Obesity

The World Health Organization^{( 2 )} defines obesity as an abnormal or excessive fat accumulation that can harm an individual’s health. One of the major problems of obesity is being both a disease and a risk factor for several other disorders, such as cardiovascular diseases, type 2 diabetes mellitus, respiratory diseases, musculoskeletal disorders and some cancers, which have a great impact on quality of life and longevity of people^{( 2 ,} Reference Knight ^{5 ,} Reference Bhaskaran, Douglas and Forbes ^{54 )}.

Changes in dietary patterns and physical activity in recent decades, such as excessive consumption of food or high-energy foods, and sedentary lifestyle are major factors that contribute to the genesis of obesity^{( 2 )}. If the total daily energy intake surpasses the amount of energy spent, the excess is stored as TAG in cells called adipocytes, which form the white adipose tissue⁽ Reference Coelho, Oliveira and Fernandes ^{8 )}. Adipose tissue responds rapidly to excess nutrient consumption by adipocyte hypertrophy and hyperplasia⁽ Reference Halberg, Wernstedt-Asterholm and Scherer ^{55 )}. Therefore, obesity is characterised by increased storage of fatty acids in an expanded mass of adipose tissue⁽ Reference Galic, Oakhill and Steinberg ^{3 )}.

White adipose tissue, along with its important role in energy storage, is an important endocrine organ⁽ Reference Galic, Oakhill and Steinberg ^{3 ,} Reference Coelho, Oliveira and Fernandes ^{8 )}. This tissue produces many bioactive molecules, such as cytokines (when secreted by adipose tissue, the cytokines can be called adipokines or adipocytokines), which not only serve as regulators of systemic metabolism but also have immunoregulatory properties⁽ Reference Galic, Oakhill and Steinberg ^{3 ,} Reference Ouchi, Parker and Lugus ^{56 ,} Reference Exley, Hand and O’Shea ^{57 )}.

Obesity leads to changes in adipokine secretion. Progressive increase of the adipocytes and consequent expansion of the adipose tissue leads to reduced blood supply, with consequent hypoxia, which is related to necrosis and infiltration of macrophages into adipose tissue. Infiltrated macrophages form crown-like structures surrounding adipocytes, leading to adipokine overproduction, which includes pro-inflammatory mediators such as TNF-α, IL-6 and IL-1β ⁽ Reference Galic, Oakhill and Steinberg ^{3 ,} Reference Johnson, Milner and Makowski ^{10 ,} Reference Maury and Brichard ^{58 ,} Reference Mathis ^{59 )}.

Obesity is characterised by a low-grade chronic inflammation, once the elevation of inflammatory markers and cytokines, as well as the presence of macrophages infiltrated into the white adipose tissue, can be detected⁽ Reference Johnson, Milner and Makowski ^{10 )}. This low-grade chronic inflammation in adipose tissue spreads to systemic inflammation and contributes to the onset and progression of associated metabolic disorders, such as insulin resistance, type 2 diabetes mellitus, hyperlipidaemia, atherosclerosis and metabolic syndrome⁽ Reference Ouchi, Parker and Lugus ^{56 ,} Reference Donath and Shoelson ^{60 ,} Reference Moore and Tabas ^{61 )}. Although most of the changes were initially identified and reported in peripheral tissues, studies conducted in the past decade have shown that obesity is also related to brain changes⁽ Reference Convit ^{6 ,} Reference Shefer, Marcus and Stern ^{7 ,} Reference Cai ^{62 )}.

Obesity and the brain

Studies have shown that both excessive consumption of saturated fats and obesity can lead to brain damage⁽ Reference Convit ^{6 ,} Reference Shefer, Marcus and Stern ^{7 ,} Reference Thaler, Yi and Schur ^{63 )}. More specifically, although little is known about the effects of obesity on the brain⁽ Reference Freeman, Zhang and Nair ^{64 )}, studies have found associations between obesity and abnormalities in the hypothalamus⁽ Reference Thaler, Yi and Schur ^{63 ,} Reference Milanski, Degasperi and Coope ^{65 ,} Reference Moraes, Coope and Morari ^{66 )}, hippocampus, prefrontal cortex and striatum⁽ Reference Boitard, Cavaroc and Sauvant ^{67 ,} Reference Nummenmaa, Hirvonen and Hannukainen ^{68 )}.

