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Distinct effects of oleic acid and its trans-isomer elaidic acid on the expression of myokines and adipokines in cell models

Published online by Cambridge University Press:  05 January 2011

Nuria Granados
Affiliation:
Laboratory of Molecular Biology, Nutrition and Biotechnology, Universitat de les Illes Balears, CIBER de Fisiopatología de la Obesidad y Nutrición (CIBERobn), Cra. Valldemossa, Km 7.5, 07122 Palma deMallorca, Spain
Jaume Amengual
Affiliation:
Laboratory of Molecular Biology, Nutrition and Biotechnology, Universitat de les Illes Balears, CIBER de Fisiopatología de la Obesidad y Nutrición (CIBERobn), Cra. Valldemossa, Km 7.5, 07122 Palma deMallorca, Spain
Joan Ribot
Affiliation:
Laboratory of Molecular Biology, Nutrition and Biotechnology, Universitat de les Illes Balears, CIBER de Fisiopatología de la Obesidad y Nutrición (CIBERobn), Cra. Valldemossa, Km 7.5, 07122 Palma deMallorca, Spain
Andreu Palou*
Affiliation:
Laboratory of Molecular Biology, Nutrition and Biotechnology, Universitat de les Illes Balears, CIBER de Fisiopatología de la Obesidad y Nutrición (CIBERobn), Cra. Valldemossa, Km 7.5, 07122 Palma deMallorca, Spain
M. Luisa Bonet
Affiliation:
Laboratory of Molecular Biology, Nutrition and Biotechnology, Universitat de les Illes Balears, CIBER de Fisiopatología de la Obesidad y Nutrición (CIBERobn), Cra. Valldemossa, Km 7.5, 07122 Palma deMallorca, Spain
*
*Corresponding author: Professor A. Palou, fax +34 971 173426, email andreu.palou@uib.es
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Abstract

Trans-fatty acids (TFA) and cis-monounsaturated fat appear to exert detrimental and beneficial effects, respectively, on glucose metabolism and insulin sensitivity. Adipose tissue and skeletal muscle are a source of signalling proteins (adipokines and myokines), some of which have been related to the control of insulin sensitivity. Here, we investigated the possible differential effects of elaidic acid (EA; trans-9-18 : 1) – the major component in industrially produced TFA – and oleic acid (OA; cis-9-18 : 1) – its cis-isomer naturally present in food – on cellular glucose uptake and the expression of selected myokines and adipokines using cell models. Differentiated C2C12 myotubes and 3T3-L1 adipocytes were pretreated with the vehicle (control cells) or fatty acids for 24 h, after which basal and insulin-stimulated 2-deoxyglucose uptake and the expression of selected signalling proteins were measured. In C2C12 myotubes, pretreatment with OA, but not with EA, led to increased insulin-stimulated 2-deoxyglucose uptake and IL-6 expression levels, while pretreatment with EA, but not with OA, led to reduced IL-15 mRNA levels and increased TNF-α expression levels. In 3T3-L1 adipocytes, exposure to OA, but not to EA, resulted in reduced resistin gene expression and increased adiponectin gene expression. The results show evidence of distinct, direct effects of OA and EA on muscle glucose uptake and the expression of target myokines and adipokines, thus suggesting novel mechanisms by which cis- and trans-monounsaturated fat may differentially affect systemic functions.

Type
Full Papers
Copyright
Copyright © The Authors 2011

Trans-fatty acids (TFA) are unsaturated fatty acids with at least one double bond in the trans, rather than the typical cis, configuration. A major source of TFA in food is industrially produced trans-fatty acids (IP-TFA) generated during partial hydrogenation (‘hardening’) of vegetable fat. Industrial hydrogenation involves heating the raw oils to about 400°C under high pressure and with the addition of different catalysts, and in this process, trans-double bonds are undesirably formed along with fatty acid molecules from position six and higher, at the same time as SFA are formed. Partially hydrogenated fat is used in a variety of food products, especially spreads, baked goods and fast food. Elaidic acid (EA; trans-9-18 : 1), the trans-isomer of oleic acid (OA), is the predominant IP-TFA(Reference Mensink1, Reference Stender, Astrup and Dyerberg2).

