Abstract
Accumulation of excess saturated free fatty acids such as palmitate (PAL) in skeletal muscle leads to reductions in mitochondrial integrity, cell viability and differentiation. Omega-3 polyunsaturated fatty acids (n-3 PUFAs) such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) counteract PAL-induced lipid accumulation. EPA and DHA, as well as the n-3 PUFA docosapentaenoic acid (DPA), may therefore mitigate PAL-induced lipotoxicity to promote skeletal muscle cell survival and differentiation. C2C12 myoblasts were treated with 50 μM EPA, DPA, or DHA in the absence or presence of 500 μM PAL for 16 h either prior to myoblast analysis or induction of differentiation. Myoblast viability and markers of apoptosis, endoplasmic reticulum (ER) stress and myotube differentiation capacity were investigated using fluorescence microscopy and immunoblotting. High-resolution respirometry was used to assess mitochondrial function and membrane integrity. PAL induced cell death via apoptosis and increased protein content of ER stress markers BiP and CHOP. EPA, DPA, and DHA co-treatment maintained cell viability, prevented PAL-induced apoptosis and attenuated PAL-induced increases in BiP, whereas only DPA prevented increases in CHOP. PAL subsequently reduced protein content of the differentiation marker myogenin and inhibited myotube formation, and all n-3 PUFAs promoted myotube formation in the presence of PAL. Furthermore, DPA prevented PAL-induced release of cytochrome c and maintained mitochondrial integrity. These findings demonstrate the n-3 PUFAs EPA, DPA and DHA elicit similar protective effects against PAL-induced impairments in muscle cell viability and differentiation. Mechanistically, the protective effects of DPA against PAL lipotoxicity are attributable in part to its ability to maintain mitochondrial respiratory capacity via mitigating PAL-induced loss of mitochondrial membrane integrity.
Highlights
Excess cellular accumulation of lipids such as palmitate (PAL) is well appreciated to negatively impact skeletal muscle cell viability
There were no detectable changes between any of the PUFA treatments alone or PAL-PUFA cotreatments relative to CON in terms of nuclei number or brightness (Figures 1D,E). Both eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) attenuated PALinduced decreases in nuclei area (p < 0.05; Figure 1F) and perimeter (p < 0.05; Figure 1G) relative to CON-PAL, these indices were decreased relative to CON and PUFA alone
CON myoblasts treated with PAL exhibited a significant increase in mitochondrial respiration when exogenous cytochrome c was added (∼60%, p < 0.05), which was not observed when cells were co-treated with docosapentaenoic acid (DPA) (Figure 5B). This response occurred independently of changes in mitochondrial content, as there were no detectable differences in protein content of any of the mitochondrial OXPHOS complexes in myoblasts treated with PAL and/or DPA (Figures 6A–F). This is the first study to compare the protective effects of EPA, DHA, as well as DPA against the lipotoxic effects of PAL in skeletal muscle cells
Summary
Excess cellular accumulation of lipids such as palmitate (PAL) is well appreciated to negatively impact skeletal muscle cell viability. PAL exposure in vitro negatively impacts skeletal muscle cell metabolism by impairing insulin sensitivity (Hage Hassan et al, 2012), suppressing protein synthesis (Perry et al, 2018), and upregulating proteolytic machinery (Woodworth-Hobbs et al, 2014). The n-3 PUFA docosahexaenoic acid (DHA) has been shown to ameliorate lipotoxic effects of PAL in skeletal muscle cell models by restoring insulin sensitivity (Bryner et al, 2012) and preventing activation of the UPR in differentiated skeletal muscle myotubes (Woodworth-Hobbs et al, 2014). In contrast to DHA, eicosapentaenoic acid (EPA) is the only n-3 PUFA shown to protect against the deleterious effects of inflammation (Magee et al, 2008) and PAL exposure (Saini et al, 2017) by partially restoring the regenerative capacity of skeletal muscle. EPA and DHA are commonly found in fish oil supplements and have been demonstrated to improve markers of myogenic differentiation (i.e., myosin 4 expression and myotube fusion index) (Briolay et al, 2013)
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