IL-15 Mediates Mitochondrial Activity through a PPARδ-Dependent-PPARα-Independent Mechanism in Skeletal Muscle Cells

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.


Introduction
Obesity has become a modern epidemic and is growing in both prevalence and severity throughout the world. Obesity is one of the leading causes of preventable death, with more than one-third of all Americans considered overweight or obese [1,2]. Current treatments for obesity include a calorie restricted diet and physical exercise, but sustaining long-term weight loss has proven to be a challenge for many [3]. A mainstay for treating obesity has been to increase metabolic rate, particularly through induction of mitochondrial activity in skeletal muscle (SKM). SKM is considered the largest organ in the body and acts to carry out bodily movement through generation of ATP, primarily via mitochondrial respiration [4].
Recently, SKM has attracted attention due to its newly identified ability to release cytokines, termed "myokines," into circulation that act to increase overall energy expenditure [5][6][7][8][9]. Many myokines (FGF-21, Irisin, BDNF5, interleukin-6, and interleukin-15, among others) increase in circulation following physical exercise, owing to their potential to reduce adiposity [9][10][11]. However, the downstream effectors of myokine signaling that positively influence metabolism remain elusive. In this regard, studies aimed at elucidating myokine signaling pathways, for the potential treatment and/or prevention of obesity, have come to the forefront of metabolic research.
PPARs are important transcriptional regulators linked to numerous beneficial metabolic effects, such as induction of mitochondrial biogenesis and fatty acid oxidation [30,31]. Among the three isoforms, PPAR is highly expressed in oxidative tissues, such as liver, heart, and type I SKM fibers, while PPAR appears to be ubiquitously expressed and acts to induce mitochondrial activity and lipid metabolism [30,32,33]. Both PPAR and have been suggested to promote induction of mitochondrial activity in many cell types and have garnered attention as potential antiobesogenic factors [30,[34][35][36][37]. PPAR is most highly expressed in adipose tissue, both brown and white, and it plays an integral part in lipogenesis and adipogenesis [38]. The antidiabetic drug class, thiazolidinediones, acts to bind to and activate PPAR to clear circulating lipids for the restoration of insulin sensitivity [39]. Much is known regarding the positive metabolic effects of PPARs but it is not known if myokines, such as IL-15, act to upregulate their transcriptional activity [30,36]. Interestingly, PPAR expression levels have been strongly linked to IL-15 signaling in SKM [26,28]. However, the direct relationship between IL-15 and PPAR transcriptional activity, to modulate mitochondrial processes, has yet to be firmly established. On the other hand, there are reports that IL-15 acts to increase PPAR expression levels in adipose tissue [29], but little is known regarding an IL-15-PPAR relationship in SKM. Taken together, it is clear that a relationship between IL-15 and PPAR and/or exits, but the depth of these relationships to induce metabolism, thereby, reducing adiposity is unknown.
Here we aimed to determine the necessity of PPAR and/or as downstream mediators of the metabolically beneficial effects of IL-15 action on mitochondrial activity in SKM. Here, we show that PPAR is required for IL-15 mediated induction of mitochondrial activity independent of PPAR in SKM cells.

Western Blotting.
Western blotting was performed as previously described [40]. Briefly, C2C12 cells were lysed in a modified RIPA buffer supplemented with protease inhibitors (Pierce). Approximately 20 g of protein from the cell homogenate preparations was separated on a 4-12% gradient gel (GenScript) via SDS-PAGE. Proteins were transferred onto Immobilon-P polyvinylidene difluoride (PVDF) membranes and blocked with 5% BSA in Tween-TBS for 1 hour. The membranes were then incubated (4 ∘ C) in 5% BSA in Tween-TBS with antibodies (1 : 1000) against PPAR , PPAR , or GAPDH (Sigma). Following overnight incubation, the membranes were then probed with a secondary antibody (GenScript, 1 : 2000; or Thermo, 1 : 10,000). Blots were then washed and subjected to enhanced chemiluminescence (Pierce). Membranes were stripped in 0.5 M NaOH and probed for total proteins and subsequently GAPDH (Sigma) was used as a loading control.

