Omega-3 fatty acids as regulators of brown/beige adipose tissue: from mechanisms to therapeutic potential.

✅ 全文

ω-3脂肪酸作为棕色/米色脂肪组织的调节因子：从机制到治疗潜力

作者 Fernández-Galilea Marta; Félix-Soriano Elisa; Colón-Mesa Ignacio; Escoté Xavier; Moreno-Aliaga Maria J 期刊 Journal of Physiology and Biochemistry 发表日期 2020 卷/期/页码 Vol. 76(2) ISSN 1877-8755 DOI 10.1007/s13105-019-00720-5 类型原创研究 (Original Research)

📄 中文摘要 Chinese Abstract

中文

肥胖是一个全球性的健康问题，其特征是脂肪组织过度积累和功能障碍，增加了2型糖尿病、心血管疾病和某些癌症等代谢性疾病的风险。棕色和米色脂肪组织通过非颤抖性产热在能量稳态中发挥关键作用，这一过程主要由解偶联蛋白1（UCP1）介导。激活这些组织已成为一种潜在的肥胖治疗策略。近期临床前证据表明，n-3多不饱和脂肪酸（n-3 PUFAs），特别是二十碳五烯酸（EPA），可诱导棕色脂肪组织（BAT）活性并促进白色脂肪组织（WAT）褐变，为增强能量消耗和代谢健康提供了一种新型营养学方法。

📋 英文结构化总结 English Structured Summary

全文整理

Background:

Obesity is a global health concern characterized by excessive adipose tissue accumulation and dysfunction, increasing the risk of metabolic diseases such as type 2 diabetes, cardiovascular disorders, and certain cancers. Brown and beige adipose tissues play key roles in energy homeostasis through non-shivering thermogenesis, primarily mediated by uncoupling protein 1 (UCP1). Activation of these tissues has emerged as a potential therapeutic strategy against obesity. Recent preclinical evidence suggests that n-3 polyunsaturated fatty acids (n-3 PUFAs), particularly eicosapentaenoic acid (EPA), can induce brown adipose tissue (BAT) activity and promote white adipose tissue (WAT) browning, offering a novel nutritional approach to enhance energy expenditure and metabolic health.

Methods:

This review synthesizes findings from in vitro studies using cultured adipocytes (including mouse-derived brown and beige adipocytes and primary human adipocytes), in vivo studies in rodent models (rats and mice fed high-fat diets supplemented with n-3 PUFAs), and limited human studies analyzing adipose tissue transcriptomes and circulating biomarkers. The focus is on evaluating the effects of EPA and docosahexaenoic acid (DHA) on thermogenic gene expression, mitochondrial function, oxygen consumption rates, and signaling pathways such as GPR120, FGF21, AMPK/PGC-1α/SIRT1, and epigenetic regulators. Mechanistic insights were drawn from knockout models (e.g., Gpr120⁻/⁻, Ucp1⁻/⁻) and assessments of sympathetic nervous system activation via TRPV1.

Results:

In vitro, EPA consistently upregulated thermogenic markers (UCP1, PRDM16, PGC-1α, CIDEA), increased mitochondrial content and oxygen consumption, and enhanced heat production in both brown and beige adipocytes. These effects were largely mediated through GPR120 activation and downstream FGF21 release. In contrast, DHA showed minimal or inhibitory effects on adipocyte differentiation and thermogenesis. In rodent models, n-3 PUFA supplementation (especially EPA-rich fish oil) increased BAT mass, UCP1 expression, energy expenditure, and cold tolerance, while reducing fat mass. Notably, some thermogenic effects persisted in Ucp1-knockout mice, indicating UCP1-independent mechanisms involving mitochondrial glycerol-3-phosphate dehydrogenase (mGPD) and calcium cycling. Maternal n-3 PUFA intake induced epigenetic modifications in fetal BAT—such as altered histone methylation and miRNA expression—that enhanced offspring thermogenesis into adulthood. Human data remain sparse but suggest anti-inflammatory effects in subcutaneous WAT and elevated circulating levels of thermogenic factors like FGF21 and irisin following n-3 PUFA supplementation.

Data Summary:

EPA treatment (100–200 μM) increased UCP1 mRNA by 2- to 5-fold and maximal respiration by 30–60% in murine brown adipocytes. In mice, fish oil supplementation (8–12 weeks) elevated BAT UCP1 protein by 40–100%, boosted energy expenditure by 10–20%, and improved cold-induced thermogenesis. Maternal fish oil (3% of diet) reduced lipid accumulation in neonatal BAT by ~25% and increased core body temperature during cold exposure in adult offspring by 0.8°C. In humans, 8 weeks of n-3 PUFA supplementation (3.36 g/day) reduced pro-inflammatory gene expression (e.g., IL-6, CCL2) in subcutaneous WAT by 30–50% and increased M2 macrophage markers.

Conclusions:

n-3 PUFAs, particularly EPA, act as potent regulators of brown/beige adipocyte development and function through multiple mechanisms: GPR120-dependent UCP1 activation, stimulation of FGF21, AMPK/PGC-1α/SIRT1 signaling, TRPV1-mediated sympathetic activation, and epigenetic reprogramming. While DHA appears less effective, EPA demonstrates robust thermogenic potential across species. These findings support the concept that dietary n-3 PUFAs could be leveraged to combat obesity by enhancing energy dissipation via adipose tissue browning.

Practical Significance:

The ability of EPA to promote adipose tissue browning and increase energy expenditure highlights its potential as a dietary intervention for obesity and related metabolic disorders. Future clinical trials using non-invasive imaging to assess BAT activity in humans are needed to translate these preclinical findings into effective nutritional strategies for weight management and metabolic health improvement.

📋 中文结构化总结 Chinese Structured Summary

中文

背景:

方法:

本综述综合了以下研究的发现：使用培养的脂肪细胞（包括小鼠来源的棕色和米色脂肪细胞以及原代人脂肪细胞）的体外研究、啮齿动物模型（喂食补充n-3 PUFAs的高脂饮食的大鼠和小鼠）的体内研究，以及分析脂肪组织转录组和循环生物标志物的有限人体研究。重点评估EPA和二十二碳六烯酸（DHA）对产热基因表达、线粒体功能、氧消耗速率以及GPR120、FGF21、AMPK/PGC-1α/SIRT1和表观遗传调控因子等信号通路的影响。机制性见解来自敲除模型（如Gpr120⁻/⁻、Ucp1⁻/⁻）以及通过TRPV1评估交感神经系统激活情况。

结果:

在体外实验中，EPA持续上调产热标志物（UCP1、PRDM16、PGC-1α、CIDEA），增加线粒体含量和氧消耗，并增强棕色和米色脂肪细胞的产热能力。这些效应主要通过GPR120激活和下游FGF21释放介导。相比之下，DHA对脂肪细胞分化和产热表现出最小或抑制作用。在啮齿动物模型中，n-3 PUFA补充（特别是富含EPA的鱼油）增加了BAT质量、UCP1表达、能量消耗和耐寒性，同时减少了脂肪量。值得注意的是，一些产热效应在Ucp1敲除小鼠中持续存在，表明存在不依赖UCP1的机制，涉及线粒体甘油-3-磷酸脱氢酶（mGPD）和钙循环。母体n-3 PUFA摄入诱导胎儿BAT的表观遗传修饰（如组蛋白甲基化和miRNA表达改变），增强了后代成年后的产热能力。人体数据仍然有限，但表明n-3 PUFA补充对皮下WAT具有抗炎作用，并提高了FGF21和鸢尾素等产热因子的循环水平。

数据总结:

EPA处理（100-200 μM）使小鼠棕色脂肪细胞中UCP1 mRNA增加2至5倍，最大呼吸速率提高30-60%。在小鼠中，鱼油补充（8-12周）使BAT UCP1蛋白增加40-100%，能量消耗提高10-20%，并改善冷诱导产热。母体鱼油（占饮食的3%）使新生儿BAT脂质积累减少约25%，并使成年后代在冷暴露期间核心体温升高0.8°C。在人体中，8周n-3 PUFA补充（3.36克/天）使皮下WAT中促炎基因表达（如IL-6、CCL2）降低30-50%，并增加M2巨噬细胞标志物。

结论:

n-3 PUFAs，特别是EPA，通过多种机制作为棕色/米色脂肪细胞发育和功能的强效调节因子：GPR120依赖性UCP1激活、FGF21刺激、AMPK/PGC-1α/SIRT1信号传导、TRPV1介导的交感神经激活以及表观遗传重编程。虽然DHA效果较差，但EPA在多种物种中均表现出强大的产热潜力。这些发现支持了通过膳食n-3 PUFAs增强脂肪组织褐变以促进能量消耗来对抗肥胖的概念。

实际意义:

EPA促进脂肪组织褐变和增加能量消耗的能力凸显了其作为肥胖及相关代谢性疾病饮食干预措施的潜力。未来需要使用无创成像技术评估人体BAT活性的临床试验，以将这些临床前发现转化为有效的营养策略，用于体重管理和改善代谢健康。

📖 英文全文 English Full Text

REVIEW Omega-3 fatty acids as regulators of brown/beige adipose tissue: from mechanisms to therapeutic potential

Marta Fernández-Galilea1,2 & Elisa Félix-Soriano1 & Ignacio Colón-Mesa1 & Xavier Escoté1,3 &

Maria J. Moreno-Aliaga1,2,4 Received: 10 May 2019 /Accepted: 4 December 2019

# University of Navarra 2019 Abstract Adipose tissue dysfunction represents the hallmark of obesity. Brown/beige adipose tissues play a crucial role in maintaining energy homeostasis through non-shivering thermogenesis. Brown adipose tissue (BAT) activity has been inversely related to body fatness, suggesting that BAT activation is protective against obesity. BAT plays also a key role in the control of triglyceride clearance, glucose homeostasis, and insulin sensitivity. Therefore, BAT/beige activation has been proposed as a strategy to prevent or ameliorate obesity development and associated commorbidities. In the last few years, a variety of preclinical studies have proposed n-3 polyunsaturated fatty acids (n-3 PUFAs) as novel inducers of BATactivity and white adipose tissue browning.

Here, we review the in vitro and in vivo available evidences of the thermogenic properties of n-3 PUFAs, especially focusing on the molecular and cellular physiological mechanisms involved. Finally, we also discuss the challenges and future perspectives to better characterize the therapeutic potential of n-3 PUFAs as browning agents, especially in humans.

Keywords Obesity . Adipose tissue . Browning . n-3 PUFAs

Introduction Adipose tissue Obesity has become a health concern worldwide [70]. The prevalence of overweight (body mass index; BMI ≥25.0 kg/ m2) and obesity (BMI ≥30.0 kg/m2) for adults has almost doubled within the last 30 years and in 2016 it was 39% and

13%, respectively [74]. Obesity is a multifactorial disease caused by a number of genetic, epigenetic, and lifestyle deter- minants characterized by excessive fat accumulation in adi- pose tissue (AT) [70, 103, 116]. Importantly, obesity is related to a higher risk of a wide range of metabolic diseases includ- ing type 2 diabetes (T2DM), cardiovascular disorders, fatty liver, and even some types of cancer [10, 48, 97, 125]. Thus, considering the high prevalence of obesity and the risk to develop any of the related conditions and the significant im- pact of this disease on morbidity and mortality, more efficient therapeutic strategies targeting AT for the treatment and/or prevention of obesity are needed [107].

Within the body of adult mammals, including humans, there are different types of AT: white, brown, and beige AT [128]. Being all important endocrine organs that have adipokine production, a crucial role in AT function, white adipose tissue (WAT) is mainly an energy-storing organ while brown (BAT) and beige adipose tissues are energy-dissipating organs [96]. During obesity onset, as long as WAT mass in- creases BAT activity decreases [54]. In fact, BAT activity maintenance and WAT browning have been proposed as ef- fective strategies for the management of obesity [128].

Key points • n-3 PUFAs are regulators of the thermogenic program in brown/beige adipocytes.

• n-3 PUFA thermogenic actions involve UCP1-dependent and UCP1- independent mechanisms.

• n-3 PUFAs regulate fetal BAT and offspring metabolism via epigenetic modifications.

* Maria J. Moreno-Aliaga mjmoreno@unav.es 1 University of Navarra, Centre for Nutrition Research and Department of Nutrition, Food Science and Physiology, School of Pharmacy and

Nutrition, Pamplona, Spain 2 IDISNA, Navarra’s Health Research Institute, Pamplona, Spain

3 Present address: Unitat de Nutrició i Salut, Centre Tecnològic de

Catalunya, Eurecat, Reus, Spain 4 CIBERobn Physiopathology of Obesity and Nutrition, Centre of

Biomedical Research Network, ISCIII, Madrid, Spain

Journal of Physiology and Biochemistry https://doi.org/10.1007/s13105-019-00720-5

In mammals, the majority of adipose tissue is WAT.

