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Endocrinology, doi:10.1210/en.2005-0440
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Endocrinology Vol. 147, No. 1 552-561
Copyright © 2006 by The Endocrine Society

Palmitate Induces Tumor Necrosis Factor-{alpha} Expression in C2C12 Skeletal Muscle Cells by a Mechanism Involving Protein Kinase C and Nuclear Factor-{kappa}B Activation

Mireia Jové, Anna Planavila, Rosa M. Sánchez, Manuel Merlos, Juan Carlos Laguna and Manuel Vázquez-Carrera

Pharmacology Unit, Department of Pharmacology and Therapeutic Chemistry, Faculty of Pharmacy, University of Barcelona, E-08028 Barcelona, Spain

Address all correspondence and requests for reprints to: Manuel Vázquez-Carrera Unitat de Farmacologia. Facultat de Farmàcia, Diagonal 643, E-08028 Barcelona, Spain. E-mail: mvazquezcarrera{at}ub.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The mechanisms responsible for increased expression of TNF-{alpha} in skeletal muscle cells in diabetic states are not well understood. We examined the effects of the saturated acid palmitate on TNF-{alpha} expression. Exposure of C2C12 skeletal muscle cells to 0.75 mM palmitate enhanced mRNA (25-fold induction, P < 0.001) and protein (2.5-fold induction) expression of the proinflammatory cytokine TNF-{alpha}. This induction was inversely correlated with a fall in GLUT4 mRNA levels (57% reduction, P < 0.001) and glucose uptake (34% reduction, P < 0.001). PD98059 and U0126, inhibitors of the ERK-MAPK cascade, partially prevented the palmitate-induced TNF-{alpha} expression. Palmitate increased nuclear factor (NF)-{kappa}B activation and incubation of the cells with the NF-{kappa}B inhibitors pyrrolidine dithiocarbamate and parthenolide partially prevented TNF-{alpha} expression. Incubation of palmitate-treated cells with calphostin C, a strong and specific inhibitor of protein kinase C (PKC), abolished palmitate-induced TNF-{alpha} expression, and restored GLUT4 mRNA levels. Palmitate treatment enhanced the expression of phospho-PKC{theta}, suggesting that this PKC isoform was involved in the changes reported, and coincubation of palmitate-treated cells with the PKC inhibitor chelerythrine prevented the palmitate-induced reduction in the expression of I{kappa}B{alpha} and insulin-stimulated Akt activation. These findings suggest that enhanced TNF-{alpha} expression and GLUT4 down-regulation caused by palmitate are mediated through the PKC activation, confirming that this enzyme may be a target for either the prevention or the treatment of fatty acid-induced insulin resistance.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
DURING THE DEVELOPMENT of insulin resistance in skeletal muscle, an impairment of glucose utilization and insulin sensitivity has been related to the presence of elevated plasma free fatty acids (FFA). Thus, several studies have consistently demonstrated that elevations of plasma FFA produce insulin resistance in diabetic patients and in nondiabetic subjects (1, 2, 3, 4). On the other hand, accumulating evidence suggests a link between inflammation and type 2 diabetes mellitus. Markers of inflammation, including the cytokine tumor necrosis factor-{alpha} (TNF-{alpha}), have been postulated as critical mediators of insulin resistance. In fact, it has been clearly demonstrated that TNF-{alpha} is expressed in human muscle, and its level is higher in the muscle tissue of insulin-resistant and diabetic subjects (5). In these patients, muscle TNF-{alpha} expression was found to be 4-fold higher than in insulin-sensitive subjects. In addition, an inverse significant linear relationship between maximal glucose disposal rate and muscle TNF-{alpha} was also reported (5). Despite the potent inhibitory effect of TNF-{alpha} on insulin signaling in both adipose tissue and skeletal muscle, the concentrations of TNF-{alpha} in the serum of both lean and obese subjects is very low, suggesting that TNF-{alpha} secreted by muscle cells and adipocytes acts in an autocrine fashion (5, 6).

Little is known about the mechanisms responsible for the increased expression of TNF-{alpha} in skeletal muscle, but elevation of plasma FFA could be involved. Thus, FFA activate the proinflammatory transcription factor nuclear factor (NF)-{kappa}B (7, 8), which has been linked to fatty acid-induced impairment of insulin action in skeletal muscle in rodents (9, 10) and regulates the expression of TNF-{alpha}. In resting cells, NF-{kappa}B is present in the cytoplasm as an inactive heterodimer, consisting of the p50 and p65 subunits, complexed with an inhibitor protein subunit, I{kappa}B. After stimulation, a serine kinase cascade is activated leading to the phosphorylation of I{kappa}B. This event converts I{kappa}B in a substrate for ubiquitination and subsequent degradation, releasing the NF-{kappa}B heterodimer, which then translocates to the nucleus and regulates the expression of proinflammatory genes, such as TNF-{alpha}. Furthermore, it is worth noting that protein kinase C (PKC) activates NF-{kappa}B (9). Elevation of plasma FFA may lead to diacylglycerol-mediated activation of PKC (11, 12), an enzyme that has been linked to insulin resistance in a wide variety of rodent models (13, 14, 15), including rats infused with lipid (16) and massively obese humans (17, 18). Therefore, the PKC/NF-{kappa}B pathway may be crucial linking increased FFA and induction of a proinflammatory state during the development of insulin resistance and type 2 diabetes mellitus.

