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The Eicosapentaenoic Acid Metabolite 15-Deoxy-δ12,14-Prostaglandin J3 Increases Adiponectin Secretion by Adipocytes Partly via a PPARγ-Dependent Mechanism

  • Jennifer Lefils-Lacourtablaise,

    Affiliation Université de Lyon, Institut National de la Santé et de la Recherche Médicale Unité Mixte de Recherche 1060, “Laboratoire de Recherche en Cardiovasculaire, Métabolisme, Diabétologie et Nutrition”, Institut Multidisciplinaire de Biochimie des Lipides/Institut National des Sciences Appliquées-Lyon, Villeurbanne, France

  • Mairobys Socorro,

    Affiliation Laboratorio de Biología Celular del Endotelio, Instituto de Medicina Experimental-Facultad de Medicina Universidad Central de Venezuela, Caracas, Venezuela

  • Alain Géloën,

    Affiliation Université de Lyon, Institut National de la Santé et de la Recherche Médicale Unité Mixte de Recherche 1060, “Laboratoire de Recherche en Cardiovasculaire, Métabolisme, Diabétologie et Nutrition”, Institut Multidisciplinaire de Biochimie des Lipides/Institut National des Sciences Appliquées-Lyon, Villeurbanne, France

  • Patricia Daira,

    Affiliation Université de Lyon, Institut National de la Santé et de la Recherche Médicale Unité Mixte de Recherche 1060, “Laboratoire de Recherche en Cardiovasculaire, Métabolisme, Diabétologie et Nutrition”, Institut Multidisciplinaire de Biochimie des Lipides/Institut National des Sciences Appliquées-Lyon, Villeurbanne, France

  • Cyrille Debard,

    Affiliation Université de Lyon, Institut National de la Santé et de la Recherche Médicale Unité Mixte de Recherche 1060, “Laboratoire de Recherche en Cardiovasculaire, Métabolisme, Diabétologie et Nutrition”, Institut Multidisciplinaire de Biochimie des Lipides/Institut National des Sciences Appliquées-Lyon, Villeurbanne, France

  • Emmanuelle Loizon,

    Affiliation Université de Lyon, Institut National de la Santé et de la Recherche Médicale Unité Mixte de Recherche 1060, “Laboratoire de Recherche en Cardiovasculaire, Métabolisme, Diabétologie et Nutrition”, Institut Multidisciplinaire de Biochimie des Lipides/Institut National des Sciences Appliquées-Lyon, Villeurbanne, France

  • Michel Guichardant,

    Affiliation Université de Lyon, Institut National de la Santé et de la Recherche Médicale Unité Mixte de Recherche 1060, “Laboratoire de Recherche en Cardiovasculaire, Métabolisme, Diabétologie et Nutrition”, Institut Multidisciplinaire de Biochimie des Lipides/Institut National des Sciences Appliquées-Lyon, Villeurbanne, France

  • Zury Dominguez,

    Affiliation Laboratorio de Biología Celular del Endotelio, Instituto de Medicina Experimental-Facultad de Medicina Universidad Central de Venezuela, Caracas, Venezuela

  • Hubert Vidal,

    Affiliation Université de Lyon, Institut National de la Santé et de la Recherche Médicale Unité Mixte de Recherche 1060, “Laboratoire de Recherche en Cardiovasculaire, Métabolisme, Diabétologie et Nutrition”, Institut Multidisciplinaire de Biochimie des Lipides/Institut National des Sciences Appliquées-Lyon, Villeurbanne, France

  • Michel Lagarde,

    Affiliation Université de Lyon, Institut National de la Santé et de la Recherche Médicale Unité Mixte de Recherche 1060, “Laboratoire de Recherche en Cardiovasculaire, Métabolisme, Diabétologie et Nutrition”, Institut Multidisciplinaire de Biochimie des Lipides/Institut National des Sciences Appliquées-Lyon, Villeurbanne, France

  • Nathalie Bernoud-Hubac

    nathalie.bernoud-hubac@insa-lyon.fr

    Affiliation Université de Lyon, Institut National de la Santé et de la Recherche Médicale Unité Mixte de Recherche 1060, “Laboratoire de Recherche en Cardiovasculaire, Métabolisme, Diabétologie et Nutrition”, Institut Multidisciplinaire de Biochimie des Lipides/Institut National des Sciences Appliquées-Lyon, Villeurbanne, France

The Eicosapentaenoic Acid Metabolite 15-Deoxy-δ12,14-Prostaglandin J3 Increases Adiponectin Secretion by Adipocytes Partly via a PPARγ-Dependent Mechanism