RNA Extraction and Reverse Transcription.
Standard RNA isolation procedures were performed on the cells following the 6-day treatment protocol, as previously described [41,42]. Mouse gastrocnemius muscle was used to verify IL-2 receptor expression. Briefly, cells or muscle tissue was lysed with Trizol reagent and chloroform was added to separate the RNA from the DNA and protein fractions. RNA was precipitated from the clear phase of the Trizol-chloroform mixture, followed by centrifugation at 12,000 ×g at 4 ∘ C, with isopropanol. The RNA pellet was washed with 75% ethanol and centrifuged at 7,500 ×g for 5 minutes, at 4 ∘ C, and the pellets were air-dried. Using RNAse-free water, the pellets were resuspended and the RNA purity and concentration were quantified using a NanoDrop spectrophotometer. Reverse transcription of RNA to cDNA was performed on 2 g of RNA using SuperScript reverse transcriptase VILO kit.

Mitochondrial DNA Assessments.
Following the IL-15 treatment protocol, genomic DNA was isolated using a miniprep DNA isolation kit (Sigma). Briefly, 5 × 10 6 of cells in suspension, in lysis buffer, were mixed with RNase and proteinase K and incubated at 70 ∘ C for 10 minutes. Ethanol was added and then homogenates were transferred to the binding columns and subjected to a series of washes. The columns were air-dried and the DNA was eluted. DNA concentration and purity were assessed using a NanoDrop spectrophotometer (Thermo). Real time qPCR was carried out and a mitochondrial DNA marker was compared relative to a nuclear encoded marker (18S) and the corresponding sequences are displayed in Table 1.

Real Time Quantitative PCR.
Real time qPCR was performed on the cDNA, using SYBR green in a 96-well plate. The primers are displayed in Table 1. GAPDH was used as an internal control and the ddCT method was used to calculate gene expression levels.

Live Cell Mitochondrial Activation Assay.
Following the IL-15 treatment protocol, as described above, live cells were stained using a dye that becomes sequestered in active mitochondria [42]. The cells were then fixed with phosphate buffered formalin and DAPI was used as a nuclear stain. Fluorescence levels were assessed using an inverted Zeiss microscope and images were captured using an Axiovision camera. Relative and absolute fluorescence levels were calculated using Image J software. Measurements were corrected for total cell fluorescence to account for the variation in myotube size.

Citrate Synthase and ATP Assays.
Citrate synthase (CS) activity was measured using a previously described protocol with some modifications [42][43][44]. To assess CS activity, C2C12 cell lysates were added to a 96-well plate containing 100 M 5, 5 -dithio-bis (2-nitrobenzoic acid) and 250 M acetyl-CoA. To initiate the reaction, 500 M oxaloacetate was added. The reaction was monitored in a microplate reader for 5 min at an ABS of 405. The specific activity was calculated as the absorbance rate per minute divided by the mercaptide extinction coefficient and expressed per g of protein. ATP was measured on cell lysates using a fluorometric kit in a 96well plate (Sigma) and normalized to total protein.

Statistical
Analysis. Data are presented as mean ± SEM and all calculations were carried out using GraphPad Prism 6. A one-way ANOVA was calculated to determine multiple comparisons with a Fisher's post hoc analysis. For comparisons between two groups Student's -test was performed. A level of 0.05 was used to determine statistical significance.

IL-15 Induces Mitochondrial Activity in Skeletal Muscle
Cells. To verify total mitochondrial activation via IL-15 signaling, a mitochondrial activity assay was performed to analyze relative mitochondrial activity in live SKM cells, as Table 1: Primer sequences used for qPCR analysis.

