Ageing and protein turnover in skeletal muscle

The loss of skeletal muscle mass in the elderly is characterised by atrophy of type-II (fast) muscle fibres (Fig. 1(A)), fibre necrosis, fibre-type grouping and a reduction in satellite cell content in type-II muscle fibres⁽Reference Lexell^6, Reference Kadi, Charifi and Denis^9–Reference Verdijk, Koopman and Schaart¹⁴⁾. The loss of skeletal muscle mass is accompanied by the loss of muscle strength (Fig. 1(B)), a decline in functional capacity⁽Reference Bassey, Fiatarone and O'Neill^15–Reference Wolfson, Judge and Whipple²²⁾ and a reduction in whole-body and muscle oxidative capacity⁽Reference Short and Nair^4, Reference Nair^23, Reference Nair²⁴⁾. Together, these alterations at a muscle level have substantial health consequences, since they contribute to the greater risk of developing insulin resistance due to the reduced capacity for blood glucose disposal and a greater likelihood of excess lipid deposition in liver and skeletal muscle tissue leading to hyperlipidaemia, hypertension and cardiovascular co-morbidities.

Fig. 1.

Muscle fibre cross-sectional area in young (about 20 year) and elderly (about 76 year) men (A). Note the smaller type-II muscle fibres in the elderly men compared with the young controls (adapted from ⁽Reference Verdijk, Koopman and Schaart¹⁴⁾). (B) Correlation between age and one-repetition maximum (1RM) leg press strength (adapted from⁽Reference Verdijk, van Loon and Meijer¹⁴⁶⁾).

The progressive muscle wasting with ageing must be due to a disruption in the regulation of skeletal muscle protein turnover, leading to a chronic imbalance between muscle protein synthesis and degradation. Although it was originally reported that healthy older adults had decreased rates of basal muscle protein synthesis⁽Reference Short, Vittone and Bigelow^5, Reference Short, Vittone and Bigelow^25–Reference Yarasheski, Zachwieja and Bier³¹⁾, more recent studies have failed to reproduce these findings and generally show little or no differences in basal muscle protein synthesis rates between young and old adults⁽Reference Cuthbertson, Smith and Babraj^32–Reference Kumar, Selby and Rankin³⁹⁾. These discrepancies may be due to the standardisation of prior physical activity⁽Reference Nair²⁴⁾, selection of subjects⁽Reference Yarasheski, Welle and Nair³⁰⁾ or the selection of different precursor pools to calculate muscle protein synthesis⁽Reference Tipton⁴⁰⁾. It seems unlikely that basal muscle protein fractional synthesis rates are diminished by 20–30% as reported previously⁽Reference Balagopal, Rooyackers and Adey^25, Reference Hasten, Pak-Loduca and Obert^26, Reference Welle, Thornton and Jozefowicz^28, Reference Welle, Thornton and Statt²⁹⁾ and/or that muscle protein breakdown is elevated by as much as 50% in the elderly compared to younger adults⁽Reference Trappe, Williams and Carrithers⁴¹⁾. Such opposing alterations in the rates of protein synthesis and breakdown would be accompanied by more rapid muscle wasting than what is typically observed (3–8% per decade⁽Reference Lindle, Metter and Lynch^20, Reference Lynch, Metter and Lindle⁴²⁾), and it therefore seems unlikely that basal muscle protein fractional synthesis rates could be diminished by 20–30% during ageing as reported previously⁽Reference Balagopal, Rooyackers and Adey^25, Reference Hasten, Pak-Loduca and Obert^26, Reference Welle, Thornton and Jozefowicz^28, Reference Welle, Thornton and Statt²⁹⁾. The relatively slow rate of muscle loss during ageing must mean that the mismatch between the average diurnal rate of muscle protein synthesis and breakdown is small. It is currently accepted that basal fasting protein synthesis and/or breakdown rates are not (substantially) different between young and elderly human subjects⁽Reference Hasten, Pak-Loduca and Obert^26, Reference Cuthbertson, Smith and Babraj^32–Reference Kumar, Selby and Rankin^39, Reference Rasmussen, Fujita and Wolfe⁴³⁾. To better understand the skeletal muscle wasting in the elderly, researchers have started to focus on the muscle anabolic response to anabolic stimuli such as physical activity, food intake and anabolic hormones such as insulin. It was well established that the protein turnover in skeletal muscle is highly responsive to exercise and nutrient intake in healthy young individuals⁽Reference Koopman, Saris and Wagenmakers⁴⁴⁾. Interestingly, data from recent studies suggest that the muscle protein synthetic response to resistance exercise⁽Reference Kumar, Selby and Rankin³⁹⁾ and following the ingestion of a small amount of amino acids (AA) with⁽Reference Volpi, Mittendorfer and Rasmussen^36, Reference Guillet, Prod'homme and Balage⁴⁵⁾ or without carbohydrate⁽Reference Cuthbertson, Smith and Babraj^32, Reference Katsanos, Kobayashi and Sheffield-Moore³³⁾ is reduced in the elderly when compared with young controls. The latter is believed to represent a key factor responsible for the age-related decline in skeletal muscle mass⁽Reference Rennie⁴⁶⁾.

