Oxidative stress impairs nuclear proteins binding to the insulin responsive element in the GLUT4 promoter

Abstract

Aims/hypothesis. Substantial evidence suggests an important role for the expression of GLUT4 in adipocytes, in the pathogenesis of insulin resistance and Type II (non-insulin-dependent) diabetes mellitus. We investigated whether oxidative stress decreases GLUT4 expression by impairing DNA binding of nuclear proteins to the insulin responsive element in the GLUT4 promoter. Methods. 3T3-L1 adipocytes were exposed to micromolar H2O2 concentrations and GLUT4 expression and binding of nuclear proteins to defined DNA sequences were assessed. Results. GLUT4 mRNA was decreased after at least 4 h exposure to H2O2, without a major change in the stability of GLUT4 transcripts. Nuclear protein extracts prepared from oxidized cells showed decreased binding to the insulin responsive element of the GLUT4 promoter but not to other DNA sequences. The direct effect of oxidation on the binding to the insulin response element was shown by the observation that in vitro oxidation of nuclear extracts with H2O2, N-ethylmaleimide or diamide decreased protein DNA complex formation. This, and decreased binding capacity observed in nuclear extracts from oxidized cells, were partly reversible by subsequent treatment with a reducing agent. Protein binding to a consensus DNA sequence for nuclear factor 1 transcription factors was decreased 16% by oxidation, whereas no change was observed in the protein content of several isoforms of these proteins. Conclusion/interpretation. Oxidative stress causes decreased GLUT4 expression, associated with impaired binding of nuclear proteins to the insulin responsive element in the GLUT4 promoter.