EPA prevents fat mass expansion and metabolic disturbances in mice fed with a Western diet
2016; Elsevier BV; Volume: 57; Issue: 8 Linguagem: Inglês
10.1194/jlr.m065458
ISSN1539-7262
AutoresAlexandre Pinel, Elodie Pitois, Jean‐Paul Rigaudière, Chrystèle Jouve, Sarah de Saint Vincent, Brigitte Laillet, Christophe Montaurier, Alain Huertas, Béatrice Morio, Frédéric Capel,
Tópico(s)Adipose Tissue and Metabolism
ResumoThe impact of alpha linolenic acid (ALA), EPA, and DHA on obesity and metabolic complications was studied in mice fed a high-fat, high-sucrose (HF) diet. HF diets were supplemented with ALA, EPA, or DHA (1% w/w) and given to C57BL/6J mice for 16 weeks and to Ob/Ob mice for 6 weeks. In C57BL/6J mice, EPA reduced plasma cholesterol (−20%), limited fat mass accumulation (−23%) and adipose cell hypertrophy (−50%), and reduced plasma leptin concentration (−60%) compared with HF-fed mice. Furthermore, mice supplemented with EPA exhibited a higher insulin sensitivity (+24%) and glucose tolerance (+20%) compared with HF-fed mice. Similar effects were observed in EPA-supplemented Ob/Ob mice, although fat mass accumulation was not prevented. By contrast, in comparison with HF-fed mice, DHA did not prevent fat mass accumulation, increased plasma leptin concentration (+128%) in C57BL/6J mice, and did not improve glucose homeostasis in C57BL/6J and Ob/Ob mice. In 3T3-L1 adipocytes, DHA stimulated leptin expression whereas EPA induced adiponectin expression, suggesting that improved leptin/adiponectin balance may contribute to the protective effect of EPA. In conclusion, supplementation with EPA, but not ALA and DHA, could preserve glucose homeostasis in an obesogenic environment and limit fat mass accumulation in the early stage of weight gain. The impact of alpha linolenic acid (ALA), EPA, and DHA on obesity and metabolic complications was studied in mice fed a high-fat, high-sucrose (HF) diet. HF diets were supplemented with ALA, EPA, or DHA (1% w/w) and given to C57BL/6J mice for 16 weeks and to Ob/Ob mice for 6 weeks. In C57BL/6J mice, EPA reduced plasma cholesterol (−20%), limited fat mass accumulation (−23%) and adipose cell hypertrophy (−50%), and reduced plasma leptin concentration (−60%) compared with HF-fed mice. Furthermore, mice supplemented with EPA exhibited a higher insulin sensitivity (+24%) and glucose tolerance (+20%) compared with HF-fed mice. Similar effects were observed in EPA-supplemented Ob/Ob mice, although fat mass accumulation was not prevented. By contrast, in comparison with HF-fed mice, DHA did not prevent fat mass accumulation, increased plasma leptin concentration (+128%) in C57BL/6J mice, and did not improve glucose homeostasis in C57BL/6J and Ob/Ob mice. In 3T3-L1 adipocytes, DHA stimulated leptin expression whereas EPA induced adiponectin expression, suggesting that improved leptin/adiponectin balance may contribute to the protective effect of EPA. In conclusion, supplementation with EPA, but not ALA and DHA, could preserve glucose homeostasis in an obesogenic environment and limit fat mass accumulation in the early stage of weight gain. Obesity is a complex disorder involving an excessive amount of body fat. It has several health consequences, which are frequently linked to metabolic syndrome (MetS). Metabolic syndrome (MetS) is defined as a cluster of several risk factors for type 2 diabetes and cardiovascular diseases (1Eckel R.H. Grundy S.M. Zimmet P.Z. The metabolic syndrome.Lancet. 2005; 365: 1415-1428Abstract Full Text Full Text PDF PubMed Scopus (4900) Google Scholar, 2Reaven G.M. Banting lecture 1988. Role of insulin resistance in human disease.Diabetes. 