The hypothalamus is a brain structure that plays a central role in the regulation of energy homoeostasis, integrating multiple metabolic signals from peripheral organs and modulating eating behaviour and energy metabolism⁽ Reference Velloso and Schwartz ^{69 –} Reference Drougard, Fournel and Valet ^{72 )}. Therefore, changes at this site may cause neural control loss and make room for obesity onset and worsening⁽ Reference Williams ^{70 )}.

The hypothalamus rapidly responds to metabolic challenges (e.g. hyperenergetic diet)⁽ Reference Kälin, Heppner and Bechmann ^{73 )}. Overnutrition causes hypothalamic inflammation, which disrupts the normal homoeostasis of energy intake and expenditure, as well as alters insulin secretion and sensitivity⁽ Reference Shefer, Marcus and Stern ^{7 ,} Reference Thaler, Yi and Schur ^{63 )}. Unlike inflammation in peripheral tissues – a process that develops over weeks of high-fat-diet feeding in rodent models – markers of hypothalamic inflammation are elevated within 1 to 3 d of high-fat-diet exposure, before weight gain⁽ Reference Thaler, Yi and Schur ^{63 ,} Reference Kälin, Heppner and Bechmann ^{73 )}. Therefore, the excess nutrients, as well as circulating inflammatory cytokines seen in obesity, such as TNF-α, IL-1β and IL-6, activate intracellular inflammatory pathways in a variety of target cells⁽ Reference Thaler, Choi and Schwartz ^{74 –} Reference Gregor and Hotamisligil ^{76 )}.

By using MRI in humans, Thaler et al.⁽ Reference Thaler, Yi and Schur ^{63 )} have found evidence of increased gliosis in the mediobasal hypothalamus of obese individuals, suggesting that, similarly to what occurs in rodents with obesity induced by a high-fat diet, obesity in humans is associated with the neuronal injury in the hypothalamus, which is a crucial brain area for body weight control. Thereby, the hypothalamus functions may be altered when inflamed, leading to an imbalance between food intake and energy expenditure⁽ Reference Cai ^{62 ,} Reference Milanski, Degasperi and Coope ^{65 )}.

The hippocampus, the brain structure involved in cognition, memory, learning and emotions, is also vulnerable to the inflammatory process present in obesity⁽ Reference Shefer, Marcus and Stern ^{7 ,} Reference Boitard, Cavaroc and Sauvant ^{67 ,} Reference Kanoski and Davidson ^{77 –} Reference Miller and Spencer ^{79 )}. Excessive consumption of food rich in saturated fat and sugar, besides being related to obesity, is associated with hippocampal impairment⁽ Reference Davidson, Hargrave and Swithers ^{78 )}. Therefore, cognitive function may also be impaired by high-fat intake and by obesity⁽ Reference Miller and Spencer ^{79 ,} Reference Park, Park and Choi ^{80 )}.

In rodent studies, Moroz et al.⁽ Reference Moroz, Tong and Longato ^{81 )} showed that high-fat-diet feeding increased lipid peroxidation in the hippocampus, as indicated by high 4-hydroxynonenal levels. Park et al.⁽ Reference Park, Park and Choi ^{80 )} also showed that consumption of a high-fat diet leads to increased lipid peroxidation and impairs neurogenesis in the hippocampus. Jeon et al.⁽ Reference Jeon, Jeong and Shin ^{82 )} showed that a high-fat diet promotes an increase in hippocampal TNF-α expression and activates microglia. Boitard et al.⁽ Reference Boitard, Cavaroc and Sauvant ^{67 )} reported that the intake of a high-fat diet increases the expression of pro-inflammatory cytokines in the hippocampus.

