Given the well-known beneficial effects of LC n-3 PUFA on health (see the Introduction), it is of the utmost importance to define the optimal lipid form for delivering these fatty acids into the organism. We show here that dietary DHA and EPA supplemented as phospholipids were more efficient than triglycerides in ameliorating obesity-associated pathologies, including impaired glucose homeostasis, hepatic steatosis and dyslipidemia, as well as adipocyte hypertrophy and low-grade inflammation of WAT in mice fed a high-fat diet.
It is relatively difficult to compare the biological effectiveness of individual components of complex mixtures, as in case of the LC n-3 PUFA concentrates from marine fish used here. Thus, in the ‘prevention study’, assuming that LC n-3 PUFA are the major active constituents, cHF+ω3TG and cHF+ω3PL treatments were compared using diets matched for total DHA/EPA content (30 g per kg diet). Due to a higher DHA/EPA level in the triglyceride than in the phospholipid concentrate, a higher portion of dietary lipids had to be replaced by the phospholipid concentrate in order to achieve an equal dietary DHA/EPA content. Despite this fact, the ratios of total n-6 PUFA to n-3 PUFA in the two diets were quite similar. In order to eliminate a possible confounding effect of differential dilutions of corn oil (high in LA, i.e. n-6 PUFA) as in the case of triglyceride and phospholipid concentrates, the cHF+ω3PL-treatment was also performed at a 3-fold lower dietary DHA/EPA content (10 g DHA/EPA per kg diet), i.e. ~30% less dietary lipids were replaced by the phospholipid concentrate as compared with the cHF+ω3TG diet containing 30 g DHA/EPA per kg diet. Irrespectively of the DHA/EPA content, both cHF+ω3PL treatments preserved glucose tolerance and prevented hepatic steatosis, while the cHF+ω3TG-treatment did not. This made it possible to exclude the possibility that a higher efficacy of the phospholipid-based treatment was the result of a higher content of the concentrate in the diet rather than of qualitative differences between the LC n-3 PUFA concentrates. Moreover, when using the DHA/EPA-matched diets (i.e. 30 g DHA/EPA per kg), the cHF+ω3PL-treatment tended to exert stronger beneficial effects on weight gain, adiposity and fat cell size, plasma triglycerides, fasting glycemia and insulinemia.
In the ‘reversal study’, body weight, hepatic steatosis, adipocyte hypertrophy and low-grade inflammation of WAT were more potently reduced by the cHF+ω3PL-treatment. In contrast to the prevention study, blood glucose levels in obese mice were lowered to a similar extent by both treatments. However, only the cHF+ω3PL-treatment significantly decreased plasma insulin. Future experiments should therefore focus on detailed characterisation of changes in insulin sensitivity brought about by dietary phospholipids.
In line with previous studies 
, our results document better bioavailability of both DHA and EPA when supplemented as dietary phospholipids and this effect can largely be attributed to a marked improvement in EPA bioavailability (see also ref. 
). Accordingly, in humans, most orally administered PC is hydrolysed to lysoPC before absorption 
. This in turn is well absorbed and may augment LC n
-3 PUFA delivery 
. However, similar ratio between EPA and DHA content was found in the PC and the lysoPC fraction from the marine phospholipid concentrate used in our study, suggesting that the differences in EPA and DHA bioavailability could not be explained by their absorption via the lysoPC molecule.
In agreement with the previous observation in both humans 
and mice 
, saturable incorporation of DHA into total plasma lipids was observed, while a linear relationship between dietary concentration of EPA and its plasma level was found, which contributed to a relatively high bioavailability of EPA from the phospholipid concentrate. The difference in bioavailability was not reflected in changes in DHA and EPA in the composition of either liver or WAT triglycerides. However, in total liver phospholipids, EPA accumulated more and AA less in the cHF+ω3PL-treated mice than in the cHF+ω3TG-treated mice. The differences between the two treatments became apparent when the TOF-SIMS analysis was conducted. PE, PI and PC phospholipid species, such as PE 38
6, PE 40
6 and PI 38
5 in the liver and PE 40
6 in WAT, were the discriminating analytes. It is likely that these phospholipid species are enriched with DHA, since it has previously been shown that, in egg phospholipids from hens fed on seal blubber oil 
, there is a preferential incorporation of DHA into the PE, PI, and PC fraction 
, especially in the sn-2 position of the phospholipid molecule 
. These data are consistent with the notion that cell membrane phospholipids represent an important reservoir of DHA and EPA and their processing into lipid signalling molecules 
, and they suggest that the difference in the effectiveness of the treatments is largely explained by differential accumulation of DHA and EPA in specific phospholipid molecules (see also 
). It is also likely that the more potent reductions of AA in the phospholipid and diacylglycerol pools associated with (i) AA replacement in membrane phospholipids by LC n
-3 PUFA (for reviews, see 
), and (ii) lower dietary content of LA, which is the precursor of AA, contribute to the superior anti-inflammatory effects of the cHF+ω3PL-treatment. This could be due to a lower formation of both, the pro-inflammatory mediators and endocannabinoids derived from AA.