The purpose of this study was to investigate the contribution of FFA to the expression of the proinflammatory cytokine TNF-{alpha} and the mechanisms involved. Using mouse skeletal muscle C2C12 myotubes, we examined the effects of the saturated FFA palmitate on TNF-{alpha} gene expression. Exposure of the cells to palmitate led to increased TNF-{alpha} gene expression through mechanisms involving activation of PKC and NF-{kappa}B. These data suggest that palmitate-mediated PKC activation leads to increased expression of the proinflammatory cytokine TNF-{alpha} and that this mechanism may be involved in fatty acid-induced insulin resistance in skeletal muscle. Furthermore, these findings also suggest that drugs targeting PKC may be effective for reversing fatty acid-induced insulin-dependent glucose uptake in skeletal muscle cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
C2-ceramide, ISP1 or Myriocin, chelerythrine and pyrrolidine dithiocarbamate (PDTC) were from Sigma (St. Louis, MO). Calphostin C was from Biomol Research Labs, Inc. (Plymouth Meeting, PA). Other chemicals were from Sigma.

Cell culture
Mouse C2C12 myoblasts (ATCC, Manassas, VA) were maintained in DMEM supplemented with 10% fetal bovine serum, 50 U/ml penicillin, and 50 µg/ml streptomycin. When cells reached confluence, the medium was switched to the differentiation medium containing DMEM and 2% horse serum, which was changed every other day. After 4 additional days, the differentiated C2C12 cells had fused into myotubes. Lipid-containing media were prepared by conjugation of FFA with FFA-free BSA, by a method modified from that described by Chavez et al. (19). Briefly, palmitate was dissolved in ethanol and diluted in DMEM containing 2% (wt/vol) fatty acid-free BSA. Myotubes were incubated for 16 h in serum-free DMEM containing 2% BSA in either the presence or absence of palmitate. Cells were then incubated with 100 nM insulin for 10 min. After the incubation, RNA, total proteins and nuclear extracts were extracted from myotubes as described below. Inhibitors were added 30 min before the incubation with palmitate.

Measurements of mRNA
Levels of TNF-{alpha} and GLUT4 mRNA were assessed by the RT-PCR as previously described (20). Total RNA was isolated by using the Ultraspec reagent (Biotecx, Houston, TX). The total RNA isolated by this method is undegraded and free of protein and DNA contamination. The sequences of the sense and antisense primers used for amplification were: Tnf-{alpha}, 5'-TACTGAACTTCGGGGTGATTGGTCC-3' and 5'-CAGCCTTGTCCCTTGAAGAGAACC-3'; Glut4, 5'-GATGCCGTCGGGTTTCCAGCA-3' and 5'-TGAGGGTGCCTTGTGGGATGG-3'; and Aprt (adenosyl phosphoribosyl transferase), 5'-GCCTCTTGGCCAGTCACCTGA-3' and 5'-CCAGGCTCACACACTCCACCA-3'. Amplification of each gene yielded a single band of the expected size (Tnf-{alpha}, 284 bp; Glut4, 232 bp; and Aprt, 329 bp). Preliminary experiments were carried out with various amounts of cDNA to determine nonsaturating conditions of PCR amplification for all the genes studied. Therefore, under these conditions, relative quantification of mRNA was assessed by the RT-PCR method used in this study (21). Radioactive bands were quantified by video-densitometric scanning (Vilbert Lourmat Imaging, Torcy, France). The results for the expression of specific mRNAs are always presented relative to the expression of the control gene (Aprt).

Determination of glucose uptake by C2C12 skeletal muscle cells
Glucose uptake was assayed using [3H]2-deoxyglucose. Glucose uptake measurements were performed in duplicate and in three independent experiments. After 16 h of 0.5 mM palmitate treatment, cells were incubated in the presence or in the absence of 100 nM insulin for 30 min and then washed two times with wash buffer [20 mM HEPES (pH 7.4), 140 mM NaCl, 5 mM KCl, 2.5 mM MgSO4, and 1 mM CaCl2]. Cells were then incubated in buffer transport solution (wash buffer containing 0.5 mCi [3H]2-DG/ml and 10 µM 2-DG) for 10 min. Nonspecific uptake was determined incubating the cells in the presence or in the absence of 5 µM cytochalasin B. Uptake was terminated by aspiration of the solution. Cells were then washed three times, and radioactivity associated with the cells was determined by cell lysis in 0.05 M NaOH, followed by scintillation counting. Aliquots of cell lysates were used for protein content determination by the Bradford method. 2-DG uptake was expressed as picomoles per minute per milligram of protein.

