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Increased triacylglycerol - Fatty acid substrate cycling in human skeletal muscle cells exposed to eicosapentaenoic acid.

ABSTRACT: It has previously been shown that pretreatment of differentiated human skeletal muscle cells (myotubes) with eicosapentaenoic acid (EPA) promoted increased uptake of fatty acids and increased triacylglycerol accumulation, compared to pretreatment with oleic acid (OA) and palmitic acid (PA). The aim of the present study was to examine whether EPA could affect substrate cycling in human skeletal muscle cells by altering lipolysis rate of intracellular TAG and re-esterification of fatty acids. Fatty acid metabolism was studied in human myotubes using a mixture of fatty acids, consisting of radiolabelled oleic acid as tracer (14C-OA) together with EPA or PA. Co-incubation of myotubes with EPA increased cell-accumulation and incomplete fatty acid oxidation of 14C-OA compared to co-incubation with PA. Lipid distribution showed higher incorporation of 14C-OA into all cellular lipids after co-incubation with EPA relative to PA, with most markedly increases (3 to 4-fold) for diacylglycerol and triacylglycerol. Further, the increases in cellular lipids after co-incubation with EPA were accompanied by higher lipolysis and fatty acid re-esterification rate. Correspondingly, basal respiration, proton leak and maximal respiration were significantly increased in cells exposed to EPA compared to PA. Microarray and Gene Ontology (GO) enrichment analysis showed that EPA, related to PA, significantly changed i.e. the GO terms "Neutral lipid metabolic process" and "Regulation of lipid storage". Finally, an inhibitor of diacylglycerol acyltransferase 1 decreased the effect of EPA to promote fatty acid accumulation. In conclusion, incubation of human myotubes with EPA, compared to PA, increased processes of fatty acid turnover and oxidation suggesting that EPA may activate futile substrate cycling of fatty acids in human myotubes. Increased TAG-FA cycling may be involved in the potentially favourable effects of long-chain polyunsaturated n-3 fatty acids on skeletal muscle and whole-body energy metabolism.

SUBMITTER: Lovsletten NG

PROVIDER: S-EPMC6264501 | biostudies-literature | 2018

REPOSITORIES: biostudies-literature

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Increased triacylglycerol - Fatty acid substrate cycling in human skeletal muscle cells exposed to eicosapentaenoic acid.

Løvsletten Nils G NG Bakke Siril S SS Kase Eili T ET Ouwens D Margriet DM Thoresen G Hege GH Rustan Arild C AC

PloS one 20181129 11

It has previously been shown that pretreatment of differentiated human skeletal muscle cells (myotubes) with eicosapentaenoic acid (EPA) promoted increased uptake of fatty acids and increased triacylglycerol accumulation, compared to pretreatment with oleic acid (OA) and palmitic acid (PA). The aim of the present study was to examine whether EPA could affect substrate cycling in human skeletal muscle cells by altering lipolysis rate of intracellular TAG and re-esterification of fatty acids. Fatt ...[more]

PMID: 30496314

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Project description:Sarcopenic obesity is characterised by high fat mass, low muscle mass and an elevated inflammatory environmental milieu. We therefore investigated the effects of elevated inflammatory cytokine TNF-α (aging/obesity) and saturated fatty acid, palmitate (obesity) on skeletal muscle cells in the presence/absence of EPA, a-3 polyunsaturated fatty acid with proposed anti-inflammatory, anti-obesity activities. In the present study we show that palmitate was lipotoxic, inducing high levels of cell death and blocking myotube formation. Cell death under these conditions was associated with increased caspase activity, suppression of differentiation, reductions in both creatine kinase activity and gene expression of myogenic factors; IGF-II, IGFBP-5, MyoD and myogenin. However, inhibition of caspase activity via administration of Z-VDVAD-FMK (caspase-2), Z-DEVD-FMK (caspase-3) and ZIETD-KMK (caspase 8) was without effect on cell death. By contrast, lipotoxicity associated with elevated palmitate was reduced with the MEK inhibitor PD98059, indicating palmitate induced cell death was MAPK mediated. These lipotoxic conditions were further exacerbated in the presence of inflammation via TNF-α co-administration. Addition of EPA under cytotoxic stress (TNF-α) was shown to partially rescue differentiation with enhanced myotube formation being associated with increased MyoD, myogenin, IGF-II and IGFBP-5 expression. EPA had little impact on the cell death phenotype observed in lipotoxic conditions but did show benefit in restoring differentiation under lipotoxic plus cytotoxic conditions. Under these conditions Id3 (inhibitor of differentiation) gene expression was inversely linked with survival rates, potentially indicating a novel role of EPA and Id3 in the regulation of apoptosis in lipotoxic/cytotoxic conditions. Additionally, signalling studies indicated the combination of lipo- and cyto-toxic effects on the muscle cells acted through ceramide, JNK and MAPK pathways and blocking these pathways using PD98059 (MEK inhibitor) and Fumonisin B1 (ceramide inhibitor) significantly reduced levels of cell death. These findings highlight novel pathways associated with in vitro models of lipotoxicity (palmitate-mediated) and cytotoxicity (inflammatory cytokine mediated) in the potential targeting of molecular modulators of sarcopenic obesity.

Project description:Polyunsaturated fatty acids are important nutrients for human health, especially omega-3 fatty acids such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), which have been found to play positive roles in the prevention of various diseases. However, previous studies have reported that excessive omega-3 fatty acids supplement during pregnancy caused side effects such as slower neural transmission times and postnatal growth restriction. In this study, we investigated the effect of EPA and DHA on mitochondrial function and gene expression in C2C12 myoblasts during skeletal muscle differentiation. C2C12 myoblasts were cultured to confluency and then treated with differentiation medium that contained fatty acids (50-µM EPA and DHA). After 72?h of myogenic differentiation, mRNA was collected, and gene expression was analyzed by real-time PCR. Microscopy was used to examine cell morphology following treatment with fatty acids. The effect of EPA and DHA on cellular oxygen consumption was measured using a Seahorse XF24 Analyzer. Cells treated with fatty acids had fewer myotubes formed (P ? 0.05) compared with control cells. The expression of the genes related to myogenesis was significantly lower (P ? 0.05) in cells treated with fatty acids, compared with control cells. Genes associated with adipogenesis had higher (P ? 0.05) expression after treatment with fatty acids. Also, the mitochondrial biogenesis decreased with lower (P ? 0.05) gene expression and lower (P ? 0.05) mtDNA/nDNA ratio in cells treated with fatty acids compared with control cells. However, the expression of genes related to peroxisome biosynthesis was higher (P ? 0.05) in cells treated with fatty acids. Moreover, fatty-acid treatment reduced (P ? 0.05) oxygen consumption rate under oligomycin-inhibited (reflecting proton leak) and uncoupled conditions. Our data imply that fatty acids might reduce myogenesis and increase adipogenesis in myotube formation. Fatty acids may also decrease cell metabolism by reducing mitochondrial biogenesis as well as respiration rate. This study suggests that the maternal overdosage of EPA and DHA may influence fetal muscle development, increase intramuscular adipose tissue deposition in offspring, and have a long-term effect on the development of metabolic diseases such as obesity and diabetes in adult offspring.

Dataset Information

Increased triacylglycerol - Fatty acid substrate cycling in human skeletal muscle cells exposed to eicosapentaenoic acid.

Publications

Increased triacylglycerol - Fatty acid substrate cycling in human skeletal muscle cells exposed to eicosapentaenoic acid.

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