Graduate Thesis Or Dissertation
 

Age-related alterations in transcriptional regulation of hepatic glutathione synthesis : remediation by R-alpha-lipoic acid

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  • Glutathione (GSH) is the predominant low molecular weight thiol antioxidant in liver tissue. GSH plays an important role in maintaining the intracellular thiol redox ratio as well as detoxification of electrophiles and xenobiotics. Aging leads to a significant decline (35%; P ≤ 0.05) in hepatocellular GSH levels. Using young (2-4 months corresponding to an adolescent human) and old (24-28 months corresponding to a 60-75 year old person) male Fischer 344 rats, we determined that the age-related loss of GSH levels were due to lower activity (53 + 6%; P ≤ 0.05) and levels of the rate-limiting enzyme, γ-glutamate cysteine ligase (GCL). GCL is composed of a catalytic subunit (GCLC) and also a modulatory subunit (GCLM) that affects the KM of its substrate. Since GCLC levels are regulated by transcription, we sought to elucidate its precise transcriptional mechanism and whether aging alters the transcriptome of the enzyme subunit. A cis-acting DNA sequence called the antioxidant response element (ARE) has been previously implicated in the transcriptional regulation of Phase II enzymes, including GCLC and GCLM. Computer-based analysis of the promoter region of Gclc revealed the presence of three putative AREs and a single cis element (ARE-like) containing the core but not the flanking nucleotides of the ARE. Results from experiments where H4IIE rat hepatoma cells were transfected with luciferase reporter constructs containing individual Gclc ARE elements revealed that only the ARE element 3.9 kb upstream of the transcriptional start site (ARE3) possessed basal transcriptional activity. Electromobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP) experiments on liver tissue and primary hepatocytes in culture showed that NF-E2-related factor 2 (Nrf2) was the predominant transcription factor bound to ARE3 and was partnered with small maf proteins, c-Jun, c-Fos and the histone acetyltransferase CREB-Binding Protein (CBP). In aging, nuclear steady-state levels of Nrf2 showed a profound 51 + 7% (P ≤ 0.0001) decline leading to lower Nrf2-ARE3 binding (40%) and transcriptional activity (70 + 10%; P ≤ 0.05), consistent with the loss in GCLC levels. Concomitantly, the transcriptional repressor Bach1 was enriched at the ARE3 site and was accompanied by a loss of CBP. These results show that a negative remodeling of the active Gclc transcriptional complex occurs in the liver of old rats. Furthermore, Nrf2 was detected at the ARE-like site, which was not transcriptionally active in hepatocytes from young rats. Thus, a promoter switching mechanism may occur with age. In previously published reports, we demonstrated that administration of the dithiol compound R-alpha-lipoic acid (LA; 40 mg/kg body weight; intraperitoneal injection) to old rats reversed the age-related decline in hepatic glutathione levels. LA admininstration both to old rats and to hepatocytes in primary cell culture (100 μM) replenished nuclear Nrf2 levels lost during aging. Additionally, LA increased Nrf2 enrichment and activity of both the ARE3 and ARE-like promoters albeit, to a greater extent at the ARE-like promoter (60 + 10%; P ≤ 0.05). This was accompanied by reversal of the age-related decline in GCLC expression, protein levels and GCL activity. Thus, LA maintains hepatic GSH status during aging by permitting normal ARE-mediated GCLC expression, suggesting that it would be a good therapeutic agent to restore GSH-dependent detoxification systems during aging.
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