N-3 fatty acids improve body composition and insulin sensitivity during energy restriction in the rat

Prostaglandins, Leukotrienes and Essential Fatty Acids 91 (2014) 203–211

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Prostaglandins, Leukotrienes and Essential Fatty Acids journal homepage: www.elsevier.com/locate/plefa

N-3 fatty acids improve body composition and insulin sensitivity during energy restriction in the rat N. Guelzim a,b, J.-F. Huneau a,b, V. Mathé a,b, S. Tesseraud c, J. Mourot d, N. Simon e, D. Hermier a,b,n a

INRA, UMR914 Nutrition Physiology and Ingestive Behavior, AgroParisTech, 16 rue Claude Bernard, F-75005 Paris, France AgroParisTech, UMR914 Nutrition Physiology and Ingestive Behavior, F-75005 Paris, France c INRA, UR83 Recherches Avicoles, F-37380 Nouzilly, France d INRA, UMR1348 PEGASE, F- 35590 Saint Gilles, France e ONIDOL, 11 rue de Monceau, CS 60003, F-75008 Paris, France b

art ic l e i nf o

a b s t r a c t

Article history: Received 19 March 2014 Received in revised form 7 July 2014 Accepted 9 July 2014

The hypothesis that n-3 polyunsaturated fatty acids (PUFA) could contribute to maintain muscle mass during energy restriction aiming to weight loss was tested in the rat, with special attention paid to insulin signalling. After 10 weeks on a diet rich in lipids and sucrose, male rats were energy restricted and fed diets rich in 18:1 n-9 (OLE), 18:3 n-3 (ALA) or n-3 long-chain (LC, 418 carbons) PUFA. After 4 weeks, they were killed after an insulin injection. Red blood cells, liver, and Gastrocnemius muscle were enriched in ALA in the ALA group, and in LC-PUFA in the ALA and LC groups. The LC diet resulted in a higher weight loss, without negative impact on the muscle weight. In parallel, hepatic phosphorylation of insulin receptor and IRS1 was the highest in this group. This suggests that the trend we observed in the preservation of protein homeostasis in the LC group is mediated, at least partly, by an enhancement of the early steps of insulin signalling resulting from cell membrane enrichment in n-3 PUFA. & 2014 Elsevier Ltd. All rights reserved.

Keywords: N-3 fatty acids Insulin signalling Protein homeostasis Weight loss

1. Introduction The excess of body weight concerns over 50% people in Europe, and often clusters into the metabolic syndrome (MetSynd), which makes these subjects at higher risk of type 2 diabetes and cardiovascular diseases [1]. Among changes in lifestyle recommended to overweight people, weight loss cannot be achieved without a more or less drastic reduction in energy intake, resulting in a catabolic status which affects essentially adipose tissues, but may also alter protein homeostasis, leading to a more or less severe loss of fat-free mass (mostly muscle) [2–5]. As a consequence, nutritional strategies aiming to weight loss should also consider the conservation of lean mass as a priority and include nutrients likely to preserve protein homeostasis. Among those nutrients, n-3 polyunsaturated fatty acids (PUFA) are good candidates. Indeed, in various situations of protein homeostasis challenge, n-3 PUFA have been repeatedly shown to beneficially regulate insulin signalling and/or insulin sensitivity. In growing animal models, the providing of n-3 PUFA favored amino acid accretion into body proteins [6–8]. The resulting n Corresponding author at: UMR914, Nutrition Physiology and Ingestive Behavior, INRA, AgroParisTech, 16 rue Claude Bernard, F-75005 Paris, France. Tel.: þ 33 144087246; fax: þ33 144081858. E-mail address: [email protected] (D. Hermier).

http://dx.doi.org/10.1016/j.plefa.2014.07.007 0952-3278/& 2014 Elsevier Ltd. All rights reserved.

enhancement in protein anabolism was associated with an improvement of insulin sensitivity, via an increased phosphorylation of Akt and downstream proteins such as mTOR, P70S6K1 and 4EBP1 [7,8]. In young, middle-aged and older adults, dietary n-3 fatty acid (FA) supplementation stimulated protein synthesis under a hyperinsulinemic–hyperaminoacidemic condition [9,10]. Again, this enhancement of protein anabolism was accompanied by an increase in mTOR and P70S6K1 phosphorylation in the muscle of these subjects [9,10]. On the other side, in rodent models of protein catabolism, n-3 PUFA supplementation contributed to limit muscle loss resulting from various proteolytic situations, such as ageing, limb immobilization, starvation, cancer, or sepsis, through mechanisms involving enhanced insulin signalling pathway via Akt/P70S6K1 activation, and/or reduced proteolysis via downregulation of the ubiquitin–proteasome pathway [11–16]. Yet, a potentially beneficial role of dietary n-3 FA on the preservation of protein homeostasis during energy restriction aiming to weight loss is poorly documented, and the results remain conflicting. In a number of studies, obese patients on a low or very low energy diet lost fat-free mass, without a protective effect of added n-3 PUFA [17–20]. In contrast, in obese patients consuming isolipidic diets containing 25% FA in the form of saturated fatty acids (SFA), n-6 PUFA, or n-3 PUFA, all patients lost fat-free mass, but only those on the n-3-rich diet showed a trend towards a preservation of the fat-free mass [21]. In addition,

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overweight or obese people often exhibit an insulin resistance, and n-3 fatty acids have been shown to improve insulin sensitivity in these subjects [22–24]. However, to our knowledge, the mechanistic links between n-3 PUFA supplementation, insulin sensitivity, and a possible preservation of the muscle mass during energy restriction in obese or overweight patients have not been explored so far. Noteworthy, with the exception of one of our previous studies [8], all available mechanistic studies on the effect of n-3 fatty acids on protein homeostasis used long-chain (LC, 418 carbons) n-3 fatty acids provided as fatty fish, fish oil supplementation, or individual fatty acids. For economical, environmental, and nutritional reasons, the possibility that some of the beneficial effects attributed to LC n-3 PUFA could be achieved by their vegetable precursor, α-linolenic acid (ALA, 18:3 n-3) is important to consider [25]. This question has been addressed in a number of studies in the human and in animal models [17,26–32], reporting beneficial effects of dietary ALA on markers and risk factors of the MetSynd. However the effects differ in response to either ALA or n-3 LC derivatives, and may not involve the same metabolic and regulatory pathways [33]. This may be also true as regards protein homeostasis, which necessitates a comparison of possible differential effects of ALA and n-3 LC derivatives. The present study was performed in rats with diet-induced obesity and insulin resistance, which were submitted to energy restriction. Our first aim was to investigate whether n-3 PUFA, when provided during energy restriction, could help to maintain the fat-free mass during weight loss, and to what extent insulin signalling was involved. As a second objective, we explored the potential interest of vegetable ALA by comparing diets containing either ALA or LC derivatives to a diet rich in monounsaturated fatty acids (MUFA). Beyond the comparison with the ALA-rich diet, the group fed n-3 LC-PUFA was also used as a positive control to validate whether their known effects on the metabolic syndrome markers were preserved during weight loss in the rat.

