We have reported here that dietary supplementation to obese men of DHA and EPA, in the form of krill powder, efficiently and persistently reduces high triglycerides independently from any overall effect on body weight, and at the same time lowers plasma AEA levels, i.e. a marker of obesity thought to contribute, via peripheral CB1 receptor overactivation, to obesity-associated dysmetabolism .
The effect of krill powder on high triglyceride levels appeared to be maximal already after 12 weeks of supplementation, and persisted until the end of the study, i.e. at 24 weeks. This suggests that this effect was of a relatively rapid onset, sustained and not merely due to subjects undergoing periodic medical control.
Unlike triglycerides, the effect on plasma levels of the endocannabinoid AEA was already significant after 12 weeks, but was maximal after 24 weeks of supplementation. Thus, possibly also due to the relatively low number of subjects recruited for this study, no correlation was found between plasma AEA and triglyceride levels, either before, in the middle or at the end of the dietary supplementation. Interestingly, previous studies showed that, of the two endocannabinoids, AEA and 2-AG, it is rather the plasma levels of the latter compound that, in overweight/obese individuals, directly correlate with plasma triglycerides, as well as with other anthropometric and metabolic parameters, such as visceral adipose tissue, insulin resistance and low HDL-cholesterol [5, 6]. Furthermore, weight loss in abdominally obese men after one year of lifestyle (caloric restriction and exercise) intervention caused a reduction of the levels of both AEA and 2-AG, but only the latter: 1) correlated with weight loss-induced reduction in visceral adipose tissue, plasma triglycerides, insulin resistance and low HDL-cholesterol, and 2) through multivariate analyses was found to be an independent predictor of some of the observed reduction in plasma triglycerides .
Krill powder supplementation in this study: 1) reduced AEA, but not 2-AG, levels, this effect becoming maximal after the effect on triglyceride levels; and 2) produced only a trend towards the reduction of intra-abdominal fat. Therefore, it is tempting to speculate that krill oil-induced reduction of AEA levels is an effect that either occurs independently from the effect on triglycerides, or contributes, via reduced CB1 activation in peripheral organs, only to the sustained reduction of triglyceride levels. On the other hand, it was surprising to find that a reduction in plasma triglyceride levels (~20-22%) similar to the one (22.2%) previously shown  to be accompanied by reduced 2-AG levels, did not produce a similar effect in the present study, despite the fact that, as mentioned above, 2-AG is considered an independent predictor of at least part of plasma triglyceride levels in intra-abdominally obese men. It is possible that such correlation holds true only after weight loss, an effect not observed here, or following stronger reductions in intra-abdominal fat, or only in intra-abdominally obese men, as were the subjects of the previous study (who exhibited an average BMI of ~30 at the beginning of the study). Alternatively, it is also possible that the low number of obese men who underwent krill powder treatment (n = 11), a limitation of the present study, or the shorter duration of the present treatment as compared to the 1 year lifestyle intervention used in the previous study , prevented us from observing a reduction in 2-AG levels. However, it must be pointed out that a statistically significant, albeit small, effect on waist/hip ratio and visceral fat (if normalized to skeletal muscle mass) was observed here with krill powder. The former effect is in agreement with the previous finding of an association between high plasma AEA levels and waist circumference in mixed gender (predominantly female) non-obese and obese cohorts with average BMI of 21 and 42, respectively .