1988; 37: 1595-1607Crossref PubMed Scopus (11139) Google Scholar). MetS is closely related to the progression of insulin resistance (IR) and the accumulation of fat mass, notably in the visceral area (2Reaven G.M. Banting lecture 1988. Role of insulin resistance in human disease.Diabetes. 1988; 37: 1595-1607Crossref PubMed Scopus (11139) Google Scholar). Thus, alteration in the cross-talk between key metabolic tissues, such as the liver, adipose tissue (AT), skeletal muscle, and intrinsic dysfunctions in these organs probably represent a key step in the progression of MetS (3Cusi K. The role of adipose tissue and lipotoxicity in the pathogenesis of type 2 diabetes.Curr. Diab. Rep. 2010; 10: 306-315Crossref PubMed Scopus (212) Google Scholar, 4Argilés J.M. López-Soriano J. Almendro V. Busquets S. López-Soriano F.J. Cross-talk between skeletal muscle and adipose tissue: a link with obesity?.Med. Res. Rev. 2005; 25: 49-65Crossref PubMed Scopus (156) Google Scholar). The impairment of lipid storage capacity in subcutaneous AT has been related to the alteration of the endocrine function of the tissue, the increased free FA release in the circulation, visceral fat accumulation (5Alligier M. Gabert L. Meugnier E. Lambert-Porcheron S. Chanseaume E. Pilleul F. Debard C. Sauvinet V. Morio B. Vidal-Puig A. et al.Visceral fat accumulation during lipid overfeeding is related to subcutaneous adipose tissue characteristics in healthy men.J. Clin. Endocrinol. Metab. 2013; 98: 802-810Crossref PubMed Scopus (77) Google Scholar), and the deposition of ectopic fat in other organs contributing to IR (6Després J.P. Lemieux I. Bergeron J. Pibarot P. Mathieu P. Larose E. Rodés-Cabau J. Bertrand O.F. Poirier P. Abdominal obesity and the metabolic syndrome: contribution to global cardiometabolic risk.Arterioscler. Thromb. Vasc. Biol. 2008; 28: 1039-1049Crossref PubMed Scopus (1118) Google Scholar). Lifestyle changes are recommended for the prevention and the management of MetS. Several reports from the literature have suggested that metabolic abnormalities and alterations of AT biology could be prevented by increasing the intake of n-3 PUFAs (7Pinel A. Morio-Liondore B. Capel F. n-3 Polyunsaturated fatty acids modulate metabolism of insulin-sensitive tissues: implication for the prevention of type 2 diabetes.J. Physiol. Biochem. 2014; 70: 647-658Crossref PubMed Scopus (36) Google Scholar). n-3 PUFAs, especially those of marine origin (i.e., C20:5n-3, EPA and C22:6n-3, DHA), were identified as potent positive regulators of insulin sensitivity in vitro (8Pinel A. Rigaudière J.P. Laillet B. Pouyet C. Malpuech-Brugère C. Prip-Buus C. Morio B. Capel F. N-3PUFA differentially modulate palmitate-induced lipotoxicity through alterations of its metabolism in C2C12 muscle cells.Biochim. Biophys. Acta. 2016; 1861: 12-20Crossref PubMed Scopus (32) Google Scholar) and in animal models (9Storlien L.H. Kraegen E.W. Chisholm D.J. Ford G.L. Bruce D.G. Pascoe W.S. Fish oil prevents insulin resistance induced by high-fat feeding in rats.Science. 1987; 237: 885-888Crossref PubMed Scopus (566) Google Scholar). However, epidemiological studies aiming at determining the beneficial effects of those n-3 PUFAs and their common precursor alpha linolenic acid (C18:3n-3, ALA), led to inconclusive results (10Wu J.H. Micha R. Imamura F. Pan A. Biggs M.L. Ajaz O. Djousse L. Hu F.B. Mozaffarian D. Omega-3 fatty acids and incident type 2 diabetes: a systematic review and meta-analysis.Br. J. Nutr. 2012; 107: S214-S227Crossref PubMed Scopus (260) Google Scholar). This may be mainly due to the fact that different experimental protocols were used for these studies. Furthermore, most of the interventional studies in humans or animal models examined the impact of a mixture of EPA and DHA on IR (11Lorente-Cebrián S. Costa A.G. Navas-Carretero S. Zabala M. Martínez J.A. Moreno-Aliaga M.J. Role of omega-3 fatty acids in obesity, metabolic syndrome, and cardiovascular diseases: a review of the evidence.J. Physiol. Biochem. 