FoxO家族含有4名成員(FoxO1/FKHR, FoxO3/FKHRL1, FoxO4/AFX, FoxO6)，這些成員上均有一段consensus DNA 序列 TTGTTTAC，其功能相當於轉錄因子。FoxOs轉錄因子在調節細胞的分化、增殖、代謝及生存等相關功能上扮演重要的角色。近來研究發現FoxOs可經由PI3K的磷酸化來調控下游基因的表現，而影響肌肉的分化。一些研究指出FoxO3在骨骼肌當中表現會造成肌肉萎縮，但其他成員對肌肉造成相關性影響的功能及機制並未深入探討。為了觀察FoxOs在肌肉細胞生長及分化中所扮演的角色，因此在本研究中利用反轉錄病毒來穩定的大量表達FoxOs在肌肉纖維母細胞中。在大量表達FoxOs的肌肉細胞株中經誘導細胞分化後，觀察其型態上的差異，發現在大量表現FoxO1-AAA (FoxO1基因持續表現的突變體)、FoxO6的細胞株中，其分化的功能有被抑制的現象，其他細胞株與控制組對照之下並無明顯差異。進而抽取其不同時期 (細胞快速生長時期、細胞聚合時期、細胞分化時期) 的RNA並做RT-PCR來觀察各個FoxOs對肌肉分化相關性的特殊基因的影響。從實驗數據中發現在大量表現FoxO1-AAA的細胞株在經誘導分化之後，MRFs家族 (MyoD、MEF2C、Myf5及Myogenin) 的RNA表現量均有下降之情形，MRFs家族是調控肌肉特定基因轉錄的因子。因此可推測造成大量表現FoxO1-AAA細胞株不分化的原因可能是藉由調控MRFs基因來控制細胞生長及分化。要確定造成細胞不分化的相關機制是經由哪些訊息所影響的，因此我們在這些大量表現FoxOs而不分化的細胞株中，給予胰島素，Y27632及A23187處理，來觀察它們對於肌肉細胞末期分化所造成的影響，在實驗中發現當大量表現FoxO1-AAA時原本經誘導而不分化的型態下，在加入了胰島素時肌肉細胞反而走向分化。在實驗中觀察到葡萄糖濃度對肌肉細胞分化有差異性影響，因此我們想了解這些大量表現FoxOs並不分化的細胞株在處理胰島素之後對葡萄糖代謝是否會造成影響，所以利用glucose uptake來進行初步的實驗，並且利用流式細胞儀來觀察FoxOs在cell cycl中所造成的影響。

FoxO family contains four members (FoxO1/FKHR, FoxO3/FKHRL1, FoxO4/AFX, FoxO6) that function as transcriptional activators by binding as monomer to the consensus DNA sequence TTGTTTAC. FoxO transcription factors have been implicated in regulating diverse cellular functions including differentiation, metabolism, proliferation, and survival. Recent studies have revealed that PI3K can directly regulate gene expression through phosphorylation. Some studies have shown that FoxO3 can promote atrophy in skeletal muscle. However, their regulation and function in skeletal muscle have not been thoroughly understood. In this study, we have constructed stable clones of FoxO-overexpressed myoblasts by retrovirus transduction. FoxO1-AAA、FoxO6 over-expressed cells lose their ability to differentiate into myotubes, but not the other FoxOs over-expressed stable clones. The gene expression patterns of the later after differentiation were not significantly different from those of control cells. To identify how the differentiation of FoxO1-AAA overexpressed myoblasts were inhibited, we treated them with insulin, Y27632, and A23187, and then observed their influence on terminal myogenic differentiation. Our results revealed that treating the undifferentiated FoxO1-AAA over-expressed cells with insulin would provoke their differentiation. Moreover, we also found the maturity of myotube was changed when treated with different glucose concentrations. Finally we investigated whether well-differentiation of FoxO1-AAA over-expressed cells by insulin treatment was due to effects on glucose metabolism. We also examined the glucose uptake by 2-[1,2-3H(N)]-Deoxy-D-Glucose (2-DG) and the change of cell cycle by flow cytometry.

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