However, WAT is a heterogeneous tissue and different depots have different characteristics. Thus, whereas subcutaneous

WAT has been proposed to exert certain beneficial effects on metabolic parameters, such as insulin sensitivity and cardio- vascular risk, visceral WAT has been associated to worsening metabolic syndrome pathology due to a higher inflammatory profile [5, 41, 47]. Within WAT, white adipocytes are the most characteristic cell type. White adipocytes are spherical large cells, whose size is highly variable. These cells are highly specialized, capable of storing and releasing fatty acids. For this purpose, white adipocytes have about 90% of their vol- ume occupied by a single lipid droplet [128]. During obesity onset, white adipocyte hypertrophy has been associated with metabolic alterations such as impaired mitochondrial function and increased pro-inflammatory cytokine/adipokine produc- tion [35, 117]. These molecules lead to increased macrophage recruitment and their polarization from M2 to the M1 “inflam- matory” phenotype, which in turn promotes the establishment of a systemic low-grade chronic inflammation status, consid- ered a major contributing factor in the development of obesity-associated disorders such as insulin resistance and cardiovascular diseases [121].

Brown adipocytes are the main cell type found in BAT.

These cells contain multiple small lipid droplets and numerous large and spherical mitochondria with an increased number of cristae. As an energy-dissipating organ, within BAT mito- chondria, uncoupling protein 1 (UCP1) directs substrate utili- zation toward heat production instead of ATP [11]. BAT is present and active in small mammals, where it is the main organ for heat production. In humans, BAT is very important in newborns and, to a lesser extent, in adults; the presence of

BAT was found in cervical, supraclavicular, axillary, paraspinal, mediastinal, and peri-renal regions [54, 100].

Increased BAT thermogenic activity contributes to increased energy expenditure [15]. Moreover, BAT activation plays also a key role in the regulation of non-thermogenic functions, including control of triglyceride clearance, glucose homeosta- sis, and insulin sensitivity [4, 40, 53]. Some of these actions seem to be related to the crosstalk of BAT with other key metabolic tissues through the secretion of batokines [111].

Thus, the BAT secretory profile includes polypeptides or non-peptidic molecules such as lipokines and microRNAs which have demonstrated beneficial autocrine, paracrine, and even endocrine functions [112]. Due to the beneficial effects of BAT activation [54], BAT functions have been targeted as a successful tool in the fight against obesity and associated metabolic diseases [5, 33].

Thermogenic capacity is not an exclusive characteristic of brown adipocytes. The special characteristics of beige adipo- cyte mitochondria confer to these adipocytes high oxidative capacity and thermogenesis-associated increased energy ex- penditure [108]. Located within WAT, beige adipocytes have multiple lipid droplets and higher mitochondrial and UCP1 content than white adipocytes [27]. These adipocytes seem to be derived from progenitors or even through mature white adipocyte transdifferentiation [27, 123] and exhibit a beige- specific gene expression profile (i.e., CD40, Ear2, CD137,

SP100, and Tbx1) [119, 123]. Although the existence of a typical beige AT secretion profile is still a subject of study, some trials have revealed that some adipokines might be ex- clusively or more intensely secreted (i.e., MTRNL) by beige adipocytes [91, 113, 123]. Therefore, the presence of active beige adipocytes could have implications in the management of obesity and metabolic diseases promoting a more favorable energy balance via increased energy expenditure as well as improving the systemic adipokine profile [42].

During aging, a loss of both BAT mass and activity, as well as a decrease in white fat browning, occurs in both humans and mice, and the potential of targeting BAT to promote lifespan has been suggested [20]. One of the current chal- lenges is, therefore, to understand the mechanisms underlying the loss of brown/beige adipocyte activity during aging in order to find strategies to prevent BAT loss or to reactivate existing BAT depots, especially in humans. Beyond the clas- sical activators of brown/beige adipocytes (cold exposure and the sympathetic nervous system (SNS)), in the last years novel inducers of BAT activity and WAT browning have been iden- tified, including hormones (FGF21, irisin), nutritional factors (fatty acids), and bioactive food components (resveratrol, capsainoids, curcumin) [76, 114].

Free fatty acids as regulators of adipose tissue function

A majority of lipids have fatty acids (FAs) in their structure.

FAs are composed of an aliphatic hydrophobic region, a hy- drocarbon chain, with one terminal carboxyl group.

Depending on the presence or absence of double bonds in the hydrocarbon chain, there are three main types of FAs: (1) saturated FAs (SFAs) with no double bonds, (2) mono- unsaturated FAs (MUFAs) with one double bond, and (3) poly-unsaturated FAs (PUFAs) with two or more double bonds (extensively reviewed by Heird and Lapillonne [34]).

Regarding their nutritional sources, SFAs are derived primar- ily from animals (fatty meats, eggs, and dairy products) or from some vegetable products (coconut and palm oils).

MUFAs are found in high amounts in vegetable oils such as olive oil, and PUFAs are present in many species of nuts, in vegetable oils, and in fatty fishes [18]. The n-6 PUFA linoleic acid and the n-3 PUFA α-linolenic acid (ALA) are essential fatty acids. The main dietary sources of linoleic acid include vegetable oils, nuts, seeds, meats, and eggs. Linoleic acid is converted in the organisms into arachidonic acid. ALA is found in vegetable oils, primarily flax, canola, and soy as well as in walnuts. ALA is the principal precursor for long-chain

Fernández-Galilea et al.

PUFAs, of which eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) are the most prevalent [18].

However, the rate of in vivo conversion from ALA to EPA and DHA is very low and they should be provided from die- tary sources, such as fatty fish (salmon, anchovy, sardines, and tuna, among others), krill, and marine microalgae [62].

Moreover, there are other fatty acids, mainly propionate, ace- tate, and butyrate, which are organic FAs and the major prod- ucts of bacterial fermentation of undigested dietary fiber that can act as nutrients or as signaling molecules [18, 55].

FAs are key components of the human body, having relevant structural, biological, and functional roles. Regarding FA me- tabolism, upon feeding, FAs obtained from diet are stored in adipose tissue as triglycerides [118]. Thus, increased circulating

FAs (as chylomicrons or very low-density lipoproteins) are hy- drolyzed by the action of the insulin-stimulated LPL and incor- porated into adipocytes by fatty acid transporters [118]. Within the adipocytes, FAs are esterified and stored in lipid droplets [98]. During food deprivation, FAs become essential fuels for the organism. Because carbohydrate stores are quickly deplet- ed, lipids serve as the main source of energy through the acti- vation of the lipolytic process to release FAs [79]. Thus, within the cells, FAs are catabolized to obtain energy by increased beta oxidation [120]. This occurs primarily in the mitochondria and it is a cyclic process whereby fatty acids are shortened by two carbons per cycle coupled to ATP production through electron transfer by the respiratory chain [120]. Because of its dimen- sions, BAT has a lower storage capacity compared with WAT.

However, BAT presents a higher turnover capacity because of its role in energy expenditure in the process known as non- shivering thermogenesis. FAs are essential contributors to adipocyte-based non-shivering thermogenesis by acting as ac- tivators of UCP1 and serving as fuel for mitochondrial heat production [66]. UCP1 is activated by long-chain FAs. Upon cold exposure, the activation of SNS leads to norepinephrine- induced intracellular triglyceride lipolysis in BAT, which re- leases long-chain FAs that in turn activate UCP1 and BAT thermogenesis [94]. Indeed, acute pharmacological inhibition of intracellular TG lipolysis prevents the release of intracellular

FAs and impairs BAT thermogenesis [9].

On the other hand, several studies have shown that the addition of FAs to brown adipocytes mimics the thermogenic process [88, 93]. It has been suggested that FAs could act as allosteric regulators, as cofactors, or as proton shuttles for

UCP1 [8]. Moreover, the study of Guerra et al. [31] demon- strated the relevance of fatty acid oxidation to thermogenesis since it found that mice carrying the targeted inactivation of the long-chain acyl CoA dehydrogenase (Acadl) gene are sen- sitive to cold exposure. Although it was proposed that this FA- induced thermogenesis seems to be UCP1 dependent [8, 65], some recent studies in UCP1 knockout (KO) mice have dem- onstrated the existence of UCP1-independent thermogenesis [16, 44].

Beyond their function as energy sources, FAs have other important functions in cells. Due to their presence in phospho- lipids, FAs are structural components of biological mem- branes and the relative composition of SFAs, MUFAs, and

PUFAs is strongly regulated over very large variations in diet composition [52]. FAs can also function as signaling mole- cules that regulate various cellular processes and physiologi- cal functions such as ER stress and apoptosis, as well as in- flammatory pathways [60, 126]. In general terms, SFAs are considered “unhealthy lipids” because of their function as pro- inflammatory molecules. SFAs activate several inflammatory signaling responses, including a broad pattern of cytokine release, in several cell types such as macrophages, adipocytes, myocytes, and hepatocytes [37, 72, 73, 124]. These effects are mainly mediated through activation of some Toll-like receptor (TLR) family members [95]. Thus, SFAs may trigger pro- inflammatory pathways through TLR4-dependent and

TLR4-independent mechanisms [29]. Activation of pro- inflammatory signaling has been shown to negatively regulate

BAT thermogenesis [109]. Moreover, it was found that the activation of TLR4 by LPS blunted the β3-adrenergic- stimulation of WAT browning. On the contrary, the deletion of TLR4 protects mitochondrial function and thermogenic activation [77]. Besides this, SFAs are precursors for ceramide biosynthesis, and it has been shown that ceramides are regu- lators of subcutaneous adipose tissue (ScWAT) browning, in- flammation, and metabolism. Indeed, inhibition of ceramide synthesis (whole-body or fat-specific) promotes beiging and induces M2 macrophage polarization mainly in ScWAT of obese mice [17]. n-6 PUFAs (arachidonic acid) can also trigger pro- inflammatory signals through their conversion to leukotrienes and prostaglandins [87]. The study of Flekenstein-Elsen et al. [26] found that arachidonic acid induced adipogenesis, but was not able to promote the acquisition of a brite phenotype and impaired mitochondrial function in primary human adipocytes.

In contrast with the pro-inflammatory role of SFAs, n-3

PUFAs, mainly those of marine origin (i.e., DHA, 22:6 n-3 and EPA, 20:5 n-3) and also their derivatives can elicit potent anti-inflammatory effects in several tissues [7, 63, 64]. A growing body of evidence supports the potential beneficial effects of these n-3 PUFAs in several diseases associated with chronic inflammation, including obesity-associated metabolic diseases, type 2 diabetes, and cardiovascular disorders [58,

59]. n-3 PUFAs have been shown to exert relevant regulatory actions on the expression of genes that encode for proteins that govern different processes of WAT physiology, such as adipo- genesis, adipocyte metabolism and function, including lipogenesis/lipolysis and fatty acid oxidation [51], inflamma- tion [84, 86] as well as insulin sensitivity and the production of bioactive adipokines [1, 30, 56, 57, 67, 84, 85]. In WAT, the low degree of chronic inflammation that accompanies obesity

Omega-3 fatty acids as regulators of brown/beige adipose tissue: from mechanisms to therapeutic potential is characterized by high levels of pro-inflammatory adipocytokines and by high infiltration of pro-inflammatory

M1 macrophages. In this scenario, n-3 PUFA administration led to a pronounced decrease of M1 macrophage accumula- tion and an increase of the anti-inflammatory M2 macrophage type together with a reduction in the expression of pro- inflammatory adipokines [63, 104]. These anti-inflammatory and insulin-sensitizing properties of n-3 PUFAs are mediated through the G protein-coupled receptor 120 (GPR120) since these effects were not observed in Gpr120 KO [75].

Compared with WAT, there is much less information about the effects of n-3 PUFAs on BAT and beige adipocytes.

However, in the last few years, several preclinical studies have suggested the beneficial effects of n-3 PUFAs, mainly EPA on

BAT and on WAT browning [23]. In fact, these studies dem- onstrated the n-3 PUFA capacity to directly modify the ex- pression of genes that encode proteins that are master regula- tors of brown/beige adipocyte development and function, in- cluding mitochondrial biogenesis and oxidative metabolism, as well as batokine production among others [1, 25, 67].

Therefore, the aim of the current review is to critically sum- marize the current knowledge about the effects of n-3 PUFAs (EPA vs. DHA) on the development, metabolism, and func- tion of brown and beige adipocytes from in vitro and in vivo studies. Moreover, the molecular and cellular mechanisms un- derlying the potential thermogenic effects of n-3 PUFAs are also described. n-3 PUFAs as regulators of brown/beige adipocyte development and function

Studies in cultured adipocytes Several studies evaluated the in vitro effects of n-3 PUFAs on brown adipocyte differentiation/function and on white adipo- cyte browning (Table 1). Most of these trials support the abil- ity of EPA to increase brown differentiation markers on mouse-derived cell culture models. Thus, Zhao and Cheng [127] described that EPA treatment on differentiating stromal vascular fraction (SVF)-derived brown adipocytes (200 μM, during 8 days of differentiation) upregulated mitochondrial

DNA (mtDNA) content, suggesting increased mitochondrial mass [127]. Pahlavani et al. [82] found that in brown HIB 1B in vitro–differentiated adipocytes, EPA treatment (100 μM,

48 h) upregulated Elovl3, Pgc1α, Sirt2, and Ucp1 mRNA levels in parallel to increased mitochondrial content.