2013; 69: 633-651Crossref PubMed Scopus (277) Google Scholar, 12Fedor D. Kelley D.S. Prevention of insulin resistance by n-3 polyunsaturated fatty acids.Curr. Opin. Clin. Nutr, Metab. Care. 2009; 12: 138-146Crossref PubMed Scopus (197) Google Scholar), and the impact of ALA has been poorly investigated. Finally, the specific roles of each n-3 PUFA on IR during obesity still remain poorly known because a mixture of EPA and DHA was generally used to study the effects of n-3 PUFAs on adiposity and MetS. Overall, nutritional supplementations with EPA and DHA had a beneficial effect on adiposity and fat cell production of adipokines or FA metabolites (13Ruzickova J. Rossmeisl M. Prazak T. Flachs P. Sponarova J. Veck M. Tvrzicka E. Bryhn M. Kopecky J. Omega-3 PUFA of marine origin limit diet-induced obesity in mice by reducing cellularity of adipose tissue.Lipids. 2004; 39: 1177-1185Crossref PubMed Scopus (249) Google Scholar, 14Flachs P. Mohamed-Ali V. Horakova O. Rossmeisl M. Hosseinzadeh-Attar M.J. Hensler M. Ruzickova J. Kopecky J. Polyunsaturated fatty acids of marine origin induce adiponectin in mice fed a high-fat diet.Diabetologia. 2006; 49: 394-397Crossref PubMed Scopus (292) Google Scholar, 15Kuda O. Rombaldova M. Janovska P. Flachs P. Kopecky J. Cell type-specific modulation of lipid mediator's formation in murine adipose tissue by omega-3 fatty acids.Biochem. Biophys. Res. Commun. 2016; 469: 731-736Crossref PubMed Scopus (23) Google Scholar). Only one study examined the impact of EPA-, ALA-, or DHA-enriched oils in rats fed with a high-fat, high-carbohydrate diet for 8 weeks (16Poudyal H. Panchal S.K. Ward L.C. Brown L. Effects of ALA, EPA and DHA in high-carbohydrate, high-fat diet-induced metabolic syndrome in rats.J. Nutr. Biochem. 2013; 24: 1041-1052Crossref PubMed Scopus (121) Google Scholar). This study suggested that EPA and DHA, but not ALA, could partially protect animals from whole body IR and limit abdominal adiposity, but without any exploration in AT. Therefore, the present study aimed at analyzing the specific effects of supplementing the diet with pure ALA, EPA, or DHA preparations on AT biology and whole body and tissue IR in a murine model of diet-induced obesity. Diet preparations were purchased as powder form from Brogaarden (Denmark). Free FAs were used for dietary supplementation; ALA and DHA were purchased from Nu-Chek-Prep Inc. (MN), and EPA and a mixture of oleic acid, palmitic acid, stearic acid, and linoleic acid (50:25:15:10) were purchased from Larodan (Malmö, Sweden). Primary antibodies were obtained from Cell Signaling Technology (Leiden, Netherlands) and Sigma Aldrich (Saint-Quentin Fallavier, France). Secondary antibodies were from Bethyl Laboratories (Montgomery, TX); ECL and PierceTM BCA protein assay kit were purchased from Thermo Scientific (Villebon sur Yvette, France). DMEM was from Sigma Aldrich (Saint-Quentin Fallavier, France). Calf and FBS, FA-free BSA, PBS, and penicillin/streptomycin mix were from PAA (Velizy-Villacoublay, France). For cell culture experiments, ALA, EPA, and DHA (catalog numbers 90210, 90110, and 90310, respectively) were from Cayman Chemicals (Ann Arbor, MI). The [1-14C] palmitate (catalog number NEC075H001MC) was from Perkin Elmer (Courtaboeuf, France). When not specified, chemicals were from Sigma Aldrich. Five-week-old WT male C57BL/6J mice and Ob/Ob male mice were obtained from Janvier Laboratories (Le Genest Saint Isle, France). WT and Ob/Ob mice were housed four/five and one per cage, respectively, in a room maintained at 22°C–24°C with an alternating 12 h light/dark cycle with free access to food and water. Mice were weighed, and food consumption was measured every 2 weeks. Body