise combined with protein feeding both in the presence or absence of prior (8–16 weeks) n-3 PUFA supplementation^{(Reference McGlory, Wardle and Macnaughton61,Reference Lalia, Dasari and Robinson64)} . In terms of experimental setup, study participants were confined to the laboratory setting for the duration of the trial while connected to an infusion pump, hence it could be argued that these studies^{(Reference McGlory, Wardle and Macnaughton61,Reference Lalia, Dasari and Robinson64)} were low in ecological validity and thus primarily served as proof-of-concept from an efficacy perspective. Nevertheless, in healthy older (76 (sd 5) years) men (n 5) and women (n 7), n-3 PUFA supplementation (3⋅9 g/d) was shown to markedly (30 %) augment the acute (3-h) response of MPS (myofibrillar and mitochondrial protein fractions) to resistance exercise combined with the ingestion of two standardised mixed macronutrient (41.84 kJ/kg of 50 % carbohydrate, 30 % fat and 20 % protein) meals^{(Reference Lalia, Dasari and Robinson64)}. Conversely, in resistance exercise trained young men, 8 weeks of n-3 PUFA (5 g/d) supplementation failed to elicit any measurable changes in the acute response of myofibrillar-MPS to a large bolus of whey protein ingested immediately post-exercise in comparison to a coconut oil control condition^{(Reference McGlory, Wardle and Macnaughton61)}. These seemingly discrepant findings may be attributed to several methodological considerations. First, due to financial constraints, measurements of MPS were only conducted post n-3 PUFA or coconut oil supplementation in the study by McGlory et al.^{(Reference McGlory, Wardle and Macnaughton61)}. Hence, we sacrificed a degree of statistical power afforded by utilising a more robust repeated-measures design that may have detected statistical differences in MPS between n-3 PUFA and coconut oil supplementation groups. Secondly, we cannot discount the possibility that ingestion of 30 g whey protein saturated MPS rates in our cohort of resistance-trained and ‘anabolic sensitive’ young men^{(Reference McGlory, Wardle and Macnaughton61)}. Thus, future studies are warranted to investigate the potential anabolic action of n-3 PUFA under conditions of sub-optimal protein intake (<20 g) in both young and older adults. Finally, in the study by Lalia et al., data values for MPS were remarkably high (i.e. about 2-fold greater than values presented by multiple comparable studies across various laboratories using an identical tracer protocol in older adults^{(Reference Smith, Atherton and Reeds52,Reference Bukhari, Phillips and Wilkinson67–Reference Tipton, Ferrando and Phillips69)} ) and an open-label design was implemented without placebo control. Therefore, the validity of this dataset should be considered with caution. Nevertheless, taken together, these acute metabolic data indicate that n-3 PUFA ingestion may serve as a more effective intervention to increase the response of MPS to nutritional and exercise stimuli in ‘anabolically resistant’ older adults than ‘anabolically sensitive’ young adults.

The reintroduction of deuterium oxide tracer methodology into the field of nutrition, exercise and muscle protein metabolism^{(Reference Wilkinson, Brook and Smith70)} has provided a unique platform for laboratories worldwide to conduct measurements of free-living MPS rates that integrate post-absorptive and post-prandial physiological states over several days or weeks. Accordingly, a series of recent randomised, placebo-controlled, repeated measures studies have investigated the influence of chronic (6–24 weeks) n-3 PUFA ingestion on free-living integrative rates of MPS in healthy young^{(Reference McGlory, Gorissen and Kamal45)} and older adults^{(Reference Da Boit, Sibson and Sivasubramaniam60,Reference Murphy, Flanagan and De Vito62,Reference Brook, Din and Tarum63)} . Given the relatively longitudinal nature of these study designs, whole-body measurements of muscle mass, strength and function have been conducted simultaneously with tissue-specific measurements of MPS, alongside other metabolic and molecular parameters understood to regulate muscle mass and quality such as surrogate markers of MPB^{(Reference Brook, Din and Tarum63)}, myonuclei number^{(Reference Brook, Din and Tarum63)}, satellite cell activation^{(Reference Brook, Din and Tarum63)} and transcriptional changes in genes related to extracellular matrix organisation^{(Reference Lalia, Dasari and Robinson64,Reference Yoshino, Smith and Kelly71)} . In theory, the measurement of MPS over this intermediate time period (rather than an acute 3–4-h period) should better translate to clinically relevant chronic changes in muscle mass (and by extension strength and function) with direct application to the maintenance of muscle mass in later life.