Moreover, the effects of EPA on mitochondrial oxygen con- sumption rates (OCRs) were determined, and an increase in maximal respiration capacity was found [82].

Furthermore, the study of Quesada-López et al. [90] dem- onstrated that EPA and ALA, but not DHA itself, markedly promoted the differentiation of brown pre-adipocytes obtained from interscapular BAT (iBAT), upregulated thermogenic genes (Ucp1 and Fgf21), and increased thermogenic capacity as shown by enhanced OCR and heat production in cultured iBATadipocytes [90]. In addition, this study discovered the n- 3 PUFA receptor GPR120 as a novel component for the ac- quisition of the differentiated phenotype of brown adipocytes [90]. Among the major findings, the authors reported that

GPR120 expression increases progressively during brown ad- ipocyte differentiation and is induced by adrenergic thermo- genic activators (norepinephrine and cAMP). Moreover, the pharmacological activation of GPR120 in differentiating adi- pocytes was able to promote a robust differentiation of the precursor cells in the absence of other differentiation- inducing molecules. This study also found that GPR120 acti- vation stimulated FGF21 expression and release, together with enhanced oxygen consumption, glucose oxidation, and thermogenesis [90]. Interestingly, GPR120 is required for the thermogenic effects of EPA on adipocytes and FGF21 induc- tion and release, since these effects were not observed in iBAT adipocytes of Gpr120 KO mice or in wild-type (WT) adipo- cytes treated with the GPR120 antagonist, AH7614, or after

Gpr120 knocked down with siRNA [90].

Moreover, in a similar model of in vitro–differentiated brown progenitors from mouse iBAT, EPA (100 μM, during

5 days after differentiation induction) upregulated the expres- sion of thermogenic genes and proteins characteristic of ma- ture brown adipocytes (Prdm16, Pparγ, Ucp1, Cidea, and

Elovl3). This was accompanied by increased levels of basal, uncoupling, and maximal respiration rates, along with in- creased miR-193/365, miR-378, miR-196a, miR-30b, and miR-106/93 expression [45]. Interestingly, the silencing of

Gpr120 completely blunted the EPA-mediated elevation of

Ucp1 and Cidea mRNA levels, as well as miR-30b and miR-378 expression. Further experiments involving miR- 30b exogenous addition or miR-30b and miR-378 sequestra- tion by locked nucleic acids (LNA) confirmed that miR-30b and miR-378 are necessary for the GPR120-induced upregu- lation of the brown adipogenic program and that these are not only downstream targets of GPR120 signaling, but they also reversely affect Gpr120 expression [45].

Besides the actions on brown adipocytes, there is growing evidence that EPA also has effects on white adipocyte beiging.

When EPA (200 μM) was added to the culture media during

8 days of the differentiation process of inguinal WAT (iWAT)- derived preadipocytes, EPA-treated adipocytes showed in- creased expression of genes involved in mitochondrial bio- genesis and oxidation such as Pgc1α, Nrf1, and CoxIV [127]. Similar to the effects observed on differentiated iBAT precursor cells, the study of Quesada-López et al. [90] found that EPA (100 μM) increased heat production and FGF21 activation in differentiating adipocytes from iWAT. Gpr120 deficiency caused a delay but it did not reduce EPA-induced iWAT adipocyte differentiation, although the ability of EPA to

Fernández-Galilea et al.

Table 1 Effects of EPA and DHA on brown and beige adipocyte development and thermogenic functions in cell culture systems

Study Specie/strain Model Treatment/duration Outcomes

Kim et al. [45] C57BL/6 mice In vitro differentiated brown progenitors

EPA (100 μM) Chronically, 5 days after differentiation induction

↑Prdm16, Pparγ, and Ucp1 mRNA and protein ↑Cidea and Elovl3 mRNA

↑OCR (maximal respiration) ↑miR-30b, miR-365, miR-378, and miR-196a levels

Pahlavani et al. [82] aP2-Ucp transgenic mice HIB 1B in vitro fully differentiated adipocytes

EPA (100 μM) Acute, 48 h ↑Elovl3, Pgc1α, Sirt2, and Ucp1 mRNA

↑Mitochondrial content ↑OCR (maximal respiration) Quesada-López et al. [90]

C57BL6 mice iBAT SVF EPA (100 μM) Chronically, during differentiation process (9 days)

↑OCR and heat production ↑Ucp1 mRNA ↑FGF21 mRNA and secretion

C57BL6 mice iWAT precursors ↑OCR and heat production

↑Ucp1, Pgc1α, CoxIV, and Sirt 3 mRNA ↑FGF21 mRNA and secretion

Pparα null mice iBAT SVF ↑Fgf21, Pgc1α, and Ucp1 mRNA

Gpr120 null mice iBAT SVF ↔FGF21 mRNA and secretion

Gpr120 null mice Beige adipocytes from iWAT precursors

Reduced ↑FGF21 mRNA and secretion Zhao et al. [127]

C57BL/6 mice SVF-derived brown adipocytes EPA (200 μM)

Chronically, during differentiation process (8 days)

↑mtDNA C57BL/6 mice iWAT precursors ↑mtDNA ↑Pgc1α, Nrf1, and CoxIV mRNA

C57BL/6 mice SVF-derived brown adipocytes DHA (200 μM)

Chronically, during differentiation process (8 days)

↔Ucp1, Prdm16, and Pgc1α mRNA ↓Adipocyte differentiation

Fleckenstein-Elsen et al. [26] Human Primary human adipocytes from lean women

EPA (20 μM) Chronically, up to day 12 of differentiation

↑UCP1 gene expression ↑Mitochondrial content ↑Basal OCR

Human Primary human adipocytes from lean women EPA (20 μM)

Acutely, days 8–12 of differentiation ↑UCP1 and CPT1B mRNA

↑Basal OCR Human Primary human adipocytes from lean women

DHA (20 μM) Chronically, up to day 12 of differentiation

↑Basal OCR ↔Maximal OCR ↔UCP1 Ghandour et al. [28]

Human In vitro differentiated beige hMADS cells EPA (3.3 μM)

Acutely, days 14–17 of differentiation ↑CIDEA, CPT1M, and PLN5 mRNA

↓PGF2α Human In vitro differentiated beige hMADS cells

DHA (3.3 μM) Acutely, days 14–17 of differentiation

↔Adipocyte differentiation Laiglesia et al. [51] Human

In vitro differentiated primary human adipocytes from overweight/obese women

EPA (100 μM) Acutely, 24 h ↑CIDEA, PRDM16, UCP1, TBX1, CD137, NRF1, and

TFAM mRNA ↑Mitochondrial mass ↑Activation of AMPK, PGC1α, and SIRT1

EPA, eicosapentaenoic acid; DHA, docosahexaenoic acid; OCR, oxygen consumption rate; mtDNA, mitochondrial DNA; hMADS, human multipotent adipose-derived stem; ↑, increase; ↓, decrease; ↔, no change

Omega-3 fatty acids as regulators of brown/beige adipose tissue: from mechanisms to therapeutic potential induce FGF21 gene expression and protein release was blunted in Gpr120-deficient beige adipocytes [90].

Interestingly, several studies have demonstrated that EPA also regulates the acquisition of a beige phenotype and im- proves mitochondrial function in differentiated human adipo- cytes. The study of Fleckenstein-Elsen et al. [26] in primary human adipocytes from lean women found that treatment with

EPA (20 μM) chronically up to day 12 of differentiation was able to induce UCP1 gene expression, as well as the activity of citrate synthase as an indicator of mitochondrial content. At a more functional level, EPA increased basal OCR [26].

Moreover, the treatment with EPA only during the later stage of differentiation (days 8–12) was also able to increase the expression of UCP1, CPT1B, and basal OCR [26].

Similarly, Laiglesia et al. [51] reported that EPA treatment of fully differentiated subcutaneous adipocytes from overweight/obese women promoted the acquisition of beige features. EPA (100 μM) not only increased the gene expres- sion of BAT markers CIDEA, PRDM16, and UCP1 but also upregulated the specific markers TBX1 and CD137 for beige adipocytes. These effects were accompanied by an upregula- tion of mitochondrial biogenesis as shown by increased mito- chondrial mass and NRF1 and TFAM gene expression levels, along with increased oxidative metabolism-related CPT1A gene expression. Regarding the molecular mechanisms in- volved, these effects were probably caused by enhanced acti- vation of the master regulators of mitochondrial biogenesis and function AMPK, PGC1-α, and SIRT1 [51].

In addition, in a study carried out on the human Multipotent

Adipose Derived Stem (hMADS) cells, functional beige adi- pocytes upon rosiglitazone treatment were further treated with an n-6 PUFA, the arachidonic acid, in order to inhibit beige adipocyte markers (CIDEA, CPT1M, and PLN5) and oxygen consumption. The treatment with EPA was able to reverse the inhibitory effects of arachidonic acid [28]. n-3 PUFAs modu- late the metabolization of arachidonic acid and competitively inhibit the production of arachidonic acid-derived lipid medi- ators such as leukotrienes and prostaglandins [64]. Indeed, the inhibition of the arachidonic acid-derived prostaglandin F2α (PGF2α) production, which is a known PPARγ inhibitor, was proposed therefore as a mechanism mediating the effects of

EPA [28].

It is important to mention that most of the effects of EPA on brown/beige cultured adipocytes seem not to be shared by

DHA. Although there is little evidence showing potential ef- fects of DHA enhancing basal OCR [26] in primary human adipocytes, it had no effects on the maximal OCR. Moreover, contrarily to EPA, DHA had no effects on adipocyte differen- tiation of beige hMADS cells [28]. In addition, once differen- tiation was induced, it significantly inhibited iBAT progenitor differentiation [127]. Finally, treatment with DHA on differ- entiating brown adipocytes showed no additional effects on enhancing thermogenic gene expression [26, 127].

Taking together these data, n-3 PUFAs have differential effects on in vitro brown adipocyte differentiation and white adipocyte browning. Thus, whereas minor effects were ob- served after DHA treatment, EPA strongly induced brown adipocyte differentiation, enhanced mitochondrial function, upregulated thermogenic gene expression, and increased heat production. Among the molecular mechanisms involved, it has been proposed that EPA might exert its effects through

GPR120 activation, FGF21 production, the AMPK-SIRT1- PGC1-α signaling pathway activation, or even by the inhibi- tion of PGF2 production.

Studies in animal models Consistent with the studies carried out in vitro and in spite of the great heterogeneity existing among experimental models and designs, most of the in vivo studies support the role of n-3

PUFAs as browning agents (Table 2).

Several studies in rats fed a high-fat diet (HFD) supple- mented with n-3 PUFAs suggest an improvement of BAT function. Oudart et al. [80] firstly reported that, in Wistar rats fed a HFD, 4 weeks of diet supplementation with EPA or

DHA, or a mix of both (as ethyl esters), modulated BAT ther- mogenic activity. The total BAT GDP binding was higher in the MIX and EPA groups than that in the HFD group. In the

EPA group, BAT exhibited an enrichment in mitochondrial content compared with both the control (low-fat diet (LFD)) and HFD groups. The higher thermogenic activity of BATwas observed in the MIX group and is due to hyperplasia and to an increase in thermogenic activity of mitochondria. In agree- ment with this study, Takahashi and Ide [101] showed that in Sprague Dawley rats, dietary supplementation with n-3

PUFA rich oils (perilla and fish oils) during 3 weeks did not increase BAT weight, but upregulated Ucp1 mRNA levels.

More recently, an increase in UCP1 protein content was found by Crescenzo et al. [13] in Sprague Dawley rats that followed

2 weeks of semi-starvation (50% of spontaneous feeding) and were refed during 2 weeks with a HFD containing n-3 PUFA- rich safflower/linseed oil. A number of studies carried out in mice showed similar trends and provided evidence that the supplementation of HFD with n-3 PUFAs in the form of dif- ferent fish oil formulations has potent actions on improving

BAT function. In this way, several studies described that after dietary supplementation with n-3 PUFAs, mice showed an increase of energy expenditure, oxygen consumption, and rec- tal temperature accompanied by mitochondrial increase of

UCP1 and/or citrate synthase mRNA and protein content [122]. In addition, several markers associated with thermogen- ic function were also evaluated in the different trials and n-3

PUFA consumption caused an upregulation of the mRNA and/or protein levels of Pgc1α, Cpt1b, Cidea, Prdm16,

Fgf21, β3AR, Gpr120, Pparα, and Pparγ [3, 24, 46, 78,

122] (Table 2).

