Hypertrophy following resistance training requires net protein synthesis of the myofibrillar proteins, and hence, a maximal stimulation of protein synthesis is favourable for the development of muscle hypertrophy in the elderly. Mixed muscle protein synthesis rate is increased in humans after bouts of resistance training provided the stimulus is of a sufficient magnitude (Chesley et al. 1992). This transient increase in protein synthesis is marked and surpasses the increase in degradation rate, and persists for up to 48 h following an acute exercise bout (Phillips et al. 1997). Evidence exists in favour of the balance between protein synthesis and degradation in skeletal muscle being tipped in favour of protein synthesis by protein intake and hyperaminoacidaemia during rest (Bennet et al. 1989; Biolo et al. 1997; Smith et al. 1998), and net protein balance remains negative after training if individuals remain postabsorptive (Biolo et al. 1995; Phillips et al. 1997). Yet, amino acid supplementation postexercise has been shown to have a synergistic effect upon the muscle contraction-induced augmentation of muscle protein synthesis, when provided both intravenously (Biolo et al. 1997) and orally (Tipton et al. 1999). The stimulation of protein synthesis after bouts of resistance exercise probably follows a specific time course. Thus, it has been observed that protein synthesis is greater 3 h compared to 24 and 48 h postexercise (Phillips et al. 1997). As protein administration is crucial for an optimal effect on net protein synthesis, an early intake of protein after exercise is likely to be important. Recently, it was observed that young individuals had identical acute protein synthesis responses to an amino acid-carbohydrate intake during the first hour following ingestion, irrespective of the supplement being administered 1 or 3 h after resistance exercise (Rasmussen et al. 2000). However, in a resistance training study on rats the timing of a mixed meal ingestion after each training session influenced net protein synthesis over a 10 week training period, as the group that was fed immediately postexercise increased hindlimb muscle mass more than the group fed 4 h later (Suzuki et al. 1999). Yet, it is not known whether ingestion of amino acids immediately postexercise will have a greater effect on the net protein synthesis compared to a later ingestion in elderly males during a period of resistance training.
Hence, the purpose of this study was to investigate the importance of the timing of protein intake after exercise upon the development of muscle hypertrophy and strength during a period of resistance training in elderly individuals. Muscle hypertrophy was evaluated by MRI and from muscle biopsy samples, and muscle strength was determined using both dynamic and isokinetic strength measurements. The acute glucose, insulin and catecholamine responses to exercise and supplementation were also determined for 4 h after training.
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The major finding of the present study is that the timing of protein intake after resistance training bouts in elderly males is of importance for the development of hypertrophy in skeletal muscle. Thus, over a 12 week resistance training period, the CSA of m. quadriceps femoris and MFA-tot increased by 7 and 22 %, respectively, when protein was ingested immediately after exercise (P0), whereas no significant changes were observed when protein was supplemented 2 h postexercise (P2) (Fig. 2 and Fig. 3). The degree of hypertrophy found in P0 was similar to the findings of other studies investigating the effects of resistance training in the elderly where no specific dietary restrictions were reported (Frontera et al. 1988; Brown et al. 1990; Fiatarone et al. 1990; Welle et al. 1996). In addition to the difference in hypertrophy development between the two groups, it is interesting to note the absence of any detectable hypertrophy in P2 despite 12 weeks of resistance exercise identical to the P0 group. This points to the importance of the early timing of protein intake in recovery from resistance exercise in terms of the amount of net protein synthesis in skeletal muscle. Such a hypothesis somewhat resembles the findings of the importance of early carbohydrate intake for the magnitude of glycogen resynthesis after exercise-induced depletion of muscle glycogen (Ivy et al. 1988).
The increase in MFA was more pronounced (22 %) than that in CSA of the whole muscle (7 %). This is in accordance with the findings of other groups (Frontera et al. 1988) and indicates that a concomitant reduction in the relative amount of non-muscular tissue (fat and connective tissue) to CSA takes place in response to training. This is supported by findings in resistance-trained fragile elderly women, where a 10 % increase in CSA of quadriceps was measured when corrected for fat and connective tissue versus only a 5 % increase when not corrected for this (Harridge et al. 1999). Furthermore, since the angle of pennation has been shown to increase with resistance training in young individuals (Aagaard et al. 2001) it is likely that this contributes to the discrepancy between the two measurements of the relative increase of the muscle mass, and thus to the fact that the anatomical CSA of the muscle (determined by MRI) underestimated the true increase in physiological CSA (determined by muscle biopsy).