Studies have shown that obesity may also be related to changes in the reward system, highlighting abnormalities in the prefrontal cortex and striatum⁽ Reference Nummenmaa, Hirvonen and Hannukainen ^{68 ,} Reference Tomasi and Volkow ^{83 ,} Reference Farr, Li and Mantzoros ^{84 )}. It has been shown that dopamine levels in the medial prefrontal cortex and in the striatum of rats prone to obesity have been reduced⁽ Reference Geiger, Behr and Frank ^{85 )}. In addition, a study on rodents has shown hypofunction of the dopaminergic system in obese rats⁽ Reference Mathes, Nehrenberg and Gordon ^{86 )}. Dysregulation of the dopaminergic system is associated with changes that include addiction and hyperphagia⁽ Reference Mathes, Nehrenberg and Gordon ^{86 )}. Regarding humans, a link has been found between the elevation of BMI and low dopaminergic activation in the striatum in obese women⁽ Reference Stice, Spoor and Bohon ^{87 )}. Furthermore, MRI of the brain of young women showed striated hypofunction in those who were prone to weight gain⁽ Reference Stice, Yokum and Blum ^{88 )}.

Obesity treatment

Obesity treatment is extremely important. One should not only seek weight loss but the individual’s metabolic health as well. Lifestyle modification (intervention focusing on diet and exercise) constitutes the conventional and first-choice treatment for obesity. The failure of these measures, mainly with regard to the maintenance of the results, has emphasised the need for adjuvant therapeutic resources⁽ Reference Pucci and Finer ^{89 )}.

Obesity treatment remains a major challenge. Food re-education therapy and exercise often do not achieve satisfactory results. Pharmacological therapy has been shown to be fraught with serious adverse effects, and many drugs have been withdrawn from the market because of unfavourable risk–benefit relationships⁽ Reference Pucci and Finer ^{89 )}.

In view of increasing obesity rates in the world, as well as the recurrent treatment difficulties currently observed, several studies are required to broaden treatment options. In this context, studies have shown that n-3 PUFA have the potential to help treat obesity⁽ Reference Golub, Geba and Mousa ^{16 ,} Reference Campbell and Bello ^{17 ,} Reference Itariu, Zeyda and Hochbrugger ^{38 ,} Reference Flachs, Ruhl and Hensler ^{90 ,} Reference Hensler, Bardova and Jilkova ^{91 )}.

n-3 PUFA

n-3 Fatty acids (ω-3, n-3 or w-3) are a family of PUFA, which are vital for the functioning of the body, termed essential fatty acids⁽ Reference Surette ^{92 ,} Reference Gómez Candela, Bermejo López and Loria Kohen ^{93 )}. The term ‘n’ refers to the position of the first double bond, and n-3 PUFA have the first double bond between the third and fourth carbon atoms⁽ Reference Arbex, Bizarro and Santos ^{94 )}. The major n-3 PUFA are α-linolenic acid (ALA) (eighteen carbons, three double bonds), EPA (twenty carbons, five double bonds) and DHA (twenty-two carbons, six double bonds)⁽ Reference Gómez Candela, Bermejo López and Loria Kohen ^{93 –} Reference Anderson and Ma ^{95 )}.

Lipid components, especially fatty acids, play an important role in the structure of cell membranes and in metabolic processes⁽ Reference Arbex, Bizarro and Santos ^{94 )}. Because n-3 PUFA cannot be synthesised by the body, they need to be obtained from dietary sources⁽ Reference Surette ^{92 ,} Reference Arbex, Bizarro and Santos ^{94 )}. These sources may be foods of plant origin, in the form of ALA, or from some species of fish, in the form of EPA and DHA⁽ Reference Ellulu, Khaza’ai and Abed ^{13 ,} Reference Surette ^{92 ,} Reference Anderson and Ma ^{95 )}.