Anabolic response to exercise

Exercise is a powerful stimulus to promote net muscle protein anabolism, resulting in specific metabolic and morphological adaptations in skeletal muscle. Endurance training can increase whole-body and muscle oxidative capacity and endurance⁽Reference Wilkinson, Phillips and Atherton⁴⁷⁾, whereas resistance exercise training can increase muscle mass and strength, and thus improve physical performance and functional capacity⁽Reference Evans⁴⁸⁾. It generally takes weeks to months before training-induced changes in skeletal muscle mass become apparent⁽Reference Rennie and Tipton⁴⁹⁾. The prolonged time course for hypertrophy is a reflection of the slow turnover rate of muscle proteins, i.e. about 1% per day for contractile proteins⁽Reference Balagopal, Rooyackers and Adey^25, Reference Nair, Halliday and Griggs^50, Reference Volpi, Ferrando and Yeckel⁵¹⁾. Although muscle hypertrophy occurs at a slow rate, a single bout of resistance exercise can rapidly (within 2–4 h⁽Reference Phillips, Tipton and Aarsland⁵²⁾) stimulate muscle protein synthesis, and increase protein synthesis rates, particularly the myofibrillar protein synthesis⁽Reference Welle, Thornton and Jozefowicz^28, Reference Yarasheski, Zachwieja and Bier^31, Reference Wilkinson, Phillips and Atherton⁴⁷⁾, which persist for up to 16 h in trained⁽Reference Tang, Perco and Moore⁵³⁾ and 24–48 h in untrained individuals⁽Reference Phillips, Tipton and Aarsland^52–Reference MacDougall, Tarnopolsky and Chesley⁵⁴⁾. Muscle protein breakdown is also stimulated following exercise, albeit to a lesser extent than protein synthesis⁽Reference Phillips, Tipton and Aarsland^52, Reference Biolo, Maggi and Williams⁵⁵⁾, and results in an improved net muscle protein balance that persists for up to 48 h in untrained individuals⁽Reference Phillips, Tipton and Aarsland⁵²⁾.

It has been generally accepted that the increase in protein synthesis following exercise is due to increased mRNA translation⁽Reference Laurent, Sparrow and Millward⁵⁶⁾. Many laboratories have shown that the signalling pathway involving a mammalian target of rapamycin (mTOR) complex I (mTORCI) plays a crucial role in the control of mRNA translation initiation and elongation⁽Reference Bodine, Stitt and Gonzalez^57–Reference Dreyer, Fujita and Cadenas⁵⁹⁾. The activity of mTORCI determines the activity of downstream effectors such as the 70-kDa S6 protein kinase (S6K1) and the eukaryotic initiation factor 4E-binding protein⁽Reference Kimball, Farrell and Jefferson⁶⁰⁾. Both play key regulatory roles in modulating translation initiation, and control the binding of mRNA to the 40S ribosomal subunit⁽Reference Kimball, Farrell and Jefferson⁶⁰⁾. Studies have shown that the mTORCI signalling pathway is activated after acute resistance exercise in healthy human subjects⁽Reference Wilkinson, Phillips and Atherton^47, Reference Dreyer, Fujita and Cadenas^59, Reference Drummond, Dreyer and Pennings^61, Reference Koopman, Zorenc and Gransier⁶²⁾. Moreover, Drummond et al. ⁽Reference Drummond, Fry and Glynn⁶³⁾ showed elegantly that early acute contraction-induced increase in human protein synthesis in human subjects can be blocked with rapamycin treatment indicating that mTORCI signalling is crucial during the early post-exercise recovery. In addition, it was shown that the phosphorylation status of S6K1 following resistance exercise is a good marker for the long-term increase in skeletal muscle mass in rats⁽Reference Baar and Esser⁶⁴⁾ and human subjects⁽Reference Terzis, Georgiadis and Stratakos⁶⁵⁾. Moreover, significant correlations were reported between S6K1 phosphorylation/activation and muscle protein synthesis following exercise in young healthy human subjects⁽Reference Kumar, Selby and Rankin³⁹⁾, highlighting the importance of this signalling pathway in the adaptive response to resistance exercise.