  • Jennifer Lefils-Lacourtablaise, 
  • Mairobys Socorro, 
  • Alain Géloën, 
  • Patricia Daira, 
  • Cyrille Debard, 
  • Emmanuelle Loizon, 
  • Michel Guichardant, 
  • Zury Dominguez, 
  • Hubert Vidal, 
  • Michel Lagarde
PLOS
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Abstract

The intake of ω-3 polyunsaturated fatty acids (PUFAs), which are abundant in marine fish meat and oil, has been shown to exert many beneficial effects. The mechanisms behind those effects are numerous, including interference with the arachidonic acid cascade that produces pro-inflammatory eicosanoids, formation of novel bioactive lipid mediators, and change in the pattern of secreted adipocytokines. In our study, we show that eicosapentaenoic acid (EPA) increases secreted adiponectin from 3T3-L1 adipocytes and in plasma of mice as early as 4 days after initiation of an EPA-rich diet. Using 3T3-L1 adipocytes, we report for the first time that 15-deoxy-δ12,14-PGJ3 (15d-PGJ3), a product of EPA, also increases the secretion of adiponectin. We demonstrate that the increased adiponectin secretion induced by 15d-PGJ3 is partially peroxisome proliferator-activated receptor-gamma (PPAR-γ)-mediated. Finally, we show that 3T3-L1 adipocytes can synthesize 15d-PGJ3 from EPA. 15d-PGJ3 was also detected in adipose tissue from EPA-fed mice. Thus, these studies provide a novel mechanism(s) for the therapeutic benefits of ω-3 polyunsaturated fatty acids dietary supplementation.

Introduction

Eicosapentaenoic (EPA) and docosahexaenoic (DHA) acids are ω-3 polyunsaturated fatty acids (PUFA), found primarily in marine lipids, that display many health benefits, such as the improvement of insulin sensitivity with beneficial effects against obesity and the prevention of cardiovascular diseases [1–4]. The American Heart Association recommends eating fish rich in ω-3 fatty acids. Despite numerous studies suggesting protective actions of EPA and DHA, the cellular and molecular rational for their intake remains of considerable interest.

It is assumed that these beneficial effects are linked to the ability of both acids to inhibit the production of ω-6 PUFA-derived prostaglandins and leukotrienes [5]. Additionally, recent studies have shown that a series of novel ω-3 PUFA-derived compounds could be responsible for eliciting their beneficial effects [6–8]. Resolvins and protectins have been shown for example to display potent anti-inflammatory and immunoregulatory actions [9, 10].

Among bioactive lipid mediators, prostaglandins (PG) exert a plethora of biological activities. PGs of the 2-series are formed by cyclooxygenase (COX)-1 and COX-2 from arachidonic acid (AA). COX converts AA (released from membrane phospholipids through the activity of several phospholipases, mainly phospholipases A2) to the unstable cyclic endoperoxide intermediates PGG2/H2 [11]. PGH2 is subsequently metabolized to several prostanoids, PGD2, PGE2, PGF, PGI2 and thromboxane A2 (TXA2) through the action of synthases (prostaglandin D synthase [PGDS], PGES, PGFS, PGIS and TXAS) [12,13]. In vitro, PGD2 spontaneously dehydrates to PGJ2 [14] which is converted to 15-deoxy-δ12,14-PGJ2 (15d-PGJ2) in the absence of albumin [15]. 15d-PGJ2 has been detected in vivo [15,16] and has been shown to exhibit in vitro and in vivo anti-inflammatory and anti-proliferative effects [15,17]. The anti-inflammatory cyclopentenone PGs exert their effects, in part, by binding and activating the peroxisome proliferator-activated receptor-gamma (PPAR-γ) [18,19].

EPA can also be enzymatically converted by cyclooxygenase into PGH3 which in turn is converted to the 3-series PGs, e.g., PGD3, PGE3, PGF and PGI3 [20–22]. The eicosanoids derived from EPA have less inflammatory activities compared with those produced from AA [23–25].

Another mechanism by which ω-3 PUFA may exert beneficial effects is by modulating the secretion of adipocytokines [26, 27]. Adiponectin is one of the most abundant plasma protein adipocytokines that shows anti-inflammatory, anti-atherogenic and insulin-sensitizing properties [28, 29]. The potential mechanism by which ω-3 PUFA modulate adiponectin secretion is not fully understood, but may partially involve PPAR-γ [30-33] which has been shown to play an important role in the transcriptional activation of the adiponectin gene [34].

A recent study showed the formation of J-series PGs from EPA [35]. The pathway by which 15d-PGJ3 could be generated is shown in Fig. 1. PGD3 would be first dehydrated to 15d-PGD3 and PGJ3 and then the latter compound would be directly converted to 15d-PGJ3.

