Palmitate-Induced Interleukin 6 Production Is Mediated by Protein Kinase C and Nuclear-Factor B Activation and Leads to Glucose Transporter
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内分泌学杂志 2005年第7期
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@ub.edu.
Abstract
The mechanisms by which elevated levels of free fatty acids cause insulin resistance are not well understood. In addition, accumulating evidence suggests a link between inflammation and type 2 diabetes. Here, we report that exposure of C2C12 skeletal muscle cells to 0.5 mM palmitate results in increased mRNA levels (3.5-fold induction; P < 0.05) and secretion (control 375 ± 57 vs. palmitate 1129 ± 177 pg/ml; P < 0.001) of the proinflammatory cytokine IL-6. Palmitate increased nuclear factor-B activation and coincubation of the cells with palmitate and the nuclear factor-B inhibitor pyrrolidine dithiocarbamate prevented both IL-6 expression and secretion. Furthermore, incubation of palmitate-treated cells with calphostin C, a strong and specific inhibitor of protein kinase C, and phorbol myristate acetate, that down-regulates protein kinase C in long-term incubations, abolished induction of IL-6 production. Finally, exposure of skeletal muscle cells to palmitate caused a fall in the mRNA levels of glucose transporter 4 and insulin-stimulated glucose uptake, whereas in the presence of anti-IL-6 antibody, which neutralizes the biological activity of mouse IL-6 in cell culture, these reductions were prevented. These findings suggest that IL-6 may mediate several of the prodiabetic effects of palmitate.
Introduction
INSULIN RESISTANCE IS a major characteristic of type 2 diabetes mellitus and is also associated with obesity, hypertension, and cardiovascular disease (1). Skeletal muscle accounts for the majority of insulin-stimulated glucose use and is, therefore, the major site of insulin resistance. During the development of insulin resistance in skeletal muscle, an impairment of glucose use and insulin sensitivity has been related to the presence of elevated plasma free fatty acids (FFA). Different evidence supports that elevated FFA are responsible for much of the insulin resistance present in type 2 diabetic patients. Thus, several studies have consistently demonstrated that elevations of plasma FFA produce insulin resistance in diabetic patients and in nondiabetic subjects (2, 3, 4, 5). In addition, elevation of plasma FFA may lead to diacylglycerol-mediated activation of protein kinase C (PKC) (6, 7), an enzyme that has been linked to insulin resistance in a wide variety of rodent models (8, 9, 10), including rats infused with lipid (11) and massively obese humans (12, 13). Furthermore, one of these FFA, palmitate, once activated to palmitoyl-coenzyme A (palmitoyl-CoA) by acyl-CoA synthase, is the precursor of de novo synthesis of ceramides, which can attenuate insulin signaling pathways leading to insulin resistance (14). Despite these data, the mechanisms by which elevated levels of FFA cause insulin resistance are not well understood.
Accumulating evidence suggests a link between inflammation and type 2 diabetes. Markers of inflammation, including proinflammatory cytokines (such as TNF, IL-1, interferon-, and IL-6) have been reported to be elevated in type 2 diabetes (15, 16). Of these cytokines, IL-6 presents the strongest correlation with insulin resistance and type 2 diabetes (15, 16, 17), and its plasma levels are increased 2- to 3-fold in patients with obesity and type 2 diabetes compared with lean control subjects (16). Until recently, the main source of IL-6 production was thought to be macrophages and peripheral mononuclear cells. However, recent evidence suggests that adipose and skeletal muscle cells are important sites of IL-6 production. This cytokine is expressed in resting human skeletal muscle, and contraction rapidly increases its gene expression (18, 19). In addition, recent studies have reported that insulin increases IL-6 gene expression in insulin-resistant, but not healthy, skeletal muscle, suggesting that IL-6 expression in skeletal muscle is sensitive to unknown changes associated with insulin resistance (20). Regarding the molecular pathways responsible for the induction of IL-6 gene expression in skeletal muscle, it has been reported that both reactive oxygen species (21) and lipopolysaccharide (22) can up-regulate IL-6 in skeletal muscle, probably through a mechanism involving activation of nuclear factor (NF)-B. There is also evidence to suggest that increased transcription of IL-6 occurs via activation of MAPK (23).
The purpose of this study was to investigate the mechanisms responsible for FFA-induced insulin resistance. Using mouse skeletal muscle C2C12 myotubes, we examined the effects of the saturated FFA palmitate on IL-6 gene expression and protein secretion. Preincubation of the cells with palmitate led to increased IL-6 gene expression and secretion through mechanisms involving activation of NF-B and PKC. Furthermore, by using an antibody that neutralizes the biological activity of mouse IL-6 in cell culture we demonstrate that IL-6 is responsible for the down-regulation of glucose transporter 4 (GLUT4) and glucose uptake in palmitate-incubated skeletal muscle cells. These results suggest that secretion of the proinflammatory IL-6 may be involved in fatty acid-induced insulin resistance in skeletal muscle.
Materials and Methods
Materials
Anti-IL-6 antibody, C2-ceramide, ISP1, and pyrrolidine dithiocarbamate (PDTC) were from Sigma Chemical Co. (St. Louis, MO). PD98059 and calphostin C were from Biomol Research Labs Inc. (Plymouth Meeting, PA). Other chemicals were from Sigma.
Cell culture
Mouse C2C12 myoblasts (American Type Culture Collection, Rockville, MD) 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. (14). Briefly, FFA were dissolved in ethanol and diluted 1:100 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 (FFA-treated cells) or absence (control cells) of FFA. Cells were then incubated with 100 nM insulin for 10 min. After the incubation, RNA was extracted from myotubes as described below.
IL-6 measurements
Levels of IL-6 mRNA were assessed by RT-PCR (24) as previously described (25). 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 as follows: IL-6, 5'-TCCAGCCAGTTGCCTTCTTGG-3' and 5'-TCTGACAGTGCATCATCGCTG-3'; GLUT4, 5'-GATGCCGTCGGGTTTCCAGCA-3' and 5'-TGAGGGTGCCTTGTGGGATGG-3'; and adenosyl phosphoribosyl transferase (APRT), 5'-GCCTCTTGGCCAGTCACCTGA-3' and 5'-CCAGGCTCACACACTCCACCA-3'. Amplification of each gene yielded a single band of the expected size (IL-6, 229 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 (26). 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).