Anabolic response to food intake

Protein turnover in skeletal muscle is highly responsive to nutrient intake⁽Reference Rennie, Edwards and Halliday⁷⁰⁾. Ingestion of AA and/or protein strongly stimulates muscle protein synthesis⁽Reference Paddon-Jones, Sheffield-Moore and Zhang^35, Reference Volpi, Mittendorfer and Wolf^37, Reference Volpi, Ferrando and Yeckel^51, Reference Rennie, Edwards and Halliday^70, Reference Paddon-Jones, Sheffield-Moore and Katsanos⁷¹⁾. Besides serving as a substrate for polypeptide biosynthesis, AA were shown to directly activate regulatory proteins in mRNA translation, while non-essential AA do not induce a substantial increase in muscle protein synthesis. In contrast, essential amino acids (EAA) increase muscle protein synthesis in the absence of increased non-essential AA availability. The branched-chain amino acid, leucine, is of particular interest since it has the unique ability to directly increase signalling through mTOR and its downstream targets 4E-binding protein and S6K1 and ribosomal S6. The EAA⁽Reference Tipton, Gurkin and Matin^72, Reference Volpi, Kobayashi and Sheffield-Moore⁷³⁾, and leucine in particular⁽Reference Smith, Barua and Watt^74, Reference Norton and Layman⁷⁵⁾, seem to represent the main anabolic signals responsible for the post-prandial increase in muscle protein synthesis. The observations that EAA show a dose-dependent stimulation of muscle protein synthesis without increasing plasma insulin⁽Reference Bohe, Low and Wolfe⁷⁶⁾, and that carbohydrate ingestion does not affect protein synthesis⁽Reference Borsheim, Cree and Tipton⁷⁷⁾, suggest that insulin is rather permissive instead of modulatory⁽Reference Rennie^46, Reference Bohe, Low and Wolfe^76, Reference Greenhaff, Karagounis and Peirce⁷⁸⁾. Greenhaff et al. ⁽Reference Greenhaff, Karagounis and Peirce⁷⁸⁾ showed that insulin in the range of 30–150 μU/ml does not further stimulate muscle protein synthesis. In contrast to protein synthesis, muscle protein degradation seems to be very responsive to relatively small changes in insulin concentrations. Insulin levels of 15 μU/ml can almost maximally reduce muscle protein breakdown⁽Reference Wilkes, Selby and Atherton⁷⁹⁾ and there seems to be no further inhibition above 30 μU/ml⁽Reference Greenhaff, Karagounis and Peirce⁷⁸⁾. These data suggest that protein breakdown can be already maximally reduced by slightly increased insulin concentrations which can be achieved by the intake of a small breakfast in healthy young men⁽Reference Rennie⁴⁶⁾.

Ageing and the anabolic response to food intake

Data from recent studies suggest that the muscle protein synthetic response to the ingestion of a small amount of EAA⁽Reference Cuthbertson, Smith and Babraj^32, Reference Katsanos, Kobayashi and Sheffield-Moore³³⁾ is attenuated in the elderly, and is now believed to represent one of the key factors responsible for the age-related decline in skeletal muscle mass. The so-called ‘anabolic resistance’ in elderly human subjects was demonstrated by a rightward and downward shift of the dose–response relationship between myofibrillar protein synthesis and the availability of leucine in the plasma⁽Reference Cuthbertson, Smith and Babraj³²⁾. Cuthbertson et al. ⁽Reference Rennie⁴⁶⁾ showed that even a very large (40 g) dose of EAA is not able to bring the curve back to values for young subjects, suggesting that supplementation with extra protein, EAA or leucine will not be sufficient to restore the rate of muscle protein synthesis in older adults, relative to those found in the young.