While there is evidence pointing to favourable effects of OA-enriched diets and of OA itself on CVD risk, weight maintenance and insulin sensitivity(Reference Covas3Reference Riserus, Willett and Hu5), dietary TFA is generally associated with detrimental effects on health(Reference Stender, Astrup and Dyerberg2, Reference Mozaffarian, Aro and Willett6, Reference Teegala, Willett and Mozaffarian7). Importantly, deleterious effects are well documented for IP-TFA, but not for TFA of ruminant origin, in the low amounts usually consumed(Reference Stender, Astrup and Dyerberg2, Reference Mozaffarian, Aro and Willett6). In fact, health benefits have been suggested for conjugated linoleic acid and vaccenic acid (trans-11-18 : 1), the major TFA naturally present in beef and dairy products(Reference Bhattacharya, Banu and Rahman8, Reference Field, Blewett and Proctor9).

Adverse effects of IP-TFA consumption on multiple cardiovascular risk factors are well established, including promotion of a pro-atherogenic serum lipid/lipoprotein profile, endothelial dysfunction and an increased production of pro-inflammatory cytokines(Reference Mozaffarian, Aro and Willett6, Reference Teegala, Willett and Mozaffarian7, Reference Mozaffarian10). The impact of IP-TFA consumption on insulin resistance and the risk of type 2 diabetes is more controversial, since detrimental effects have been reported in some, but not all, human observational studies and short-term intervention trials on the topic(Reference Lovejoy, Smith and Champagne11Reference Tardy, Lambert-Porcheron and Malpuech-Brugere14). Likewise, in rats, the effects of TFA-enriched diets (fed for 8–12 weeks) increasing insulin resistance and impairing systemic and tissue-specific glucose disposal have been reported in some studies(Reference Natarajan and Ibrahim15Reference Dorfman, Laurent and Gounarides17), but not in others(Reference Tardy, Giraudet and Rousset18). Nevertheless, in a long-term, 5-year controlled study in monkeys, TFA feeding (v. OA) caused insulin resistance and impaired glucose metabolism along with abdominal obesity(Reference Kavanagh, Jones and Sawyer19). Two long-term prospective observational studies in humans have also suggested that TFA consumption promotes weight gain, and particularly accumulation of abdominal fat(Reference Koh-Banerjee, Chu and Spiegelman20, Reference Field, Willett and Lissner21). Abdominal obesity, insulin resistance and CVD risk are intimately linked(Reference Ritchie and Connell22).

Mechanisms underlying the relationship between dietary fatty acids and metabolic health are incompletely understood and seem to include the effects on cell-membrane fatty acid composition and function, as well as on gene expression and enzyme activity in key tissues(Reference Riserus, Willett and Hu5). Both adipose tissue and skeletal muscle produce and secrete signalling proteins (named adipokines and myokines, respectively), which may affect metabolic health, and particularly glucose metabolism and insulin sensitivity locally and systemically. Studies examining the possible differential, direct effects of concrete species of cis- and trans-MUFA on the cellular production of such signals are lacking, yet of potential interest to support the biological plausibility of observations derived from epidemiological studies and studies using complex mixtures. Therefore, the aim of the present study was to compare the direct effects of EA, the major IP-TFA, and its cis-isomer, OA, naturally present in foods on the expression of myokines and adipokines possibly involved in the control of insulin sensitivity, along with their effects on basal and insulin-stimulated glucose uptake, in muscle and adipocyte cell models.

Experimental methods

Cell culture and fatty acid treatment

All reagents used for cell culture and treatment including insulin from bovine pancreas (product no. I4011), OA (product no. O1008) and EA (product no. E4637) were from Sigma (St Louis, MO, USA), unless otherwise indicated. The purity of the fatty acids was at least 99 %. 3T3-L1 mouse pre-adipocytes (ATTC CL-173™; LGC Deselaers SL, Barcelona, Spain) were grown to confluence in basal medium – Dulbecco's modified Eagle's medium with penicillin (50 U/ml), streptomycin (50 μg/ml) and 4 mm-l-glutamine – supplemented with 10 % newborn calf serum (Linus, Madrid, Spain) under 8 % CO2 and 92 % air at 37°C in six- or twelve-well culture plates. At 2 d after the cells reached confluence (referred to as day 0), they were induced to differentiate in basal medium containing 10 % fetal bovine serum (FBS; Invitrogen, Carlsbad, CA, USA), dexamethasone (1 μm), methylisobutylxanthine (0·5 mm) and insulin (1 μg/ml) for 48 h, followed by 48 h in basal medium containing 10 % FBS and insulin (1 μg/ml). The cells were subsequently refed every other day with fresh basal medium supplemented with 10 % FBS (without insulin). Adipogenesis was monitored by morphological examination of the cells for lipid accumulation using phase contrast microscopy. The cells were treated with fatty acids along with fresh medium on days 8–10 of culture, when more than 90 % had acquired the adipose phenotype. For treatment, stock solutions of fatty acids in dimethyl sulphoxide as the vehicle were prepared at 500 ×  the desired final working concentration and diluted 1:500 with basal medium containing 10 % FBS. This mixture was pre-warmed for 45 min at 37°C with vortex every 15 min before being added to the differentiated cells(Reference Amri, Teboul and Vannier23). Control cells received the same medium incubated with the corresponding volume of the vehicle (0·2 % dimethyl sulphoxide). Experimental parameters were measured following 24 h treatment with the fatty acids or the vehicle.