Isolation of nuclear extracts
Nuclear extracts were isolated according to Andrews et al. (22). Cells were scraped into 1.5 ml of cold PBS, pelleted for 10 sec and resuspended in 400 µl of cold Buffer A [10 mM HEPES-KOH (pH 7.9) at 4 C, 1.5 mM MgCl2, 10 mM KCl, 0.5 mM dithiothreitol (DTT), 0.2 mM phenylmethylsulfonyl fluoride (PMSF), 5 µg/ml aprotinin, and 2 µg/ml leupeptin] by flicking the tube. Cells were allowed to swell on ice for 10 min, and then vortexed for 10 sec. Then, samples were centrifuged for 10 sec and the supernatant fraction discarded. Pellets were resuspended in 50 µl of cold Buffer C [20 mM HEPES-KOH (pH 7.9) at 4 C, 25% glycerol, 420 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 0.5 mM DTT, 0.2 mM PMSF, 5 µg/ml aprotinin, and 2 µg/ml leupeptin] and incubated on ice for 20 min for high-salt extraction. Cellular debris was removed by centrifugation for 2 min 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.

EMSA
EMSA was performed using double-stranded oligonucleotides (Promega, Madison, WI) for the consensus binding site of the NF-{kappa}B nucleotide (5'AGTTGAGGGGACTTTCCCAGGC-3') and Oct-1 (5'-TGTCGAATGCAAATCACTAGAA-3'). Oligonucleotides were labeled in the following reaction: 2 µl of oligonucleotide (1.75 pmol/µl), 2 µl of 5x kinase buffer, 1 µl of T4 polynucleotide kinase (10 U/µl), and 2.5 µl of [{gamma}-32P] ATP (3000 Ci/mmol at 10 mCi/ml) incubated at 37 C for 1 h. The reaction was stopped by adding 90 µl of TE buffer [10 mM Tris-HCl (pH 7.4) and 1 mM EDTA]. To separate the labeled probe from the unbound ATP the reaction mixture was eluted in a Nick column (Pharmacia, Sant Cugat, Spain) according to the manufacturer’s instructions. Five micrograms of crude nuclear proteins were incubated for 10 min on ice in binding buffer [10 mM Tris-HCl (pH 8.0), 25 mM KCl, 0.5 mM DTT, 0.1 mM EDTA (pH 8.0), 5% glycerol, 5 mg/ml BSA, 100 µg/ml tRNA and 50 µg/ml poly(deoxyinosine-deoxycytosine)], in a final volume of 15 µl. Labeled probe (~60,000 cpm) was added and the reaction was incubated for 15 min at room temperature. Where indicated, specific competitor oligonucleotide was added before the labeled probe and incubated for 10 min on ice. p65 Antibody was added 15 min before incubation with the labeled probe at 4 C. Protein-DNA complexes were resolved by electrophoresis at 4 C on a 5% acrylamide gel and subjected to autoradiography.

Immunoblotting
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, 5.4 µg/ml aprotinin]. The homogenate was centrifuged at 10,000 x g for 30 min at 4 C. For obtaining total membranes from C2C12 myotubes, cells were scraped into 5 ml of cold PBS, pelleted for 10 sec and resuspended in ice-cold HES-buffer containing proteinase inhibitors [250 mmol/liter sucrose, 1 mmol/liter EDTA, 1 mmol/liter PMSF, 1 µmol/liter aprotinin, 1 µmol/liter leupeptin, and 20 mmol/liter HEPES (pH 7.4)] and subsequently homogenized with 20 strokes in a glass Dounce homogenizer (Selecta, Barcelona, Spain) at 4 C. After centrifugation at 1000 x g for 3 min at 4 C to remove large cell debris and unbroken cells, the supernatant was then centrifuged at 245,000x g for 90 min at 4 C to yield a pellet of total cellular membranes and a supernatant representing the cytosolic fraction. The resulting pellet representing the total cellular membrane fraction was resuspended in HES buffer before use. Protein concentration was measured by the Bradford method. Proteins (30 µg) were separated by SDS-PAGE on 10% separation gels and transferred to Immobilon polyvinylidene diflouride membranes (Millipore, Bedford, MA). Western blot analysis was performed using antibodies against I{kappa}B{alpha}, I{kappa}Bß, phospho-PKC{epsilon}, total Akt (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), ß-tubulin (Sigma), TNF-{alpha} (R&D Systems, Minneapolis, MN), phospho-PKCßII (Ser660), phospho-PKC{delta} (Ser463), phospho-PKC{theta} (Thr538) and phospho-Akt (Ser473) (Cell Signaling Technology Inc., Beverly, MA). Detection was achieved using the EZ-ECL chemiluminescence detection kit (Biological Industries, Beit Hemeek Ltd., Jerusalem, Israel). Size of detected proteins was estimated using protein molecular mass standards (Invitrogen, Barcelona, Spain).

