Impact of External Stimuli on AKT Phosphorylation and Myokine Secretion in C2C12 Muscle Cells

Background: Insulin resistance (IR) in skeletal muscle is a key feature of metabolic disorders like type 2 diabetes, marked by impaired insulin-stimulated glucose uptake and disrupted signaling. Muscle contraction promotes insulin sensitivity through myokine secretion. To analyze secretome changes in insulin-resistant C2C12 muscle cells, various IR-inducing protocols were developed. Additionally, the effects of Electrical Pulse Stimulation (EPS) on the secretome of insulin-resistant myotubes were examined. Prior studies showed that different IR-inducing agents similarly affect AKT phosphorylation but lead to distinct myokine secretion patterns

Research question: The objective of this study was to characterize the effects of IR induced by different compounds on insulin- and contraction-regulated signaling pathways using phosphoproteomic profiling and to evaluate whether combining IR-inducing compounds further reduces AKT phosphorylation.

Methodology: Murine C2C12 myotubes as a skeletal muscle model were treated with Activin A, Chemerin, TNF-α, Palmitate, or by chronic exposure to high insulin concentrations to induce IR. Validation of the IR protocols involved assessing phosphorylation of AKT (a key target in the insulin signalling pathway) via immunoblotting and evaluating glycogen synthesis as a functional readout. Secretome analyses of insulin-resistant murine myotubes were then performed to identify alterations in intracellular signaling pathways.

Results: All tested compounds significantly reduced insulin-stimulated AKT phosphorylation, confirming their role in inducing insulin resistance, with reductions of up to 62%, thereby validating the protocols. Secretome analysis identified over 2500 potential myokines in the conditioned medium. Bioinformatic classification revealed that 500 proteins were classically secreted (SignalP, SP+), while 1000 were categorized as non-classical secreted proteins (SecretomeP, Outcyte, SP-). EPS treatment of insulin-resistant cells led to the significant upregulation of more than 100 myokines. Notably, the composition of the secretome varied depending on the specific stimulus used to induce insulin resistance, indicating distinct regulatory mechanisms.

Conclusion: These findings demonstrate that IR induced by diverse stimuli leads to distinct alterations in myokine secretion and probably on insulin- and contraction-regulated signaling pathways in skeletal muscle. The results underscore the intricacy of IR-related signaling and furnish significant experimental models for investigating various forms of insulin resistance with respect to the underlying intracellular cell signaling. To further elucidate the signaling alterations underlying these differences, phosphoproteomic analyses are planned

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Artikel online veröffentlicht:
28. Mai 2025