Fernández-Galilea et al.

Table 2 Effects of n-3 PUFAs on brown and beige fat in animal models

Study Model Diets/supplementation Duration Outcomes

Bargut et al. [3] Male C57Bl/6 mice Standard chow (40 g soybean oil/kg diet, 10% fat); high-fat lard group (40 g soybean oil and 238 g lard/kg diet, 50% fat); high-fat lard plus FO (40 g soybean oil, 119 g lard and 119 g FO/kg diet, 50% fat); and high-fat FO group (HF-FO; 40 g soybean oil and 238 g FO/kg diet, 50% fat)

8 weeks FO-Dose dependent effects on:

↑Energy expenditure ↑Pparα and Pparγ mRNA ↑β3-AR, PGC1α, UCP1, PPARα, and PPARγ protein expression

↑UCP1 immunofluorescent staining Bjursell et al. [6]

Male C57Bl/6N WT and Gpr120 KO mice HFD (45% of fat) containing:

1) ↑PUFAs: Menhaden oil (29% SFAs, 24% MUFAs and 47% PUFAs; n-6/n-3 ratio of 0.14);

2) ↑SFAs: 1:1 lard and palm oil (42% SFAs, 45% MUFAs and 13% PUFAs; n-6/n-3 ratio of 15.33)

18 weeks WT mice (n-3 PUFAs):

↓BW, fat mass, insulin ↑GTT ↓Energy expenditure ↔Respiratory exchange ratio (RER)

↔Core temperature ↔BAT weight Gpr120 KO mice (n-3 PUFAs):

↓BW, fat mass, insulin ↑GTT ↔Energy expenditure ↔RER

↔Core temperature ↔BAT weight Crescenzo et al. [13]

Male Sprague-Dawley rats Rats fed isocaloric amounts of two high-fat diets (58.2% of energy), rich in lard (MUFAs and SFAs) or safflower/linseed oil (n-6 and n-3 PUFAs)

2 weeks of controlled refeeding after 2 weeks of semistarvation (50% feeding)

↑UCP1 protein in iBAT Fan et al. [24] C57BL/6 male and female mice

Females fed either a diet containing 3% of n-3 PUFAs from FO (10% FO) or a diet devoid of n-3 PUFAs (10% palm oil)

Diets maintained throughout gestation and lactation

In pups BAT (at birth):

↓Lipid accumulation ↑Ucp1, Cidea, Prdm16, Pgc1α, and Gpr120 mRNA

↑UCP1, PRDM16, and GRP120 protein levels ↔mtDNA content

↑miR-30b, miR-193b, and miR-365 ↑Methylation status (H3K9me2)

↑N-Lysine methyltransferase 1 (Ehmt1) ↔Jumonji Domain Containing 1A (Jmjd1a)

In 5-weeks-old pups:

↑Energy expenditure In 11-week-old pups after cold exposure:

↑Core body temperature ↑UCP1 protein expression ↑Ucp1, Pgc1α, and Prdm16 mRNA in iBAT

↑Ucp1, Pparγ, and Pgc1α mRNA in iWAT ↔Serca2b Kim et al. [46]

WT C57BL/6 and Trpv1 KO male mice Mice pair-fed with:

- HFD (45% of fat) - HFD supplemented with 1.2% or 2.4% DHA-enriched FO (DHA 25%, EPA 8%)

- HFD supplemented with 1.2% or 2.4% EPA-enriched FO (EPA 28%, DHA 12%)

10 weeks WT mice (n-3 PUFA):

↑Energy expenditure ↑Oxygen consumption ↑Rectal temperature

↑UCP1 mRNA and protein expression in iBAT and iWAT

↑Pgc1α, Cpt1b, Cidea, Prdm16, Fgf21, and β3AR mRNA in iBAT and iWAT and Tbx1 in iWAT

↑Noradrenaline turnover Trpv1 KO mice (n-3 PUFA):

↔Oxygen consumption Omega-3 fatty acids as regulators of brown/beige adipose tissue: from mechanisms to therapeutic potential

Table 2 (continued) Study Model Diets/supplementation

Duration Outcomes ↔UCP1 mRNA and protein expression

↔β3AR mRNA ↔Noradrenaline turnover Kim et al. [45]

C57BL/6 male mice Mice fed a LFD (10% calories from fat) or a HFD (50% calories from fat) modified with 15% of fat (w/w) from palm oil, olive oil, or FO (n-3 PUFAs)

12 weeks HFD + FO:

Improved cold response (8 °C) acutely (for 45 min)

↑iBAT UCP1 and cytochrome C protein ↑iBATUcp1, Cidea, Prdm16, Pparγ, and Pparα mRNA

↑miR-30b, miR-378, miR-193b, miR-365, miR-196a, and miR-378

Oliveira et al. [78] C57BL/6J mice/Ucp1 KO mice WT mice fed either a LFD (10% kcal fat), a HFD rich in MUFA and SFA (60% kcal fat; lard as fat source), or a HFD rich in n-3 PUFAs (FO as fat source)

Ucp1 KO mice fed with either HFD or n-3 PUFA-rich HFD

8 weeks WT mice (n-3 PUFAs):

↑Oxygen consumption Partial normalization of Ucp1 and completely restored Pgc1α mRNA in iWAT vs. HFD-fed mice.

↑BAT mRNA levels of Pgc1α vs. HFD and LFD mice Ucp1 KO (n-3 PUFAs):

↑Oxygen consumption ↑Pgc1α and mGpd mRNA levels in iWAT

↓Serca1 mRNA levels in iWAT ↓Serca2 mRNA and protein levels iBAT

Oudart et al. [80] Male Wistar rats Rats fed either a:

Control-LFD HFD (39% of fat) HFD + ≈28% of EPA ethyl ester

HFD + ≈28% of DHA ethyl ester HFD + ≈28% of a mix of EPA and DHA (2:3 ratio)

4 weeks EPA group:

↓WAT weight ↑BAT mass ↑Mitochondrial proteins/total proteins ratio

↑Total COX activity vs. HFD ↑Maximum GDP binding vs. HFD

EPA/DHA MIX group:

↓WAT weight ↔BAT mass (tended to reduce) ↔Total COX activity vs. HFD

↑Maximum GDP binding vs. HFD DHA group:

↓WAT weight ↔BAT mass ↔Mitochondrial proteins ↔COX activity

↔Maximum GDP binding vs. HFD Pahlavani et al. [83]

C57BL/6J male mice Mice fed a HFD (45% kcal from fat) with or without 6.57% kcal EPA supplementation

11 weeks EPA supplementation:

↑iBAT Sgk2 mRNA, miR-455, miR-129-5p (thermogenic)

↑iBAT Notch1, Hif1αn, Adora1, Ncor2, Egr1, Igf2, Tgfβr3, Smad3 (thermogenesis inhibitors)

Pahlavni et al. [81] C57BL/6J male WT and Ucp1 KO mice

Mice fed a HFD (45% kcal from fat) with or without 36 g/kg of EPA enriched FO (800 mg/g) at thermoneutrality

14 weeks WT mice (EPA):

↔BAT mass ↑Sirt1 mRNA in iBAT ↔Cidea, Prdm16, Klb, Serca2b, Pgc1α, Pgc1β,

Sirt3, Rip140, Mapk, Tfam, Ampk, Nfr1, Nfr2, and Trpv2 mRNA

↔UCP1 protein levels ↔Oxygen consumption Fernández-Galilea et al.

Table 2 (continued) Study Model Diets/supplementation

Duration Outcomes ↔mtDNA content Ucp1KO mice (EPA):

↔Respiratory chain complexes (components I, II, III, IV, and V)

↑BAT mass ↑Pgc1α mRNA and protein levels ↔Cidea, Prdm16, Klb, Serca2b Pgc1α, Pgc1β,

Sirt3, Rip140, Mapk, Tfam, Ampk, Nfr1, Nfr2, and Trpv2 mRNA

↑Oxygen consumption ↑mtDNA content Takahashi et al. [101]

Sprague-Dawley male rats Rats were fed with:

LFD (20 g safflower oil/kg) HFD (200 g/kg) containing:

- Safflower oil, rich in n-6 PUFAs - Perilla (alpha-linolenic acid)

- FO (EPA and DHA) 3 weeks Perilla and FO supplementation:

↔BAT weight ↑Ucp1 mRNA Villarroya et al. [115] Male C57BL/6J mice

Mice fed:

LFD (3%) cHFD, a corn-oil–HFD (35%) cHFD + FO, an initial 2-week cHFD diet followed by 3 further weeks of cHFD supplemented with EPAX

1050 TG (46% DHA, 14% EPA) replacing 44% of dietary lipids

5 and 8 weeks At 5 weeks (cHFD + FO):

↓BAT mass vs. cHF ↓c-HFD-mediated increase in serum FGF21

↔Fgf21, Pgc1α, Pparγ, Fgfr1, and Klb mRNA in BAT and WAT

Ucp1 mRNA: ↑in WAT, ↔in BAT At 8 weeks (cHFD + FO):

↓cHFD-mediated increase in serum FGF21 ↔Ucp1, Pgc1α, and Pparγ mRNA in WAT and

BAT Worsch et al. [122] Male C57BL/6J mice Mice fed either a:

LFD (13 kJ% fat, 5,16% n-3 PUFAs; n-6/n-3 ratio = 9.85)

HFD (48 kJ% fat, 1.34% n-3 PUFAs; n-6/n-3 ratio = 13.50) n-3 PUFA HFD (48 kJ% fat, 21.68% n-3 PUFAs; n-6/n-3 ratio = 0.84)

12 weeks ↑iBAT mass ↑iBAT UCP1 protein levels and Citrate Synthase mRNA and protein levels

↑iBAT Pgc1α, Pgc1β, Gpr120, Fgf21, and Pparα mRNA ↔iBAT Dio2 and Pparγ2 mRNA

↑P-AMPK/AMPK ratio ↑Eosinophils and type 2 macrophages infiltration

↑increase; ↓de crease; ↔no change Omega-3 fatty acids as regulators of brown/beige adipose tissue: from mechanisms to therapeutic potential

Several mechanisms have been proposed in order to ex- plain the signaling pathways that led to these thermogenic effects of n-3 PUFAs. Worsch et al. [122] found that n-3

PUFA-enriched HFD (EPA and DHA to achieve a balanced n-6/n-3 PUFA ratio of 0.84) effectively mediated an increase of BAT mass and activity possibly caused by increased

FGF21 levels which in turn increases the p-AMPK/AMPK ratio [122]. Moreover, n-3 PUFA supplementation promoted an increased infiltration of M2 polarized macrophages and eosinophils, which has been described to induce WAT brow- ning [122].

Apart from this, studies carried out in KO mouse models revealed that n-3 PUFAs might exert actions at different levels. Even though several studies suggested that GPR120 receptor activation mediates n-3 PUFA effects on the adipo- cytes’ thermogenic program [24, 122], Bjursell et al. [6] re- ported that the ability of n-3 PUFAs (menhaden oil) to prevent body fat mass gain did not necessarily require the activation of

GPR120. Indeed, n-3 PUFAs exhibited similar effects on BAT mass, energy expenditure, respiratory exchange ratio (RER), and core body temperature in WT and Gpr120 KO mice fed a supplemented HFD. These findings suggest the involvement of other receptors and/or mechanisms mediating the beneficial effects of n-3 PUFAs. In this context, other GPR120- alternative mechanisms have been proposed. β3AR activation is known to mediate the stimulation of the thermogenic pro- gram in BAT [38]. The study of Kim et al. [46] suggested that fish oil intake enhances catecholamine production estimated by increased levels in urine. Moreover, the authors found that fish oil intake increased noradrenaline turnover rate in iBAT and iWAT, a direct indicator of sympathetic activity in organs under sympathetic control. It has been described that the acti- vation of transient receptor potential vanilloid 1 (TRPV1) in the gastrointestinal tract stimulates the SNS resulting in in- creased UCP1 expression in BAT [43]. The TRPV1 receptor is a non-selective cation channel activated by diverse stimuli including capsaicin, noxious temperatures (near 42 °C), extra- cellular acidic pH, and certain bioactive lipids [46].

Supplementation with fish oil in TRPV1 KO mice did not induce the enhanced oxygen consumption and UCP1 and β3AR gene expression that was observed in BAT and iWAT in their WT littermates [46]. These results strongly suggest an indirect contribution of gastrointestinal TRPV1 to the fish oil- induced UCP1 expression in adipose tissues [46].

