Omegavie® Tuna oil 25 DHA flavorless was provided by Polaris (Pleuven, France) as a source of triacylglycerol rich in LC n-3 fatty acids. A lecithin rich in DHA was enzymatically synthesized by Polaris (Pleuven, France) and further purified by precipitation with cold acetone to eliminate all traces of triacylglycerols and ethyl esters 30 . As shown by HPLC paired with evaporative light-scattering detector and external calibration curves, PL-DHA was composed of lysophosphatidylcholine (lysoPC: 49.2 ± 0.7 wt%), phosphatidylcholine (PC; 33.5 ± 0.4 wt%), phosphatidylinositol (PI; 9.0 ± 0.3 wt%), phosphatidylethanolamine (PE: 4.8 ± 0.7 wt%) and sphingomyelin (3.5 ± 0.2 wt%). Lard was supplied by Celys (Rezé, France) and kiwi seed oil, as additional source of alpha-linolenic acid, by Polaris (Pleuven, France). Sunflower oil (Lesieur®) was purchased from a local supermarket and oleic sunflower oil from Olvéa (Marseille, France). The vegetable lecithin rich in linoleic acid 18:2 n-6 (PL-LA) was sold by Lipoid (Ludwigshafen, Germany) and was composed exclusively of phosphatidylcholine and antioxidants (no TAG).

Low fat (LF), HF and the two HF lipid blends containing LC n-3 PUFA in the form of PL or TG were prepared at the labscale. DHA and, in lower amount, EPA were supplied either in the form of triacylglycerols (HF-ω3TG) or phospholipids (HF-ωPL). The fatty acid compositions of both the tuna oil and the lecithin rich DHA product are reported in Additional file 1. The four blends were prepared by direct mixing of the different oils and fat sources, including the lard in the proportions indicated in Table 1. Then they were stored under a flux of nitrogen at −20°C and sent to SAFE (Augy, France) for preparing the mice diets. The composition of the four diets is reported Table 1. HF-ω3TG and HF-ω3PL are the diets that served as unoxidized n-3 diet controls in our other study about the impact of PUFA oxidation 30 .

^aConsidering the difference observed in tocopherols in lipid mixtures, which can affect their metabolic impact 31 , care was taken to supplement lipid mixtures with α-tocopherol during formulation to achieve iso-tocopherol diets.

Male C57BL/6 mice (8 wk, 20 g) from Janvier SA (Le Genest Saint-Isle, France) were housed in a temperature-controlled room (22°C) with a 12 h light/12 h dark cycles. After 2 weeks of chow diet, mice were randomly divided into four groups fed one of the four following diets for 8 weeks: LF, HF, HF-ω3TG and HF-ω3PL diets. Animal experiments were performed under the authorization n°69-266-0501 (Direction Départementale des Services Vétérinaires du Rhône, France). All experiments were carried out in compliance with the French Ministry of Agriculture guidelines (n° 87–848) and the E.U. Council Directive for the Care and Use of Laboratory Animals of November 24th, 1986 (86/609/EEC), in conformity with the Public Health Service (PHS) Policy on Humane Care and Use of Laboratory Animals. COS holds a special licence (n° 69266257) to experiment on living vertebrates issued by the French Ministry of Agriculture and Veterinary Service Department. Body weight was measured twice a week and food intake was measured weekly. After 8 weeks, mice were euthanized by intraperitoneal (IP) injection of sodium pentobarbital and blood was collected by cardiac puncture under pyrogen-free conditions on heparin-containing tubes. Plasma, liver, WAT, muscles, duodenum and jejunum were collected. For analysis of 4-hydroxy-2-alkenals, due to plasma volume constraints, 3 pools of 300 μL obtained from 3 mice (100 μL per mice) were used for each group.

