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.
Innovative Drug Research Center for Metabolic and Inflammatory Disease, College of Pharmacy and Research Institute of Pharmaceutical Sciences, Seoul National University, Seoul, Korea.
S H
SH
Ki
S G
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Kim
D000818:Animals; D022762:CCAAT-Enhancer-Binding Proteins / Q000378:metabolism*; D002460:Cell Line; D004790:Enzyme Induction; D005982:Glutathione Transferase / Q000096:biosynthesis* / Q000235:genetics; D022781:Hepatocytes / Q000378:metabolism*; D007328:Insulin / Q000378:metabolism*; D007527:Isoenzymes / Q000096:biosynthesis* / Q000235:genetics; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism*; D051381:Rats; D015398:Signal Transduction; D008063:Thioctic Acid / Q000494:pharmacology*; D015533:Transcriptional Activation
D022762:CCAAT-Enhancer-Binding Proteins; D007328:Insulin; D007527:Isoenzymes; D008063:Thioctic Acid; D005982:Glutathione Transferase; C119995:glutathione S-transferase alpha; D019869:Phosphatidylinositol 3-Kinases