ALA is found in flaxseed, soyabean, chia, rapeseed and walnuts, and it can be metabolised in EPA and DHA via elongase and desaturase enzymes⁽ Reference Arbex, Bizarro and Santos ^{94 ,} Reference Simopoulos ^{96 )}. However, humans are inefficient in performing this synthesis from ALA⁽ Reference Anderson and Ma ^{95 ,} Reference Barceló-Coblijn and Murphy ^{97 ,} Reference Brenna, Salem and Sinclair ^{98 )}, as these enzymes are influenced by innumerable aspects, such as smoking, alcohol consumption, diabetes, stress and ageing⁽ Reference Anderson and Ma ^{95 ,} Reference Simopoulos ^{96 ,} Reference Brenna, Salem and Sinclair ^{98 ,} Reference Calder ^{99 )}. Sea fish, such as sardines, salmon, tuna, mackerel and herring, are the main sources of EPA and DHA⁽ Reference Arbex, Bizarro and Santos ^{94 ,} Reference Calder ^{99 )}.

n-3 PUFA are present in cell membranes, particularly in the lipid bilayer of the plasma membrane, and depending on their proportion in the membranes they may undergo changes in fluidity and, therefore, in their functions⁽ Reference Gómez Candela, Bermejo López and Loria Kohen ^{93 )}. DHA, in particular, is one of the most abundant components in the brain’s structural lipids⁽ Reference Simopoulos ^{96 ,} Reference Agrawal and Gomez-Pinilla ^{100 )} and is a key component of neuronal membranes at signal transduction sites, which indicates that its action is vital for brain function⁽ Reference Gomez-Pinilla ^{14 ,} Reference Agrawal and Gomez-Pinilla ^{100 )}. The incorporation of EPA and DHA into the diet can influence not only lipid composition and structure of cell membranes but also the physiological responses that depend on these membranes, as is the case with cell signalling mechanisms⁽ Reference Farooqui ^{101 )}. There is evidence that inadequate intake of maternal n-3 PUFA may lead to abnormal development and function of the CNS⁽ Reference Sun, Simonyi and Fritsche ^{102 )}. DHA-derived lipid mediators are neuroprotective and ameliorate neurological disorders⁽ Reference Sun, Simonyi and Fritsche ^{102 )}. There is also evidence that DHA may ameliorate cognitive decline and affect behavioural symptoms in major neuropsychiatric disorders such as dementia, schizophrenia and depression⁽ Reference Lauritzen, Brambilla and Mazzocchi ^{103 )}.

n-3 PUFA derived from fish oil (EPA and DHA) exerts important effects on the inflammatory pathways, acting as anti-inflammatory agents⁽ Reference Wall, Ross and Fitzgerald ^{12 ,} Reference Ellulu, Khaza’ai and Abed ^{13 ,} Reference Chapkin, Kim and Lupton ^{104 ,} Reference Yates, Calder and Ed Rainger ^{105 )}, because of its ability to interact with the major inflammatory signalling pathways, as well as its suppressive effect on the production of cytokines⁽ Reference Yates, Calder and Ed Rainger ^{105 ,} Reference Lorente-Cebrián, Costa and Navas-Carretero ^{106 )}. The anti-inflammatory mechanisms of action exerted by EPA and DHA include suppression of NF-κB, reduction of eicosanoid production, as well as alteration of membrane organisation, particularly those related to the functions of Toll-like receptors and T-lymphocyte signalling⁽ Reference Chapkin, Kim and Lupton ^{104 ,} Reference Flock, Rogers and Prabhu ^{107 )}. In addition, the anti-inflammatory properties of EPA and DHA are mediated also through the inhibition of TLR signalling pathways⁽ Reference Flock, Rogers and Prabhu ^{107 )}.