4-hydroxy-2-alkenals were derivatized from 300 μl of plasma as described previously 32 . Deuterated 4-hydroxy-2-nonenal (4-HNE) and 4-hydroxy-2-hexenal (4-HHE) (20 ng) used as internal standards were added to the samples. Briefly, they were treated with O-2,3,4,5,6-pentafluorobenzyl hydroxylamine hydrochloride. After acidification with H₂SO₄, pentafluorobenzyloxime derivatives were extracted with methanol and hexane. The hydroxyl group was then converted into trimethylsilylether after an overnight treatment with N,O-bis(trimethylsilyl)trifluoroacetamide at room temperature. The pentafluorobenzyloxime trimethylsilylether derivatives of 4-HHE (O-PFB-TMS-4-HHE) and 4-HNE (O-PFB-TMS-4-HNE) were then analyzed by GC-MS using negative ion chemical ionization (NICI) mode on a Hewlett-Packard quadripole mass spectrometer interfaced 32 with a Hewlett-Packard gas chromatograph (Les Ullis, France). For the determination of 4-hydroxy-2-alkenals in liver, the tissue was firstly minced in liquid nitrogen with PIPES, then centrifuged and the supernatant treated as previously described. Regarding WAT, a protocol has been adapted to the extraction and derivatization of 4-hydroxy-2-alkenals in lipophilic media. Briefly the o-pentafluorobenzyl hydroxylamine was prepared by dilution in hexane containing pure triethylamine leading to the formation of a precipitate, which was removed. Oxime derivatives were then prepared and extracted with hexane. Lipids were remove by solid phase extraction on silica cartridge, and the oxime derivatives were eluated with hexane/diethyl ether (50/50, vol/vol). The second derivatization (silylation) was achieved as previously described. The method allowed the quantification of free 4-HHE and 4-HNE.

MCP-1, IL-6 (Clinisciences, France), sCD14, LBP (Enzo Life Sciences), leptin, adiponectin (SpiBio, Montigny Le Bretonneux, France) and insulin (Crystal Chem Inc., USA) plasma levels were assayed by ELISA kits according to the manufacturer’s instructions. Blood glucose levels were measured on animals in mouse-tail blood using a glucometer (Accu-Chek ®, Roche, France).

Plasma triacyglycerols (TAG) were measured with the triglyceride PAP kit (BioMérieux France) as previously described 33 . Plasma TAG concentration was calculated by subtracting the free glycerol in plasma measured with the glycerol UV-method (R-Biopharm/Boehringer, Mannheim, Germany). Plasma NEFA was measured using NEFA-C kit (Wako Chemicals, Neuss, Germany).

Total lipids were extracted from 35 μL of plasma as described previously 33 and from tissues according to Folch et al. 34 . The organic phase (solvent) was evaporated under N₂ and total fatty acids were transesterified using boron trifluoride in methanol (BF₃/methanol) and in the presence of heptadecanoic acid (C17:0, Sigma, France) as an internal standard 33 . The FA methyl esters were then analyzed by GC using a DELSI instrument model DI 200 equipped with a fused silica capillary SP-2380 column (60 m × 0.22 mm).

Tocopherols in epipidymal WAT (eWAT) and retroperitoneal WAT (rWAT) were quantified without saponification according to a method described by 35 . An aliquot of lipid extract was dried under N_2, then solubilized in n-hexane and analyzed by HPLC paired with a fluorimeter detector (λex = 295 nm and λem = 330 nm). Separation of the different isomers of tocopherols and tocotrienols was achieved on a polar column (Acclaim Polar advantage II, Dionex, 3 μm; 250 × 3 mm) in isocratic mode at 0.5 ml/min with hexane/methyl-terbutyl-ether 90/10, vol/vol. The quantification was realized by comparison of the peak areas with calibration curves performed with external standard solutions of α, γ, β and δ tocopherols (Calbiochem, tocopherol set) and γ tocotrienol (Sigma, France). An aliquot of each diet was saponified for 30 min at 70°C in a mixture containing potassium hydroxide (50% w/vol in water), pyrogallol in ethanol (1% w/vol). Tocopherols were extracted with hexane containing 0.005% (w/vol) of BHT. After centrifugation and washing, the organic phase was mixed. The solvent was removed under vacuum, and then under a stream of nitrogen. Tocopherols were finally dissolved in n-hexane and then quantified as described previously.