Culture supernatants were collected, and the secretion of IL-6 was assessed by ELISA (Amersham Biosciences, Little Chalfont, UK). All determinations were performed in triplicate.
Isolation of nuclear extracts
Nuclear extracts were isolated according to Andrews et al. (27). 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-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 [-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(dI-dC)], in a final volume of 15 μl. Labeled probe (approximately 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 PMSF, 1 mM sodium orthovanadate, and 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 collected into 10 ml of ice-cold HES buffer (250 mmol/liter sucrose, 1 mmol/liter EDTA, 1 mmol/liter PMSF, 1 μmol/liter pepstatin, 1 μmol/liter aprotinin, 1 μmol/liter leupeptin, and 20 mmol/liter HEPES, pH 7.4) and subsequently homogenized 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 245,000 x 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 μg) were separated by SDS-PAGE on 10% separation gels and transferred to Immobilon polyvinylidene difluoride membranes (Millipore, Bedford, MA). Western blot analysis was performed using antibodies against inhibitor B (IB) and IB? (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). Detection was achieved using the EZ-ECL chemiluminescence detection kit (Biological Industries, Beit Hemeek Ltd., Jerusalem, Israel). Equal loading of proteins was assessed by red phenol staining. Size of detected proteins was estimated using protein molecular-mass standards (Invitrogen, Barcelona, Spain).
Determination of glucose uptake by C2C12 skeletal muscle cells
Glucose uptake was assayed using [3H]2-deoxyglucose (2-DG). 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 by 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.
Statistical analyses
Results are expressed as means ± SD of four separate experiments. Differences between group means were determined by a one-way ANOVA using the computer program GraphPad Instat (version 2.03; GraphPad Software Inc., San Diego, CA). When significant variations were found, the Tukey-Kramer multiple comparisons test was performed. Differences were considered significant at P < 0.05.
Results
Palmitate, but not oleate, induces IL-6 expression and secretion in skeletal muscle cells
To study the effects of FFA on IL-6 production by C2C12 skeletal muscle cells, we chose the saturated FFA palmitate (16:0) and the monounsaturated FFA oleate (18:1 n-9), which are among the most common fatty acids found in skeletal muscle (27). FFA concentrations up to 2 mM are within the physiological serum range in humans and in rodents (28). C2C12 cells were treated with 0.5 mM oleate and 0.5 mM palmitate for 16 h. When we analyzed the effects of these FFA on IL-6 expression, a 3.5-fold induction (P < 0.05) was observed in the mRNA levels of IL-6 after palmitate treatment, whereas oleate reduced the expression of this cytokine, although differences did not reach significance (Fig. 1A). Similarly, incubation of C2C12 myotubes with palmitate caused a 3-fold induction in the levels of IL-6 protein secreted into the culture media (control 375 ± 57 vs. palmitate 1129 ± 177 pg/ml; P < 0.001) (Fig. 1B) and oleate reduced secretion (control 375 ± 57 vs. oleate 196 ± 25 pg/ml) (Fig. 1B).
FIG. 1. Palmitate (Palm.), but not oleate, induces IL-6 expression and secretion in skeletal muscle cells. C2C12 myotubes were incubated in the presence or in the absence of the indicated FFA (16 h, 0.5 mM). A, Analysis of the mRNA levels of IL-6; 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. B, Determination by ELISA of IL-6 secretion to the culture media. Data are expressed as mean ± SD of three different experiments. *, P < 0.05; ***, P < 0.001 vs. control.
Ceramides and the ERK-MAPK pathway are not involved in palmitate-induced IL-6 expression and secretion in skeletal muscle cells
Because palmitoyl-CoA is a precursor of sphingolipid synthesis, palmitate treatment may result in enhanced ceramide synthesis and apoptosis (29). Thus, to gain further insight into the mechanism by which palmitate up-regulates IL-6 mRNA levels and secretion, we tested the effects of one inhibitor of de novo ceramide synthesis. The initial step in ceramide synthesis is the formation of 3-ketodihydrosphingosine from palmitoyl-CoA and L-serine. This step is inhibited by the sphingosine analog ISP1 at picomole concentrations (30). Although a small reduction was observed, ISP1 treatment affected neither IL-6 mRNA (Fig. 2A) nor IL-6 secretion (Fig. 2B) reached by palmitate. To further clarify the potential involvement of ceramides in the up-regulation of IL-6 caused by palmitate, we treated C2C12 skeletal muscle cells with C2-ceramide, a cell-permeable ceramide analog. Addition of 50 μM C2-ceramide modified neither IL-6 mRNA levels (Fig. 2A) nor IL-6 secretion (Fig. 2B). These data suggest that de novo ceramide synthesis is not involved in the effects of palmitate on IL-6 production.
FIG. 2. Ceramides are not involved in palmitate-induced IL-6 expression and secretion in skeletal muscle cells. C2C12 myotubes were incubated (16 h) with either 0.5 mM palmitate (Palm.) in the presence or in the absence of 100 nM ISP1, an inhibitor of de novo ceramide synthesis. Cells were also treated with 50 μM C2-ceramide, a cell-permeable ceramide analog. A, Analysis of the mRNA levels of IL-6; 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. B, Determination by ELISA of IL-6 secretion to the culture media. Data are expressed as mean ± SD of three different experiments. *, P < 0.05; **, P < 0.001; ***, P < 0.001 vs. control.
Because activation of MAPK has been associated with increased IL-6 expression, we assayed the effects of PD98059 and U0126, two known inhibitors of the ERK-MAPK pathway, on IL-6 expression and secretion. In the presence of either 100 μM PD98059 or 10 μM U0126, no changes were observed either in the expression (Fig. 3A) or in the secretion (Fig. 3B) to the culture media of this cytokine.
FIG. 3. The ERK-MAPK pathway is not involved in palmitate-induced IL-6 expression and secretion in skeletal muscle cells. C2C12 myotubes were incubated (16 h) with 0.5 mM palmitate in the presence or in the absence of 100 μM PD98059 or 10 μM U0126, inhibitors of the ERK-MAPK pathway. A, Analysis of the mRNA levels of IL-6; 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. B, Determination by ELISA of IL-6 secretion to the culture media. Data are expressed as mean ± SD of three different experiments. ***, P < 0.001 vs. control.
