Impact of dietary ω3 polyunsaturated fatty acid supplementation on brown and brite adipocyte function[S]

The recent characterization of functional brown adipose tissue in adult humans has opened new perspectives for regulation of energy expenditure with respect to obesity and diabetes. Furthermore, dietary recommendations have taken into account the insufficient dietary intake of ω3 PUFAs and the concomitant excessive intake of ω6 PUFA associated with the occurrence of overweight/obesity. We aimed to study whether ω3 PUFAs could play a role in the recruitment and function of energy-dissipating brown/brite adipocytes. We show that ω3 PUFA supplementation has a beneficial effect on the thermogenic function of adipocytes. In vivo, a low dietary ω6:ω3 ratio improved the thermogenic response of brown and white adipose tissues to β3-adrenergic stimulation. This effect was recapitulated in vitro by PUFA treatment of hMADS adipocytes. We pinpointed the ω6-derived eicosanoid prostaglandin (PG)F2α as the molecular origin because the effects were mimicked with a specific PGF2α receptor agonist. PGF2α level in hMADS adipocytes was reduced in response to ω3 PUFA supplementation. The recruitment of thermogenic adipocytes is influenced by the local quantity of individual oxylipins, which is controlled by the ω6:ω3 ratio of available lipids. In human nutrition, energy homeostasis may thus benefit from the implementation of a more balanced dietary ω6:ω3 ratio.

adipocytes (22)(23)(24). Several studies have demonstrated the involvement of oxylipins in fat mass development through the modulation of white and brown/brite adipocyte differentiation and activity (25). Mice exposed to diets with high levels of 6 PUFA during the perinatal period display progressive accumulation of body fat across generations (26). This observation is in agreement with findings that overweight and obesity have increased and develop earlier in life within a given population consuming Westernized diets rich in 6 PUFAs during the last decades (26). Moreover, oxylipins derived from 6 PUFA inhibit brite and brown adipocyte activity both in vitro and in vivo (17). ARA lowers the expression and function of UCP1, thus affecting the dissipation of energy in these thermogenic adipocytes. These effects are mediated via COX activities that lead to increased synthesis and release of prostaglandin (PG)E2 and PGF2, which act as inhibitors of Ucp1-mediated thermogenesis via a calcium-dependent pathway.
It has been shown that healthy adult humans exhibit BAT in the cervical and thoracic part of the body, which consists of islets of energy-dissipating thermogenic adipocytes (27)(28)(29)(30)(31). These adipocytes display a gene expression signature comparable to either rodent brown or brite adipocytes depending on the localization and the depth of the analyzed tissue (21,(32)(33)(34). As brown and brite adipocytes represent important candidates for controlling body weight, investigations in humans of the regulation of brown/ brite adipocyte recruitment and activation are in demand, particularly from a nutritional point of view, because quantitative and qualitative issues of dietary lipids are relevant to increased body weight (3). Because, in mammals, oxylipins govern a large part of the biology of adipose tissue, any dysregulation in their levels may disrupt tissue homeostasis. Thus, it is tempting to assume that prevention of excessive consumption of 6 fatty acids or reestablishment of a balanced 6:3 PUFA ratio may contribute to reduce excessive adipose tissue development by controlling white adipocyte formation and enhancing brite adipocyte recruitment.
Herein, we aimed to study whether the inhibitory effect of 6-PUFA LA (via local action of ARA) on brite adipocyte recruitment could be reversed by supplementation with the 3-PUFA -linolenic acid associated to its metabolites EPA and DHA. We show, in vivo in mice and in vitro in human cells, that adjustment of a 6:3 PUFA ratio from 30 to 3.7 by supplementation of 6 PUFA-enriched diet with 3 PUFAs rescues the inhibitory effect of 6-derived oxylipins on the activity of brown and brite adipocytes. These effects are mediated through oxylipins via the decreased level of PGF2.