EPA and DHA exert their anti-inflammatory effects by decreasing the production of pro-inflammatory eicosanoids derived from arachidonic acid, as well as by serving as substrates for production of lipid mediators with pro-resolution properties, such as resolvins, protectins and maresins⁽ Reference Flock, Rogers and Prabhu ^{107 ,} Reference Martínez-Fernández, Laiglesia and Huerta ^{108 )}. These specialised pro-resolving lipid mediators have potent immunoregulatory actions as they activate specific mechanisms to promote resolution of inflammation⁽ Reference Flock, Rogers and Prabhu ^{107 ,} Reference Martínez-Fernández, Laiglesia and Huerta ^{108 )}. Mediators derived from EPA are known as E series resolvins, and those derived from DHA are named as D series resolvins, whereas protectins and maresins are derived only from DHA⁽ Reference Flock, Rogers and Prabhu ^{107 ,} Reference Martínez-Fernández, Laiglesia and Huerta ^{108 )}.

Furthermore, other documented n-3 PUFA effects include cardiovascular benefits such as reduction of TAG, cardiac arrhythmias, blood pressure and platelet aggregation⁽ Reference Flock, Rogers and Prabhu ^{107 ,} Reference Lee, O’Keefe and Lavie ^{109 ,} Reference Bermúdez Menéndez de la Granda and Sinclair ^{110 )}. Studies have also shown that an n-3 PUFA-enriched diet may be able to inhibit neuroinflammation and delay oxidative stress and cell apoptosis⁽ Reference Farooqui ^{101 ,} Reference Trépanier, Hopperton and Orr ^{111 )}. Therefore, considering that inflammation and related changes are strongly linked to many chronic diseases (among them obesity), the ingestion of n-3 PUFA (mainly in the form of EPA and DHA) can play a fundamental role in preventing and treating these diseases⁽ Reference Campbell and Bello ^{17 ,} Reference Simopoulos ^{96 ,} Reference Flock, Rogers and Prabhu ^{107 )}.

n-3 PUFA and obesity

n-3 PUFA has the potential to induce a number of effects that may be useful for the treatment of obesity as this substance can attenuate weight gain and reduce body fat⁽ Reference Buckley and Howe ^{15 )}. Most studies on n-3 PUFA and obesity have focused on peripheral tissues, as well as on plasma and serum. n-3 PUFA have shown several beneficial effects on obesity-related changes in animals, such as reduced body-fat gain, reduced fat accumulation in visceral region, improved lipid profile, insulin resistance, glucose intolerance and hepatic steatosis, as well as inflammation reduction in peripheral tissues⁽ Reference Rokling-Andersen, Rustan and Wensaas ^{20 ,} Reference Kalupahana, Claycombe and Newman ^{22 ,} Reference Liu, Xue and Liu ^{24 ,} Reference Rossmeisl, Medrikova and van Schothorst ^{27 ,} Reference Bargut, Mandarim-de-Lacerda and Aguila ^{28 )}. The effect of n-3 PUFA in the brain alterations related to obesity was less studied, with hypothalamus being the major focus of the investigation. n-3 PUFA showed beneficial effects on modulating inflammation and hypothalamic function in rodents⁽ Reference Cintra, Ropelle and Moraes ^{23 ,} Reference Pimentel, Lira and Rosa ^{26 ,} Reference Viggiano, Mollica and Lionetti ^{33 )} and was able to reverse the negative effect on the brain-derived neurotrophic factor (BDNF) gene expression caused by obesity in the hypothalamus of rats⁽ Reference Abdel-Maksoud, Hassanein and Gohar ^{31 )}. A review of animal studies on n-3 PUFA for obesity (and related alterations) is summarised in Table 1.

In humans with overweight or obesity, studies have shown that n-3 PUFA treatment can modulate adipose tissue, reduce adiposity, improve inflammation (adipose tissue and serum), reduce TAG and reduce oxidative stress in plasma, indicating that n-3 PUFA may be beneficial to help treat obesity⁽ Reference Itariu, Zeyda and Hochbrugger ^{38 ,} Reference Kiecolt-Glaser, Belury and Andridge ^{40 ,} Reference Kiecolt-Glaser, Epel and Belury ^{41 )}. A review of human studies on n-3 PUFA for obesity (and related alterations) is summarised in Table 2.

