Induction of lipid oxidation by polyunsaturated fatty acids of marine origin in small intestine of mice fed a high-fat diet

Background Dietary polyunsaturated fatty acids (PUFA), in particular the long chain marine fatty acids docosahexaenoic (DHA) and eicosapentaenoic (EPA), are linked to many health benefits in humans and in animal models. Little is known of the molecular response to DHA and EPA of the small intestine, and the potential contribution of this organ to the beneficial effects of these fatty acids. Here, we assessed gene expression changes induced by DHA and EPA in the wildtype C57BL/6J murine small intestine using whole genome microarrays and functionally characterized the most prominent biological process. Results The main biological process affected based on gene expression analysis was lipid metabolism. Fatty acid uptake, peroxisomal and mitochondrial beta-oxidation, and omega-oxidation of fatty acids were all increased. Quantitative real time PCR, and -in a second animal experiment- intestinal fatty acid oxidation measurements confirmed significant gene expression differences and showed in a dose-dependent manner significant changes at biological functional level. Furthermore, no major changes in the expression of lipid metabolism genes were observed in the colon. Conclusion We show that marine n-3 fatty acids regulate small intestinal gene expression and increase fatty acid oxidation. Since this organ contributes significantly to whole organism energy use, this effect on the small intestine may well contribute to the beneficial physiological effects of marine PUFAs under conditions that will normally lead to development of obesity, insulin resistance and diabetes.


both control and intervention group.
To investigate the effects of EPA&DHA on gene expression in the intestinal tract, we isolated RNA from scrapings from small intestine of mice following a 4-week dietary intervention.
We compared, using whole genome microarray analysis as initial step, the mice fed the control sHF diet, which was rich in ALA and free of EPA or DHA, with the mice fed the isocaloric sHFf-F2 diet, in which 44 % of lipids were replaced by an EPA and DHA concentrate.
In total, 1474 probesets showed a significant change in expression by EPA&DHA compared to control diet (p<0.0027, Affymetrix criteria -see methods section-; downregulated probesets with -9.78 ≤ fold change (FC) ≤ -1.19 and upregulated probesets with 1.11 ≤ FC ≤ 6.96). As dietary intervention at physiological levels is known to generally give small effects on gene expression changes ( [1,2], own unpublished observations), we performed initial pathway analyses using an absolute FC ≥ 1.5 for all significantly regulated genes, which resulted in a total of 621 probesets (420 increased, 201 decreased). Metabolism, and especially lipid metabolism, was identified as the major regulated process. A similar approach using a more stringent condition, FC ≥ 2.0, resulted in 155 probesets (Supplementary Table 1), of which 130 were annotated genes. Increased expression was observed for 99 annotated probesets (73 unique genes), while 31 annotated probesets (27 unique genes) showed decreased expression (Table 1). Pathway analysis using those 130 probesets resulted in a similar list of differentially regulated pathways when we compared these results with the list obtained using FC ≥ 1.5 (data not shown). Again, metabolism was highly regulated (49% of differentially expressed genes) and involved changes in fatty acid uptake, fatty acid oxidation and cholesterol biosynthesis, amongst others. Of note, most, if not all, pathways selected were linked together by the energy molecule acetyl-CoA. Based on the sufficiently large group of genes with FC ≥ 2.0, we focussed on those 100 unique, differentially expressed genes (see Table 1).
Detailed inspection of the expression data (see Additional file 1; see also for full names of the genes) revealed that EPA&DHA induced expression of the genes mediating fatty acid uptake from the lumen into intestinal tissue, namely of Cd36 (FC=2.3) and Scarb1 (FC=2.2). However, expression of the class of fatty acid binding proteins (Fabp1,2,4-6), including intestinal-specific Fabp2, was not changed. Remarkably, small intestine of the EPA&DHA mice showed also increased expression of the genes of fatty acid β-oxidation (both in peroxisomes and mitochondria) and fatty acid ω-oxidation, the latter is indicated by the increased expression of Cyp4a10 (FC=5.6).
In more detail, the genes involved in both branched chain and straight chain fatty acid β-oxidation with a more than two fold increase due to the intake of EPA&DHA were Acox2 increased as well, suggesting increased transport of fatty acids across the mitochondrial membrane. Expression of cytosolic Acot1 (FC=5.1) and Acot12 (FC=1.6) was also increased.
Importantly, also expression of Pdk4 was increased by EPA&DHA (FC=3.0), which strongly suggested a switch from glycolysis to fatty acid oxidation [3]. Many of the aforementioned genes are targets of peroxisome proliferator activated receptor (PPAR) alpha [4], which itself was also upregulated by EPA&DHA (FC=2.0). Clearly, such a cooperative inhibition of the majority of the genes in this pathway suggests an orchestrated function within the small intestine. However, the main transcription factor regulating this pathway, Srebf1 (the mouse counterpart of human SREBP), did not show differential expression. Interestingly, elastase 3B (FC=8.7) and ScarB1 (FC=2.2), two putative transporters of cholesterol, were upregulated, as was the known cholesterol transporter Abca1 (FC=1.4). Regulation by n-3 PUFAs was likely given the fact that we could not detect a difference in the cholesterol content between the two diets (data not shown), but the mechanisms involved remain unexplained. Finally, downstream steroid hormone biosynthesis was upregulated as shown by a few family members of Hsd3b (Hsd3b2: FC=2.3; Hsd3b3 FC=2.0) and Hsd17b (Hsd17b4 two probesets: FC=1.6 and 2.0; Hsd17b13 FC=2.4).