The Effect of Marine Derived n-3 Fatty Acids on Adipose Tissue Metabolism and Function

Adipose tissue function is key determinant of metabolic health, with specific nutrients being suggested to play a role in tissue metabolism. One such group of nutrients are the n-3 fatty acids, specifically eicosapentaenoic acid (EPA; 20:5n-3) and docosahexaenoic acid (DHA; 22:6n-3). Results from studies where human, animal and cellular models have been utilised to investigate the effects of EPA and/or DHA on white adipose tissue/adipocytes suggest anti-obesity and anti-inflammatory effects. We review here evidence for these effects, specifically focusing on studies that provide some insight into metabolic pathways or processes. Of note, limited work has been undertaken investigating the effects of EPA and DHA on white adipose tissue in humans whilst more work has been undertaken using animal and cellular models. Taken together it would appear that EPA and DHA have a positive effect on lowering lipogenesis, increasing lipolysis and decreasing inflammation, all of which would be beneficial for adipose tissue biology. What remains to be elucidated is the duration and dose required to see a favourable effect of EPA and DHA in vivo in humans, across a range of adiposity.


Introduction
Adipose tissue, the largest organ in the human body, was historically considered to be metabolically inert. However, white adipose tissue is now considered an endocrine organ as it secretes adipokines (and hormones) which act locally and distally through autocrine, paracrine and endocrine effects [1]. Although adipose tissue is comprised of several cell types, including pre-adipocytes, adipocytes, endothelial cells, fibroblasts, leukocytes and macrophages [2], all of which may impact on tissue function, one of the main functions of adipocytes/adipose tissue is to store fatty acids [3]. Adipose tissue acts to "buffer" the influx of dietary fat into the circulation [3,4], with excess dietary fat being stored in adipose tissue rather than "overflowing" to non-adipose organs. Ectopic fat deposition has been proposed to underlie obesity-associated metabolic diseases [5]. An increase in adipose tissue mass may alter the function of the tissue. For example, when adipose tissue starts to expand (such as with excess nutrition) macrophages infiltrate and orchestrate inflammatory responses via molecules such as tumor necrosis factor α (TNFα), interleukin 6 (IL-6) and monocyte chemoattractant protein-1 (MCP-1), all of which have been implicated in the development of pathological changes in adipose tissue physiology [6][7][8][9]. Intriguingly, a proportion of overweight/obese individuals remain metabolically healthy even with further weight gain, whilst others do not; it has recently been suggested this is due to an increased capacity of adipose tissue for lipogenesis [10]. Multiple factors have been suggested to impact on the function of adipose tissue, however as the tissue is the primary site for dietary fat storage and reflects dietary fat intakes [11] it is reasonable to suggest that the composition or type of fat that the tissue is exposed to may also influence the function.
A class of fatty acids that has received a lot of attention over the last 30 years is the n-3 (or ω-3) fatty acids, specifically those derived from marine sources. n-3 fatty acids have been suggested to lower the risk of a number of non-communicable metabolic diseases including cardiovascular disease, obesity and diabetes [12][13][14]. Here we review the effect of long chain n-3 polyunsaturated fatty acids (LCPUFA), specifically eicosapentaenoic acid (EPA, 20:5n-3) and docosahexaenoic acid (DHA, 22:6n-3) on white adipose tissue metabolism and function. Although other n-3 fatty acids such as α-linolenic acid (ALA, 18:3n-3) and docosapentanoic acid (DPA, 22:5n-3) are of potential interest, data are limited. A number of reviews on the effect of fish oil or n-3 fatty acids on adipose tissue have previously been undertaken [15][16][17][18][19][20], therefore we have chosen to review the evidence from human, animal (rodent and fish) and in vitro cellular studies regarding the specific effects EPA and DHA have on the metabolism and function of white adipose tissue from different depots. Specifically, we will discuss the mechanisms by which EPA and DHA are proposed to reduce adiposity along with discussion regarding how n-3 fatty acids may influence markers of adipose tissue inflammation and cytokine production.

Dietary Sources of Eicosapentaenoic Acid (EPA) and Docosahexaenoic Acid (DHA)
EPA and DHA, commonly referred to as fish oil fatty acids, are not synthesized de novo by fish. Fish accumulate them through consumption of water plants, such as plankton and algae, which are part of the marine food chain [21]. Therefore, if plankton and algae are not a dietary component or if fish oil is replaced by other feed sources, such as in fish farming where a vegetable-oil based diet rich in linoleic acid (18:2n-6) and oleic acid (18:1n-9) may be given, the EPA and DHA content of the fish will decrease [22,23]. Marine fish tend to have higher amounts of EPA and DHA than freshwater fish. Fish typically store EPA and DHA mainly as triacylglycerol, at the middle position (sn-2) of the glycerol backbone however, in krill, a shrimp-like crustacean that feed off algae in deep ocean waters, 30%-65% of EPA and DHA is in phospholipids [24].
