Scientia Agricultura Sinica ›› 2014, Vol. 47 ›› Issue (1): 154-160.doi: 10.3864/j.issn.0578-1752.2014.01.016

• ANIMAL SCIENCE·VETERINARY SCIENCERE·SOURCE INSECT • Previous Articles     Next Articles

Inhibition of PI3K/AKT Pathway Suppressing Porcine Skeletal Muscle Sattelite Differentiation Through Activation of FoxO1 Transcription Factor

 SHI  Xin-E, WU  Guo-Fang, SONG  Zi-Yi, LU  Hong-Chao, JIA  Long, ZHU  Jia-Yu, YANG  Gong-She   

  1. Laboratory of Animal Fat Deposition and Muscle Development, College of Animal Science and Technology, Northwest A&F University, Yangling 712100, Shaanxi
  • Received:2013-05-02 Online:2014-01-01 Published:2013-06-20

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Abstract: 【Objective】Skeletal satellite cells are activated by some specific stresses such as development and trauma, and differentiate and form myotubes to participate in the development or repair of skeletal muscle. FoxO1 negatively controls the genesis of skeletal muscle, but the molecular mechanisms by which FoxO1 funcions in the differentiation of satellite cells have not been reported so far. This experiment was conducted to explore the effects of FoxO1 on porcine skeletal muscle satellite cells differentiation, aiming to provide new theoretical reference for further research. 【Method】Extensor digitorum longus of 1 to 3-day-old piglets were used to isolate the skeletal muscle satellite cells and the cells were observed and pictures were taken by inverted microscope on day 2, day 4 and day 6, respectively. The cells were stained by immunofluorescence staining and DAPI nuclear staining on day 8 of differentiation, and observed under a fluorescence microscope. Meanwhile, the medium was replaced with differentiation medium containing 50 nmol•L-1 wortmannin (wortmannin, WM) differentiation medium when the cells density reached 70% -80% confluence, the cells were collected on day 0, day 4, and day 8, respectively. Total RNA and total protein were extracted, and Real-time qPCR and Western blotting were performed to measure the alterations in the expression of FOXO1 and myogenic differentiation marker genes caused by WM supplement. 【Result】Porcine skeletal muscle satellite cells became adherent to the dish bottem, spindle-shaped on day 2. Cell number increased on day 4 and some cells started to fuse. On day 6, cell started to grow with directivity. On day 8, cell further fused to form myotubes, further fused to form myotubes. There was no significant difference in mRNA expression level of FoxO1 between WM treatment group and the control (P＞0.05), unphosphorylated FoxO1 increased significantly (P＜0.05) with WM treatment, whereas phosphorylation level of FoxO1 dropped drastically (P＜0.05). Although on day 8 the cells displayed an alveolate morphology after treated with WM, they failed to show directional growth and formation of myotubes. Moreover, Western blotting results demonstrated that WM decreased the protein level of MyoD (early myogenic marker), MyoG (middle-stage marker), and MyHC (late marker) significantly.【Conclusion】Results of the study suggest that inhibition of PI3K signaling pathway by WM blocks results in FoxO1 phosphorylation, suppression of porcine skeletal muscle satellite cell differentiation, delay of the formation of myotubes, and down-regulation of myogenic differentiation marker genes, such as MyoD, MyoG, and MyHC. Take together, blockade of PI3K signaling pathway suppresses porcine skeletal muscle satellite cell differentiation through the activation of FoxO1.

Key words: PI3K/AKT signaling , FoxO1 , skeletal muscle satellite , myogenic differentiation , porcine

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