Animals and diets
The experiments were conducted in accordance with the French and European regulations (directive 2010/63/EU) for the care and use of research animals and were approved by national experimentation committees (MESR 01947.03). Ten-week-old C57BL/6J male mice from Janvier Laboratory (Le Genest Saint Isle, France) were maintained at thermoneutrality (28 ± 2°C) and 12:12 h light-dark cycles, with ad libitum access to food and water and euthanized between 10 and 11 AM. Mice were fed for 12 weeks with isocaloric 6or 3-supplemented diets (12% energy content as lipids). These experimental diets were prepared from standard chow diets (ref. 2016, Harlan Laboratories, Madison WI). The 6-supplemented diet was enriched with 0.5% linoleateethyl-ester (LA) and 0.7% oleate-ethyl-ester (ratio 6:3 = 30); the 3-supplemented diet comprised 0.5% LA, 0.54% -linolenateethyl-ester (LNA), 0.08% eicosapentaenoate-ethyl-ester and 0.08% docosahexaenoate-ethyl-ester (ratio 6:3 = 3.7) (see supplemental Table S1 for details) to follow human nutritional recommendations. Safflower oil (0.5%) was added to favor dispersion of ethyl esters in the diet. Fatty acid ethyl esters were from Nu-Chek-Prep (Waterville, MN) and diets were produced by Harlan. Chronic  3 -adrenergic receptor stimulation was carried out during the last week of the diet treatment by daily intra-peritoneal injections of CL316,243 (1 mg/kg in saline solution). Control mice were injected with vehicle only. Blood, interscapular BAT (iBAT), epididymal WAT (eWAT), and inguinal subcutaneous WAT (scWAT) were sampled and used for different analyses.

hMADS cell culture
The establishment and characterization of hMADS cells has been described (35)(36)(37). In the experiments reported herein, hMADS-3 cells were used between passages 14 and 20. All experiments were performed at least three times using different cultures. Cells were cultured and differentiated as previously described (17,35). Briefly, cells were induced to differentiate at day 2 postconfluence (designated as day 0) in DMEM/Ham's F12 media supplemented with 10 µg/ml transferrin, 10 nM insulin, 0.2 nM triiodothyronine, 1 µM dexamethasone, and 500 µM isobutylmethylxanthine. Two days later, the medium was changed (dexamethasone and isobutyl-methylxanthine omitted) and 100 nM rosiglitazone was added. At day 9, rosiglitazone was withdrawn to enable white adipocyte differentiation but was again included between days 14 and 17 to promote white-to-brite adipocyte conversion as previously described (17,35). Fatty acids were bound to BSA (0.04% for 15 min at 37°C) prior to addition to culture media.

Oxylipin quantification
In vitro analysis of secreted PGF2 was performed on differentiated cells at day 17 after incubation for 1 or 24 h in fresh culture media. PGF2 was quantified by Elisa Immuno Assay following the manufacturer's instructions (Cayman, BertinPharma, Montigny le Bretonneux, France).
Quantification of oxylipins was performed at the METATOUL platform (MetaboHUB, INSERM UMR 1048, I2MC, Toulouse, France) by mass spectrometry analysis. All tissues were snap-frozen with liquid nitrogen immediately after collection and stored at 80°C until extraction. Extraction and analysis were performed as previously described (17,38).

Measurement of oxygen consumption
Oxygen consumption rate (OCR) of hMADS adipocytes was determined at day 17 using an XF24 Extracellular Flux Analyzer (Seahorse Bioscience, Agilent). ATP synthase uncoupled OCR was determined by the addition of 1.2 µM oligomycin A (ATP synthase inhibitor) and maximal OCR by addition of 1 µM FCCP [Carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone as a mitochondrial oxidative phosphorylation uncoupling agent]. Rotenone and Antimycin A (2 µM each) were used to inhibit Complex Iand Complex III-dependent respiration, respectively. Parameters were measured for each individual well using the OCR values as previously described (39).

