PKCε Regulates Contraction-Stimulated GLUT4 Traffic In C2C12 Skeletal Muscle Cells
Bibliographic record
Abstract
The signaling pathways that stimulate glucose uptake in response to muscle contraction are not well defined. We recently established an in vitro cellular model to study GLUT4 traffic using nicotinic acetylcholine receptor-mediated contraction of cultured C2C12 skeletal muscle myotubes expressing a myc-tagged GLUT4 (GLUT4myc) reporter (Niu W., et al AJP-Endocrinol Metab 298:E1058-1071). We osbserved the cell-surface increase of GLUT4 was stimulated by carbachol and sensitive to inhibition by ·-bungarotoxin and not atropine and only partly required AMPK activation. PURPOSE: To search for AMPK-independent pathways which regulate contraction-stimulated GLUT4 traffic. METHODS: C2C12 myotubes were treated siRNA targeting PKCε (72 h) or with chemical inhibitors of novel and/or conventional PKC or a peptidic myristoylated translocation inhibitor (Myr- EAVSLKPT) of PKCε (30 min) prior to 20 min stimulation with the acetylcholine analog, carbachol (100 μM), followed by measurement of cell-surface GLUT4myc levels, signal transduction or membrane translocation of PKC isoforms. RESULTS: Cell surface carbachol-induced GLUT4myc levels were partly inhibited by the conventional/novel PKC inhibitors GF-109203X, Gö6983, and Ro-31-8425 but not by the conventional PKC inhibitor Gö6976. However, PMA-stimulated GLUT4myc traffic to the cell surface was fully inhibited by the novel/conventional PKC inhibitors. C2C12 myotubes express several novel isoforms of PKC with PKCδ and PKCε in greatest abundance. Carbachol stimulated the PKC phosphorylation of and translocation of PKCδand PKCε to membranes from the cytosol within 5 min. However, only a peptidic inhibitor of PKCε translocation, but not one of PKCδ(Myr-SFNSYELGSL), prevented the GLUT4myc response to carbachol. Lastly, siRNA-mediated PKCε protein knockdown inhibited the carbachol-induced gain of GLUT4myc at the cell surface by approximately 50%. CONCLUSIONS: PMA-stimulated GLUT4myc traffic to the cell surface provided proof-of-concept that PKC can regulate GLUT4 traffic. Our findings with carbachol-induced contractions of C2C12 myotubes, supports a role for novel PKC isoforms, especially PKCε, in contraction-stimulated GLUT4 traffic in muscle cells. Supported by a joint grant from CIHR-NSFC to AK (# FRN-82420) and WN (# 30611120532) and by a NSFC grant (# 30570912) to WN.
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How this classification was reachedexpand
Full frame machine prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. The Gemma side is a direct model label for every work in the frame, read from the title-only record. The Codex side is a classifier learned from the 10,348 direct Codex labels and calibrated to design-weighted sample rates; fields without enough sample support carry no Codex call. Candidate is the union of the two sides; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels.
Distilled classifier scores by category (both heads)
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.000 | 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) | 0.001 | 0.001 |
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 source (direct Gemma or distilled Codex), 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".