n-3 PUFA, especially EPA and DHA, have been shown to play an important role in the treatment of obesity⁽ Reference Flock, Rogers and Prabhu ^{107 ,} Reference Flachs, Rossmeisl and Bryhn ^{112 ,} Reference Lorente-Cebrián, Costa and Navas-Carretero ^{113 )}. A systematic review with meta-analysis conducted by Bender et al.⁽ Reference Bender, Portmann and Heg ^{114 )} showed evidence that the consumption of fish or encapsulated fish oil (rich in n-3 PUFA) is related to slight reductions in body weight and waist circumference. However, the authors of that study concluded that further research is needed to clarify the possible mechanisms by which n-3 PUFA can lead to weight reductions⁽ Reference Bender, Portmann and Heg ^{114 )}.

A meta-analysis conducted by Du et al.⁽ Reference Du, Jin and Fang ^{115 )} revealed that fish oil had no effect on the reduction of body weight and BMI in overweight or obese individuals. However, waist circumference and hip:waist ratio were significantly reduced in individuals taking fish oil supplementation, especially when combined with life modification intervention. The authors conclude that current evidence does not prove that fish oil intake may decrease body weight in overweight or obese adults, but that these individuals may benefit from reduced abdominal fat. They have also emphasised that the results should be treated with caution and suggested a large-scale investigation over a long period to draw definitive conclusions⁽ Reference Du, Jin and Fang ^{115 )}.

A more recent meta-analysis conducted by Zhang et al.⁽ Reference Zhang, Liu and Zhao ^{116 )} also showed that a statistically non-significant difference was revealed in weight loss between n-3 PUFA and placebo, whereas n-3 PUFA might effectively reduce waist circumference and TAG levels in overweight and obese adults. The authors also suggested more studies to explore and clarify this issue⁽ Reference Zhang, Liu and Zhao ^{116 )}.

n-3 PUFA, especially EPA and DHA, can modulate adipocyte number by regulating adipocyte proliferation, differentiation and apoptosis. n-3 PUFA also regulates pathways related to fat storage and fat mobilisation, decreasing lipid accumulation processes and favouring adipocyte oxidative metabolism by promoting mitochondrial biogenesis and fatty acid oxidation. In addition, n-3 PUFA can modulate adipocyte insulin sensitivity and glucose utilisation⁽ Reference Martínez-Fernández, Laiglesia and Huerta ^{108 )}.

Other mechanisms by which EPA and DHA can help to treat obesity are related to the capacity of this substance to inhibit NF-κB activity and TLR-mediated inflammatory signalling⁽ Reference Flock, Rogers and Prabhu ^{107 )}. n-3 PUFA can also reduce adipose tissue inflammation by regulating the production of pro-inflammatory cytokines, by decreasing M1 macrophage infiltration and by reducing the formation of n-6-derived pro-inflammatory lipid mediators⁽ Reference Martínez-Fernández, Laiglesia and Huerta ^{108 )}. Fish oil supplementation can inclusively polarise macrophages and microglia towards the anti-inflammatory phenotype⁽ Reference Bashir, Sharma and Elahi ^{117 ,} Reference Inoue, Tanaka and Masuda ^{118 )}. Moreover, the treatment with n-3 PUFA can increase the production of lipid mediators with pro-resolution properties (resolvins, protectins and maresins) in adipose tissue⁽ Reference González-Périz, Horrillo and Ferré ^{119 ,} Reference Neuhofer, Zeyda and Mascher ^{120 )} and promote the reduction of adipose tissue and systemic inflammation⁽ Reference Itariu, Zeyda and Hochbrugger ^{38 )}.

Inflammation also links obesity with the development of insulin resistance and hepatic steatosis⁽ Reference Flock, Rogers and Prabhu ^{107 ,} Reference González-Périz, Horrillo and Ferré ^{119 )}. The adverse metabolic changes associated with insulin resistance occur as a result of inflamed adipose tissue. Thus, EPA and DHA also may improve insulin sensitivity by generating pro-resolving lipid mediators and promoting alternatively activated macrophages⁽ Reference Flock, Rogers and Prabhu ^{107 )}. González-Périz et al.⁽ Reference González-Périz, Horrillo and Ferré ^{119 )} showed that dietary intake of n-3 PUFA, by triggering the formation of n-3 PUFA-derived resolvins and protectins, had insulin-sensitising actions in adipose tissue and liver and improved insulin tolerance in obese mice, as well as alleviated hepatic steatosis⁽ Reference González-Périz, Horrillo and Ferré ^{119 )}.