Palmitate-induced IL-6 expression and secretion is mediated through NF-B activation
Recent studies have reported that activation of the redox transcription factor NF-B plays an important role in IL-6 production from myocytes (21). To test whether incubation of C2C12 cells with palmitate led to increased NF-B activity we performed EMSA studies. NF-B formed three complexes with nuclear proteins (complexes I–III) (Fig. 4A). Specificity of the three DNA-binding complexes was assessed in competition experiments by adding an excess of unlabeled NF-B oligonucleotide. NF-B binding activity, mainly of specific complex II, increased in nuclear extracts from palmitate-treated cells. Addition of antibody against the p65 subunit of NF-B completely supershifted complex II, indicating that this band was mainly constituted of 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-B probe was specific (Fig. 4B).
FIG. 4. Palmitate treatment induces NF-B activation in C2C12 myotubes. C2C12 myotubes were incubated in the presence or in the absence of 0.5 mM palmitate (Palm) for 16 h. A, Autoradiograph of EMSA performed with a 32P-labeled NF-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-kB is also shown. B, Autoradiograph of EMSA performed with a 32P-labeled Oct-1 nucleotide. C, Protein extracts from C2C12 myotubes were assayed for Western blot analysis with IB and IB? antibodies. A representative blot is shown. Data normalized to ?-tubulin levels are expressed as mean ± SD of three different experiments.
NF-B is located in the cytosol bound to IB, and inflammatory signals cause phosphorylation and ubiquitination of IB, thus liberating and activating NF-B. We next assessed whether palmitate resulted in changes in the content of IB (Fig. 4C). Palmitate addition to cells caused approximately a 49% (P < 0.01) decrease in the abundance of IB, whereas the 16% reduction achieved in the levels of IB? were not statistically significant. To evaluate whether NF-B activation was involved in palmitate induction of IL-6, we took advantage of the use of two NF-B inhibitors, PDTC (31) and parthenolide (32). The 5-fold induction in the expression of IL-6 mRNA levels attained by palmitate was prevented when C2C12 cells were coincubated with either PDTC (1.3-fold induction; P < 0.001 vs. palmitate) or parthenolide (2.6-fold induction; P < 0.01 vs. palmitate) (Fig. 5A). Likewise, the 4-fold induction in the IL-6 secretion to the culture medium caused by palmitate was reduced by coincubation of cells with PDTC (1.7-fold induction; P < 0.01 vs. palmitate) and parthenolide (1.5-fold induction; P < 0.001 vs. palmitate) (Fig. 5B). These results suggest that expression and secretion of IL-6 are regulated by NF-B activation.
FIG. 5. Palmitate-induced IL-6 expression is mediated through NF-B activation. C2C12 myotubes were incubated (16 h) with 0.5 mM palmitate in the presence or in the absence of 5 mM PDTC or 10 μM parthenolide, two inhibitors of NF-B. A, Analysis of the mRNA levels of IL-6; 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. B, Determination by ELISA of IL-6 secretion to the culture media. Data are expressed as mean ± SD of three different experiments. *, P < 0.05; ***, P < 0.001 vs. control; #, P < 0.05; ##, P < 0.01; ###, P < 0.001 vs. palmitate.
Palmitate-induced IL-6 expression and secretion is mediated through PKC activation
Because elevation of plasma FFA may lead to diacylglycerol-mediated activation of PKC (6), and this enzyme is known to activate NF-B (33), we next investigated whether palmitate-induced IL-6 expression and secretion involved PKC activation. To test this hypothesis, we used two strategies. First, we verified the effect of calphostin C, a strong and specific inhibitor of PKC (34), on palmitate-induced IL-6 expression and secretion in C2C12 myotubes. Cells were preincubated with calphostin C (100 μM) for 30 min and subsequently stimulated with 0.5 mM palmitate for 16 h. Furthermore, we pretreated cells with 0.5 μM phorbol myristate acetate (PMA) for 24 h before stimulation with palmitate. This long-term pretreatment with PMA causes PKC down-regulation (35, 36). As shown in Fig. 6, calphostin C did not affect palmitate-induced IL-6 mRNA levels, whereas it abolished IL-6 secretion to the culture media. Pretreatment of C2C12 cells with PMA completely abolished both IL-6 expression and secretion. These data indicate that PKC activation is involved in palmitate-induced IL-6 secretion.
FIG. 6. Palmitate-induced IL-6 expression and secretion is mediated through PKC activation. C2C12 myotubes were incubated (16 h) with 0.5 mM palmitate (Palm) in the presence or in the absence of either 100 μM calphostin C or 500 nM PMA. A, Analysis of the mRNA levels of IL-6; 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. B, Determination by ELISA of IL-6 secretion to the culture media. Data are expressed as mean ± SD of three different experiments. *, P < 0.05; ***, P < 0.001 vs. control; #, P < 0.05; ###, P < 0.001 vs. palmitate.
IL-6 secretion is responsible for the down-regulation of GLUT4 induced by palmitate
Finally, we examined whether some metabolic effects associated with elevated FFA in skeletal muscle cells were mediated by IL-6 secretion. Thus, to verify the contribution of IL-6 to the effects caused by elevated FFA, we took advantage of using an anti-IL-6 antibody. Previous studies have reported that IL-6 induces insulin resistance in 3T3-L1 adipocytes, causing reduction in the expression of GLUT4 (37). Treatment with palmitate caused a 57% reduction in the mRNA levels of GLUT4 in C2C12 myotubes (P < 0.001), but coincubation with anti-IL-6 antibody prevented this change (Fig. 7A). Finally, we tested whether these changes 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), and this was prevented by incubating the cells in the presence of anti-IL-6 antibody (Fig. 7B).
FIG. 7. IL-6 secretion is responsible for the down-regulation of GLUT4 induced by palmitate. C2C12 myotubes were incubated (16 h) with 0.5 mM palmitate (Palm) in the presence or in the absence of 5 μg/ml anti-IL-6 antibody (Ab). 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 three different experiments. **, P < 0.01 vs. control. B, 2-DG uptake in the presence or in the absence of 100 nM insulin (Ins). 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; , P < 0.05 vs. insulin-stimulated cells incubated with palmitate.