Perhaps the most comprehensive of these deuterium oxide tracer studies, albeit relatively small-scale in cohort size (n 16 participants, eight per group), was conducted by Brook et al. in healthy older women that embarked on a 6-week programme of structured and progressive resistance exercise training^{(Reference Brook, Din and Tarum63)}. The treatment regimen consisted of 3⋅7 g/d of n-3 PUFA supplementation and the control group was assigned to a dose-matched n-6-rich corn oil supplementation regimen. Integrated rates of MPS were determined during the early (0–2 week) and latter (4–6 week) phases of resistance training. No effect of n-3 PUFA ingestion on MPS rates was observed during the early phase of training. However, a trend for an increased stimulation of MPS was observed during the latter phase of training in the n-3 PUFA group that was accompanied by an upregulation of eukaryotic initiation factor 4E binding protein 1 as a downstream (of mTORC1) anabolic signalling protein. Moreover, thigh fat-free mass and type II muscle fibre CSA increased following short-term resistance training in the n-3 PUFA group only, whereas no differences in myonuclear number, satellite cell number and static markers of MPB (calpain, MAFbx and ubiquitin) measured 60–90 min after the final exercise training bout were observed between n-3 PUFA and corn oil regimens. Consistent with this observation^{(Reference Brook, Din and Tarum63)}, McGlory et al. reported higher free-living integrated rates of MPS during 2 weeks of leg immobilisation (used as a model of muscle disuse) and 2 weeks of remobilisation (resistance exercise training) following n-3 PUFA supplementation in a cohort of physically active young women^{(Reference McGlory, Gorissen and Kamal45)}. This increased stimulation of MPS with n-3 PUFA ingestion translated to an attenuated decline in muscle (quadriceps) volume and CSA during the muscle disuse period and an accelerated restoration of muscle mass during rehabilitation^{(Reference McGlory, Gorissen and Kamal45)}. Hence, taken together the augmentation of resistance exercise training-induced muscle mass in older adults, specifically of type II fibres, with n-3 PUFA ingestion observed by Brook et al. and others^{(Reference Bischoff-Ferrari, Vellas and Rizzoli72)} appears to be mediated by an increased stimulation of MPS rather than suppression of MPB and/or via satellite cell recruitment^{(Reference Brook, Din and Tarum63)}. Larger-scale, follow-up studies are warranted to investigate the impact of n-3 PUFA ingestion on free-living integrated MPS rates and muscle mass in response to discrete (injury-induced leg immobilisation) or chronic (illness-induced hospitalisation) periods of muscle disuse in older adults.

Two larger-scale (≥50 participants) randomised placebo-controlled trials also investigated the impact of chronic (18–24 week) n-3 PUFA supplementation on free-living integrated rates of MPS in combination with validated readouts of muscle mass (i.e. muscle anatomical CSA, appendicular lean mass) in healthy older adults^{(Reference Da Boit, Sibson and Sivasubramaniam60,Reference Murphy, Flanagan and De Vito62)} . Based on previous evidence highlighting the interaction of bioavailable n-3 PUFA and amino acids in modulating MPS^{(Reference Smith, Atherton and Reeds52,Reference Kamolrat and Gray55,Reference Kamolrat and Gray73)} , Murphy et al. administered a treatment condition consisting of a leucine-rich whey protein-based beverage that was fortified with n-3 PUFA (3⋅8 g/d + vitamin D) v. protein/leucine and isoenergetic controls twice daily over a 24-week period^{(Reference Murphy, Flanagan and De Vito62)}. No exercise training stimulus was included in this study. In comparison, Da Boit et al. combined n-3 PUFA supplementation with an 18-week progressive resistance exercise training programme^{(Reference Da Boit, Sibson and Sivasubramaniam60)}. As a note of caution, neither study stipulated MPS as the primary endpoint measurement and only a relatively small subset of participants (i.e. five participants in the n-3 PUFA group^{(Reference Murphy, Flanagan and De Vito62)}) volunteered for muscle biopsies, thus allowing for measurement of MPS. Hence, by the authors' own admission, the MPS datasets from these studies should be interpreted with caution. Nonetheless, studies were consistent in reporting no beneficial impact of chronic (18–24-week) n-3 PUFA supplementation on integrated MPS rates, appendicular lean mass^{(Reference Murphy, Flanagan and De Vito62)} and muscle anatomical CSA^{(Reference Da Boit, Sibson and Sivasubramaniam60)} in older adult men and women. These findings may be considered somewhat surprising given that a comparable large-scale (n 60) study by Smith et al.^{(Reference Smith, Julliand and Reeds74)} previously demonstrated that supplementation with a similar dose (3⋅4 g) of n-3 PUFA over the same 24-week period increased thigh muscle volume in a cohort of healthy older adults. Moreover, this favourable change in muscle mass with n-3 PUFA supplementation was accompanied by an increase in skeletal muscle gene expression of pathways involved in mitochondrial function (i.e. uncoupling protein 3, ubiquinol-cytochrome c reductase core protein 1) and extracellular matrix organisation, and a decreased gene expression of pathways related to calpain and ubiquitin-mediated proteolysis and the inhibition of mTORC1^{(Reference Yoshino, Smith and Kelly71)}. Hence, in these studies^{(Reference Yoshino, Smith and Kelly71,Reference Smith, Julliand and Reeds74)} the favourable effects of n-3 PUFA on muscle mass and function appeared to be transcriptionally regulated. The inconsistent findings regarding the efficacy of n-3 PUFA ingestion in modulating muscle mass are difficult to reconcile given the subtle, yet potentially important methodological differences in study design (i.e. participant characteristics including sex, n-3 PUFA dosing regimen, inclusion or absence of resistance training), but may be attributed to inter-individual variability in the skeletal muscle adaptive response to n-3 PUFA ingestion^{(Reference Murphy, Connolly and Flanagan75)}. Follow-up studies designed to investigate the anabolic effects of n-3 PUFA should include omics (i.e. metabolomics, transcriptomics and proteomics) analysis to further interrogate the magnitude of inter-individual variability in response of MPS, muscle mass, strength and function to n-3 PUFA ingestion in healthy and more compromised older adult populations.

