Decline in transcriptional activity of Nrf2 causes age-related loss of glutathione synthesis, which is reversible with lipoic acid

Abstract

Glutathione (GSH) significantly declines in the aging rat liver. Because GSH levels are partly a reflection of its synthetic capacity, we measured the levels and activity of gamma-glutamylcysteine ligase (GCL), the rate-controlling enzyme in GSH synthesis. With age, both the catalytic (GCLC) and modulatory (GCLM) subunits of GCL decreased by 47% and 52%, respectively (P < 0.005). Concomitant with lower subunit levels, GCL activity also declined by 53% (P < 0.05). Because nuclear factor erythroid2-related factor 2 (Nrf2) governs basal and inducible GCLC and GCLM expression by means of the antioxidant response element (ARE), we hypothesized that aging results in dysregulation of Nrf2-mediated GCL expression. We observed an approximately 50% age-related loss in total (P < 0.001) and nuclear (P < 0.0001) Nrf2 levels, which suggests attenuation in Nrf2-dependent gene transcription. By using gel-shift and supershift assays, a marked reduction in Nrf2/ARE binding in old vs. young rats was noted. To determine whether the constitutive loss of Nrf2 transcriptional activity also affects the inducible nature of Nrf2 nuclear translocation, old rats were treated with (R)-alpha-lipoic acid (LA; 40 mg/kg i.p. up to 48 h), a disulfide compound shown to induce Nrf2 activation in vitro and improve GSH levels in vivo. LA administration increased nuclear Nrf2 levels in old rats after 12 h. LA also induced Nrf2 binding to the ARE, and, consequently, higher GCLC levels and GCL activity were observed 24 h after LA injection. Thus, the age-related loss in GSH synthesis may be caused by dysregulation of ARE-mediated gene expression, but chemoprotective agents, like LA, can attenuate this loss.

Fig. 1.

Age-related decline in total hepatic GSH is due to loss in GCL activity and expression. Hepatic GSH levels in young (3 mo; n = 4) and old (24 mo; n = 4) F344 rats are shown in A. Results show a 35% decline in total GSH [GSH + 2 glutathione disulfide (GSSG)] in old relative to young rats. (B) Measurement of GCL activity reveals a significant 54.8% decline with age. Western analysis of GCL subunits shows that decreased GCL activity was, in part, due to the lower levels of GCLC and GCLM in old relative to young rats (C). Results are expressed as the mean ± SEM.

Fig. 2.

Aged rats display a significant loss in nuclear Nrf2 content and ARE-binding activity. Western analysis of Nrf2 present in nuclear extracts prepared from young and old rats are shown in A. Results are also graphically presented relative to Histone H1 loading controls and show that basal nuclear Nrf2 levels are 51% lower on an age basis. (B) EMSA analysis using nuclear extracts from young and old rats shows an overall age-related loss of transcription factor binding to the ARE consensus sequence. Lane 1 is the migration of free probe in absence of nuclear extract. A negative control using excess unlabeled cold probe is shown in lane 2. (C) Supershift analysis reveals that Nrf2 binding to the consensus ARE sequence declines with age. The two distinct supershifted bands are denoted by brackets whereas the lower band as indicated by braces denotes band shifts. Lane 1 is a competitive control where cold unlabeled probe was added in 100-fold excess. Results shown are representative of three independent experiments.

Fig. 3.

LA induces nuclear Nrf2 levels and increases its ARE binding activity. The time-dependent changes in nuclear Nrf2 levels after LA injection (40 mg/kg of body weight) in old rats were determined by Western blot analysis (A). The histone H1 normalized values are graphically represented and show an increase in nuclear Nrf2, with maximum induction seen within 24 h after LA injection (A). (B) EMSA analysis of nuclear extracts shows increased transcription factor binding to the ARE consensus sequence within 12 h, which was maintained for 48 h. Lane 1 is a competition with cold unlabeled probe. (C) Results from supershift assays, which indicate that LA increases Nrf2 binding to the ARE in a time-dependent manner and show maximal binding at 24 h after LA injection. Lane 1 shows a negative control using an antibody against the P65 subunit of NF-κB. Results are representative of three independent experiments. †, The group that is significantly (P < 0.05) different from old control rats.

Fig. 4.

LA improves GSH synthetic capacity and hepatic GSH levels. Western analysis of GCL subunits was performed after LA injection at times indicated in A. Results show that GCLC levels in old rats were maximally increased in response to LA within 24 h (A). GCLM also exhibited a similar time-dependent increase, but the difference observed was not statistically significant (A). Paralleling the increase in GCL levels, enzyme activity increased by two-fold within 24 h after LA injection (B). The changes in GCL levels and enzyme activity resulted in an overall increase in hepatic GSH 24 h post LA injection (C). The dashed lines in B and C indicate mean experimental values seen in young animals. Results are expressed as the mean ± SEM. †, Groups that are significantly (P < 0.05) different from old controls.