DHA Inhibits Protein Degradation More Efficiently than EPA by Regulating the PPARγ/NFκB Pathway in C2C12 Myotubes

This study was conducted to evaluate the mechanism by which n-3 PUFA regulated the protein degradation in C2C12 myotubes. Compared with the BSA control, EPA at concentrations from 400 to 600 µM decreased total protein degradation (P < 0.01). However, the total protein degradation was decreased when the concentrations of DHA ranged from 300 µM to 700 µM (P < 0.01). DHA (400 µM, 24 h) more efficiently decreased the IκBα phosphorylation and increased in the IκBα protein level than 400 µM EPA (P < 0.01). Compared with BSA, 400 µM EPA and DHA resulted in a 47% or 68% induction of the NFκB DNA binding activity, respectively (P < 0.01). Meanwhile, 400 µM EPA and DHA resulted in a 1.3-fold and 2.0-fold induction of the PPARγ expression, respectively (P < 0.01). In C2C12 myotubes for PPARγ knockdown, neither 400 µM EPA nor DHA affected the levels of p-IκBα, total IκBα or NFκB DNA binding activity compared with BSA (P > 0.05). Interestingly, EPA and DHA both still decreased the total protein degradation, although PPARγ knockdown attenuated the suppressive effects of EPA and DHA on the total protein degradation (P < 0.01). These results revealed that DHA inhibits protein degradation more efficiently than EPA by regulating the PPARγ/NF-κB pathway in C2C12 myotubes.


Introduction
Nuclear factor-kappa B (NF B) is one of the most important signaling pathways linked to the loss of skeletal muscle mass in normal physiological and pathophysiological conditions [1]. NF B expresses constitutively and exists in the cytosol as part of a heterotrimeric complex [2]. This complex typically comprises the DNA-binding proteins p50 and p65 plus the inhibitory protein I B . Activation of NF B requires phosphorylation of I B , followed by ubiquitin conjugation and proteolysis of I B by the 26S proteasome [3,4]. The activated NF B dimer is then translocated to the cell nucleus, which is a feed-forward signal that leads to the upregulation of pathway components including ubiquitin, E 2 /E 3 proteins, and proteasome subunits, subsequently increasing activity of the ubiquitin/proteasome pathway and enhancing skeletal muscle protein degradation [5][6][7].
There is growing evidence suggesting that long chain eicosapentaenoic acid (EPA, C20:5n-3) can inhibit NF B activation by preventing the degradation of I B in skeletal muscle [8,9]. Remarkably, most of the previous researches focused on that EPA can decrease muscle protein degradation in cancer cachexia [10][11][12]. In addition, it was further observed that long chain EPA, but not -linolenic acid (ALA, C18:3n-3), can inhibit the NF B activation in C2C12 myotubes by activating transcription factors peroxisome proliferators-activated receptor-(PPAR ) [13]. The above results demonstrated that the effect of n-3 polyunsaturated fatty acid (n-3 PUFA) on the I B /NF B pathway may be different because of the variety of fatty acids. Therefore, we hypothesized that longer chain docosahexaenoic acid (C22:6n-3) can more efficiently inhibit the protein degradation by regulating PPAR /NF B pathway in C2C12 myotubes than EPA.
In the present study, C2C12 myotubes were treated with EPA and DHA for 24 h, respectively. Meanwhile, knockdown of PPAR in C2C12 myotubes was achieved by RNA interference (RNAi). C2C12 myotubes for PPAR knockdown were also treated with EPA or DHA for 24 h, respectively. The actions of EPA or DHA were compared with those of a fatty 2 BioMed Research International acid-free control (containing BSA). The aim of this study was to investigate whether DHA can more efficiently inhibit protein degradation than EPA by regulating I B /NF B pathway in C2C12 myotubes in a PPAR -dependent manner.

