Differential localization and anabolic responsiveness of mTOR complexes in human skeletal muscle in response to feeding and exercise

Abstract

Mechanistic target of rapamycin (mTOR) resides as two complexes within skeletal muscle. mTOR complex 1 [mTORC1-regulatory associated protein of mTOR (Raptor) positive] regulates skeletal muscle growth, whereas mTORC2 [rapamycin-insensitive companion of mTOR (Rictor) positive] regulates insulin sensitivity. To examine the regulation of these complexes in human skeletal muscle, we utilized immunohistochemical analysis to study the localization of mTOR complexes before and following protein-carbohydrate feeding (FED) and resistance exercise plus protein-carbohydrate feeding (EXFED) in a unilateral exercise model. In basal samples, mTOR and the lysosomal marker lysosomal associated membrane protein 2 (LAMP2) were highly colocalized and remained so throughout. In the FED and EXFED states, mTOR/LAMP2 complexes were redistributed to the cell periphery [wheat germ agglutinin (WGA)-positive staining] (time effect; P = 0.025), with 39% (FED) and 26% (EXFED) increases in mTOR/WGA association observed 1 h post-feeding/exercise. mTOR/WGA colocalization continued to increase in EXFED at 3 h (48% above baseline) whereas colocalization decreased in FED (21% above baseline). A significant effect of condition (P = 0.05) was noted suggesting mTOR/WGA colocalization was greater during EXFED. This pattern was replicated in Raptor/WGA association, where a significant difference between EXFED and FED was noted at 3 h post-exercise/feeding (P = 0.014). Rictor/WGA colocalization remained unaltered throughout the trial. Alterations in mTORC1 cellular location coincided with elevated S6K1 kinase activity, which rose to a greater extent in EXFED compared with FED at 1 h post-exercise/feeding (P < 0.001), and only remained elevated in EXFED at the 3 h time point (P = 0.037). Collectively these data suggest that mTORC1 redistribution within the cell is a fundamental response to resistance exercise and feeding, whereas mTORC2 is predominantly situated at the sarcolemma and does not alter localization.

Keywords: Raptor; Rictor; lysosome; mTORC1; mTORC2.

Fig. 1.

Rictor and Raptor antibody validation and ribosomal protein S6 kinase 1 (S6K1) and Akt kinase activity. A–C: immunofluorescent staining of each protein was performed in muscle-specific knockout (mKO) and littermate wild-type (WT) samples, in addition to staining of each sample with primary antibodies omitted (CON). Rictor/Raptor is displayed in green and wheat germ agglutinin (WGA; cell membrane) is stained in blue. Representative images of staining in each condition are displayed (A) alongside the corresponding quantification for Rictor (B) and Raptor (C). Scale bars, 50 µm. Data presented as means ± SE. *Significantly different WT (P < 0.001). D and E: S6K1 (D) and Akt (E) kinase activity following unilateral resistance exercise and/or protein-carbohydrate feeding (FED). Black bars denote FED condition and open bars denote resistance exercise plus protein-carbohydrate feeding (EXFED) condition. Data presented as means ± SE. *Significantly different from baseline (P < 0.05), ^¥significant difference between conditions at this time point (P < 0.001).

Fig. 2.

The effect of resistance exercise and/or protein carbohydrate feeding on mechanistic target of rapamycin (mTOR)-lysosomal associated membrane protein 2 (LAMP2) and mTOR-WGA colocalization. A: representative images of mTOR-LAMP2 and mTOR-WGA colocalization at rest, and following resistance exercise and/or protein-carbohydrate feeding. Orange/yellow regions denote areas of mTOR localization with the marker of the lysosome in images on the top row. mTOR-positive staining is shown in red, LAMP2-positive in green, and WGA-positive in blue. B–D: quantification of LAMP2 fluorescence intensity (B), mTOR-LAMP2 colocalization (C), and mTOR-WGA colocalization (D) at each time point. Scale bars, 50 µm. Data presented as means ± SE. ^¥Significant difference between conditions at this time point (P < 0.05), ^#significantly different compared with baseline when conditions combined (P = 0.008).

Fig. 3.

The effect of resistance exercise and/or protein carbohydrate feeding on Rictor-mTOR and Rictor-WGA colocalization. A: representative images of Rictor-mTOR and Rictor-WGA colocalization at rest, and following resistance exercise and/or protein-carbohydrate feeding. Orange/yellow regions denote areas of Rictor localization with mTOR on top row. mTOR-positive staining is shown in red, Rictor-positive in green, and WGA-positive in blue. B and C: quantification of Rictor-mTOR (B) and Rictor-WGA (C) colocalization at each time point. Scale bars, 50 µm. Data presented as means ± SE.

Fig. 4.

The effect of resistance exercise and/or protein carbohydrate feeding on Raptor-mTOR and Raptor-WGA colocalization. A: representative images of Raptor-mTOR and Raptor-WGA colocalization at rest, and following resistance exercise and/or protein-carbohydrate feeding. Orange/yellow regions denote areas of Raptor localization with mTOR. mTOR-positive staining is shown in red, Raptor-positive in green, and WGA-positive in blue B and C: quantification of Raptor-mTOR (B) and Raptor-WGA (C) colocalization at each time point. Scale bar, 50 µm. Data presented as means ± SE. ^¥Significant difference between conditions at this time point (P = 014), ^#significantly different compared with baseline when conditions combined (P = 0.007).