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Figure 1. Proposed pathway for PGD3 metabolism

(adapted from Ref Shibata et al., 2002 for PGD2).

https://doi.org/10.1371/journal.pone.0063997.g001

We concomitantly raised the possibility that 3-series PGs, PGD3 and J3 PGs might influence the production of adipokines. Our studies show that EPA, PGD3 and 15d-PGJ3 increased adiponectin secretion by 3T3-L1 and that this partly occurred via a PPAR-γ-dependent mechanism. Moreover, we present evidence that 15d-PGJ3 is formed in significant amount after incubation of cells with EPA.

Materials and Methods

Ethics Statement. This study was carried out in strict accordance with the European Communities Council Guidelines (November 24, 1986, 86/609/EEC) and all animal experiments followed a strict protocol. This study was specifically approved by the Committee on the Ethics of Animal Experiments of the INSA of Lyon CETIL (permit Number: 012012). All efforts were made to minimize suffering.

Materials

3T3-L1 cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). Dexamethasone, 3-isobutyl-1-methyl-xanthine and GW9662 were purchased from Sigma-Aldrich (Saint Quentin Fallavier, France). Insulin was obtained from Novo Nordisk Actrapid and rosiglitazone from Molekula (La Tour du Pin, France). EPA, as the synthetic triglyceride, Omegavie 90, was purchased from Polaris (Pleuven, France). Mouse adiponectin EIA was purchased from SpiBio (Montigny Le Bretonneux, France). EPA, d5-EPA, PGD3 and PGD2 were purchased from Cayman Europe (Tallinn, Estonia). All solvents used were of HPLC quality. RNeasy mini kit and rotor-Gene Q were from Qiagen (Courteboeuf, France). Superscript II was from Invitrogen (Eragny, France). Random hexamers and oligo (dT) primers were from Promega (Charbonnières, France). XBridge™ columns were from Waters (St Quentin, France).

Cell Culture

3T3-L1 preadipocytes were cultured in a 5% CO2 atmosphere at 37°C in a growth medium containing the following constituents: Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal calf serum, 4 mM L-glutamin and antibiotics. Differentiation of the cells was induced after confluence using the growth medium containing 0.5 mM 3-isobutyl-1-methyl-xanthine, 5 µg/mL insulin, 10 µmol/L rosiglitazone and 0.25 µmol/L dexamethasone. On day 2, the media was replaced by the growth medium containing 5 µg/mL insulin and 10 µmol/L rosiglitazone for 2 days. The fully differentiated phenotype was controlled by observing the cells using light microscopy for the existence of the typical appearance of extensive accumulation of lipid droplets. Insulin was removed on day 4 by changing the media to growth medium containing 10 µmol/L rosiglitazone and cells were maintained thereafter in this medium. Day 10 differentiated 3T3-L1 adipocytes were used for the experiments.

Effects of Eicosapentaenoic Acid and Prostaglandins of the 3 Series on Adiponectin Secretion

Preceding the different treatments, 3T3-L1 cells were washed with phosphate-buffered saline (PBS) and incubated under serum-free culture medium for 4 h. Cells were then incubated in fresh DMEM for 2 and 4 h with EPA (1 µM or 10 µM) complexed with bovine serum albumin (50 µM) or with PGD3 (1 µM) or 15d-PGJ3 (100 nM) in an ethanolic solution in the presence or absence of 10 µM GW9662, a PPAR-γ antagonist. Control cells received vehicle (bovine serum albumin or ethanol alone).

Secreted Adiponectin Protein Measurement

Concentrations of adiponectin in the cell culture media and in sera were measured by enzyme-linked immunosorbent assay (ELISA) following the manufacturer’s instructions. In brief, samples were incubated in a 96-well pre-coated plate with a monoclonal antibody specific of mouse adiponectin for 1 h at room temperature. After washing the plate, anti-adiponectin antibody horseradish peroxidase tracer was added. Incubation was carried out for 1 h and substrate solution was then added for 10 min after washing the plate. The reaction was arrested by addition of stop solution. The concentration of the adiponectin was determined by spectrophotometry.