Cell
Culture. Mouse C2C12 myoblasts (American Type Culture Collection, Manassas, VA, USA) were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, penicillin (50 units/mL), and streptomycin (50 mg/mL). When cells reached confluence, the medium was transferred to the differentiation medium containing Dulbecco's modified Eagle's medium and 2% horse serum, which was changed every other day. After four additional days, the differentiated C2C12 cells had fused into myotubes.

Transfection of Stealth RNAi for PPAR Knockdown in C2C12
Myotubes. Transfection of Stealth RNAi for PPAR knockdown in C2C12 myotubes was according to Kim et al. [14]. The PPAR Stealth Select RNAi oligonucleotide (Target Accession nos. NM 138712.1, M015869.2, NM138711.1, and NM 005037.3) was synthesized by Invitrogen. The Stealth RNAi negative control duplex (Invitrogen Corp., Carlsbad, CA, USA) was used as a control oligonucleotide. Transfection efficiency was monitored using a fluorescent oligonucleotide (BLOCK-iT Fluorescent Oligonucleotide; Invitrogen) and estimated to be 40% in C2C12 cells. The Stealth RNAi molecules were transfected into C2C12 myotubes using Lipo-fectAMINE 2000 by following Invitrogen's protocols. The final concentration of 50 nM PPAR Stealth Select RNAi oligonucleotide was selected for C2C12 myotubes, and the Stealth RNAi oligonucleotides were transfected to the cells 48 h before the treatment of fatty acids. The ability of the Stealth RNAi oligonucleotide to knockdown PPAR expression was analyzed by western blot and real-time quantitative PCR on whole-cell extract.

Treatment of Cells.
Lipid-containing media was prepared by conjugation of free fatty acids (DHA or EPA) with FFA-free bovine serum albumin, by a method modified from that described by Chavez et al. [15]. Briefly, FFAs were dissolved in ethanol and diluted 1 : 100 in DMEM containing 2% (w/v) fatty-acid-free bovine serum albumin. 3T3-L1 adipocytes and C2C12 myotubes were cocultured in serum free medium overnight and then washed with fresh medium, refed with medium containing the different treatments (BSA or EPA, resp.), and incubated at 37 ∘ C in 5% CO 2 for 24 h before analysis. Cell viability was measured via trypan blue exclusion. At the end of the incubation, culture supernatant was collected and stored at −20 ∘ C until assayed.

Measurement of Protein Degradation in C2C12 Myotubes.
Cells were analyzed for protein degradation using procedures adapted from Desler [16]. C2C12 myotubes were labeled on day 4 for 24 h with 1 mCi/mL of [ 3 H]tyrosine before cells were washed, and the chase medium (DMEM + 2 mM tyrosine) was then added to wells and allowed to incubate for 4 h. Test medium was added and left for 20 h, and medium was sampled and counted. Remaining medium was aspirated off, and 1 mL of 0.5 M NaOH + 0.1% Triton was added. Plates were put in a cold room at 4 ∘ C overnight to allow the cells to dissolve. The cell suspension was placed in scintillation vials containing 5 mL of scintillation fluid, and wells were washed with 250 mL of 1 N acetic acid. This solution was added to scintillation vials and counted.
2.6. RNA Isolation. Total RNA was extracted using the TRIzol reagent (Invitrogen Corp., Carlsbad, CA, USA) according to the manufacturer's specifications. The RNA samples were quantified spectrophotometrically at 260 and 280 nm. The ratio of light absorbance at 260 nm to that at 280 nm was between 1.8 and 2.0, indicating that they were pure and clean. The quality of RNA was also checked by 1.0% agarose gel electrophoresis and staining with 1 g/mL ethidium bromide.