Statistical analyses
Results are expressed as means ± SD of four separate experiments. Significant differences were established by Student’s t test or one-way ANOVA, according to the number of groups compared, using the computer program GraphPad Instat (GraphPad Software version 2.03) (GraphPad Software Inc., San Diego, CA). In the latter case, when significant variations were found, the Tukey-Kramer multiple comparisons test was performed. Differences were considered significant at P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Palmitate induces TNF-{alpha} expression in skeletal muscle cells
To characterize the effects of FFA on the expression of TNF-{alpha}, we chose the saturated FFA palmitate (16:0), which is among the most common fatty acids (23). C2C12 myotubes were treated for 16 h with up to 0.75 mM palmitate, a concentration previously used in studies performed with these cells (19, 24) because concentrations of FFA up to 2 mM are found in serum. Incubation of skeletal muscle cells with 0.5 mM caused a 6.8-fold induction (P < 0.01) in the mRNA levels of TNF-{alpha} (Fig. 1AGo). When cultured cells were incubated with 0.75 mM palmitate, the mRNA levels of TNF-{alpha} were strongly induced (25-fold induction, P < 0.001), suggesting that small increases above 0.5 mM in the concentration of palmitate result in a strong induction in the expression of TNF-{alpha}. In agreement with the induction of the mRNA levels, enhanced TNF-{alpha} protein levels were observed in myotubes exposed to palmitate (Fig. 1BGo). No changes in the TNF-{alpha} mRNA levels were observed when cells were incubated with 0.5 mM oleate (Fig. 1CGo), suggesting that the effect of palmitate on TNF-{alpha} expression was specific for this saturated fatty acid.



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  		FIG. 1. Palmitate induces TNF-{alpha} expression in skeletal muscle cells. C2C12 myotubes were incubated in the presence or in the absence of palmitate for 16 h. A, Analysis of the mRNA levels of TNF-{alpha}. Total RNA (0.5 µg) was analyzed by RT-PCR. A representative autoradiogram and the quantification normalized to the APRT mRNA levels are shown. Data are expressed as mean ± SD of five different experiments. **, P < 0.01; ***, P < 0.001 vs. control. B, Protein extracts from C2C12 myotubes incubated in the presence or in the absence of 0.75 mM palmitate for 16 h were assayed for Western blot analysis with TNF-{alpha} and ß-tubulin antibodies. The blot data are representative of three separate experiments. C, Analysis of the mRNA levels of TNF-{alpha} in C2C12 myotubes incubated in the presence or in the absence of either 0.5 mM palmitate or oleate for 16 h. Total RNA (0.5 µg) was analyzed by RT-PCR. A representative autoradiogram and the quantification normalized to the APRT mRNA levels are shown. Data are expressed as mean ± SD of five different experiments. **, P < 0.01 vs. control. CT, Control.

 
Induced TNF-{alpha} expression negatively correlates with down-regulation of GLUT4 in palmitate-exposed skeletal muscle cells
Because impairment in insulin sensitivity has been associated to an increase in the mRNA levels of TNF-{alpha} and to a fall in GLUT4 mRNA in human skeletal muscle, and the expression of both genes were inversely correlated (25), we examined whether this association was present in the conditions used in this study. Treatment with palmitate caused a 57% reduction (P < 0.001) in the mRNA levels of GLUT4 in C2C12 myotubes (Fig. 2Go A). Interestingly, when we studied the relationship between GLUT4 and TNF-{alpha} mRNA levels we found a significant correlation (r2 = 0.85, P = 0.008, n = 8) (Fig. 2BGo). We also tested whether exposure to palmitate affected the uptake of glucose. A 16-h incubation period with 0.5 mM palmitate decreased absolute insulin-stimulated 2-DG uptake by 34% (P < 0.001 vs. insulin-stimulated cells incubated with BSA alone) (Fig. 2CGo). These findings suggest that the activation of the same pathway is responsible for the reduction of GLUT4 and the induction of TNF-{alpha}, linking both changes with the reduction in insulin sensitivity caused by pamitate treatment.



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  		FIG. 2. Palmitate reduces GLUT4 expression in skeletal muscle cells. C2C12 myotubes were incubated in the presence or in the absence of 0.75 mM palmitate for 16 h. A, Analysis of the mRNA levels of GLUT4. 0.5 µg of total RNA was analyzed by RT-PCR. A representative autoradiogram and the quantification normalized to the APRT mRNA levels are shown. Data are expressed as mean ± SD of five different experiments. **, P < 0.01 vs. control. B, Correlation between TNF-{alpha} and GLUT4 mRNA levels in control and palmitate-exposed myotubes. Eight pairs of data were analyzed. C, 2-DG uptake in the presence or in the absence of 100 nM insulin. Data are expressed as mean ± SD of four different experiments. *, P < 0.05 vs. non-insulin-treated cells. ###, P < 0.001 vs. insulin-stimulated cells. CT, Control; Ins, insulin.