Discussion
Skeletal muscle insulin resistance is the major characteristic of type 2 diabetes mellitus (1). The mechanisms responsible for the reduced sensitivity of muscle to insulin still remain unclear, but there is a strong correlation between insulin resistance and elevated FFA. In addition, accumulating evidence suggests a link between inflammation and type 2 diabetes. The proinflammatory cytokine IL-6 presents a strong correlation with insulin resistance and type 2 diabetes (16, 17). Previous recent reports have suggested that IL-6 impairs insulin sensitivity and glucose regulation in adipocytes (39, 40) and hepatocytes (41, 42, 43). In this study, we have used the myoblast C2C12 cell line, which develops biochemical and morphological properties characteristic of skeletal muscle and has been proven useful for studies of skeletal muscle metabolism (44). Exposure of these skeletal muscle cells to palmitate results in increased IL-6 expression and secretion. The current results provide direct strong evidence that IL-6 secretion may be responsible for part of the effects of the saturated fatty acid palmitate on insulin resistance in skeletal muscle cells. Moreover, data presented indicate that PKC and NF-B activation are involved in palmitate-induced IL-6 production. It is worth noting that, in contrast to palmitate, oleate reduced IL-6 expression and secretion. These findings suggest that palmitate-induced IL-6 expression and secretion were specific for this saturated fatty acid. Additional studies are necessary to confirm whether this monounsaturated fatty acid may counteract the effects of palmitate on IL-6 expression and secretion.
Elevated FFA presumably increases FFA uptake, exceeding its oxidation, which in turns leads to increased intramuscular triglycerides and diacylglycerol, a potent allosteric activator of both conventional and novel PKC isoforms. Activation of PKC could lead to insulin resistance by several mechanisms. This enzyme can phosphorylate both the insulin receptor (13, 45) and insulin receptor substrate-1 (37), leading to impaired insulin signaling; or it can increase oxidative stress and activate NF-B. The results presented here show that a long treatment with PMA, which results in PKC down-regulation (35, 36), completely abolishes both palmitate-induced IL-6 expression and secretion. Pretreatment with calphostin C, a strong and specific inhibitor of PKC (36), also prevented palmitate-induced IL-6 secretion to the culture media. In contrast, this PKC inhibitor did not affect IL-6 expression caused by palmitate. Previous studies have reported abolishment of induced IL-6 expression by calphostin C in osteoblastic cells after 3 h (36), suggesting that the lack of inhibitory effect of calphostin C in C2C12 cells after 16 h of treatment could be attributed to recovery of PKC activity that led to increased mRNA expression of IL-6, but not protein, because secretion of this cytokine was still blocked.
As stated above, PKC activation can increase oxidative stress and NF-B activation. Itani et al. (46) reported that lipid infusion in humans during a euglycemic-hyperinsulinemic clamp increases PKC activity and degradation of the mass of the NF-B inhibitor IB. Activation of PKC can lead to the activation of this transcription factor by directly phosphorylating IB (47) or by causing the generation of reactive oxygen species that can secondarily activate IB-kinase. In fact, phosphorylation by IB-kinase is considered the main pathway by which IB is released from NF-B and subsequently subjected to ubiquitination and proteosomal degradation. The result is a decrease in IB mass and movement of NF-B from cytosol to the nucleus. In the present study, palmitate increased NF-B activation as demonstrated by EMSA studies. Activation of this proinflammatory transcription factor seems to be mediated by degradation of IB. The mouse IL-6 promoter contains a consensus sequence for the redox-sensitive transcription factor NF-B, supporting a role for NF-B in the palmitate-mediated induction of IL-6. This role was confirmed by pretreating cells with inhibitors of NF-B, which prevented the palmitate-mediated induction of IL-6 mRNA levels and secretion to the culture media. Recent studies support the findings that palmitate leads to NF-B activation (48) and that palmitate-induced insulin resistance is prevented by inhibition of this transcription factor (38).
The data of this study also discard the involvement of several processes in the palmitate-induced production of IL-6 in skeletal muscle cells. Thus, ceramides, which are palmitate-derived lipid metabolites, seem not to be involved in the induction of IL-6 in skeletal muscle cells. There is also evidence showing that activation of the MAPK signaling cascade increases transcription of IL-6 (20, 23). However, coincubation of cells with inhibitors of the ERK-MAPK pathway did not affect either expression or secretion of IL-6, making unlikely the involvement of this signaling cascade in IL-6 changes mediated by palmitate.
Finally, we evaluated the metabolic effects caused by palmitate-induced IL-6 secretion. To clearly differentiate between the effects caused by either palmitate or IL-6, we used an anti-IL-6 antibody. A previous study performed in adipocytes reported a reduction in the expression of GLUT4 after IL-6 treatment (40). In the skeletal muscle cells used in this study, palmitate addition to the culture media caused a reduction in the expression of GLUT4 and insulin-stimulated glucose uptake, changes that were prevented in the presence of the antibody against IL-6, clearly involving this cytokine in some of the prodiabetic effects of palmitate. The data shown here do not permit us to establish which is the IL-6-dependent mechanism responsible for the reduction in GLUT4 expression. Because palmitate impairs serine/threonine protein kinase Akt phosphorylation (14), it is tempting to speculate that IL-6 may mediate changes in the phosphorylation of the Akt, which promotes translocation of glucose transporter GLUT4 to the plasma membrane. However, the reduction in GLUT4 mRNA levels achieved after palmitate treatment also suggests that IL-6 may cause changes in the transcriptional rate of this glucose transporter.
In summary, here we report that palmitate treatment of skeletal muscle cells induces IL-6 expression and secretion through mechanisms involving the activation of the axis PKC-NF-B. Furthermore, IL-6 production seems to be responsible for the reduction in GLUT4 expression in skeletal muscle exposed to palmitate. These results support recent reports showing that IL-6 is involved in insulin resistance and converts this cytokine and the mechanisms responsible for its induction in a target for the treatment of lipid-mediated insulin resistance.