below and was briefly: 16.2 70.8% SFA, 25.4 70.4% MUFA, and 58.47 1.3% PUFA for the STD diet; 39.6 70.2% SFA, 44.3 70.1% MUFA, and 16.2 70.1% PUFA for the HFSI diet. For the second period (energy restriction phase), 3 high fat/high sucrose restriction (HFSR) diets were designed. These diets had the same energy content and protein/lipid/carbohydrate ratios as the HFSI diet (Supplementary Table 1), but differed in the lipids provided and thus in their fatty acid composition (Table 1). OLE diet was rich in monounsaturated fatty acids (MUFA, 18:1 n-9 essentially), whereas the n-3 FA-rich ALA and LC diets were designed to provide similar amounts of n-3 fatty acids as ALA and n-3 LC-PUFA, respectively. Because the proportion of 18:2 n-6 (linoleic acid, LA) was twice higher in the ALA diets than in the two other diets, and because the OLE and LC diets were very poor in ALA, the LA/ALA ratio of the later (90.1 and 26.8, respectively) was considerably higher than in the ALA diet (2.41). 2.3. Study design (Fig. 1) 2.3.1. Induction phase Following 1-week adaptation to housing conditions, 60 rats were allowed free access to the HFSI diet for 10 weeks. Six other rats, with the same mean body weight as those fed the HFSI diet, were kept as controls and fed the STD diet. Body weight was monitored weekly, and food intake every other weeks. During the 10th week of the induction phase and after an overnight fast, 300 μL of blood was collected from the tail vein in tubes containing EDTA 10% (5 μL solution/ml of blood) and centrifuged at 4 1C for 10 min at 1700g. Blood glucose, as well as plasma insulin, triglyceride, and cholesterol were assayed immediately, and distribution curves were drawn for the parameters, as well as for body weight. At the end of the restriction phase, among the 60 rats fed the HFSI diet, the 21 rats with the highest or lowest body weight, HOMA index, triglyceridemia, or cholesterolemia Table 1 Fatty acid composition (mol/100 mol) of the HFSR experimental diets.

2. Materials and methods 2.1. Animals and diets Male Wistar rats (66 rats) (Harlan, Gannat, France), initially weighing 200–224 g, were individually housed in a room maintained at 2371 1C with an artificial 12:12 h light–dark cycle (lights on at 05:00 a.m.). They were fed ad libitum a standard pelleted diet (SAFE A04, Teklad 20-18S, Harlan) and acclimated to local conditions for 1 week. Body weight was monitored weekly during all the experimental period. The study was performed according to the guidelines of the French Ministry of Agriculture for the use and care of laboratory animals, and received the agreement of the local animal ethics committee for animal experimentation (COMETHEA, approval number 11/016).

16:0 18:0 Total SFA 18:1n 9 Total MUFA 18:2n 6 Total n  6 18:3n 3 20:5n  3 22:5n  3 22:6n  3 Total n  3

HFSR-OLE

HFSR-ALA

HFSR-LC

3.89 7 0.13 3.32 7 0.06 8.96 7 0.20 80.84 7 0.19 81.92 70.17 9.01 7 0.03 9.01 7 0.03 0.10 70.00 ND ND ND 0.10 70.00

4.69 7 0.08 1.74 7 0.05 7.737 0.09 60.617 0.08 65.647 0.09 18.75 70.04 18.75 70.04 7.79 7 0.02 0.017 0.01 ND ND 7.82 7 0.04

6.90 7 0.11 3.447 0.06 13.647 0.16 65.377 0.21 69.727 0.16 7.34 7 0.03 7.777 0.02 0.29 7 0.00 4.54 7 0.01 0.54 7 0.01 2.83 7 0.01 8.82 7 0.03

2.2. Diets

HFSR, hypercaloric restriction diet. Values are mean 7standard deviations (SD) for three aliquots of each diet. Only major or nutritionally important fatty acids are given; ND, not detected (detection limit 0.1–0.2% of total FA); OLE, rich in 18:1 n 8; ALA, rich in 18:3 n  3; LC, rich in n  3 long-chain PUFA; SFA, saturated fatty acids; MUFA, monounsaturated fatty acids.

All diets were manufactured by UPAE (INRA, Jouy-en-Josas, France). High-oleic sunflower and rapeseed oils were provided by ONIDOL (Paris, France), and fish oil by Seah International (Wimille, France). During the first period (induction of the MetSynd), rats were fed either a standard normocaloric diet (STD) or a high fat/ high sucrose induction diet (HFSI). The STD diet provided, as calculated energy %: protein 20.0, lipid 12.9, and carbohydrate 67.1, whereas the HFSI diet was rich in sucrose (14.3%) and saturated fatty acids (SFA) (lard 28.0%), and provided, as calculated energy %: protein 20.0, lipid 55.4, and carbohydrate 24.6. (Supplementary Table 1). Their fatty acid composition was determined as described

Fig. 1. Experimental design. STD, standard normocaloric diet; HFSI, high fat/high sucrose induction diet; HFSR, high fat/high sucrose restriction diet. OLE, rich in 18:1 n-8; ALA, rich in 18:3 n-3; LC, rich in n-3 long-chain PUFA ( 418 carbons). The number of rats is in parentheses. Vertical arrows indicate the times of blood sampling, ✝ indicates the times of sacrifice and dissection.

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were excluded, in order to keep the 39 rats homogenous on the basis of these parameters. Among these 39 rats, 30 rats were kept for the restriction phase, and the 9 remaining rats were placed in their cages and fed ad libitum the same HFSI diet. Two days after the first blood sampling, these 9 rats from the HFSI group and the 6 rats of the STD group were fasted overnight, deeply anesthetized by an intraperitoneal injection of 150 mg sodium pentobarbital/ 100 g body weight (Sanofi Santé Animale, Paris, France) then sacrificed by exsanguination. The following tissues were dissected and weighed: liver, visceral adipose tissues (VAT, composed of epididymal, mesenteric, retroperitoneal, and perirenal white adipose tissues), and subcutaneous white adipose tissues (SCAT). Finally, remaining viscera, tail, head, and paws were removed, and the remaining carcass was weighed and stored at 20 1C.

