Abstract
1. α-Lipoic acid (α-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 α-LA treatment promoted phase II antioxidant gene induction through C/EBP activation and, if so, whether combined treatment of α-LA and insulin synergistically increased target gene expression.3. α-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, α-LA treatment increased luciferase reporter-gene activity. Immunoblot, immunocytochemistry, and gel shift assays identified the nuclear translocation and DNA binding of C/EBPα and C/EBPβ, but not C/EBPδ, in α-LA-treated cells. Deletion of the C/EBP binding site abolished the ability of α-LA to promote the luciferase gene activity.4. α-LA, when combined with low concentrations of insulin, transactivated the GSTA2 gene to a greater extent compared with α-LA or a higher concentration of insulin treatment alone. Combined treatment of α-LA with insulin not only potentiated insulin signalling, but also enhanced PI3K-dependent activation of C/EBPα and C/EBPβ forms. α-LA in combination with insulin substantially increased haemoxygenase-1, microsomal epoxide hydrolase and β-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 α-LA and insulin was also dependent on C/EBPs.5. The results demonstrate that α-LA induces phase II enzymes via PI3K-dependent C/EBPα and C/EBPβ activation, and enhances the ability of insulin to promote target gene induction.
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