composition of WT mice was measured during the 16th week of dietary experiment and during the 6th week for Ob/Ob mice, using EchoMRITM (EchoMRI®, Houston, TX). All protocols followed animal care guidelines of the European Union and were approved by the local research ethics committee (CEMEAA, CE91-12 and 00845.02). After 1 week of acclimation, five groups of WT mice and Ob/Ob mice of similar body weight (BW) were randomly constituted. Animals were then assigned to receive five diets as follows: 1) CTRL, low-fat (LF) diet (Research Diet D12450H); 2) HF, high-fat, high-sucrose (HF) diet (Research diet D12451, providing 45 kcal% and 17 kcal% from fat and sucrose, respectively) supplemented with 1% w/w of an FA mixture of the four main FAs in plasma triglycerides (oleic acid, palmitic acid, stearic acid, and linoleic acid; 50:25:15:10%, respectively); 3) HF-A, HF diet supplemented with 1% w/w ALA; 4) HF-E, HF diet supplemented with 1% w/w EPA; 5) HF-D, HF diet supplemented with 1% w/w DHA. Each FA and FA mix were dissolved in a minimal volume of high oleic sunflower oil before incorporation in diet powder. Diets were stored at −20°C and provided fresh every 2 days for 16 weeks (WT mice) or 6 weeks (Ob/Ob mice). Food intake per cage was evaluated every 2 weeks, averaged, and expressed as grams per mouse per day or kilojoules per mouse per day. Dioxygen consumption (VO2), carbon dioxide production (VCO2), and activity of mice were measured during 24 h using a four-cage TSE System Pheno-Master/LabMaster (Bad Homburg, Germany). Energy expenditure was calculated using Weir's equation (17Weir J.B. New methods for calculating metabolic rate with special reference to protein metabolism. 1949.Nutrition. 1990; 6: 213-221PubMed Google Scholar). The respiratory quotient (RQ) was calculated as the ratio of VCO2 to VO2. Spontaneous activity was measured using a three dimensions meshing of light beams. Ambient temperature was maintained at 22°C, the light was on from 8 AM to 8 PM, and mice had free access to food and water. Data were collected after 24 h of acclimation, and the O2 and CO2 analysers were calibrated before each measurement period. Insulin tolerance test (ITT) and glucose tolerance test (GTT) were performed on 10 WT mice per group during the 16th week of diet. All Ob/Ob mice were submitted to ITT and GTT, with a 3 day recovery between them. After 6 h of fasting, animals received an intraperitoneal injection of insulin (1.2 mIU/g) or glucose (2 mg/g for WT and 1.5 mg/g for Ob/Ob mice), and blood samples were collected from the tail vein 0, 15, 30, 45, 60, and 120 min later. Blood glucose levels were determined using a commercial glucometer (One Touch®Vita®, Issy les Moulineaux, France) for the calculation of the area under the curve. Sixteen-hour fasted mice were anesthetized by intraperitoneal administration of ketamine-xylazine mix (20 mg/kg:4 mg/kg). To explore insulin sensitivity, eight WT mice per group and all Ob/Ob mice were subjected to insulin injection (1.2 mIU/g) 30 min before anesthesia. Cardiac puncture was performed to collect blood samples, and cervical dislocation was then done to euthanize mice. Blood was sampled in EDTA-coated tubes to avoid coagulation and was processed for plasma collection. Red blood cells (RBCs) were separately collected from WT mice for FA composition analyses. Blood samples, heart, epididymal white adipose tissue (eWAT), gastrocnemius, and quadriceps were removed, weighed, frozen in liquid nitrogen, and stored at −80°C until analyses. Lipid extracts from RBCs or other tissues were prepared using 4 ml chloroform-methanol (2:1, v/v; Sigma Aldrich) and 1 ml 0.9% NaCl. Extracts were centrifuged to separate lipid phase to aqueous phase. Methylation was then performed before FA methyl ester separation by GC as previously described (18Capel F. Acquaviva C. Pitois E. Laillet B. Rigaudiere J.P. Jouve C. Pouyet C. Gladine C. Comte B. Vianey Saban C. et al.DHA at nutritional doses restores insulin sensitivity in skeletal muscle by preventing lipotoxicity and inflammation.J. Nutr. Biochem. 