Within the human diet, EPA and DHA can be produced from ALA but the capacity of conversion is low in humans, although higher in women of child-bearing age than men [25]. Thus, it is likely that the majority of EPA and DHA within the body, for most individuals are derived from fish and fish oil intakes. Fish oil is often considered to be the best source of EPA and DHA however, as mentioned above, the amount of EPA and DHA varies amongst species and within a species according to environmental variables such as diet, temperature and salinity of the water.

Fatty Acid Composition of Adipose Tissue
As the fatty acid composition of adipose tissue has a half-life between 6 months and 2 years, it reflects long-term dietary intake along with endogenous metabolism [11]. The abundance of EPA and DHA in human subcutaneous adipose tissue is low, typically less than 0.2 for EPA and up to 1.0 mol% for DHA [11]. The amount of EPA and DHA in adipose tissue has been reported to increase or remain unchanged with increasing age [26][27][28], which is suggested to be an age-dependent effect independent of dietary intake [28].
Studies measuring the change in adipose tissue fatty acid composition, as a marker of compliance to n-3 supplementation are limited and findings inconsistent with some [29][30][31][32] but not all [33,34] noting small but significant increases in the abundance of adipose tissue EPA and DHA after varying periods of fish oil supplementation ( Table 1). The inconsistency in findings may in part be explained by differences in: duration of supplementation, amount of EPA and DHA consumed, participant age, sex and adiposity, or site where the adipose biopsy was taken. Elegant work by Katan et al. [30] clearly demonstrated that the levels of DHA rose more rapidly in subcutaneous abdominal compared to gluteal adipose tissue depots whilst differences between the depots for EPA were not as obvious ( Table 1). The difference in the appearance of DHA in subcutaneous abdominal compared to gluteal adipose tissue, may be explained by the fact that dietary fat extraction (from chylomicron-triacylglycerol) occurs to a greater extent in subcutaneous abdominal than gluteal adipose tissue [35]. Of note, Katan et al. [30] found that the proportion of EPA and DHA in subcutaneous abdominal and gluteal adipose tissue was approximately one-sixth and one-third respectively of dietary intake. It would be of interest to determine the extent to which the fatty acid composition of visceral (intra-abdominal) adipose tissue changed with n-3 fatty acid supplementation. However, as visceral adipose tissue samples are often obtained during elective surgery, it would be challenging to undertake a well-controlled study. Taken together, the data presented in Table 1 clearly demonstrate that even with supplementation the abundance of EPA and DHA in adipose tissue does not increase notably. This suggests that EPA and DHA are not preferentially stored in adipose tissue triacylglycerol long-term, rather they may be partitioned to oxidation pathways or to storage in other lipid fractions, such as phospholipids; red blood cell and plasma phospholipids have a notably higher abundance of both EPA and DHA than adipose tissue [11]. However, a change in adipose tissue fat mass and therefore dilution of EPA and DHA abundance cannot be ruled out as the majority of studies do not indicate if there were changes in participants' body weight over the course of the study. Changes in fatty acid composition of adipose tissue have been reported with weight loss, notably there was not change in EPA abundance but an increase in DHA abundance, without a reported change in n-3 fatty acid intake, over the weight maintenance period [36]. These changes highlight the importance of weight/fat mass stability in subjects participating in intervention studies where adipose tissue fatty acid composition is being measured as a marker of compliance.