The mechanisms responsible for the proposed anabolic resistance to protein and/or AA administration in the elderly are yet to be elucidated fully. Cuthbertson et al. ⁽Reference Cuthbertson, Smith and Babraj³²⁾ reported decrements in amounts of signalling protein in the protein kinase B/mTORCI pathway in old muscle and showed an attenuated rise in the activation of key signalling proteins in this pathway after ingesting 10 g EAA in the elderly v. the young. These findings seem to be consistent with previous observations by Guillet et al. ⁽Reference Guillet, Prod'homme and Balage⁴⁵⁾ who showed reduced S6K1 phosphorylation following combined AA and glucose infusions in the elderly. Combined, these data suggest that anabolic signalling is impaired in skeletal muscles of older compared to younger adults⁽Reference Cuthbertson, Smith and Babraj^32, Reference Bohe, Low and Wolfe⁷⁶⁾, which may be in part due to insulin resistance in the elderly. Recent data suggest that muscle protein breakdown is not strongly inhibited by insulin in the elderly⁽Reference Wilkes, Selby and Patel⁸⁰⁾, whereas other reports suggested that muscle protein synthesis is resistant to the anabolic action of insulin in the elderly⁽Reference Volpi, Mittendorfer and Rasmussen^36, Reference Rasmussen, Fujita and Wolfe⁴³⁾. It has been proposed that the anabolic resistance can be attributed to a less responsive impact of physiological hyperinsulinemia on the increase in skeletal muscle blood flow and subsequent AA availability in aged muscle⁽Reference Rasmussen, Fujita and Wolfe^43, Reference Fujita, Rasmussen and Cadenas⁸¹⁾, which would agree with the reduced activation of the phosphatidylinositol-3 kinase–protein kinase B–mTOR signalling pathway and with the lesser increase in the muscle protein synthetic rate after AA/protein ingestion in the elderly⁽Reference Cuthbertson, Smith and Babraj³²⁾.

Another mechanism that has been suggested to contribute to the anabolic resistance to food intake in elderly men is an impairment in dietary protein digestion and/or absorption⁽Reference Boirie, Dangin and Gachon⁸²⁾. Recent data show that the digestion rate of protein is an independent regulating factor of post-prandial protein anabolism⁽Reference Dangin, Boirie and Garcia-Rodenas⁸³⁾. As such, it seems plausible to assume that any impairment in protein digestion and/or absorption will reduce the appearance rate of dietary AA in the bloodstream, thereby reducing AA delivery to the muscle and subsequently attenuating the muscle protein synthetic response. To accurately assess the appearance rate of AA derived from dietary protein, the labelled AA need to be incorporated in the dietary protein source⁽Reference Beaufrere, Dangin and Boirie^84–Reference Dangin, Boirie and Guillet⁸⁶⁾. As free AA and protein-derived AA exhibit a different timing and efficiency of intestinal absorption⁽Reference Boirie, Gachon and Corny⁸⁵⁾, simply adding labelled free AA to a drink containing protein does not provide an accurate measure of the digestion and absorption kinetics of the ingested dietary protein⁽Reference Boirie, Fauquant and Rulquin⁸⁷⁾. These methodological restrictions represent the main reasons why only a few researchers have investigated the differences in digestion and absorption kinetics of specific dietary protein sources and the disparity in anabolic response between young and elderly human subjects. These studies have suggested that AA utilisation in the splanchnic area is elevated in the elderly⁽Reference Boirie, Dangin and Gachon⁸²⁾, which would imply that less of the ingested AA are available for muscle protein synthesis⁽Reference Boirie, Dangin and Gachon⁸²⁾. We have recently repeated similar experiments, comparing the appearance rate of dietary L-[1-¹³C]phenylalanine in the circulation following the intake of 35 g intact intrinsically labelled casein protein⁽Reference Koopman, Walrand and Beelen⁸⁸⁾. Our data clearly show that splanchnic extraction is not altered significantly in elderly men, and that over a 3 and 6 h period the same amount of dietary phenylalanine appears in the circulation⁽Reference Koopman, Walrand and Beelen⁸⁸⁾. Although we did not observe any impairment in digestion and absorption in the elderly, we observed substantially (about 12%) lower rates of whole-body protein synthesis and phenylalanine hydroxylation following protein ingestion in the elderly men compared to the young men (Fig. 2), calculated over the first 3 h, subsequent 3 h or total 6 h time period after protein ingestion. Consistent with these observations, we observed a 14% difference in muscle protein synthesis rates between young and elderly men over the 6 h period, although this difference did not reach statistical significance⁽Reference Koopman, Walrand and Beelen⁸⁸⁾. Not all researchers have found impaired muscle protein synthetic response to protein intake in the elderly as similar protein synthetic rates were observed in young and elderly human subjects after ingestion of large amounts of carbohydrate and proteins⁽Reference Koopman, Verdijk and Manders⁸⁹⁾, and following ingestion of large⁽Reference Symons, Sheffield-Moore and Wolfe⁹⁰⁾ and small amount of beef⁽Reference Symons, Sheffield-Moore and Wolfe^90, Reference Symons, Schutzler and Cocke⁹¹⁾. Discrepancies may arise from differences in timing of biopsy collection, the precursor pool used to calculate muscle protein synthesis or the age of the elderly volunteers studied. Clearly, more research is warranted to determine the extent of an anabolic resistance to food (i.e. intact protein) intake that exists in elderly human subjects.

