Effects of Rhizome Extract of Dioscorea batatas and Its Active Compound, Allantoin, on the Regulation of Myoblast Differentiation and Mitochondrial Biogenesis in C2C12 Myotubes

Abstract

With the aging process, a loss of skeletal muscle mass and dysfunction related to metabolic syndrome is observed in older people. Yams are commonly use in functional foods and medications with various effects. The present study was conducted to investigate the effects of rhizome extract of Dioscorea batatas (Dioscoreae Rhizoma, Chinese yam) and its bioactive compound, allantoin, on myoblast differentiation and mitochondrial biogenesis in skeletal muscle cells. Yams were extracted in water and allantoin was analyzed by high performance liquid chromatography (HPLC). The expression of myosin heavy chain (MyHC) and mitochondrial biogenesis-regulating factors, peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α), sirtuin-1 (Sirt-1), nuclear respiratory factor-1 (NRF-1) and transcription factor A, mitochondrial (TFAM), and the phosphorylation of AMP-activated protein kinase (AMPK) and acetyl-CoA carboxylase (ACC) were determined in C2C12 myotubes by reverse transcriptase (RT)-polymerase chain reaction (RT-PCR) or western blot. The glucose levels and total ATP contents were measured by glucose consumption, glucose uptake and ATP assays, respectively. Treatment with yam extract (1 mg/mL) and allantoin (0.2 and 0.5 mM) significantly increased MyHC expression compared with non-treated myotubes. Yam extract and allantoin significantly increased the expression of PGC-1α, Sirt-1, NRF-1 and TFAM, as well as the phosphorylation of AMPK and ACC in C2C12 myotubes. Furthermore, yam extract and allantoin significantly increased glucose uptake levels and ATP contents. Finally, HPLC analysis revealed that the yam water extract contained 1.53% of allantoin. Yam extract and allantoin stimulated myoblast differentiation into myotubes and increased energy production through the upregulation of mitochondrial biogenesis regulators. These findings indicate that yam extract and allantoin can help to prevent skeletal muscle dysfunction through the stimulation of the energy metabolism.

Keywords: C2C12 cells; Chinese yam; Dioscorea batatas; Dioscoreae Rhizoma; allantoin; mitochondrial biogenesis; myoblast differentiation.

Figure 1

Effects of yam extract and allantoin on the expression of MyHC protein and mRNA in C2C12 myotubes. C2C12 myoblasts were differentiated with DMEM containing 2% HS for 5 days, then treated with or without yam extract (0.5 and 1.0 mg/mL) or allantoin (0.2 and 0.5 mM) for 24 h. Metformin (2.5 mM) was used as a positive control. The expression of MyHC mRNA (A) and protein (B) was determined by RT-PCR and western blot, respectively. GAPDH and β-actin were used as internal controls. All data were presented as the means ± SEM of three independent experiments. Y, yam extract; A, allantoin; and M, metformin. * p < 0.05 and *** p < 0.001 vs. non-treated cells; (C) the myotubes were stained with anti-MyHC antibody and DAPI, then observed by fluorescence microscopy (original magnification = 200×). Green, MyHC-positive cells; and blue, DAPI-positive nuclei.

Figure 2

Effects of yam extract and allantoin on the expression of mitochondrial biogenesis-regulating factors in C2C12 myotubes. Differentiated myotubes were treated with or without yam extract (0.5 and 1.0 mg/mL) or allantoin (0.2 and 0.5 mM) for 24 h, after which the expression of PGC1α (A,B), NRF-1 (C,D), TFAM (E,F) and Sirt-1 (G,H) mRNA (A,C,E,G) and protein (B,D,F,H) was analyzed by RT-PCR (A,C,E) and western blot (B,D,F), respectively. Metformin (2.5 mM) was used as a positive control. GAPDH and β-actin were used as internal controls. Each band was presented as a representative figure and the histogram was calculated from the band density value of each experiment. All data were presented as the means ± SEM of three independent experiments. Y, yam extract; A, allantoin; and M, metformin. * p < 0.05, ** p < 0.01 and *** p < 0.001 vs. non-treated negative control.

Figure 3

Effects of yam extract and allantoin on the phosphorylation of AMPK and ACC protein inC2C12 myotubes. Differentiated C2C12 myotubes were treated with or without yam extract (0.5 and 1.0 mg/mL) or allantoin (0.2 and 0.5 mM) for 24 h and the phosphorylation of AMPK (A) and ACC (B) protein was investigated by western blot. Metformin (2.5 mM) was used as a positive control. Each band was presented as a representative figure and a histogram was calculated from the band density value of each experiment. β-actin were used as an internal control. All data were presented as the means ± SEM of three independent experiments. Y, yam extract; A, allantoin; and M, metformin. * p < 0.05, ** p < 0.01 and *** p < 0.001 vs. non-treated negative control.

Figure 4

Effects of yam extract and allantoin on the expression of GLUT-4 and the levels of glucose in C2C12 myotubes. Differentiated myotubes were treated with or without yam extract (0.5 and 1 mg/mL) or allantoin (0.2 and 0.5 mM) for 24 h. (A) The expression of GLUT-4 protein was determined by western blot. Metformin (2.5 mM) was used as a positive control and β-actin was used as an internal control. Each band was presented as a representative figure and a histogram was calculated from the band density value of each experiment. The levels of glucose in culture medium (B) and in the cells (C) were measured by a glucose consumption assay and glucose uptake assay, respectively. The contents of ATP in the myotubes were measured using an ATP assay kit (D). All data were presented as the means ± SEM of three independent experiments. Y, yam extract; A, allantoin; and M, metformin. * p < 0.05 and ** p < 0.01 vs. non-treated negative control.

Figure 5

HPLC analysis of allantoin in the yam extract: (A) allantoin as a standard compound, and (B) allantoin in the water extract. a, allantoin (retention time: 3.253 min).