The Involvement of PPARs in IL-15
Signaling. To verify the efficiency of the PPAR inhibitor, GW, we confirmed a reduction in PPAR mRNA expression levels by 57% when compared to vehicle control cells ( < 0.05; Figure 4(a)). PPAR mRNA levels were assessed to determine the specificity of GW and there were no reductions with PPAR inhibition ( > 0.05; Figure 4(b)). Although PPAR was inhibited, the stimulatory effects of IL-15 on PGC1 and UCP2 mRNA expression levels were maintained ( < 0.05; Figure 4(c)). Conversely, the IL-15 induced increases in PGC1 and Nrf1 mRNA levels were abolished with PPAR inhibition when compared to vehicle control cells ( > 0.05; Figure 4(c)). Next, PPAR mRNA levels were confirmed to be reduced by 60%, when compared to vehicle control cells, with its inhibitor (GSK) ( < 0.05; Figure 5(a)). Conversely the PPAR inhibitor had no effects on PPAR mRNA levels, confirming the specificity of GSK ( > 0.05; Figure 5(b)). Unlike the PPAR experiments, inhibition of PPAR signaling resulted in a loss of IL-15 induced increases in PGC1 , PGC1 , UCP2, and Nrf1 when compared to vehicle control cells ( > 0.05, Figure 5(c)). cells, ( < 0.05) and these stimulatory effects were eliminated with PPAR inhibition ( > 0.05; Figure 6(a)). Likewise, ATP content was elevated (30%) with IL-15 treatment and PPAR inhibition abolished these effects ( < 0.05; Figure 6(b)). IL-15 induced increases in mRNA levels of cytochrome C oxidase (Cox) isoforms 5b, 7a1, and 8b were dependent on PPAR activity ( < 0.05; Figure 6