Fig. 2.

Whole-body protein synthesis rates, calculated over 3 h or 6 h periods, following the ingestion of 35 g of casein protein in young (about 23 year, n 10) and elderly (about 64 year, n 10) men. Whole-body protein synthesis rates, calculated per kg body weight, are significantly lower in the elderly compared to the young controls (*P<0·05). Adapted from Koopman et al. ⁽Reference Koopman, Walrand and Beelen⁸⁸⁾.

Early work from the laboratory of Yves Boirie⁽Reference Boirie, Dangin and Gachon^82–Reference Beaufrere, Dangin and Boirie^84, Reference Dangin, Boirie and Guillet^86, Reference Dangin, Guillet and Garcia-Rodenas⁹²⁾ showed that ingestion of a slowly digested protein (casein) led to a more positive whole-body protein balance (averaged over a 7 h period) when compared with the ingestion of a fast digestible protein (whey) or a mixture of free AA in healthy, young subjects⁽Reference Dangin, Boirie and Garcia-Rodenas⁸³⁾. In contrast, ingestion of a fast protein resulted in greater (whole-body) net protein retention compared to a slow protein when provided to healthy, older men⁽Reference Boirie, Dangin and Gachon^82, Reference Beaufrere, Dangin and Boirie^84, Reference Dangin, Boirie and Guillet^86, Reference Dangin, Guillet and Garcia-Rodenas⁹²⁾. The latter response might be attributed to the reported anabolic resistance of the muscle protein synthetic machinery to become activated in the elderly. In accordance with the fast v. slow protein concept, we tested the hypothesis that the ingestion of a casein protein hydrolysate, i.e. enzymatically pre-digested casein, would enhance protein digestion and the absorption rate in elderly men⁽Reference Koopman, Crombach and Gijsen⁹³⁾. We expected that this enhanced AA uptake in the gut would result in a greater increase in plasma AA availability and might improve the post-prandial muscle protein synthetic response. Elderly men ingested 35 g intrinsically L-[1-¹³C]phenylalanine labelled casein or casein hydrolysate and we assessed the appearance rate of dietary phenylalanine in the circulation and the subsequent muscle protein synthetic response. The ingestion of casein hydrolysate accelerated the appearance rate of dietary phenylalanine in the circulation, lowered splanchnic phenylalanine extraction, increased post-prandial plasma AA availability and tended to augment the subsequent muscle protein synthetic response in vivo in human subjects, compared to the ingestion of intact casein⁽Reference Koopman, Crombach and Gijsen⁹³⁾. The difference in the appearance rate of dietary protein between intact and hydrolysed casein was particularly evident in the first 3 h after the protein ingestion, with about 50% more dietary phenylalanine appearing in the circulation after ingestion of the casein hydrolysate⁽Reference Koopman, Crombach and Gijsen⁹³⁾. Consistent with these findings, it was reported that protein pulse feeding (providing up to 80% of daily protein intake in one meal) leads to greater protein retention than ingesting the same amount of protein provided over four meals throughout the day (spread-feeding) in elderly women⁽Reference Arnal, Mosoni and Boirie^94, Reference Arnal, Mosoni and Boirie⁹⁵⁾. These findings may indicate that part of the proposed anabolic resistance in the elderly might be compensated for, in part, by enhancing AA availability during the post-prandial period.

