Phase II enzyme induction by alpha-lipoic acid through phosphatidylinositol 3-kinase-dependent C/EBPs activation.

1. alpha-Lipoic acid (alpha-LA) activates antioxidant pathways and exerts insulin-like actions via phosphatidylinositol 3-kinase (PI3K), and previous studies from the authors' laboratory support the essential role of PI3K-dependent CCAAT/enhancer binding protein (C/EBP) activation in antioxidant defence responses. 2. The study investigated whether alpha-LA treatment promoted phase II antioxidant gene induction through C/EBP activation and, if so, whether combined treatment of alpha-LA and insulin synergistically increased target gene expression. 3. alpha-LA treatment induced GSTA2 in H4IIE cells in a concentration- and time-dependent manner. In cells transfected with the regulatory region of the GSTA2 gene, alpha-LA treatment increased luciferase reporter-gene activity. Immunoblot, immunocytochemistry, and gel shift assays identified the nuclear translocation and DNA binding of C/EBPalpha and C/EBPbeta, but not C/EBPdelta, in alpha-LA-treated cells. Deletion of the C/EBP binding site abolished the ability of alpha-LA to promote the luciferase gene activity. 4. alpha-LA, when combined with low concentrations of insulin, transactivated the GSTA2 gene to a greater extent compared with alpha-LA or a higher concentration of insulin treatment alone. Combined treatment of alpha-LA with insulin not only potentiated insulin signalling, but also enhanced PI3K-dependent activation of C/EBPalpha and C/EBPbeta forms. alpha-LA in combination with insulin substantially increased haemoxygenase-1, microsomal epoxide hydrolase and beta-nicotinamide adenine dinucleotide phosphate (NADPH) quinone oxidoreductase expression, verifying the enhanced induction of other phase II enzymes. A dominant negative C/EBP transfection experiment indicated that GSTA2 gene induction by simultaneous treatment of alpha-LA and insulin was also dependent on C/EBPs. 5. The results demonstrate that alpha-LA induces phase II enzymes via PI3K-dependent C/EBPalpha and C/EBPbeta activation, and enhances the ability of insulin to promote target gene induction.