Histology
Histological analysis was performed as previously described (17). Sections (4 µm) were dewaxed and treated in boiling citrate buffer (10 mM, pH 6.0) for 6 min. Cooled sections were rinsed and then permeabilized in PBS 0.2% triton X-100 at room temperature for 20 min. Sections were saturated in the same buffer containing 3% BSA for 30 min, incubated with perilipin antibody (#RDI-PROGP29, Research Diagnostic Inc., Flanders, NJ) for 1 h, and TRITC-coupled anti-guinea pig antibody for 45 min. Nuclear staining was performed with DAPI.

Isolation and analysis of RNA
These procedures follow MIQE (Minimum Information for Publication of Quantitative Real-Time PCR Experiments) recommendations (41). Total RNA was extracted using TRI-Reagent kit (Euromedex, Souffelweyersheim, France) according to the manufacturer's instructions. For RNA isolation from organs, tissues were homogenized in TRI-Reagent using a dispersing instrument (ULTRA TURRAX T25, Ika, Germany). RT-PCR was performed using M-MLV-RT (Promega). SYBR qPCR premix Ex TaqII from Takara (Ozyme, France) was used for quantitative PCR (qPCR), and assays were run on a StepOne Plus ABI real-time PCR machine (PerkinElmer Life and Analytical Sciences, Waltham, MA). The expression of selected genes was normalized to that of the TATA-box binding protein (TBP) and 36B4 housekeeping genes for human genes, and 36B4 and GAPDH for mouse genes, and then quantified using the comparative-Ct method. Primer sequences are available upon request.

Statistical analysis
Data are expressed as mean values ± SEM and were analyzed using InStat software (GraphPad Software). Data were analyzed by one-way ANOVA followed by a Student-Newman-Keuls posttest, or Student's t-test to assess statistical differences between experimental groups. Differences were considered statistically significant with P < 0.01.

RESULTS
3 PUFA supplementation makes mice sensitive to 3-adrenergic receptor agonist treatment Ten-week-old mice were fed for 12 weeks with an isocaloric standard diet enriched in 6 PUFAs (6 diet, 6:3 = 30), or supplemented with 3 PUFAs (3 diet, 6:3 = 3.7) (supplemental Table S1). There was no difference in body weight (Fig. 1A) and food intake during 12 weeks (6 diet, 4.49 g/day vs. 3 diet, 4.46 g/day per mouse). During the last week, mice received daily injections of the agonist CL316,243 (1 mg/kg) to activate the 3-adrenergic receptor pathway. As expected, such treatment decreased body weight ( Fig. 1A) as well as iBAT and eWAT mass (Fig. 1B). Interestingly, 3 diet-fed mice showed a larger decrease in body mass and adipose tissue mass on CL316,243-treatment compared with 6 diet-fed mice (Fig. 1A,B). This observation is in line with higher plasma glycerol levels (Fig. 1C). Together, these results indicate a higher sensitivity of 3 diet-fed mice to CL316,243 treatment.