The "Anti-Obesity" Effect of EPA and DHA
Measuring an anti-obesity effect of increased EPA and DHA consumption in humans is challenging, not least as there are many other factors to control for (e.g., exercise and other dietary components) and methodology sensitive to small changes in adipose tissue mass needs to be used. In 2009, Buckley and Howe [37] reviewed the available evidence for an anti-obesity effect of EPA and DHA. They suggested from the limited human studies, that increased consumption of EPA and DHA may reduce body fat; the majority of these studies were short-term, with a small number of subjects. It remains unclear if similar conclusions can be drawn from longer-term studies. A recent meta-analysis by Du and colleagues [38] identified randomised, placebo controlled trials where adults were assigned to either fish oil/marine group for a period of greater than 4 weeks and had reported at least one anthropometric measure of body composition (i.e., body weight, BMI, waist circumference or waist to hip ratio). From the 21 studies (a total of 1329 individuals) they found no evidence to support an anti-obesity role of n-3 LCPUFA [38]. It is plausible that changes were not detected due to the non-specific and insensitive methods used to assess changes in body fat. By using computed tomography Sato et al. [39] noted that 6 months supplementation with EPA only (1800 mg/day) resulted in a significant decrease in epicardial and visceral adipose tissue mass, with no change in subcutaneous abdominal adipose tissue, in individuals with confirmed coronary artery disease. It is possible that subcutaneous abdominal adipose tissue mass did decrease however it was only measured in a single slice at the level of the umbilicus, thus changes in other depots would not have been detected. Results from some, but not all animal studies have suggested EPA and DHA consumption to have an anti-obesity effect with a lack of increase in fat mass even when an obesogenic diet is consumed [40], as well as a reduction in body weight if already obese [41]. Moreover, these studies, along with cellular studies have been used to tease out the mechanisms involved in this process, as discussed below with data from human [42][43][44][45][46][47][48], animal [40,41,[49][50][51][52][53][54][55] and cellular [56][57][58][59][60][61][62][63][64][65][66][67][68][69] studies provided in Tables 2-4.

Suppression of Fat Deposition and Adipogenesis
A decrease in fatty acid deposition within adipose tissue may occur due to a decrease in triacylglycerol synthesis via decreased de novo lipogenesis or re-esterification of fatty acids within the tissue; alternatively it may occur due to a lower flux of fatty acids to the tissue. In the latter situation, fatty acids could be repartitioned to other tissues (such as muscle) for disposal, rather than going to adipose tissue for storage. In humans, the absolute contribution of de novo synthesized fatty acids to adipose tissue triacylglycerol is potentially small [70] and measuring adipose tissue de novo lipogenesis (or fatty acid esterification/re-esterification) in vivo in humans is challenging. Therefore, it is not surprising that studies have not been undertaken investigating how EPA and DHA supplementation influence these processes in humans. Although not a direct measure of fatty acid synthesis or esterification/re-esterification within the tissue, the measurement of the expression of genes related to these processes provides some insight to the effect of EPA and DHA on these processes. Camargo et al. [42] reported that consumption of 4 g/day of fish oil (containing a total of 1.24 g EPA and DHA) for 12 weeks significantly decreased the expression of genes related to fatty acid uptake and storage in subcutaneous obese adipose tissue ( Table 2).
Work in animal models has typically found EPA and DHA to limit lipid accumulation in adipose tissue ( Table 3). The majority of studies have reported lower fad pad mass and adipocytes number and size which was suggested to occur via suppression of lipogenic genes and, in some studies, a concomitant activation of lipolytic genes after supplementation with EPA and DHA (Table 3). Despite reporting a significant decrease in inguinal retroperitoneal fat pad mass Hainault et al. [52] did not find any significant change in fatty acid synthase (FAS) activity or mRNA expression in these depots. Of note, one study reported that EPA and DHA consumption resulted in higher total and perigonadal fat mass than control group [55]. This discrepancy in findings maybe in part explained that this study used an LDL receptor deficient (LDLR´{´) mouse model whilst others have typically used C57Bl/6 mice or Wistar rats.

wk AT inflammation
Wild-type animals: FO group had decreased mRNA expression of genes related to inflammation and macrophage infiltration in AT.
FO supplementation had no effect in GPR120 knockout. Studies investigating the effects of dietary EPA and DHA on adipose tissue function have also been undertaken in fish (Table 3). Todorcevic et al. [40] demonstrated that a diet supplemented with EPA and DHA (20% of total fatty acids) for 21 weeks repressed the development of adiposity, regulating triacylglycerol accumulation in visceral adipose tissue of Atlantic salmon. A positive influence of dietary EPA and DHA on lipid accumulation in adipose tissue was also reported in grass carp [71]. Diet containing EPA and DHA, (12% of total fatty acids for 75 days), suppressed lipid accumulation in intraperitoneal adipose tissue and significantly up-regulated the expression lipolytic genes including: lipoprotein lipase (LPL), stearoyl-CoA desaturase 1 (SCD1) and peroxisome proliferator activated receptor α (PPARα) [71]. Furthermore, similar results were reported by Liu et al. [72] in grass carp treated with dietary EPA and DHA (11% of total fatty acids) for 95 days.
