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| The Effects of N-Oleoyl Glycine on the Proliferation, Differentiation and Protein Deposition of C2C12 Cells |
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Abstract Fatty acyl amino acids, an endogenous compounds from the dehydration synthesis of fatty acids and amino acids, plays an important role of anti-inflammatory, cell proliferation and cell apoptosis. Skeletal muscle protein deposition is promoted by extracellular signal-regulated kinase (ERK) in mitogen-activated protein kinase (MAPK) signaling pathway and ribosomal protein (rpS6) in mammalian target of rapamycin (mTOR) signaling pathway, while muscle RING finger 1 (MuRF1) and muscle atrophy F-box (MAFBx) in forkheadbox O1 (FoxO1) signaling pathway increase protein degradation. In addition, fatty acyl amino acids could be detected in skeletal muscle, which also express their receptors. To identify the effects of N-Oleoyl glycine (OLGly) on the proliferation, differentiation and protein deposition of mouse (Mus musculus) myoblast cell line (C2C12), cell counting kit-8 (CCK-8) was used to calculate cell numbers and detected creatine kinase activity to identify the differentiation level of C2C12. Moreover, qRT-PCR was adopted to test the mRNA expression level of proliferating cell nuclear antigen (PCNA), cell cycle protein dependent kinase inhibiting factors (p27 and p21), myogenic detemination gene 5 (Myf-5) and myogenic regulatory factor (MyOG), which were the marker genes of proliferation and differentiation of C2C12 cells. In this study, total protein content and the protein synthesis level (puromycin incorporation) were tested. Western blot was used to detect the expression level of mTOR, ERK, S6, FoxO1, MAFBx and MuRF1. The results showed that 0.2, 2 or 20 μmol/L of OLGly had no significant effect on the cell numbers and the activity of creatine kinase, as well as the marker genes expression of proliferation and differentiation of C2C12 cells. Interestingly, OLGly could significantly enhance the protein contents of C2C12 myotubes in dose and time-dependent manners. 20 μmol/L of OLGly significantly enhanced the total contents of protein and the incorporation contents of puromycin (P＜0.05). However, the same dose of the mixture of oleic acid and glycine had no significant effect on protein synthesis. Together, these results indicated that the effect of OLGly on protein synthesis in C2C12 myotubes was not mediated by its metabolites. Furthermore, the protein expression of the p-ERK and p-S6 were significantly elevated in response to 20 μmol/L OLGly treatment for 30 and 60 min (P＜0.05). However, both OLGly and the mixture of its motabolites, oleic acid and glycine, unable to alter the protein expression of p-mTOR. For the protein degradation, OLGly had no effects on the protein expression of p-FoxO1, MAFBx and MuRF1 and the mRNA expression level of MAFBx and MuRF1. In summary, the present evidences confirmed that OLGly promoted protein synthesis, while had no effect on the proliferation and differentiation of C2C12 cells. The upregulation of ERK and S6 might be involved in the process for OLGly induced protein synthesis. The present results will provide the experimental basis for the development of novel drugs to promote skeletal muscle growth and protein deposition.
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Received: 29 December 2015
Published: 20 May 2016
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