2.3.2. Restriction phase To induce a weight loss, the 30 remaining rats were then allocated to one of three restriction (HFSR) diets for 4 weeks (10 rats per group) (Fig. 1). Overall means and 95% CI (n¼30) at the beginning of the energy restriction were: body weight, 476 g [467–486]; glycemia, 102 mg/dL [98–106]; insulinemia, 122 pmol/L [94–149], triglyceridemia, 77 mg/dL[70–84]; cholesterolemia, 65 mg/dL [61–69]. All values were within what would be considered a healthy range and the three groups did not differ in any of the selection parameters. The mean spontaneous energy intake during the 10th week of the induction period (139 kJ/100 g body weight) was used to determine the amount of food provided during each week of the restriction period, using the following restriction pattern: 70% of the spontaneous energy intake during the first restriction week, then 60% during the three following weeks. The amount of food was the same for all rats, and adjusted every week according to the actual body weight, i.e. 11.8 g, 10.1 g, 9.8 g and 9.6 g during the 1st, 2nd, 3rd, and 4th week, respectively. At the end point of 4 weeks of restriction, rats were fasted overnight. After a blood sample has been taken from the tail vein, rats were deeply anesthetized (see above). The abdominal cavity was opened and 0.5 IU/kg body weight of insulin (Actrapid, Novo Nordisk, Bagsvaerd, Denmark) was injected in the inferior vena cava. Skin was incised and the left Tibialis anterior muscle was exposed. Five minutes after insulin injection, 50 mg liver and the whole Tibialis anterior muscle were taken, immediately homogenized in a lysis buffer containing 20 mM Tris–HCl (pH 7.5), 130 mM NaCl, 1 mM EDTA, 1 mM EGTA, 50 mM sodium fluoride, 1 mM sodium β-glycerophosphate, 2.5 mM sodium pyrophosphate, 1 mM sodium orthovanadate and 1% (vol/vol) Triton X-100, supplemented with a protease inhibitor cocktail (Roche Diagnostics, Mannheim, Germany) and antiphosphatase (Sigma-Aldrich, Saint Quentin Fallavier, France). After 30 min, samples were centrifuged for 10 min at 15,000g and 4 1C and the supernatants were sampled and stored at  80 1C for quantification of proteins involved in insulin signalling. Rats were then sacrificed by exsanguination by section of the inferior vena cava, and 2 mL blood was taken and centrifuged as described above. Plasma was sampled and stored at  80 1C, and red blood cells (RBC) were rinsed in sterile saline then stored at 20 1C. Meanwhile, the following tissues were dissected and weighed: liver, right Gastrocnemius muscle, VAT and SCAT (as described above). Samples of the liver and the Tibialis anterior muscle were snap frozen in liquid nitrogen and stored at  80 1C for determination of the mRNA levels. Another liver sample (3 g) and the right Gastrocnemius muscle were frozen at  20 1C for the determination of fatty acid composition. Finally, the remaining carcass was prepared as described above, weighed and stored at 20 1C.

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2.4. Analyses 2.4.1. Chemical analyses After lipid extraction [34] and BF3 derivatization [35], FA of diets, RBC, liver, and Gastrocnemius muscle were analyzed on a Perkin-Elmer Autosystem XL gas chromatograph (Waltham, Massachusetts, USA) equipped with fused silica gel capillary column (0.25 mm i.d. 330 m), filled with stationary phase (80% biscyanopropyl and 20% cyanopropylphenyl), using margaric acid (C17) as internal standard. The chromatography conditions used were: temperature program from 45–240 1C at 20–35 1C/min. The injector and detector temperatures were maintained at 220 1C and 280 1C, respectively. Results were expressed as percentages of the total FA content. After carcasses have been grinded and freezedried, their protein and lipid contents were determined according to the methods of Kjeldahl and of Soxhlet, respectively [36]. 2.4.2. Plasma parameters Both after induction and restriction, glucose concentration was determined immediately on whole blood taken from conscious rats with an Accu-Cheks glucometer (Roche Diagnostics, Meylan, France). Plasma triglyceride and cholesterol concentrations were determined by colorimetric enzymatic methods using the kits provided by Randox (Roissy, France) and BioMérieux (Craponne, France), respectively [37,38]. Plasma insulin was determined by enzyme immunoassay using a specific kit (Mercodia, Upsala, Sweden). HOMA index was calculated using the formula: [fasting insulin (pmol/L)  fasting glucose (mmol/L)]/22.5. At the end of the restriction period only, urea and HDL-cholesterol (HDL-C, after precipitation of non-HDL cholesterol, non-HDL-C) concentrations were determined by using the corresponding reagents provided by BioMérieux [39]. Since the Friedewald formula is not valid in non-human species for the calculation of LDL-cholesterol, non-HDLC concentration in plasma, including cholesterol in VLDL, IDL, and LDL, was calculated by subtracting HDL-C concentration from that of total cholesterol. Plasma free amino acids were determined by ion-exchange chromatography with post-column ninhydrine derivatization on an Aminotac JLC-500/V (Jeol, Tokyo, Japon). Plasma concentrations of interleukin 1β (IL-1β), plasminogen activator inhibitor-1 (PAI-1), and monocyte chemotactic protein-1 (MCP-1) were determined using multiplexed-immunoassays (Merck-Millipore, Molsheim, France) on a Bioplex-200 analyzer (Bio-Rad Laboratories, Marnes-laCoquette, France). 2.4.3. Proteins involved in insulin signalling The following total and phosphorylated proteins were quantified in the liver and the Tibialis anterior muscle using PathScans ELISA kits provided by Cell Signaling Technology (Denver, USA): total insulin receptor (IR) (Kit # 7069S), Tyr 1150/1151 phosphorylated IR (Kit # 7258S), total IR substrate 1 (IRS1) (Kit #7328), pan Tyr phosphorylated IRS1 (Kit #7133S), total Akt2 (Kit # 7930S) and Ser-474 phosphorylated Akt2 (Kit #7932S). Protein concentration was determined with Protein Assay (Bio-Rad). 2.4.4. Gene expression Total RNA was extracted from liver and muscle samples using Trizol reagent (Invitrogen, Carlsbad, USA) and synthesis of cDNA was performed on 400 ng of total RNA using a high capacity cDNA reverse transcription kit, based on the use of both oligodT and hexamers (Applied Biosystems, Foster City, USA). Target genes were those coding for insulin signalling pathway proteins (i.e. IR, IRS1, IRS2, Akt1, Akt2), proteolysis (calpain 2, ubiquitinconjugating enzyme E2B, E3 ubiquitin ligase, atrogin-1), and IGF1. The primers listed in Supplementary Table 2 were used for

