Krill oil significantly decreases 2-arachidonoylglycerol plasma levels in obese subjects
- Sebastiano Banni1, 2Email author,
- Gianfranca Carta1, 2,
- Elisabetta Murru1, 2,
- Lina Cordeddu1, 2,
- Elena Giordano1, 2,
- Anna Rita Sirigu1, 2,
- Kjetil Berge3,
- Hogne Vik3,
- Kevin C Maki4,
- Vincenzo Di Marzo5 and
- Mikko Griinari6
© Banni et al; licensee BioMed Central Ltd. 2011
Received: 7 October 2010
Accepted: 30 January 2011
Published: 30 January 2011
We have previously shown that krill oil (KO), more efficiently than fish oil, was able to downregulate the endocannabinoid system in different tissues of obese zucker rats.
We therefore aimed at investigating whether an intake of 2 g/d of either KO or menhaden oil (MO), which provides 309 mg/d of EPA/DHA 2:1 and 390 mg/d of EPA/DHA 1:1 respectively, or olive oil (OO) for four weeks, is able to modify plasma endocannabinoids in overweight and obese subjects.
The results confirmed data in the literature describing increased levels of endocannabinoids in overweight and obese with respect to normo-weight subjects. KO, but not MO or OO, was able to significantly decrease 2-arachidonoylglycerol (2-AG), although only in obese subjects. In addition, the decrease of 2-AG was correlated to the plasma n-6/n-3 phospholipid long chain polyunsaturated fatty acid (LCPUFA) ratio. These data show for the first time in humans that relatively low doses of LCPUFA n-3 as KO can significantly decrease plasma 2-AG levels in obese subjects in relation to decrease of plasma phospholipid n-6/n-3 LCPUFA ratio. This effect is not linked to changes of metabolic syndrome parameters but is most likely due to a decrease of 2-AG biosynthesis caused by the replacement of 2-AG ultimate precursor, arachidonic acid, with n-3 PUFAs, as previously described in obese Zucker rats.
The endocannabinoid system is deeply involved in the regulation of the homeostasis of body composition by regulating food intake and energy expenditure. An overactive endocannabinoid system was suggested to contribute to increased fat mass and to several features of metabolic syndrome . In fact, an increase of anandamide (AEA) and 2-arachidonoylglycerol (2-AG) in overweight and obese subjects has been described [2–4].
A therapeutic approach aimed at re-establishing a physiological tone of the endocannabinoid system mainly relies on using antagonists of the one of their targets that is mostly responsible for their metabolic effects, i.e. the cannabinoid CB1 receptor . However, it has been shown that the use of these antagonists in obese individuals is accompanied by psychiatric side effects such as increased incidence of depression and anxiety [5, 6].
Endocannabinoids are ultimately derived from arachidonic acid incorporated in the sn-1 or sn-2 position of phospholipids, and their biosynthesis was shown to be affected by dietary fatty acids and in particular by EPA and DHA .
Recently , we have shown that in Zucker rats, an animal model of obesity, both krill oil (KO) and fish oil similarly increased EPA and DHA plasma levels, with KO being more effective than fish oil in improving some parameters of metabolic syndrome such as fatty liver and fatty heart. This might most probably be related to the stronger inhibitory effect of KO on endocannabinoid levels in these tissues and, particularly, in the visceral adipose tissue. In addition, we have recently reported that administration of 2 g/d of KO or menhaden oil (MO) for four weeks, significantly increased EPA and DHA levels in plasma in normal, overweight and obese subjects . This KO dose provided 216 mg/d EPA and 90 mg/d DHA, while MO provided 212 mg/d EPA and 178 mg/d DHA. The International Society for the Study of Fatty Acids and Lipids (ISSFAL) reported as a recommendation  for cardiovascular health a minimum intake of combined EPA and DHA of 500 mg/day, based on several studies showing a significant reduction of cardiovascular risk with this dose or higher. In addition, the effect of n-3 long chain polyunsaturated fatty acids (LCPUFA) on metabolic syndrome parameters has been shown to be effective at much higher doses .
To our knowledge, the effect of dietary n-3 fatty acids on AEA and 2-AG concentrations in human plasma has never been investigated. This issue is not trivial, since it is well established that plasma 2-AG levels in obese individuals strongly correlate with several parameters of the metabolic syndrome, including visceral adipose tissue, high triglyceride levels, low HDL-cholesterol levels and indices of insulin resistance. Therefore, in this study we aimed at verifying whether or not four-week dietary intake of KO, FO or olive oil (OO), is able to modify endocannabinoid levels in the plasma of normo-weight, overweight and obese subjects.
