3.2. Plasma Leucine, Branched-Chain Amino Acids, Insulin, and Glucose
Fasting and peak concentrations, time to peak concentration, and AUC values for all plasma variables in each age group are shown in
Table 3.
Leucine. For time series data in the pooled sample, analysis showed that there were significant main effects of time, condition, and a time x condition interaction (all
p < 0.01). Plasma leucine tended to be higher 90 min after ingestion of EAA compared to baseline (382 ± 54 vs. 114 ± 17 µM;
p = 0.09,
d = 2.11). In RE, plasma leucine did not significantly change from baseline (all
p > 0.99). In COM, plasma leucine tended to be elevated at 60 min post-ingestion, compared with baseline (139 ± 25 vs. 394 ± 66 µM;
p = 0.07,
d = 1.62), and was significantly greater at 90 min (426 ± 50 µM;
p < 0.01,
d = 2.30) and 120 min (449 ± 76 µM;
p = 0.04,
d = 1.74), compared with baseline. When data were split by age group, a significant main effect was observed for condition (
p < 0.001), but not age (
p = 0.88) or age x condition interaction (
p = 0.96). In the younger group, plasma leucine tended to be higher 90 min following EAA compared with baseline (
p = 0.08,
d = 3.81;
Figure 1A), whereas all other comparisons were non-significant (
p > 0.23). In the older group, decreased plasma leucine was observed 30 min after the conclusion of RE (
p = 0.02,
d = 0.29;
Figure 1B), with no other significant differences observed (
p > 0.56).
Peak postprandial leucine concentrations (C
max) in the pooled sample differed significantly (
p < 0.01) with EAA (436 ± 49 µM;
p < 0.01,
d = 2.23) and COM (545 ± 83 µM;
p < 0.01,
d = 1.97) greater than RE (167 ± 22 µM). EAA and COM did not significantly differ (
p = 0.27,
d = 0.51). After splitting for age, leucine C
max was significantly different between conditions (
p < 0.001;
Table 3). In the young, EAA and COM were similar (
p = 0.52,
d = 0.45), and higher than RE (
p = 0.02,
d = 4.30;
p = 0.06,
d = 2.03; respectively). In the older group, COM tended to be higher compared with RE (
p = 0.06,
d = 1.74) and was similar to EAA (
p = 0.43,
d = 0.51). EAA did not differ from RE despite a large effect (
p = 0.13,
d = 1.51).
Time to peak plasma leucine concentration (Tmax) in the pooled sample did not differ between conditions (p = 0.14). After splitting by age, Tmax differed significantly (age x condition interaction: p = 0.01), occurring earlier in COM compared with RE in the older group (p = 0.01, d = 1.70). Tmax did not differ significantly by condition in the younger group.
Leucine AUC in the pooled sample significantly differed between conditions (p < 0.01) with EAA (39272 ± 5299 µM; p < 0.01, d = 1.51) and COM (43094 ± 6612 µM; p < 0.01, d =1.29) greater than RE (16022 ± 1844 µM). EAA and COM did not significantly differ (p = 0.62, d = 0.20). After splitting by age, leucine AUC differed by condition (p = 0.002) but there were no main effects for age (p = 0.87) or age x condition interaction (p = 0.86). In the young, leucine AUC was greater in EAA compared with RE (p = 0.03, d = 3.82) and was similar to COM (p = 0.96, d = 0.02). In the older group, leucine AUC tended to be greater in COM compared with RE (p = 0.07, d = 1.73), with no other significant effects.
