Figure 7.

Glucose uptake in C2C12 cells. C2C12 cells were previously incubated in serum-free and glucose-free DMEM medium, and were treated with either insulin, Ric4, or indicated Ric4 derived peptide (100 µM; Table 3), in the presence of 2-[N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxy-d-glucose (2-NBDG; 80 µM). Insulin was used as the positive control. Before the assays, the culture medium was removed from each well and replaced with 100 μL of culture medium containing fluorescent d-glucose analog 2-NBDG (80 µM; for standardization concentrations of 25, 50, 80, and 100 µM were used, data not shown) in the absence or presence of the indicated compound (insulin, Ric4 or Ric4-1/16 derived peptides). Plates were incubated at 37 °C with 5% CO2 for a period of 30 min, and after that were lysed with 50 μL of 0.1 N NaOH and fluorescence of aliquots from the lysate was measured. The statistical comparisons were performed using Student’s t-test or analysis of variance (ANOVA), followed by ad-hoc Tukey’s test using GraphPad Prism software * p < 0.05; ** p < 0.001; *** p <0.0001.