The process of adipogenesis (or an increase in fat mass) involves the differentiation of preadipocytes to mature adipocytes, is a complex and tightly regulated process involving a cascade of transcription factors which are sensitive to the nutritional environment [73]. In a comprehensive review by McMillen and Robinson [74] the role of the nutritional environment an individual is exposed to before birth and in early infancy impacts on risk of obesity and obesity-related diseases later in life was discussed. Evidence from animal studies shows that offspring of mothers fed a diet high in calories or high in fat before birth are heavier and have a higher percentage body fat throughout life [75,76]. Findings from human studies are compelling; children born to mothers who are obese during their pregnancy have an increased incidence of obesity over the life course [75]. Therefore, it has been suggested that targeting maternal nutrition during pregnancy may reduce risk of obesity in subsequent generations [77]; n-3 fatty acids may decrease adipogenesis and lipogenesis and thus exposure in utero to these fatty acids may lower the risk of obesity in offspring. In 2011 Muhlhausler and colleagues [77] reviewed animal studies to determine the effects of n-3 LCPUFA supplementation during pregnancy and lactation on postnatal body composition of offspring. Although 13 potential studies were identified, only four met the inclusion criteria and the authors found from albeit limited data that there was a suggestion that the offspring from n-3 LCPUFA supplemented dams had a lower fat mass [77]. In contrast, supplementation of dams with a high DHA diet (5% fat of which DHA was 0.95% total fatty acids) during pregnancy and lactation resulted in offspring that had significantly higher total and subcutaneous fat mass (as percentage of total body weight) at 6 weeks of age, compared to control animals fed a diet containing the same amount of fat but devoid of n-3 LCPUFA [78]. Thus it remains unclear if increased exposure in utero to n-3 fatty acids decreases adipogenesis and lipogenesis and is an area that warrants further investigation.
To date, the majority of in vitro evidence regarding the mechanistic effects of EPA and DHA on triacylglycerol accumulation/lipid deposition comes from the clonal murine cell line, 3T3-L1 (Table 4). This cell line offers advantages over primary cells, as they are homogenous with regards to cellular population and stage of differentiation; however, their ability to reflect human adipose tissue function and metabolism remains to be clarified. Primary pre-adipocyte cultures have been shown to better reflect in vivo adipose function, than cell lines because they can be isolated from different species and fat depots. The latter is of interest as there are distinct molecular and biochemical hallmarks between different adipose tissue depots and at a cellular level; pre-adipocytes isolated from different adipose tissue depots and cultured in vitro retain depot-specific functional properties [79][80][81]. Unlike cell lines, the function and metabolism of primary cells will be influenced by the age, sex, and genetics of the donor and therefore consideration is needed when comparing across studies.
Results from in vitro cellular studies that have added EPA and DHA to media for periods of 24 h to 3 weeks are mixed; some suggest EPA and DHA to be anti-adipogenic whilst others find a pro-adipogenic response. EPA and DHA have been found to inhibit, promote or have no effect on the differentiation of pre-adipocytes (Table 4). Typically the markers of adipocytes adipogenesis that have been measured include: the accumulation of triacylglycerol, lipid droplet formation, expression of master adipogenic transcription factors, and lipid genes. Using 3T3-L1 pre-adipocytes, Kim et al. [59] investigated the effects of DHA alone (6 days) on lipogenesis and lipolysis and found mean lipid droplet size, percent lipid area, as well as glycerol-3-phosphate dehydrogenase (GPDH) activity all significantly decreased whilst basal lipolysis increased in fully differentiated adipocytes. The results from this work demonstrate the anti-adipogenic effects of DHA via inhibition of triacylglycerol accumulation and increased lipolysis [59]. When comparing the effects of EPA and DHA on lipid droplet formation in 3T3-L1 cells it was found that although both fatty acids reduced the presence of lipid droplets, DHA was more potent than EPA [56]. In addition to the decreased lipid droplet formation, there were notable reductions in the expression of key protein involved in this process, including perilipin A, caveolin-1 and Cidea [56], however there was no effect of DHA on PPARγ expression [56]. In contrast, Murali et al. [62] reported that incubating 3T3-L1 with EPA and DHA induced adipogenesis; DHA being more potent than EPA in inducing the differentiation process. The authors suggested the differential effects of EPA and DHA on adipognesis