Figure 4. Downstream biological effects of insulin, glargine and its metabolites M1 and M2 in MCF-7 cells.

(A) Effect of insulin, IGF1, glargine and its metabolites M1 and M2 on Akt and Erk1/2 phosphorylation in MCF-7 cells. MCF-7 cells were starved overnight and then incubated for 5 min in presence of 10 nM of insulin, glargine, M1, M2 or IGF1. Ligand-induced phosphorylation of Erk1/2 and Akt was evaluated by western blotting. (B) Dose-dependent effect of insulin, IGF1, glargine and its metabolites M1 and M2 on Akt and Erk1/2 phosphorylation in MCF-7 cells. Cells were stimulated for 20 min and ligand-induced phosphorylation of Akt and Erk1/2 was evaluated by in-cell western. Results correspond to mean ± SEM of 4 to 6 independent experiments. (C) MCF-7 cells were incubated for 18 h in serum free medium in the presence or absence of 10 nM of insulin, glargine, M1, M2 or IGF1. mRNA expression level was measured by qRTPCR. Results are normalized to the expression of cyclophilin A mRNA and correspond to the mean ± SEM of 4 to 8 independent experiments (*, **, p<0.05 or p<0.01 respectively, when compared to the control condition). (D) Subconfluent MCF-7 cells cultured in Cytostar-T scintillation microplates were starved for 4 h and then incubated for 19 h with increasing concentrations of IGF-1, insulin or analogues in serum free medium. [¹⁴C]thymidine was added for an additional 6 h and the radioactivity measured in a Wallac 1450 Micro Beta Trilux Scintillation counter. Data are means ± SEM of at least 6 independent experiments. EC50 for insulin, IGF1, glargine and its metabolites on Akt and Erk1/2 phosphorylation and on thymidine incroporation are given in Table 4.