C2C12 mouse myoblasts (ATTC CRL-1772™; LGC Promochem SL, Barcelona, Spain) were cultured at 37°C with humidified 95 % air and 5 % CO2 in Dulbecco's modified Eagle's medium supplemented with 10 % FBS, penicillin (50 U/ml), streptomycin (50 μg/ml) and 3 mm-l-glutamine (growth medium) in twelve-well culture plates. To induce differentiation, C2C12 myoblasts were allowed to reach 80 % confluence in growth medium and then changed to Dulbecco's modified Eagle's medium containing 2 % horse serum (differentiation medium). After ten additional days, the cells had differentiated and fused into myotubes, as assessed by microscopic examination. Differentiated myotubes were treated with the fatty acids or the vehicle for 24 h using the same procedure described above for adipocytes, before RNA isolation or the measurement of glucose uptake.

RNA isolation and analysis

Total RNA was extracted from 3T3-L1 adipocytes and C2C12 myotubes using Tripure reagent (Roche, Barcelona, Spain). Isolated RNA was quantified using the NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies, Inc., Wilmington, DE, USA), and its integrity was confirmed using agarose gel electrophoresis. A260:A280 ratios were between 1·9 and 2 for all samples, indicating good RNA purity. Retrotranscription followed by real-time quantitative PCR was used to measure the mRNA levels of resistin, adiponectin, retinol-binding protein 4, IL-6, IL-15, TNF-α, GLUT1, GLUT4 and β-actin as an internal control. In brief, 0·25 μg of total RNA (in a final volume of 5 μl) was denatured at 65°C for 10 min and then reverse transcribed to complementary DNA using murine leukaemia virus RT (Applied Biosystems, Madrid, Spain) in the presence of 50 pmol of random primers in an Applied Biosystems 2720 Thermal Cycler. Sense and antisense primers used in the PCR were designed with specific primer analysis software Primer3 (Whitehead Institute for Biomedical Research, Cambridge, MA, USA), and their specificity was analysed by the ENTREZ and BLAST database utilities (National Center for Biotechnology Information, Bethesda, MD, USA). The primers were produced by Sigma (Madrid, Spain), and their sequences are shown in Table 1. Each PCR was performed from diluted (1:20) complementary DNA template, forward and reverse primers (1 μm each), and Power SYBR Green PCR Master Mix (Applied Biosystems, Madrid, Spain) on a StepOnePlusTM Real-Time PCR System (Applied Biosystems). In order to verify the purity of the products, a melting curve was produced after each run according to the manufacturer's instructions. The relative expression of each mRNA was calculated according to Pfaffl(Reference Pfaffl24), using β-actin as a reference gene.

Table 1 Primers used in the quantitative PCR

F, forward; R, reverse.

Quantification of cytokines

IL-6 and TNF-α concentrations in the cell's conditioned culture medium were measured using ELISA kits (both from Pierce, Rockford, IL, USA), following the manufacturer's instructions.

2-Deoxy-d-glucose uptake

Analysis of 2-deoxy-d-glucose (2-DOG) uptake was performed immediately after completion of the 24 h incubation with fatty acids, essentially as described previously(Reference Kumar and Dey25). After washing with Krebs–Ringer phosphate buffer (10 mm-KH2PO4, pH 7·4, containing 136 mm-NaCl, 4·7 mm-KCl, 1·25 mm-CaCl2, 1·25 mm-MgSO4 and 0·05 % bovine serum albumin), cells were incubated with Krebs–Ringer phosphate buffer without glucose in the absence of insulin or in the presence of 100 nm-insulin for 15 min at 37°C. The transport assay was then initiated by the addition of 3H-2-DOG (3700 Bq/ml in 1 μm unlabelled 2-DOG), and the cells were further incubated for 10 min with or without 100 nm-insulin at 37°C. Cells were then washed three times with ice-cold PBS and lysed with 0·1 m-NaOH. Radioactivity in cell lysates was counted in Hi-safe 3 scintillant (Perkin Elmer, Shelton, CT, USA) using a Beckman Coulter LS 6500 multi-purpose liquid scintillation counter (Beckman Coulter, Brea, CA, USA). Counting efficiency was about 47 %. Protein concentration in cell lysates was evaluated via the bicinchoninic acid protein assay kit (Pierce, Rockford, IL, USA).