could be due to differential accumulation of n-3 fatty acids in membrane phospholipids [62]. In line with Murali et al. [62], Wojcik et al. [69] reported increased accumulation of neutral lipids in mature 3T3-L1 adipocytes; however, others have reported no effect on triacylglycerol accumulation at any stage of maturation in 3T3-L1 adipocytes [65]. A reduced expression of both adipogenic and lipogenic genes, including sterol regulatory element-binding protein 1 (SREBP1), FAS, and peroxisome proliferator-activated receptor γ (PPARγ) after EPA and DHA treatment of mature adipocytes has been report by some [69] but not by others [67]. Using human breast adipocytes as a cell model, Wang et al. [68] demonstrated exposure of DHA for 24 h decreased the expression of lipogenic genes, including FAS, LPL and PPARγ, whilst expression of lipolytic genes was increased. Lee et al. [82] found EPA to stimulate glycerol and free fatty acids release which was associated with induction of lipolytic gene expression and suppression of adipogenic gene expression in 3T3-L1 adipocytes. Treatment of fish primary adipocytes with EPA and DHA (for 3 weeks) resulted in decreased triacylglycerol accumulation in mature adipocytes [83]. In an acute study, using mature adipocytes isolated from grass carp Liu et al. [72] found that 6 h of incubation with EPA and DHA was sufficient to notably decreased triacylglycerol accumulation, significantly increased glycerol release and the expression of genes involved in lipolysis (e.g., adipose triglyceride lipase (ATGL), hormone-sensitive lipase (HSL)). The findings from in vitro cellular studies, notably those using primary adipocytes demonstrate that EPA and DHA inhibit triacylglycerol accumulation, which may be the result of effects mediated through genes related to lipogenesis and lipolysis.

Adipocyte Apoptosis
To our knowledge, there have been no studies in humans investigating the effect of n-3 fatty acids on adipocyte apoptosis and only limited work has been undertaken in animal and in vitro cellular models. Although outside the scope of this review, there have been a large number of studies investigating the effect of n-3 fatty acids and cancer in relation to apoptosis, as reviewed by Wendel and Heller [84].
Limiting findings from human in vitro and in vivo studies have reported apoptosis in white adipose tissue along with alternations in adipose tissue mass. Thus consideration is required when looking at adipose tissue mass in relation to cell number as they might be partly regulated by pre-adipocyte/adipocyte apoptosis [85,86]. Nelson-Dooley et al. [87] have suggested targeting apoptotic pathways in adipocytes as a novel way of treating obesity. Apoptosis is often assessed by cytomorphological alterations, DNA fragmentation and condensation, detection of caspases, protein cleavage at specific locations, cell membrane alterations and and increased mitochondrial membrane permeability [88]. In 2004 Ruszickova et al. [41] were the first to suggest the concept of n-3 fatty acids and regulation of cellularity in adipose tissue. Using a rodent model the authors suggested that increased intakes of EPA and DHA (Table 3) reduced high-fat diet-induced obesity by decreasing the number of adipocytes in adipose tissue, which could be interpreted as evidence of a pro-apoptotic effect. Perez-Matute et al. [54] demonstrated increased levels of histone-associated DNA oligonucleosomal fragments, classical markers of apoptosis in the white adipose tissue of rats fed a standard diet with additional oral administration EPA ethyl ester (1 g/kg per day) daily for 5 weeks. Moreover, they found a cafeteria diet strongly impaired the apoptotic action induced by EPA and suggested that EPA-induced apoptosis depends on the nutritional and metabolic status of the animals [54]. High dietary-n-3 fatty acid levels are at increased susceptibility to fatty acid peroxidation which has been reported to occur in different tissues within a fish model [89] including adipose tissue [40]. Fish contain a greater amount of more highly unsaturated fatty acids than mammals which makes them more prone to fatty acid peroxidation leading to apoptosis [90]. Todorcevic et al. [40] were the first to demonstrate that high dietary intakes of EPA and DHA induced oxidative stress and apoptosis in the visceral adipose tissue in Atlantic salmon. Salmon was fed with diets containing 50% EPA and 55% DHA of total fatty acid for 21 weeks and found increased activity of caspase-3, indicative of apoptosis occurring in white adipose tissue. The authors concluded that decreased adipocytes cell number due to apoptosis, may be one factor explaining the lower triacylglycerol accumulation occurring in fish white adipose tissue when diets enriched with EPA and DHA are fed [40]. On the basis of these finding, it would be prudent to suggest the measurement of adipose tissue apoptotic markers when EPA and DHA, notably at high dietary doses, are given.