Acknowledgments
We thank the Language Advisory Service of the University of Barcelona for helpful assistance.
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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@ub.edu.
Abstract
The mechanisms by which elevated levels of free fatty acids cause insulin resistance are not well understood. In addition, accumulating evidence suggests a link between inflammation and type 2 diabetes. Here, we report that exposure of C2C12 skeletal muscle cells to 0.5 mM palmitate results in increased mRNA levels (3.5-fold induction; P < 0.05) and secretion (control 375 ± 57 vs. palmitate 1129 ± 177 pg/ml; P < 0.001) of the proinflammatory cytokine IL-6. Palmitate increased nuclear factor-B activation and coincubation of the cells with palmitate and the nuclear factor-B inhibitor pyrrolidine dithiocarbamate prevented both IL-6 expression and secretion. Furthermore, incubation of palmitate-treated cells with calphostin C, a strong and specific inhibitor of protein kinase C, and phorbol myristate acetate, that down-regulates protein kinase C in long-term incubations, abolished induction of IL-6 production. Finally, exposure of skeletal muscle cells to palmitate caused a fall in the mRNA levels of glucose transporter 4 and insulin-stimulated glucose uptake, whereas in the presence of anti-IL-6 antibody, which neutralizes the biological activity of mouse IL-6 in cell culture, these reductions were prevented. These findings suggest that IL-6 may mediate several of the prodiabetic effects of palmitate.
Introduction
INSULIN RESISTANCE IS a major characteristic of type 2 diabetes mellitus and is also associated with obesity, hypertension, and cardiovascular disease (1). Skeletal muscle accounts for the majority of insulin-stimulated glucose use and is, therefore, the major site of insulin resistance. During the development of insulin resistance in skeletal muscle, an impairment of glucose use and insulin sensitivity has been related to the presence of elevated plasma free fatty acids (FFA). Different evidence supports that elevated FFA are responsible for much of the insulin resistance present in type 2 diabetic patients. Thus, several studies have consistently demonstrated that elevations of plasma FFA produce insulin resistance in diabetic patients and in nondiabetic subjects (2, 3, 4, 5). In addition, elevation of plasma FFA may lead to diacylglycerol-mediated activation of protein kinase C (PKC) (6, 7), an enzyme that has been linked to insulin resistance in a wide variety of rodent models (8, 9, 10), including rats infused with lipid (11) and massively obese humans (12, 13). Furthermore, one of these FFA, palmitate, once activated to palmitoyl-coenzyme A (palmitoyl-CoA) by acyl-CoA synthase, is the precursor of de novo synthesis of ceramides, which can attenuate insulin signaling pathways leading to insulin resistance (14). Despite these data, the mechanisms by which elevated levels of FFA cause insulin resistance are not well understood.
Accumulating evidence suggests a link between inflammation and type 2 diabetes. Markers of inflammation, including proinflammatory cytokines (such as TNF, IL-1, interferon-, and IL-6) have been reported to be elevated in type 2 diabetes (15, 16). Of these cytokines, IL-6 presents the strongest correlation with insulin resistance and type 2 diabetes (15, 16, 17), and its plasma levels are increased 2- to 3-fold in patients with obesity and type 2 diabetes compared with lean control subjects (16). Until recently, the main source of IL-6 production was thought to be macrophages and peripheral mononuclear cells. However, recent evidence suggests that adipose and skeletal muscle cells are important sites of IL-6 production. This cytokine is expressed in resting human skeletal muscle, and contraction rapidly increases its gene expression (18, 19). In addition, recent studies have reported that insulin increases IL-6 gene expression in insulin-resistant, but not healthy, skeletal muscle, suggesting that IL-6 expression in skeletal muscle is sensitive to unknown changes associated with insulin resistance (20). Regarding the molecular pathways responsible for the induction of IL-6 gene expression in skeletal muscle, it has been reported that both reactive oxygen species (21) and lipopolysaccharide (22) can up-regulate IL-6 in skeletal muscle, probably through a mechanism involving activation of nuclear factor (NF)-B. There is also evidence to suggest that increased transcription of IL-6 occurs via activation of MAPK (23).
The purpose of this study was to investigate the mechanisms responsible for FFA-induced insulin resistance. Using mouse skeletal muscle C2C12 myotubes, we examined the effects of the saturated FFA palmitate on IL-6 gene expression and protein secretion. Preincubation of the cells with palmitate led to increased IL-6 gene expression and secretion through mechanisms involving activation of NF-B and PKC. Furthermore, by using an antibody that neutralizes the biological activity of mouse IL-6 in cell culture we demonstrate that IL-6 is responsible for the down-regulation of glucose transporter 4 (GLUT4) and glucose uptake in palmitate-incubated skeletal muscle cells. These results suggest that secretion of the proinflammatory IL-6 may be involved in fatty acid-induced insulin resistance in skeletal muscle.
Materials and Methods
Materials
Anti-IL-6 antibody, C2-ceramide, ISP1, and pyrrolidine dithiocarbamate (PDTC) were from Sigma Chemical Co. (St. Louis, MO). PD98059 and calphostin C were from Biomol Research Labs Inc. (Plymouth Meeting, PA). Other chemicals were from Sigma.
Cell culture
Mouse C2C12 myoblasts (American Type Culture Collection, Rockville, MD) 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. (14). Briefly, FFA were dissolved in ethanol and diluted 1:100 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 (FFA-treated cells) or absence (control cells) of FFA. Cells were then incubated with 100 nM insulin for 10 min. After the incubation, RNA was extracted from myotubes as described below.
IL-6 measurements
Levels of IL-6 mRNA were assessed by RT-PCR (24) as previously described (25). 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 as follows: IL-6, 5'-TCCAGCCAGTTGCCTTCTTGG-3' and 5'-TCTGACAGTGCATCATCGCTG-3'; GLUT4, 5'-GATGCCGTCGGGTTTCCAGCA-3' and 5'-TGAGGGTGCCTTGTGGGATGG-3'; and adenosyl phosphoribosyl transferase (APRT), 5'-GCCTCTTGGCCAGTCACCTGA-3' and 5'-CCAGGCTCACACACTCCACCA-3'. Amplification of each gene yielded a single band of the expected size (IL-6, 229 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 (26). 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).
