Protein blend and casein supplementations before inactive phase similarly activate mechanistic target of rapamycin signaling in rat skeletal muscle

Following anabolic stimuli (e.g. mechanical loading and/or amino acid provision) the mechanistic target of rapamycin complex 1 (mTORC1), a master regulator of protein synthesis, translocates toward the cell periphery. However, it is unknown if mTORC1 activity occurs prior to or following this translocation. We therefore aimed to determine the cellular location of mTORC1 activity in human skeletal muscle following anabolic stimuli. Fourteen young, healthy males either ingested a protein-carbohydrate beverage (0.25g/kg protein, 0.75g/kg carbohydrate) alone (n=7, 23±5yrs, 76.8±3.6kg, 13.6±3.8%BF, FED) or following a whole-body resistance exercise bout (n=7, 22±2yrs, 78.1±3.6kg, 12.2±4.9%BF, EXFED). Vastus lateralis muscle biopsies were obtained at rest (PRE) and 120 and 300min following anabolic stimuli. The spatial regulation of mTORC1 activity was assessed through immunofluorescent staining of p-RPS6Ser240/244, an mTORC1-specific phosphorylation event. p-RPS6Ser240/244 measured by immunofluorescent staining or immunoblot was positively correlated (r=0.76, p<0.001). Peripheral staining intensity of p-RPS6Ser240/244 increased above PRE in both FED and EXFED at 120min (~54% and ~138% respectively, p<0.05) but was greater in EXFED at both post-stimuli time points (p<0.05). The peripheral-central ratio of p-RPS6240/244 staining was displayed a similar pattern, suggesting mTORC1 activity occurs predominantly in the periphery of fibers. Moreover, p-RPS6Ser240/244 intensity within paxillin-positive regions, a marker of focal adhesion complexes, was elevated at 120min irrespective of stimulus (p=0.006) before returning to PRE at 300min. These data confirm that mTORC1 activity occurs in the region of human muscle fibers to which mTORC1 translocates following anabolic stimuli and identifies focal adhesion complexes as a potential site of mTORC1 activation in vivo.

Download Full-text

Resistance exercise initiates mechanistic target of rapamycin (mTOR) translocation and protein complex co-localisation in human skeletal muscle

Scientific Reports ◽

10.1038/s41598-017-05483-x ◽

2017 ◽

Vol 7 (1) ◽

Cited By ~ 39

Author(s):

Zhe Song ◽

Daniel R. Moore ◽

Nathan Hodson ◽

Carl Ward ◽

Jessica R. Dent ◽

...

Keyword(s):

Skeletal Muscle ◽

Resistance Exercise ◽

Protein Complex ◽

Human Skeletal Muscle ◽

Target Of Rapamycin ◽

Mechanistic Target Of Rapamycin

Download Full-text

Differential regulation of mTORC1 activation by leucine and β-hydroxy-β-methylbutyrate in skeletal muscle of neonatal pigs

Journal of Applied Physiology ◽

10.1152/japplphysiol.00332.2019 ◽

2020 ◽

Vol 128 (2) ◽

pp. 286-295 ◽

Cited By ~ 3

Author(s):

Agus Suryawan ◽

Marko Rudar ◽

Marta L. Fiorotto ◽

Teresa A. Davis

Keyword(s):