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quantitative PCR on a 7300 real-time PCR system (Applied Biosystems), as described previously [40]. Primer sequences were designed using Primer ExpressTM (Applied Biosystems) software and were from Eurogentec (Eurogentec, Seraing, Belgium). Gene Δ expression was determined using the 2  Ct formula using 18S as the reference (ΔCt ¼ Ct target gene  Ct 18S). 2.4.5. Calculations and statistical analyses Statistical analysis of the data was carried out using one-way Anova (GLM procedure, SAS 9.1, SAS Institute, Cary, USA) with diet as a fixed effect. Orthogonal contrasts were computed to compare the OLE group to LC and ALA groups taken together (n-3 effect). Statistical analysis of the post-energy-restriction body composition was performed after adjustment for the pre-energy-restriction body weight, used a covariable. For body weight, HOMA and plasma glucose, insulin, triglycerides, total cholesterol, post-energy-restriction data were analyzed after adjustment for the corresponding pre-energy-restriction value, used as a covariable. Means and LS-means comparisons were performed between the three groups using Tukey–Kramer adjustment.

3. Results 3.1. Body weight and body composition During the 10 weeks of the induction phase, body weight, body gain, and food intake (as energy) were identical in STD- or HFSIfed rats (Supplementary Fig. 1). However, rats on the HFSI diet exhibited a higher proportion of white adipose tissue, especially at the visceral sites (þ 27%, P o0.05), and a lower proportion of carcass, an indicator of the lean mass (  7%, P o0.05); besides, after 10 weeks, insulin concentration (1147 65 pmol/L) and HOMA (28.7 716.7) were twice higher in the HFSI group compared to controls (51 722 pmol/L and 12 74.5, respectively, Po 0.05), whereas glycemia was the same in both groups (1027 11 mg/dL in the HFSI group and 104 710 mg/dL in the STD group). In response to food restriction, all rats lost weight (P o0.001), with a more pronounced weight reduction with the n-3 PUFA-rich diets (ALA and LC groups, with P of orthogonal contrast o0.01) (Fig. 2). After adjustment for body weight at the end of the induction period, the body weight at the end of the restriction period was significantly lower in the LC group compared to the OLE group, while the ALA group neither differed from LC nor OLE group. During the 4 weeks, weight losses (in g and % of body weight at the end of the induction period) were 31.6 g and 6.5% (OLE group), 35.6 g and 7.5% (ALA group) and 46.0 g and 9.5% (LC group). They were significantly higher in the LC group than in the two other ones (statistical analysis not shown). Absolute weights of carcass, adipose tissues and liver were not affected by the nature of the restriction diet (Table 2). This was also true for the relative amounts of adipose tissues (as % body weight),

Body weight (g)

600

a

500

ab

b

400 300 200 100

Table 2 Body composition (g) after a 4-week dietary restriction. P values for effects Diet

Carcass VAT SCAT Liver G. muscle

OLE

1707 10 51.9 7 6.2 31.2 7 6.5 10.6 7 0.9

ALA

1747 17 52.7 7 8.0 30.9 7 3.3 10.8 7 0.7

LC

1827 15 47.2 7 9.8 26.5 7 10.8 11.5 7 1.1

Initial body weight

Diet

N-3 vs OLE

0.0045 0.8996 0.1653 0.0288

0.6740 0.4813 0.2106 0.1244

0.7200 0.5584 0.5136 0.0831

2.34 7 0.11ab 2.177 0.18b 2.43 7 0.24a 0.2826

0.0551 0.6387

Values are mean 7 standard deviations (SD) for 10 rats per group. Carcass, remaining of the body after removal of head, tail, paws, skin, internal organs, and all adipose tissues; VAT, visceral adipose tissue, including epididymal, mesenteric, retroperitoneal and perirenal adipose tissues; SCAT, subcutaneous adipose tissue; G. muscle, right Gastrocnemius muscle. Mean values within a column sharing a same superscript letter, or without superscript letter, were not significantly different at P o0.05, according to post-hoc Tukey–Kramer analysis.

showing that adiposity was the same in all groups (data not shown). In contrast, the weight of the Gastrocnemius muscle was sensitive to the nature of dietary fatty acids during energy restriction, being higher in the LC group than in the ALA group. Finally, lipid and protein contents of the carcasses were similar in all groups, and did not differ significantly (lipid %: 7.78 71.60, 8.33 71.37, 7.02 71.91; protein %: 19.8 7 0.7, 19.7 70.1, 19.8 70.8, in the OLE, ALA and LC groups, respectively). 3.2. FA composition and lipid content In RBC, FA are present almost exclusively in phospholipids, and thus RBC FA profile is a good marker of bioconversion and incorporation of dietary FA into cell membranes (Table 3). Indeed, in all groups, RBC FA profile reflected that of the diet, with the highest levels of linoleic acid (18:2 n-6, LA) and ALA in the ALA group, and the highest level of n-3 LC-PUFA in the LC group. Besides, as a consequence of conversion of ALA into its LC derivatives, 20:5 n-3 and 22:5 n-3 (but not 22:6 n-3) contents were significantly higher in the ALA group than in the OLE one. The effect of diet composition on the FA profile of the Gastrocnemius muscle resembles that observed in red blood cells, but was less pronounced (Table 4). Taken as a whole, SFA and MUFA proportions were the same in all groups. The proportion of 18:2 n-6 and ALA was the highest in the muscles of the ALA group, while muscles of the LC group were richer in all n-3 LC-PUFA. Compared to RBC and muscle, a diet effect was more pronounced in the liver (Table 5). When considering the main FA classes, livers from the LC group were the poorest in SFA, whereas those from the ALA group were the richest in n-6 PUFA and the lowest in MUFA. N-3 fatty acid composition of liver lipids was markedly affected by the diet. As expected, ALA content was significantly higher in the ALA group than in the two other dietary groups. Besides, ALA content in this group was twice higher in the liver than in the two other tissues. All n-3 LC-PUFA were significantly higher in the ALA group than in the OLE one, but remained lower than in the LC group. Lipid content of the Gastrocnemius muscle and the liver was significantly higher in the LC group than in the OLE and ALA groups (Tables 4 and 5).