2015; 26: 949-959Crossref PubMed Scopus (52) Google Scholar). One hundred to 150 mg of gastrocnemius, liver, or eWAT were ground three times in a mini-bead beater in the presence of 0.7 ml lysis buffer (50 mM HEPES, 150 mM sodium chloride, 10 mM EDTA, 10 mM sodium pyrophosphate tetrabasic anhydrous, 25 mM β-glycerophosphate, 100 mM sodium fluoride, 10% glycerol anhydrous) supplemented with phosphatase inhibitors cocktail (Sigma Aldrich) respecting 2 min timeout between each session. Successive centrifugations were done to collect supernatant. Protein quantification was performed using a BCA protein assay kit (Pierce). BSA standard curve and sample preparation and analysis were realized according to the manufacturer's instructions. For protein immunoblotting, 20 µg of proteins loaded for separation by SDS-PAGE electrophoresis. Proteins were transferred on polyvinylidene difluoride membranes. These membranes were then immunoblotted with the appropriate antibody to detect GAPDH, serine 473 phosphorylated Akt (also called protein kinase B), and total Akt. Antibody binding was detected using HRP-conjugated secondary antibodies and ECL Western blotting substrate (Thermo Scientific). Immunoblots were visualized by chemiluminescence imaging system (MF ChemiBIS 2020; DNR Bio-Imaging Systems, Jerusalem, Israel) and quantified using MultiGauge V3.2 software. ELISA on plasma samples were performed according to the manufacturer's instructions (BioRad, Marnes-la-Coquette, France). Leptin, insulin, monocyte chemoattractant protein-1 (MCP-1), resistin, and hormones of appetite [ghrelin, glucagon-like peptide-1 (GLP-1), gastric inhibitory polypeptide (GIP), and glucagon] were quantified using the Luminex® technology (Bioplex® 200, BioRad). Adiponectin was quantified using an ELISA assay (Eurobio, Courtaboeuf, France). Plasma levels of glucose, nonesterified FA, glycerol, triglyceride, and total cholesterol were measured using KonelabTM 20 analyzer (Thermo Electron SA, Cergy-Pontoise, France), according to the manufacturer's instruction of each assay. For tissue or cell culture gene expression assays, RNA extraction was performed using TRIzol® (Thermo Scientific) according to the manufacturer's instructions. Chloroform was added (0.2 ml/ml of TRIzol®), and samples were mixed and centrifuged for 15 min at 12,000 g and 4°C. Aqueous phase containing RNA was collected, mixed with isopropanol to precipitate RNA, and centrifuged (12,000 g, 4°C, 15 min). After centrifugation, the pellet was washed with ethanol 70% (v/v), dried, and suspended in water. RNA quantification and integrity were verified by measuring the ratio of optical density at 260 nm and 280 nm and by agarose gel migration, respectively. Two micrograms of total RNA was used to realize reverse transcription. The products of reverse transcription were used for reverse transcription quantitative polymerase chain reaction (RT-qPCR) to evaluate gene expression. TaqMan low density array was used for liver and skeletal muscle samples using 384-well format plates on a 7900HT Fast Real Time PCR system (Applied Biosystems). AT gene expression was performed using specific primers (sequences available on request) and Rotor-Gene SYBR Green PCR master mix on a Rotor-Gene Q system (Qiagen, Courtaboeuf, France). mRNA quantification was assayed using the ddCT method. Hypoxanthine guanine phosphoribosyltransferase (Hprt) or non-POU-domain-containing octamer binding protein (NoNo) gene were used as the housekeeping gene in