Statistical analysis

Data are expressed as means with their standard errors of at least three individual experiments, each run in triplicate or quadruplicate. Differences between treatments were assessed by one-way ANOVA, followed by a least significant differences post hoc comparison. Student's t test was used to assess the effect of insulin on glucose uptake. The threshold of significance was set at P < 0·05.

Results

Distinct effects of oleic acid and elaidic acid on myokine expression and insulin-stimulated 2-deoxy-D-glucose uptake in C2C12 myotubes

Differentiated C2C12 myotubes were pretreated for 24 h with 20–500 μm-OA, 20–500 μm-EA or the vehicle (0·2 % dimethyl sulphoxide), and the changes elicited by fatty acid treatment on IL-6, TNF-α and IL-15 expression were analysed. Treatment with OA caused a dose-dependent increase in cellular IL-6 mRNA levels (Fig. 1(A)) and in IL-6 protein levels in the cell's conditioned culture medium (Fig. 1(B)), whereas treatment with EA had no effect on IL-6 expression either at the mRNA or at the secreted protein level (Fig. 1(C) and (D)). TNF-α mRNA levels were unaffected by OA treatment (Fig. 2(A)) and were dose dependently increased following treatment with EA by 69 % at the 100 μm dose and by 90 % at the 500 μm dose (Fig. 2(B)). In good concordance with the mRNA results, TNF-α protein was undetectable in the conditioned culture medium of the vehicle- and OA-treated cells, yet it could be consistently detected, though at very low levels, in the medium of cells treated with the highest EA doses (Fig. 2(C)). IL-15 mRNA levels were unaffected by OA treatment (Fig. 3(A)) and were significantly reduced by about 25 % following treatment with 100 or 500 μm-EA (Fig. 3(B)).

Fig. 1 Effects of 24 h exposure to varying doses of oleic acid or elaidic acid on IL-6 gene expression and secreted levels in C2C12 myotubes. Gene expression of IL-6 ((A) and (B)) was measured by real-time quantitative PCR and normalised to β-actin. IL-6 protein in the medium ((C) and (D)) was measured by ELISA. Values are means of at least three experiments (n 3–7) each run in triplicate, with standard errors represented by vertical bars, and are expressed relative to the mean value in the vehicle dimethyl sulphoxide (DMSO)-treated control cells, which was set at 100 %. The absolute mean value of IL-6 in the medium of the vehicle-treated cells was 429 (se 62) pg/mg of cell protein. Data were analysed by one-way ANOVA followed by the least significant difference post hoc test. a,b,c Mean values with unlike letters were significantly different (P < 0·05).

Fig. 2 Effects of 24 h exposure to varying doses of oleic acid or elaidic acid on TNF-α gene expression and secreted levels in C2C12 myotubes. Gene expression of TNF-α ((A) and (B)) was measured by real-time quantitative PCR and normalised to β-actin, and is expressed relative to the mean value in the vehicle dimethyl sulphoxide (DMSO)-treated control cells, which was set at 100 %. TNF-α protein in the medium (C) was measured by ELISA and is expressed as absolute values (n.d., not detected). Values are means of at least three experiments (n 3–7) each run in triplicate, with standard errors represented by vertical bars. Gene expression data were analysed by one-way ANOVA followed by the least significant difference post hoc test. a,b Mean values with unlike letters were significantly different (P < 0·05).

Fig. 3 Effects of 24 h exposure to varying doses of (A) oleic acid or (B) elaidic acid on IL-15 gene expression in C2C12 myotubes. Values are means of at least three experiments (n 3–7) each run in triplicate, with standard errors represented by vertical bars, and are expressed relative to the mean value in the vehicle dimethyl sulphoxide (DMSO)-treated control cells, which was set at 100 %. Gene expression of IL-15 was measured by real-time quantitative PCR and normalised to β-actin. Data were analysed by one-way ANOVA followed by the least significant difference post hoc test. a,b Mean values with unlike letters were significantly different (P < 0·05).

