In this pilot study we have confirmed data in the literature showing that overweight and obese subjects exhibit increased plasma levels of the endocannabinoids, AEA and 2-AG [2–4]. In our cohort of subjects, however, plasma 2-AG levels were increased significantly only in overweight individuals, whereas AEA levels were increased significantly only in obese subjects. This finding agrees with previous results suggesting that increased plasma AEA levels are associated with high BMI , whereas increased plasma 2-AG levels are associated with high visceral adipose tissue and not necessarily with high BMI [3, 18, 19]. It is possible that the cohort of obese subjects of the present study might have been characterized by a higher proportion of subcutaneous adipose tissue than in other cohorts previously investigated. As we did not acquire data on the adipose distribution in the obese subjects of a present study, this remains only a speculative hypothesis, to be specifically addressed in future studies. In addition, fat distribution in overweight premenopausal women may be different from that in postmenopausal women and in men for a given level of waist circumference. Thus, the small number of subjects in the present study and the heterogeneous nature of the sample (i.e., men as well as pre- and postmenopausal women) do not permit a meaningful assessment of the correlation of 2-AG levels with visceral fat distribution.
The novel finding of the present study is that KO, more efficiently than MO, was able to reduce endocannabinoid levels in the plasma despite the fact that the effects of the two dietary treatments on EPA and DHA plasma concentrations were comparable and even slightly lower in the KO group than in the MO group . Comparable results were obtained in the visceral adipose tissue, liver and heart of obese Zucker rats . However, in this previous study, 2-AG concentrations were decreased significantly by KO, and to a smaller extent by fish oil, only in the visceral adipose tissue. One possible explanation for the different effects of KO and fish oil might be, as previously suggested , the more efficient incorporation of n-3 LCPUFAs into visceral adipose tissue phospholipids, and subsequent decrease in arachidonic acid incorporation associated with KO supplementation, hence leading to impaired endocannabinoid biosynthesis.
Thus, it is tempting to suggest that plasma 2-AG mainly derives from this tissue, possibly because of its relatively high concentrations in this adipose depot. This hypothesis is in agreement with the strong correlations previously described between the amount of visceral adipose tissue and plasma 2-AG levels in overweight and obese subjects [18, 19]. By contrast, in the subcutaneous adipose tissue of obese animals  and obese subjects with type 2 diabetes , 2-AG levels seem to be rather decreased, indicating that the 2-AG levels in the plasma cannot be predicted from those in the subcutaneous fat, and vice versa.
The positive correlation between 2-AG and the plasma phospholipid n-6/n-3 LCPUFA ratio, and not with the absolute plasma phospholipid concentrations of n-3 or n-6 LCPUFA, suggests that at least 2-AG levels are strongly influenced by fatty acid metabolism involving the balance between n-6 and n-3 LCPUFA. Interestingly, it has been demonstrated that the n-6/n-3 LCPUFA ratio, rather than absolute values of n-6 and n-3 PUFA, is correlated to cardiovascular disease , which is also directly associated with many of the metabolic disorders that positively correlate with plasma 2-AG levels [3, 18, 19]. Thus, it is tempting to hypothesize that KO ameliorates cardiovascular disorders in overweight and obese subjects, at least in part, by re-establishing a physiological endocannabinoid tone at CB1 receptors, via decrease of the n-6/n-3 phospholipid LCPUFA ratio and, hence, reduction of the ultimate biosynthetic precursors of 2-AG, the up-regulation of which is instead associated with visceral obesity, dyslipidemia, insulin resistance and atherogenic inflammation . Since AEA is derived from AA esterified on the sn-1 position, and 2-AG from that esterified on the sn-2 position in the phospholipids, and since a reduction of the n-6/n-3 LCPUFA ratio would mostly affect the latter, this hypothesis, which is also based on the results from our previous study in Zucker rats, would also explain why KO only affected 2-AG and not AEA levels in the plasma. However, in the present study no significant differences in lipid metabolism, body weight or metabolic syndrome parameters were detected among the 3 groups of dietary treatments . Therefore, the hypothesis that KO-induced reduction of plasma 2-AG levels may result in an amelioration of the metabolic dysfunctions associated with overweight and obesity will require further investigation. Even though a recent report  showed that plasma phospholipid n-3 PUFA was inversely associated with the metabolic syndrome, the lack of changes in metabolic syndrome parameters in the subjects that were administered with KO may suggest that four weeks of such treatment, and of the consequent KO-induced inhibition of 2-AG levels, is not sufficient to exert any beneficial metabolic effects. Indeed, even the direct antagonism of CB1 with rimonabant (20 mg/day) in obese subjects starts reducing body weight and ameliorating dyslipidemia and insulin resistance only after 2-3 months from the beginning of treatment . Moreover, the lack of effect on triglyceride levels after treatment might be related to the fact that the participants in the study were normo-lipidemic. The lipid-lowering property of omega-3 fatty acids such as EPA and DHA is more pronounced in subjects with elevated triglycerides [23, 24].
Future studies will have to investigate whether longer dietary interventions and higher dietary levels of KO, apart from still down-regulating the endocannabinoid system, also improve the metabolic syndrome, thus possibly representing an alternative to CB1 antagonists/inverse agonists for the treatment of this disorder.