Figure 3
Effect of ucOCN on insulin signal transduction is mediated via PI3K/Akt/NF-κB signaling in HUVECs. Tunicamycin (Tun) was used to induce insulin resistance. For insulin signaling, cells were stimulated with 10 nM of insulin for 10 min. Cells were cultured in the presence or absence of uncarboxylated osteocalcin with or without specific signaling pathway inhibitors such as 10 μM wortmannin (a PI3K inhibitor), 10 μM Akti-1/2 (an AKT inhibitor) or 10 μM U0126 (a MAPK inhibitor) for 4 h. PI3K binding activity was determined by an in vitro kinase assay. NF-κB p65-DNA binding activity was determined by Elisa. The relative quantity of proteins was analyzed Quantity One software. (A) PI3K activity in HUVECs. (B) Phosphorylation of PERK and IRS-1 in HUVECs. (C) Densitometric analyses of PERK and IRS-1 in HUVECs. (D) NF-κB p65 DNA binding activity in HUVECs. A representative blot from three independent experiments is shown and the data expressed as mean ± SEM in each bar graph represent the average of three independent experiments. *P < 0.05 (Tun/ucOcn vs. Tun). # P < 0.05 (Tun/ucOcn/inhibitor vs. Tun/ucOcn). ΔP < 0.05 (Tun vs. control). IB, immunoblot; IP, immunoprecipitation.
