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What is the central question of this study? Are myofibre protein signalling responses to ex vivo dynamic contractions altered by accustomization to voluntary endurance training in rats? What is the main finding and its importance? In response to ex vivo dynamic muscle contractions, canonical myofibre protein signalling pertaining to metabolic transcriptional regulation, as well as translation initiation and elongation, was not influenced by prior accustomization to voluntary endurance training in rats. Accordingly, intrinsic myofibre protein signalling responses to standardized contractile activity may be independent of prior exercise training in rat skeletal muscle.
Skeletal muscle training status may influence myofibre regulatory protein signalling in response to contractile activity. The current study employed a purpose-designed ex vivo dynamic contractile protocol to evaluate the effect of exercise-accustomization on canonical myofibre protein signalling for metabolic gene expression and for translation initiation and elongation. To this end, rats completed 8 weeks of in vivo voluntary running training versus no running control intervention, whereupon an ex vivo endurance-type dynamic contraction stimulus was conducted in isolated soleus muscle preparations from both intervention groups. Protein signalling response by phosphorylation was evaluated by immunoblotting at 0 and 3 h following ex vivo stimulation. Phosphorylation of AMP-activated protein kinase α-isoforms and its downstream target, acetyl-CoA carboxylase, as well as phosphorylation of eukaryotic elongation factor 2 (eEF2) was increased immediately following the dynamic contraction protocol (at 0 h). Signalling for translation initiation and elongation was evident at 3 h after dynamic contractile activity, as evidenced by increased phosphorylation of p70 S6 kinase and eukaryotic translation initiation factor 4E-binding protein 1, as well as a decrease in phosphorylation of eEF2 back to resting control levels. However, prior exercise training did not alter phosphorylation responses of the investigated signalling proteins. Accordingly, protein signalling responses to standardized endurance-type contractions may be independent of training status in rat muscle during ex vivo conditions. The present findings add to our current understanding of molecular regulatory events responsible for skeletal muscle plasticity.

Performing resistance exercise with heavier loads is often proposed to be necessary for the recruitment of larger motor units and activation of type II muscle fibres, leading to type II fibre hypertrophy. Indirect measures [surface electromyography (EMG)] have been used to support this thesis, although we propose that lighter loads lifted to task failure (i.e. volitional fatigue) result in the similar activation of type II fibres. In the present study, participants performed resistance exercise to task failure with heavier and lighter loads with both a normal and longer repetition duration (i.e. time under tension). Type I and type II muscle fibre glycogen depletion was determined by neither load, nor repetition duration during resistance exercise performed to task failure. Surface EMG amplitude was not related to muscle fibre glycogen depletion or anabolic signalling; however, muscle fibre glycogen depletion and anabolic signalling were related. Performing resistance exercise to task failure, regardless of load lifted or repetition duration, necessitates the activation of type II muscle fibres.
Heavier loads (>60% of maximal strength) are considered to be necessary during resistance exercise (RE) to activate and stimulate hypertrophy of type II fibres. Support for this proposition comes from observation of higher surface electromyography (EMG) amplitudes during RE when lifting heavier vs. lighter loads. We aimed to determine the effect of RE, to task failure, with heavier vs. lighter loads and shorter or longer repetition durations on: EMG-derived variables, muscle fibre activation, and anabolic signalling. Ten recreationally-trained young men performed four unilateral RE conditions randomly on two occasions (two conditions, one per leg per visit). Muscle biopsies were taken from the vastus lateralis before and one hour after RE. Broadly, total time under load, number of repetitions, exercise volume, EMG amplitude (at the beginning and end of each set) and total EMG activity were significantly different between conditions (P < 0.05); however, neither glycogen depletion (in both type I and type II fibres), nor phosphorylation of relevant signalling proteins showed any difference between conditions. We conclude that muscle fibre activation and subsequent anabolic signalling are independent of load, repetition duration and surface EMG amplitude when RE is performed to task failure. The results of the present study provide evidence indicating that type I and type II fibres are activated when heavier and lighter loads are lifted to task failure. We propose that our results explain why RE training with higher or lower loads, when loads are lifted to task failure, leads to equivalent muscle hypertrophy and occurs in both type I and type II fibres.

Cellular stress or injury induces release of endogenous danger signals such as ATP, which plays a central role in activating immune cells. ATP is essential for the release of nonclassically secreted cytokines such as IL-1β but, paradoxically, has been reported to inhibit the release of classically secreted cytokines such as TNF. Here, we reveal that ATP does switch off soluble TNF (17 kDa) release from LPS-treated macrophages, but rather than inhibiting the entire TNF secretion, ATP packages membrane TNF (26 kDa) within microvesicles (MVs). Secretion of membrane TNF within MVs bypasses the conventional endoplasmic reticulum- and Golgi transport-dependent pathway and is mediated by acid sphingomyelinase. These membrane TNF-carrying MVs are biologically more potent than soluble TNF in vivo, producing significant lung inflammation in mice. Thus, ATP critically alters TNF trafficking and secretion from macrophages, inducing novel unconventional membrane TNF signaling via MVs without direct cell-to-cell contact. These data have crucial implications for this key cytokine, particularly when therapeutically targeting TNF in acute inflammatory diseases.-Soni, S., O\'Dea, K. P., Tan, Y. Y., Cho, K., Abe, E., Romano, R., Cui, J., Ma, D., Sarathchandra, P., Wilson, M. R., Takata, M. ATP redirects cytokine trafficking and promotes novel membrane TNF signaling via microvesicles.

The phosphoinositide-3-kinase (PI3K) pathway is the most commonly activated pathway in cancers due to mutations at multiple nodes and loss of PTEN. Furthermore, in endometrial cancer (EC), PI3K and RAS/RAF/MEK/MAPK (RAS/MAPK herein) pathway mutations frequently co-exist. We examined the role of PI3K and RAS/MAPK pathway mutations in determining responsiveness to therapies targeted to these pathways in vitro in EC.
13 EC cell lines were profiled for their PI3K pathway and KRAS mutational and PTEN protein status and treated with one MEK- and two PI3K- targeted inhibitors alone and in combination. Expression and phosphorylation of 66 proteins were evaluated by Reverse-Phase-Protein-Array (RPPA) in 6 EC cell lines to identify signalling changes in these pathways in response to therapy.
PTEN protein loss and the absence of any tested pathway mutations are dominant negative predictors of sensitivity to MEK inhibition. KRAS-mutated cells were most sensitive to MEK inhibition, but significantly more resistant to PI3K inhibition than KRAS-wild-type cell lines. Combinations of PI3K and MEK inhibitors showed synergy or additivity in all but two cell lines tested. Treatment of KRAS-mutated cells with PI3K inhibitors and treatment of PTEN-low cells with a MEK inhibitor were most likely to induce activation of MEK/MAPK and AKT, respectively, likely indicative of feedback-loop regulation.
MEK inhibition may be a promising treatment modality, not just for ECs with mutated KRAS, but also for those with retained PTEN. Up-regulation of MEK/MAPK signalling by PI3K inhibition, and up-regulation of AKT activation by MEK inhibition may serve as potential biomarkers of likely responsiveness to each inhibitor.