Materials and methods
This was a 4-week, randomized, double-blind, controlled, parallel clinical trial conducted at 2 clinical research sites in the United States (Provident Clinical Research, Bloomington, IN, and Meridien Research, St. Petersburg, FL). The study included 3 visits: 2 screening/baseline visits (weeks -1 and 0) and 1 end-of-treatment visit (week 4). An independent institutional review board, Quorum Review, Inc. (Seattle, Wash), approved the protocol before initiation of the study, and written informed consent was obtained from all subjects before protocol-specific procedures were performed.
Baseline demographic and anthropometric characteristics of subjects by treatment group.
(n = 19)
(n = 23)
(n = 21)
Normoweight, n, male/female
Overweight, n, male/female
Obese, n, male/female
Age, year, mean ± SEM
47.4 ± 8.5
49.6 ± 8.7
49.4 ± 8.5
Body mass index, kg/m2, mean ± SEM
30.6 ± 1.3
31.6 ± 0.9
30.1 ± 1.0
At baseline, eligible subjects were randomly assigned to 1 of 3 groups: 2 g/d of either KO (Superba krill oil, Aker BioMarine ASA, Oslo, Norway), MO (Omega-Pure, Houston, Tex), or olive oil (control). Subjects were instructed to consume four 500 mg capsules per day, preferably 2 capsules with each of 2 meals, for 4 weeks. Four capsules of the KO supplement provided 216 mg/d EPA and 90 mg/d DHA, and the MO supplement provided 212 mg/d EPA and 178 mg/d DHA. Stratification of the subjects has been carried out by BMI values: normoweight BMI <25; overweight 25 < BMI <30; obese 30 < BMI <35.
Further details of the study are described in .
Total lipids were extracted from plasma using chloroform/methanol 2:1 (v/v) . Separation of phospholipids (PL) from total lipids was performed as previously reported . Aliquots were mildly saponified as previously described  in order to obtain free fatty acids for HPLC analysis. Separation of fatty acids was carried out with an Agilent 1100 HPLC system (Agilent, Palo Alto, Calif., USA) equipped with a diode array detector as previously reported . N-arachidonoylethanolamine (anandamide, AEA) and 2-arachidonoylglycerol (2-AG) were measured as previously described .
One way ANOVA with the Bonferroni test for post-hoc analyses was applied to evaluate statistical differences between groups. Whereas t-student test for paired samples was applied to detect significant differences between before and after treatment.
No changes in BMI, waist circumference, glycemia and insulinemia were detected after any of the treatments (data not shown).
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.
The Authors thank Aker Biomarine ASA, Oslo, Norway, for supporting in part the study.
- Matias I, Petrosino S, Racioppi A, Capasso R, Izzo AA, Di Marzo V: Dysregulation of peripheral endocannabinoid levels in hyperglycemia and obesity: Effect of high fat diets. Mol Cell Endocrinol. 2008, 286: S66-78. 10.1016/j.mce.2008.01.026.View Article
- Annuzzi G, Piscitelli F, Di Marino L, Patti L, Giacco R, Costabile G, Bozzetto L, Riccardi G, Verde R, Petrosino S, Rivellese AA, Di Marzo V: Differential alterations of the concentrations of endocannabinoids and related lipids in the subcutaneous adipose tissue of obese diabetic patients. Lipids Health Dis. 2010, 9: 43-10.1186/1476-511X-9-43.View Article
- Di Marzo V, Cote M, Matias I, Lemieux I, Arsenault BJ, Cartier A, Piscitelli F, Petrosino S, Almeras N, Despres JP: Changes in plasma endocannabinoid levels in viscerally obese men following a 1 year lifestyle modification programme and waist circumference reduction: associations with changes in metabolic risk factors. Diabetologia. 2009, 52: 213-217. 10.1007/s00125-008-1178-6.View Article
- Sipe JC, Scott TM, Murray S, Harismendy O, Simon GM, Cravatt BF, Waalen J: Biomarkers of endocannabinoid system activation in severe obesity. PLoS One. 2010, 5: e8792-10.1371/journal.pone.0008792.View Article
- Di Marzo V, Despres JP: CB1 antagonists for obesity--what lessons have we learned from rimonabant?. Nat Rev Endocrinol. 2009, 5: 633-638. 10.1038/nrendo.2009.197.View Article
- Moreira FA, Grieb M, Lutz B: Central side-effects of therapies based on CB1 cannabinoid receptor agonists and antagonists: focus on anxiety and depression. Best Pract Res Clin Endocrinol Metab. 2009, 23: 133-144. 10.1016/j.beem.2008.09.003.View Article
- Banni S, Di Marzo V: Effect of dietary fat on endocannabinoids and related mediators: consequences on energy homeostasis, inflammation and mood. Mol Nutr Food Res. 2010, 54: 82-92. 10.1002/mnfr.200900516.View Article
- Batetta B, Griinari M, Carta G, Murru E, Ligresti A, Cordeddu L, Giordano E, Sanna F, Bisogno T, Uda S, Collu M, Bruheim I, Di Marzo V, Banni S: Endocannabinoids may mediate the ability of (n-3) fatty acids to reduce ectopic fat and inflammatory mediators in obese Zucker rats. J Nutr. 2009, 139: 1495-1501. 10.3945/jn.109.104844.View Article
- Maki KC, Reeves MS, Farmer M, Griinari M, Berge K, Vik H, Hubacher R, Rains TM: Krill oil supplementation increases plasma concentrations of eicosapentaenoic and docosahexaenoic acids in overweight and obese men and women. Nutr Res. 2009, 29: 609-615. 10.1016/j.nutres.2009.09.004.View Article
- International Society for the Study of Fatty Acids and Lipids: [http://www.issfal.org.uk/images/stories/pdfs/PUFAIntakeReccomdFinalReport.pdf]
- Carpentier YA, Portois L, Malaisse WJ: n-3 fatty acids and the metabolic syndrome. Am J Clin Nutr. 2006, 83: 1499S-1504S.
