Metformin Delays Satellite Cell Activation and Maintains Quiescence

Figure 3

Metformin delays SC differentiation. (a) SCs were isolated from C57BL/6 mice, attached to a plastic dish, and treated with 2 mM metformin for 2, 4, and 8 days. The SCs were analyzed by immunofluorescence microscopy for the expression of the early myogenic marker myogenin. (b) The percentage of cells expressing myogenin after 2, 4, and 8 days of treatment with metformin was averaged from the results of three independent cell isolations and experimental replicates (). Statistical significance was evaluated by the ANOVA test (). (c) SCs were treated upon attachment for 2, 4, and 8 days with 2 mM metformin and analyzed by fluorescence microscopy for the expression of the myosin heavy chain (MyHC). (d) The percentage of cells expressing MyHC after 2, 4, and 8 days of treatment with metformin was analyzed after three independent cell isolations and experimental replicates (). Statistical significance was evaluated by the ANOVA test (). (e) The fusion index was calculated as the % of nuclei inside myotubes over the total number of nuclei. A myotube is defined as a cell expressing MyHC and containing at least three nuclei inside a continuous cell membrane. Statistical significance was evaluated by the ANOVA test () after three independent cell isolations and experimental replicates (). (f) Western blot analysis for the expression of MyHC of control and metformin-treated SCs after 2, 4, 6, and 8 days of treatment (t2, t4, t6, and t8, respectively). Vinculin was used as a loading control. (g) Quantitation graph of MyHC protein levels monitored by western blot in four independent cell isolations and biological replicates (). Statistical significance was evaluated by the ANOVA test ().

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