Culture supernatants were collected, and the secretion of IL-6 was assessed by ELISA (Amersham Biosciences, Little Chalfont, UK). All determinations were performed in triplicate.
Isolation of nuclear extracts
Nuclear extracts were isolated according to Andrews et al. (27). 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-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 [-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(dI-dC)], in a final volume of 15 μl. Labeled probe (approximately 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 PMSF, 1 mM sodium orthovanadate, and 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 collected into 10 ml of ice-cold HES buffer (250 mmol/liter sucrose, 1 mmol/liter EDTA, 1 mmol/liter PMSF, 1 μmol/liter pepstatin, 1 μmol/liter aprotinin, 1 μmol/liter leupeptin, and 20 mmol/liter HEPES, pH 7.4) and subsequently homogenized 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 245,000 x 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 μg) were separated by SDS-PAGE on 10% separation gels and transferred to Immobilon polyvinylidene difluoride membranes (Millipore, Bedford, MA). Western blot analysis was performed using antibodies against inhibitor B (IB) and IB? (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). Detection was achieved using the EZ-ECL chemiluminescence detection kit (Biological Industries, Beit Hemeek Ltd., Jerusalem, Israel). Equal loading of proteins was assessed by red phenol staining. Size of detected proteins was estimated using protein molecular-mass standards (Invitrogen, Barcelona, Spain).
Determination of glucose uptake by C2C12 skeletal muscle cells
Glucose uptake was assayed using [3H]2-deoxyglucose (2-DG). 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 by 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.
Statistical analyses
Results are expressed as means ± SD of four separate experiments. Differences between group means were determined by a one-way ANOVA using the computer program GraphPad Instat (version 2.03; GraphPad Software Inc., San Diego, CA). When significant variations were found, the Tukey-Kramer multiple comparisons test was performed. Differences were considered significant at P < 0.05.
Results
Palmitate, but not oleate, induces IL-6 expression and secretion in skeletal muscle cells
To study the effects of FFA on IL-6 production by C2C12 skeletal muscle cells, we chose the saturated FFA palmitate (16:0) and the monounsaturated FFA oleate (18:1 n-9), which are among the most common fatty acids found in skeletal muscle (27). FFA concentrations up to 2 mM are within the physiological serum range in humans and in rodents (28). C2C12 cells were treated with 0.5 mM oleate and 0.5 mM palmitate for 16 h. When we analyzed the effects of these FFA on IL-6 expression, a 3.5-fold induction (P < 0.05) was observed in the mRNA levels of IL-6 after palmitate treatment, whereas oleate reduced the expression of this cytokine, although differences did not reach significance (Fig. 1A). Similarly, incubation of C2C12 myotubes with palmitate caused a 3-fold induction in the levels of IL-6 protein secreted into the culture media (control 375 ± 57 vs. palmitate 1129 ± 177 pg/ml; P < 0.001) (Fig. 1B) and oleate reduced secretion (control 375 ± 57 vs. oleate 196 ± 25 pg/ml) (Fig. 1B).
FIG. 1. Palmitate (Palm.), but not oleate, induces IL-6 expression and secretion in skeletal muscle cells. C2C12 myotubes were incubated in the presence or in the absence of the indicated FFA (16 h, 0.5 mM). A, Analysis of the mRNA levels of IL-6; 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. B, Determination by ELISA of IL-6 secretion to the culture media. Data are expressed as mean ± SD of three different experiments. *, P < 0.05; ***, P < 0.001 vs. control.
Ceramides and the ERK-MAPK pathway are not involved in palmitate-induced IL-6 expression and secretion in skeletal muscle cells
Because palmitoyl-CoA is a precursor of sphingolipid synthesis, palmitate treatment may result in enhanced ceramide synthesis and apoptosis (29). Thus, to gain further insight into the mechanism by which palmitate up-regulates IL-6 mRNA levels and secretion, we tested the effects of one inhibitor of de novo ceramide synthesis. The initial step in ceramide synthesis is the formation of 3-ketodihydrosphingosine from palmitoyl-CoA and L-serine. This step is inhibited by the sphingosine analog ISP1 at picomole concentrations (30). Although a small reduction was observed, ISP1 treatment affected neither IL-6 mRNA (Fig. 2A) nor IL-6 secretion (Fig. 2B) reached by palmitate. To further clarify the potential involvement of ceramides in the up-regulation of IL-6 caused by palmitate, we treated C2C12 skeletal muscle cells with C2-ceramide, a cell-permeable ceramide analog. Addition of 50 μM C2-ceramide modified neither IL-6 mRNA levels (Fig. 2A) nor IL-6 secretion (Fig. 2B). These data suggest that de novo ceramide synthesis is not involved in the effects of palmitate on IL-6 production.
FIG. 2. Ceramides are not involved in palmitate-induced IL-6 expression and secretion in skeletal muscle cells. C2C12 myotubes were incubated (16 h) with either 0.5 mM palmitate (Palm.) in the presence or in the absence of 100 nM ISP1, an inhibitor of de novo ceramide synthesis. Cells were also treated with 50 μM C2-ceramide, a cell-permeable ceramide analog. A, Analysis of the mRNA levels of IL-6; 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. B, Determination by ELISA of IL-6 secretion to the culture media. Data are expressed as mean ± SD of three different experiments. *, P < 0.05; **, P < 0.001; ***, P < 0.001 vs. control.
Because activation of MAPK has been associated with increased IL-6 expression, we assayed the effects of PD98059 and U0126, two known inhibitors of the ERK-MAPK pathway, on IL-6 expression and secretion. In the presence of either 100 μM PD98059 or 10 μM U0126, no changes were observed either in the expression (Fig. 3A) or in the secretion (Fig. 3B) to the culture media of this cytokine.
FIG. 3. The ERK-MAPK pathway is not involved in palmitate-induced IL-6 expression and secretion in skeletal muscle cells. C2C12 myotubes were incubated (16 h) with 0.5 mM palmitate in the presence or in the absence of 100 μM PD98059 or 10 μM U0126, inhibitors of the ERK-MAPK pathway. A, Analysis of the mRNA levels of IL-6; 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. B, Determination by ELISA of IL-6 secretion to the culture media. Data are expressed as mean ± SD of three different experiments. ***, P < 0.001 vs. control.
