Unpicking the Molecular Basis of n-3 Fatty-Acid Action in Skeletal Muscle with Systems Based Approaches.

Abstract

PURPOSE: Supplementation of omega3 fatty acids from fish oil improves the anabolic response of skeletal muscle to amino acid infusion and also leads to improvements in muscle function following resistance training. These effects are thought to be mediated partly by the incorporation of the essential fatty acids eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) into the skeletal muscle. Supplementation of EPA in cultured skeletal muscle cells improves metabolic flexibility. However, little is known about the mechanism of action of these fatty acids and in the context of muscle anabolism EPA seems to be more potent than DHA. We find that EPA but not DHA increases the protein content of C2C12 myotubes and basal /insulin stimulated glucose uptake. The differential response of cultured muscle to EPA and DHA allowed us to take a subtractive approach with systems based approaches to dissect out the mechanism of action of EPA. The purpose of this study was therefore to determine the molecular basis of EPA’s effects on muscle metabolism. METHODS: We carried out fatty acid profiling, global lipidomics, targeted lipidomics and SILAC based proteomics in conjunction with assessing glucose uptake and protein accretion. Where appropriate we used multivariate analysis to determine significant changes and we subtracted the effects of DHA from those of EPA to understand how EPA regulates muscle metabolism. RESULTS: Fatty acid profiling revealed that EPA and DHA are effectively incorporated into the cultured myotubes and that n-9, n-6, and MUFA’s are displaced. Fatty acid profiling showed that DPA was the most differentially regulated fatty acid suggesting that the elongation of EPA to DPA may be a key event in the action of EPA. Global lipidomic profiling revealed that many of the lipid species incorporating EPA/DPA or DHA with EPA/DHA treatment were in the phospholipid pool. Phospholipidomic screening revealed that the most differentially regulated species were those that had incorporated EPA or DPA and this effect seemed to occur largely in the phosphatidylethanolamine pool. We hypothesised that these treatments would alter the proteomic profile. We find that EPA and DHA differentially regulate the expression of collagens, membrane binding proteins and ubiquitination proteins. CONCLUSION: These data indicate that EPA but not DHA restructures the lipid and protein landscape of the muscle cell to enhance protein accretion and glucose transport.