Quantitative PCR

RNAs were prepared using the RNeasy mini kit according to the method recommended by the manufacturer. First-strand cDNAs were synthesized from 1 µg of total RNA in the presence of 100 units of Superscript II using a mixture of random hexamers and oligo (dT) primers. Real-time PCR assays were performed using a Rotor-Gene Q as described previously [36]. The following primers were used : for FAS: forward 5′-GTGCACCCCATTGAAGGTTCC-3′, reverse 5′- GGTTTGGAATGCTGTCCAGGG-3′; for adiponectin: forward 5′- AGGCCGTGATGGCAGAGATG-3′, reverse 5′- CTTCTCCAGGTTCTCCTTTCCTGC-3′; for FABP4 forward 5′-CAGAAGTGGGATGGAAAGTCG-3′, reverse 5′- CGACTGACTATTGTAGTGTTTGA-3′; for PPAR-γ: forward 5′-TCTCTCCGTAATGGAAGACC-3′, reverse 5′-GCATTATGAGACATCCCCAC-3′; for PDK4: forward 5′-AGTGTGCAAAGATGCTCTGC-3′, reverse 5′- AGAGCATGTGGTGAAGGTGTG-3′. Glucuronidase, beta (Gusb) mRNA level was used to normalize the data.

Animals and Diets

Ethics Statement. Mice were treated in accordance with the European Communities Council Guidelines (November 24, 1986, 86/609/EEC) and all animal experiments followed a strict protocol, approved by the Committee on the Ethics of Animal Experiments of the INSA of Lyon CETIL (permit Number: 012012). Animals were kept on a 12∶12-h light/dark cycle and were allowed an unrestricted access to diet and water. Animals were randomised into groups of 8 and assigned to a standard control diet A03 (SAFE, Augy, France) containing 5% lipids, or to an EPA-enriched diet reconstituted with lipid-free powder (SAFE, Augy, France), 4.5% sunflower oil (Lesieur, Asnières-sur-Seine, France) and 0.5% 1,2,3-trieicosapentaenoylglycerol (Polaris, Pleuven, France) for 4 days. The complete nutrient and fatty acid composition of each of the diets are listed in Table 1. The diet was replaced every day for a new one to avoid EPA peroxidation. Mice were killed by lethal intraperitoneal injection of pentobarbital on day 0 and day 4 and blood was collected and immediately centrifuged. Plasma aliquots were sampled and stored at −80°C before measurement of adiponectin. Adipose tissue was dissected and immediately frozen in liquid nitrogen and then stored at −80°C until analysis of 15d-PGJ3.

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Table 1. Nutrient composition and fatty acid composition of standard (A03) and EPA-rich diet.

https://doi.org/10.1371/journal.pone.0063997.t001

Dehydration/isomerization of PGD2 and PGD3

PGD2 or PGD3 (1 mM) were incubated in PBS at 37°C. After acidification to pH 3, samples were extracted three times with 4 volumes of ethyl acetate and extracts were dried under nitrogen gas. The samples were reconstituted in ethanol.

HPLC Separation of PGD2 and PGD3 Metabolites

Products from PGD2 or PGD3 were isolated by HPLC using a 4.6×250 mm C18 XBridge™ column. The solvent system used was a gradient consisting of acetonitrile/water acidified to pH 3 (2/8, v/v)) (solvent A) to acetonitrile (solvent B). The flow rate was 1 mL/min beginning at 100% A, followed by an increase to 100% B over 30 min. The column was then washed with 100% B for 10 more min. The elution profiles were monitored by UV absorbance at 195 nm.

Analysis of 15d-PGJ3 by Gas Chromatography (GC)-mass Spectrometry (GC-MS) and GC/tandem MS

15d-PGJ3 putative compound eluting as a single peak from HPLC was collected. The compound was converted to a pentafluorobenzyl ester derivative, with pentafluorobenzyl bromide and diisopropylethylamide in acetonitrile. A Thermo Trace GC connected to a Thermo PolarisQ MS operated in negative ion chemical ionization (NICI) mode was used to analyze the sample. 15d-PGJ3 was detected by GC-MS using selected ion monitoring for the [M-CH2C6F5]- ion (m/z 313). The molecular ion m/z 313 of putative 15d-PGJ3 was subjected to collision-induced dissociation (CID).

Analysis of 15d-PGJ3 and d5-15d-PGJ3 in the Culture Medium of 3T3-L1 Cells Incubated with EPA or d5-EPA

Cells were incubated with EPA or d5-EPA. Products were separated by HPLC; the HPLC fraction supposed to contain 15d-PGJ3 or d5-15d-PGJ3 was collected and was analyzed by GC-MS and GC-MS/MS as described above. The molecular ions m/z 313 of putative 15d-PGJ3 and m/z 318 of putative d5-15d-PGJ3 were subjected to collision-induced dissociation (CID). Spectra that are shown were obtained at 2 eV. Quantifications were performed using d4-15d-PGJ2.

Analysis of 15d-PGJ3 in Adipose Tissue from EPA Fed-mice

Epididymal adipose tissues were obtained from previously mentioned feeding study. Sample was extracted using a silica Sep-Pak cartridge. 15d-PGJ3 was purified by HPLC as described above, converted into a pentafluorobenzyl ester derivative, and analyzed by GC-MS/MS as described above.