the liver/skeletal muscle and AT, respectively. DNA was extracted using TRIzol® after separation of aqueous phase containing RNA and organic phase. Five hundred microliters of back extraction buffer (guanidine thiocyanate 4 M, sodium citrate 50 mM, and Tris base 1 M) were added to the organic phase before centrifugation at 16,000 g for 30 min. Superior phase was then used for DNA precipitation by addition 400 µl of isopropanol (Sigma Aldrich) and centrifugation at 16,000 g for 15 min. DNA pellet was then washed twice with ethanol 75% (v/v) and dissolved in DNA RNA-free water. DNA content was quantified by measuring the ratio of optical density at 260 nm and 280 nm. Ratio between total DNA content and tissue sample weight was used to calculate cellularity, as DNA content is proportional to the number of cells. 3T3-L1 cells were purchased from ATCC (LGC Standards, Molsheim, France) and grown in DMEM supplemented with 10% calf serum and 100 U/ml penicillin and 100 mg/ml streptomycin in 5% CO2/humidified atmosphere at 37°C. Differentiation to adipocytes was induced 2 days post confluency (day 0) by incubating the cells in DMEM supplemented with 10% fetal bovine serum, 100 U/ml penicillin and 100 mg/ml streptomycin, 0.5 mM 3-isobutyl-1-methylxanthine (IBMX), 1 µM dexamethasone, and 10 µg/ml insulin for 48 h. Cells were maintained in the same medium without IBMX and dexamethasone for an additional 48 h. Insulin was removed, and cells were maintained until day 8 (medium was replaced at day 6). FAs were added to cell culture medium at 50 µM from days 0 to 8. Stock solutions of FAs were prepared in ethanol and further diluted at 1:1,000 in DMEM containing 2% of FA-free BSA. Control cells were also exposed to 0.1% ethanol. Cells were harvested every 2 days from day 0 (undifferentiated-untreated and confluent cells) up to day 8. Before RNA isolation and purification, cells were washed in ice-cold PBS and harvested by scrapping in TRIzol®. All data are presented as mean ± SEM. One-way ANOVA (ANOVA) was used to compare each treatment. If significant (P < 0.05), one-way ANOVA was followed by Fisher's least significant difference post hoc test with the Benjamini-Hochberg multiple testing correction. All statistical analyses were performed using R (Bioconductor). In some cases, a Student's t-test was performed. Daily energy intake (J/day) was not statistically different between groups from weeks 1 to 14 (ANOVA, P = NS; Fig. 1A). However, energy intake at week 10 tended to be lower in mice from the HF-D group compared with control, HF-A, or HF-E groups (ANOVA, P = 0.05; post hoc analysis showed a significant difference between HF-D vs. LF, HF-A, and HF-E groups at P < 0.05). Food intake expressed in grams per day was similar in the four groups receiving an HF diet (i.e., HF, HF-A, HF-E, and HF-D) during the first 8 weeks of the intervention. Thereafter, mice consuming the HF-D diet ate significantly less food from weeks 8 to 14 (Fig. 1B), compared with HF-A and HF-E groups. Unsurprisingly and because of a lower caloric density of the diet, mice fed with the control diet ate more food than mice receiving the four HF diets with or without n-3 PUFA. No changes in fasting GLP-1, GIP, or ghrelin plasma levels were detected between groups (data not shown). Despite this, in animals fed with the HF-D diet, BW gain between weeks 6 and 14 was comparable to mice from the HF, HF-A, and HF-E groups (Fig. 1C). At the end of the dietary intervention, animals fed with the HF diet gained significantly more weight than control mice (Table 1). Final BW of HF, HF-A, HF-E, and HF-D mice was not statistically different. Supplementation with EPA significantly limited fat mass accumulation compared with HF and HF-D diets. Due to a higher variability in the HF-A group, fat