Palmitate-induced IL-6 expression and secretion is mediated through NF-B activation
Recent studies have reported that activation of the redox transcription factor NF-B plays an important role in IL-6 production from myocytes (21). To test whether incubation of C2C12 cells with palmitate led to increased NF-B activity we performed EMSA studies. NF-B formed three complexes with nuclear proteins (complexes I–III) (Fig. 4A). Specificity of the three DNA-binding complexes was assessed in competition experiments by adding an excess of unlabeled NF-B oligonucleotide. NF-B binding activity, mainly of specific complex II, increased in nuclear extracts from palmitate-treated cells. Addition of antibody against the p65 subunit of NF-B completely supershifted complex II, indicating that this band was mainly constituted of 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-B probe was specific (Fig. 4B).
FIG. 4. Palmitate treatment induces NF-B activation in C2C12 myotubes. C2C12 myotubes were incubated in the presence or in the absence of 0.5 mM palmitate (Palm) for 16 h. A, Autoradiograph of EMSA performed with a 32P-labeled NF-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-kB is also shown. B, Autoradiograph of EMSA performed with a 32P-labeled Oct-1 nucleotide. C, Protein extracts from C2C12 myotubes were assayed for Western blot analysis with IB and IB? antibodies. A representative blot is shown. Data normalized to ?-tubulin levels are expressed as mean ± SD of three different experiments.
NF-B is located in the cytosol bound to IB, and inflammatory signals cause phosphorylation and ubiquitination of IB, thus liberating and activating NF-B. We next assessed whether palmitate resulted in changes in the content of IB (Fig. 4C). Palmitate addition to cells caused approximately a 49% (P < 0.01) decrease in the abundance of IB, whereas the 16% reduction achieved in the levels of IB? were not statistically significant. To evaluate whether NF-B activation was involved in palmitate induction of IL-6, we took advantage of the use of two NF-B inhibitors, PDTC (31) and parthenolide (32). The 5-fold induction in the expression of IL-6 mRNA levels attained by palmitate was prevented when C2C12 cells were coincubated with either PDTC (1.3-fold induction; P < 0.001 vs. palmitate) or parthenolide (2.6-fold induction; P < 0.01 vs. palmitate) (Fig. 5A). Likewise, the 4-fold induction in the IL-6 secretion to the culture medium caused by palmitate was reduced by coincubation of cells with PDTC (1.7-fold induction; P < 0.01 vs. palmitate) and parthenolide (1.5-fold induction; P < 0.001 vs. palmitate) (Fig. 5B). These results suggest that expression and secretion of IL-6 are regulated by NF-B activation.
FIG. 5. Palmitate-induced IL-6 expression is mediated through NF-B activation. C2C12 myotubes were incubated (16 h) with 0.5 mM palmitate in the presence or in the absence of 5 mM PDTC or 10 μM parthenolide, two inhibitors of NF-B. A, Analysis of the mRNA levels of IL-6; 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. B, Determination by ELISA of IL-6 secretion to the culture media. Data are expressed as mean ± SD of three different experiments. *, P < 0.05; ***, P < 0.001 vs. control; #, P < 0.05; ##, P < 0.01; ###, P < 0.001 vs. palmitate.
Palmitate-induced IL-6 expression and secretion is mediated through PKC activation
Because elevation of plasma FFA may lead to diacylglycerol-mediated activation of PKC (6), and this enzyme is known to activate NF-B (33), we next investigated whether palmitate-induced IL-6 expression and secretion involved PKC activation. To test this hypothesis, we used two strategies. First, we verified the effect of calphostin C, a strong and specific inhibitor of PKC (34), on palmitate-induced IL-6 expression and secretion in C2C12 myotubes. Cells were preincubated with calphostin C (100 μM) for 30 min and subsequently stimulated with 0.5 mM palmitate for 16 h. Furthermore, we pretreated cells with 0.5 μM phorbol myristate acetate (PMA) for 24 h before stimulation with palmitate. This long-term pretreatment with PMA causes PKC down-regulation (35, 36). As shown in Fig. 6, calphostin C did not affect palmitate-induced IL-6 mRNA levels, whereas it abolished IL-6 secretion to the culture media. Pretreatment of C2C12 cells with PMA completely abolished both IL-6 expression and secretion. These data indicate that PKC activation is involved in palmitate-induced IL-6 secretion.
FIG. 6. Palmitate-induced IL-6 expression and secretion is mediated through PKC activation. C2C12 myotubes were incubated (16 h) with 0.5 mM palmitate (Palm) in the presence or in the absence of either 100 μM calphostin C or 500 nM PMA. A, Analysis of the mRNA levels of IL-6; 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. B, Determination by ELISA of IL-6 secretion to the culture media. Data are expressed as mean ± SD of three different experiments. *, P < 0.05; ***, P < 0.001 vs. control; #, P < 0.05; ###, P < 0.001 vs. palmitate.
IL-6 secretion is responsible for the down-regulation of GLUT4 induced by palmitate
Finally, we examined whether some metabolic effects associated with elevated FFA in skeletal muscle cells were mediated by IL-6 secretion. Thus, to verify the contribution of IL-6 to the effects caused by elevated FFA, we took advantage of using an anti-IL-6 antibody. Previous studies have reported that IL-6 induces insulin resistance in 3T3-L1 adipocytes, causing reduction in the expression of GLUT4 (37). Treatment with palmitate caused a 57% reduction in the mRNA levels of GLUT4 in C2C12 myotubes (P < 0.001), but coincubation with anti-IL-6 antibody prevented this change (Fig. 7A). Finally, we tested whether these changes 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), and this was prevented by incubating the cells in the presence of anti-IL-6 antibody (Fig. 7B).
FIG. 7. IL-6 secretion is responsible for the down-regulation of GLUT4 induced by palmitate. C2C12 myotubes were incubated (16 h) with 0.5 mM palmitate (Palm) in the presence or in the absence of 5 μg/ml anti-IL-6 antibody (Ab). 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 three different experiments. **, P < 0.01 vs. control. B, 2-DG uptake in the presence or in the absence of 100 nM insulin (Ins). 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; , P < 0.05 vs. insulin-stimulated cells incubated with palmitate.
