mTORC1 Activity Occurs Predominantly in the Periphery of Human Skeletal Muscle Fibers Following Anabolic Stimuli
Bibliographic record
Abstract
The mechanistic target of rapamycin complex 1 (mTORC1) is regarded as the master regulator of protein synthesis in skeletal muscle and is essential for optimal responses to acute anabolic stimuli. We have shown mTORC1 to translocate toward the cell periphery where it colocalizes with upstream activators, downstream effectors and the microvasculature after protein feeding and muscle contraction. These events occur at similar times to elevated whole muscle mTORC1 kinase activity and protein synthesis; however, whether mTORC1 activity occurs in a region‐specific pattern is yet to be studied. Therefore, the aim of the current study was to develop an immunofluorescent staining (IF) protocol to measure mTORC1 activation in a region‐specific manner following anabolic stimuli in human skeletal muscle. Fourteen young, healthy males (22±4yrs, 14±4% body fat, mean±SD) participated in the study and were randomized to complete trials where a protein‐carbohydrate beverage was ingested at rest (FED, n=7) or following a whole‐body resistance exercise session (EXFED, n=7). Vastus lateralis biopsy samples were obtained prior to feeding/exercise (basal) and 120 and 300min following anabolic stimuli. Phosphorylated ribosomal protein S6 Ser240/244 (p‐RPS6 Ser240/244 ) was chosen as the marker of mTORC1 activation as this phosphorylation event is rapamycin‐sensitive. We first optimized and validated an IF protocol for p‐RPS6 Ser240/244 through the omission of primary and secondary antibodies. Using this protocol, we then showed that RPS6 Ser240/244 phosphorylation was elevated at 120min (p<0.04) with EXFED being greater than FED (p=0.02). p‐RPS6 Ser240/244 remained elevated above basal values at 300min in EXFED only (p=0.04). Importantly, a strong positive correlation was observed between RPS6 Ser240/244 phosphorylation measured by immunoblotting and IF (r=0.85, p<0.001) showing our novel IF method is a valid readout of mTORC1 activity. In central regions of skeletal muscle fibers, p‐RPS6 Ser240/244 phosphorylation was only elevated in EXFED at 120min. In the outer 5.5µm of fibers (periphery), mTORC1 activity increased at 120min in both conditions (p<0.03) but to a greater extent in EXFED (p=0.002), remaining elevated above basal levels at 300min only in EXFED (p=0.02). Similarly, the peripheral‐to‐central ratio of RPS6 Ser240/244 phosphorylation was elevated above basal values in both conditions at 120min and remained elevated only in EXFED at 300min, suggesting mTORC1 activity occurs to a greater extent in the periphery of skeletal muscle fibers. In conclusion, we have optimized and validated an IF technique to investigate mTORC1 activity in a region‐specific manner in human skeletal muscle. We show that, following physiological anabolic stimuli, mTORC1 activity occurs to a greater extent in the periphery of fibers, regions where mTORC1 is commonly visualized in humans and protein synthesis is observed in animal models. These findings extend our knowledge regarding the spatial regulation of mTORC1 in human skeletal muscle and provide a novel method to investigate mTORC1 dynamics in various populations.
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How this classification was reachedexpand
Full frame distilled prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. Learned from the 10,348 direct Codex labels and 10,348 direct Gemma labels. Candidate is the union of thresholded teacher heads; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels or direct frontier model labels.
Codex and Gemma teacher scores by category
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.002 | 0.000 |
| Meta-epidemiology (narrow) | 0.000 | 0.000 |
| Meta-epidemiology (broad) | 0.000 | 0.001 |
| Bibliometrics | 0.000 | 0.000 |
| Science and technology studies | 0.000 | 0.000 |
| Scholarly communication | 0.000 | 0.000 |
| Open science | 0.000 | 0.000 |
| Research integrity | 0.000 | 0.001 |
| Insufficient payload (model declined to judge) | 0.000 | 0.000 |
Machine scores (provisional)
The two teacher heads of the student model, read on this work. A score orders the frame for review; it never asserts a category, and the validation status ships verbatim with every row.
Baseline scores from an immature model (maturity gate not passed, 7 training rounds). Scores rank; they never assert a category.
score_only:v0-immature-baseline · verbatim from the scoring run: score_only means the number may rank works, and no category label ships from itClassification
machine, unvalidatedMachine predicted; a candidate call from one teacher head, not a consensus.
How this classification was reached, model by model and score by score, is at the end of the page under "How this classification was reached".