Whilst dietary supplementation of DHA and EPA in the form of fish oil had been already shown to reduce triglycerides in human subjects with signs of the metabolic syndrome [22–26], before the present report no such human data existed with regard to krill preparations. Importantly, in order to obtain a decrease of triglycerides similar to that found in the present study, higher doses of EPA plus DHA (3–4 g/day) in the form of fish oil are required . It has been claimed that fish oil exerts its effect through PPARα activation in the liver by DHA, thus strongly increasing fatty acid β-oxidation and reducing hepatic triglyceride formation and release . Krill oil and krill powder, however, differ from fish oil as they contain DHA and EPA mostly esterified to PLs rather than triglycerides. More than 80% of the EPA and DHA in krill oil/krill powder are associated with PLs, and the remaining is in triglyceride form . Interestingly, both dietary krill oil and n-3 LCPUFAs supplemented as PLs were recently shown to produce stronger beneficial effects than fish oil or n-3 LCPUFAs supplemented as triglycerides, respectively, on ectopic triglyceride accumulation, inflammation and other metabolic dysfunctions in obese Zucker rats and DIO mice [15, 30]. In these previous animal studies, krill oil or n-3 LCPUFAs supplemented as PLs were also found to be more effective at reducing endocannabinoid levels in peripheral organs, and, in Zucker rats, a stronger effect was found on AEA rather than 2-AG levels [15, 29]. On the other hand, in a cohort of mostly women with average BMI of ~31, administered with dietary krill oil at a dose of 2.5 g/day, the endocannabinoid the plasma levels of which were reduced after 4 weeks treatment was 2-AG and not AEA. This effect was more efficacious than with an n-3 LCPUFA-equivalent amount of fish oil, which, in fact, did not produce any effect . Therefore, it is possible that treatments with different formulations of krill oil, in different cohorts of subjects, and for a shorter duration, may produce different effects on plasma endocannabinoid levels. However, regardless of the type of formulation, protocol of administration and subjects or animal model of obesity undergoing the supplementation, the effect of krill oil on the peripheral levels of AEA and/or 2-AG is always accompanied by a reduction in the levels of esterified AA relative to EPA and DHA levels. This effect was observed also in the present study and we hypothesize that it may lead to an overall reduction of AEA biosynthetic precursor, N-arachidonoyl-phosphatidylethanolamine, which in turn would be responsible for the observed reduction in plasma AEA levels. Indeed, in the previous study in which n-3 LCPUFAs were supplemented as PLs to DIO mice , the reduction in white adipose tissue AEA levels was found to be accompanied by an equivalent elevation of the levels of its DHA- and EPA-derived congeners. Therefore, rather than, or in addition to, activation of PPARα, dietary EPA and DHA, especially in the form of krill preparations, might produce their effects on fasting triglycerides in obesity by re-equilibrating endocannabinoid levels and CB1 receptor tone. Interestingly, a decrease of the LCPUFA n-6/n-3 ratio in the plasma of hypercholesterolemic and overweight subjects, obtained with a different dietary intervention, was recently reported to be still accompanied by a 40% decrease in plasma AEA levels, but to be associated, instead, with a significant decrease of LDL-cholesterol . Moreover, krill powder also contains astaxanthin which could theoretically cause biological effects, but the concentration is believed to be too low to be of any relevance (approximately 200 μg in 4 g krill powder).
It was interesting to find here how the levels of two AEA-related metabolites, OEA and PEA, changed in the same direction as AEA following dietary krill powder supplementation. These two compounds are inactive at cannabinoid receptors but can stimulate peroxisome proliferator-activated receptor-α and transient receptor potential vanilloid type-1 channels , two lipid-sensitive proteins which have been involved in the control of metabolism. In a previous study in human lean, obese and type 2 diabetes obese subjects, the plasma levels of AEA, OEA and PEA were found to correlate with each other , which is not surprising since these three compounds share similar, although not fully overlapping, biosynthetic and degrading routes and enzymes (see [18, 19] and references cited therein). Therefore, we hypothesize that the small but significant reduction in the plasma levels of PEA and OEA might be due not only to the decrease of esterified palmitic and oleic acid relative to DHA and EPA, but also to alterations in the expression of their metabolic enzymes, as previously observed in osteoblasts , and that this might have contributed also to AEA, but not 2-AG, level reduction.
In conclusion, we have shown here that dietary supplementation with a krill powder persistently ameliorates high triglycerides without weight loss in obese men, to an extent similar to that observed following lifestyle-induced weight loss in a previous study in dyslipidemic overweight/obese men , and in a shorter period of time (12–24 weeks vs. one year). The understanding of whether or not this metabolic effect of dietary DHA and EPA is due to its concomitant reduction of plasma AEA levels, i.e. a potential index of peripheral CB1 receptor activity in humans, and of its subsequent action on lipid metabolism and triglyceride clearance evidenced in rodents [9, 10], will probably require further investigations with larger cohorts of subjects, varying doses of krill powder and several different time points. At any rate, given the ever increasingly established role of high triglycerides as an early determinant of insulin resistance, type 2 diabetes, atherosclerosis and cardiovascular risk in overweight and obese subjects, the present data suggest that dietary krill powder supplementation might represent a novel preventive strategy for these disorders.