Preparation of adipose tissue for determination of cell size was performed essentially as described previously 36 . Briefly, 30–40 mg of eWAT or rWAT was immediately fixed in osmium tetroxide for 96 hours at room temperature. After washing, adipose cell-size distribution was then assessed using a Beckman Coulter Multisizer IV (Beckman Coulter) with a 400 μm aperture. The range of cell-sizes that can effectively be measured using this aperture is 20–240 μm. The instrument was set to count 1000 particles per run, and the fixed-cell suspension was diluted so that coincident counting was less than 10%. After collection of pulse sizes, the data were expressed as particle diameters and displayed as histograms of counts against diameter using linear bins and a linear scale for the cell diameter. Cell-size distributions were drawn from measurement of at least 12 000 cell diameters per animal. Mean cell weight was calculated as measured Cell volume × TG density (0.9); cell number were calculated as Cell weight/WAT mass (eWAT or rWAT).

Total RNA was extracted from 50 mg of duodenum and jejunum of mice using the NucleoSpin® RNA/Protein kit (Macherey Nagel, Duren, Germany), and from 50 mg of eWAT or rWAT with TRIzol (Invitrogen, Eragny, France). cDNAs were synthesized from 1 μg of total RNA in the presence of 100 units of Superscript II (Invitrogen, France) with a mixture of random hexamers and oligo (dt) primers (Promega, Charbonnières, France). The amount of target mRNAs was measured by RT, followed by real-time PCR, using a Rotor-Gene Q (Qiagen, France). The amount of target mRNAs was measured by RT, followed by real-time PCR, using a Rotor-Gene Q (Qiagen, France). Primer sequence and RT-quantitative PCR conditions are available upon request (cyrille.debard@univ-lyon1.fr). Hypoxanthine guanine phosphoribosyl transferase (HPRT) mRNA was used to normalize data of duodenum, jejunum and WAT of mice.

All data are presented as means ± SEM and were analysed using Statview 5.0 software (Abacus Concept, Berkeley). One-way ANOVA followed by Fisher PLSD was used to compare the four groups. Differences were considered significant at P < 0.05.

Table 2 shows that we succeeded in producing diets equilibrated in terms of n-3 PUFA supplies and n-6/n-3 ratios, with LC n-3 PUFA in the form of PL or TG. Importantly, HF-ω3TG diet, HF diet and LF diet also contained PL in the form of PL-LA lecithin, so that effects of PL-ω3 can be attributed to the molecular form of LC n-3 PUFA in the diet, not to the presence of phospholipids.

(*P < 0.05 vs HF); (^$P < 0.05 vs LF), (^£P < 0.05 vs HF-w3TG). ANOVA followed by Fisher test. Data are mean ± SEM for n = 3. Abbreviations: FA, fatty acids; MUFA, monounsaturated fatty acids; SFA, saturated fatty acids.

The HF-ω3PL diet contained slightly less n-3 PUFA than the HF-ω3TG diet, as frequently found in dietary n-3 PUFA sources. In n-3 PUFA enriched diets, n-3 PUFA contents and n-6/n-3 ratios were consistent with dietary recommendations, whereas n-6/n-3 ratio was nearly 15 in HF diet as observed in human Western diets.

As shown in Table 3, the supplementation of HF diets with LC n-3 PUFA decreased the body weight gain in HF-ω3PL fed mice compared to HF mice but not in HF-ω3TG mice. Liver weight was significantly lower in mice fed HF-ω3PL or HF-ω3TG diets compared to HF group, and even lower than LF group regarding HF-ω3PL. White adipose tissue weight (total of retroperitoneal, epididymal and subcutaneous adipose tissues) was also lower in HF-ω3PL group than in HF group but no significant difference was observed between HF-ω3PL and HF-ω3TG groups. However, the lean mass estimated by the weight of gastrocnemius muscle was not different between the groups (Table 3).

(*P < 0.05 vs HF); (^$P < 0.05 vs LF), ANOVA followed by Fisher test.