Reverse Transcription PCR and Real-Time Quantitative PCR Analysis.
Reverse transcription (20 L) of total RNA (1 g) was performed using avian myeloblastosis virus reverse transcriptase with a first-strand cDNA synthesis kit for reverse transcription PCR. Aliquots (2 L) of the reverse transcription reactions were then submitted in duplicate to online quantitative PCR with the LightCycler 480 Real-Time PCR System (Roche Applied Science, Mannheim, Germany) with SYBR green using the FastStart DNA Master SYBR Green I. Initial real-time amplifications were examined by agarose gel electrophoresis, followed by ethidium bromide staining to verify that the primer pairs amplified a single product of the predicted size. Subsequent aliquots of the PCR reaction were checked by melting curve analysis as provided by the LightCycler system. Primer sequences and optimal PCR annealing temperatures (ta) are listed in Table 1. The PCR was performed in a volume of 20 L: 2 L of FastStart DNA Master SYBR Green I, 3 mM MgCl 2 and primers according to a primer concentration of 1 M. The instrument settings were denaturing at 95 ∘ C for 10 min, 45 × denaturing at 95 ∘ C for 30 s, annealing at 59 ∘ C for 30 s, and elongation at 72 ∘ C for 8 min for (PPAR and -actin). Quantification was performed by online monitoring for identification of the exact time point at which the logarithmic linear phase was distinguishable from the background. Serially diluted samples obtained by PCR with the above-mentioned primers from human myotubes were used as external standards in each run. The cycle numbers of the logarithmic linear phase were plotted against the logarithm of the concentration of the template DNA, and the concentration of cDNA in the different samples was calculated with the LightCycler software (version 5.32).

Isolation of Nuclear Extracts.
Nuclear extracts were isolated according to Andrews and Faller [17]. Cells were scraped into 1.5 mL of cold phosphate-buffered saline, pelleted for 10 seconds, and resuspended in 400 L of cold BufferA (10 mM HEPES-KOH pH 7.9 at 4 ∘ C, 1.5 mM MgCl 2 , 10 mM KCl, 0.5 mM DTT, 0.2 mM PMSF, 5 g/mL aprotinin, and 2 g/mL leupeptin) by flicking the tube. Cells were allowed to swell on ice for 10 minutes and then vortexed for 10 seconds. Then, samples were centrifuged for 10 seconds and the supernatant fraction was discarded. Pellets were resuspended in 50 L of cold BufferC (20 mM HEPES-KOH pH 7.9 at 4 ∘ C, 25% glycerol, 420 mM NaCl, 1.5 mM MgCl 2 , 0.2 mM EDTA, 0.5 mM DTT, 0.2 mM PMSF, 5 g/mL aprotinin, and 2 g/mL leupeptin) and incubated on ice for 20 minutes for high-salt extraction. Cellular debris was removed by centrifugation for 2 minutes at 4 ∘ C and the supernatant fraction (containing DNA binding proteins) was stored at −80 ∘ C. Nuclear extract concentration was determined by using the Bradford method. , dried under a vacuum at 80 ∘ C for 1 h, and exposed to photographic film at −70 ∘ C with an intensifying screen.

2.10.
Immunoblotting. In order to obtain total proteins C2C12, myotubes were homogenized in cold lysis buffer (5 mM Tris-HCl (pH 7.4), 1 mM EDTA, 0.1 mM phenylmethylsulfonyl fluoride, 1 mM sodium orthovanadate, and 5.4 g/mL aprotinin). The homogenate was centrifuged at 10,000 ×g for 30 min at 4 ∘ C. For obtaining total membranes from C2C12 myotubes, cells were collected into 10 mL of ice cold HES buffer (250 mmol/L sucrose, 1 mmol/L EDTA, 1 mmol/L phenylmethylsulfonyl fluoride, 1 mol/L pepstatin, 1 mol/L aprotinin, 1 mol/L leupeptin, and 20 mmol/L HEPES, pH 7.4) and subsequently homogenized at 4 ∘ C. After centrifugation at 1,000 ×g for 3 minutes at 4 ∘ C to remove large cell debris and unbroken cells, the supernatant was then centrifuged at 245,000 ×g for 90 min at 4 ∘ C to yield a pellet of total cellular membranes. Protein concentration was measured by the Bradford method. Proteins (30 g) were separated by SDS-PAGE on 10% separation gels and transferred to Immobilon Polyvinylidene difluoride membranes (Millipore, Bedford, MA, USA). Western blot analysis was performed using antibodies against phospho I B (Ser32), I B , and PPAR (Santa Cruz Biotechnology, Inc.). Detection was achieved using the EZ-ECL chemiluminescence detection kit (Biological Industries, Beit Haemek Ltd., Israel). Equal loading of proteins was assessed by red phenol staining. Size of detected proteins was estimated using protein molecularmass standards (Invitrogen, Barcelona, Spain).