0

OLE

ALA

LC

Fig. 2. Changes in body weight in response to a 4-week food restriction. Values are mean 7standard deviations for 10 rats per group, before (open bars) and after (full bars) energy restriction. Body weights after energy restriction were compared after adjustment for the corresponding values before energy restriction used as a covariable. Bars sharing a same superscript letter were not significantly different at Po 0.05, according to post-hoc Tukey–Kramer analysis.

3.3. Markers of MetSynd There was no overall effect of energy restriction on glycemia, insulinemia or HOMA index of insulin resistance (Fig. 3). However, contrasting evolutions were observed during the restriction period between the different groups regarding the changes in blood

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Table 3 Fatty acid composition (mol/100 mol) of red blood cells after a 4-week energy restriction.

16:0 18:0 22:0 Total SFA 18:1n  9 18:1n  7 20:1n 9 22:1n 9 20:12n  9 Total MUFA 18:2n  6 20:3n 6 20:4n 6 22:4n 6 22:5n 6 Total n  6 LC n 6 18:3n  3 20:3n 3 20:5n 3 22:5n 3 22:6n 3 Total n  3 LC n 3 Unsaturation index

OLE

ALA

LC

16.60 7 1.04 16.86 7 3.95 1.647 0.63 36.077 4.92 25.80 7 10.82 2.19 70.28b 0.46 7 0.22 1.18 70.65 0.247 0.27 30.82 7 11.04 6.79 7 1.70b 0.277 0.21b 20.517 6.82 1.277 0.94 0.79 7 0.37a 29.96 7 6.48 22.83 7 7.87 0.13 70.18b 0.58 7 0.59 ND 0.75 7 0.30c 1.40 70.45b 2.91 70.88b 2.78 7 0.92b

17.25 7 0.83 15.64 74.40 1.647 0.62 35.45 74.98 25.007 12.03 2.98 7 0.25a 0.75 7 0.51 1.477 0.72 0.177 0.27 31.12 7 12.41 9.727 2.39a 0.2470.23b 18.167 8.50 ND 0.477 0.16b 28.95 7 6.53 18.87 7 8.76 0.85 7 0.48a 0.157 0.33 0.38 7 0.32b 1.32 7 0.56b 1.617 0.73b 4.317 1.13b 3.477 1.53b

17.13 71.65 15.917 3.74 1.64 71.13 35.87 25.58 24.167 11.42 2.22 7 0.26b 0.50 7 0.14 1.22 7 1.05 0.20 7 0.20 29.38 7 11.40 7.34 7 1.69b 0.517 0.20a 16.29 7 5.56 0.69 7 0.38 0.57 70.27ab 25.777 4.78 18.067 6.14 0.137 0.14b 0.477 0.34 3.107 0.75a 1.94 7 0.57a 3.157 0.96a 8.82 7 2.28a 8.687 2.40a

1.52

1.49

1.63

Values are mean 7 standard deviations (SD) for 10 rats per group. SFA, saturated fatty acid; MUFA, monounsaturated fatty acid; LC, long chain; ND, not detected (detection limit 0.1–0.2% of total FA). With the exception of LC n-3 PUFA, values lower than 0.20% were not reported in the table. Mean values within a row sharing a same superscript letter, or without superscript letter, were not significantly different at Po 0.05, according to post-hoc Tukey–Kramer analysis.

Table 4 Fatty acid composition (mol/100 mol) of the Gastrocnemius muscle after a 4-week energy restriction. OLE

ALA

LC

Lipids (g/100 g) 14:0 16:0 18:0 22:0 Total SFA 16:1n  9 16:1n  7 18:1n  9 18:1n  7 20:1n 9 Total MUFA 18:2n  6 20:3n 6 20:4n 6 22:4n 6 22:5n 6 Total n  6 LC n 6 18:3n  3 20:5n 3 22:5n 3 22:6n 3 Total n  3 LC n 3

2.497 0.48b 0.56 7 0.14 18.89 7 0.57 10.36 71.73 0.43 7 0.42 30.32 7 1.58 0.687 0.52 0.90 7 0.65 31.37 77.42 2.86 7 0.21b 0.29 7 0.16ab 36.09 7 8.03 13.45 70.64b 0.32 7 0.09 9.917 3.38 0.42 7 0.10a 0.52 7 0.21 24.617 3.85 11.177 3.68a 0.28 7 0.07b ND 1.187 0.31 7.417 2.59b 8.88 7 2.82b 8.59 7 2.88b

1.977 0.12b 0.57 70.13 18.95 70.72 9.737 1.59 0.767 0.64 30.03 7 2.08 0.90 7 0.55 0.50 7 0.49 30.56 7 7.86 3.36 7 0.13a 0.43 7 0.19a 35.707 8.08 15.577 0.40a 0.22 7 0.16 7.87 7 2.94 0.277 0.14b 0.05 7 0.09 23.90 7 93.00 8.42 7 2.96ab 0.90 7 0.24a 0.187 0.21b 1.50 7 0.62 7.40 7 3.02b 10.107 3.42b 9.247 3.64b

3.03 7 0.62a 0.65 7 0.10 19.85 7 1.20 8.26 7 4.43 0.72 70.64 29.55 7 3.45 0.777 0.41 0.74 70.64 29.05 7 5.99 3.03 7 0.16b 0.22 7 0.16b 33.817 6.23 13.717 0.78b 0.27 70.16 7.137 2.29 0.077 0.13c 0.167 0.12 21.357 2.98 7.63 7 2.52b 0.317 0.13b 0.95 7 0.41a 1.71 70.51 12.30 7 3.71a 15.277 4.28a 14.977 4.34a

Unsaturation index

1.60

1.57

1.80

Values are mean 7 standard deviations (SD) for 10 rats per group. SFA, saturated fatty acid; MUFA, monounsaturated fatty acid; LC, long chain; ND, not detected (detection limit 0.1–0.2% of total FA). With the exception of LC n-3 PUFA, values lower than 0.20% were not reported in the table. Mean values within a row sharing a same superscript letter, or without superscript letter, were not significantly different at Po 0.05, according to post-hoc Tukey–Kramer analysis.