An interesting and remarkably consistent observation across published studies investigating the efficacy of n-3 PUFA ingestion to stimulate MPS and/or elicit favourable changes in muscle mass, strength and function relates to evidence of a sex-specific response. This notion is supported by several lines of scientific evidence. First, whereas fish oil-derived (3 g/d) n-3 PUFA supplementation was shown to enhance improvements in maximal isometric strength and muscle quality (calculated as strength per unit of muscle area) following 18 weeks of resistance exercise training in older women, a comparable response was not observed in older men^{(Reference Da Boit, Sibson and Sivasubramaniam60)}. Secondly, and consistent with this observation, 12 weeks of fish oil-derived (2 g/d) n-3 supplementation was shown to enhance muscle strength (increases in maximal torque and rate of torque development) and functional (sit-to-stand performance) outcomes following 90 d of resistance training in a large cohort (n 45) of older women^{(Reference Rodacki, Rodacki and Pereira76)}. Thirdly, a relatively large-scale (n 44) study by Smith et al. reported that daily ingestion of n-3 PUFA (1⋅86 g EPA and 1⋅50 g DHA) over 6 months elicited clinically relevant improvements in muscle volume (about 3⋅5 %) and function (about 6 %) in an older adult cohort that consisted primarily (67 %) of females^{(Reference Smith, Julliand and Reeds74)}. These remarkable findings, particularly in light of the absence of resistance training, imply that 6 months of n-3 PUFA ingestion has the potential to offset 2–3 years of the ‘expected’ age-associated decline in skeletal muscle mass (about 0⋅5–1⋅0 %/year) and function (about 2–3 %/year)^{(Reference Goodpaster, Park and Harris19)}.

As alluded to earlier (see ‘Incorporation of ո-3 PUFA into skeletal muscle membranes’ section), the most intuitive mechanism to explain these sex-specific observations relates to sex differences in the incorporation of n-3 PUFA into the phospholipid membrane. However, to this end, although a greater uptake of EPA in plasma, whole blood and erythrocytes^{(Reference Metherel, Armstrong and Patterson50,Reference Caslake, Miles and Kofler77)} following n-3 PUFA supplementation has been reported in women than men, the propensity for EPA and DHA incorporation into the muscle phospholipid membrane has been shown to be comparable between sexes^{(Reference Da Boit, Sibson and Sivasubramaniam60)}, at least in older adults. Alternatively, the apparent sex-specific response of skeletal muscle adaptation to n-3 PUFA ingestion may merely be a function of a higher dose relative to body mass administered to female participants. In all previous studies^{(Reference McGlory, Gorissen and Kamal45,Reference Smith, Atherton and Reeds52,Reference Da Boit, Sibson and Sivasubramaniam60,Reference Brook, Din and Tarum63,Reference Lalia, Dasari and Robinson64,Reference Yoshino, Smith and Kelly71,Reference Smith, Julliand and Reeds74)} , the dose of ingested n-3 PUFA was administered on an absolute basis (typically between 3 and 5 g/d), irrespective of body mass. Assuming a lower body mass in older adult women than men, the higher relative dose of n-3 PUFA administered to females provides a plausible explanation for the observation of a more favourable muscle adaptive response to n-3 PUFA in older women. Clearly, further studies are warranted to confirm this apparent sex difference in muscle adaptive response to n-3 PUFA ingestion and fully elucidate the mechanism(s) that underpin any sexual dimorphism in response to n-3 PUFA ingestion.