IL‐15 Promotes Mitochondrial Activity Through an ERK1/2‐PPARδ Signaling Axis in C2C12 Skeletal Muscle Cells

Myokines are specialized factors that are secreted from skeletal muscle (SKM). Many myokines have been shown to increase in circulation following exercise. In particular, interleukin‐15 (IL‐15) is one such myokine and its expression and secretion is induced with exercise. It has been postulated that IL‐15 induces SKM mitochondrial activity, via activation of PPARδ. However, the direct downstream mediators linking IL‐15 to PPARδ activity have yet to be elucidated. Here we aimed to identify the direct downstream signaling molecules of IL‐15 and their roles in mediating mitochondrial activity in SKM. The SKM cell line, C2C12, was cultured and cells were induced to differentiate into mature myotubes. Upon induction of differentiation, C2C12 cells were treated every other day, for 6 days, with vehicle (DMSO), IL‐15 (100 ng/ml), a PPARδ inhibitor (GSK), or IL‐15 + GSK. Western blotting and qPCR analysis were performed to verify inhibition of PPARδ. To assess mitochondrial activity, cells were stained with Mitotracker, fixed, and imaged at the end of the 6 day protocol. Additional short term experiments were performed on fully differentiated cells that were treated with or without IL‐15 (100 ng/ml) for 24 hours. Subsequently, proteins were extracted and western blotting was performed to assess phosphorylation levels of potential downstream signals of IL‐15 (Jak1, Akt, Akt substrates, and ERK1/2). Additionally, following the 24 hour protocol proteins were extracted and ERK1/2 was immunoprecipitated and western blotting procedures were performed to determine potential PPARδ interaction. PPARδ protein and mRNA expression were increased with IL‐15 treatment and these effects were abolished with GSK. Further, IL‐15 stimulated mitochondrial activity, as indicated by increases in Mitoctracker staining, and IL‐15 + GSK blunted these effects. IL‐15 treatment, for 24 hours, had no effect on phosphorylation or total protein expression of Jak1, Akt, or Akt substrates. However, ERK1/2 phosphorylation was increased with IL‐15, without a concomitant increase in total ERK1/2 expression levels. Moreover, when ERK1/2 was immunoprecipitated, there were higher levels of PPARδ interaction with ERK1/2 (34%; P <0.05), in the IL‐15 treated cells when compared to cells without IL‐15 treatment. Taken together, IL‐15 mediated increases in mitochondrial activity are dependent on PPARδ activity. Here we show, for the first time, that IL‐15 activates ERK1/2 in C2C12 SKM cells. Further, our data indicate that IL‐15 induces interaction between ERK1/2 and PPARδ. Overall, it is clear that IL‐15 acts to regulate mitochondrial activity via an ERK1/2‐PPARδ signaling axis in SKM cells. Support or Funding Information Crean College of Health and Behavioral Sciences, Chapman University, Start‐Up Funding