Statistical
Analysis. Data were presented as means ± S.E. Differences between group means were determined by a oneway ANOVA using the computer program GraphPad Instat (version 2.03; GraphPad Software Inc., San Diego, CA, USA). When significant variations were found, the Tukey-Kramer multiple comparisons test was performed. Differences were considered significant at < 0.05.

Effect of Increasing Concentrations of n-3PUFA on Total
Protein Degradation in C2C12 Myotubes. In the present study, two long chain n-3 PUFA were chosen for study: eicosapentaenoic acid (EPA), a C20:5n-3 n-3 polyunsaturated

Effect of 24 h Treatment with 400 M n-3PUFA on I B /NF-B Pathway in C2C12 Myotubes.
After being treated with 400 M EPA, and DHA for 24 h in C2C12 myotubes, respectively, the levels of p-I B and total I B were measured by western blot method. The effect of 400 M EPA and DHA on the I B protein level in C2C12 myotubes was present in Figure 2(a). As expected, compared with the BSA control, EPA (400 M, 24 h) decreased the I B phosphorylation and caused approximately the 72% increase in the I B protein level ( < 0.01). In addition, a 24 h incubation of C2C12 myotubes with 400 M DHA also decreased the I B phosphorylation and caused approximately the 89% increase in the I B protein level ( < 0.01). Taken together, these data suggested that 400 M DHA more effectively prevented the degradation of I B by decreasing the phosphorylation of I B and increased the I B protein level in C2C12 myotubes than 400 M EPA.
To test whether incubation of C2C12 cells with 400 M EPA or DHA for 24 h affected NF B activity, we performed EMSA studies (Figure 2(b)). Compared with BSA, incubation of C2C12 myotubes with 400 M EPA for 24 h decreased the I B phosphorylation and resulted in a 47% induction of the NF B DNA binding activity ( < 0.01). Furthermore, C2C12 myotubes incubated in the presence of 400 M DHA for 24 h decreased the I B phosphorylation and caused a 68% reduction in the levels of the NF B DNA binding activity ( < 0.01). These results demonstrated that the inhibitory effect of 400 M DHA on the NF B DNA binding activity in C2C12 myotubes was greater than that of 400 M EPA.

