Effect of ethanol and high-fat feeding on hepatic gamma-glutamylcysteine synthetase subunit expression in the rat

Glutathione (GSH) is important in antioxidant defense. A major determinant of the rate of GSH synthesis is the activity of the rate-limiting enzyme, g amma-glutamylcysteine synthetase (GCS), A heavy (HS) and light subunit (LS) make up GCS; oxidative stress regulates both transcriptionally Cis-acting elements important for the oxidative stress-induced transcriptional up-regu lation of both subunits are antioxidant response element (ARE) and activato r protein-1 (AP-1), Nuclear factor-kappa B (NF-kappa B) may also regulate t he heavy subunit, Chronic ethanol ingestion causes oxidative stress, increa ses AP-1 expression, and depletes hepatic GSH. Data conflict regarding GSH synthesis and are lacking regarding GCS subunit gene expression. We examine d the effect of chronic ethanol ingestion on ARE, AP-1, and NF-kappa B acti vity and GCS subunit expression. Male Wistar rats were fed an ethanol and h igh-fat (28.7% cal) diet intragastrically for 9 weeks. Liver GSH level fell by 40%, although GCS activity doubled. GCS-HS mRNA level doubled, whereas GCS-LS mRNA level remained unchanged. Electrophoretic mobility shift assay (EMSA) showed that binding to ARE, AP-1, and NF-kappa B probes all increase d. In conclusion, chronic ethanol ingestion increased GCS-HS expression and GCS activity by activating cis-acting elements important for transcription al up-regulation of GCS-HS. GCS-LS mRNA level remained unchanged despite ac tivation of ARE and AP-1, suggesting that negative transcriptional factors may be involved or the mRNA may be unstable. Despite induction in GCS activ ity, GSH level fell because of alterations in the other factors important i n determining the steady-state GSH level.