Abstract
Maternal dietary supplementation of n−3 polyunsaturated fatty acids (n−3 PUFAs), especially eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), is considered to play positive roles in fetal neuro system development. However, maternal n−3 PUFAs may induce molecular reprogramming of uncommitted fetal myoblasts into adipocyte phenotype, in turn affecting lipid metabolism and energy expenditure of the offspring. The objective of this in vitro study was to investigate the combined effects of EPA and DHA on C2C12 cells undergoing brown adipogenic differentiation. C2C12 myoblasts were cultured to confluency and then treated with brown adipogenic differentiation medium with and without 50 μM EPA and 50 μM DHA. After differentiation, mRNA and protein samples were collected. Gene expression and protein levels were analyzed by real-time PCR and western blot. General Proteomics analysis was conducted using mass spectrometric evaluation. The effect of EPA and DHA on cellular oxygen consumption was measured using a Seahorse XFP Analyzer. Cells treated with n−3 PUFAs had significantly less (P < 0.05) expression of the brown adipocyte marker genes PGC1α, DIO2, and UCP3. Expression of mitochondrial biogenesis-related genes TFAM, PGC1α, and PGC1β were significantly downregulated (P < 0.05) by n−3 PUFAs treatment. Expression of mitochondrial electron transportation chain (ETC)-regulated genes were significantly inhibited (P < 0.05) by n−3 PUFAs, including ATP5J2, COX7a1, and COX8b. Mass spectrometric and western blot evaluation showed protein levels of enzymes which regulate the ETC and Krebs cycle, including ATP synthase α and β (F1F0 complex), citrate synthase, succinate CO-A ligase, succinate dehydrogenase (complex II), ubiquinol-cytochrome c reductase complex subunits (complex III), aconitate hydratase, cytochrome c, and pyruvate carboxylase were all decreased in the n−3 PUFAs group (P < 0.05). Genomic and proteomic changes were accompanied by mitochondrial dysfunction, represented by significantly reduced oxygen consumption rate, ATP production, and proton leak (P < 0.05). This study suggested that EPA and DHA may alter the BAT fate of myoblasts by inhibiting mitochondrial biogenesis and activity and induce white-like adipogenesis, shifting the metabolism from lipid oxidation to synthesis.
Highlights
Worldwide obesity prevalence is increasing, in the United States (Shin et al, 2018)
To confirm changing C2C12 cells’ genetic profiles and losing their myogenic ability, we investigated the combined effect of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) on the relative expression of genes regulating the terminal differentiation of myoblast into mature multinucleated myotubes
We found that the expression level of genes regulating myogenesis including Myf5, MyoD, MyoG, and MRF4 (Myf6) was significantly down-regulated in EPA/DHA treated group in comparison with control group (35 ± 4.5%, P = 0.02; 89 ± 2.3%, P = 0.00001; 61 ± 4.4%, P = 0.02; and 92 ± 0.71%, P = 0.0019, respectively) (Figure 1A)
Summary
Worldwide obesity prevalence is increasing, in the United States (Shin et al, 2018). Other important mechanisms may exert a profound impact on the thermogenic function of BAT and relevant overall body energy expenditure independence of any induction of UCP1 leading to promoting substrates oxidation and preventing lipid accumulation. Examples of such methods are pharmacological activation of AMPK induced fat oxidation (Gaidhu et al, 2011), suppressing adipocytes re-esterification capacity (Gaidhu et al, 2011), and enhancing cytochrome oxidase activity in UCP1-ablated upon cold stimulus (Meyer et al, 2010). It was reported that mitochondrial dysfunction-induced impairing oxidative capacity in brown adipocytes even without any change in UCP1 level is strongly linked to diet-induced obesity (Feldmann et al, 2009)
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