Effect of n-3PUFA on the I B /NF-B Signaling Pathway and Total Protein Degradation in C2C12 Myotubes for PPAR
Knockdown. To confirm that the inhibition of the I B /NF-B signaling pathway and total protein degradation by n-3PUFA is mediated via activating the PPAR mRNA expression, we examined the effect of n-3PUFA on the I B /NF-B pathway in C2C12 myotubes for PPAR knockdown.
The C2C12 myotubes transfected with either negative control Stealth RNAi oligonucleotide or PPAR Stealth RNAi oligonucleotide were incubated for 48 h, respectively. Transfection of Stealth RNAi for PPAR knockdown in C2C12 myotubes significantly decreased protein expression (Figures 4(a) and 4(b)) and PPAR mRNA (Figure 4(c)) to approximately 82% and 86% ( < 0.01), respectively. Negative control Stealth RNAi treatment had no influence on PPAR mRNA and protein expression ( > 0.05).
In C2C12 myotubes transfected with PPAR Stealth RNAi oligonucleotide, treatment with 400 M EPA or 400 M DHA for 24 h did not affect the levels of p-I B or total I B ( Figure 5(a)), NF B DNA binding activity ( Figure 5(b)), compared with BSA ( > 0.05). Remarkably, Figure 6 showed that in C2C12 myotubes transfected with either negative control Stealth RNAi oligonucleotide, 24 h incubation period with 400 M EPA and DHA, respectively, caused 30% and 49% reduction in the rate of total protein degradation compared with BSA ( < 0.01). However, in C2C12 myotubes transfected with PPAR Stealth RNAi oligonucleotide, 24 h incubation period with 400 M EPA still caused a 17% reduction in the rate of total protein degradation ( < 0.01), whereas 24 h incubation period with 400 M DHA resulted  (Figure 2(a)). The band on the western blot represented a protein with a molecular mass of ∼37 kDa as determined by the molecular mass markers included in the experiment. Total nuclear protein was subsequently isolated and analyzed by EMSA for NF B DNA binding activity using a 32 P-labeled double-stranded oligonucleotide of the NF B (Figure 2(b)). An additional unlabeled probe was added in the competition assay (cold). Data are representative of three experiments. BSA = bovine serum albumin, EPA = eicosapentaenoic acid, DHA = docosahexaenoic acid, and p-I B = phosphorylated I B . in a 29% reduction in the rate of total protein degradation, compared with BSA ( < 0.01).