Basal and insulin-stimulated 2-DOG uptake was measured in differentiated C2C12 myotubes following 24 h exposure to 100 μm-OA, 100 μm-EA or the vehicle (Fig. 4(A)). Basal 2-DOG uptake was slightly but significantly lower in the cells that had been treated with either fatty acid compared with the vehicle-treated cells. Stimulation of the cells with 100 nm-insulin produced, as expected, a significant increase in 2-DOG uptake. Insulin-stimulated 2-DOG uptake was approximately 35 % higher (P < 0·05) in the OA-treated cells compared with the vehicle- or EA-treated cells. Reduced basal 2-DOG uptake in fatty acid-treated myotubes was not related to reduced GLUT1 mRNA expression levels, which did not show differences between treatments (data not shown). Likewise, increased insulin-stimulated 2-DOG uptake in the OA-treated cells was not related to the differences in GLUT4 mRNA levels in these cells compared with the vehicle- or EA-treated cells (data not shown).

Fig. 4 Effects of 24 h exposure to 100 μm-oleic or -elaidic acid on basal and insulin-stimulated 2-deoxy-d-glucose (2-DOG) uptake in (A) C2C12 myotubes and (B) 3T3-L1 adipocytes. 2-DOG uptake was measured in the absence of insulin (basal) and in the presence of 100 nm-insulin as described in the Experimental methods. Values are means of at least three experiments (n 3–8) each run in quadruplicate, with standard errors represented by vertical bars, and are expressed relative to the mean value in the vehicle dimethyl sulphoxide (DMSO)-treated control cells, which was set at 100 %. One-way ANOVA followed by the least significant difference post hoc test was used to analyse the significance of differences among treatments under the basal and insulin-stimulated conditions; a,b,c for each condition, mean values with unlike letters were statistically different (P < 0·05). * Statistically significant difference (P < 0·05) v. the basal condition assessed by Student's t test. □, Vehicle (DMSO); ■, 100 μm-oleic acid; , 100 μm-elaidic acid; dpm, disintegrations per min.

Distinct effects of oleic acid and elaidic acid on adipokine expression in 3T3-L1 adipocytes

Adipokine mRNA levels and 2-DOG uptake were measured in differentiated 3T3-L1 adipocytes following 24 h exposure to 100 μm-OA, 100 μm-EA or the vehicle (0·2 % dimethyl sulphoxide), and the changes elicited by fatty acid treatment relative to the vehicle were analysed. Treatment with EA did not affect resistin or adiponectin mRNA levels, whereas treatment with OA resulted in a significant 28 % decrease in resistin mRNA levels and a significant 25 % increase in adiponectin mRNA levels (Fig. 5). Similar results were obtained in a separate experiment in which the fatty acids were assayed at 500 μm concentration (data not shown). The mRNA levels of the other cytokines/adipokines assayed, namely IL-6, IL-15, TNF-α and retinol-binding protein 4, were very low in 3T3-L1 adipocytes, both under the control condition and after 24 h treatment with OA or EA up to 500 μm (crossing points ≥ 30 in the real-time quantitative PCR). Low basal levels of expression of retinol-binding protein 4 and IL-15 mRNA in 3T3-L1 adipocytes are in agreement with previous reports(Reference Mercader, Granados and Bonet26, Reference Quinn, Strait-Bodey and Anderson27). No differential effects of OA and EA on 2-DOG uptake in 3T3-L1 adipocytes were found: pretreatment with either fatty acid similarly slightly lowered basal 2-DOG uptake relative to the vehicle, and insulin-stimulated 2-DOG uptake was equal in adipocytes pre-exposed to OA, EA or the vehicle (Fig. 4(B)).

Fig. 5 Effects of 24 h exposure to 100 μm-oleic or -elaidic acid on the gene expression of (A) resistin and (B) adiponectin in 3T3-L1 adipocytes. Values are means of four experiments (n 4) each run in triplicate, with standard errors represented by vertical bars, and are expressed relative to the mean value in the vehicle dimethyl sulphoxide (DMSO)-treated control cells, which was set at 100 %. Gene expression was measured by real-time quantitative PCR and normalised to β-actin. Data were analysed by one-way ANOVA followed by the least significant difference post hoc test. a,b Mean values with unlike letters were significantly different (P < 0·05). □, Vehicle (DMSO); ■, 100 μm-oleic acid; , 100 μm-elaidic acid.