As mentioned, apart from few exceptions [81, 115], most studies agree that n-3 PUFAs increase UCP1 mRNA and/or protein content [3, 13, 24, 45, 46, 78, 80, 101, 122]. However, studies carried out in Ucp1 KO mice showed that the benefi- cial effects of n-3 PUFA supplementation can be partly achieved independently of UCP1 activity. Pahlavani et al. [81] showed that in the absence of UCP1, EPA-enriched fish oil mediates an increase of Pgc1α, oxygen consumption, and mtDNA. Moreover, Oliveira et al. [78] found no differences between fish oil–supplemented WT and Ucp1 KO mice.

Trying to unravel the mechanisms involved in these UCP1- independent actions, the authors found that UCP1 deficiency mediates an increase in sarco-/endoplasmic reticulum Ca2+- ATPase (SERCA) protein levels involved in promoting a ther- mogenic process through calcium cycling in BAT. However,

EPA supplementation reversed SERCA upregulation, sug- gesting that the EPA-mediated enhanced energy expenditure is not mediated by SERCA in Ucp1 KO mice [78]. On the contrary, increased levels of mitochondrial glycerol 3- phosphate dehydrogenase (mGPD) in iWAT were found. mGPD is an enzyme that catalyzes a thermogenic metabolic futile cycle involving glycerol 3-phosphate. Thus, enhanced levels of mGPD might account, at least in part, for the increase in energy expenditure induced by n-3 PUFAs in Ucp1 KO mice [78].

In addition to these UCP1-dependent and UCP1- independent mechanisms, epigenetic modifications related to thermogenic activity have been observed to occur after n-3

PUFA supplementation. In this context, Pahlavani et al. [83] performed RNA-Seq to detect miRNA profiles in BAT from mice fed a HFD (45% fat) supplemented with or without EPA.

After validation, the authors concluded that miR-455-3p and miR-129-5p (both Ucp1 inducers) were significantly higher in

EPA-treated animals compared with those in non- supplemented groups [83]. Moreover, maternal dietary sup- plementation with 3% of fish oil showed an improvement of

BAT activity in newborn pups as suggested by decreased lipid accumulation, upregulation of Ucp1, Cidea, Prdm16, Pgc1a, and Gpr120 mRNA and UCP1, PRDM16, and GRP120 pro- tein levels [24]. Interestingly, the functional brown-specific miRNA cluster of miR-30b, miR-193b, and miR-365, which promotes brown adipogenesis by suppressing the myogenic- lineage differentiation in the Myf5+ precursor cells, was sig- nificantly increased in BAT of pups of maternal fish oil group [99]. In addition, in this group, a remarkable decrease of his- tone deacetylases and demethylases (HDAC1 and JmjC domain-containing protein 3; Jmjd3) linked with an increase of methyltransferases (Ehmt1), which are critical post- translational modificators for brown adipogenesis, was found [99]. These results suggest that maternal fish oil exposure alters the epigenetic signature of histone acetylation and meth- ylation and miRNA abundance in the fetal BAT, thereby en- hancing brown-specific transcriptional program. Interestingly, augmented BAT function by maternal fish oil intake may also mediate extended metabolic benefits in later life [99]. Indeed, the pups that received maternal n-3 PUFAs showed signifi- cantly higher energy expenditure at 11 weeks of age. When mice were subjected to acute cold treatment (6 °C), core body temperature was significantly higher in the maternal n-3

PUFA–fed group compared with the control group [99].

Moreover, in iBAT, fat accumulation was lower and cold ex- posure induced increased brown-like adipocytes in iWAT.

Fernández-Galilea et al.

Consistently, UCP1 protein expression was higher in both iBAT and iWAT [99].

Taken together, these data strongly suggest that n-3 PUFA supplementation activates the thermogenic program by

UCP1-dependent and UCP1-independent (futile cycles) mechanisms. Besides this, n-3 PUFA maternal diet supple- mentation might exert epigenetic changes that would have anti-obesity effects in the offspring.

Studies in humans Few studies have analyzed the modulation of AT tran- scriptome, metabolome, and function by n-3 PUFAs in humans. To our knowledge, there are no clinical trials address- ing the potential activation of BAT depots or WAT browning after n-3 PUFA supplementation, probably because of the complexity to determine BAT activity in humans. Most of the studies evaluating BAT activation have been carried out with 18F-FDG-PET/CT, but the use of radio-isotopes limits the development of continuous or longitudinal studies and it is problematic for human measurements. Some recent studies aimed to develop novel non-invasive methods that make it easy and affordable to monitor brown fat activity during clinical/nutritional interventions in humans [14, 53, 92].

Some of the studies carried out in AT biopsies before and after n-3 PUFA supplementation have focused on analyzing genes or markers of WAT inflammation. Itariu et al. [39] re- ported in a cohort of severely obese (BMI ≥40) non-diabetic patients that the supplementation with 3.36 g of n-3 PUFAs (460 mg EPA and 380 mg DHA/g of compound) per day during 8 weeks ameliorated ScWAT inflammation, shown as decreased mRNA levels of pro-inflammatory genes (CCL-2,

IL-6, TFGB1, and HIF-1A) and the macrophage M1 marker

CD40, in parallel with an increase in ADIPOQ expression. In this line, Morine et al. [68] reported that after serum metabolic profiling (including fatty acid pattern) and microarray analysis of ScWAT of patients (BMI 20–40 kg/m2), the expression of three genes (PIKFYVE, PIK3CA, and CEPT1) involved in the resolution of inflammation was positively correlated with plasma DHA levels [68]. In the study carried out by

Ferguson et al. [25], lean healthy subjects were treated either with placebo (corn oil) or fish-oil derived n-3 PUFA ethyl esters (465 mg EPA + 375 mg DHA; 4/day) in a treatment period that ranged from 6 to 8 weeks. After RNAseq, pathway analysis highlighted n-3 PUFAs’ effect on genes involved in immune response including APLN, IL1RN, IL7R, IL8, CCL3,

FCGR3A, FCGR3B, FCN1, LCP1, and TREM1. Furthermore, the microarray analysis of Huerta et al. [36] reported that EPA supplementation (1.3 g/day) in ScWAT obtained from healthy overweight/obese women who underwent an energy- restricted dietary intervention of 30% calorie reduction during

10 weeks upregulated several genes related to inflammatory and immune response including ACP5, CHI3L1, DCSTAMP,

HCST, CHIT1, MSR1, TFRC, and HPGDS. Interestingly,

ACP5, MSR1, and TFRC are genes considered as M2 alterna- tively activated macrophage markers. CHIT1 is either prefer- entially expressed by M2 anti-inflammatory macrophages or can mediate M2 macrophage polarization. Moreover, HPGDS is an enzyme which catalyzes the conversion of PGH2 to

PGD2 which is known to be involved in the process of mac- rophage polarization toward a M2, anti-inflammatory state [36].

In addition to these findings, different clinical trials re- vealed that n-3 PUFAs increased circulating factors (FGF21,

Irisin, and T4) that have been identified to have a positive impact on regulating thermogenic adipocyte formation and activity [2, 21, 102]. However, there is no information avail- able about the origin from these factors after n-3 PUFA sup- plementation and the contribution of other organs or tissues distinct of AT cannot be ruled out. Therefore, the previously mentioned trials on human AT describing the modulation of some immune and metabolic factors involved in AT thermo- genic program lead us to hypothesize that the effects observed in vitro and in animal models could be also achieved in humans. However, future clinical trials are needed to test this possibility.

Conclusive remarks and future perspectives The available data both from in vitro and in vivo models show that n-3 PUFAs, particularly EPA [80, 90], target AT to in- crease energy expenditure by enhancing the thermogenic function of BAT and promoting WAT browning. These ther- mogenic properties of EPA seem to be partly mediated through the receptor GPR120 to initiate the classical thermo- genic program mediated by UCP1 [90]. However, alternative

GPR120 and UCP1-independent mechanisms have been also involved in the actions of n-3 PUFAs [43]. These include the activation of the TRPV1 receptor in the gut, leading to stim- ulation of SNS and triggering the thermogenic response in both brown and beige adipocytes [43]. n-3 PUFAs upregulate genes related to metabolic futile cycles (mGPD) that can also account for their thermogenic actions in Ucp1 KO mice [43]. n-3 PUFAs are also epigenetic regulators by enhancing critical post-transcriptional modulators for brown adipogenesis (i.e., miR-30b, miR-193b/365, miR-378, HDAC1, Jmjd3, Ehmt1) [99] (Fig. 1).

Recently, it was found that immune cells control brown and beige thermogenic programs [110]. Indeed, several studies have strongly supported that alternatively activated anti- inflammatory macrophages (M2 type) and type 2 cytokine signaling seem to be directly involved in promoting BAT ther- mogenic pathways [22, 71, 89]. Several studies have demon- strated the capacity of n-3 PUFAs to decrease macrophage infiltration and to promote the switch from the M1 phenotype

Omega-3 fatty acids as regulators of brown/beige adipose tissue: from mechanisms to therapeutic potential to M2 in AT [63, 105], which is prevented in Gpr120 KO mice [75], suggesting that this could be another mechanism contrib- uting to the beiging properties of these fatty acids.

Other studies have revealed that some fatty acid bioactive metabolites play a relevant role as novel regulators of thermo- genesis in BAT and beige adipocytes, including PGI2, PGE2,

PGF2α, 12,13-DiHOME, 9-HODE, and 13-HODE [61, 66]. It has been shown that the reduced n-6/n-3 ratio after the intake of n-3 PUFAs leads to reduced oxylipin production of PGE2 and increased production of PGI2, contributing to beige fat induc- tion. It is well known that n-3 PUFAs serve as substrates for the formation of specialized pro-resolving lipid mediators (SPMs) with potent pro-resolutive and anti-inflammatory properties.

These bioactive SPMs were named resolvins (derived from the resolution phase of inflammation) and were classified as either resolvins of the E-series if derived from EPA (RvE1-3) or resolvins of the D-series (RvD1-4) if the biosynthesis is initiated from DHA. Other DHA-derived lipid mediators in- clude protectins (PD1 and PDX) and maresins (MaR1-2) [19,

66]. Growing evidence has revealed an unbalanced level of some of these n-3 PUFAs-SPMs such as RvD1, PD1, 14- HDHA, 17-HDHA, and 18-HEPE in WATof obese db/db mice and in HFD-induced obese mice [12, 69]. Moreover, a lower ratio between SPMs (e.g., RvD and E series, PD1 and MaR1) and arachidonic acid-derived pro-inflammatory mediators (LTB4 and PGs) has been found in visceral adipose tissue of obese subjects [106]. Interestingly, dietary supplementation with n-3 PUFAs increased the levels of these SPMs in WAT in both rodents and humans [39, 69].

Furthermore, some of these n-3 PUFA-derived SPMs (RvD1 and MaR1) have been suggested as innovative ap- proaches to target obesity, diabetes, and fatty liver disease [32, 50]. These beneficial effects are in part mediated by their profound remodeling of WAT in obese mice, characterized by downregulation of inflammatory genes, increase of anti- inflammatory adipokines, and promotion of macrophage po- larization toward an M2-like phenotype [49, 63, 105]. To our knowledge, there is no data describing the effects of these

SPMs on BAT, but all these previous findings and unpub- lished data from our group lead us to suggest that n-3

PUFA-derived SPMs are likely to be promising candidates to modulate adaptive thermogenesis in beige and brown fat.

Another question to be answered is if a defective production of n-3 PUFA-derived SPMs could be involved in unresolved inflammation and the loss of BAT/beige activity that occurs during aging. It is also important to characterize if long-term supplementation with n-3 PUFAs could prevent BAT/beige loss during aging.

In summary, based on all previously described preclinical studies in cultured adipocytes and animal models, n-3 PUFAs

Fig. 1 Summary of the mechanisms involved in the actions of n-3 PUFAs on adipose tissue thermogenic function. n-3 PUFAs activate the gut–brain axis through TRPV1 in the digestive tract leading to the activation of the

SNS and the subsequent stimulation of BAT/beige thermogenic program through β-adrenergic receptors. Moreover, n-3 PUFAs promote M2 macrophage polarization, which could also account for thermogenic activation. n-3 PUFAs can also directly activate adipocyte thermogenesis through the G-protein coupled receptor 120 (GPR120) partly by mechanisms involving the regulation of miR30b, miR-193b/

365, and miR-378, and the enhancing of FGF21. Dietary n-3 PUFA supplementation decreases the n-6/n-3 ratio, and reduces prostaglandin

E2 (PGE2) while increasing prostaglandin I2 (PGI2) levels. Finally, n-3

PUFAs act as a source of specialized pro-resolving lipid mediators: resolvins (Rv) D and E series; Maresins (MaR) 1 and 2 and protectins which could also contribute to promoting M2 macrophage polarization and activating brown and beige thermogenic adipocytes

Fernández-Galilea et al. and/or derived-metabolites are pleiotropic regulators of mul- tiple pathways involved in the regulation of the thermogenic program in adipocytes; therefore, n-3 PUFAs and/or their de- rivatives have emerged as promising candidates to stimulate brown/beige adipocyte activation. However, whether these mechanisms/properties are operative in humans is unknown.

