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

机译：肝谷胱甘肽合成转录调控中与年龄有关的改变：R-α-硫辛酸的修复。

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摘要

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, gamma-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 muM) 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.
机译：谷胱甘肽（GSH）是肝脏组织中主要的低分子量硫醇抗氧化剂。 GSH在维持细胞内硫醇氧化还原比以及亲电试剂和异种生物的解毒中起着重要作用。衰老导致肝细胞谷胱甘肽水平显着下降（35％； P≤0.05）。我们使用年轻的（2-4个月相当于一个青春期的人）和年龄较大的（24-28个月相当于一个60-75岁的人）雄性Fischer 344大鼠，我们确定了与年龄相关的GSH水平降低是由于降低活性（53 +/- 6％； P≤0.05）和限速酶γ-谷氨酸半胱氨酸连接酶（GCL）的水平。 GCL由催化亚基（GCLC）和影响其底物KM的调节亚基（GCLM）组成。由于GCLC的水平受转录调控，我们试图阐明其精确的转录机制以及衰老是否会改变酶亚基的转录组。先前已将称为抗氧化剂反应元件（ARE）的顺式作用DNA序列牵涉II期酶（包括GCLC和GCLM）的转录调控。基于计算机的Gclc启动子区域分析表明存在三个推定的ARE和一个仅包含ARE核心而不是侧翼核苷酸的顺式元件（类似ARE）。用含有单个Gclc ARE元件的萤光素酶报告基因构建体转染H4IIE大鼠肝癌细胞的实验结果表明，仅转录起始位点（ARE3）上游3.9 kb的ARE元件具有基础转录活性。在培养的肝组织和原代肝细胞中进行的电迁移率测定（EMSA）和染色质免疫沉淀（ChIP）实验表明，NF-E2相关因子2（Nrf2）是与ARE3结合的主要转录因子，并与小的maf蛋白c -Jun，c-Fos和组蛋白乙酰转移酶CREB结合蛋白（CBP）。在衰老过程中，Nrf2的核稳态水平显着下降51 +/- 7％（P≤0.0001），从而导致Nrf2-ARE3结合降低（40％）和转录活性（70 +/- 10％； P≤0.05 ），这与GCLC水平的下降相一致。同时，转录阻遏物Bach1在ARE3位点富集，并伴有CBP丢失。这些结果表明，活性Gclc转录复合物的负重塑在老大鼠的肝脏中发生。此外，在ARE样位点检测到Nrf2，该位点在幼鼠的肝细胞中没有转录活性。因此，启动子转换机制可能随着年龄而发生。在以前发表的报告中，我们证明了对老大鼠施用二硫醇化合物R-α-硫辛酸（LA； 40 mg / kg体重；腹膜内注射）可以逆转与年龄相关的肝谷胱甘肽水平下降。对老大鼠和原代细胞培养物中的肝细胞（100μM）的LA补充补充了衰老过程中丢失的核Nrf2水平。另外，LA在ARE样启动子上更大程度地增加了ARE3和ARE样启动子的Nrf2富集和活性（60 +/- 10％；P≤0.05）。这伴随着与年龄相关的GCLC表达，蛋白质水平和GCL活性下降的逆转。因此，LA通过允许ARE介导的GCLC正常表达来维持衰老过程中肝脏GSH的状态，这表明它是恢复衰老过程中GSH依赖性排毒系统的良好治疗剂。

著录项

  • 作者

    Shenvi, Swapna V.;

  • 作者单位

    Oregon State University.;

  • 授予单位 Oregon State University.;
  • 学科 Biology Molecular.; Biology Animal Physiology.; Health Sciences Nutrition.
  • 学位 Ph.D.
  • 年度 2007
  • 页码 216 p.
  • 总页数 216
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类 分子遗传学;生理学;预防医学、卫生学;
  • 关键词

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