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Table 5 Fatty acid composition (mol/100 mol) of the liver after a 4-week energy restriction. OLE

ALA

LC

Lipids (g/100 g) 14:0 16:0 18:0 22:0 Total SFA 16:1n  9 16:1n  7 18:1n  9 18:1n  7 20:1n  9 20:2n  9 Total MUFA 18:2n  6 18:3n  6 20:3n  6 20:4n  6 22:4n  6 22:5n  6 Total n  6 LC n  6 18:3n  3 20:5n  3 22:5n  3 22:6n  3 Total n  3 LC n  3

7.417 0.94b 0.28 7 0.05 15.05 70.80 12.86 7 1.18a 0.487 0.48 28.81 7 0.85ab 0.65 7 0.08 0.487 0.14b 35.617 2.25a 1.94 7 0.09b 0.4 17 0.16a 0.20 7 0.10ab 39.107 2.45a 10.78 70.74c 0.36 7 0.13a 0.50 7 0.10 15.9 71.40a 0.417 0.09a 0.447 0.15a 28.56 7 1.83b 17.707 1.54a 0.20 7 0.02b 0.117 0.03c 0.197 0.07c 2.80 7 0.24c 3.30 7 0.28c 3.157 0.27c

6.60 70.31b 0.29 70.06 15.30 7 0.70 12.86 71.63 a 0.53 70.55 29.14 71.87a 0.717 0.15 0.56 70.17ab 24.867 2.87c 2.58 70.18a 0.53 70.07a 0.22 70.09a 29.277 3.12c 15.34 7 1.24a 0.43 70.03a 0.64 70.11 15.45 7 1.87a 0.28 70.18a 0.177 0.13b 32.34 71.19a 16.91 72.05a 1.65 7 0.28a 1.017 0.16b 1.217 0.19b 5.04 70.66b 9.04 70.75b 7.43 7 0.78b

9.20 71.32a 0.30 70.05 15.32 7 1.03 10.78 7 2.47b 0.16 70.10 26.737 2.77b 0.707 0.12 0.69 70.11a 30.647 3.87b 1.6770.10c 0.277 0.05b 0.117 0.02b 33.977 3.97b 12.170.76b 0.247 0.01b 0.58 70.11 8.42 72.15b 0.14 70.04b 0.177 0.02b 21.73 72.23c 9.55 72.22b 0.32 70.05b 5.09 70.94a 2.82 70.61a 9.15 70.80a 17.46 7 1.45a 17.147 1.43a

Unsaturation index

1.51

1.74

1.91

Values are mean 7standard deviations (SD) for 10 rats per group. SFA, saturated fatty acid; MUFA, monounsaturated fatty acid; LC, long chain; ND, not detected (detection limit 0.1–0.2% of total FA);. With the exception of LC n-3 PUFA, values lower than 0.20% were not reported in the table. Mean values within a row sharing a same superscript letter, or without superscript letter, were not significantly different at P o0.05, according to post-hoc Tukey–Kramer analysis.

glucose and HOMA values. During this period, glycemia increased in the OLE group (þ 15.8 mg/dL, P o0.005), but not in the ALA and LC groups (n-3 PUFA-rich diets). In the same time, the change in HOMA value was significantly different between rats fed the OLE and the LC groups ( þ7.87 and  9.91 respectively, P o0.05). Plasma TG concentration decreased in the three groups of energy-restricted rats (P o0.0001 for changes in values before and after restriction) and was affected by the lipid composition of the restriction diet (Fig. 4A), being significantly lower, after restriction, in the LC group than in the control OLE group, and intermediary in the ALA group. Total cholesterol concentration decreased during energy restriction in the LC group only (Fig. 4B). The non-HDL-C/HDL-C ratio was only determined as the end of the restriction period and was significantly lower in the ALA group than in the OLE one, whereas it was intermediary in the LC group (Fig. 4C). At the end of the restriction period, plasma concentrations of inflammatory cytokines (IL-1β, PAI-1, and MCP-1) were not significantly affected by dietary treatments (data not shown). 3.4. Markers of protein metabolism The plasma concentration of most amino acids was not affected by the diet (data not shown). When a significant difference occurred between groups, it was almost always due to a lower value in the ALA and/or LC groups: when compared to the OLE group, concentration was decreased for alanine in the ALA and LC groups (by 17 and 24%, respectively) and for threonine, serine, αaminobutyric acid and proline (by 12, 16, 48, and 17%, respectively) in the LC group only. Plasma concentrations of 1-methyl- and

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P values for effects Initial value: 0.5906 Diet: 0.1726 n-3 vs OLE: 0.0746

120

Triglyceride (mg/dL)

Glycemia (mg/dL)

140

100 80 60 40 20 0 OLE

100 90 80 70 60 50 40 30 20 10 0

a

ab

OLE

LC

200 150 100 50 0 OLE

ALA

LC

P values for effects Initial value: 0.0097 Diet: <0.0001 n-3 vs OLE: 0.0030

LC

Cholesterol (mg/dL)

250

b

ALA

P values for effects Initial value: 0.0157 Diet: 0.0463 n-3 vs OLE: 0.4393

300

Insulinemia (mg/dL)

ALA

P values for effects Initial value: 0.0058 Diet: 0.0006 n-3 vs OLE: 0.0010

80

a a

60

b 40 20 0 OLE

P values for effects Initial value: 0.0451 Diet: 0.0397 n-3 vs OLE: 0.1407

70

a

1.60

50 40 30 20 10 0 OLE

ALA

LC

Fig. 3. Effect of energy restriction on plasma glucose and insulin concentrations, and on HOMA. Values are mean7standard deviations for 10 rats per group, before (open bars) and after (full bars) energy restriction. Values for glycemia (2A), insulinemia (2B) and HOMA index (2C) after energy restriction were compared after adjustment for the corresponding values before energy restriction used as a covariable.

3-methyl-histidine, and of urea, which are markers of proteolysis, were not affected by the diet. As concerns insulin signalling, the total amounts of IR, IRS1 and Akt2 in both muscle and liver, as well as the amounts of their respective phosphorylated forms, when adjusted for their respective total forms, did not differ between groups in muscle (data not shown). In contrast, the amounts of p-IR and p-IRS1 in the liver were higher in the LC group than in the OLE one (Table 6). Besides, the contrast analysis showed significantly higher values for both IR and IRS1 in the n-3 PUFA-rich groups (ALA and LC) than in the MUFA-rich group (OLE). In the liver and the muscle, mRNA levels of the corresponding genes did not differ between groups, as well as the mRNA levels of proteins involved in proteolysis in the muscle (calpain 2, ubiquitinconjugating enzyme E2B, E3 ubiquitin ligase, atrogin-1) (data not shown).

