Figure 6

Effects of metformin on EPS-inducible upregulation of human R-spondin 3 (RSPO3) and other factors. EPS (1 Hz, 4-ms, 20 V/25 mm) was applied to the differentiated myotubes in the presence or absence of the indicated concentrations of metformin. Total RNA samples were prepared and subjected to RT-PCR analysis to evaluate mRNA levels for RSPO3 (A), IL-6 (B), IL-8 (C), CXCL1(D), and striated muscle activator of Rho signaling (STARS) (E). Data normalized using RPLP0 transcript were averaged over 3 independent experiments. Multi factor ANOVA with Tukey’s multiple comparison test was used for statistical analyses; the effect of EPS is denoted as *P < 0.05; the effect of metformin is denoted as # < 0.05. (F) Whole cell lysates of the differentiated human myotubes (day8) with and without a total of 24 h of EPS (1 Hz, 4-ms, 20 V/25 mm) in the presence of metformin (0, 2.5, 5 mM) were subjected to Western blot analysis using anti-phospho-acetyl-CoA carboxylase (pACC), anti-phospho-AMP-activated kinase (pAMPK), and anti-β-actin antibodies. Three independent experiments were performed; representative results are shown.