Discussion
Data from this study solidify the notion that IL-15 is directly involved in mediating mitochondrial activity in SKM cells. Importantly, our data indicate that PPAR activation is required for IL-15 signaling to carry out its stimulatory effects on mitochondrial activity. Further, based on our findings, we have ruled out PPAR as a potential modulator In line with other reports in adipose tissue [25], treatment with IL-15 increased mitochondrial associated processes in SKM cells, as indicated by increased activity of CS and ATP production. Our data confirm other reports showing that IL-15 has the ability to induce activity of the key Krebs Cycle factor, CS, in SKM from mice [25]. It has been postulated that one route that IL-15 acts to reduce adiposity is through its ability to increase lipolysis and mitochondrial activity in adipocytes [25]. Additionally, IL-15 has been shown to increase the activity of mitochondrial processes, such as fatty acid oxidation [24,46]. Here we show, for the first time, that IL-15 acts to directly increase overall mitochondrial activity in live SKM cells. On the other hand, it does not appear that IL-15 induces increases in mitochondrial biogenesis as indicated by the mtDNA and Tfam assessments in the C2C12 cells.
Likewise, in the current study, activity of CS, ATP production, and Cox isoform expression were fully dependent on PPAR activity with IL-15 stimulation in the SKM cells. Further, it has previously been established that IL-15 increases expression levels of key factors that function to increase mitochondrial activity and biogenesis in both white and brown adipose tissue as well as in SKM [22,26,27,29,47]. Our results are in line with those findings, as PPAR , PPAR , PGC1 , PGC1 , UCP2, and Nrf1 expression levels were all elevated with IL-15 stimulation. Conversely, our data do not support the notion that IL-15 stimulates mitochondrial biogenesis, which is in agreement with previously reported data in mouse SKM [48]. Unlike other studies, our treatment with IL-15 failed to increase SIRT1 expression levels [27,28]. Here we employed an in vitro model to study the effects of IL-15 on SKM cells and the other reports linking IL-15 to SIRT1 were carried out in a transgenic mouse model overexpressing IL-15 [27,28]. Therefore, it is a possibility that IL-15 induced SIRT1 expression levels are secondary to direct activation of the IL-15 signaling pathway in SKM. On the other hand, it cannot be ruled out that SIRT1 activity is regulated by IL-15, as we have only assessed mRNA expression levels. Taken together, it is clear that PPAR is required for the IL-15 induced effects on mitochondrial associated factors and activity.
Here, our data point to a strong link between IL-15 and PPAR in SKM cells, but PPAR activity involvement had not been fully assessed in SKM [24,[26][27][28]. PPAR has been associated with IL-15 signaling in adipose tissue and, with this in mind, we attempted to elucidate a potential IL-15-PPAR relationship in C2C12 cells [29]. Interestingly, IL-15 induced PPAR expression levels nearly 4-fold, while PPAR expression was induced only 2-fold, suggesting that PPAR may be a more direct target of IL-15 signaling. However, with inhibition of PPAR , PGC1 and UCP2 mRNA expression levels were maintained with IL-15 treatment. Conversely, Nrf1 mRNA expression levels were equivocal to the vehicle control cells with IL-15 treatment when PPAR was inhibited. Nrf1 has been shown to be directly regulated by PPAR , with a greater affinity than PPAR , which may explain the reduction in its expression levels with IL-15 treatment and PPAR inhibition [49]. Furthermore, PGC1 expression levels were not statistically different from vehicle control cells with both IL-15 and PPAR inhibitor. On the other hand, with PPAR inhibition, the stimulatory effect of IL-15 on mitochondrial activity was maintained. Although we show a potential connection between IL-15-PPAR mediated increases in some mitochondrial associated factors, these relationships do not appear to translate to functional assays, such as mitochondrial activity. It should be noted that addition of the vehicle control (DMSO) in the inhibitor studies yielded alterations in baseline mRNA expression levels when compared to mRNA levels in the absence of DMSO. Further, our data is dependent on pharmacological inhibitors of PPARs. Genetic knockdown studies would provide additional support for our data. However, the functionality of IL-15 induced increases in mitochondrial activity is relevant in mature fully differentiated SKM cells. Therefore, methodological constraints do not allow for genetic knockdown studies on fully differentiated cells with repeated treatments. Even though our data indicate that PPAR activity is not required for IL-15 mediated mitochondrial activity in SKM cells, we definitively show that PPAR activity is a requirement. Indeed, the master mitochondrial regulators, PGC1 and PGC1 , mRNA expression levels were both reduced with PPAR inhibition in combination with IL-15 stimulation. Both PGC1 and PGC1 are responsible for the numerous beneficial effects of exercise on mitochondrial processes and biogenesis and signal in concert with PPARs [50,51]. Additionally, PGC1 is responsible for regulating mitochondrial uncoupling, via UCP2, and our data are in support of this pathway, as indicated by the reductions of UCP2 expression with inhibition of PPAR with IL-15 treatment [52]. The importance of IL-15 induced PPAR activation for the regulation of mitochondrial activity is further supported by the reduction of Nrf1 mRNA expression levels with PPAR inhibition and IL-15 treatment. In this regard, not only does IL-15 signal directly through PPAR but also its effects initiate a master metabolic regulation pathway, including UCP2 and Nrf1 as downstream targets.

Conclusions
It is widely accepted that PPARs play an important role in mediating mitochondrial processes to prevent and/or treat metabolic disorders [30,32,34,53,54]. We provide evidence for the requirement of PPAR as a direct target of IL-15 signaling to carry out mitochondrial processes in SKM. Additionally, our data indicate that PPAR is not necessary for the beneficial effects of IL-15 signaling on mitochondrial activation in SKM. Although we have shown the importance of PPAR in IL-15 signaling, the signals directly downstream the IL-2 receptor remain unknown in SKM. Therefore, examining the effects of IL-15 on IL-2R targets such as Akt and the Jak/STAT pathway is required in SKM. In order to define the complex relationship of IL-15 signaling and PPARs further in vivo studies are warranted. Overall, understanding the players involved in IL-15 signaling will give rise to potential therapies for obesity and its associated disorders.