Branched-chain amino acids. Time series data for plasma BCAA concentrations in the pooled sample showed that there were significant main effects of time, condition, and a time x condition interaction (all
p < 0.01). In the EAA condition, plasma BCAA concentrations were significantly elevated at 60 min (815 ± 78 µM;
p = 0.02,
d = 2.63), 90 min (779 ± 74 µM;
p = 0.01,
d = 2.53), and 120 min (729 ± 74 µM;
p = 0.03,
d = 2.25) postprandially compared with baseline (329 ± 29 µM). In the RE condition, plasma BCAA did not significantly change during the trial (all
p > 0.99). Following COM, plasma BCAA were elevated at 60 min (686 ± 77 µM;
p = 0.03,
d = 1.99) and 90 min (733 ± 60 µM;
p < 0.001,
d = 2.77) postprandially compared with baseline (327 ± 26 µM). Age group time series data are shown in
Figure 1C and 1D, respectively. Concurrent with plasma leucine, BCAA concentrations were significantly different between conditions (
p < 0.001) but there was no effect of age (
p = 0.97) or age x condition interaction (
p = 0.22). Although post-hoc testing was performed, no significant differences were found (all P > 0.24).
Peak plasma BCAA concentrations in the pooled sample differed significantly (
p < 0.01), with EAA (889 ± 56 µM;
p < 0.01,
d = 2.86) and COM (953 ± 142 µM;
p < 0.01,
d = 1.48) greater than RE (467 ± 35 µM). COM and EAA did not significantly differ (
p = 0.66,
d = 0.19). After splitting data for age, BCAA C
max was significantly different between conditions (
p = 0.002), but there were no effects for age (
p = 0.59) or age x condition interaction (
p = 0.94). In the young, peak BCAA did not differ between groups (all
p > 0.11;
Table 3), whereas in the older group BCAA C
max was higher in EAA compared with RE (
p = 0.01,
d = 3.54) and tended to be higher in COM (
p = 0.06,
d = 2.16), with no difference between EAA and COM (
p = 0.84,
d = 0.14).
For time to peak concentration, in pooled data a trend was found between conditions (p = 0.07). When data were split by age group, significant main effects for condition (p = 0.01) and age x condition interaction (p = 0.03) were found. However, post-hoc testing could not detect these differences (all p > 0.10).
For BCAA AUC, a significant main effect was observed for condition (p < 0.01), with EAA (84023 ± 6838 µM; p < 0.01, d = 2.70) and COM (78347 ± 8221 µM; p < 0.01, d = 2.00) significantly greater than RE (39653 ± 2681 µM). EAA and COM did not significantly differ (p = 0.58, d = 0.24). After splitting by age group, a significant main effect was found for condition (p < 0.001), but not age (p = 0.78) or age x condition interaction (p = 0.53). AUC was higher in the younger group following EAA, compared with RE (p = 0.05, d = 2.80) and was not different to COM (p = 0.29, d = 0.62). In the older group, AUC was significantly higher after EAA, compared to RE (p = 0.04, d = 2.36) and tended to be higher in COM (p = 0.06, d = 2.50), with no difference between COM or EAA (p = 0.77, d = 0.20).
Insulin. For the pooled sample, significant main effects of time, condition, and time x condition interaction were found (p < 0.01). In EAA and RE, plasma insulin did not significantly differ from baseline values (9.1 ± 1.7 and 8.3 ± 1.8 µIU/mL, respectively; all p > 0.12). In COM, plasma insulin was significantly elevated at 0 min (17.8 ± 2.0 µIU/mL; p = 0.01; d = 2.79), at 30 min (14.2 ± 1.6 µIU/mL; p = 0.03, d = 2.11) and 60 min (12.7 ± 1.5 µIU/mL; p = 0.04, d = 1.74) postprandially, compared with baseline (6.7 ± 1.6 µIU/mL).
After splitting data by age group, significant main effects were found for age (p = 0.01) and condition (p < 0.001), but not age x condition interaction (p = 0.14). Post-hoc testing revealed no significant change in the younger group in EAA and RE, whereas in COM, insulin was significantly increased immediately post-condition (i.e., 0 min), compared to basal (p = 0.02, d = 3.51). In the older group, insulin was significantly elevated at 0 min (p = 0.01, d = 3.72) and higher at 30 min (p = 0.05, d = 1.91) compared with baseline in COM. Between groups, insulin was significantly greater in the older group at 60 min in EAA (p < 0.001, d = 4.57) and 0 min in COM (p = 0.01, d = 2.63).
