(R)-α-Lipoic acid reverses the age-related loss in GSH redox status in post-mitotic tissues: evidence for increased cysteine requirement for GSH synthesis

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Abstract

Age-related depletion of GSH levels and perturbations in its redox state may be especially deleterious to metabolically active tissues, such as the heart and brain. We examined the extent and the mechanisms underlying the potential age-related changes in cerebral and myocardial GSH status in young and old F344 rats and whether administration of (R)-α-lipoic acid (LA) can reverse these changes. Our results show that GSH/GSSG ratios in the aging heart and the brain declined by 58 and 66% relative to young controls, respectively (p<0.001). Despite a consistent loss in GSH redox status in both tissues, only cerebral GSH levels declined with age (p<0.01). To discern the potential mechanisms underlying this differential loss, the levels and the activities of γ-glutamylcysteine ligase (GCL) and cysteine availability were determined. There were no significant age-related changes in substrate or enzyme levels, or GCL activity when saturating amounts of substrates were provided. However, kinetic analysis of GCL in brains of old rats displayed a significant increase (p<0.05) in the apparent Km for cysteine (Kmcys) vs. young rats (84.3 ± 25.4 vs. 179.0 ± 49.0; young and old, respectively), resulting in a 40% loss in apparent catalytic turnover of the enzyme. Thus, the age-related decline in total GSH appears to be mediated, in part, by a general decrement in GCL catalytic efficiency. Treating old rats with LA (40 mg/kg body wt; by i.p.) markedly increased tissue cysteine levels by 54% 12 h following treatment and subsequently restored the cerebral GSH levels. Moreover, LA improved the age-related changes in the tissue GSH/GSSG ratios in both heart and the brain. These results demonstrate that LA is an effective agent to restore both the age-associated decline in thiol redox ratio as well as increase cerebral GSH levels that otherwise decline with age.

Section snippets

Materials

(R)-α-lipoic acid (LA) was a gift from Asta Medica (Frankfurt, Germany). Rabbit polyclonal antibodies to GCLC (catalytic subunit) were obtained as a reference [18]. All high performance liquid chromatography (HPLC) solvents were HPLC grade reagents from Fisher Scientific (Houston, TX). All other chemicals were of reagent grade or the highest quality available from Sigma (St. Louis, MO).

Animals

Rats (Fischer 344 [F344], virgin male, outbred albino), both young (2–5 mo, N=15) and old (24–28 mo, N=15;

Age-related changes in cerebral and myocardial thiol redox state

To gauge the extent of age-related changes in thiol redox status, the GSH/GSSG ratios in brains and hearts of young and old rats were examined. The cerebral GSH/GSSG ratios declined from 28.3 ± 3.8 to 13.8 ± 0.3 with age, a significant (p<0.01) 50% loss in this important redox parameter (Table 1). Paralleling the patterns observed in the brain, myocardial GSH/GSSG ratios were also markedly lower on an age basis (18.2 ± 0.3 vs. 7.7 ± 0.8 in young compared to old rats, respectively; p<0.01; Table 1),

Discussion

Maintaining the ability to respond to oxidant challenges is critical for overall survival of organisms and for ameliorating increased risks for morbidity and mortality associated with aging. In particular, a robust and responsive GSH synthesizing capacity is needed in aging cells to protect against endogenous and exogenous oxidative insults [15], [29]. Thus, it is not surprising that the expression and activities of enzymes critical for GSH synthesis are sensitively controlled to ensure timely

Acknowledgements

This work was supported by NIH Grants RO1 AG17141A and P01 AT002034-01 to T.M.H. and RO1 ES11831-01 to RLU. This work was also supported by the Environmental Health Sciences Center, Oregon State University (ES00210). The authors thank Ms. Swapna Shenvi and Mrs. Du Heath for their expert technical assistance. We are also grateful to Stephen Lawson for careful review of the manuscript.

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