Translocation and protein complex co-localization of mTOR is associated with postprandial myofibrillar protein synthesis at rest and after endurance exercise

Abstract

Translocation and colocalization of mechanistic target of rapamycin complex 1 (mTORC1) with regulatory proteins represents a critical step in translation initiation of protein synthesis in vitro. However, mechanistic insight into the control of postprandial skeletal muscle protein synthesis rates at rest and after an acute bout of endurance exercise in humans is lacking. In crossover trials, eight endurance-trained men received primed-continuous infusions of L-[ring-² H₅ ]phenylalanine and consumed a mixed-macronutrient meal (18 g protein, 60 g carbohydrates, 17 g fat) at rest (REST) and after 60 min of treadmill running at 70% VO_2peak (EX). Skeletal muscle biopsies were collected to measure changes in phosphorylation and colocalization in the mTORC1-pathway, in addition to rates of myofibrillar (MyoPS) and mitochondrial (MitoPS) protein synthesis. MyoPS increased (P < 0.05) above fasted in REST (~2.1-fold) and EX (~twofold) during the 300 min postprandial period, with no corresponding changes in MitoPS (P > 0.05). TSC2/Rheb colocalization decreased below fasted at 60 and 300 min after feeding in REST and EX (P < 0.01). mTOR colocalization with Rheb increased above fasted at 60 and 300 min after feeding in REST and EX (P < 0.01), which was consistent with an increased phosphorylation 4E-BP1^Thr37/46 and rpS6^ser240/244 at 60 min. Our data suggest that MyoPS, but not MitoPS, is primarily nutrient responsive in trained young men at rest and after endurance exercise. The postprandial increase in MyoPS is associated with an increase in mTOR/Rheb colocalization and a reciprocal decrease in TSC2/Rheb colocalization and thus likely represent important regulatory events for in vivo skeletal muscle myofibrillar mRNA translation in humans.

Keywords: mTOR; Amino acids; immunofluorescence; mRNA translation; muscle protein synthesis.

Figure 1

Schematic of the experimental infusion protocol during the rest (REST; (A) and exercise (EX; (B) trials. Trials were randomized and counterbalanced. EX trial involved 60 min of treadmill running at 70% VO_2peak. Vertical arrows indicate blood and muscle biopsy sampling points. The drink was a mixed macronutrient beverage that consisted of 18 g of egg protein +60 g of carbohydrates +17 g of fat.

Figure 2

Plasma L‐[ring‐²H₅]phenylalanine enrichment (mole percent excess; MPE) during the REST and EX trials. Circles represent REST, open circles represent EX, gray area represents exercise bout (60 min treadmill running at 70% VO_2peak), and dotted line refers to mixed meal ingestion. Data were analyzed using linear regression. Plasma L‐[ring‐²H₅]phenylalanine: linear regression, P = 0.54; r ² = 0.003. Data presented as mean ± SEM.

Figure 3

Myofibrillar (A) and mitochondrial (B) fractional synthetic rates (FSR) in the fasted state and after meal ingestion at rest (REST) and after 60 min of treadmill running at 70% of VO_2peak (EX) throughout a 0–300 min postprandial period in young men (n = 8). Data were analyzed using a two‐way ANOVA. Data presented as mean ± SEM. *Different from Fasted, P < 0.05.

Figure 4

Immunofluorescence quantification of TSC2 (Red) and Rheb (Green) interaction, displayed as a composite image (Merge) and WGA (Blue). Yellow/orange regions represent TSC2 and Rheb interaction. (A) Each panel represents one subject from REST and EX across the experimental time course. (B) Group data are quantified and reported; circles represent REST, open circles represent EX. All data are presented relative to REST Fasted. Scale bar = 100 μm area. Data were analyzed using a two‐way repeated measures ANOVA. Data presented as mean ± SEM. *Different from Fasted in REST and EX (P < 0.01). ^†Different from REST at same time point (P < 0.001).

Figure 5

Immunofluorescence quantification of mTOR (Red) and Rheb (Green) interaction, displayed as a composite image (Merge) and WGA (Blue). Yellow/orange regions represent mTOR and Rheb interaction. (A) Each panel represents one subject from REST and EX across the experimental time course (B) Group data are quantified and reported; circles represent REST, open circles represent EX. All data presented relative to REST Fasted. Scale bar = 100 μm area. Data were analyzed using a two‐way repeated measures ANOVA. Data presented as mean ± SEM. *Different from Fasted in REST and EX (P < 0.01).

Figure 6

Immunofluorescence quantification of TSC2 (Red) and WGA (Blue) interaction, displayed as individual images. (A) Each panel represents one subject from REST and EX across the experimental time course. (B) Group data are quantified and reported. Immunofluorescence quantification of mTOR (Red) and WGA (Blue) interaction, displayed as individual images (C). Each panel represents one subject from REST and EX across the experimental time course (D). Group data are quantified and reported; circles represent REST, open circles represent EX. All data presented relative to REST Fasted. Scale bar = 100 μm. Data were analyzed using a two‐way repeated measures ANOVA. Data presented as mean ± SEM. *Different from Fasted in REST and EX (P < 0.01).

Figure 7

Change in (a) 4E‐BP1^Thr37/46 and (b) rpS6^ser240/244 phosphorylation before (Fasted) and after meal ingestion at rest (REST) and after 60 min of treadmill running at 70% of VO_2peak (EX) in young men (n = 8). Data were analyzed using a two‐way repeated‐measures ANOVA. Data are expressed in arbitrary units (AU). Data presented as mean ± SEM. *Different from Fasted, P < 0.01.