Endocannabinoids derived from n-3 PUFAs - Formation, release and possible roles in inflammation and obesity

The fatty acid composition of our daily diet is considered a major determinant of long-term health risk and the development of disease. Several lines of evidence point toward a state of chronic ‘low-grade’ inflammation as an overarching process that is modulated by fatty acids and their different metabolites. Diets rich in omega-3 polyunsaturated fatty acids (PUFAs), among which docosahexaenoic acid (22:6n-3; DHA) have been found to be associated with a reduction of inflammatory activity. However, the mechanisms underlying these immune-modulatory effects of n-3 PUFAs are only partly known. Earlier data from our group and from other labs have provided evidence for an as yet largely unexplored mechanism involving the formation of DHA-derived fatty acid amides. Fatty acid amides (FAAs) are a group of lipids formed from fatty acids and biogenic amines, which are widely occurring in nature. An increasing number of FAAs, including conjugates of fatty acids with neurotransmitters and mono-amines, have been detected as endogenous molecules in different cells and tissues. However, their bioactivities have remained largely unknown so far.

In the first experimental part (chapter 2 and 3) of this thesis, we explored the immune-modulatory profiles of two relatively unknown DHA-derived FAAs conjugates with dopamine and serotonin, respectively.  In chapter 2, we enzymatically synthesised the dopamine conjugate of DHA, N-docosahexaenoyl dopamine (DHDA), and demonstrated that DHDA significantly suppressed the production of several mediators involved in (neuro-)inflammation. We showed that these immune-modulatory effects involved the enzyme cyclooxygenase-2 (COX-2), as its gene-expression and (or) production of its metabolite PGE2 were down-regulated by DHDA in both activated macrophages as well as microglia. Additionally, the immune-modulatory activities of DHDA were compared with those of N-arachidonoyl dopamine (NADA) and similar potencies were found in the cell types tested. In chapter 3, we investigated the effects of docosahexaenoyl serotonin (DHA-5-HT), the serotonin conjugate of DHA on inflammatory processes in human PBMCs. By comparing the immune-modulatory potencies of 6 serotonin-conjugates with palmitic acid (PA-5-HT), stearic acid (SA-5-HT), oleic acid (OA-5-HT), arachidonic acid (AA-5-HT), eicosapentaenoic acid (EPA-5-HT) and docosahexaenoic acid (DHA-5-HT),  DHA-5-HT turned out to exert the strongest inhibitory effects on the production of IL-17 from ConA-stimulated human PBMCs. Furthermore, DHA-5-HT concentration-dependently inhibited the production of IL-17 and CCL-20, two important Th17 mediators involved in the pathogenesis of IBD. Also, we demonstrated the in vivo presence of N-acyl serotonins in human intestine. Taken together, we revealed the immune-modulatory effects of two n-3 PUFA-derived fatty acid amides with thus far largely unknown functions and showed that these compounds were far more potent than its parent compound DHA.  These findings were shown not only for innate inflammatory processes in stimulated mouse macrophages, but were also found to be present in human PBMCs and likely involved the adaptive CD4+ Th17 response.

In order to study the effects of dietary omega-3 fatty acids on endocannabinoid tone in relation to obesity and metabolic health, a parallel-designed, randomized human study was conducted in the second part of the thesis. In this 12 weeks intervention hundred men and women with abdominal obesity were assigned to either a Western type energy restricted (ER) diet, a Targeted ER diet or a control group. The two ER diet groups were both subjected to energy restriction but their diets differed in nutrient composition. The traditional, more Western-style diet (Western ER diet) included both saturated as well as unsaturated fats, whereas the Targeted ER diet was amongst others enriched with monounsaturated fatty acids (MUFAs) and polyunsaturated fatty acids (PUFAs). This intervention resulted in significant weight loss and significant improvements of metabolic parameters in both energy restriction (ER) dietary intervention groups. In chapter 4, we revealed that the two weight loss regimes (ER-diets) with different fatty acid composition did not significantly affect fasting peripheral levels of AEA and 2-AG in both plasma and abdominal adipose tissues. By contrast, plasma DHEA was found to be significantly decreased in the Western ER group compared with the Targeted ER group. Additionally, circulating EC-related compounds DHEA, DHAGly, PEA and SEA were significantly decreased in the Western ER diet group after intervention. Furthermore, decreased levels of DHEA were positively associated with body weight reduction. In chapter 5, by performing a high calorie mixed meal test (MMT) before and after the intervention, we found that postprandial AEA and 2-AG levels were significantly reduced in the targeted ER group upon the intervention. By contrast, the DHA-derived compounds DHEA and DHAGly showed a significant increase in the Targeted ER group after 12 weeks of intervention. Additionally, all measured endocannabinoids and related compounds, with the exception of 2-AG, showed a similar characteristic time curve in response to the MMT, with EC levels reaching their highest concentration as early as 5 minutes after food intake (T=10min in experiment).

In conclusion, we showed here that two largely unknown amidated DHA conjugates are more potent mediators of inflammatory processes than their parent compound DHA. These findings support our previously proposed idea that DHA-derived FAAs play a role in the underlying mechanism of the beneficial health effects of DHA. We further uncovered that a combination of ER and n-3 PUFAs in the diet alters the postprandial endocannabinoid tone. Given the fact that the endocannabinoid system (ECS) plays an important role in both the central and peripheral regulation of food intake and energy homeostasis, these findings provide new insights in the potentially mechanisms involved in an over-activated endocannabinoid system during obesity.