Discussion

Skeletal muscle plays a major role in insulin-stimulated glucose disposal(Reference Parish and Petersen28), and is nowadays recognised as a secretory organ that produces and releases bioactive proteins exerting autocrine, paracrine and endocrine effects, including local effects on muscle glucose uptake(Reference Pedersen and Febbraio29). Among these is IL-6, a cytokine produced by many cell types and by muscle fibres in response to contraction(Reference Pedersen and Febbraio29, Reference Hoene and Weigert30). IL-6 enhances glucose uptake by human and rat skeletal muscle cells(Reference Carey, Steinberg and Macaulay31Reference Glund, Deshmukh and Long33), and secretion of IL-6 is reduced in human skeletal muscle cells that are made insulin-resistant by incubation with adipocyte-conditioned medium(Reference Sell, Eckardt and Taube34). These and other findings, including reports of central and peripheral anti-obesity effects of IL-6, have challenged the classical view of IL-6 as a pro-inflammatory cytokine associated with obesity and reduced insulin sensitivity(Reference Pedersen and Febbraio29, Reference Hoene and Weigert30). Our finding that incubation with OA increased both IL-6 expression and insulin-stimulated 2-DOG uptake in C2C12 myotubes supports this novel scenario for muscle-derived IL-6. The present results are in concordance with reported stimulatory effects of IL-6 on skeletal muscle glucose uptake(Reference Carey, Steinberg and Macaulay31Reference Glund, Deshmukh and Long33) and with reported beneficial effects of OA on insulin sensitivity, systemically and at the skeletal muscle level(Reference Schroder4, Reference Riserus, Willett and Hu5, Reference Gao, Griffiths and Bailey35). In particular, OA has been shown to protect against palmitate-induced insulin resistance at the level of glucose uptake in rat L6 myotubes(Reference Gao, Griffiths and Bailey35). In that study(Reference Gao, Griffiths and Bailey35), however, OA alone did not increase insulin-stimulated glucose uptake as in ours; the reasons for this difference remain unclear and may relate to differences in the cell model and treatment conditions used. Importantly, we found that the stimulatory effects of OA on insulin-stimulated 2-DOG uptake and IL-6 expression at both the mRNA and secreted protein levels in skeletal muscle cells were not reproduced by its trans-isomer, EA.

Treatment with either OA or EA caused in our hands a small, but consistent, reduction in cellular basal 2-DOG glucose. The present results indicate that the observed effects of fatty acids on 2-DOG uptake in muscle cells were unrelated to changes in GLUT1 or GLUT4 mRNA levels. Muscle has a high potential to adjust fuel selection depending on energy supply and demand. Fatty acids have been shown to induce the expression in muscle cells (including C2C12 myotubes) of key genes involved in their catabolism, which may facilitate the transition from a more glycolytic to a more lipid-oxidative metabolism during episodes of heavy influx of fatty acids to skeletal muscle, such as in fasting(Reference Rodriguez, Sanchez and Tobaruela36). It is possible, therefore, that reduced basal glucose uptake may, by default, result as a consequence of increased cellular fatty acid catabolism. In any case, the significance and mechanistic basis of fatty acid effects on glucose uptake observed in the present study requires further investigation.

IL-15 is another myokine possibly involved in the control of muscle glucose disposal. IL-15 is highly expressed in skeletal muscle compared with other tissues and plays important regulatory roles in skeletal muscle itself, where it promotes protein synthesis, fatty acid oxidation, and glucose uptake and utilisation(Reference Busquets, Figueras and Almendro37, Reference Argiles, Lopez-Soriano and Busquets38). In addition to anti-diabetogenic effects, anti-adiposity effects have been proposed for muscle-derived IL-15 acting on multiple target tissues including adipose tissue(Reference Argiles, Lopez-Soriano and Busquets38, Reference Quinn, Anderson and Strait-Bodey39). We report here, for the first time to our knowledge, a down-regulatory effect of EA on IL-15 gene expression in skeletal muscle cells, which could have implications for the effects of TFA promoting insulin resistance and obesity evidenced in previous studies(Reference Odegaard and Pereira12, Reference Riserus13, Reference Natarajan and Ibrahim15Reference Dorfman, Laurent and Gounarides17, Reference Kavanagh, Jones and Sawyer19Reference Field, Willett and Lissner21).

