Age Related Changes in NAD+ Metabolism Figure 8. Reduced Sirt1 activity induces p53 acetylation in the liver, heart, kidney and lung with aging. Acetylated p53 and total p53 levels were determined by Western blotting in (A) liver, (B) heart, (C) kidney, and (D) lung with aging using anti-acetylated p53 and anti-total p53 antibodies. The blots shown are representative tracings of an experiment done eight times. Graphs are mean 6 S.E brains from tissue obtained from eight different rats for each age group. Each bar of the quantification graph represents the corresponding band for each age group. Significance *p,0.01 compared to 3 month old rats. doi:10.1371/journal.pone.0019194.g008 phosphorylation of histone H2AX at Serine139 residue. H2AX phosphorylation is considered a specific reporter of double- stranded DNA damage [39]. The level of DNA damage increased with age in all organs consistent with an increase in protein carbonyls and lipid peroxidation. Experimental data from other laboratories have shown a similar increase in DNA damage, (e.g. 8-oxoguanine) in the heart, lung, liver and kidney of aging rats [51]. The DNA damage associated with oxidative stress is known to activate the NAD+-dependent DNA repair enzyme, PARP, which assists in combination with the enzymes of the base- excision-and-repair complex of the cell in energy-consuming repair processes [12,52,53]. The vital role of PARP activation in several diseases has been obtained from experimental studies using PARP inhibitors, PARP knock-down mice, and clinical trials. Recent studies have shown the protective effects of PARP inhibitors on ischaemic heart and liver, diabetic kidney disease, and endotoxin-induced acute lung injury, arguably due to the preservation of NAD+ levels [20,21,22,23,24]. PARP activation can also promote apoptosis by stabilising p53, and mediating the translocation of apoptosis-inducing factor from mitochondria to the nucleus [19,54]. Several lines of evidence reported here show that PARP activation increases in the aging heart, lung, liver, and kidney (Fig. 5), and depletes cellular NAD+ levels (Fig. 6). Theoretically this should lead to a reduction in Sirt1 deacetylase activity and accumulation of acetylated p53. Consistent with this prediction we observed that increased PARP activation (Fig. 5) was associated with NAD+ depletion (Fig. 6) which was accompanied by a decrease in Sirt1 deacetylase activity (Fig. 7). It is well established that genotoxic stress can kill cells by depleting the intracellular NAD+ pools due to extensive use of NAD+ as a substrate for PARP [12,13,16,19]. Results from the present study, consistent with others, suggests that both PARP and Sirt1 compete for the same limiting intracellular NAD+ pool [55]. One study showed that PARP activation depleted intracellular NAD+ levels and reduced Sirt1 deacetylase activity in DNA damaged in cardiac myocytes [55]. Replenishing intracellular NAD+ levels by increasing NAD+ levels was able to restore cellular viability but only in the presence of functional Sirt1 [55]. As NAD+ serves as a cofactor for key glycolytic and mitochondrial enzymes, vulnerable cells subsequently lose their ability to carry out energy dependent functions including the maintenance of cell wall integrity and DNA/RNA synthesis and consequently die [56]. We demonstrated, for the first time, that aging in the rat is + associated with decreased tissue levels of NAD . This may lower the rate of cell survival in response to several endogenous and exogenous stressors. As NAD+ is the only substrate for both PARP and Sirt1 activities, maintaining optimal NAD+ content is crucial to counteract the age-related degeneration observed during the aging process [56,57]. As a substrate for the sirtuin family of NAD+ dependent deacetylases, known as silent information regulators of gene function [27]. This discovery suggests that gene silencing by this system might be related to metabolic rate and hence to NAD+ concentrations, whose levels therefore may be directly proportional to a longer lifespan [28]. While several studies have demonstrated the protective roles of sirtuin activators during aging [30,34,35,58,59,60,61,62,63], little is known regarding role of Sirt1 in the aging heart, lung, liver, and kidney. The present results show that aging increases the amount of available Sirt1 protein and activities of Sirt1 in nuclear extracts in the heart, lung, liver, and kidney. The activity per molecule of Sirt1 appears to become lower in these organs as the animal ages, suggesting the presence of more inactive or less active molecules in aged animals. These observations are in line with a previous study which reported a decrease in Sirt1 activity but not Sirt1 protein expression in skeletal muscle of aged rats [64].Two possibilities, which are not mutually exclusive, may explain this observation: 1) Oxidative damage may potentially inhibit Sirt1 activity in a similar way to several other proteins [65] and/or 2) It is also likely that the age-associated drop in NAD+ content due to increased demand by PARP in the DNA repair process limits the Sirt1 substrate NAD+ thereby limiting Sirt1 activity. A compensatory mechanism which increases the production of the Sirt1 protein [64] would increase its relative success when competing for the limited NAD+ pool.. Sirt1 deficiency has been shown to accentuate apoptosis of renal medullary cells [66] and cardiac mycocytes [55]. Over- expression of Sirt1 has been shown to promote the resistance of these cells to oxidative insult, both in vitro and in vivo [67]. The protective function of Sirt1 has been reported in other organs such as the heart, neurons, lung, liver [67,68,69,70]. Given the pivotal role of Sirt1 as an anti-stress and anti-aging protein, targeting Sirt1 by either Sirt1 activators or increasing its substrate (i.e. NAD+) availability may prove therapeutically beneficial for the prevention of age-related cardiovascular, respiratory, hepatic, and renal dysfunction. Because Sirt1 activity is affected by the NAD+:NADH ratio [71], we determined the NAD+ and NADH content in aging heart, lung, liver, and kidney. Our results show a significant decline in the NAD+:NADH ratio with aging, consistent with a decline in NAD+ levels and an increase in NADH levels. This indicates that age related metabolic changes may influence NAD+ and NADH upstream of Sirt1 similar to what was observed in yeast Sir2a and in the skeletal muscle [71]. To examine the downstream effect of Sirt1 deacetylase activity in cellular processes, we measured the expression of both total and acetylated p53 in aging rat tissue. The main tumour suppressor protein, p53 regulates the expression of several gene products that either lead to cell cycle arrest in G1 phase and prevent DNA replication immediately before the repair of damage, or cause cell death via an apoptotic mechanism [72]. We observed a significant increase in acetylated p53 protein while no change was observed in total p53 protein in all organs was apparent. Higher acetylation levels of p53 have been reported in cardiac myocytes following treatment with the oxidant H2O2, probably resulting from decreased NAD+ and impaired Sirt1 deacetylase activity [55]. A recent report showed that microRNA 34a (miR-34a), a tumour suppressor gene inhibiting Sirt1 expression also increases acetylated p53 levels, thus stimulating transcriptional targets of p53 responsible for promoting cell-cycle arrest, and apoptosis PLoS ONE | www.plosone.org 14 April 2011 | Volume 6 | Issue 4 | e19194