Peak insulin concentration differed between conditions in the pooled sample (
p < 0.01). EAA was higher than COM (24.2 ± 2.0 vs. 18.6 ± 1.2 µIU/mL;
p < 0.01,
d = 1.07) and RE (14.7 ± 1.6 µIU/mL;
p < 0.01,
d = 1.64). COM was also significantly higher than RE (
p < 0.01,
d = 0.88). After splitting for age, significant main effects were found for age (
p = 0.006) and condition (
p < 0.001), but there was no age x condition interaction (
p = 0.66;
Table 3). In the young, peak insulin concentration tended to be higher in EAA and COM, compared with RE (both
p = 0.07,
d = 0.96 and
d = 1.98, respectively) but did not differ between COM and EAA (
p = 0.20). In the older group insulin C
max was higher in EAA compared with RE and COM (both
p = 0.03;
d = 2.29 and
d = 2.01, respectively), and higher in COM compared with RE (
p = 0.03,
d = 0.71). In all conditions, insulin C
max was higher in the older group compared with the younger group (all
p < 0.04;
d = 1.59 - 2.10).
No main effects were found for insulin Tmax in the pooled sample (p = 0.87) or age groups (all p > 0.13).
For insulin AUC, significant differences were found between conditions in the pooled sample (p < 0.01). EAA was greater than COM (1927.8 ± 161.8 vs. 1608.3 ± 124.2 µIU/mL; p = 0.03, d = 0.70) and RE (1188.5 ± 132.6 µIU/mL; p < 0.01, d = 1.58), respectively. COM was also greater than RE (p < 0.01, d = 1.03). After splitting groups by age, main effects were found for age (p = 0.03), condition (p < 0.001), and age x condition interaction (p = 0.02). In the younger group, there was a tendency for insulin AUC to be greater in EAA and COM, compared with RE (both p = 0.08; d = 2.60 and d = 2.40, respectively). In the older group, insulin AUC was greater in EAA (p = 0.003, d = 2.23) and COM (p = 0.003, d = 0.84), compared with RE and was greater in EAA compared with COM (p = 0.004, d = 1.38). Insulin AUC was significantly greater in the older group, compared with the younger group in EAA (p = 0.004, d = 3.06) but was not different for RE and COM (both p = 0.26, d = 0.96 and d = 1.07).
Glucose. In pooled data, a significant main effect was found for time (
p < 0.01) but not condition (
p = 0.86) or time x condition interaction (
p = 0.66). However, post-hoc testing revealed no significant changes in plasma glucose between any time point. When data were split by age group (
Table 3), no significant main or interaction effects were found for plasma glucose in time series values, peak concentration, time to peak concentration, or AUC (all
p > 0.20).
3.4. mTORC1 Signaling
The phosphorylation activity of key mTORC1 pathway components in younger and older adults is shown in
Figure 4. For mTOR
Ser2481 phosphorylation, there was a tendency towards a main effect of time (
p = 0.08), a significant main effect for condition (
p = 0.04), and an interaction effect (
p = 0.04). However, post-hoc testing did not reveal any significant differences between EAA (1.05 ± 0.11;
d = 0.13), RE (1.07 ± 0.11;
d = 0.17), and COM (1.74 ± 0.35;
d = 0.77) in relation to PRE (1.00 ± 0.11). When data were split by age group, significant main effects were found for condition (
p = 0.02), but not age (
p = 0.59), or age x condition interaction (
p = 0.36;
Figure 4A). Subsequent post-hoc testing did not detect any significant differences (all
p > 0.25). In the younger group, a large increase following COM was observed (
d = 0.81), but trivial effects after EAA (
d = 0.01) and RE (
d = 0.11), in comparison with baseline. The same pattern was found in the older group, with a large increase following COM (
d = 1.65), but small following EAA (
d = 0.49) and RE (
d = 0.30).