The MFA of fibre-type 1 (MFA-1) was found to be larger than MFA-2 in P-tot (as well as in P0) in agreement with previous observations in the elderly (Lexell et al. 1983; Aniansson et al. 1992). However, with training MFA-2 increased significantly more than MFA-1 in P0, and concomitantly the distribution of MHC-II increased in P0 (Fig. 4). These changes with resistance exercise are similar to findings in both young (Andersen & Aagaard, 2000) and elderly individuals (Charette et al. 1991) and could indicate that training induces a larger increment in stress of the type 2 fibres than type 1 fibres compared to normal daily living (Henneman et al. 1965). Further, it was only the area of fibre-type 2a that increased, as MFA-2b determined histochemically was unaffected by training. In addition, lean body mass increased more in P0 than in P2 with training, in agreement with a larger net muscle protein synthesis in P0 over the 12 week period of resistance training compared to P2.
In line with the observed disparity in the degree of hypertrophy between the two groups, the muscle strength was also affected differently by the 12 weeks of training in P0 compared to P2. Thus, isokinetic strength increased at both measured velocities (60 and 180 deg s−1) in P0, whereas no significant increase was observed in P2 at any velocity (see Fig. 1B). However, both groups showed an increase in dynamic training strength assessed as 5 RM (see Fig. 1A), but this is more likely to reflect the neural factors of learning and coordination resulting from training (Rutherford & Jones, 1986). Thus, differences between P0 and P2 were observed only in the isolated non-trained isokinetic knee extensions, where the increase in strength in P0 was in agreement with other studies investigating the effect of 12 weeks of resistance training in the elderly using knee extension as the exercise mode (Frontera et al. 1988; Lexell et al. 1995).
No significant differences were observed between P0 and P2 for any anthropometrical, dietary, muscle or strength parameter before training (see Table 1). Hence, both P0 and P2 fulfilled the recommended daily allowance for energy and protein intake (WHO/FAO/UNU, 1985) and also met the more extensive protein recommendations for the elderly (0.9 g kg−1 day−1; Campbell et al. 1994). Moreover, in the elderly it has been found that the protein requirements probably are not increased above normal dietary intake on non-training days as the myofibrillar protein synthesis was found to be similar in an exercised leg 23 h postexercise whether high-protein (28 E %) or isocaloric low-protein meals (7 E %) were ingested (Welle & Thornton, 1998). Therefore, when subjects are fulfilling the dietary recommendations, it appears only to be necessary to provide protein supplementation on training days in order to maximise the net protein synthesis stimulated by the bout of resistance exercise. However, some controversy exists as to whether or not a daily nutritional supplement combined with resistance training has an additive effect on hypertrophy in the elderly. Whereas one group has observed an additive effect (Meredith et al. 1992), another group did not (Fiatarone et al. 1994). In both studies supplementation was given on top of normal adequate nutrition, however, and furthermore food recordings in both studies were not optimal. In the present study subjects in the two groups received the same controlled protein supplementation per body weight (0.13 g kg−1) and per lean body mass (0.19 g kg−1) after every training bout. Hence, the findings do seem to indicate that the timing of the protein intake is of utmost importance for protein synthesis and muscle hypertrophy.
In the present study muscle protein synthesis was not determined, but it is evident that the resulting hypertrophy after training is a product of an accumulation of net synthesis of structural muscle proteins after each resistance exercise bout. As the synthesis of mixed muscle, myofibrillar and MHC proteins has been shown to increase in response to resistance training in the elderly (Yarasheski et al. 1993; Welle et al. 1999; Hasten et al. 2000), resulting in net protein synthesis over a period of resistance training (Frontera et al. 1988; Brown et al. 1990; Fiatarone et al. 1990; Welle et al. 1996), it might be surprising that in the present study one of the groups, P2, did not show any significant increase in muscle CSA. However, two studies have failed to show an acute response in muscle protein synthesis to resistance exercise (Tipton et al. 1996; Roy et al. 1997), although in these studies, trained subjects were used suggesting that the training stimulus could have been insufficient (Rennie & Tipton, 2000). In the present study all individuals were untrained prior to the programme, and furthermore relative loads at 75 % of RM were used during the last 6 weeks, making it unlikely that loading was insufficient to stimulate muscle protein synthesis. Nevertheless, no hypertrophy was observed in P2 despite the fact that comparable studies with very similar (Hakkinen & Hakkinen, 1995) or slightly heavier training protocols (Frontera et al. 1988; Fiatarone et al. 1990; Welle et al. 1996) have found an increase in muscle mass. However, we have no well-founded reason for believing that these studies were carried out in circumstances in which the subjects ate ‘early’ though the changes in muscle mass resemble our findings in P0. Yet, none of these studies report the dietary habits in association with the training. Alternatively, it could be speculated that the time of day when the training was carried out was important. The diurnal hormonal profile could influence the anabolic response to resistance exercise. Thus, in the present study the subjects always trained in the morning between 08.00 and 10.00 h with no differences in the specific time points between the groups P0 and P2. This was done in order to standardise the conditions, which were best controlled at this time of the day. Hence, the subjects in the present study may have had a disadvantage in training compared with other studies, and this could explain the lack of hypertrophy in P2. Unfortunately, no previous studies report when the training was carried out; further investigations are required to elucidate this point.