 
Ceramides are not involved in palmitate-induced TNF-{alpha} expression in skeletal muscle cells
Because palmitoyl-coenzyme A is a precursor of sphingolipid synthesis, palmitate treatment may result in enhanced synthesis of ceramides (26), which can attenuate insulin signaling pathways leading to insulin resistance (19). Thus, to gain further insight into the mechanism by which palmitate up-regulates TNF-{alpha} mRNA levels, we examined the effects of one inhibitor of de novo ceramide synthesis. The initial step in ceramide synthesis is the formation of 3-ketodihydrosphingosine from palmitoyl-coenzyme A and L-serine. This step is inhibited by the sphingosine analog ISP1 (27). Induction of TNF-{alpha} mRNA expression (Fig. 3Go) caused by exposure to palmitate was not significantly affected by ISP1 treatment, although a 25% increase was observed compared with cells incubated only with palmitate. To further clarify the potential involvement of ceramides in the up-regulation of TNF-{alpha} caused by palmitate, we treated C2C12 skeletal muscle cells with C2-ceramide, a cell-permeable ceramide analog. Addition of 50 µM C2 ceramide did not cause any induction in the TNF-{alpha} mRNA levels. These data suggest that de novo ceramide synthesis is not involved in the effects of palmitate on TNF-{alpha} induction.



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  		FIG. 3. Ceramides are not involved in palmitate-induced TNF-{alpha} expression in skeletal muscle cells. C2C12 myotubes were incubated (16 h) with either 0.75 mM palmitate in the presence or in the absence of 100 nM ISP1, an inhibitor of de novo ceramide synthesis, or with 50 µM C2-ceramide, a cell-permeable ceramide analog. A, Analysis of the mRNA levels of TNF-{alpha}. Total RNA (0.5 µg) was analyzed by RT-PCR. A representative autoradiogram and the quantification normalized to the APRT mRNA levels are shown. Data are expressed as mean ± SD of five different experiments. ***, P < 0.001 vs. control.

 
The ERK-MAPK pathway is involved in palmitate-induced TNF-{alpha} expression but not in GLUT4 down-regulation
Because activation of the ERK-MAPK cascade has been reported to be activated by palmitate (27), we used pharmacological inhibitors to block upstream regulators of ERK1/2 to evaluate the involvement of the ERK-MAPK cascade in the induction of TNF-{alpha} after palmitate exposure. We assayed the effects of PD98059, a more general inhibitor of this pathway, and U0126, a more potent and specific ERK1/2 inhibitor, which binds to MAPK kinase, thereby inhibiting its catalytic activity and phosphorylation of ERK1/2. In the presence of PD98059, a 36% reduction (P < 0.001 vs. palmitate-treated cells) in the mRNA levels of TNF-{alpha} was observed, whereas U0126 nearly abolished (89% reduction, P < 0.001) the expression of TNF-{alpha} caused by palmitate treatment (Fig. 4AGo). Furthermore, we examined the effects of these inhibitors on the palmitate-mediated reduction of GLUT4 mRNA expression (Fig. 4BGo). Neither PD98059 nor U0126 significantly modified the fall in the expression of GLUT4 caused by palmitate. However, palmitate-exposed cells incubated with these inhibitors showed a tendency toward the recovery of the GLUT4 mRNA levels, as demonstrated by the 2-fold increase in the expression of GLUT4 compared with cells exposed only to palmitate.



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  		FIG. 4. The ERK-MAPK cascade is involved in palmitate-induced TNF-{alpha} expression in skeletal muscle cells. C2C12 myotubes were incubated (16 h) with either 0.75 mM palmitate in the presence or in the absence of 100 µM PD98059 or with 10 µM U0126. Analysis of the mRNA levels of TNF-{alpha} (A) and GLUT4 (B). Total RNA (0.5 µg) was analyzed by RT-PCR. A representative autoradiogram and the quantification normalized to the APRT mRNA levels are shown. Data are expressed as mean ± SD of five different experiments. Data are expressed as mean ± SD of five different experiments. ***, P < 0.001 vs. control; ###, P < 0.001 vs. palmitate-treated cells.

 
Palmitate-induced TNF-{alpha} expression is mediated through NF-{kappa}B and PKC activation
Because elevation of plasma FFA may lead to diacylglycerol-mediated activation of PKC (11), and this enzyme is known to activate NF-{kappa}B (9), we next investigated whether palmitate-induced TNF-{alpha} expression was mediated through activation of this pathway. To test whether incubation of C2C12 cells with palmitate led to increased NF-{kappa}B activity, we performed EMSA studies. NF-{kappa}B formed three complexes with nuclear proteins (complexes I to III) (Fig. 5AGo). Specificity of the three DNA-binding complexes was assessed in competition experiments by adding an excess of unlabeled NF-{kappa}B oligonucleotide. NF-{kappa}B binding activity, mainly of specific complexes II and III, increased in nuclear extracts from palmitate-treated cells. However, in the presence of PDTC, an inhibitor of NF-{kappa}B (28), the activation of this transcription factor was prevented. Addition of an antibody against the p65 subunit of NF-{kappa}B completely supershifted the three complexes, indicating that these bands were mainly contained this subunit. No changes were observed in the DNA binding of nuclear proteins from control and palmitate-treated cells to an Oct-1 probe, indicating that the increase observed for the NF-{kappa}B probe was specific (data not shown). NF-{kappa}B is located in the cytosol bound to the inhibitor {kappa}B (I{kappa}B) and inflammatory signals cause phosphorylation and ubiquitination of I{kappa}B{alpha}, thus liberating and activating NF-{kappa}B. Activation of PKC can lead to the activation of this transcription factor by directly phosphorylating I{kappa}B{alpha} (29). We next assessed whether palmitate resulted in changes in the content of I{kappa}B{alpha} (Fig. 5BGo). Palmitate addition to cells caused a 49% decrease (P < 0.01) in the abundance of I{kappa}B{alpha}, whereas I{kappa}Bß was only slightly reduced. To directly evaluate whether NF-{kappa}B activation was involved in palmitate-induction of TNF-{alpha}, we determined mRNA levels of this cytokine either in the presence or in the absence of the NF-{kappa}B inhibitors PDTC and parthenolide. The former is a potent antioxidant, whereas the second specifically inhibits activation of NF-{kappa}B by preventing I{kappa}B degradation (30). The 25.5-fold induction in the expression of TNF-{alpha} mRNA levels attained by palmitate were reduced by 62% (P < 0.001 vs. palmitate-treated cells) when C2C12 cells were coincubated with PDTC and by 63% (P < 0.001 vs. palmitate-treated cells) in cells coincubated with parthenolide (Fig. 6AGo). We next examined the effects of these NF-{kappa}B inhibitors on the fall of GLUT4 expression caused by exposure to palmitate (Fig. 6BGo). The 67% reduction (P < 0.01) in GLUT4 mRNA levels achieved by palmitate was not significantly modified by coincubation with either PDTC or parthenolide, although treatment with this latter inhibitor caused a reduction of less intensity (55%, P < 0.05 vs. control cells) in GLUT4 mRNA levels.



