Glucose control with insulin results in reduction of NF-κB-binding activity in mononuclear blood cells of patients with recently manifested type 1 diabetes

Aim: Chronic elevated blood glucose levels are associated with the formation of advanced glycation endproducts (AGEs). Hyperglycaemia and AGEs have been shown to induce activation of the redox-sensitive transcription factor nuclear factor-kappa B (NF-kappa B). To validate the hypothesis that the maintenance of normal glucose levels results in the reduction of NF-kappa B-binding activity in vivo, the redox-sensitive transcription factor NF-kappa B was used as marker of hyperglycaemia-induced mononuclear cell activation in patients who recently developed type 1 diabetes. Methods: Twelve patients with recently manifested type 1 diabetes mellitus were examined in our study. After sampling blood for determination of baseline glucose values, the 12 patients were treated with insulin, and blood samples were taken 4 and 12 weeks later. Mononuclear cells were isolated and assayed in a tissue culture-independent electrophoretic mobility shift assay (EMSA)-based detection system for NF-kappa B-binding activity. Western blot analysis was used to determine nuclear and cytoplasmic localization of NF-kappa B-p65 and cytoplasmic content of inhibitor of kappa B-alpha (I kappa B-alpha). In addition, we determined the concentration of heme oxygenase-1 (HO-1) from cytoplasmic extract as a marker of oxidative stress. Results: Normalization of blood glucose levels resulted in a highly significant reduction of NF-kappa B activation in EMSA. Before and after glucose normalization, there were no differences in binding by the members of the NF-kappa B family to the NF-kappa B consensus sequence oligonucleotide. Similar data were obtained by Western blot analysis showing NF-kappa B-p65 localization in the nucleus, while p65 levels increased in the cytoplasm. I kappa B-alpha increased in the cytoplasm after glucose normalization. HO-1 antigen consistently decreased, as expected from the decrease in NF-kappa B activation. Conclusion: Thus, we conclude that normalization of blood glucose levels results in the reduction of NF-kappa B activation and gene products controlled by this transcription factor.