Leucine-enriched essential amino acid and carbohydrate ingestion following resistance exercise enhances mTOR signaling and protein synthesis in human muscle

Abstract

We recently showed that resistance exercise and ingestion of essential amino acids with carbohydrate (EAA+CHO) can independently stimulate mammalian target of rapamycin (mTOR) signaling and muscle protein synthesis in humans. Providing an EAA+CHO solution postexercise can further increase muscle protein synthesis. Therefore, we hypothesized that enhanced mTOR signaling might be responsible for the greater muscle protein synthesis when leucine-enriched EAA+CHOs are ingested during postexercise recovery. Sixteen male subjects were randomized to one of two groups (control or EAA+CHO). The EAA+CHO group ingested the nutrient solution 1 h after resistance exercise. mTOR signaling was assessed by immunoblotting from repeated muscle biopsy samples. Mixed muscle fractional synthetic rate (FSR) was measured using stable isotope techniques. Muscle protein synthesis and 4E-BP1 phosphorylation during exercise were significantly reduced (P < 0.05). Postexercise FSR was elevated above baseline in both groups at 1 h but was even further elevated in the EAA+CHO group at 2 h postexercise (P < 0.05). Increased FSR was associated with enhanced phosphorylation of mTOR and S6K1 (P < 0.05). Akt phosphorylation was elevated at 1 h and returned to baseline by 2 h in the control group, but it remained elevated in the EAA+CHO group (P < 0.05). 4E-BP1 phosphorylation returned to baseline during recovery in control but became elevated when EAA+CHO was ingested (P < 0.05). eEF2 phosphorylation decreased at 1 and 2 h postexercise to a similar extent in both groups (P < 0.05). Our data suggest that enhanced activation of the mTOR signaling pathway is playing a role in the greater synthesis of muscle proteins when resistance exercise is followed by EAA+CHO ingestion.

Figures

Fig. 1

Study design and time line. Schematics display the study design used to measure the combined effect of resistance exercise plus leucine-enriched essential amino acids with carbohydrate (EAA+CHO) solution on the regulation of muscle protein synthesis in human subjects. The study design consisted of a basal period (hours 2–3), an exercise period (hours 3–4), and 2 postexercise periods (hours 4–5 and 5–6, respectively). Marking the beginning of the second hour postexercise, an EAA+CHO solution was ingested by each subject. Indocyanine green (ICG) was infused to measure blood flow in each of the periods. Blood samples were collected to measure blood glucose uptake, lactate, and pH. Muscle biopsies were used to measure muscle protein synthesis and components of the mammalian target of rapamycin (mTOR) signaling pathway involved in translation initiation and elongation.

Fig. 2

Muscle protein synthesis (fractional synthesis rate, FSR) and net balance across the leg. All subjects were treated identically during baseline, exercise, and 1 h postexercise (1 h post). One-half of the subjects ingested a nutrient solution at the end of the first hour of postexercise recovery [2 h post (EAA+CHO)] compared with control subjects [2 h post (control)]. A: FSR increased in all subjects following exercise but was further increased with the addition of a leucine-enriched EAA+CHO solution (EAA + CHO). B: net balance across the leg, comparing baseline with control and subjects ingesting the leucine-enriched EAA+CHO solution. Data are means ± SE; n = 13 (baseline), n = 7 (exercise alone), n = 6 (exercise and EAA+CHO). *P < 0.05 vs. baseline. #P < 0.05 vs. 2 h post (control).

Fig. 3

Akt and TSC2. All subjects were treated identically during baseline, exercise, and 1 h postexercise. One-half of the subjects ingested a nutrient solution at the end of the first hour of postexercise recovery [2 h post (EAA+CHO)] compared with control subjects [2 h post (control)]. Data are from each biopsy taken at baseline, immediately following resistance exercise (exercise), 1 h following exercise (1 h post), and 2 h following resistance exercise [2 h post (control) and 2 h post (EAA + CHO)]. A: Akt phosphorylation and total Akt. B: TSC2 phosphorylation and total TSC2. Data are means ± SE in arbitrary units (AU). *P < 0.05 vs. baseline. #P < 0.05 vs. 2 h post (control). Insets show representative blots for the loading control (LC) and for each time point. B, baseline; Ex, exercise alone; Con, control.

Fig. 4

mTOR and the eukaryotic initiation factor 4E-binding protein (4E-BP1). All subjects were treated identically during baseline, exercise, and 1 h postexercise. One-half of the subjects ingested a nutrient solution at the end of the first hour of postexercise recovery [2 h post (EAA+CHO)] compared with control subjects [2 h post (control)]. A: mTOR phosphorylation and total mTOR. B: 4E-BP1 phosphorylation and total 4E-BP1. Data are means ± SE. *P < 0.05 vs. baseline. #P < 0.05 vs. 2 h post (control). Insets show representative blots for the loading control and for each time point.

Fig. 5

p70 ribosomal S6 kinase 1 (S6K1) and eukaryotic elongation factor-2 (eEF2). All subjects were treated identically during baseline, exercise, and 1 h postexercise. One-half of the subjects ingested a leucine-enriched EAA+CHO solution at the end of the first hour of postexercise recovery [2 h post (EAA+CHO)] compared with control subjects [2 h post (control)]. A: S6K1 phosphorylation and total S6K1. B: eEF2 phosphorylation and total eEF2. Data are means ± SE. *P < 0.05 vs. baseline. Insets show representative blots for the loading control and for each time point.