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  		FIG. 5. Palmitate treatment induces NF-{kappa}B activation in C2C12 myotubes. C2C12 myotubes were incubated in the presence or in the absence of 0.75 mM palmitate for 16 h. A, Autoradiograph of EMSA performed with a 32P-labeled NF-{kappa}B nucleotide and crude nuclear protein extract (NE). Three specific complexes (I–III), based on competition with a molar excess of unlabeled probe, are shown. A supershift analysis performed by incubating NE with an antibody directed against the p65 subunit of NF-{kappa}B is also shown. B, Protein extracts from C2C12 myotubes were assayed for Western blot analysis with I{kappa}B{alpha}, I{kappa}Bß, and ß-tubulin antibodies. Data are expressed as mean ± SD of three different experiments. **, P < 0.01 vs. control cells.

 


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  		FIG. 6. Palmitate-induced TNF-{alpha} expression is mediated through NF-{kappa}B activation. C2C12 myotubes were incubated for 16 h with 0.75 mM palmitate in the presence or in the absence of 5 mM PDTC and 10 µM parthenolide, two inhibitors of NF-{kappa}B. A, Analysis of the mRNA levels of TNF-{alpha} (A) and GLUT4 (B). Total RNA (0.5 µg) was analyzed by RT-PCR. A representative autoradiogram and the quantification normalized to the APRT mRNA levels are shown. Data are expressed as mean ± SD of five different experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001 vs. control. ###, P < 0.001 vs. palmitate-treated cells.

 
The involvement of PKC on palmitate-induced TNF-{alpha} expression was verified by using calphostin C, a strong and specific inhibitor of this enzyme (31). Cells preincubated with calphostin C (100 µM) for 30 min and subsequently exposed to 0.75 mM palmitate for 16 h, showed no induction in TNF-{alpha} expression (Fig. 7AGo). In addition, incubation of C2C12 cells with palmitate in the presence of calphostin C prevented the fall in the expression of GLUT4 expression caused by exposure to palmitate (Fig. 7BGo). These data indicate that PKC activation is involved in palmitate-induced TNF-{alpha} expression. Overall, these findings show that activation of the PKC-NF-{kappa}B pathway is responsible for the palmitate-induced TNF-{alpha} expression and the fall in GLUT4 mRNA levels in skeletal muscle cells.



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  		FIG. 7. Palmitate-induced TNF-{alpha} and GLUT4 down-regulation is mediated through PKC activation. C2C12 myotubes were incubated for 16 h with 0.75 mM palmitate in the presence or in the absence of 100 µM calphostin C. Analysis of the mRNA levels of TNF-{alpha} (A) and GLUT4 (B). Total RNA (0.5 µg) was analyzed by RT-PCR. A representative autoradiogram and the quantification normalized to the APRT mRNA levels are shown. Data are expressed as mean ± SD of five different experiments. **, P < 0.01 vs. control; #, P < 0.05 vs. palmitate-treated cells.