Long-term interventions

The clinical relevance of nutritional and/or exercise intervention in the elderly stems from the long-term impact on skeletal muscle mass and strength, and the implications for functional capacity. In accordance with the previously discussed findings, the muscle protein synthetic machinery is able to respond to anabolic stimuli, albeit maybe to a lesser extent⁽Reference Rennie⁴⁶⁾, until very old age⁽Reference Fiatarone, Marks and Ryan^97, Reference Frontera, Meredith and O'Reilly⁹⁸⁾. Although it was suggested previously that elderly human subjects need more protein⁽Reference Campbell, Trappe and Jozsi⁹⁹⁾, more recent studies by Campbell et al.⁽Reference Campbell, Johnson and McCabe¹⁰⁰⁾, who performed very comprehensive nitrogen balance experiments, clearly showed that dietary protein requirements did not increase with age, and that a dietary protein allowance of 0·85 g/kg per day is adequate. Some researchers believe that the attenuated muscle protein synthetic response to food intake in the elderly can, at least partly, be compensated for by increasing the leucine content of a meal⁽Reference Katsanos, Kobayashi and Sheffield-Moore^34, Reference Rieu, Balage and Sornet¹⁰¹⁾. However, we have shown previously that additional leucine intake does not further increase muscle protein synthesis after resistance exercise when ample protein is ingested by elderly men⁽Reference Koopman, Verdijk and Beelen¹⁰²⁾. In addition, we investigated the effect of 3 months of leucine supplementation with each main meal (7·5 g/d) on skeletal muscle mass and strength and on glycemic control in healthy elderly men⁽Reference Verhoeven, Vanschoonbeek and Verdijk¹⁰³⁾. Consistent with our observations from our acute post-exercise study, we did not observe any effect of leucine supplementation on skeletal muscle mass and strength. In addition, no improvements in indices of whole-body insulin sensitivity blood-glycated Hb content, or the plasma lipid profile were observed. We concluded that long-term leucine supplementation (7·5 g/d) does not augment skeletal muscle mass or strength and does not improve glycaemic control or the blood lipid profile in healthy elderly men.

Resistance exercise training interventions were shown effective in augmenting skeletal muscle mass, increasing muscle strength and/or improving functional capacity in the elderly⁽Reference Fiatarone, Marks and Ryan^97, Reference Frontera, Meredith and O'Reilly^98, Reference Ades, Ballor and Ashikaga^104–Reference Verdijk, Gleeson and Jonkers¹¹⁹⁾. In addition, endurance⁽Reference Fiatarone, Marks and Ryan^97, Reference Frontera, Meredith and O'Reilly^98, Reference Ades, Ballor and Ashikaga^104–Reference Vincent, Braith and Feldman¹¹⁰⁾ exercise was shown to enhance the skeletal muscle oxidative capacity, resulting in greater endurance capacity⁽Reference Short, Vittone and Bigelow^5, Reference Short, Vittone and Bigelow¹²⁰⁾. Although the muscle regenerative capacity seems to decline at a more advanced age, the reduced satellite cell pool size⁽Reference Verdijk, Gleeson and Jonkers¹¹⁹⁾ does not compromise the capacity for muscle hypertrophy to occur even at an advanced age⁽Reference Dedkov, Borisov and Wernig^121–Reference Thornell, Lindstrom and Renault¹²³⁾ and resistance exercise training was shown to increase muscle fibre size⁽Reference Kadi, Schjerling and Andersen^124–Reference Olsen, Aagaard and Kadi¹²⁷⁾. Recently, Verdijk et al.⁽Reference Verdijk, Gleeson and Jonkers¹¹⁹⁾ assessed the effects of 12 weeks of leg resistance exercise training on fibre-type specific hypertrophy and satellite cell content in healthy, elderly men. Prolonged training resulted in a 28% increase in the size of type-II muscle fibres and a concomitant 76% increase in type-II muscle fibre satellite cell content in elderly males⁽Reference Verdijk, Gleeson and Jonkers¹¹⁹⁾. The apparent differences in fibre size and/or satellite cell content between type-I and type-II muscle fibres prior to intervention were no longer evident after 12 weeks of training. Overall, these findings suggest that satellite cells are instrumental in the generation of new myonuclei to facilitate muscle fibre hypertrophy⁽Reference Snijders, Verdijk and van Loon¹²⁸⁾.