3 diet-fed mice showed an improved response to thermogenic stimulation in BAT and scWAT
After CL316,243 treatment, mice fed the 3-diet displayed an increase in BAT mass loss and in plasma glycerol level, suggesting a higher lipolysis capacity. As expected, histological analysis of BAT showed clear activation of brown adipocytes after CL316,243 treatment as indicated by adipocyte morphological changes in the two groups of mice ( Fig. 2A). This observation was confirmed by immunostaining for perilipin 1 (PLN1), a known lipid droplet surface protein, which allowed the measurement of droplet diameters. The data showed smaller lipid droplets in BAT of mice treated with CL316,243 (Fig. 2C). Interestingly, the droplet size was smaller in 3 compared with 6 diet-fed mice of both treatment groups (Fig. 2C). These morphological modifications were in line with elevated Fig. 1. A low dietary 6:3 ratio enhances body weight loss in response to a 3-adrenergic receptor agonist. Mice maintained at 28°C were fed with diets supplemented with 6 PUFAs or 3 PUFAs for 12 weeks. CL316,243 or vehicle (NaCl) treatment was performed daily during the last week of feeding. A: Body weight development of mice (left); body weight loss of mice after CL316,243 treatment relative to vehicle treatment (right). B: Interscapular brown adipose tissue (iBAT) and epidydimal white adipose tissue (eWAT) weight loss after CL316,243 treatment. C: Plasma glycerol levels after NaCl or CL316,243 treatment. Data are mean ± SEM, n = 12 mice/group. a, P < 0.01 6 versus 3; b, P < 0.01 NaCl versus CL316,243. expression of brown adipocyte marker genes in 3 versus 6 diet-fed mice (Fig. 2B, D).
The 3-adrenergic agonist CL316,243 is a potent inducer of brite adipocytes and therefore we hypothesized a contribution of these thermogenic cells to CL316,243-induced plasma glycerol levels and body weight loss. No morphological or gene expression differences were found in scWAT between the two groups of mice in response to vehicle treatment (Fig. 3A, B). However, after CL316,243 treatment, visual examination of sections suggested increased Ucp1-positive adipocytes in scWAT of 3 diet-fed mice compared with 6 diet-fed mice (Fig. 3A). This observation was supported by the analysis of Ucp1 mRNA expression, which tended to be increased in the 3 diet-fed group after CL316,243 treatment. Gene expression of other markers in response to CL316,243 was not affected by the diet (Fig. 3B). As shown in supplemental Fig. S1, eWAT did not display the multiloculated adipocytes that are characteristic of UCP1+ adipocytes. However, a decrease of adipocyte mean diameter after CL316,243 treatment was found in both groups, in agreement with eWAT weight decrease (Fig. 1B).
Together, these data indicate that the dietary 6:3 ratio affects the induction/activation of thermogenic adipocytes in BAT and scWAT upon stimulation with a -adrenergic agonist. As dietary fatty acid composition can considerably influence the quality and quantity of fatty acid metabolites (2,5), this differential response to thermogenic stimulation may be the consequence of modulated eicosanoid level in adipose tissues.

Dietary 3 PUFA supplementation controls the level of 6-derived oxylipins
To investigate diet-induced differences in the quantity of 3and 6-derived oxylipins, we quantified the levels of more than 30 selected metabolites in scWAT and iBAT from 3 and 6 diet-fed mice. Depending on their origin, oxygenated metabolites derived from 6 (ARA) or 3 (EPA and/or DHA) PUFAs, due to COX, LOX, and CYP450 activities (supplemental Fig. S2A, B, left panels, oxylipins) or to COX activity only (right panels, eicosanoids: a group of oxylipins) were analyzed (analyzed oxylipins are detailed in supplemental Table S2).
As expected, 3 PUFA supplementation allowed higher quantities of their metabolites in BAT and scWAT (supplemental Fig. S2A, B, black columns). In BAT, where the molecular effect of a 3 diet was the most important, we did not find any significant impact of the diet on 6-derived oxylipin levels (supplemental Fig. S2A, gray columns). In scWAT, similar data were obtained in untreated mice; however, when the mice were treated with the 3-adrenergic receptor agonist, the decrease in the levels of 6-derived oxylipins was higher in 3 diet-fed mice than in 6-diet fed mice (supplemental Fig. S2).
Analysis of COX-derived eicosanoids showed an increase in EPA-derived eicosanoids in the 3 diet-fed group and no significant decrease of 6-derived eicosanoids, except after CL316,243 treatment (supplemental Fig. S2A, B, right panels). Of note, the treatment with CL316,243, a situation where UCP1 activity is increased via the release of fatty acids, led to a decrease in the levels of the various oxygenated metabolites, possibly reflecting the preferential use of PUFAs as fuel for thermogenesis, thus limiting PUFA availability for oxylipin production ( Fig. 4 and supplemental Fig. S2).
Previous work from our laboratory has demonstrated that COX-derived oxylipins are crucial for the formation and activation of thermogenic adipocytes (17,42). Accordingly, further analyses focused on the levels of individual COX-derived oxylipins [i.e., 6kPGF1 (representative of PGI2), PGF2, PGE2, TXB2, PGD2, and 15dPGJ2] in BAT and scWAT. The levels of most individual metabolites were similar in iBAT and scWAT of vehicle-treated 6 diet-and 3 diet-fed mice, except for a decrease in TXB2 levels in BAT and in PGE2 levels in scWAT (Fig. 4A, B). Treatment with CL316,243 resulted in decreased quantity of these oxylipins in both diet groups in BAT and scWAT, although downregulation of TXB2 and PGE2 was more pronounced in scWAT than iBAT. Interestingly, PGF2 was exempt from this mode of regulation: CL316,243-treatment resulted in a significant decrease in PGF2 level in 3 diet-fed but not in 6 diet-fed mice. Thus, the level of this eicosanoid in response to CL316,243-treatment is inversely correlated with Ucp1 expression in BAT and scWAT.
Our previous results demonstrated (17) that PGF2 is a negative regulator of thermogenic adipocyte recruitment. Indeed, the 6-enriched diet PGF2 level was not affected by CL316,243 treatment (Fig. 4A, B) and correlated with inhibition of brown and brite adipocyte recruitment and activation (Figs. 2, 3). In contrast, for an 3-enriched diet after CL316,243 treatment, no alteration in brown and brite adipocyte recruitment and activation was found, although a striking decrease in the PGF2 level was observed (Fig. 4A, B).
Taken together, these results demonstrated that diet supplementation with 3 PUFAs reversed the inhibitory effect of a 6-enriched diet. This effect could be due to competition between 6 and 3 PUFAs at the COX activity level leading in turn to a decrease in PGF2 synthesis. To further investigate the effect of PUFAs and eicosanoids on adipocyte function, we used brite adipocytes derived from hMADS cells as a model system.