 
Palmitate activates PKC{theta} in C2C12 myotubes
To identify which PKC isoform was involved in the activation of NF-{kappa}B activation in palmitate-exposed myotubes, we performed Western blot assay using phospho-PKC-specific antibodies. Cells exposed to palmitate did not show changes in the phosphorylation status of PKC isoforms ßII, {delta} (Fig. 8AGo) or {epsilon} (data not shown). In contrast, palmitate induced phosphorylation of PKC{theta} (Fig. 8AGo). Then, we evaluated whether PKC activation was involved in the impairment of insulin signal transduction caused by palmitate by assessing insulin-stimulated Akt phosphorylation. As expected, insulin stimulated Akt phosphorylation, and this process was inhibited by palmitate (Fig. 8BGo). Interestingly, when palmitate-exposed cells were coincubated with chelerythrine, an inhibitor of the PKC catalytic site (32), the reduction in insulin-stimulated Akt phosphorylation was prevented, indicating that PKC activation by palmitate inhibited insulin signal transduction. Finally, we tested whether PKC activation was involved in palmitate-mediated NF-{kappa}B activation. Because palmitate seems to activate NF-{kappa}B through a mechanism involving I{kappa}B{alpha} degradation, we assessed the effect of chelerythrine on the expression of this NF-{kappa}B inhibitor. As shown in Fig. 8CGo, chelerythrine treatment prevented the fall in the expression of I{kappa}B{alpha} induced by palmitate, suggesting that PKC was involved in NF-{kappa}B activation by palmitate in myotubes.



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  		FIG. 8. Palmitate induces PKCq activation in C2C12 myotubes. A, Total membrane protein extracts from C2C12 myotubes incubated in the presence or in the absence of 0.75 mM palmitate for 16 h were assayed for Western blot analysis with specific phospho-PKC antibodies: phospo-PKCbII, phospho-PKCd and phospho-PKCq. The blot data are representative of three separate experiments. B, Prevention of PKC activation by palmitate improves insulin-stimulated Akt activation. C2C12 myotubes were incubated for 16 h with 0.75 mM palmitate in the presence or in the absence of 10 µM chelerytrine before stimulation with insulin (100 nM, 10 min). Cell lysates were assayed for Western blot analysis with anti-Akt and anti-phospho-Akt. The blot data are representative of three separate experiments. C, Protein extracts from C2C12 myotubes incubated for 16 h with 0.75 mM palmitate in the presence or in the absence of 10 µM chelerytrine were assayed for Western blot analysis with I{kappa}B{alpha} and ß-tubulin antibodies. Data are expressed as mean ± SD of three different experiments. **, P < 0.01 vs. control cells.

 

   Discussion
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The present study provides new insights into the mechanisms by which increased FFA availability may result in insulin resistance. Our data indicate that the saturated FFA palmitate causes a strong induction in the expression of the TNF-{alpha} gene in skeletal muscle cells, which is not expressed in untreated cells. This induction seems to involve the activation of the PKC-NF-{kappa}B pathway, linking increased plasma FFA with a proinflammatory state in diabetic patients. Furthermore, the inverse correlation between fatty acid-induced TNF-{alpha} expression and the down-regulation in GLUT4 mRNA levels is in agreement with Randle’s hypothesis of glucose/fatty acid fuel competition (33).

Previous studies have suggested that adipose-released TNF-{alpha} plays an important role in the development of insulin resistance (5, 6). However, the fact that circulating TNF-{alpha} in insulin-resistant obese subjects was undetectable in different clinical studies (34, 35) casts doubts about the contribution of adipose-derived TNF-{alpha} to muscle metabolism. Our findings open a new potential mechanism. According to the results here presented, an increase in the levels of circulating FFA, usually associated to the presence of visceral obesity, may lead to an increase in the expression of muscle TNF-{alpha}, which then can function in an autocrine fashion to cause insulin resistance (36). Similar results have been previously reported by Fabris et al. (37). They showed that high levels of circulating FFA directly increased TNF-{alpha} mRNA levels in red fiber-type muscle. However, because skeletal muscle contains other cells besides myocytes, it was necessary to clearly demonstrate the effects of FFA on TNF-{alpha} expression in cultured skeletal muscle cells to discard any potential contamination with adipose cells. It is worth noting that the kind of fatty acid may have a key role in the development of changes leading to insulin resistance. In this study we have used a saturated fatty acid, palmitate. Intramuscular triglycerides and the palmitate fraction of these triglycerides, but not the oleate fraction, were negatively correlated with insulin-stimulated glucose uptake (38). These data suggest that the palmitate fraction of fatty acids present in im triglycerides, which was increased in the muscle of obese patients (38) compared with lean subjects, may have deleterious effects on muscle metabolism.

Because an inverse linear relationship between the maximum glucose disposal rate and muscle TNF-{alpha} has been reported (5), this association may conform a critical key in the development of insulin resistance. Thus, besides the induction in the expression of TNF-{alpha}, palmitate treatment led to a simultaneous reduction in the expression of GLUT4 in skeletal muscle cells. In fact, it has been shown that TNF-{alpha} impairs insulin receptor signaling (36, 39, 40) and that TNF-{alpha} knockout mice have more GLUT4 protein expressed in muscle tissue (41). These findings suggest that exposure of skeletal muscle cells to increased FFA may shift skeletal muscle metabolism to preferential use of lipids rather than glucose as fuel substrates. This may be partially due to the induction of TNF-{alpha} and the inhibition of GLUT4 expression. Furthermore, the induction in the expression of TNF-{alpha} in skeletal muscle has been directly associated with im triglycerides content (25), which is the factor that correlates more tightly with insulin resistance (42). Thus, bariatric surgery in severely obese patients, which resulted in increased glucose disposal rate, was associated with a reduction in the expression of TNF-{alpha} and in the content of im triglycerides levels in skeletal muscle and with an increase in the mRNA levels of GLUT4 (25). All of these findings suggest that all the changes in the expression of TNF-{alpha}, GLUT4, and im triglycerides form part of the same process, which finally results in insulin resistance and type 2 diabetes mellitus. Thus, it is likely that all these changes may result from a common mechanism. Therefore, we studied the mechanism leading to the induction in TNF-{alpha} expression after palmitate treatment.