Even though there is a growing literature on the studying the mechanisms for the inhibitory effects of n-3 fatty acids on proliferation of various tumor cells (reviewed by [91]) but also on non-cancerous cells [92], there are surprisingly very few in vitro studies that have investigated the effect of EPA and DHA on adipocyte apoptosis. Kim et al. [59] reported significant DHA-induced apoptosis in 3T3-L1 post-confluent pre-adipocytes after 48 h incubation with 200 µM/L compared to 100 µM/L DHA, demonstrating the inhibitory effects of DHA on adipocyte differentiation. Todorcevic et al. [93] treated primary antioxidant glutathione (GSH) depleted salmon adipocytes with high doses of EPA and DHA (600 µM for 6 days) in present or absence of α tocopherol and showed increased expression of genes encoding a set of well-known apoptotic markers in the groups with no added α tocopherol, suggesting that the induction of adipocyte cell death by EPA and DHA likely plays an important part in the adipose tissue homeostasis especially in animals exposed to high dietary n-3 fatty acids.
Taken together, the available data from animal and in vitro studies suggests that high doses of EPA and DHA may induce adipocyte apoptosis. How targeting the apoptotic pathway in white adipose tissue would decrease obesity and influence adipose tissue function and overall metabolic health in humans remains to be elucidated.

Increased Fatty acid Oxidation (Energy Expenditure)
Although an increase in fatty acid oxidation, via β-oxidation has been suggested to play a role in a reduction of triacylglycerol accumulation in adipocytes, evidence for this in white adipose tissue is limited; fatty acid oxidation and mitochondrial function has been studied more often in brown adipose tissue. The number and activity of mitochondria within adipocytes has been suggested to contribute to insulin resistance and type 2 diabetes [94]. Changes in the expression of genes related to insulin-signaling have been reported to increase, whilst the expression of genes related to glycolysis, gluconeogenesis and glyceroneogenesis decreased in subcutaneous abdominal adipose tissue after 12 weeks supplementation with n-3 fatty acids [45] (Table 2). On the basis of these changes, the authors suggested that a low-fat (fat 28% total energy (TE)) high complex carbohydrate diet supplemented with 1.24 g/day n-3 fatty acids (EPA and DHA) improved adipose tissue insulin sensitivity compared to diets high saturated or monounsaturated fat in individuals with the metabolic syndrome [45]. To lower the risk of obesity-mediated diseases such as the metabolic syndrome, weight loss is often encouraged to decrease fat mass; weight loss by calorie restriction has been suggested to increase subcutaneous abdominal adipose tissue capacity for lipid oxidation [95]. Whether similar changes occur in subcutaneous gluteal or visceral adipose tissue remains to be determined. Moreover, it would be of interest to determine if calorie restriction in combination with EPA and DHA supplementation has an additive effect on up-regulating adipose tissue fatty acid oxidation in different adipose tissue depots. The amount of EPA and DHA has varied between studies, with higher doses tending to be used in animal and in vitro studies, translation to the appropriate dose, along with duration required to see an effect in humans needs to be elucidated.
In vivo or in vitro cellular studies investigating the effects of EPA and DHA on adipose tissue fatty acid oxidation and/or mitochondrial function are sparse. Specifically measuring markers of adipose tissue fatty acid oxidation in vivo in humans has not, to our knowledge, been undertaken. This is most likely to be due to the challenges associated with assessing adipose tissue fatty acid β-oxidation directly. Surprisingly no study in humans has yet investigated changes in the expression of relevant genes in adipose tissue before and after supplementation with EPA and DHA. Fasting whole-body fatty acid oxidation (assessed by indirect calorimetry) has been reported to increase in young, healthy men (n = 5) after 3 weeks of supplementation with fish oil (6 g/day) when compared to a control diet containing equal amounts of total dietary fat [96]. Only a few animal studies have investigated the effect of EPA and DHA on fatty acid β-oxidation in white adipose tissue (Table 3). Flachs et al. [51] reported that feeding mice for 4 weeks with diet containing increasing amounts of EPA and DHA, preferentially up-regulated several mitochondrial regulatory genes, increased β-oxidation and suppressed lipogenesis in white abdominal fat. Using a fish model, Atlantic salmon, Todorcevic et al. [40] reported an increase in adipose tissue fatty acid β-oxidation after fish consumed fish oil rich in EPA and DHA for 21 weeks.