EPA reversed the ARA-inhibitory effect in vitro
hMADS cells are a human stem cell model that is able to differentiate into white adipocytes and to convert into functional brite adipocytes upon rosiglitazone treatment [ Fig. 5A (17, 35)]. As described previously (17), this process can be modified by 6-PUFA ARA; treatment of hMADS adipocytes during the conversion to brite adipocytes with ARA inhibited the expression of UCP1 and other brite adi-pocyte markers (CIDEA, CPT1M, PLN5) (Fig. 5A). Such treatment did not affect adipogenesis per se, as the expression of PLN1 and ADIPQ was not affected (Fig. 5A). Treatment of hMADS adipocytes with 3-PUFA EPA (molar ARA:EPA ratio of 3) reversed the inhibitory effect of ARA Fig. 5. EPA reversed the effect of ARA on adipocyte browning in vitro. hMADS cells were differentiated into white or brite adipocytes. Brite hMADS adipocytes were treated during the last 3 days of differentiation with 10 µM ARA in the presence or absence of 3.3 µM EPA. A: Expression of adipocyte markers was determined by RT-qPCR and is expressed as fold increase relative to "brite" group values. B: Basal, ATP synthase uncoupled respiration (% of residual respiration after addition of ATP synthase inhibitor oligomycin), and maximal respiration (obtained after addition of the chemical uncoupling agent FCCP) were assessed at the end of treatment to determine the oxygen consumption rate (OCR) of mitochondria. The spare respiratory capacity represents a measure for the full respiratory potential of mitochondria and is calculated as the difference between maximal and basal respiration. Data are mean ± SEM of three (A) or six (B) independent experiments. a, P < 0.01 versus brite; b, P < 0.01 versus brite + ARA. expression of the major enzymes involved in PGF2 synthesis. Indeed, the expression of COX-1, COX-2 (allowing the metabolization of ARA to PGG2), AKR1B1, and AKR1C3 (allowing the metabolization of PGG2 to PGF2) mRNAs was not altered by EPA treatment (Fig. 6C). As both ARA and EPA can be recognized and metabolized by COX-1 and COX-2, EPA most likely blocks PGF2 synthesis by competing with ARA.