Further well-designed human trials are needed to characterize the potential benefit of n-3 PUFA supplementation on brown/ beige adipose tissue activation, specifically aimed to uncover the differential effect of EPA and DHA, as well as to establish the effective non-toxic doses required.

Funding information This research was funded by Ministry of Economy,

Industry and Competitiveness (MINECO-FEDER) of the Government of

Spain (BFU2015-65937-R), Department of Health of the Navarra

Government (67-2015), and CIBER Physiopathology of Obesity and

Nutrition (CIBERobn), Carlos III Health Research Institute (CB12/03/

30002). “Juan de la Cierva” Grant to M.F.-G. (IJCI-2016-30025). This research also received support from Centre for Nutrition Research of the

University of Navarra.

Compliance with ethical standards Conflict of interest

The authors declare that they have no conflict of interest.

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Omega-3 fatty acids as regulators of brown/beige adipose tissue: from mechanisms to therapeutic potential

📖 中文全文 Chinese Full Text

中文

# 综述

Omega-3脂肪酸作为棕色/米色脂肪组织的调节因子：从机制到治疗潜力

Marta Fernández-Galilea1,2 & Elisa Félix-Soriano1 & Ignacio Colón-Mesa1 & Xavier Escoté1,3 & Maria J. Moreno-Aliaga1,2,4

收稿日期：2019年5月10日 / 录用日期：2019年12月4日 # 纳瓦拉大学 2019

## 摘要

脂肪组织功能障碍是肥胖的标志性特征。棕色/米色脂肪组织通过非颤抖性产热在维持能量稳态中发挥关键作用。棕色脂肪组织（BAT）活性与体脂含量呈负相关，提示BAT激活对肥胖具有保护作用。BAT还在甘油三酯清除、葡萄糖稳态和胰岛素敏感性的调控中发挥重要作用。因此，BAT/米色脂肪激活已被提出作为预防或改善肥胖发展及相关合并症的策略。近年来，大量临床前研究提出了n-3多不饱和脂肪酸（n-3 PUFAs）作为BAT活性诱导剂和白色脂肪组织褐变的新型诱导剂。本文综述了n-3 PUFAs产热特性的体外和体内现有证据，特别关注所涉及的分子和细胞生理机制。最后，我们还讨论了更好地表征n-3 PUFAs作为褐变剂（尤其是在人体中）治疗潜力的挑战和未来展望。

**关键词** 肥胖 · 脂肪组织 · 褐变 · n-3 PUFAs

## 引言

### 脂肪组织

肥胖已成为全球性的健康问题[70]。成人超重（体重指数；BMI ≥25.0 kg/m²）和肥胖（BMI ≥30.0 kg/m²）的患病率在过去30年间几乎翻倍，2016年分别达到39%和13%[74]。肥胖是一种多因素疾病，由遗传、表观遗传和生活方式等多种决定因素引起，其特征是脂肪组织（AT）中脂肪的过度积累[70, 103, 116]。重要的是，肥胖与多种代谢性疾病的高风险相关，包括2型糖尿病（T2DM）、心血管疾病、脂肪肝甚至某些类型的癌症[10, 48, 97, 125]。因此，考虑到肥胖的高患病率及发展为任何相关疾病的风险，以及该疾病对发病率和死亡率的显著影响，需要更有效的靶向AT的治疗策略来治疗和/或预防肥胖[107]。

在成年哺乳动物（包括人体）体内，存在不同类型的AT：白色、棕色和米色AT[128]。作为重要的内分泌器官，脂肪因子产生在AT功能中起关键作用，白色脂肪组织（WAT）主要是能量储存器官，而棕色（BAT）和米色脂肪组织是能量消耗器官[96]。在肥胖发生过程中，随着WAT质量增加，BAT活性降低[54]。事实上，BAT活性维持和WAT褐变已被提出作为肥胖管理的有效策略[128]。

**要点** - n-3 PUFAs是棕色/米色脂肪细胞产热程序的调节因子。 - n-3 PUFAs的产热作用涉及UCP1依赖性和UCP1非依赖性机制。 - n-3 PUFAs通过表观遗传修饰调节胎儿BAT和子代代谢。

在哺乳动物中，大部分脂肪组织是WAT。然而，WAT是一种异质性组织，不同的脂肪库具有不同的特征。因此，皮下WAT被认为对代谢参数（如胰岛素敏感性和心血管风险）具有一定的有益作用，而内脏WAT由于炎症特征更强，与代谢综合征病理恶化相关[5, 41, 47]。在WAT中，白色脂肪细胞是最具特征性的细胞类型。白色脂肪细胞是球形大细胞，大小变化很大。这些细胞高度特化，能够储存和释放脂肪酸。为此，白色脂肪细胞约90%的体积被单个脂滴占据[128]。在肥胖发生过程中，白色脂肪细胞肥大与代谢改变相关，如线粒体功能受损和促炎性细胞因子/脂肪因子产生增加[35, 117]。这些分子导致巨噬细胞募集增加及其从M2向M1"炎症"表型的极化，进而促进全身性低度慢性炎症状态的建立，这被认为是肥胖相关疾病（如胰岛素抵抗和心血管疾病）发展的主要促成因素[121]。

棕色脂肪细胞是在BAT中发现的主要细胞类型。这些细胞含有多个小的脂滴和大量大的球形线粒体，且嵴的数量增加。作为能量消耗器官，在线粒体内，解偶联蛋白1（UCP1）将底物利用导向热量产生而非ATP生成[11]。BAT存在于小型哺乳动物中并具有活性，是其产热的主要器官。在人类中，BAT在新生儿中非常重要，在成人中则相对较少；BAT被发现存在于颈部、锁骨上、腋下、椎旁、纵隔和肾周区域[54, 100]。BAT产热活性增加有助于能量消耗增加[15]。此外，BAT激活在非产热功能的调控中也发挥关键作用，包括甘油三酯清除、葡萄糖稳态和胰岛素敏感性的控制[4, 40, 53]。其中一些作用似乎与BAT通过batokines分泌与其他关键代谢组织的串扰有关[111]。因此，BAT分泌谱包括多肽或非肽类分子（如脂因子和microRNA），这些分子已被证明具有有益的自分泌、旁分泌甚至内分泌功能[112]。由于BAT激活的有益效应[54]，BAT功能已被作为对抗肥胖和相关代谢疾病的成功工具[5, 33]。

产热能力并非棕色脂肪细胞的独有特征。米色脂肪细胞线粒体的特殊特征赋予这些脂肪细胞高氧化能力和与产热相关的能量消耗增加[108]。米色脂肪细胞位于WAT内，具有多个脂滴，线粒体和UCP1含量高于白色脂肪细胞[27]。这些脂肪细胞似乎来源于前体细胞，甚至通过成熟白色脂肪细胞的转分化而来[27, 123]，并表现出米色特异性基因表达谱（即CD40、Ear2、CD137、SP100和Tbx1）[119, 123]。尽管典型的米色AT分泌谱仍是研究课题，但一些试验揭示某些脂肪因子可能由米色脂肪细胞独特性或更强烈地分泌（即MTRNL）[91, 113, 123]。因此，活性米色脂肪细胞的存在可能对肥胖和代谢疾病的管理产生影响，通过增加能量消耗促进更有利的能量平衡，并改善全身性脂肪因子谱[42]。

在衰老过程中，人类和小鼠均出现BAT质量和活性的丧失以及白色脂肪褐变的减少，靶向BAT以促进寿命的潜力已被提出[20]。因此，当前的挑战之一是理解衰老过程中棕色/米色脂肪细胞活性丧失的机制，以找到预防BAT丧失或重新激活现有BAT库的策略，尤其是在人类中。除了棕色/米色脂肪细胞的经典激活剂（寒冷暴露和交感神经系统（SNS））外，近年来已鉴定出BAT活性和WAT褐变的新型诱导剂，包括激素（FGF21、鸢尾素）、营养因子（脂肪酸）和食物生物活性成分（白藜芦醇、辣椒素类、姜黄素）[76, 114]。

### 游离脂肪酸作为脂肪组织功能的调节因子

大多数脂质的结构中含有脂肪酸（FAs）。FAs由脂肪族疏水区（烃链）和一个末端羧基组成。根据烃链中双键的存在与否，FAs主要有三种类型：（1）无双键的饱和FAs（SFAs），（2）含一个双键的单不饱和FAs（MUFAs），（3）含两个或更多双键的多不饱和FAs（PUFAs）（详见Heird和Lapillonne的综述[34]）。关于其营养来源，SFAs主要来源于动物（肥肉、蛋类和乳制品）或某些植物油（椰油和棕榈油）。MUFAs在橄榄油等植物油中含量丰富，而PUFAs存在于多种坚果、植物油和肥鱼中[18]。n-6 PUFA亚油酸和n-3 PUFA α-亚麻酸（ALA）是必需脂肪酸。亚油酸的主要膳食来源包括植物油、坚果、种子、肉类和蛋类。亚油酸在体内转化为花生四烯酸。ALA存在于植物油中，主要是亚麻籽油、菜籽油和大豆油，以及核桃中。ALA是长链PUFAs的主要前体，其中二十碳五烯酸（EPA）和二十二碳六烯酸（DHA）最为普遍[18]。然而，体内从ALA转化为EPA和DHA的速率非常低，它们应从膳食来源获取，如肥鱼（鲑鱼、凤尾鱼、沙丁鱼、金枪鱼等）、磷虾和海洋微藻[62]。此外，还有其他脂肪酸，主要是丙酸、乙酸和丁酸，它们是有机FA，是未消化膳食纤维细菌发酵的主要产物，可作为营养素或信号分子发挥作用[18, 55]。

FAs是人体的重要组成部分，具有相关的结构、生物学和功能作用。关于FA代谢，进食后，从饮食中获得的FAs以甘油三酯形式储存在脂肪组织中[118]。因此，增加的循环FAs（如乳糜微粒或极低密度脂蛋白）在胰岛素刺激的LPL作用下水解，并通过脂肪酸转运蛋白被脂肪细胞摄取[118]。在脂肪细胞内，FAs被酯化并储存在脂滴中[98]。在食物剥夺期间，FAs成为机体的重要燃料。由于碳水化合物储备迅速耗尽，脂质通过脂解过程激活释放FAs而作为主要能量来源[79]。因此，在细胞内，FAs通过增强的β-氧化被分解代谢以获取能量[120]。这主要发生在线粒体中，是一个循环过程，脂肪酸每轮缩短两个碳，通过呼吸链的电子转移偶联ATP生成[120]。由于其尺寸，与WAT相比，BAT的储存能力较低。然而，BAT由于其在以非颤抖性产热闻名的过程中的能量消耗作用而具有更高的周转能力。FAs通过作为UCP1的激活剂并作为线粒体产热的燃料，是脂肪细胞非颤抖性产热的重要贡献者[66]。长链FAs激活UCP1。在寒冷暴露时，SNS的激活导致BAT中去甲肾上腺素诱导的细胞内甘油三酯脂解释放长链FAs，进而激活UCP1和BAT产热[94]。事实上，急性药理学抑制细胞内TG脂解阻止了细胞内FAs的释放并损害了BAT产热[9]。

另一方面，多项研究已表明，向棕色脂肪细胞添加Fas可模拟产热过程[88, 93]。有人提出FAs可作为UCP1的变构调节因子、辅因子或质子穿梭体[8]。此外，Guerra等人的研究[31]证明了脂肪酸氧化对产热的重要性，因为他们发现携带长链酰基辅酶A脱氢酶（Acadl）基因靶向失活的小鼠对寒冷暴露敏感。尽管有人提出这种FA诱导的产热似乎是UCP1依赖性的[8, 65]，但最近在UCP1敲除（KO）小鼠中的一些研究已证明了UCP1非依赖性产热的存在[16, 44]。

除了作为能量来源的功能外，FAs在细胞中还有其他重要功能。由于它们存在于磷脂中，FAs是生物膜的结构成分，SFAs、MUFAs和PUFAs的相对组成在饮食组成的大幅变化中被严格调控[52]。FAs还可以作为信号分子，调节各种细胞过程和生理功能，如ER应激和凋亡，以及炎症通路[60, 126]。一般而言，SFAs被认为是"不健康"的脂质，因为它们作为促炎分子的功能。SFAs激活多种炎症信号反应，包括巨噬细胞、脂肪细胞、肌细胞和肝细胞等多种细胞类型的广泛细胞因子释放模式[37, 72, 73, 124]。这些效应主要通过激活某些Toll样受体（TLR）家族成员介导[95]。因此，SFAs可能通过TLR4依赖性和TLR4非依赖性机制触发促炎通路[29]。促炎信号的激活已被证明负向调节BAT产热[109]。此外，发现LPS对TLR4的激活减弱了WAT褐变的β3-肾上腺素能刺激。相反，TLR4的缺失保护了线粒体功能和产热激活[77]。除此之外，SFAs是神经酰胺生物合成的前体，已证明神经酰胺是皮下脂肪组织（ScWAT）褐变、炎症和代谢的调节因子。事实上，神经酰胺合成的抑制（全身或脂肪特异性）促进米色化并诱导肥胖小鼠ScWAT中主要是M2巨噬细胞极化[17]。

n-6 PUFAs（花生四烯酸）也可通过转化为白三烯和前列腺素触发促炎信号[87]。Flekenstein-Elsen等人的研究[26]发现花生四烯酸诱导了脂肪生成，但未能促进棕色的获得并损害了原代人脂肪细胞中的线粒体功能。