For SK61
Thr421/Ser424 phosphorylation, in the pooled sample there was a main effect of time (
p = 0.01), but not condition (
p = 0.98) or time x condition interaction (
p = 0.98). However, post-hoc testing did not reveal any significant differences (all
p > 0.17) between EAA (1.82 ± 0.41;
d = 0.71), RE (1.74 ± 0.38;
d = 0.68), and COM (1.88 ± 0.40;
d = 0.77) in relation to PRE (1.00 ± 0.16). When data were split by age group, a main effect was found for age (
p = 0.01), but not condition or age x condition interaction (both
p > 0.28;
Figure 4B). Post-hoc testing did not detect any significant differences (all
p > 0.16). However, a large effect was found between age groups after COM (
d = 1.23) and EAA (
d = 0.90), but not RE (
d = 0.27). Within groups, a moderate effect was found after RE in the younger group, compared with baseline (
d = 0.77), with small and trivial effects after EAA (
d = 0.23) and COM (
d = 0.15), respectively. Comparatively, in the older group large effects were found following COM (
d = 1.24) and EAA (
d = 1.07), with moderate after RE (
d = 0.65).
For rpS6
Ser235/236 phosphorylation, in the pooled sample there were significant main effects of time, condition, and time x condition interaction (all
p < 0.001). Compared with PRE (1.00 ± 0.17), rpS6
Ser235/236 phosphorylation was greater after EAA (2.04 ± 0.32;
p = 0.05;
d = 1.08), RE (1.99 ± 0.31;
p = 0.05;
d = 1.06), and COM (8.13 ± 1.32;
p < 0.001;
d = 2.03). COM was also greater compared to RE (
p < 0.01;
d = 1.72) and EAA (
p < 0.01;
d = 1.70). When data were split by age group, a main effect was found for condition (
p < 0.001), but not age or age x condition interaction (both
p > 0.73;
Figure 4C). Post-hoc testing revealed no significant differences between age groups (all
p > 0.52), but a large effect was found between groups following EAA (
d = 0.87), moderate after RE (
d = 0.44), and trivial after COM (
d = 0.02). In the younger group, a large and significant effect was found after COM compared with baseline (
p = 0.05;
d = 2.37), with no other statistically significant effects (
p > 0.30). Large and moderate effects were noted following RE (
d = 1.30) and EAA (
d = 0.50), respectively. In the older group, a large and significant effect was found following EAA (
p = 0.05;
d = 1.99), with no other statistically significant differences (
p > 0.10). However, large effects were observed following COM (
d = 1.70) and RE (
d = 0.79), respectively.
For 4E-BP1
Thr37/46 phosphorylation, in the pooled sample there was a significant main effect of time (
p = 0.05), but not condition (
p = 0.52) or time x condition interaction (
p = 0.52). Post-hoc testing revealed no significant differences (all
p > 0.43) between EAA (1.25 ± 0.16;
d = 0.52), RE (1.50 ± 0.28;
d = 0.64), and COM (2.26 ± 0.81;
d = 0.58) in relation to PRE (1.00 ± 0.08). When data were split by age group, a main effect for age was found (
p = 0.02), but not condition or age x condition interaction (both
p > 0.20;
Figure 4D). Subsequent post-hoc testing did not detect any significant differences (all
p > 0.19). However, large effects were found between age groups following RE (
d = 1.24) and COM (
d = 0.85), with moderate after EAA (
d = 0.76). In the younger group, large effects were found for 4E-BP1
Thr37/46 phosphorylation after RE (
d = 1.21), COM (
d = 0.88) and EAA (
d = 0.82). By comparison, in the older group only trivial effects were found for each condition (
d = 0.13 – 0.18).