Data are mean ± SEM for n = 8 per group. Abbreviations: WAT, white adipose tissue; TAG, plasma triacyglycerols; NEFA, non-esterified fatty acids; 4-HHE, 4-hydroxy-2-hexenal; 4-HNE, 4-hydroxy-2-nonenal.

For 4-hydroxy-2-alkenals analysis, 3 pools of 300 μL obtained from 3 mice (100 μL per mice) were used for each group (n = 3).

Energy intake was evaluated during the 8 weeks of feeding trial. Table 3 shows that it was higher in the three HF groups than in LF group. However, results did not show any difference between HF-ω3PL and HF-ω3TG groups. After 8 weeks feeding, free fatty acid and triacyglycerol concentrations were lower (P < 0.05) in the plasma of the three groups of mice fed HF diets, regardless of supplementation with n-3 PUFA, than in LF group (Table 3). Plasma insulin and glucose levels did not differ among the groups. Neither plasma free fatty acid nor triacyglycerol concentrations were significantly different between the three groups of mice fed HF diets. Hepatic lipid accumulation was similar among groups.

Fatty acid composition of total plasma, liver and WAT were characterized to investigate whether long-term consumption of different diets could change PUFA metabolism. In fact, the profile of total FA in plasma (Table 4), in liver and eWAT (See Additional file 2) reflected the composition of ingested dietary fats (Table 2). FA profile of rWAT was similar to that of eWAT (See Additional file 3).

(*P < 0.05 vs HF), (^$P < 0.05 vs LF), (^£P < 0.05 vs HF-ω3TG). Data are mean ± SEM for n = 5 per group. Abbreviations: Tr, traces FA; fatty acids; MUFA, monounsaturated fatty acids; SFA, saturated fatty acids.

Table 4 shows that the plasma arachidonic acid (20:4 n-6), an n-6 FA precursor of pro-inflammatory mediators in biological membranes, was significantly higher in HF group than in three other groups. EPA proportion was higher in plasma of mice fed the HF diets supplemented with n-3 PUFA compared with HF group, and higher in HF-ω3TG group than in HF-ω3PL group. Importantly, plasma DHA was higher in the two HF groups supplemented with LC n-3 PUFA in the form of PL or TG compared with LF and HF groups. Additionally, the proportions of LC n-3 PUFA (EPA and DHA) were higher in the HF-ω3TG group than in the HF-ω3PL group as observed in the diets.

In liver like in eWAT, DHA and EPA levels were significantly increased in both HF-ω3PL and HF-ω3TG groups compared to LF and HF groups. A significant difference of LC n-3 PUFA levels was observed between HF-ω3PL and HF-ω3TG groups (See Additional file 2), as observed in the plasma. The n-6/n-3 ratio, related to a risk of inflammation derived from PUFA metabolites, was higher in mice fed HF diet than the three other groups. This ratio was not different between HF-ω3PL and HF-ω3TG groups (Table 4 and Additional file 2). The relative difference of n-6/n-3 ratio that existed between the high-fat diets and the LF diet was altogether still observed in plasma and tissues after 8 weeks of diet (Figure 1). Altogether, both diets enriched in LC n-3 PUFA resulted in proper accretion of these FA in tissues.

High-fat diets are known to induce a metabolic inflammation related to (i) the concentrations of different types of adipokines and (ii) endotoxin transport. Regarding inflammation, we show higher concentrations in plasma of the chemokine MCP-1 (Figure 2A) in HF group than in the three other groups. Plasma interleukin IL-6 was also significantly higher in HF group than in HF-ω3PL group (Figure 2B) and tended to be higher in HF group than in HF-ω3TG mice (P = 0.06). Both n-3 diets even resulted in plasma concentrations of inflammatory markers similar to the LF diet. Among plasma markers involved in the transport of the pro-inflammatory endotoxins, the lipopolysaccharide binding protein (LBP) level was higher in plasma of HF mice than in LF and HF-ω3PL groups (Figure 2C). However, no difference was found between HF and HF-ω3TG groups or between HF-ω3PL and HF-ω3TG groups. The plasma level of soluble cluster of differentiation 14 (sCD14), an endotoxin receptor, was also higher in HF mice than in the two HF groups supplemented with LC n-3 PUFA (Figure 2D). Regarding adipokines in plasma, results showed an increased leptin concentration in the HF group vs all other groups (Figure 2E). Supplementation with LC n-3 PUFA in both forms allowed recovering leptin concentrations similar to LF group. In contrast, there was no difference in plasma adiponectin concentration among the groups (Figure 2F).