Protein/AA ingestion before, during and/or after exercise acutely stimulates muscle protein synthesis and reduces muscle protein breakdown to facilitate muscle fibre hypertrophy. Remarkably, little evidence exists that dietary interventions can further augment the adaptive response to prolonged exercise training in the elderly. The proposed importance of ample dietary protein intake in the long-term adaptive response to resistance training in the elderly has been a topic of intense debate⁽Reference Campbell and Evans^129–Reference Campbell and Leidy¹³¹⁾. Some researchers suggest that the current RDA for habitual protein intake of 0·8 g/kg per day⁽Reference Rand, Pellett and Young^132, Reference Trumbo, Schlicker and Yates¹³³⁾ is marginal to allow lean mass accretion following resistance exercise training⁽Reference Campbell, Trappe and Jozsi⁹⁹⁾ or even insufficient for long-term maintenance of skeletal muscle mass in sedentary elderly human subjects⁽Reference Campbell, Trappe and Wolfe¹³⁴⁾. However, others have shown that when habitual dietary protein intake is standardised at 0·9 g/kg per day, exercise-induced increases in muscle mass become apparent and further increases in protein intake does not provide any additional effect⁽Reference Iglay, Thyfault and Apolzan¹¹⁴⁾. In addition, data from Walrand et al. ⁽Reference Walrand, Short and Bigelow¹³⁵⁾ indicated that although increased protein intake in the elderly further improved nitrogen balance (by increasing AA oxidation), no beneficial effects on muscle protein synthesis and muscle function were observed. These observations might explain why most studies fail to observe any additional benefit of nutritional co-intervention on the skeletal muscle adaptive response to prolonged resistance exercise training in the elderly⁽Reference Fiatarone, Marks and Ryan^97, Reference Frontera, Meredith and O'Reilly^98, Reference Fiatarone, O'Neill and Ryan^106, Reference Godard, Williamson and Trappe^113, Reference Iglay, Thyfault and Apolzan^114, Reference Verdijk, Jonkers and Gleeson^117, Reference Haub, Wells and Tarnopolsky^118, Reference Campbell, Crim and Young^136–Reference Welle and Thornton¹³⁹⁾. However, it has been suggested that it is not the total protein amount per se, but the timing of protein intake that is crucial for its stimulatory effect on muscle protein synthesis and muscle fibre hypertrophy. Esmarck et al. ⁽Reference Esmarck, Andersen and Olsen¹⁴⁰⁾ concluded that the intake of a protein supplement immediately after each bout of resistance-type exercise was required for skeletal muscle hypertrophy to occur with a 12-week intervention in the elderly. Although the absence of any hypertrophy in the control group seems to conflict with previous studies that show muscle hypertrophy following resistance training without any dietary intervention, the proposed importance of nutrient timing is supported by more recent studies investigating the impact of AA or protein co-ingestion prior to, during and/or after exercise on the acute muscle protein synthetic response⁽Reference Beelen, Koopman and Gijsen^141, Reference Tipton, Rasmussen and Miller¹⁴²⁾. Verdijk et al.⁽Reference Verdijk, Jonkers and Gleeson¹¹⁷⁾ compared increases in skeletal muscle mass and strength following 3 months of resistance exercise training with or without protein ingestion prior to and immediately after each exercise session in elderly males. Timed protein supplementation prior to and after each exercise bout did not further increase skeletal muscle hypertrophy in healthy, elderly men who habitually consumed about 1·0 g protein/kg per day. Taken together, the available data suggest that sufficient habitual protein intake (about 0·9 g/kg per day) combined with a normal meal pattern (i.e. providing ample protein three times daily) will allow for substantial gains in muscle mass and strength with resistance exercise training in the elderly. Additional protein supplementation does not seem to provide large surplus benefits to the exercise intervention in healthy, elderly males. Additional protein intake may reduce subsequent voluntary food consumption in the elderly⁽Reference Fiatarone Singh, Bernstein and Ryan¹⁴³⁾ and consequently some have suggested that supplementation with EAA would be more efficient⁽Reference Timmerman and Volpi¹⁴⁴⁾. Clearly, acute studies have shown benefits of timed supplementation with small (7–15 g) amounts of EAA on muscle protein synthesis⁽Reference Katsanos, Kobayashi and Sheffield-Moore^33, Reference Paddon-Jones, Sheffield-Moore and Zhang^35, Reference Paddon-Jones, Sheffield-Moore and Katsanos⁷¹⁾. However, well-designed, double-blind, placebo-controlled long-term studies to investigate beneficial and adverse effects of long-term EAA supplementation in the elderly are yet to be performed⁽Reference Henderson, Irving and Nair¹⁴⁵⁾.