Most studies utilise a combination of DHA and EPA as n-3 PUFA supplement, which may mask the effects of each individual fatty acid. The individual effect of EPA and DHA must be considered⁽ Reference Huang, Chien and Chen ^{121 )}. In a rodent study that compared EPA and DHA supplementation, Bargut et al.⁽ Reference Bargut, Santos and Machado ^{36 )} showed that DHA had the most prominent action in white adipose tissue metabolism, modulating pro- and anti-inflammatory pathways and alleviating adipocyte abnormalities (caused by a high-fructose diet). In human studies comparing supplementation of EPA v. DHA, Allaire et al.⁽ Reference Allaire, Couture and Leclerc ^{51 )} showed that, related to modulation of specific markers of inflammation and blood lipids, DHA is more effective than EPA. However, Vors et al.⁽ Reference Vors, Allaire and Marin ^{53 )} showed that EPA or DHA has similar effects on the expression of many inflammation-related genes in immune cells of men and women at risk for cardiometabolic diseases.

As EPA and DHA can exert different effects, the ratio of EPA:DHA could affect the outcomes. It is observed that there is considerable variability in the ratio of EPA:DHA in the studies, and this should be taken into account. Shang et al.⁽ Reference Shang, Liu and Zhou ^{122 )} investigated diets with different ratios of DHA:EPA (2:1, 1:1 and 1:2) and showed that a lower DHA:EPA ratio seems to be more beneficial for alleviation of high-fat-diet-induced liver damage in mice, and a DHA:EPA ratio of 1:2 mitigated the inflammatory risk factors.

The form of n-3 PUFA formulations (as TAG, ethyl ester, free fatty acid or phospholipid) could also affect the bioavailability and actions of n-3 PUFA. Tang et al. ⁽ Reference Tang, Li and Xu ^{123 )} compared the effects ofTAG, ethyl ester, free fatty acid and phospholipid forms of n-3 PUFA (DHA) on lipid metabolism in mice (fed high-fat or low-fat diet) and showed that DHA-bound phospholipid showed effective bioactivity in decreasing hepatic and serum total cholesterol, TAG levels and increasing n-3 PUFA concentration in liver and brain. Rossmeisl et al.⁽ Reference Rossmeisl, Jilkova and Kuda ^{124 )} showed that, compared with TAG, n-3 PUFA (DHA and EPA) administered as phospholipids are superior in preserving a healthy metabolic profile under obesogenic conditions, possibly reflecting better bioavailability and improved modulation of the endocannabinoid system activity in white adipose tissue.

Another important topic that should be mentioned is the ratio n-6/n-3 PUFA. As vegetable oils (soyabean, corn, sunflower, safflower and cottonseed oils), which are rich in n-6 PUFA, became popularised, there was a significant increase in n-6 PUFA intake⁽ Reference Huang, Chien and Chen ^{121 )}. Western diets contain excessive levels of n-6 PUFA and very low levels of n-3 PUFA, leading to an unhealthy n-6/n-3 ratio of 20:1, instead of 1:1 during evolution⁽ Reference Simopoulos ^{125 )}. A high n-6 intake and a high n-6:n-3 ratio is pro-thrombotic, pro-inflammatory and are associated with weight gain in both animal and human studies, whereas a high n-3 PUFA intake decreases the risk for weight gain⁽ Reference Simopoulos ^{125 )}. Therefore, a balanced n-6:n-3 ratio is also important for the prevention and management of obesity (it is essential to decrease the n-6 PUFA in the diet while increasing the n-3 PUFA)⁽ Reference Simopoulos ^{125 )}.