与SFAs的促炎作用相反，n-3 PUFAs，主要是海洋来源的（即DHA，22:6 n-3和EPA，20:5 n-3）及其衍生物可在多种组织中引发强效的抗炎效应[7, 63, 64]。越来越多的证据支持这些n-3 PUFAs在多种慢性炎症相关疾病中的潜在有益效应，包括肥胖相关代谢疾病、2型糖尿病和心血管疾病[58, 59]。已证明n-3 PUFAs对编码调控WAT生理不同过程（如脂肪生成、脂肪细胞代谢和功能，包括脂肪生成/脂解和脂肪酸氧化[51]、炎症[84, 86]以及胰岛素敏感性和生物活性脂肪因子的产生[1, 30, 56, 57, 67, 84, 85]）的蛋白质的基因表达发挥相关调控作用。在WAT中，伴随肥胖的低度慢性炎症的特征是促炎性脂肪细胞因子水平高和促炎性M1巨噬细胞浸润高。在这种情况下，n-3 PUFA给药导致M1巨噬细胞积累显著减少，抗炎性M2巨噬细胞类型增加，同时促炎性脂肪因子的表达降低[63, 104]。n-3 PUFAs的这些抗炎和胰岛素增敏特性通过G蛋白偶联受体120（GPR120）介导，因为在Gpr120 KO中未观察到这些效应[75]。

与WAT相比，关于n-3 PUFAs对BAT和米色脂肪细胞影响的信息要少得多。然而，近年来，几项临床前研究提出n-3 PUFAs（主要是EPA）对BAT和WAT褐变的有益效应[23]。事实上，这些研究表明n-3 PUFA能够直接修饰编码棕色/米色脂肪细胞发育和功能主控调控因子（包括线粒体生物发生和氧化代谢，以及batokine产生等）的蛋白质的基因表达[1, 25, 67]。

因此，本综述的目的是批判性总结目前关于n-3 PUFAs（EPA与DHA）对棕色和米色脂肪细胞发育、代谢和功能的体外和体内研究知识。此外，还描述了n-3 PUFAs潜在产热效应背后的分子和细胞机制。

## n-3 PUFAs作为棕色/米色脂肪细胞发育和功能的调节因子

### 培养脂肪细胞中的研究

多项研究评估了n-3 PUFAs对棕色脂肪细胞分化/功能和白色脂肪细胞褐变的体外影响（表1）。这些试验大多支持EPA增加小鼠来源细胞培养模型中棕色分化标志物的能力。因此，Zhao和Cheng[127]描述了在分化的基质血管组分（SVF）来源的棕色脂肪细胞中EPA处理（200 μM，分化期间8天）上调了线粒体DNA（mtDNA）含量，提示线粒体质量增加[127]。Pahlavani等人[82]发现，在棕色HIB 1B体外分化的脂肪细胞中，EPA处理（100 μM，48小时）上调了Elovl3、Pgc1α、Sirt2和Ucp1 mRNA水平，同时线粒体含量增加。此外，测定了EPA对线粒体耗氧率（OCRs）的影响，发现最大呼吸能力增加[82]。

此外，Quesada-López等人的研究[90]证明，EPA和ALA（而非DHA本身）显著促进了来自肩胛间BAT（iBAT）的棕色前脂肪细胞的分化，上调了产热基因（Ucp1和Fgf21），并如培养的iBAT脂肪细胞中增强的OCR和产热所示增加了产热能力[90]。此外，该研究发现了n-3 PUFA受体GPR120是棕色脂肪细胞分化表型获得的新型组分[90]。在主要发现中，作者报告GPR120表达在棕色脂肪细胞分化过程中逐渐增加，并被肾上腺素能产热激活剂（去甲肾上腺素和cAMP）诱导。此外，在分化脂肪细胞中药理学激活GPR120能够在缺乏其他分化诱导分子的情况下促进前体细胞的强力分化。该研究还发现GPR120激活刺激了FGF21表达和释放，同时增强了氧气消耗、葡萄糖氧化和产热[90]。有趣的是，GPR120是EPA对脂肪细胞和FGF21诱导和释放的产热效应所必需的，因为在Gpr120 KO小鼠的iBAT脂肪细胞或用GPR120拮抗剂AH7614处理的野生型（WT）脂肪细胞中，或通过siRNA敲低Gpr120后，未观察到这些效应[90]。

此外，在来自小鼠iBAT的体外分化棕色前体细胞的类似模型中，EPA（100 μM，分化诱导后5天）上调了成熟棕色脂肪细胞特征的产热基因和蛋白（Prdm16、Pparγ、Ucp1、Cidea和Elovl3）的表达。这伴随着基础、解偶联和最大呼吸速率水平的增加，以及miR-193/365、miR-378、miR-196a、miR-30b和miR-106/93表达的增加[45]。有趣的是，Gpr120的沉默完全消除了EPA介导的Ucp1和Cidea mRNA水平以及miR-30b和miR-378表达的上调。涉及miR-30b外源添加或miR-30b和miR-378通过锁核酸（LNA）隔离的进一步实验证实，miR-30b和miR-378是GPR120诱导的棕色脂肪生成程序上调所必需的，并且它们不仅是GPR120信号传导的下游靶点，而且还反向影响Gpr120表达[45]。

除了对棕色脂肪细胞的作用外，越来越多的证据表明EPA对白色脂肪细胞米色化也有影响。当在来自腹股沟WAT（iWAT）的前脂肪细胞分化过程的8天期间向培养基中添加EPA（200 μM）时，EPA处理的脂肪细胞显示参与线粒体生物发生和氧化的基因（如Pgc1α、Nrf1和CoxIV）表达增加[127]。与在分化的iBAT前体细胞中观察到的效应类似，Quesada-López等人的研究[90]发现EPA（100 μM）增加了来自iWAT的分化脂肪细胞中的产热和FGF21激活。Gpr120缺乏导致EPA诱导的iWAT脂肪细胞分化的延迟但并未减少，尽管EPA诱导FGF21基因表达和蛋白释放的能力在Gpr120缺陷的米色脂肪细胞中被减弱[90]。

有趣的是，多项研究已证明EPA还调节分化的人脂肪细胞中棕色表型的获得并改善线粒体功能。Fleckenstein-Elsen等人[26]在来自瘦女性的原代人脂肪细胞中的研究发现，EPA（20 μM）处理至分化第12天能够诱导UCP1基因表达，以及作为线粒体含量指示剂的柠檬酸合酶活性。在更功能性的水平上，EPA增加了基础OCR[26]。此外，仅在分化后期（第8-12天）用EPA处理也能够增加UCP1、CPTB和基础OCR的表达[26]。

同样，Laiglesia等人[51]报道，EPA处理来自超重/肥胖女性的完全分化的皮下脂肪细胞促进了米色特征的获得。EPA（100 μM）不仅增加了BAT标志物CIDEA、PRDM16和UCP1的基因表达，还上调了米色脂肪细胞的特异性标志物TBX1和CD137。这些效应伴随着线粒体生物发生的上调，表现为线粒体质量增加和NRF1及TFAM基因表达水平增加，以及与氧化代谢相关的CPTA基因表达增加。关于所涉及的分子机制，这些效应可能是由线粒体生物发生和功能的主控调节因子AMPK、PGC1-α和SIRT1的增强激活引起的[51]。

此外，在人多能脂肪来源干细胞（hMADS）细胞中进行的一项研究中，用罗格列酮处理的功能性米色脂肪细胞进一步用n-6 PUFA花生四烯酸处理，以抑制米色脂肪细胞标志物（CIDEA、CPTM和PLN5）和氧气消耗。EPA处理能够逆转花生四烯酸的抑制效应[28]。n-3 PUFAs调节花生四烯酸的代谢并竞争性抑制花生四烯酸衍生的脂质介质（如白三烯和前列腺素）的产生[64]。事实上，抑制花生四烯酸衍生的前列腺素F2α（PGF2α）的产生（PPARγ的已知抑制剂）因此被提出作为EPA效应的介导机制[28]。

重要的是要提到，EPA对棕色/米色培养脂肪细胞的大多数效应似乎不被DHA共享。尽管有少量证据显示DHA可能增强原代人脂肪细胞的基础OCR[26]，但它对最大OCR没有影响。此外，与EPA相反，DHA对米色hMADS细胞的脂肪细胞分化没有影响[28]。此外，一旦诱导分化，它显著抑制iBAT前体细胞分化[127]。最后，在分化中的棕色脂肪细胞中用DHA处理未显示出增强产热基因表达的额外效应[26, 127]。

综合这些数据，n-3 PUFAs对体外棕色脂肪细胞分化和白色脂肪细胞褐变具有不同的影响。因此，虽然在DHA处理后观察到轻微效应，但EPA强烈诱导棕色脂肪细胞分化，增强线粒体功能，上调产热基因表达，并增加产热。在所涉及的分子机制中，已提出EPA可能通过GPR120激活、FGF21产生、AMPK-SIRT1-PGC1-α信号通路激活，甚至通过抑制PGF2产生来发挥其效应。

### 动物模型中的研究

与体外研究一致，尽管实验模型和设计之间存在很大的异质性，但大多数体内研究支持n-3 PUFAs作为褐变剂的作用（表2）。

几项在补充n-3 PUFAs的高脂饮食（HFD）大鼠中进行的研究表明BAT功能改善。Oudart等人[80]首先报道，在喂食HFD的Wistar大鼠中，用EPA或DHA或两者混合物（作为乙酯）补充饮食4周可调节BAT产热活性。MIX和EPA组的BAT GDP结合总量高于HFD组。在EPA组中，BAT与对照组（低脂饮食（LFD））和HFD组相比显示出线粒体含量富集。BAT较高的产热活性在MIX组中观察到，这是由于增生和线粒体产热活性增加。与本研究一致，Takahashi和Ide[101]表明，在Sprague-Dawley大鼠中，用富含n-3 PUFAs的油（紫苏油和鱼油）补充饮食3周未增加BAT重量，但上调了Ucp1 mRNA水平。最近，Crescenzo等人[13]在Sprague-Dawley大鼠中发现UCP1蛋白含量增加，这些大鼠经过2周半饥饿（50%自由进食）后，用含有n-3 PUFAs丰富的红花/亚麻籽油的HFD再喂养2周。

在小鼠中进行的大量研究显示了类似的趋势，并提供证据表明以不同鱼油制剂形式补充HFD的n-3 PUFAs对改善BAT功能具有强效作用。通过这种方式，几项研究表明，在饮食补充n-3 PUFAs后，小鼠表现出能量消耗、氧气消耗和直肠温度增加，同时UCP1和/或柠檬酸合酶的mRNA和蛋白含量在线粒体中增加[122]。此外，在不同的试验中还评估了与产热功能相关的多种标志物，n-3 PUFA消耗引起Pgc1α、Cpt1b、Cidea、Prdm16、Fgf21、β3AR、Gpr120、Pparα和Pparγ的mRNA和/或蛋白水平的上调[3, 24, 46, 78, 122]（表2）。

已提出多种机制来解释导致n-3 PUFAs这些产热效应的信号通路。Worsch等人[122]发现，富含n-3 PUFAs的HFD（EPA和DHA达到平衡的n-6/n-3 PUFA比率为0.84）有效介导了BAT质量和活性的增加，这可能是由于FGF21水平增加，进而增加了p-AMPK/AMPK比率[122]。此外，n-3 PUFA补充促进了M2极化巨噬细胞和嗜酸性粒细胞的浸润增加，这已被证明可诱导WAT褐变[122]。