Data from this study seem to discard the involvement of ceramides in the palmitate-induced expression of TNF-{alpha} in skeletal muscle cells. Thus, ceramides, which are palmitate-derived lipid metabolites, seem not to be involved in the induction of TNF-{alpha} in skeletal muscle cells.

Elevated FFA presumably increases FFA uptake, exceeding its oxidation, a fact that in turns leads to increased im triglycerides and diacylglycerol, a potent allosteric activator of both conventional and novel PKC isoforms. Interestingly, in this study we demonstrate that palmitate treatment activated PKC{theta}, which is the most abundant PKC isoform in skeletal muscle (16, 18). Activation of PKC{theta} by palmitate could lead to insulin resistance by several mechanisms. This PKC isoform can phosphorylate insulin receptor substrate-1 (43), the major mediator of the insulin response in the muscle (44), leading to impaired insulin signaling. In fact, it has been reported that PKC{theta} knockout mice are protected from fat-induced insulin resistance (45). In agreement with these data, we report here that PKC inhibition prevents the fall in insulin-stimulated Akt phosphorylation caused by palmitate. Furthermore, PKC{theta} has the unique ability among the PKC isoforms to activate NF-{kappa}B (16), which has been linked to fatty acid-induced impairment of insulin action in skeletal muscle in rodents (9, 10) and regulates the expression of TNF-{alpha}. In humans, Itani et al. (7) reported that lipid infusion during a euglycemic-hyperinsulinemic clamp increased PKC activity and degradation of the mass of the NF-{kappa}B inhibitor I{kappa}B{alpha}. Activation of PKC can lead to the activation of NF-{kappa}B by directly phosphorylating I{kappa}B{alpha} (29) or by causing the generation of reactive oxygen species that can secondarily activate I{kappa}B-kinase. In fact, phosphorylation by I{kappa}B-kinase is considered the main pathway by which I{kappa}B{alpha} is released from NF-{kappa}B and subsequently subjected to ubiquination and proteosomal degradation. The result is a decrease in I{kappa}B{alpha} mass and movement of NF-{kappa}B from cytosol to the nucleus. In the present study, palmitate increased NF-{kappa}B activation as demonstrated by EMSA studies. Activation of this proinflammatory transcription factor seems to be mediated by degradation of I{kappa}B{alpha}. Furthermore, NF-{kappa}B binding sites are present in the TNF-{alpha} promoter (46, 47), supporting a role for NF-{kappa}B in the palmitate-mediated induction of this cytokine. This role was confirmed by pretreating cells with two inhibitors of NF-{kappa}B, PDTC and parthenolide, which reduced palmitate-mediated induction of TNF-{alpha}. As suggested above, activation of NF-{kappa}B can be attained by PKC. Our data confirm this possibility because pretreatment of the cells exposed to palmitate with PKC inhibitors completely abolished the changes in TNF-{alpha} and GLUT4 expression and prevented the I{kappa}B{alpha} degradation caused by palmitate. PKC is also an upstream regulator of the MAPK/ERK cascade (48, 49). The findings here presented suggest that activation the MAPK/ERK cascade is involved in the palmitate-induced expression of TNF-{alpha}, suggesting that PKC activation leads to the activation of this cascade. However, inhibitors of the MAPK/ERK cascade did not prevent the fall in GLUT4 expression, indicating that blockade of upstream targets, i.e. PKC, are necessary to achieve this objective.

In summary, the findings here reported suggest that blocking activation of PKC after palmitate treatment prevents some of the main changes involved in fatty acid-mediated insulin resistance. These data convert PKC in a potential drug-target for either the prevention or the treatment of fatty acid-induced insulin resistance.


   Acknowledgments
 
We thank the Language Advisory Service of the University of Barcelona for his helpful assistance.


   Footnotes
 
This study was partly supported by grants from the Fundació Privada Catalana de Nutrició i Lipids (FPCNL), Fundación Ramón Areces, Ministerio de Ciencia y Tecnología of Spain (SAF2003-01232), and European Union Fondos Europeos de Desarrollo funds. We also thank the Generalitat de Catalunya for grant 2001SGR00141. M.J. and A.P. were supported by grants from the Ministerio de Educación y Ciencia of Spain.

First Published Online October 13, 2005

Abbreviations: Aprt, Adenosyl phosphoribosyl transferase; DTT, dithiothreitol; FFA, free fatty acids; I{kappa}B, inhibitor protein subunit; NF, nuclear factor; PDTC, pyrrolidine dithiocarbamate; PKC, protein kinase C; PMSF, phenylmethylsulfonyl fluoride.

Received April 15, 2005.

Accepted for publication September 30, 2005.


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