mass accumulation only tended to be lower in the HF-E group compared with HF-A animals (P = 0.1; Table 1). Although absolute lean mass was similar between controls and all HF groups (P = NS; Table 1), lean body mass expressed in % of BW was ∼4% higher in HF-E mice compared with the other HF groups (P < 0.05) and was similar to controls (Table 1). By contrast, lean body mass in % BW was ∼3.5% lower in the HF, HF-A, and HF-D groups compared with controls (P < 0.05; Table 1).TABLE 1Anthropometric and calorimetric parameters of C57BL/6J miceParametersControlHFHF-AHF-EHF-DBW, g27.0 ± 0.4b29.6 ± 0.3aIndividual comparison versus LF, HF, and HF-E found a difference at P = 0.1 (t-test).29.8 ± 0.5aIndividual comparison versus LF, HF, and HF-E found a difference at P = 0.1 (t-test).29.8 ± 0.5aIndividual comparison versus LF, HF, and HF-E found a difference at P = 0.1 (t-test).29.2 ± 0.4aIndividual comparison versus LF, HF, and HF-E found a difference at P = 0.1 (t-test).Fat mass, g2.66 ± 0.13b3.86 ± 0.38aIndividual comparison versus LF, HF, and HF-E found a difference at P = 0.1 (t-test).3.82 ± 0.38aIndividual comparison versus LF, HF, and HF-E found a difference at P = 0.1 (t-test).2.74 ± 0.28b3.77 ± 0.31aIndividual comparison versus LF, HF, and HF-E found a difference at P = 0.1 (t-test).Lean mass, g23.13 ± 0.4922.85 ± 0.4423.13 ± 0.5123.81 ± 0.7422.65 ± 0.48Fat mass, %9.5 ± 0.4c13.4 ± 1.2aIndividual comparison versus LF, HF, and HF-E found a difference at P = 0.1 (t-test).13.0 ± 1.2ab10.3 ± 1.1bc13.3 ± 1.1aIndividual comparison versus LF, HF, and HF-E found a difference at P = 0.1 (t-test).Lean mass, %82.6 ± 0.4aIndividual comparison versus LF, HF, and HF-E found a difference at P = 0.1 (t-test).79.6 ± 1.2b79.5 ± 1.1b82.8 ± 1.1aIndividual comparison versus LF, HF, and HF-E found a difference at P = 0.1 (t-test).79.8 ± 1.0beWAT, %BW1.3 ± 0.1b2.1 ± 0.3aIndividual comparison versus LF, HF, and HF-E found a difference at P = 0.1 (t-test).2.4 ± 0.4aIndividual comparison versus LF, HF, and HF-E found a difference at P = 0.1 (t-test).1.3 ± 0.2b2.3 ± 0.2aIndividual comparison versus LF, HF, and HF-E found a difference at P = 0.1 (t-test).Gastrocnemius, %BW0.49 ± 0.010.47 ± 0.010.49 ± 0.010.49 ± 0.010.48 ± 0.01Liver, %BW3.85 ± 0.123.52 ± 0.133.65 ± 0.093.63 ± 0.083.41 ± 0.13Heart, %BW0.49 ± 0.01ab0.47 ± 0.01abc0.46 ± 0.01bc0.5 ± 0.02aIndividual comparison versus LF, HF, and HF-E found a difference at P = 0.1 (t-test).0.45 ± 0.01cLM-EE (kJ/g)1.95 ± 0.021.87 ± 0.041.89 ± 0.021.95 ± 0.031.90 ± 0.04Locomotor activity178 ± 12174 ± 19139 ± 14179 ± 19119 ± 18aIndividual comparison versus LF, HF, and HF-E found a difference at P = 0.1 (t-test).RQ0.95 ± 0.01aIndividual comparison versus LF, HF, and HF-E found a difference at P = 0.1 (t-test).0.81 ± 0.01b0.79 ± 0.01b0.80 ± 0.01b0.8 ± 0.01bFive-week-old C57BL/6J mice were placed on an HF or n-3 PUFA-supplemented HF (HF-A, HF-E, and HF-D) diets and compared with mice fed with a standard LF diet (control) for 16 weeks. Body composition, 24 h LM-EE, RQ, and locomotor activity were evaluated during weeks 14 and 15. Tissue weights were evaluated at euthanizing after 16 weeks on the diets. Data are means ± SEM (n = 13–19 and 4–6 for anthropometric and calorimetric parameters, respectively). Means within a row with different superscripted letters differ at P < 0.05 (ANOVA).a Individual comparison versus LF, HF, and HF-E found a difference at P = 0.1 (t-test). Open table in a new tab Five-week-old C57BL/6J mice were placed on an HF or n-3 PUFA-supplemented HF (HF-A, HF-E, and HF-D) diets and compared with mice fed with a standard LF diet (control) for 16 weeks. Body composition, 24 h LM-EE, RQ, and locomotor activity were evaluated during weeks 14 and 15. Tissue weights were evaluated at euthanizing after 16 weeks on the