Effect of dietary krill oil supplementation on the endocannabinoidome of metabolically relevant tissues from high-fat-fed mice

Abstract

Background: Omega-3 polyunsaturated fatty acids (ω-3-PUFA) are known to ameliorate several metabolic risk factors for cardiovascular disease, and an association between elevated peripheral levels of endogenous ligands of cannabinoid receptors (endocannabinoids) and the metabolic syndrome has been reported. We investigated the dose-dependent effects of dietary ω-3-PUFA supplementation, given as krill oil (KO), on metabolic parameters in high fat diet (HFD)-fed mice and, in parallel, on the levels, in inguinal and epididymal adipose tissue (AT), liver, gastrocnemius muscle, kidneys and heart, of: 1) the endocannabinoids, anandamide and 2-arachidonoylglycerol (2-AG), 2) two anandamide congeners which activate PPARα but not cannabinoid receptors, N-oleoylethanolamine and N-palmitoylethanolamine, and 3) the direct biosynthetic precursors of these compounds.

Methods: Lipids were identified and quantified using liquid chromatography coupled to atmospheric pressure chemical ionization single quadrupole mass spectrometry (LC-APCI-MS) or high resolution ion trap-time of flight mass spectrometry (LC-IT-ToF-MS).

Results: Eight-week HFD increased endocannabinoid levels in all tissues except the liver and epididymal AT, and KO reduced anandamide and/or 2-AG levels in all tissues but not in the liver, usually in a dose-dependent manner. Levels of endocannabinoid precursors were also generally down-regulated, indicating that KO affects levels of endocannabinoids in part by reducing the availability of their biosynthetic precursors. Usually smaller effects were found of KO on OEA and PEA levels.

Conclusions: Our data suggest that KO may promote therapeutic benefit by reducing endocannabinoid precursor availability and hence endocannabinoid biosynthesis.

Figure 1

Representative example of targeted profiling of diacylglycerols (DAGs) in lipid extracts using multi-dimensional mass spectrometry with LC-ESI-IT-ToF. A: Representative extracted ion chromatogram of a pre-purified liver lipid extract containing various DAG species (22:6-22:6, 18:2-22:6, 18:1-22:6, 16:0-22:6), including the biosynthetic 2-AG precursors that were measured here (18:1-20:4, 16:0-20:4, 18:0-20:4). B: Example of a positive-ion electrospray mass spectrum of a major component of the extract, the 18:0-20:4 DAG precursor ion (m/z 667.5273), as a sodium adduct, and the corresponding product ions for the CID of the fragment with m/z 667.5273 in the MS² spectra (m/z 327.2295 and 383.2562), which in turn correspond to the sodiated 20:4 acid and C₂₃H₃₆O₃ fragments, respectively.

Figure 2

Levels of 2-arachidonoylglycerol (2-AG, A), anandamide (AEA, B), N-palmitoylethanolamine (PEA, C) and N-oleoylethanolamine (OEA, D) in the liver of high-fat fed mice with dietary krill oil (HFKO) supplementation at different doses (1.25, 2.5 and 5% wt). Data are means ± SEM of separate determinations in N = 4-5 mice. ^## P < 0.01 and ^### P < 0.001 N vs HF; * P < 0.05 **P < 0.01 *** P < 0.001 high-fat diet (HF) vs HFKO1.25-2.5-5. N = normal diet.

Figure 3

Levels of 2-arachidonoylglycerol (2-AG, A), anandamide (AEA, B), N-palmitoylethanolamine (PEA, C) and N-oleoylethanolamine (OEA, D) in the gastrocnemius muscle of high-fat fed mice with dietary krill oil (HFKO) supplementation at different doses (1.25, 2.5 and 5% wt). Data are means ± SEM of separate determinations in N = 4-5 mice. ^# P < 0.05 N vs HF; * P < 0.05 **P < 0.01 *** P < 0.001 high-fat diet (HF) vs HFKO1.25-2.5-5. N = normal diet.

Figure 4

Levels of 2-arachidonoylglycerol (2-AG, A), anandamide (AEA, B), N-palmitoylethanolamine (PEA, C) and N-oleoylethanolamine (OEA, D) in the heart of high-fat fed mice with dietary krill oil (HFKO) supplementation at different doses (1.25, 2.5 and 5% wt). Data are means ± SEM of separate determinations in N = 4-5 mice. # P < 0.05 and ## P < 0.01 N vs HF; * P < 0.05 **P < 0.01 *** P < 0.001 high-fat diet (HF) vs HFKO1.25-2.5-5. N = normal diet.

Figure 5

Levels of 2-arachidonoylglycerol (2-AG, A), anandamide (AEA, B), N-palmitoylethanolamine (PEA, C) and N-oleoylethanolamine (OEA, D) in the epididymal adipose tissue of high-fat fed mice with dietary krill oil (HFKO) supplementation at different doses (1.25, 2.5 and 5% wt). Data are means ± SEM of separate determinations in N = 4-5 mice. ^# P < 0.05 and ^### P < 0.001 N vs HF; * P < 0.05 **P < 0.01 *** P < 0.001 high-fat diet (HF) vs HFKO1.25-2.5-5. N = normal diet.

Figure 6

Levels of 2-arachidonoylglycerol (2-AG, A), anandamide (AEA, B), N-palmitoylethanolamine (PEA, C) and N-oleoylethanolamine (OEA, D) in the inguinal adipose tissue of high-fat fed mice with dietary krill oil (HFKO) supplementation at different doses (1.25, 2.5 and 5% wt). Data are means ± SEM of separate determinations in N = 4-5 mice. ^## P < 0.01 N vs HF; * P < 0.05 and **P < 0.01 high-fat diet (HF) vs HFKO1.25-2.5-5. N = normal diet.

Figure 7

Levels of 2-arachidonoylglycerol (2-AG, A), anandamide (AEA, B), N-palmitoylethanolamine (PEA, C) and N-oleoylethanolamine (OEA, D) in the kidneys of high-fat fed mice with dietary krill oil (HFKO) supplementation at different doses (1.25, 2.5 and 5% wt). Data are means ± SEM of separate determinations in N = 4-5 mice. ^# P < 0.05 N vs HF; * P < 0.05, **P < 0.01 and *** P < 0.001 high-fat diet (HF) vs HFKO1.25-2.5-5. N = normal diet.