Statistical Analysis

Statistical analyses were performed using Student’s t-test. The difference was considered significant at p<0.05. The results are expressed as means ± sem.

Results

Increased Plasma and Media Adiponectin Concentrations in Mice Fed an EPA-enriched Diet and in 3T3-L1 Cells Treated with EPA, Respectively

We examined the effects of EPA on the production of adiponectin in mice fed an EPA-enriched diet. Plasma level of adiponectin was significantly increased (+17%) as early as 4 days after initiation of the EPA-enriched diet group compared to control mice (Fig. 2 A).

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Figure 2. (A) Plasma levels of adiponectin from mice fed a standard diet or an EPA-rich diet.

Plasma levels are expressed in µg/mL. Results are means ± sem (n = 8). *P<0.05 as compared to the control group. (B) Body weight gain (g) of mice fed a standard diet or an EPA-rich diet. Mice were killed on days 0 and 4. Results are means ± sem (n = 8). *P<0.05 as compared to the control group. (C) Effects of eicosapentaenoic acid on adiponectin secretion by 3T3-L1 adipocytes. Cells were incubated for 2 h (gray) or 4 h (black) with 1 µM or 10 µM EPA complexed with bovine serum albumin. Adiponectin in the medium was determined by ELISA. Results are means ± sem (n = 4 in triplicate), expressed as percentage of the control (□). Statistical significance is represented as *P<0.05 vs control.

https://doi.org/10.1371/journal.pone.0063997.g002

On the contrary, blood leptin secretion was significantly decreased (by 1.5-fold) after 4 days of the EPA-enriched diet feeding (not shown). We also observed that the body weight gain of mice fed the EPA-enriched diet was significantly lower than that of mice fed the standard diet (31.0 g +/−1.4 vs 33.9+/−0.7) (Fig. 2 B). We also examined the effects of EPA on the adiponectin concentration in the culture media of cells. 3T3-L1 cells were incubated for 2 h or 4 h with 1 µM or 10 µM of EPA. As shown in Fig. 2 C, EPA induced a significant increase in adiponectin secretion.

Synthesis of 15d-PGJ3 from PGD3 in Phosphate-buffered Saline

Previously, it has been shown that PGD2, a prostaglandin derived from AA, is converted sequentially to J2 prostaglandins in vitro [14, 15]. Shibata et al [15] showed that PGD2 is initially converted to the dehydration products 15d-PGD2 and PGJ2, the latter being converted to 15d-PGJ2. A recent study showed that J3 prostanoids are also formed in vitro from PGD3 [35]. PGD2 or PGD3 was incubated in PBS at 37°C for 72h and the products were analyzed by HPLC. By comparison with PGs of the 2-series (formed from PGD2, Figure 3A) or commercial (not shown), peaks I, II and III were assumed to be PGJ3, 15d-PGD3 and 15d-PGJ3 (Fig. 3B, peaks I, II and III, respectively). To further substantiate the structural identity of peak III as 15d-PGJ3, the HPLC fraction containing this peak was collected and analyzed by GC-MS and GC-MS/MS. The predicted unique [M-CH2C6F5]- ion for the pentafluorobenzyl ester derivative 15d-PGJ3 is m/z 313. The GC/NICI/MS analysis of the product chromatogram only showed a m/z 313 peak compatible with the formation of 15d-PGJ3 (Fig. 4A). This peak was analyzed by CID and the CID spectrum is shown in figure 4B. A predicted fragment, m/z 269 [M-CH2C6F5-CO2]-, was obtained.

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Figure 3. HPLC profile of metabolites formed from PGD2 (A) or PGD3 (B).

1 mM of PGD2 or PGD3 was incubated in phosphate-buffered saline at 37°C for 24 h. PGD2/PGD3 and their metabolites were chromatographed on a Waters Xbridge C18 column (4.6×250 mm, 3.5 µm) at a flow rate of 1 ml/min starting at 100% solvent A (acetonitrile/water acidified to pH 3, 2/8 v/v) to 100% solvent B (acetonitrile) from 1 to 30 min. The elution profiles were monitored by UV absorbance at 195 nm.

https://doi.org/10.1371/journal.pone.0063997.g003

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Figure 4. GC-MS (A) and GC-MS/MS (B) spectra of 15d-PGJ3 generated during incubation of PGD3 in phosphate-buffered saline.