4. Discussion In various conditions affecting protein homeostasis, such as ageing, sepsis, starvation, cancer, or immobilization, dietary

non-HDL-C/HDL-C

HOMA

60

ALA

LC

P values for effects Diet: 0.0204 n-3 vs OLE: 0.0195 ab b

1.20 0.80 0.40 0.00 OLE

ALA

LC

Fig. 4. Effect of energy restriction on plasma lipid concentrations, and of non-HDLC/HDL-C ratio. Values are mean7 standard deviations for 10 rats per group, before (open bars) and after (full bars) energy restriction. Plasma concentrations of triglyceride (TG, 3A) and total cholesterol (TC, 3B) after energy restriction were compared after adjustment for the corresponding values before energy restriction used as a covariable. Due to needed amounts of plasma, HDL-C was determined in blood taken from the vena cava, so initial values (before restriction) were not available. Bars sharing a same superscript letter were not significantly different at Po 0.05, according to post-hoc Tukey–Kramer analysis.

Table 6 Phosphorylation level of liver proteins involved in insulin signalling. P values for effects OLE

p-IR p-IRS1 p-Akt2

ALA

b

17.7 7 4.8 13.4 7 1.2b 35.2 7 9.1

LC

ab

22.3 7 6.7 15.4 7 2.4ab 38.17 9.7

a

26.6 7 7.9 17.0 7 2.7a 34.3 7 8.5

Diet

N-3 vs OLE

0.0293 0.0133 0.6430

0.0190 0.0079 0.7680

Values are mean 7standard deviations (SD) for 10 rats per group, and were adjusted for their respective total protein values. Mean values within a column sharing a same superscript letter, or without superscript letter, did not differ significantly at Po 0.05, according to post-hoc Tukey–Kramer analysis.

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supplementation with n-3 LC-PUFA has been reported to alleviate, at least partly, the loss of lean/muscle mass, in relation with an improvement of insulin signalling [11–16]. In contrast, there is a lack of animal models aiming to explore the effects of n-3 PUFA on fat-free mass or muscle mass during energy restriction in obese subjects. Thus, in the present study, we aimed to characterize the metabolic and molecular events associated with energy restriction in a model of energy-restricted rats receiving diets rich in MUFA, ALA, or LC-PUFA. Special attention was paid to protein homeostasis, in relation with insulin sensitivity. 4.1. Validation of the model There was an improvement in the metabolic profile in rats fed diets rich in n-3 PUFA, more pronounced with n-3 LC than with ALA. Indeed, in response to energy restriction, TG concentration decreased markedly in all groups, and to a higher extent in the LC group (Fig. 4A). This is consistent with studies in energy-restricted obese subjects showing that weight loss is associated with a decrease in plasma TG concentration [23,41–43], this decrease being greater in subjects supplemented with n-3 LC-PUFA [23,44]. Total cholesterol concentration decreased markedly in the LC group only, with final values lower than in the two other groups (Fig. 4B). This differs from human studies showing that weight loss is accompanied by a decrease in plasma cholesterol concentration without an additional effect of n-3 PUFA [17,23,44]. In contrast, only the ALA group showed lower values of the nonHDL-C/HDL-C ratio than the OLE group (Fig. 4C). To our knowledge, this is first evidence of a differential effect of the precursor ALA and its LC-derivatives on the main components of circulating cholesterol. By comparison with the OLE group, the reduction of the HOMA index of insulin resistance at the end of the restriction period in the LC group only is in line with the results of studies in human showing that improvement of insulin sensitivity during weight loss may be further enhanced by n-3 LC-PUFA supplementation [23,41,42], but not by ALA supplementation [17]. Altogether, these results support the validity of our energyrestricted rat model for reproducing the additional of n-3 PUFA effects on the metabolic markers of the MetSynd, during weight loss, which have been reported in humans. Therefore, this allowed using this model to explore the phenotypic and metabolic effects of dietary n-3 PUFAs during energy restriction in relation to body composition, protein homeostasis, and insulin sensitivity. 4.2. Effect of dietary fatty acids on body weight and composition in response to energy restriction In energy-restricted overweight or obese human subjects, additional effects of n-3 LC-PUFA on weight loss were observed in some studies [19,20,45] but not in others [18,21,23,46,47]. Even in the two studies performed in both genders and supporting a beneficial additive effect of n-3 LC-PUFA, one showed a significant effect in men only [20], and the other in women only [19]. The reasons for these discrepancies are unclear and may be accounted for by a number of confounding factors, leading to considerable interindividual variations in the responses to fatty acid intake. To our knowledge, the present study, implemented under strictly controlled conditions in a rodent model, is the first one showing that providing of n-3 PUFA during energy restriction resulted in a lower body weight than after a MUFA-rich diet (ALAþ LC groups vs OLE group), and that independently of the body weight before energy restriction (Fig. 2). Post-hoc comparisons indicated that the effect was significant with n-3 LC-PUFA (LC group) while only a trend was observed with ALA (ALA group). The mechanisms by which n-3 PUFA could facilitate weight loss remain unclear [48]. However, it

