The prevalence of type 2 diabetes (T2D) continues to rise despite the amount of research dedicated to finding the culprits of this debilitating disease. Skeletal muscle is arguably the most important contributor to glucose disposal making it a clear target in insulin resistance and T2D research. Within skeletal muscle there is a clear link to metabolic dysregulation during the progression of T2D but the determination of culprits vs consequences of the disease has been elusive. Emerging evidence in skeletal muscle implicates influential cross talk between a key anabolic regulatory protein, the mammalian target of rapamycin (mTOR) and its associated complexes (mTORC1 and mTORC2), and the well-described canonical signaling for insulin-stimulated glucose uptake. This new understanding of cellular signaling crosstalk has blurred the lines of what is a culprit and what is a consequence with regard to insulin resistance. Here, we briefly review the most recent understanding of insulin signaling in skeletal muscle, and how anabolic responses favoring anabolism directly impact cellular glucose disposal. This review highlights key cross-over interactions between protein and glucose regulatory pathways and the implications this may have for the design of new therapeutic targets for the control of glucoregulatory function in skeletal muscle.

The mechanistic target of rapamycin (mTOR) is a serine/threonine protein kinase with a central role in controlling cellular anabolism. By phosphorylating two downstream targets, p70‐S6 kinase (p70‐S6K) and eIF4E binding protein 1 (4EBP1), mTOR induces an increase in mRNA translation, leading to an upregulation of protein synthesis. In muscle tissue, mTOR activation in response to an exercise stimulus is a key signaling event in the anabolic response to physical activity. As studying the signaling response to exercise at the cellular level is challenging in whole organisms due to difficulty in isolating muscle cell level effects, development of a cell culture model mimicking exercise would provide an avenue for understanding the intracellular cellular signaling response to exercise in skeletal muscle. The aim of this study was to determine whether 12 hours of electrical pulse stimulation (EPS) would alter the anabolic response in L6 myotubes. METHODS Murine L6 myoblasts were cultured on 10 cm plates in standard culture medium supplemented with 1% penicillin/streptomycin, and 10% fetal bovine serum until 60% confluence. At this point, differentiation was induced by switching to medium without sodium pyruvate supplemented with 1% penicillin/streptomycin, 2% horse serum, and 0.5% insulin‐transferrin‐selenium (ITS). After 7 days of differentiation, myotubes were stimulated, in culture, for 12 hours at a low frequency (1 Hz, 2ms, 60V). Differentiation media was changed in all plates immediately before stimulation and immediately after stimulation in half of the plates. Half of the cells were harvested immediately after stimulation to assess the acute effects and the remaining plates were harvested 24 hours post stimulation and re‐feeding. Protein expression of common anabolic markers was measured using western blot techniques. RESULTS Our results indicate an increase in phosphorylated 4EBP1 immediately after stimulation (EPS‐Ea) compared to control (CON‐Ea) (p = 0.05). Additionally, phosphorylation levels return to baseline 24 hours post stimulation. In contrast, phosphorylation levels of p70‐S6K immediately after stimulation (EPS‐Ea) were not different from control (CON‐Ea) but were elevated 24 hours post stimulation (EPS‐La) (p = 0.09). Finally, we determined the ratio of phosphorylated to total levels of 4EBP1 were two‐fold greater immediately after stimulation (EPS‐Ea) compared to 24 hours post stimulation (EPS‐La) (p = 0.09). Conversely, the ratio of phosphorylated to total levels of p70‐S6K were two times greater 24 hours post stimulation (EPS‐La) compared to immediately after stimulation (EPS‐Ea) (p = 0.09). CONCLUSION We were able to determine that electrical pulse stimulation of L6 myotubes could be used as a model to better understand anabolic signaling strategies following EPS in culture. This study represents a first step in using electrical pulse stimulation as an in culture exercise mimetic and elucidating the role of mTOR and downstream targets in cell growth. Support or Funding Information This work was...

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