In line with previous data on krill oil 
, our results support the notion that the endocannabinoid system is involved in the mechanism of action of LC n
-3 PUFA. Levels of 2-AG in WAT were more efficiently reduced by LC n
-3 PUFA administered as phospholipids, which was paralleled by decreased levels of AA in the diacylglycerol fraction, attesting to the latter’s precursor role in 2-AG synthesis 
. In contrast to another report 
, the levels of AEA in WAT were relatively low and were further reduced by LC n
-3 PUFA in the diet. Therefore, as is the case in humans 
, it is unlikely that AEA contributes significantly to metabolic derangements in dietary obese mice. Dietary phospholipids also induced adipose levels of N
-acyl ethanolamines EPEA and especially DHEA, i.e. the metabolites of EPA and DHA, respectively, more strongly and effectively than triglycerides. The pathophysiological role of the endocannabinoid system in the development of adipose tissue inflammation 
or hepatic steatosis 
is well described, and anti-inflammatory effects of adipocyte-derived EPEA or DHEA have recently been suggested 
. Average daily food intake was slightly but insignificantly decreased in the cHF+ω3PL-treated mice, however the levels of N
-oleoylethanolamine, i.e. the endocannabinoid that is known to suppress food intake 
, were completely unchanged both in WAT and plasma of these mice. Thus, our results are in agreement with a specific role of the dysregulated endocannabinoid system in WAT in the pathophysiology of metabolic syndrome 
In fact, the increase in obesity prevalence in the United States during the 20th century could be in part due to the elevated consumption of LA, the precursor of AA, and hence also the precursor of the AA-derived endocannabinoids 
. Very recently, this idea was supported by dietary experiments in mice fed various diets differing in the LA content 
. This content was positively correlated with the levels of AA, 2-AG and AEA in phospholipids isolated both from the liver and erythrocytes, while dietary LA also promoted accumulation of body fat. In accordance with our results in plasma and WAT, addition of EPA and DHA to the diet resulted in a decrease of endocannabinoid levels in the liver and also in hypothalamus of mice fed experimental diets with a high LA content 
. Accordingly, we have observed before in mice that supplementation of a high-fat diet by LC n
-3 PUFA, under very similar conditions as used in the ‘prevention study’ here, reduced body fat depots when LA formed 53% of dietary lipids, but did not affect adiposity when the LA content represented 15% of dietary fat 
. Taken together, the data further support the role of dietary LA as the key factor controlling the activity of the endocannabinoid system, and the attenuation of the endocannabinoid system activity as the key mechanism underlying anti-obesity effects of dietary LC n
-3 PUFA supplementation under these conditions. However, precise involvement of various tissues in the metabolic impact of attenuated endocannabinoid system activity in response to LC n
-3 PUFA should be better characterized 
. Moreover, conflicting data exist regarding the potential role of dietary macronutrient composition in the anti-obesity effects of LC n
-3 PUFA 
. Nevertheless, the data also suggest that the experimental model of obesity induced in mice by feeding a high-fat diet containing high levels of LA, as used in our current study as well as in most of our previous studies 
, is highly relevant for understanding the beneficial metabolic effects of LC n
-3 PUFA supplementation in humans consuming an obesogenic Western diet.
Cannabinoid receptor 1 antagonist rimonabant has been used to effectively treat obesity. It had to be withdrawn from clinical practice, however, due to adverse side effects on the central nervous system. Selective blocking of the peripheral endocannabinoid system might nevertheless represent an effective and safe therapy 
. In this respect, modulation of the endocannabinoid system in the peripheral tissues by LC n
-3 PUFA administered as phospholipids should be further explored. With regard to treatment strategies for obesity-associated disorders, combined treatment using LC n
-3 PUFA as triglycerides and pharmaceuticals like thiazolidinediones has been shown to be very effective 
. The inclusion of LC n
-3 PUFA as phospholipids might increase the efficacy of such treatment by more potently modulating the activity of the peripheral endocannabinoid system.
To preserve health, most of the nutritional and cardiological societies worldwide recommend to increase intake of fatty sea fish, which contain LC n
-3 PUFA in both the triacylglycerol and phospholipid form. Nutritional supplements containing LC n
-3 PUFA as triglycerides (or ethyl-esters) are also considered 
. Our results are in favour of this strategy and they suggest that the efficacy of the supplements could be further augmented based on the use of marine-derived phospholipids.
In conclusion, compared with triglycerides, dietary LC n-3 PUFA as phospholipids from marine fish exert superior metabolic effects in the context of high-fat diet-induced obesity in mice. Dietary supplementation of relatively low doses of DHA and EPA administered as phospholipids resulted in better bioavailability, while more efficacious lowering of the AA content in cellular phospholipids resulted in reduced production of the classical endocannabinoids AEA and 2-AG and increased production of anti-inflammatory molecules such as EPEA and DHEA. By multiple mechanisms of action, dietary phospholipids of marine origin might thus substantially improve prevention and treatment strategies for obesity-associated metabolic disorders.