diets. Data are means ± SEM (n = 13–19 and 4–6 for anthropometric and calorimetric parameters, respectively). Means within a row with different superscripted letters differ at P < 0.05 (ANOVA). As expected, RQ of HF, HF-A, HF-E, and HF-D mice was lower than in the LF group (P < 0.05; Table 1). Energy expenditure adjusted for differences in lean mass (LM-EE) was not different between groups, but locomotor activity of HF-E mice was higher compared with HF-D and HF-A animals (P < 0.05, Student's t-test). The FA composition of erythrocyte phospholipids (PLs) was used to validate n-3 PUFA dietary supplementations. Consumption of the HF-A, HF-E, and HF-D diets induced a significant enrichment of the corresponding FAs in erythrocyte PLs (Table 2) compared with the HF diet. Mice consuming the HF-E diet presented the highest percentage of n-3 PUFAs incorporated into erythrocyte PLs (P < 0.05 vs. the other groups), notably due to a high accumulation of docosapentaenoic acid (DPA, 22:5n-3). Supplementation with ALA and EPA similarly increased the percentage of DHA incorporation. As a consequence, the proportion of C18, C22, C20:4 n-6, C22:4 n-6, and C22:5 n-6 FA in erythrocyte PLs was reduced in mice fed the HF-E and HF-D diets compared with both HF and control diets. Only EPA supplementation significantly decreased total n-6 PUFA content compared with the HF diet (P < 0.05; Table 2). As compared with HF group, the n-6 to n-3 ratio was improved (Table 2) in HF-E and HF-D groups (−72% and −63%, respectively, vs. HF; P < 0.05) and to a lesser extent in HF-A group (−35% vs. HF, P < 0.05). A significant increase in EPA incorporation in erythrocyte PLs from HF-D mice was detected compared with HF mice. FA profiling in skeletal muscle and liver PL extracts showed very similar effects with some exceptions, regarding n-6 to n-3 ratio and DHA retroconversion, which were respectively lower and nonexistent in skeletal muscle compared with the liver (supplemental Tables S1, S2). Although the percentage of DHA in muscle PLs was high in control and HF mice, it was further increased by EPA and DHA supplementation. Then, the total amount of n-3 PUFA in muscle PLs was strongly increased (+56% and +86%, respectively, vs. HF) following EPA and DHA supplementation compared with mice receiving the HF diet alone leading to a marked change in the relative content of n-3 and n-6 PUFAs (supplemental Table S1).TABLE 2FA composition of erythrocytes from C57BL/6J miceFatty AcidControlHFHF-AHF-EHF-DC14.00.50 ± 0.060.51 ± 0.070.44 ± 0.070.45 ± 0.070.37 ± 0.05C15.00.36 ± 0.030.40 ± 0.060.33 ± 0.050.33 ± 0.050.28 ± 0.04C16.033.22 ± 0.6931.81 ± 0.9430.13 ± 0.6530.37 ± 0.4630.93 ± 1.13C16.1n.90.75 ± 0.090.89 ± 0.170.74 ± 0.140.70 ± 0.130.58 ± 0.1C16.1n.70.67 ± 0.04a0.30 ± 0.02b0.27 ± 0.03b0.23 ± 0.03b0.29 ± 0.04bC17.00.40 ± 0.01c0.57 ± 0.03a0.53 ± 0.02ab0.51 ± 0.02ab0.50 ± 0.03bC18.012.24 ± 0.37c16.55 ± 0.43a15.41 ± 0.46ab14.83 ± 0.33b14.75 ± 0.51bC18.19cis14.31 ± 0.3015.33 ± 0.5714.70 ± 0.2314.66 ± 0.1914.00 ± 0.38C18.1n.72.69 ± 0.09a1.66 ± 0.07b1.56 ± 0.05b1.48 ± 0.05bc1.24 ± 0.18cC18.2n6cis9.34 ± 0.23d10.97 ± 0.25c12.09 ± 0.40b11.61 ± 0.30bc13.59 ± 0.47aC20.1n.90.42 ± 0.010.44 ± 0.020.43 ± 0.010.34 ± 0.010.32 ± 0.01C18.3n.30.26 ± 0.01a0.15 ± 0.00b0.29 ± 0.01a0.13 ± 0.00bc0.12 ± 0.01cC20.20.32 ± 0.01d0.62 ± 0.02b0.68 ± 0.02a0.52 ± 0.01bc0.54 ± 0.01aC22.00.40 ± 0.04b0.63 ± 0.04a0.48 ± 0.05ab0.45 ± 0.06b0.41 ± 0.09bC20.3n.61.26 ± 0.040.79 ± 0.101.35 ± 0.480.97 ± 0.121.38 ± 0.37C20.4n.614.49 ± 0.69a12.00 ± 1.18b12.42 ± 0.31b8.80 ± 0.33c7.99 ± 0.54cC20.5n.30.90 ± 0.07c0.93 ± 0.09c1.08 ± 0.07c4.92 ± 0.18a1.63 ± 0.14bC22.4n.61.45 ± 0.09a1.36 ± 0.16ab1.14 ± 0.06b0.58 ± 0.03c0.33 ± 0.03cC22.5n.60.81 ± 0.07a0.61 ± 0.09b0.25 ± 0.0
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