DISCUSSION
The obesogenic effect of 6 PUFAs, particularly ARA, is thought to originate from their metabolization to oxylipins, thus promoting fat storage and a reduction in energy expenditure (16,17,43,44). However, this system and its regulation appear to underlie an unanticipated complexity. In fact, previous studies demonstrated the 6 eicosanoid prostacyclin (PGI2, which is derived from ARA via COX-dependent metabolization) as a positive modulator of UCP1 expression and brite adipogenesis in human and murine cellular model systems (42,(45)(46)(47). In line with these findings, transgenic mice with constitutive overexpression of COX-2 (the inducible isoform of COX) show enhanced browning of WAT and resistance to diet-induced obesity (46). The complexity of this system becomes apparent in IP receptor (PGI2 receptor) knockout mice, which are protected from 6 PUFA-induced body and fat mass gain (16). In a similar way, when mice were fed a high-fat diet, the inhibition of COX activities with indomethacin prevented body weight gain, due to decreased fat storage and enhanced recruitment of brite adipocytes in scWAT (15,43). These examples emphasize the need to increase our current understanding of the pro-and anti-adipogenic properties of PUFAs on the level of oxylipin metabolism to modulate energy balance regulation.
In our previous work, we elucidated in more detail the relationship between ARA-derived eicosanoid level, browning on brite adipocyte marker expression (Fig. 5A). This effect was further investigated at the functional level. Oxygen consumption analysis of hMADS brite adipocytes revealed that ARA inhibited all mitochondrial respiration parameters (Fig. 5B), (i.e., basal, uncoupled, and maximal respiration as well as spare respiratory capacity). EPA partially reversed this inhibitory effect of ARA on mitochondrial oxygen consumption, thus affecting the overall thermogenic capacity of these cells (Fig. 5B). No change in adipogenic marker expression was observed when DHA was added as an alternative 3 PUFA (supplemental Fig. S3) indicating that EPA specifically represents the effective compound within this class of molecules.
Taken together, these results indicate that the thermogenic function of adipocytes is positively and negatively affected by EPA (3) and ARA (6), respectively.