Adipose tissue plays a relatively modest role in insulin-stimulated glucose disposal compared with skeletal muscle, yet it contributes to the control of systemic insulin sensitivity through the secretion of NEFA and adipokines that have an impact on skeletal muscle and liver metabolism, among other mechanisms. Two important adipokines in this context are resistin and adiponectin, which exert opposite effects as an insulin resistance factor(Reference Lazar40, Reference Barnes and Miner41) and an insulin-sensitising factor(Reference Kadowaki, Yamauchi and Kubota42), respectively. Resistin and adiponectin might also oppositely relate to inflammation, as there is evidence of pro-inflammatory properties of resistin(Reference Barnes and Miner41) and anti-inflammatory properties of adiponectin especially with regard to atherosclerosis(Reference Stofkova43). In our hands, exposure to OA significantly reduced gene expression of resistin and increased that of adiponectin in mature 3T3-L1 adipocytes, and these putatively beneficial effects were not reproduced by EA, which did not affect adiponectin or resistin gene expression. We also investigated the effects on retinol-binding protein 4 – which, similar to resistin, might function as an insulin resistance factor secreted by adipocytes(Reference Yang, Graham and Mody44) – and found no effect of OA or EA treatment on its (very low) gene expression in 3T3-L1 adipocytes. Repression of adipocyte resistin expression by OA has been previously reported(Reference Rea and Donnelly45), and a stimulatory effect of OA on adiponectin expression might be in line with a recent report showing a positive association between serum adiponectin levels and OA content in white adipose tissue in rats(Reference Perez de Heredia, Sanchez and Priego46). In a previous study in 3T3-L1 adipocytes, however, 48 h incubation with 250 μm-OA had no effect on adiponectin gene expression(Reference Bueno, Oyama and de Oliveira47). Studies examining the effects of TFA on adipokine expression in adipocyte cell models are lacking, to our knowledge. There are, however, reports indicating that, in rats, consumption of TFA-enriched diets is associated with increased resistin expression(Reference Saravanan, Haseeb and Ehtesham48, Reference Duque-Guimaraes, de Castro and Martinez-Botas49) and reduced adiponectin expression in white adipose tissue(Reference Duque-Guimaraes, de Castro and Martinez-Botas49, Reference Pisani, Oyama and Bueno50). Additionally, also in rats, a negative correlation between adipose adiponectin gene expression and TFA levels in the serum has been described(Reference Perez de Heredia, Sanchez and Priego46). Taken together, these in vivo studies and our cell studies suggest that the effects of TFA-enriched diets on adipokine expression observed in vivo are likely to be indirect.

TNF-α is a cytokine linked to inflammation and obesity-associated insulin resistance(Reference Ryden and Arner51). TFA intake has been reported to be associated with increased TNF system activity in observational studies in humans(Reference Mozaffarian10) and with increased TNF-α expression in white adipose tissue in experimental studies in rats(Reference Duque-Guimaraes, de Castro and Martinez-Botas49, Reference Pisani, Oyama and Bueno50). In our hands, gene expression of TNF-α was negligible in 3T3-L1 adipocytes yet readily detectable in C2C12 myotubes, in which both TNF-α mRNA levels and secreted TNF-α protein increased following exposure to EA. TNF-α activity favours insulin resistance and protein wasting in skeletal muscle(Reference Argiles, Alvarez and Carbo52), effects that are the opposite to those exerted in muscle by IL-15(Reference Argiles, Lopez-Soriano and Busquets38), and, remarkably, we found here that EA oppositely affected gene expression of these two cytokines in muscle cells.

In summary, the present study revealed the distinct, direct effects of OA and EA on the expression of target myokines and adipokines. OA, but not EA, dose dependently induced IL-6 expression in skeletal muscle cells, and repressed resistin and induced adiponectin gene expression in cultured adipocytes. EA, but not OA, repressed IL-15 gene expression and induced TNF-α expression in cultured skeletal muscle cells. Overall, these findings suggest novel mechanisms by which cis- and trans-monounsaturated fat may differentially affect systemic functions.

Acknowledgements

The present study was supported by grants from the Spanish Government (AGL2009-11277/ALI to A. P.) and the European Union (BIOCLAIMS project, FP7-244995, Coordinator A. P.). Our laboratory consists of a member of the European Nutrigenomics Organization (NuGO, European Union Contract FOOD-CT-2004-506360). CIBER de Fisiopatología de la Obesidad y Nutrición is an initiative of the ISCIII (Spanish Government). All authors contributed to the design and discussion of the study. N. G. and J. A. undertook all the experiments. N. G. drafted the manuscript. J. R., A. P. and M. L. B. supervised the experiments. M. L. B. was in charge of the final writing of the manuscript. The authors declare that there is no conflict of interest.