Discussion
Previous studies have found that eicosapentaenoic acid (EPA, C20:5n-3) decreased gene expression of the muscle RING finger 1 (MuRF1), which has been demonstrated to have ubiquitin-ligase activity [13]. In the present study, it was further observed that total protein degradation was decreased by EPA at concentrations ranging from 400 M to 600 M and docosahexaenoic acid (C22:6n-3) at a wider dose range (300 M to 700 M), respectively. These results demonstrated that the effect of EPA and DHA on total protein degradation may be dependent on the concentration of fatty acids.
Remarkably, in contrast to the fatty acid-free BSA control, the maximal effect for the inhibition of total protein degradation was observed at concentration of 500 M EPA and 400 M DHA, respectively. The results revealed that long chain DHA can more efficiently decrease total protein degradation than EPA. However, whether the suppressive effects of EPA and DHA on the total protein degradation depend on the chain length, it remains to be seen. Despite increasing evidence of the effect of long chain n-3 polyunsaturated fatty acid (n-3 PUFA) on total protein degradation, little is known concerning the mechanisms by which long chain n-3PUFAs regulate muscle protein degradation. As a result of 400 M EPA and DHA, both can inhibit total protein degradation. Therefore, in the current study, to test whether the effect of EPA or DHA on muscle protein degradation affected NF B activity, we investigated the effect of 400 M EPA or DHA on NF B activity. The key to NF B regulation is the I B protein, which retain in the NF B cytoplasm. Phosphorylation of I B by I B kinases triggers its polyubiquitinylation and degradation, thereby releasing NF B, which translocates to the nucleus. As  Figure 4: Transfection of Stealth RNAi for PPAR knockdown in C2C12 myotubes. The C2C12 myotubes transfected with either negative control Stealth RNAi oligonucleotide or PPAR Stealth RNAi oligonucleotide were incubated for 48 h, respectively. Protein extracts from C2C12 myotubes were assayed for western blot analysis with PPAR (Figure 4(a)). The band on the western blot represented a protein with a molecular mass of ∼55 kDa as determined by the molecular mass markers included in the experiment. PPAR protein expression was determined by western blot and relative abundance of protein was calculated after normalization to -actin (Figure 4(b)). The PPAR mRNA was determined using real-time PCR analysis and relative abundance of mRNA was calculated after normalization to -actin (Figure 4 Figure 5: Effect of n-3 PUFA on the I B /NF B complex in C2C12 myotubes knockdown of PPAR . The C2C12 myotubes transfected with PPAR Stealth RNAi oligonucleotide were incubated with 600 M EPA or 600 M DHA for 24 hours, respectively. BSA was used as fatty acid-free control. Protein extracts from C2C12 myotubes were assayed for western blot analysis with p-I B , I B , or -actin ( Figure 5(a)). The band on the western blot represented a protein with a molecular mass of ∼37 kDa as determined by the molecular mass markers included in the experiment. Total nuclear protein was subsequently isolated and analyzed by EMSA for NF B DNA binding activity using a 32 P-labeled double-stranded oligonucleotide of the NF B ( Figure 5(b)). An additional unlabeled probe was added in the competition assay (cold). Data are representative of three experiments. BSA = bovine serum albumin, DHA = docosahexaenoic acid, EPA = eicosapentaenoic acid, and p-I B = phosphorylated I B . in agreement with other studies showing that EPA inhibits NF B activation by preventing I B phosphorylation and further reducing degradation of the inhibitory I B protein [18,19]. We then investigated the molecular mechanism by which EPA and DHA decreased NF B activation and inhibited total protein degradation. Many researches reported that activation of the transcription factor PPAR in the skeletal muscle can inhibit NF B activity [20,21]. It was noteworthy that Li et al. [22] reported that n-3PUFA such as EPA and DHA are known natural ligands of PPAR , which were required in relatively high concentrations (approximately up to 100 mol/L) for PPAR activation. In the present study, C2C12 myotubes incubated in the presence of 400 M EPA for 24 hours resulted in a 1.3-fold induction of the PPAR gene expression, whereas 24 h incubation period with 400 M DHA resulted in a 2.0-fold induction of the PPAR gene expression. These results supposed that the effect of n-3PUFA on PPAR gene expression is likely to depend on the chain length of fatty acids, which was in line with a previous report by Huang et al. [13], who found that C2C12 myotubes incubated in the presence of EPA (600 M, 24 h) compared with the BSA control resulted in a 1.47-fold induction of the PPAR gene expression, while -linolenic acid (ALA; C18:3n-3) with shorter chain lengths was not able to affect PPAR gene expression. Taken together, these results supposed that long chain EPA and DHA can both decrease NF B activation and inhibit skeletal muscle degradation by activating PPAR gene expression in a chain length dependent manner. However, because we did not test some other fatty acids, it is premature to give this conclusion without further study.
In order to investigate whether EPA inhibited the I B /NF-B pathway and muscle protein degradation via activating the PPAR mRNA expression, PPAR knockdown by RNA interference (RNAi) decreased PPAR mRNA and protein expression to approximately 86% and 82% in C2C12 myotubes. Interestingly, it was further observed that treatment with 400 M EPA or DHA for 24 h did not affect the levels of p-I B and total I B and NF B DNA binding activity in C2C12 myotubes for PPAR knockdown. These results demonstrated that PPAR knockdown by RNAi abolished the suppressive effects of the EPA or DHA on the I B /NF B pathway in C2C12 myotubes, supporting that the EPA or DHA effects are mediated via PPAR activation.
Remarkably, in contrast to the fatty acid-free BSA control, 24 h incubation period with 400 M EPA and DHA, respectively, can cause 30% and 49% reduction in the rate of total protein degradation in C2C12 myotubes without PPAR knockdown. However, in C2C12 myotubes for PPAR knockdown, 24 h incubation period with 400 M EPA still caused a 17% reduction in the rate of total protein degradation, whereas 24 h incubation period with 400 M DHA resulted in a 29% reduction in the rate of total protein degradation. The experiments founded that EPA and DHA both still decreased the total protein degradation in C2C12 myotubes, although PPAR knockdown by RNAi attenuated the suppressive effects of the EPA or DHA on the total protein degradation. These results revealed that the mechanism by which n-3PUFA regulated total protein degradation may be involved in other signaling pathway, except for the PPAR /I B /NF B signalling pathway in C2C12 myotubes.
Taken together, these results revealed that DHA, more efficiently than EPA, inhibited the protein degradation by regulating I B /NF B signaling pathway in C2C12 myotubes by activating PPAR gene expression.