The anion at m/z 313 [M-CH2C6F5]- generated by NICI was subjected to CID. The structure of the derivative and the proposed structure of the product ion produced by CID of m/z 313 are shown in the figure.

https://doi.org/10.1371/journal.pone.0063997.g004

Exposure of 3T3-L1 Adipocytes to PGD3 and 15d-PGJ3 Leads to Increased Adiponectin Levels

We then examined whether PGD3 and 15d-PGJ3 could increase adiponectin secretion by 3T3-L1 adipocytes. Exposure of cells to 1 µM PGD3 and 100 nM 15d-PGJ3 resulted in increased adiponectin levels in 3T3-L1 adipocytes medium by 55% and 28%, respectively compared to control cells (Fig. 5). These concentrations were chosen since the increase in adiponectin secretion was observed from 1 µM EPA. We also observed an increased adiponectin secretion (+25%) after incubation of cells for 24 h with 100 nM 15d-PGJ3 compared to control cells (not shown).

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Figure 5. Effects of PGD3 and 15d-PGJ3 on adiponectin secretion by 3T3-L1 adipocytes.

Cells were incubated for 2 h with 1 µM PGD3 or 0.1 µM 15d-PGJ3. Adiponectin in the medium was determined by ELISA. Results are means ± sem (n = 4 in triplicate), expressed as percentage of the control. Statistical significance is represented as **P<0.01 vs control.

https://doi.org/10.1371/journal.pone.0063997.g005

Formation of 15d-PGJ3 in Cell Medium after Incubation of Cells with EPA

We then sought to determine whether 15d-PGJ3 could be detected in the culture medium of cells incubated with EPA. 3T3-L1 were incubated with 10 µM EPA. Products were separated by HPLC; the HPLC fraction supposed to contain 15d-PGJ3 was collected and 15d-PGJ3 was analyzed by GC-MS and GC-MS/MS. As shown in Fig. 6A, a significant amount of 15d-PGJ3 was detected in the culture medium (nanomolar concentration). The observed production, m/z 269, generated by CID of the parent ion [M-CH2C6F5]-, is consistent with fragmentation of 15d-PGJ3 ([M-CH2C6F5-CO2]-) (Fig. 6B). To further substantiate the formation 15d-PGJ3 from EPA in 3T3-L1, cells were incubated with d5-EPA. As described above, products were separated by HPLC and the HPLC fraction containing the putative d5-15d-PGJ3 was collected and then analyzed by GC-MS/MS. The predicted molecular ion for [M-CH2C6F5]- of d5-15d-PGJ3 is m/z 318. CID of m/z 318 generates the product ion at m/z 274 ([M-CH2C6F5-CO2]-) (Fig 7).

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Figure 6. GC-MS and GC-MS/MS analysis of 15d-PGJ3 in the culture medium following incubation of cells with EPA.

3T3-L1 were incubated with 10 µM EPA for 4 h. Culture medium was extracted. 15d-PGJ3 was purified by HPLC. The 15d-PGJ3 HPLC peak was collected and was analyzed by GC-MS as a pentafluorobenzyl ester derivative (m/z 313) (Fig. 6A). B. GC-MS/MS spectrum of 15d-PGJ3. The molecular ion [M-CH2C6F5]- of m/z 313 was subjected to CID. The structure of the derivative and the proposed structure of the product ion produced by CID of m/z 313 are shown in the figure.

https://doi.org/10.1371/journal.pone.0063997.g006

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Figure 7. GC-MS/MS analysis of d5-15d-PGJ3 in the culture medium following incubation of cells with d5-EPA.

3T3-L1 cells were incubated with d5-EPA for 4 h. Culture medium was extracted. d5-15d-PGJ3 was purified by HPLC. The d5-15d-PGJ3 HPLC peak was collected and was analyzed by GC-MS/MS. The anion at m/z 315 [M-CH2C6F5]- was subjected to CID. The structure of the derivative and the proposed structure of the product ion produced by CID of m/z 315 are shown in the figure.

https://doi.org/10.1371/journal.pone.0063997.g007

Formation of 15d-PGJ3 in vivo

We then undertook experiments to determine whether 15d-PGJ3 is formed in adipose tissue in vivo. Because baseline levels of EPA in animals are very low, we determined if 15d-PGJ3 is generated in vivo by examining this compound in mice fed with the EPA-enriched diet previously mentioned. A representative GC-MS/MS ion current chromatogram obtained from these analyses is shown in Fig. 8. The chromatogram based on m/z 313 detection represents endogenous 15d-PGJ3 (Fig. 8A). The observed production, m/z 269, generated by CID of the parent ion [M-CH2C6F5]-, is consistent with fragmentation of 15d-PGJ3 ([M-CH2C6F5-CO2]-) (Fig. 8B).

