Fig. 3.

Oxidative stress inhibits IGF-mediated PI3-Akt signaling but activates the JNK–p-IRS1 (Ser307) axis. (A,B) Fibroblasts from a rat model of type 2 diabetes (Diab) and control fibroblasts treated chronically with either vehicle (Cont), TNFα (4 ng/ml every day for 4 days) or dexamethasone (Dexa; 20 ng/ml every other day for 8 days) were used for the evaluation of the status of oxidative stress, as represented by the level of ROS and protein-bound carbonyls. (A) ROS generation of cells cultured in 96-well plates was measured using the fluorescence probes DCF-DA (5 μM). Values are expressed as DCF fluorescence after 1 hour of incubation normalized to total cell number derived by propidium iodide (PI) fluorescence in the presence of 160 μM digitonin (PIDI). (B) Protein-bound carbonyl levels in total cell extracts were determined using an ELISA-based technique. (C,D) Fibroblasts with phenotypic features of HSOS induced by BSO (10 μM) were used for the determination of the level of ROS and protein-bound carbonyls, and key elements of IGF1 signaling. (C) ROS and protein-bound carbonyl levels were measured as described above. (D) Whole-cell protein extracts were used for the assessment of the ratio of p-IRS1 (Ser307):IRS1 (immunoprecipitated with anti-IRS1 and probed with anti-p-IRS1 (Ser307), stripped and reprobed with ant-IRS1), the ratio of p-Akt (Ser473):Akt (western blotting) and PI3K activity (ELISA). In addition, the ratio of p-JNK:JNK in paraformaldehyde-fixed cells was quantified using FAC ELISA assays (Active Motif). EUK-134 or LA was applied at 100 μM (EUK) or 500 μM (LA), concentrations that seem to have an optimum therapeutic benefit with a minimum detrimental effect on cell viability. Experiments were performed in triplicate on each cell lines used (n=4 for each). *Significantly different from corresponding Cont values at P≤0.05; **significantly different from corresponding DMSO-treated Diab, TNFα or Dexa values at P≤0.05.