The present study tested the hypothesis that native whey would have a greater acute anabolic effect on muscle than WPC-80, when supplemented as 20 g protein doses immediately and two hours after resistance exercise in young participants. Despite a larger increase in blood leucine concentrations, native whey stimulated post-exercise (1–5 h) p70S6K-phosphorylation and increased rates of MPS to a similar extent as WPC-80 after resistance exercise. However, compared to milk, native whey induced larger increases in blood concentrations of EAA and leucine, greater phosphorylation of p70S6K, and higher rates of MPS.
As observed in previous studies, blood amino acid concentrations reached higher levels at most time points for the individual BCAAs and EAA, after the whey supplements compared to milk ingestion [16, 21]. In support of the anabolic effect of leucine we observed a moderate correlation between area under the curve for blood concentrations of leucine and the phosphorylation of p70S6K. According to the leucine threshold hypothesis, a high blood or intracellular concentration of leucine is considered a prerequisite for maximal stimulation of MPS . Changes in blood amino acid concentrations are not only due to differences in digestion and absorption rates. It is also dependent on the release of amino acids from muscle due to MPB and the ability of muscle to take up amino acids from blood. Unfortunately, we were not able to measure MPB or intracellular amino acids. Based on previous studies we can assume the intracellular changes in amino acid concentrations are less than those observed in blood [5, 14].
The time course of p70S6K phosphorylation followed the same pattern in all groups peaking at 180 min, before returning towards baseline at 300 min. This is in line with previous studies investigating the effect of protein supplementation after resistance exercise [2, 15, 32, 33]. On the group level, p70S6K phosphorylation results were consistent with the higher total MPS-rates with native whey compared to milk. However, on the individual level there were no correlations between p70S6K phosphorylation and MPS. This disassociation between blood EAA/leucine concentrations, signalling and MPS has previously been observed in similar studies [2, 14, 15].
We observed an overall decrease (only significant with native whey) in the phosphorylation of 4E–BP1 after resistance exercise and protein supplementation during the first hours after resistance exercise. Generally, the phosphorylation of 4E–BP1 is expected to increase after resistance exercise [12, 18, 23], but not all studies support this finding [1, 40]. Although, both p70S6K and 4E–BP1 are downstream of mTORC1, they do not respond similarly to rapamycin treatment. Thus, other mechanisms than mTORC1 activity are also affecting the phosphorylation state of these kinases, possibly leading to the observed differences . In opposition to the observed lack of response to rapamycin, which is believed to imitate amino acid signalling , 4E–BP1 has been shown to respond to protein intake  and it is possible that 4E–BP1 phosphorylation already was elevated at baseline, due to the standardized breakfast in our study. We failed to observe any nutrient or exercise induced changes in eEF-2 phosphorylation, this have also been reported by others [1, 23].
We did not find a significant different MPS-response to ingestion of 20 g of WPC-80 or native whey, during the 5-h post exercise period. Both WPC-80 and native whey were likely able to maximally stimulate MPS with the applied supplementation regime [25, 42]. If supplemented as a suboptimal dose or in elderly, the higher leucine content of native whey may have resulted in a greater anabolic effect than WPC-80. However, previous studies showing an effect of added leucine on MPS have applied a substantially larger difference in the leucine content of supplements [3, 9]. Consequently, whether the moderate difference in leucine content between WPC-80 and native whey will have a meaningful effect in these settings is unclear. Native whey, in contrast to WPC-80, was able to maintain MPS-rates significantly higher than baseline during the late period. Although native whey was not significantly different from the other supplements during the late period, the observed pattern is interesting. The question as to whether the optimal feeding frequency is affected by protein type should be investigated in future studies.
Few studies have directly compared the acute anabolic responses to ingestion of milk and whey proteins. As milk protein is primarily composed of casein (80%) and a smaller part of whey (20%), some information might be gained from studies comparing these purified fractions. Such studies suggests there are only minor differences, if any, in MPS response between ingesting whey and casein after resistance exercise if measured over a sufficient amount of time, e.g. 4–6 h [33, 38]. However, there seems to be a temporal difference between the effects of whey and casein, with whey inducing a faster  more transient increase in MPS, whereas casein ingestion results in a slower more prolonged increase in MPS after resistance exercise . We observed a similar pattern as that reported by Reitelseder and colleagues , with the whey supplements increasing MPS during the early post-exercise period (1–3 h), whereas milk did not. However, our second serving makes it difficult to compare the late periods between studies. As 18 g of milk protein previously have been shown to increase MPS in young men after resistance exercise  we expected our supplementation of 2 × 20 g of milk protein to elicit a measurable effect on MPS. Perhaps ingesting the milk supplement as a single serving of 40 g instead of 2 × 20 g would have elicited a greater effect. It is also possible that our standardized breakfast stimulated MPS at baseline and the change from the fasted state would have been greater.
In order to investigate the effects of slow and fast increases in blood amino acid concentrations, without the challenge of different amino acid content, West and colleagues  supplemented participants with either a bolus (25 g) or a pulse (10 × 2.5 g every 2 min; mimicking casein) of whey protein. Anabolic signalling and MPS was greater with the bolus during a 5-h post-exercise period, indicating that appearance of amino acids in blood affect the anabolic response. The milk, WPC-80 and native whey supplements in the current study all contained 2.0, 2.2 and 2.7 g of leucine per serving. Thus, all servings contained an amount of leucine above 1.8–2.0 g, which has been estimated to be needed in order to maximally stimulate MPS in young individuals . Thus, leaving the varying digestion rate of milk and whey as the most likely explanation for our observed differences in anabolic response between milk and native whey.
Our resistance exercise protocol led to a 5–30% reductions in muscle force-generating capacity (10 min after exercise). These ranges of reductions in muscle function indicate mild to moderate muscular stress, which is supported by small increases in CK across groups . In accordance with a previous study, no differences were observed in recovery of muscle function between groups . The clear effect of protein on anabolic signalling and MPS may theoretically accelerate recovery of muscle function, as has been shown in studies applying more damaging eccentric muscle contractions [6, 11]. In the current study, a workout considered more “normal” and less muscle damaging was applied, while at the same time investigating more comparable supplements, making it less likely to observe a difference.
The lack of a direct measure of muscle protein breakdown (MPB) is a limitation in this study, as both MPS and MPB are needed to calculate net protein balance in muscle. Previous studies have shown MPS to respond with greater changes than MPB [5, 30] and MPB to be a minor determinant of net muscle protein balance in the acute response to protein intake following resistance exercise . Thus, we assume that our MPS measurements to large extent reflect the major part of the net protein balance response. [2H5]phenylalanine TTR in blood was not affected by the supplements. The short-lived increase in [2H5]phenylalanine TTR was due to a bolus infusion of [13C6]phenylalanine not related to the results in this study. This increase would not affect the IRMS measures of bound protein but could potentially alter the GCMS measures of intracellular [2H5]phenylalanine. When analysed, intracellular enrichment of [2H5]phenylalanine were at steady state and did not reflect the brief fluctuations in blood. Measures of MPS should therefore not be affected.