IL-15 Mediates Mitochondrial Activity through a PPAR δ-Dependent-PPAR α-Independent Mechanism in Skeletal Muscle Cells

Abstract

Molecular mediators of metabolic processes, to increase energy expenditure, have become a focus for therapies of obesity. The discovery of cytokines secreted from the skeletal muscle (SKM), termed "myokines," has garnered attention due to their positive effects on metabolic processes. Interleukin-15 (IL-15) is a myokine that has numerous positive metabolic effects and is linked to the PPAR family of mitochondrial regulators. Here, we aimed to determine the importance of PPARα and/or PPARδ as targets of IL-15 signaling. C2C12 SKM cells were differentiated for 6 days and treated every other day with IL-15 (100 ng/mL), a PPARα inhibitor (GW-6471), a PPARδ inhibitor (GSK-3787), or both IL-15 and the inhibitors. IL-15 increased mitochondrial activity and induced PPARα, PPARδ, PGC1α, PGC1β, UCP2, and Nrf1 expression. There was no effect of inhibiting PPARα, in combination with IL-15, on the aforementioned mRNA levels except for PGC1β and Nrf1. However, with PPARδ inhibition, IL-15 failed to induce the expression levels of PGC1α, PGC1β, UCP2, and Nrf1. Further, inhibition of PPARδ abolished IL-15 induced increases in citrate synthase activity, ATP production, and overall mitochondrial activity. IL-15 had no effects on mitochondrial biogenesis. Our data indicates that PPARδ activity is required for the beneficial metabolic effects of IL-15 signaling in SKM.

Figure 1

Effect of IL-15 signaling on mitochondrial activity. (a) Representative images of mitochondrial activity assessment in live cells using a fluorescent probe sequestered into active mitochondria; (b) quantifiable fluorescence corrected for myotube size; (c) western blot of PPARα and PPARδ protein expression; (d) mRNA expression of IL-2Rγ. Assessments were carried out on differentiated C2C12 myotubes following treatment with 100 ng/mL of IL-15 every other day for 6 days during the differentiation protocol. Image J was used to quantify cell fluorescence. GAPDH was used as control for protein and mRNA expression assessments. All values are displayed as mean ± SEM, n = 3–6 per group, ^∗ P < 0.05.

Figure 2

Effects of IL-15 signaling on mitochondrial associated factors. (a) mRNA expression of PPARα, PPARδ, PGC1β, and PGC1α; (b) mRNA expression of UCP2, SIRT1, and Nrf1. Assessments were carried out on differentiated C2C12 myotubes following treatment with 100 ng/mL of IL-15 every other day for 6 days during the differentiation protocol. GAPDH was used as an internal control for qPCR analysis. All values are displayed as means ± SEM, n = 6–9 per group, ^∗ P < 0.05.

Figure 3

Effects of IL-15 signaling on mitochondrial biogenesis. (a) mitochondrial DNA (mtDNA) assessments; (b) mRNA expression of mitochondrial factor Tfam. Assessments were carried out on differentiated C2C12 myotubes following treatment with 100 ng/mL of IL-15 every other day for 6 days during the differentiation protocol. mtDNA was normalized to the total nuclearDNA (nucDNA) content. GAPDH was used as an internal control for qPCR analysis. All values are displayed as means ± SEM, n = 6.

Figure 4

Effects of PPARα inhibition on IL-15 mediated alterations of mitochondrial associated factors. (a) mRNA expression of PPARα following inhibition with GW-6471 (GW); (b) mRNA expression of PPARδ following exposure to GW; (c) mRNA expression of PGC1α, PGC1β, UCP2, and Nrf1 with IL-15 treatment in combination with GW. Throughout differentiation, cells were treated every other day, for 6 days, with either vehicle control (DMSO), IL-15 (100 ng/mL), 10 μM of the PPARα inhibitor (GW-6471), or IL-15 + GW-6471 (I + G). GAPDH was used as an internal control for qPCR analysis. All values are displayed as means ± SEM, n = 6–9 per group, ^∗different from vehicle and GW groups; ^#different from all groups; P < 0.05.

Figure 5

Effects of PPARδ inhibition on IL-15 mediated alterations of mitochondrial associated factors. (a) mRNA expression of PPARδ following inhibition with GSK-3787 (GSK); (b) mRNA expression of PPARα following exposure to GSK; (c) mRNA expression of PGC1α, PGC1β, UCP2, and Nrf1 with IL-15 treatment in combination with GSK. Throughout differentiation, cells were treated every other day, for 6 days, with either vehicle control (DMSO), IL-15 (100 ng/mL), 1 μM of the PPARδ inhibitor (GSK-3787), or IL-15 + GSK-3787 (I + G). GAPDH was used as an internal control for qPCR analysis. All values are displayed as means ± SEM, n = 6–9 per group, ^∗different from all groups; P < 0.05.

Figure 6

Involvement of PPARδ in IL-15 mediated mitochondrial activity. (a) Citrate synthase (CS) activity; (b) total intracellular ATP content; (c) mRNA expression of cytochrome C oxidase isoforms Cox5b, Cox7a1, and Cox8b. Assessments were carried out on total cell lysates from C2C12 SKM cells following treatment every other day, for 6 days, with either vehicle control (DMSO), IL-15 (100 ng/mL), 1 μM of the PPARδ inhibitor (GSK-3787), or IL-15 + GSK-3787 (I + G). All values are displayed as means ± SEM, n = 6 per group, ^∗different from vehicle control group; P < 0.05; ^∗∗different from all groups, P < 0.01; ^∗∗∗different from IL-15 group, P < 0.001; ^∗∗∗∗GSK different from IL-15 group, P < 0.001.

Figure 7

Involvement of PPARα and PPARδ in IL-15 mediated mitochondrial activity in live cells. (a) Quantifiable fluorescence corrected for myotube size. Assessments were carried out on differentiated C2C12 myotubes following treatment with vehicle control (DMSO), IL-15 (100 ng/mL), IL-15 + GW-6471 (I + GW), IL-15 + GSK-3787 (I + GSK), and IL-15 + GW + GSK (I + G + G) every other day for 6 days during the differentiation protocol. (b) Representative images of mitochondrial activity assessment in live cells using a fluorescent probe sequestered into active mitochondria; Image J was used to quantify cell fluorescence. All values are displayed as means ± SEM, n = 6 per group, ^∗different from all groups; P < 0.05.