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Figure 8. GC-MS/MS analysis of 15d-PGJ3 in epididymal adipose tissue of mice fed a EPA-rich diet.

15d-PGJ3 was purified by HPLC. The 15d-PGJ3 HPLC peak was collected and was analyzed by GC-MS as a pentafluorobenzyl ester derivative (m/z 313) (Fig. 8A). B. GC-MS/MS spectrum of 15d-PGJ3. The molecular ion [M-CH2C6F5]- of m/z 313 was subjected to CID. The structure of the derivative and the proposed structure of the product ion produced by CID of m/z 313 are shown in the figure.

https://doi.org/10.1371/journal.pone.0063997.g008

PPAR-γ Antagonism Decreases PGD3- or 15d-PGJ3-induced Increase in Adiponectin

Next, we determined whether PGD3 or 15d-PGJ3 increased adiponectin via a PPAR-γ-dependent mechanism. 3T3-L1 adipocytes were incubated with a PPAR-γ antagonist, GW9662, alone or in combination with PGD3 or 15d-PGJ3. GW9662 alone, which has been previously reported to induce irreversible loss of ligand binding activity [37], had no effect on adiponectin secretion. As shown in Fig. 9, GW9662 significantly attenuated the increase of adiponectin level in cell medium observed in response to PGD3 or 15d-PGJ3. However, the adiponectin concentration was still significantly increased when GW9662 was added with PGD3 or 15d-PGJ3 compared with control condition, suggesting that part of the prostaglandin effect was not attributable to PPAR-γ stimulation.

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Figure 9. Effect of a PPAR-γ antagonism on the PGD3 or 15d-PGJ3 increase in adiponectin secretion by cells.

3T3-L1 adipocytes were incubated for 2 h with 1 µM PGD3 or 100 nM 15d-PGJ3 in the presence or absence of 10 µM GW9662 or with GW9662 alone. Adiponectin in the medium was determined by ELISA. Results are means ± sem (n = 3), expressed as percentage of the control. Statistical significance is represented as *P<0.05, **P<0.01.

https://doi.org/10.1371/journal.pone.0063997.g009

15d-PGJ3 Mediates Induction of PPAR-γ Target Gene Expression in 3T3-L1 Adipocytes

To go further in the demonstration of the involvement of PPAR-γ, the effects of 15d-PGJ3 on the expression of characterized PPAR-γ target genes, FAS, FABP4 and adiponectin were examined. 3T3-L1 cells were incubated with or without 100 nM of 15d-PGJ3 for 2 h. As depicted in Fig. 10, 15d-PGJ3 induced increase in mRNA level of all PPAR-γ target genes tested with a significant increase in the level of FABP4 and FAS mRNA. However, treatment with 15d-PGJ3 had no effect on PDK4 mRNA transcript abundance.

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Figure 10. Effect of 15d-PGJ3 on FAS, FABP4, adiponectin, PPAR-γ and PDK4 gene expression in 3T3-L1 adipocytes.

Cells were incubated for 2 h with or without 100 nM 15d-PGJ3. FAS, FABP4, adiponectin, PPAR-γ and PDK4 mRNA levels were quantified by qPCR. Results are means ± sem (n = 3). Statistical significance is represented as *P<0.05 vs control.

https://doi.org/10.1371/journal.pone.0063997.g010

Discussion

This study demonstrates that EPA increased adiponectin secretion in vitro in 3T3-L1 adipocytes. Our finding is consistent with that of Tishinsky et al. [33] who observed stimulatory effects of EPA on adiponectin secretion in human adipocytes but contradicts the finding of Itoh et al. [38] who reported no effect of 200 µM EPA on adiponectin secretion from 3T3-L1 or those of Lorente-Cebrián et al. [39] who showed a significant decreased protein secretion in isolated rat adipocytes treated with 100–200 µM of EPA for 96h. These conflicting data may be explained by the dose of EPA used and the duration of the treatment. Indeed, it has been demonstrated that adipose cells and adiponectin production are sensitive to oxidative stress [40]. Depending on the dose, ω-3 PUFA may have bimodal effect with antioxidant activity at low intake [41] and a prooxidant one at high dose [42]. Furukawa et al. [43] also found that incubation of 3T3-L1 adipocytes with 200 µM EPA for 24h induced an oxidative stress in cells. Thus the exposure to high concentrations of PUFA may lead to lipid peroxidation with subsequent decrease of adiponectin production.

Most studies examining the beneficial effects of ω-3 PUFA in vivo have used fish oil that usually consist mainly of EPA and DHA [26, 44–46]. Recently, we demonstrated that DHA rapidly increased adiponectin secretion in mice fed a DHA-rich diet compared with mice fed a standard diet [27]. We now show that EPA supplementation also increased adiponectin secretion as early as 4 days after initiation of the EPA-rich diet. The only previous study investigating the effect of EPA on adipocytokine secretion in mice was conducted over a 2-week or 4-week feeding period [38]. The present data show that EPA effect on adiponectin is very fast.