In vitro cellular studies have found increased β-oxidation in 3T3-L1 adipocytes after incubation with 100 µM of EPA for 24 h [58]. The increase in β-oxidation was associated with increased carnitine palmitoyltransferase 1 (CPT-1) activity but mRNA and protein expression did not change [58]. As EPA treatment increased the proportion of EPA in mitochondrial membrane lipids, the authors concluded that the activity of CPT-1 and β-oxidation was due to changes in the structure or dynamics of the mitochondrial membranes [58]. EPA and DHA are reported to activate AMP-activated protein kinase (AMPK) in 3T3-L1 adipocytes, which could be a mechanism for their effect on fatty acid oxidation [97]. Todorcevic et al. [83] demonstrated that EPA and DHA increased β-oxidation in salmon primary adipocytes, which may in part explain the concomitant reduction in adipocyte triacylglycerol. A possible mechanism, by which EPA and DHA may result in increased fatty acid oxidation and therefore less body fat accumulation, is through induction of thermogenesis mediated by mitochondrial uncoupling protein-1 (UCP1); the thermogenic capacity of brown adipose tissue (BAT) is associated with uncoupling whereas white adipose tissue is typically not [98]. In 2013 Flach et al. [98] reviewed the effect of n-3 fatty acids on mitochondrial oxidative phosphorylation (OXPHOS) and fatty acid oxidation in white adipose tissue. In this comprehensive review they reported that in a murine model, supplementation with n-3 fatty acids in combination with mild calorie restriction induced mitochondrial OXPHOS in epididymal white adipose tissue only, independent of UCP1 induction; other studies in rodents have reported increased levels of UCP1 mRNA and/or protein in BAT in response to n-3 fatty acid supplementation [98]. Recently, Zhao and Chen [99] using an in vitro cellular model of isolated stromal-vascular (SV) cells from inguinal adipose tissue of suggested that EPA enhanced energy dissipation capacity by recruiting brite adipocytes to stimulate oxidative metabolism. From the limited data available it appears that EPA and DHA increase fatty acid β-oxidation in adipocytes, however the mechanisms responsible and the effect on mitochondrial OXPHOS and thermogenesis in human adipose tissue remains to be elucidated.

The "Anti-Inflammatory" Effects of EPA and DHA on Adipose Tissue
An expansion of adipose tissue mass, is often associated with macrophage infiltration which may lead to inflammatory responses, which have been implicated in the development of pathological changes in adipose tissue physiology [6][7][8][9]. These changes potentially move the tissue toward a pro-inflammatory phenotype and there is accumulating evidence suggesting pro-inflammatory processes in adipose tissue increase the risk of obesity-related disorders, such as insulin resistance [100][101][102][103]. For example, several studies reported positive associations between degree of obesity and the expression of genes related to inflammation in adipose tissue [7,9]. A number of studies have investigated the "anti-inflammatory" effect of EPA and DHA in white adipose tissue.

Suppression of Pro-Inflammatory Cytokine Production
Studies investigating the effect of n-3 fatty acid supplementation, for periods between 8 weeks up to 6 months, on the expression of genes related to inflammation in human subcutaneous white adipose tissue have been undertaken. Overall results are variable, with some suggesting consumption of EPA and DHA decreases the expression of genes related to inflammation, whilst other report no change ( Table 2). For example, Guebre-Egziabher et al. [43] noted decreased expression of metalloprotease 9 (MMP9) and CD68 in subcutaneous abdominal adipose tissue on a low not high dose of MaxEPA in a small number (n = 12) of individuals with chronic kidney disease (CKD) who were randomised to take either a low (n = 6) or high (n = 6) dose of MaxEPA for 10 weeks. In contrast, Itariu et al. [44] found that high doses of EPA and DHA (total 4 g/day) for 8 weeks significantly decreased the expression of genes related to inflammation in subcutaneous obese adipose tissue and increased production of anti-inflammatory eicosanoids in visceral adipose tissue (Table 2).
Work in murine models has found consumption of n-3 fatty acids decreased inflammatory gene expression in white adipose tissue depots (Table 3). Todoric et al. [104] investigated the effect of an n-3 fatty acid diet on macrophage infiltration in white adipose tissue of obese, diabetic mice, as well as on gene expression of several immune genes. They found that consumption of 25.1 mg of n-3 fatty acids (containing EPA and DHA) per gram of fat for 6 weeks resulted in a reduction in macrophage infiltration in combination with decreased expression of inflammatory genes in white adipose tissue [104]. Sarawathi et al. [55] used LDLR´{´mice and showed similar results to Todoric et al. [104] despite a gain in white adipose tissue mass. They reported a diet supplemented with fish oil containing 140 mg EPA and 95 mg DHA/day for 12 weeks reduced expression of macrophage markers such as MAC-1 and CD68 as well as inflammatory markers such as TNFα, metalloprotease 3 (MMP3), and serum amyloid A3 (SAA3) in white adipose tissue [55]. Taken together these data demonstrate that consumption of n-3 fatty acids have the potential to modulate immune response in adipose tissue.