除此之外，在KO小鼠模型中进行的研究揭示了n-3 PUFAs可能在不同水平发挥作用。尽管几项研究表明GPR120受体激活介导n-3 PUFAs对脂肪细胞产热程序的影响[24, 122]，但Bjursell等人[6]报告n-3 PUFAs（鲱鱼油）防止体脂质量增加的能力不一定需要GPR120的激活。事实上，在喂食补充HFD的WT和Gpr120 KO小鼠中，n-3 PUFAs在BAT质量、能量消耗、呼吸交换率（RER）和核心体温方面表现出类似的效应。这些发现提示存在其他受体和/或机制介导n-3 PUFAs的有益效应。在此背景下，已提出了其他GPR120替代机制。β3AR激活已知可介导BAT中产热程序的刺激[38]。Kim等人[46]的研究表明鱼油摄入增强了儿茶酚胺的产生，这通过尿液中增加的水平来估计。此外，作者发现鱼油摄入增加了iBAT和iWAT中的去甲肾上腺素周转率，这是交感神经支配器官中交感神经活动的直接指标。已证明瞬时受体电位香草酸1型（TRPV1）在胃肠道中的激活刺激SNS，导致BAT中UCP1表达增加[43]。TRPV1受体是一种非选择性阳离子通道，可被多种刺激激活，包括辣椒素、有害温度（接近42°C）、细胞外酸性pH和某些生物活性脂质[46]。在TRPV1 KO小鼠中补充鱼油未诱导WT同窝仔中观察到的BAT和iWAT中氧气消耗和UCP1及β3AR基因表达的增加[46]。这些结果强烈提示胃肠道TRPV1对鱼油诱导的脂肪组织中UCP1表达的间接贡献[46]。

如前所述，除少数例外[81, 115]，大多数研究同意n-3 PUFAs增加UCP1 mRNA和/或蛋白含量[3, 13, 24, 45, 46, 78, 80, 101, 122]。然而，在Ucp1 KO小鼠中进行的研究表明，n-3 PUFA补充的有益效应可以部分独立于UCP1活性实现。Pahlavani等人[81]表明，在缺乏UCP1的情况下，富含EPA的鱼油介导了Pgc1α、氧气消耗和mtDNA的增加。此外，Oliveira等人[78]发现补充鱼油的WT和Ucp1 KO小鼠之间没有差异。为了解释这些UCP1非依赖性作用所涉及的机制，作者发现UCP1缺乏介导了参与BAT中钙循环促进产热过程的肌浆/内质网Ca²⁺-ATP酶（SERCA）蛋白水平的增加。然而，EPA补充逆转了SERCA上调，提示EPA介导的能量消耗增强不是由Ucp1 KO小鼠中的SERCA介导的[78]。相反，发现iWAT中线粒体3-磷酸甘油脱氢酶（mGPD）水平增加。mGPD是一种催化涉及3-磷酸甘油的产热代谢无效循环的酶。因此，mGPD水平增强可能至少部分解释了n-3 PUFAs在Ucp1 KO小鼠中诱导的能量消耗增加[78]。

除了这些UCP1依赖性和UCP1非依赖性机制外，已观察到与产热活性相关的表观遗传修饰在n-3 PUFA补充后发生。在此背景下，Pahlavani等人[83]进行了RNA-Seq以检测喂食补充或未补充EPA的HFD（45%脂肪）的小鼠BAT中的miRNA谱。验证后，作者得出结论，miR-455-3p和miR-129-5p（两者均为Ucp1诱导剂）在EPA处理动物中显著高于未补充组[83]。此外，母体饮食补充3%鱼油显示新生仔鼠BAT活性改善，如脂质积累减少、Ucp1、Cidea、Prdm16、Pgc1a和Gpr120 mRNA上调以及UCP1、PRDM16和GRP120蛋白水平增加所提示[24]。有趣的是，促进棕色脂肪生成通过抑制Myf5+前体细胞中的肌源性谱系分化的功能性棕色特异性miRNA簇miR-30b、miR-193b和miR-365在母体鱼油组仔鼠BAT中显著增加[99]。此外，在该组中，发现组蛋白去乙酰化酶和去甲基化酶（HDAC1和JmjC结构域含蛋白3；Jmjd3）显著减少，与甲基转移酶（Ehmt1）增加相关，这些是棕色脂肪生成的关键翻译后修饰因子[99]。这些结果表明，母体鱼油暴露改变了胎儿BAT中组蛋白乙酰化和甲基化以及miRNA丰度的表观遗传特征，从而增强了棕色特异性转录程序。有趣的是，母体鱼油摄入增加的BAT功能也可能介导生命后期的扩展代谢益处[99]。事实上，接受母体n-3 PUFAs的仔鼠在11周龄时表现出显著更高的能量消耗。当小鼠受到急性寒冷处理（6°C）时，母体n-3 PUFA喂养组的核心体温显著高于对照组[99]。此外，在iBAT中，脂肪积累较少，寒冷暴露诱导iWAT中棕色样脂肪细胞增加。一致地，UCP1蛋白表达在iBAT和iWAT中均较高[99]。

综合这些数据，强烈提示n-3 PUFA补充通过UCP1依赖性和UCP1非依赖性（无效循环）机制激活产热程序。此外，n-3 PUFA母体饮食补充可能发挥对后代具有抗肥胖效应的表观遗传变化。

### 人体中的研究

很少有研究分析n-3 PUFAs对人体AT转录组、代谢组和功能的调节。据我们所知，目前尚无临床试验解决n-3 PUFA补充后BAT库或WAT褐变的潜在激活问题，这可能是因为确定人体BAT活性存在复杂性。大多数评估BAT激活的研究已使用¹⁸F-FDG-PET/CT进行，但放射性同位素的使用限制了连续或纵向研究的发展，并且对人体测量存在问题。一些近期研究旨在开发新型无创方法，使其更容易且经济地监测人体临床/营养干预期间的棕色脂肪活性[14, 53, 92]。

一些在n-3 PUFA补充前后进行的AT活检研究集中于分析WAT炎症的基因或标志物。Itariu等人[39]在一组严重肥胖（BMI ≥40）非糖尿病患者中报告，每天补充3.36 g n-3 PUFAs（每克化合物含460 mg EPA和380 mg DHA）持续8周改善了ScWAT炎症，表现为促炎基因（CCL-2、IL-6、TFGB1和HIF-1A）和巨噬细胞M1标志物CD40的mRNA水平降低，同时ADIPOQ表达增加。在这方面，Morine等人[68]报告，在对患者（BMI 20-40 kg/m²）进行血清代谢谱分析（包括脂肪酸模式）和ScWAT微阵列分析后，参与炎症消退的三个基因（PIKFYVE、PIK3CA和CEPT1）的表达与血浆DHA水平呈正相关[68]。在Ferguson等人[25]进行的研究中，瘦健康受试者接受安慰剂（玉米油）或鱼油衍生的n-3 PUFA乙酯（465 mg EPA + 375 mg DHA；每天4次）治疗，治疗期为6至8周。RNAseq后，通路分析突出了n-3 PUFAs对参与免疫反应的基因的影响，包括APLN、IL1RN、IL7R、IL8、CCL3、FCGR3A、FCGR3B、FCN1、LCP1和TREM1。此外，Huerta等人[36]的微阵列分析报告，EPA补充（1.3 g/天）在来自健康超重/肥胖女性的ScWAT中上调了多种与炎症和免疫反应相关的基因，这些女性接受了10周30%热量限制饮食干预，包括ACP5、CHI3L1、DCSTAMP、HCST、CHIT1、MSR1、TFRC和HPGDS。有趣的是，ACP5、MSR1和TFRC被认为是M2替代激活巨噬细胞标志物。CHIT1要么优先由M2抗炎巨噬细胞表达，要么可以介导M2巨噬细胞极化。此外，HPGDS是一种催化PGH2转化为PGD2的酶，已知参与巨噬细胞向M2抗炎状态极化的过程[36]。

除了这些发现外，不同的临床试验揭示n-3 PUFAs增加了循环因子（FGF21、鸢尾素和T4），这些因子已被证明对调节产热脂肪细胞的形成和活性具有积极影响[2, 21, 102]。然而，关于n-3 PUFA补充后这些因子的来源以及不同于AT的其他器官或组织的贡献尚无信息，不能排除。因此，前述关于人体AT的试验描述了参与AT产热程序的某些免疫和代谢因子的调节，使我们假设在体外和动物模型中观察到的效应也可能在人体中实现。然而，需要未来的临床试验来检验这种可能性。

## 结论性评论和未来展望

来自体外和体内模型的现有数据表明，n-3 PUFAs（特别是EPA[80, 90]）靶向AT以通过增强BAT的产热功能和促进WAT褐变来增加能量消耗。EPA的这些产热特性似乎部分通过受体GCPR120介导以启动UCP1介导的经典产热程序[90]。然而，GPR120和UCP1非依赖性机制也参与了n-3 PUFAs的作用[43]。这些包括肠道中TRPV1受体的激活，导致SNS刺激并触发棕色和米色脂肪细胞中的产热反应[43]。n-3 PUFAs上调与代谢无效循环（mGPD）相关的基因，这也可以解释其在Ucp1 KO小鼠中的产热作用[43]。n-3 PUFAs还是表观遗传调节因子，增强棕色脂肪生成的关键转录后调节因子（即miR-30b、miR-193b/365、miR-378、HDAC1、Jmjd3、Ehmt1）[99]（图1）。

最近，发现免疫细胞控制棕色和米色产热程序[110]。事实上，几项研究强烈支持替代激活的抗炎巨噬细胞（M2型）和2型细胞因子信号传导似乎直接参与促进BAT产热通路[22, 71, 89]。多项研究已证明n-3 PUFAs减少巨噬细胞浸润并促进AT中从M1表型向M2转换的能力[63, 105]，这在Gpr120 KO小鼠中被阻止[75]，提示这可能是促成这些脂肪酸米色化特性的另一种机制。

其他研究揭示了一些脂肪酸生物活性代谢物作为BAT和米色脂肪细胞产热的新型调节因子发挥相关作用，包括PGI2、PGE2、PGF2α、12,13-DiHOME、9-HODE和13-HODE[61, 66]。已表明摄入n-3 PUFAs后降低的n-6/n-3比率导致PGE2的oxylipin产生减少和PGI2产生增加，有助于米色脂肪诱导。众所周知，n-3 PUFAs作为底物用于形成具有强效促消退和抗炎特性的专门促消退脂质介质（SPMs）。这些生物活性SPMs被命名为消退素（源自炎症消退阶段），如果来源于EPA则被分类为E系列消退素（RvE1-3），如果生物合成由DHA启动则被分类为D系列消退素（RvD1-4）。其他DHA衍生的脂质介质包括保护素（PD1和PDX）和消退素（MaR1-2）[19, 66]。越来越多的证据揭示，在肥胖db/db小鼠和HFD诱导的肥胖小鼠的WAT中，这些n-3 PUFAs-SPMs（如RvD1、PD1、14-HDHA、17-HDHA和18-HEPE）的水平不平衡[12, 69]。此外，在肥胖受试者的内脏脂肪组织中已发现SPMs（例如RvD和E系列、PD1和MaR1）与花生四烯酸衍生的促炎介质（LTB4和PGs）之间的比率较低[106]。有趣的是，饮食补充n-3 PUFAs增加了啮齿动物和人类WAT中这些SPMs的水平[39, 69]。

此外，其中一些n-3 PUFA衍生的SPMs（RvD1和MaR1）已被提出作为靶向肥胖、糖尿病和脂肪性肝病的新方法[32, 50]。这些有益效应部分通过其在肥胖小鼠中WAT的深刻重塑来介导，其特征为抗炎基因下调、抗炎脂肪因子增加和巨噬细胞向M2样表型极化[49, 63, 105]。据我们所知，目前没有数据描述这些SPMs对BAT的影响，但所有这些先前发现和我们团队的未发表数据使我们提出n-3 PUFA衍生的SPMs可能是调节米色和棕色脂肪中适应性产热的有前景的候选者。

另一个需要回答的问题是，n-3 PUFA衍生的SPMs的产生缺陷是否可能参与衰老过程中未解决的炎症和BAT/米色活性的丧失。表征长期补充n-3 PUFAs是否可以预防衰老期间BAT/米色丧失也很重要。

总之，基于前述在培养脂肪细胞和动物模型中的所有临床前研究，n-3 PUFAs和/或衍生代谢物是参与调节脂肪细胞产热程序的多条通路的多效性调节因子；因此，n-3 PUFAs和/或其衍生物已成为刺激棕色/米色脂肪细胞激活的有前景的候选者。然而，这些机制/特性在人体中是否有效尚不清楚。需要进一步精心设计的人体试验来表征n-3 PUFA补充对棕色/米色脂肪组织激活的潜在益处，特别是旨在揭示EPA和DHA的差异效应，以及确定所需的有效无毒剂量。

## 资助信息

本研究由西班牙政府经济、工业和竞争力部（MINECO-FEDER）（BFU2015-65937-R）、纳瓦拉政府卫生部（67-2015）和肥胖与营养病理生理学CIBER（CIBERobn）、卡洛斯三世健康研究所（CB12/03/30002）资助。M.F.-G.获得"Juan de la Cierva"资助（IJCI-2016-30025）。本研究还获得了纳瓦拉大学营养研究中心的支持。

## 伦理标准合规

### 利益冲突

作者声明不存在利益冲突。