Different cellular and molecular mechanisms have been proposed to explain the beneficial effects of ω-3 PUFA including modification of the membrane structure and the signal transduction [47][49], modulation of gene expressions [50], [51]. Another possible mechanism involves the altered pattern of eicosanoid production, eicosanoids derived from ω-3 PUFA having in general anti-inflammatory effects while those produced from ω-6 PUFA being pro-inflammatory mediators [5]. Additionally, in recent years, ω-3 PUFA have been demonstrated to serve as substrates of novel bioactive lipid mediators named resolvins and protectins [6, 7]. We hypothesized that metabolites derived from EPA could also partly be responsible for the increased adiponectin secretion. Our studies have shown for the first time that adiponectin secretion is up-regulated by a novel class of cyclopentenone eicosanoid, 15d-PGJ3, issued from PGD3.

In the present studies, we further explored the molecular mechanism involved in the increased adiponectin secretion by 15d-PGJ3. We investigated the role of the nuclear receptor PPAR-γ which is an assumed key transcription factor that regulates the expression of adiponectin [52], [53]. Moreover, the anti-inflammatory effects of the 2-series cyclopentenone prostaglandins have been shown to be mediated, in part, through PPAR-γ [18, 19]. We initially used the GW9662, a covalent PPAR-γ antagonist [54]. We show that GW9662 partially decreased the PGD3 and 15d-PGJ3-mediated increase in adiponectin secretion. Further analysis indicated that 15d-PGJ3 enhanced the expression of transcriptionnal target genes of PPAR-γ, such as FAS and FABP4. Our results suggest that 15d-PGJ3 elicits the effect on adiponectin secretion at least partially via PPAR-γ. This is consistent with observations showing that 15dPGJ2 can act through PPAR-γ-dependent as well as PPAR-γ-independent mechanisms [19, 55–57]. 15d-PGJ3, as 15d-PGJ2, is supposed to be particularly reactive, including toward PPAR-γ [58], because of the presence of an highly electrophilic α,?β-unsaturated ketone moiety and two electrophilic carbon centers (at carbons 9 and 13) which readily reacts with substances containing nucleophilic groups. 15d-PGJ3 could thus modulate cellular responses through interaction with key intracellular protein targets as it has been shown for 15d-PGJ2. For example, it has been reported that 15d-PGJ2 directly inhibits the NF-κB-dependent gene expression by covalent binding to a critical cysteine residue in IκB kinase and the DNA-binding domains of NF-κB subunits [56], [57]. The study of Giri and colleagues [59] support the conclusion that the anti-inflammatory actions of 15d-PGJ2 include regulation of the PI3K-Akt-NF-κB pathway, independently of PPAR-γ mechanism. In further study, the 15d-PGJ3-induced increase in adiponectin through this PPAR-γ-independent mechanism should be elucidated in order to understand the whole mechanism of 15d-PGJ3-mediated secretion of adiponectin.

The studies reported herein have characterized the formation of 15d-PGJ3 in vitro and in vivo. We indeed demonstrate for the first time that 3T3-L1 adipocytes can directly synthesize 15d-PGJ3 from EPA. The accumulation of significant amount of 15d-PGJ3 in the culture medium of 3T3-L1 may be explained by an intracellular production of 15d-PGJ3 followed by its excretion to the medium and/or the excretion of PGD3 which is then converted nonenzymatically to 15d-PGJ3. We also detected 15d-PGJ3 in adipose tissue from EPA-fed mice.

In summary, our results indicate that EPA increases secreted adiponectin concentration in 3T3-L1 adipocytes and in mice as early as 4 days after initiation of the EPA-rich diet. We first demonstrate, using 3T3-L1 adipocytes, that prostaglandins of the 3-series formed from EPA also increase the secretion of adiponectin, in part through PPAR-γ-dependent mechanism. This study opens up new avenues for scientific inquiry. This provides the rational basis to explore in depth the production of 15d-PGJ3 in vivo and its biological activities. This will likely provide important new insights into the role of ω-3 PUFA and their metabolites in physiology and diseases.

Author Contributions

Conceived and designed the experiments: NBH AG HV ML. Performed the experiments: JLL MS AG PD CD ZD EL NBH. Analyzed the data: NBH JLL ML HV CD. Contributed reagents/materials/analysis tools: MG CD AG ML HV NBH. Wrote the paper: NBH JLL ML.

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