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Figure 0

Table 1 Primers used in the quantitative PCR

Figure 1

Fig. 1 Effects of 24 h exposure to varying doses of oleic acid or elaidic acid on IL-6 gene expression and secreted levels in C2C12 myotubes. Gene expression of IL-6 ((A) and (B)) was measured by real-time quantitative PCR and normalised to β-actin. IL-6 protein in the medium ((C) and (D)) was measured by ELISA. Values are means of at least three experiments (n 3–7) each run in triplicate, with standard errors represented by vertical bars, and are expressed relative to the mean value in the vehicle dimethyl sulphoxide (DMSO)-treated control cells, which was set at 100 %. The absolute mean value of IL-6 in the medium of the vehicle-treated cells was 429 (se 62) pg/mg of cell protein. Data were analysed by one-way ANOVA followed by the least significant difference post hoc test. a,b,c Mean values with unlike letters were significantly different (P < 0·05).

Figure 2

Fig. 2 Effects of 24 h exposure to varying doses of oleic acid or elaidic acid on TNF-α gene expression and secreted levels in C2C12 myotubes. Gene expression of TNF-α ((A) and (B)) was measured by real-time quantitative PCR and normalised to β-actin, and is expressed relative to the mean value in the vehicle dimethyl sulphoxide (DMSO)-treated control cells, which was set at 100 %. TNF-α protein in the medium (C) was measured by ELISA and is expressed as absolute values (n.d., not detected). Values are means of at least three experiments (n 3–7) each run in triplicate, with standard errors represented by vertical bars. Gene expression data were analysed by one-way ANOVA followed by the least significant difference post hoc test. a,b Mean values with unlike letters were significantly different (P < 0·05).

Figure 3

Fig. 3 Effects of 24 h exposure to varying doses of (A) oleic acid or (B) elaidic acid on IL-15 gene expression in C2C12 myotubes. Values are means of at least three experiments (n 3–7) each run in triplicate, with standard errors represented by vertical bars, and are expressed relative to the mean value in the vehicle dimethyl sulphoxide (DMSO)-treated control cells, which was set at 100 %. Gene expression of IL-15 was measured by real-time quantitative PCR and normalised to β-actin. Data were analysed by one-way ANOVA followed by the least significant difference post hoc test. a,b Mean values with unlike letters were significantly different (P < 0·05).

Figure 4

Fig. 4 Effects of 24 h exposure to 100 μm-oleic or -elaidic acid on basal and insulin-stimulated 2-deoxy-d-glucose (2-DOG) uptake in (A) C2C12 myotubes and (B) 3T3-L1 adipocytes. 2-DOG uptake was measured in the absence of insulin (basal) and in the presence of 100 nm-insulin as described in the Experimental methods. Values are means of at least three experiments (n 3–8) each run in quadruplicate, with standard errors represented by vertical bars, and are expressed relative to the mean value in the vehicle dimethyl sulphoxide (DMSO)-treated control cells, which was set at 100 %. One-way ANOVA followed by the least significant difference post hoc test was used to analyse the significance of differences among treatments under the basal and insulin-stimulated conditions; a,b,c for each condition, mean values with unlike letters were statistically different (P < 0·05). * Statistically significant difference (P < 0·05) v. the basal condition assessed by Student's t test. □, Vehicle (DMSO); ■, 100 μm-oleic acid; , 100 μm-elaidic acid; dpm, disintegrations per min.

Figure 5

Fig. 5 Effects of 24 h exposure to 100 μm-oleic or -elaidic acid on the gene expression of (A) resistin and (B) adiponectin in 3T3-L1 adipocytes. Values are means of four experiments (n 4) each run in triplicate, with standard errors represented by vertical bars, and are expressed relative to the mean value in the vehicle dimethyl sulphoxide (DMSO)-treated control cells, which was set at 100 %. Gene expression was measured by real-time quantitative PCR and normalised to β-actin. Data were analysed by one-way ANOVA followed by the least significant difference post hoc test. a,b Mean values with unlike letters were significantly different (P < 0·05). □, Vehicle (DMSO); ■, 100 μm-oleic acid; , 100 μm-elaidic acid.