- Folch J, Lees M, Sloane Stanley GH: A simple method for the isolation and purification of total lipides from animal tissues. J Biol Chem. 1957, 226: 497-509.
- Banni S, Carta G, Angioni E, Murru E, Scanu P, Melis MP, Bauman DE, Fischer SM, Ip C: Distribution of conjugated linoleic acid and metabolites in different lipid fractions in the rat liver. J Lipid Res. 2001, 42: 1056-1061.
- Banni S, Carta G, Contini MS, Angioni E, Deiana M, Dessi MA, Melis MP, Corongiu FP: Characterization of Conjugated Diene Fatty Acids in Milk, Dairy Products, and Lamb Tissues. J Nutr Biochem. 1996, 7: 150-155. 10.1016/0955-2863(95)00193-X.View Article
- Melis MP, Angioni E, Carta G, Murru E, Scanu P, Spada S, Banni S: Characterization of conjugated linoleic acid and its metabolites by RP-HPLC with diode array detector. Eur J Lipid Sci Technol. 2001, 103: 617-621. 10.1002/1438-9312(200109)103:9<617::AID-EJLT6170>3.0.CO;2-C.View Article
- Di Marzo V, Goparaju SK, Wang L, Liu J, Batkai S, Jarai Z, Fezza F, Miura GI, Palmiter RD, Sugiura T, Kunos G: Leptin-regulated endocannabinoids are involved in maintaining food intake. Nature. 2001, 410: 822-825. 10.1038/35071088.View Article
- Engeli S, Bohnke J, Feldpausch M, Gorzelniak K, Janke J, Batkai S, Pacher P, Harvey-White J, Luft FC, Sharma AM, Jordan J: Activation of the peripheral endocannabinoid system in human obesity. Diabetes. 2005, 54: 2838-2843. 10.2337/diabetes.54.10.2838.View Article
- Bluher M, Engeli S, Kloting N, Berndt J, Fasshauer M, Batkai S, Pacher P, Schon MR, Jordan J, Stumvoll M: Dysregulation of the peripheral and adipose tissue endocannabinoid system in human abdominal obesity. Diabetes. 2006, 55: 3053-3060. 10.2337/db06-0812.View Article
- Cote M, Matias I, Lemieux I, Petrosino S, Almeras N, Despres JP, Di Marzo V: Circulating endocannabinoid levels, abdominal adiposity and related cardiometabolic risk factors in obese men. Int J Obes (Lond). 2007, 31: 692-699.
- Izzo AA, Piscitelli F, Capasso R, Aviello G, Romano B, Borrelli F, Petrosino S, Di Marzo V: Peripheral endocannabinoid dysregulation in obesity: relation to intestinal motility and energy processing induced by food deprivation and re-feeding. Br J Pharmacol. 2009, 158: 451-461. 10.1111/j.1476-5381.2009.00183.x.View Article
- Griffin BA: How relevant is the ratio of dietary n-6 to n-3 polyunsaturated fatty acids to cardiovascular disease risk? Evidence from the OPTILIP study. Curr Opin Lipidol. 2008, 19: 57-62. 10.1097/MOL.0b013e3282f2e2a8.View Article
- Huang T, Bhulaidok S, Cai Z, Xu T, Xu F, Wahlqvist ML, Li D: Plasma phospholipids n-3 polyunsaturated fatty acid is associated with metabolic syndrome. Mol Nutr Food Res. 54: 1628-1635. 10.1002/mnfr.201000025.View Article
- Harris WS: Fish oils and plasma lipid and lipoprotein metabolism in humans: a critical review. J Lipid Res. 1989, 30: 785-807.
- Harris WS: n-3 fatty acids and serum lipoproteins: human studies. Am J Clin Nutr. 1997, 65: 1645S-1654S.
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.