Discussion
Skeletal muscle insulin resistance is the major characteristic of type 2 diabetes mellitus (1). The mechanisms responsible for the reduced sensitivity of muscle to insulin still remain unclear, but there is a strong correlation between insulin resistance and elevated FFA. In addition, accumulating evidence suggests a link between inflammation and type 2 diabetes. The proinflammatory cytokine IL-6 presents a strong correlation with insulin resistance and type 2 diabetes (16, 17). Previous recent reports have suggested that IL-6 impairs insulin sensitivity and glucose regulation in adipocytes (39, 40) and hepatocytes (41, 42, 43). In this study, we have used the myoblast C2C12 cell line, which develops biochemical and morphological properties characteristic of skeletal muscle and has been proven useful for studies of skeletal muscle metabolism (44). Exposure of these skeletal muscle cells to palmitate results in increased IL-6 expression and secretion. The current results provide direct strong evidence that IL-6 secretion may be responsible for part of the effects of the saturated fatty acid palmitate on insulin resistance in skeletal muscle cells. Moreover, data presented indicate that PKC and NF-B activation are involved in palmitate-induced IL-6 production. It is worth noting that, in contrast to palmitate, oleate reduced IL-6 expression and secretion. These findings suggest that palmitate-induced IL-6 expression and secretion were specific for this saturated fatty acid. Additional studies are necessary to confirm whether this monounsaturated fatty acid may counteract the effects of palmitate on IL-6 expression and secretion.
Elevated FFA presumably increases FFA uptake, exceeding its oxidation, which in turns leads to increased intramuscular triglycerides and diacylglycerol, a potent allosteric activator of both conventional and novel PKC isoforms. Activation of PKC could lead to insulin resistance by several mechanisms. This enzyme can phosphorylate both the insulin receptor (13, 45) and insulin receptor substrate-1 (37), leading to impaired insulin signaling; or it can increase oxidative stress and activate NF-B. The results presented here show that a long treatment with PMA, which results in PKC down-regulation (35, 36), completely abolishes both palmitate-induced IL-6 expression and secretion. Pretreatment with calphostin C, a strong and specific inhibitor of PKC (36), also prevented palmitate-induced IL-6 secretion to the culture media. In contrast, this PKC inhibitor did not affect IL-6 expression caused by palmitate. Previous studies have reported abolishment of induced IL-6 expression by calphostin C in osteoblastic cells after 3 h (36), suggesting that the lack of inhibitory effect of calphostin C in C2C12 cells after 16 h of treatment could be attributed to recovery of PKC activity that led to increased mRNA expression of IL-6, but not protein, because secretion of this cytokine was still blocked.
As stated above, PKC activation can increase oxidative stress and NF-B activation. Itani et al. (46) reported that lipid infusion in humans during a euglycemic-hyperinsulinemic clamp increases PKC activity and degradation of the mass of the NF-B inhibitor IB. Activation of PKC can lead to the activation of this transcription factor by directly phosphorylating IB (47) or by causing the generation of reactive oxygen species that can secondarily activate IB-kinase. In fact, phosphorylation by IB-kinase is considered the main pathway by which IB is released from NF-B and subsequently subjected to ubiquitination and proteosomal degradation. The result is a decrease in IB mass and movement of NF-B from cytosol to the nucleus. In the present study, palmitate increased NF-B activation as demonstrated by EMSA studies. Activation of this proinflammatory transcription factor seems to be mediated by degradation of IB. The mouse IL-6 promoter contains a consensus sequence for the redox-sensitive transcription factor NF-B, supporting a role for NF-B in the palmitate-mediated induction of IL-6. This role was confirmed by pretreating cells with inhibitors of NF-B, which prevented the palmitate-mediated induction of IL-6 mRNA levels and secretion to the culture media. Recent studies support the findings that palmitate leads to NF-B activation (48) and that palmitate-induced insulin resistance is prevented by inhibition of this transcription factor (38).
The data of this study also discard the involvement of several processes in the palmitate-induced production of IL-6 in skeletal muscle cells. Thus, ceramides, which are palmitate-derived lipid metabolites, seem not to be involved in the induction of IL-6 in skeletal muscle cells. There is also evidence showing that activation of the MAPK signaling cascade increases transcription of IL-6 (20, 23). However, coincubation of cells with inhibitors of the ERK-MAPK pathway did not affect either expression or secretion of IL-6, making unlikely the involvement of this signaling cascade in IL-6 changes mediated by palmitate.
Finally, we evaluated the metabolic effects caused by palmitate-induced IL-6 secretion. To clearly differentiate between the effects caused by either palmitate or IL-6, we used an anti-IL-6 antibody. A previous study performed in adipocytes reported a reduction in the expression of GLUT4 after IL-6 treatment (40). In the skeletal muscle cells used in this study, palmitate addition to the culture media caused a reduction in the expression of GLUT4 and insulin-stimulated glucose uptake, changes that were prevented in the presence of the antibody against IL-6, clearly involving this cytokine in some of the prodiabetic effects of palmitate. The data shown here do not permit us to establish which is the IL-6-dependent mechanism responsible for the reduction in GLUT4 expression. Because palmitate impairs serine/threonine protein kinase Akt phosphorylation (14), it is tempting to speculate that IL-6 may mediate changes in the phosphorylation of the Akt, which promotes translocation of glucose transporter GLUT4 to the plasma membrane. However, the reduction in GLUT4 mRNA levels achieved after palmitate treatment also suggests that IL-6 may cause changes in the transcriptional rate of this glucose transporter.
In summary, here we report that palmitate treatment of skeletal muscle cells induces IL-6 expression and secretion through mechanisms involving the activation of the axis PKC-NF-B. Furthermore, IL-6 production seems to be responsible for the reduction in GLUT4 expression in skeletal muscle exposed to palmitate. These results support recent reports showing that IL-6 is involved in insulin resistance and converts this cytokine and the mechanisms responsible for its induction in a target for the treatment of lipid-mediated insulin resistance.
Acknowledgments
We thank the Language Advisory Service of the University of Barcelona for helpful assistance.
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