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has been repeatedly shown in rodents that n-3 LC-PUFA downregulate SREBP1c-mediated lipogenesis and up-regulate fatty acid ß-oxidation, which may result in the mobilization of lipid store and eventually weight loss [48]. Accordingly, obese patients in whom n-3 LC-PUFA supplementation assisted weight loss showed an increased ketogenesis, which was representative of an enhancement of ß-oxidation [45]. As concerns ALA, a few studies, including ours, suggest that the same mechanisms are involved, relying mostly on the regulation of gene transcription [28,49–51]. Despite a beneficial effect of dietary n-3 PUFA on weight loss, body lipid and protein contents, as well as adipose tissues weights, were the same in all groups, suggesting that fat mass and fat-free mass were similarly affected in all groups, and that the fatty acid composition of the diet did not influence body composition during weight loss (Table 2). Similarly, most human studies showed either no effect of energy restriction on the lean mass, whatever the diet [23] or no beneficial effect on the loss of fat-free mass in subjects supplemented with LC n-3 PUFA [18–20]. A single human study showed only a trend towards a better preservation of the fat-free mass during weight loss in patients receiving a diet rich in n-3 PUFA, compared to those receiving diets rich in SFA or in n-6 PUFA [21]. Interestingly, in the present study, the higher weight loss observed in the LC group had no negative impact on the weight of the Gastrocnemius muscle or the carcass (Table 2). However, under our experimental conditions, it is not possible to generalize this beneficial effect of the LC diet to the global muscle mass or to the lean body mass. 4.3. Effect of dietary fatty acids on insulin sensitivity in relation to protein homeostasis. Possible mechanisms N-3 fatty acids have been shown to improve the insulin regulation of lipid and carbohydrate metabolism through changes in TG content, membrane fatty acid composition, generation of second messengers and lipid mediators, downstream intracellular signalling and gene regulation [33,52] [53]. In contrast, the potential role of dietary n-3 fatty acid in amino acid metabolism and protein homeostasis has been poorly investigated. The protein kinase Akt is a key player in protein homeostasis. Indeed, Akt activation (through its phosphorylation) and subsequent regulation of its downstream pathways play a central role in both activation of protein synthesis via the Akt–mTOR–p70s6k pathway and inhibition of protein catabolism via the ubiquitin– proteasome pathway [54,55]. Besides, Akt activation occurs as a consequence of insulin signalling. Thus, a major aim of the present study was to explore to what extent insulin sensitivity and insulin signalling were involved in the effects of n-3 PUFA, after energy restriction and weight loss. As discussed above, global insulin sensitivity, as assessed by HOMA index of insulin resistance, was improved in the LC group. In the muscle, insulin signalling, as assessed by the phosphorylation rate of Akt2 and upstream proteins in response to insulin, was unaffected by the nature of dietary fatty acids. In the liver, however, phosphorylation rates of insulin receptor and IRS1 were significantly higher in the LC group compared to the OLE group (Table 6). Phosphorylation rates in the ALA group were intermediary, but did not differ significantly from either the OLE or the LC group. This result could not be explained by an enhanced expression of the corresponding genes since neither the amounts of total proteins nor the mRNA levels of the corresponding genes differed between groups in the muscle or the liver. Because TG content was higher in the liver of the LC group compared to the OLE group, this difference in insulin signalling efficiency cannot be ascribed to a reduction in TG content (Table 5). Insulin sensitivity has also been shown to be impaired in low-grade inflammatory conditions, as a consequence of the activation of the NFκB pathway [56]. Under our experimental

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conditions, however, the absence of difference in the plasma concentration of various pro-inflammatory cytokines among groups does not favor the hypothesis of a beneficial role n-3 fatty acids on low-grade inflammation leading to improved insulin signalling. Rather, this improvement of insulin signalling in the liver of the LC group could result from changes in membrane fatty acid composition. The relationship between changes in fatty acid membrane composition and activation of the insulin signalling pathways is complex, and may involve effects on receptor binding/affinity, generation of second messengers, or eicosanoid balance, especially prostaglandin synthesis [57]. All these factors ultimately alter phosphorylation of the target mediators of insulin signalling. In particular, human and rodent studies repeatedly showed that an increase in membrane unsaturation, and especially in n-3 LC-PUFA, was associated with an higher insulin sensitivity [58]. Under our experimental conditions, using three diets differing in their fatty acid profile resulted in markedly different fatty acid profile in red blood cells, muscle, and liver, and that after 4 weeks only, and without possible confounding factors due to food intake. Globally, in both the ALA group (diet rich in ALA), and the LC group (diet rich in n-3 LC derivatives), tissues were enriched in the n-3 LC content whereas the n-6 LC content was unchanged or even decreased. Besides, the effects were more pronounced in the LC group, which exhibited the highest unsaturation indexes (Tables 3–5). This in agreement with human studies showing a higher n-3 PUFA content in serum/plasma and adipose tissue lipids in obese patients given LC-PUFA during energy restriction [23,45]. A major finding of the present study is that the capacity of dietary ALA to enrich tissues in LC-PUFA is preserved during energy restriction and weight loss. Besides, in the present study, the fatty acid profile of liver appeared to be the most responsive to the diet, and the hepatic unsaturation indexes were higher in the liver of the ALA and LC groups than in the corresponding muscles. These findings may explain why insulin sensitivity was improved in the LC group only, and why, in this group, this affected insulin signalling in the liver only.

5. Conclusion In conclusion, during energy restriction in the rat, a diet rich in n-3 LC-PUFA reinforces the effect of weight loss on insulin sensitivity, as assessed by lower HOMA index and enhanced activation of the early steps of insulin signalling in the liver. This overall improvement in insulin sensitivity is probably associated with the enrichment of cell membranes in n-3 LC-PUFA. Despite their higher weight loss, rats on the LC diet did not exhibit a lower muscle weight. However, the lack of dietary-induced variations in insulin signalling is consistent with the very low differences in the weight of the Gastrocnemius muscle between the three groups. Finally, data in the ALA group remain inconclusive, being most often intermediary between those of the control (OLE) and LC groups. It is noteworthy that in all groups, weight loss was less than 10% of the initial body weight. The hypothesis that longer duration and/or more drastic energy reduction is required to ultimately unveil the effects of n-3 dietary fatty acids on the preservation of lean body mass has now to be tested. Likewise, since we did not explore Akt pathways downstream, effects on proteosynthesis or on proteolysis remain to be determined more precisely on a mechanistic point of view as well.

Sources of support in the form of grants ONIDOL (Office National Interprofessionnel des Graines et Fruits Oléagineux, 11 rue de Monceau, CS 60003, F-75008, Paris France).

Acknowledgments This work was supported by a grant from ONIDOL, of which financial support is gratefully acknowledged (grant N° 910P0A). Florie Daire, MD student, Michel Lessire and Elisabeth Baeza contributed to the running of this study. The authors wish to thank Stéphanie Priez for animal care and help with dissection and sampling. All authors were involved in the design and oversight of the study. N.G. was responsible for the conduct of the study. J.F.H. was responsible for the exploration of insulin signalling and statistical analyses. J.M. was responsible for fatty acid analyses, V.M. for biochemical analyses, and S.T. for carcass analyses. D.H. wrote the paper and all authors were responsible for the final manuscript.

Appendix A. Supporting information Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.plefa.2014.07.007.

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