Skeletal Muscle ◽

Protein Synthesis ◽

Nutrient Sensing ◽

Target Of Rapamycin ◽

Neonatal Pigs ◽

Mechanistic Target Of Rapamycin ◽

Mtor Phosphorylation ◽

Dependent Protein ◽

Mtorc1 Activation ◽

Mtor Complex 1

Leucine (Leu) and its metabolite β-hydroxy-β-methylbutyrate (HMB) stimulate mechanistic target of rapamycin (mTOR) complex 1 (mTORC1)-dependent protein synthesis in the skeletal muscle of neonatal pigs. This study aimed to determine whether HMB and Leu utilize common nutrient-sensing mechanisms to activate mTORC1. In study 1, neonatal pigs were fed one of five diets for 24 h: low protein (LP), high protein (HP), or LP supplemented with 4 (LP+HMB4), 40 (LP+HMB40), or 80 (LP+HMB80) μmol HMB·kg body wt−1·day−1. In study 2, neonatal pigs were fed for 24 h: LP, LP supplemented with Leu (LP+Leu), or HP diets delivering 9, 18, and 18 mmol Leu·kg body wt−1·day−1, respectively. The upstream signaling molecules that regulate mTORC1 activity were analyzed. mTOR phosphorylation on Ser2448 and Ser2481 was greater in LP+HMB40, LP+HMB80, and LP+Leu than in LP and greater in HP than in HMB-supplemented groups ( P < 0.05), whereas HP and LP+Leu were similar. Rheb-mTOR complex formation was lower in LP than in HP ( P < 0.05), with no enhancement by HMB or Leu supplementation. The Sestrin2-GATOR2 complex was more abundant in LP than in HP and was reduced by Leu ( P < 0.05) but not HMB supplementation. RagA-mTOR and RagC-mTOR complexes were higher in LP+Leu and HP than in LP and HMB groups ( P < 0.05). There were no treatment differences in RagB-SH3BP4, Vps34-LRS, and RagD-LRS complex abundances. Phosphorylation of Erk1/2 and TSC2, but not AMPK, was lower in LP than HP ( P < 0.05) and unaffected by HMB or Leu supplementation. Our results demonstrate that HMB stimulates mTORC1 activation in neonatal muscle independent of the leucine-sensing pathway mediated by Sestrin2 and the Rag proteins. NEW & NOTEWORTHY Dietary supplementation with either leucine or its metabolite β-hydroxy-β-methylbutyrate (HMB) stimulates protein synthesis in skeletal muscle of the neonatal pig. Our results demonstrate that both leucine and HMB stimulate mechanistic target of rapamycin (mTOR) complex 1 (mTORC1) phosphorylation in neonatal muscle. This leucine-stimulated process involves dissociation of the Sestrin2-GATOR2 complex and increased binding of Rag A/C to mTOR. However, HMB’s activation of mTORC1 is independent of this leucine-sensing pathway.

Download Full-text

Assessing the mechanistic target of rapamycin complex-1 pathway in response to resistance exercise and feeding in human skeletal muscle by multiplex assay

Applied Physiology Nutrition and Metabolism ◽

10.1139/apnm-2017-0852 ◽

2018 ◽

Vol 43 (9) ◽

pp. 945-949

Author(s):

Chris McGlory ◽

Everson A. Nunes ◽

Sara Y. Oikawa ◽

Evangelia Tsakiridis ◽

Stuart M. Phillips

Keyword(s):

Skeletal Muscle ◽

Resistance Exercise ◽

Vastus Lateralis ◽

Multiplex Assay ◽

Target Of Rapamycin ◽

Leg Extension ◽

Signalling Molecules ◽

Mechanistic Target Of Rapamycin ◽

Phosphorylated Akt ◽

Protein Feeding

The mechanistic target of rapamycin complex-1 (mTORC-1) is a key nutrient and contraction-sensitive protein that regulates a pathway leading to skeletal muscle growth. Utilizing a multiplex assay, we aimed to examine the phosphorylation status of key mTORC-1-related signalling molecules in response to protein feeding and resistance exercise. Eight healthy men (age, 22.5 ± 3.1 years; mass, 80 ± 9 kg; 1-repetition maximum leg extension, 87 ± 5 kg) performed 4 sets of unilateral leg extensions until volitional failure. Immediately following the final set, all participants consumed a protein-enriched beverage. A single skeletal muscle biopsy was obtained from the vastus lateralis before (Pre) with further bilateral biopsies at 1 h (1 h exercised legs (FEDEX) and 1 h nonexercised legs (FED)) and 3 h (3 h FEDEX and 3 h FED) after drink ingestion. Phosphorylated AktSer473 was significantly elevated from Pre at 1 h FEDEX. Phosphorylated p70S6K1Thr412 was significantly increased above Pre at 1 h FEDEX and 1 h FED and was still significantly elevated at 3 h FEDEX but not 3 h FED. Phosphorylated rpS6Ser235/236 was also significantly increased above Pre at 1 h FEDEX and 1 h FED with 1 h FEDEX greater than 1 h FED. Our data highlight the utility of a multiplex assay to assess anabolic signalling molecules in response to protein feeding and resistance exercise in humans. Importantly, these changes are comparable with those as previously reported using standard immunoblotting and protein activity assays.

Download Full-text