In spite of this, the difference in hypertrophy between the two groups suggests that the isolated act of contraction is counteracted by other factors, e.g. delayed food intake. In line with this, Tipton et al. (1999) have shown that postexercise net muscle protein balance is negative when individuals are maintained in the postabsorptive state during recovery, whereas if they ingest protein and achieve hyperaminoacidaemia the protein balance becomes positive. Finally, we are confident that both groups trained properly as the training sessions were always supervised and loads adjusted at every third training session. This is supported by the increased training strength (5 RM), which was observed for both groups.
Although not directly determined in this study we do believe that the protein intake highly stimulated muscle protein synthesis, since in a recent study by Rasmussen et al. (2000) protein synthesis was elevated 3.5 times above pre-intake values when a supplement of only 6 g amino acid with 35 g carbohydrate was ingested, whereas we gave 10 g protein together with 7 g carbohydrate. Furthermore, with ageing the stimulation of protein synthesis by resistance exercise has been shown to be preserved (Welle et al. 1994). However, as the dietary restrictions ended 2 h postexercise the possibility cannot be ruled out that if P2 regularly ingested a meal shortly after the supplement then the maximal effective dose of protein was exceeded, hence the stimulatory effect of the protein supplementation was blunted compared to P0. Yet, the dose-response relationship of ingested protein and protein synthesis remains to be elucidated. Consequently, we suggest that the contraction-induced stimulation of protein synthesis was used to a lesser extent in the formation of muscle protein in P2 compared to P0, provided that the stimulation of the protein synthesis follows a time course with a rapid increase within the first few hours following exercise (Phillips et al. 1997). Moreover, since the amino acid delivery is dependent on blood flow (Biolo et al. 1995), the intracellular amino acid availability in the exercised muscle may have been larger in P0 than in P2, hence favouring an increased anabolic response in P0 as it correlates with intracellular amino acid concentration (Biolo et al. 1995). Interestingly, Rasmussen and co-workers have found that protein synthesis and breakdown are stimulated similarly by protein intake in recovery from resistance exercise whether the protein is ingested 1 or 3 h after the termination of exercise, at least in young individuals when protein synthesis in the hour following intake is compared (Rasmussen et al. 2000). However, a 1 h measurement period may be too short to determine differences that affect muscle protein synthesis for many hours.
Altogether, our findings suggest that the first 2 h of recovery after resistance exercise are important for the net protein synthesis during a strength-training programme evaluated over a period of time, and to optimise the protein synthesis the intramuscular concentration of free amino acids is critical if it is not to be a limiting factor.
In the present study muscle fibre hypertrophy was more pronounced in P0 than in P2 despite identical rises in plasma insulin after the intake of a supplement containing protein and carbohydrate (Fig. 5). However, it may be speculated whether the insulin sensitivity of protein turnover is markedly higher immediately after exercise than 2 h later. This has, however, to our knowledge not been studied in humans. Yet, the importance of hyperinsulinaemia with respect to the present study can be questioned. First of all, the effect of postexercise hyperinsulinaemia has been shown to decrease mixed muscle protein degradation whereas synthesis is unaffected (Biolo et al. 1999) and, presumably, the effect is primarily on lysosomal degradation and not myofibrillar breakdown, which follows on the ubiquitin-proteasome pathway. Second, a recent study on postabsorptive exercise in diabetic/non-diabetic rats observed that insulin only played a permissive role at low concentrations in stimulating protein synthesis (Fedele et al. 2000). Thus, it was concluded that the effect of insulin on protein synthesis was only apparent in the low range of plasma insulin, whereas a further increase in insulin did not enhance net protein synthesis additionally (Fedele et al. 2000).
Finally, we cannot exclude the possibility that our finding of a difference in hypertrophy between the groups is a result of the relatively low number of subjects. However, no subjects were systematic outliers and, furthermore, all subjects in P0 had a larger increase in the relative change of the CSA of the quadriceps than any subject in P2. Additionally, theoretically it cannot be ruled out that exercise-induced changes in tissue and serum levels of anabolic hormones such as growth hormone, cortisol or insulin-like growth factor 1, which were not determined in the present study, could contribute to the difference between P0 and P2.
In conclusion, this study investigated the importance of the timing of protein intake after each exercise bout over 12 weeks of resistance training on morphological and strength characteristics of skeletal muscle in elderly individuals. Based on the findings in the present study it appears that the timing of protein intake after strength training bouts can be important for protein synthesis and hypertrophy of skeletal muscle in elderly individuals, and that this appears not to be related to the hyperinsulinaemia in response to the intake of a protein-carbohydrate supplement. The present findings support the hypothesis that early intake of protein after resistance exercise enhances total muscle mass as well as hypertrophy of single muscle fibres in elderly humans.