In vitro studies, using cell-lines and human primary cells, have been utilised to investigate the potential cellular mechanisms and pathways involved in an n-3 fatty acid mediated alteration in immune response (Table 4). Adiponectin, an adipocyte-specific protein, is often suggested to be anti-inflammatory cytokine and it has been postulated that a change in secretion may be associated with visceral obesity [105]. In vitro cellular work has found that incubation of primary human adipocytes isolated from subcutaneous adipose tissue with either with EPA or DHA significantly increased the concentration of secreted adiponectin [106], which is in agreement with several studies performed using primary cultured rat adipocytes [107], 3T3-L1 adipocytes [64] and human adipocyte cell lines [66]. From the work of Oster et al. [64] it appears that EPA and DHA have differential effects on adiponectin secretion, which may be influenced by the cell model used. They found DHA increased adiponectin mRNA expression and secreted adiponectin protein to a greater extent than the same dose of EPA in 3T3-L1 adipocyte after 24 h incubation [64]. In contrast, Tishinsky et al. [99] found using a commercial line of human adipocytes that EPA significantly increased cellular adiponectin protein content after 48 h of treatment while DHA did not affect cellular adiponectin protein.
The effects of n-3 fatty acids on the adipokine leptin, have been investigated in vitro however results show conflicting effects of n-3 fatty acids on leptin mRNA expression and secretion. EPA has been shown to have a stimulatory effect on leptin gene expression and secretion in 3T3-L1 adipocytes [108] and primary cultured rat adipocytes [109]. Reseland et al. [110] reported an opposite effect to the work of Murata et al. [108] and Perez-Matute et al. [109], where exposure to both EPA and DHA reduced leptin mRNA expression in 3T3-L1 adipocytes. Furthermore, the effect of EPA and DHA on leptin expression has been shown to vary depending on the stage of adipocyte maturation [65]. Thus, the discrepancy in reported results could be related to differences in different cell models used (primary cells vs. cell lines) or in measuring the effects of n-3 fatty acids on leptin at different stages of adipogenesis. Culturing human primary adipocytes in either EPA or DHA resulted in a down-regulation of IL6 and TNFα secretion [63]. In contrast, differential effects of EPA and DHA were found for IL6 secretion in 3T3-L1 cells with EPA increasing and DHA decreasing secretion [65]; the underlying mechanisms for these responses were unable to be clarified by the authors. Another divergent finding is that from Wojcik et al. [69] who noted culturing 3T3-L1 cells in either EPA or DHA increased TNFα mRNA expression; it is unclear if this lead to increased secretion as it was not measured. The authors speculated that their finding would not be replicated in adipose tissue in vivo, as the anti-inflammatory effects of EPA and DHA on TNFα expression would be modulated through the direct effect of these fatty acids on macrophages; cells that were not present in their in vitro culture [69]. It remains unclear if EPA and DHA have a differential effect on anti-inflammatory markers in human and animal models as typically these fatty acids have been given together and not directly compared.

Conclusions
In recent years evidence demonstrating that an increased consumption of EPA and DHA may have a beneficial effect on white adipose tissue function and metabolism is starting to emerge. Although current literature cannot support an exact mechanistic role of EPA and DHA on adipose tissue biology it is apparent that these fatty acids have the potential to be potent modulators of adipose tissue and adipocyte function. More work has been undertaken using animal and cell models therefore consideration is required regarding the dose and duration of EPA and DHA, the animal and cell model used (e.g., primary vs. cell-lines). Moreover, in vitro cellular cells often investigate the effects of EPA and DHA on adipocytes and it is plausible a different response may be found in whole adipose tissue due to the presence of other cell types (e.g., macrophages, endothelial cells) and their interaction with adipocytes. Although the effects of n-3 fatty acid supplementation on the fatty acid composition of subcutaneous abdominal and gluteal adipose tissue have been investigated, mechanistic studies (in vivo and in vitro) appears to be limited to primarily subcutaneous abdominal adipose tissue and/or adipocytes. Evidence for an effect of n-3 fatty acids in human visceral adipose tissue is sparse and therefore not well understood. Evidence for a reduction in fat accumulation in animal models, along with an anti-inflammatory effect appears to be consistent when intakes of EPA and DHA are high (up to 20% of total fatty acids); however recommendations for human intakes are between 0.5% and 2% of total energy intake [111]. Thus, the duration and amount of dietary EPA or DHA required for beneficial effects in human subcutaneous adipose tissue depots remains to be elucidated.