LC PUFA are known to be prone to oxidation because of their high degree of unsaturation. They form oxidized end-products which contribute to oxidative stress. We measured the levels of 4-hydroxy-2-hexenal (4-HHE) and 4-hydroxy-2-nonenal (4-HNE), two markers of lipid peroxidation derived from the oxidation of n-3 PUFA and n-6 PUFA respectively. As shown in Table 3, the plasma level of 4-HHE did not differ among the groups, although there was large variation within groups. The plasma level of 4-HNE was significantly higher in HF group than in the three other groups albeit remaining low (Table 3). No differences in 4-HHE and 4-HNE concentrations were observed in the liver that is a major target organ for these alkenals (0.5 to 2 nmol/g regardless of group).

Because we noticed a significant decrease of inflammatory markers in the plasma of mice fed HF diet supplemented with LC n-3 PUFA, we examined the MCP-1 and IL-6 gene expression in WAT. MCP-1 expression in eWAT was significantly higher in HF group than in the other groups (Figure 3A, P < 0.01). The same difference between HF and HF-ω3PL groups was observed in rWAT (see Additional file 4). The expression of mRNA IL-6 was significantly decreased in HF-ω3TG group compared to HF group and tended to be decreased in HF-ω3PL group compared to HF group (Figure 3B). In addition, the level of tocopherols was higher in eWAT of mice fed HF-ω3PL and HF-ω3TG diets than LF and HF groups (Figure 3C). Noticeably, the level of tocopherols was higher in HF-ω3PL group than in HF-ω3TG group (P < 0.05) despite the similar levels found in the diets (Table 2). The concentration of the oxidation products 4-HHE and 4-HNE in eWAT and rWAT was similar among groups, in the range 0.5 to 2 nmol per g.

To know whether the high-fat diets could affect the size and the number of adipocytes in mice, we performed a cellularity analysis of WAT. As shown Figure 4, the size distribution of the epididymal adipocytes in the HF-ω3PL group showed a marked shift toward smaller sizes compared with HF and HF-ω3TG groups. The same effect was observed in retroperitoneal fat pads. The cellular characteristics of eWAT in mice fed HF and HF supplemented with LC n-3 PUFA in the form of PL or TG are shown in Table 5. A reduction of fat accretion in eWAT was observed in HF-ω3PL group vs HF group but no difference was observed between HF-ω3TG and the two other groups. Adipocyte sizes in HF-ω3PL and LF groups were actually similar. This reduction resulted from a decrease in adipose cell volume rather than from a decrease in the total number of adipocytes per fat pad, as calculated from the measured size distribution. Indeed, mean adipocyte diameter was reduced by 14% in HF-ω3PL group vs HF group (P < 0.05) resulting in a 30% decrease in calculated adipose cell volume (P = 0.03).

(*P < 0.05 vs HF); (^#P = 0.07 vs HF); (^£P < 0.05 vs HF-ω3TG).

Data are mean ± SEM for n = 4-5 per group. Abbreviations: eWAT, epididymal white adipose tissue.

The upper small intestine represents the primary defense line of the organism that can affect metabolism and inflammation. We thus examined the gene expression of gastrointestinal glutathione peroxidase 2 (GPx2), mainly expressed in the small intestine and implicated in the detoxification of lipid oxidation products including 4-HHE and 4-HNE. The expression of GPx2 in the duodenum was higher in HF mice than in LF and HF-ω3PL groups (Figure 5A). In the jejunum, the gene expression of GPx2 was significantly increased in HF group compared with the three other groups (Figure 5B).