EPA reduced PGF2 synthesis and secretion
To identify the pathway involved in the EPA effect, we used fluprostenol, an agonist of the FP receptor (PGF2 receptor), instead of the precursor ARA during the conversion of white hMADS adipocytes into brite adipocytes. In agreement with our previous work (17), fluprostenol mimicked the effect of ARA by inhibiting the expression of UCP1 mRNA (Fig. 6A). Cotreatment with EPA did not reverse this effect (Fig. 6A), indicating that the UCP1 expression of brite hMADS adipocytes was directly affected by an interaction between the FP receptor and its 6-derived, endogenous ligand PGF2. Thus, we hypothesized that EPA reversed the ARA-induced effects in brite hMADS adipocytes by modulating the availability of PGF2 as ligand for its receptor. Accordingly, we measured PGF2 secretion after ARA treatment of brite hMADS adipocytes in the absence or presence of EPA. PGF2 secretion was not altered by EPA during the first hour of treatment. However, a striking decrease in PGF2 levels was observed after 24 h (Fig.  6B). This decrease in quantity was likely not due to a lower Fig. 6. EPA competes with ARA at the cyclooxygenase level. hMADS cells were differentiated into white or brite adipocytes. A: Brite hMADS adipocytes were treated with 10 nM fluprostenol (agonist of PGF2 receptor) in the presence or absence of 3.3 µM EPA during the last 3 days of adipogenic differentiation. UCP1 and PLN1 were evaluated by RT-qPCR. B: Brite hMADS were exposed to 10 µM ARA in the presence or absence of 3.3 µM EPA for 1 or 24 h. PGF2 was quantified in culture media by Elisa Immuno Assay. C: Brite hMADS adipocytes were treated with 10 µM ARA in the presence or absence of 3.3 µM EPA during the last 3 days of differentiation. Expression of enzymes involved in the metabolization of ARA to PGF2 was evaluated by RT-qPCR. mRNA expressions are expressed as fold increase relative to "brite" values. Data are mean ± SEM of 3 independent experiments. a, P < 0.01 versus brite; b, P < 0.01 versus brite + ARA. of WAT, and energy expenditure. Mice were fed an ARAsupplemented standard diet and showed impaired brown adipocyte activation and brite adipocyte recruitment in response to CL316,243 treatment, an effect that was attributed to the abundance of PGF2 (17). Using hMADS adipocytes as an in vitro cell model, we demonstrated that this eicosanoid translates its inhibitory effect on the thermogenic capacity of adipocytes via an interaction with its natural cell surface receptor, the FP receptor, on its synthesis from ARA via COX activity (17). Herein we show that this ARA-dependent production of PGF2 is attenuated in the presence of 3-PUFAs both in vitro in brite hMADS adipocytes and in vivo in thermogenically activated brown and brite adipocytes. We conclude that Ucp1 expression in these models is less affected by the elevated quantity of 3derived metabolites but rather depends on the reduced level of individual 6-derived metabolites (such as PGF2) with EPA-treatment.
Both 6 and 3 PUFAs are transported in the bloodstream between tissues and are incorporated in plasma membrane under the form of phospholipids or as triglycerides within adipocytes (48)(49)(50). PUFAs are released into the cell by lipases and metabolized into oxylipins using similar pathways. 6 and 3 PUFAs are known to compete at different steps that modulate the availability of their respective metabolites. LA and LNA use the same -desaturases and elongases to generate PUFA metabolites such as ARA, dihomo--linolenic acid, EPA, and DHA (51). They can also be used as substrates via -oxidation, 3 PUFAs being more rapidly oxidized compared with 6 PUFAs and mono-unsaturated fatty acids (52).
Although our data support a model in which dietary PUFAs act on the level of oxylipins, this mechanism is not exclusive in the context of thermogenic adipocyte recruitment, because 3-PUFAs per se are associated with browning properties (53,54). For example, when mice or rats are fed an LA-enriched diet, the increase in fat mass can be prevented by LNA supplementation under isolipidic and isocaloric conditions (16,55,56). Using this strategy, we demonstrated herein that 3 PUFA diet supplementation ameliorates brown adipocyte function in response to -adrenergic stimulation by promoting a more oxidative phenotype and, to a lesser extent, brite adipocyte recruitment. Analysis of PUFA metabolites associated with this improvement showed a decrease in n-2 series prostaglandins levels, especially PGF2. Altogether, in vivo and in vitro data show that the competition between 6 and 3 PUFAs takes place at the level of COX activities, favoring the production of EPA-derived metabolites instead of ARA-derived metabolites. DHA treatment was inefficient. The lack of effect could be due to the fact that DHA does not compete with ARA for metabolite production; however, DHA was described to inhibit activity and expression of COX-2 (57).
Interestingly, this competition between 6 and 3 PUFAs does not appear to contribute to the regulation of body weight when mice are fed under thermoneutral conditions but rapidly initiates the mobilization of fat as an energy substrate in response to thermogenic stimulation. This observation may be of particular relevance to the treatment of overweight and obesity in human subjects as humans constantly live in a thermoneutral environment. Thus, adjusting the 6:3-ratio of human nutrition according to dietary recommendations may benefit the recruitment of brown and brite adipocytes associated with other therapeutic strategies to promote a negative energy balance. Supporting the physiological relevance of 3 PUFA-induced effects, fish oil supplementation has been reported to induce UCP1 expression via the sympathetic nervous system, although this mechanism is restricted to BAT (58,59).
In conclusion, herein we demonstrate that 3 PUFA supplementation compensates for the inhibitory effect of 6 PUFAs on the thermogenic function of adipocytes. We identified the eicosanoid PGF2 as a causal effector, the level of which is influenced by the availability of 6 and 3 PUFAs competing at the level of their metabolization. Our data indicate that the dietary 6:3 ratio is a major regulator of adaptive thermogenesis consequently affecting energy homeostasis. These findings are of particular importance in a human nutritional context as the dietary 6:3 ratio is associated with the development of obesity and cardiovascular and inflammatory diseases.