Age related changes in NAD+ metabolism oxidative stress and Sirt1 activity in wistar rats

Abstract

The cofactor nicotinamide adenine dinucleotide (NAD+) has emerged as a key regulator of metabolism, stress resistance and longevity. Apart from its role as an important redox carrier, NAD+ also serves as the sole substrate for NAD-dependent enzymes, including poly(ADP-ribose) polymerase (PARP), an important DNA nick sensor, and NAD-dependent histone deacetylases, Sirtuins which play an important role in a wide variety of processes, including senescence, apoptosis, differentiation, and aging. We examined the effect of aging on intracellular NAD+ metabolism in the whole heart, lung, liver and kidney of female wistar rats. Our results are the first to show a significant decline in intracellular NAD+ levels and NAD:NADH ratio in all organs by middle age (i.e.12 months) compared to young (i.e. 3 month old) rats. These changes in [NAD(H)] occurred in parallel with an increase in lipid peroxidation and protein carbonyls (o- and m- tyrosine) formation and decline in total antioxidant capacity in these organs. An age dependent increase in DNA damage (phosphorylated H2AX) was also observed in these same organs. Decreased Sirt1 activity and increased acetylated p53 were observed in organ tissues in parallel with the drop in NAD+ and moderate over-expression of Sirt1 protein. Reduced mitochondrial activity of complex I-IV was also observed in aging animals, impacting both redox status and ATP production. The strong positive correlation observed between DNA damage associated NAD+ depletion and Sirt1 activity suggests that adequate NAD+ concentrations may be an important longevity assurance factor.

Figure 1. Increased formation of oxidatively modified proteins in the liver, heart, kidney, and lung with age.

Increased levels of (A) o- tyrosine and (B) m- tyrosine were reported in the brain after 12 months of age compared to 3 month old rats. All values are means ± S.E from tissue obtained from eight different rats for each age group. Significance ^*p<0.01 compared to 3 month old rats.

Figure 2. Increased oxidative damage to lipids in selected brain regions with age.

(A) Liver, (B) heart, (C) kidney, and (D) lung were analysed by Western blotting using anti-4HNE antibody. The blots shown are representative tracings of an experiment done eight times. Graphs are mean ± S.E 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. (E) Immunodetection of 4-HNE in the liver, heart, kidney, and lung from 3 month, 12 month and 24 month old rats. 4-HNE (green) and DAPI (blue). Higher immunoreactivity for 4-HNE was observed in 12 and 24 month old rats compared to 3-month old rats.

Figure 3. Total antioxidant capacity significantly declined in the rat liver, heart, kidney, and lung with age.

All values are means ± S.E from tissue obtained from eight different rats for each age group. Significance ^*p<0.01 compared to 3 month old rats.

Figure 4. Increased DNA damage was reported in the rat liver, heart, kidney, and lung with age.

(A) DNA damage was determined by measuring the flourescence of phosphorylated H2AX at Serine139 using the Fluostar Optima Fluorometer (NY, USA). All values are means ± S.E from tissue obtained from eight different rats for each age group. Significance ^*p<0.01 compared to 3 month old rats. (B) Immunodetection of phosphor-H2AX-Serine139 in the liver, heart, kidney, and lung from 3 month, 12 month and 24 month old rats. Phosphor-H2AX-Serine139 (green) and DAPI (blue). Higher immunoreactivity for phosphor-H2AX-Serine139 was observed in 12 and 24 month old rats compared to 3-month old rats.

Figure 5. Increased Poly(ADP-ribose) activity in the brain with age.

(A) PARP activity was determined in aging tissue using a spectrophotometric assay. All values are means ± S.E from tissue obtained from eight different rats for each age group. Significance ^*p<0.01 compared to 3 month old rats. (B) Western blotting for poly(ADP-ribose) in (i) liver, (ii) heart, (iii) kidney, and (iv) lung with aging using anti-Poly(ADP-ribose) (10H) antibody. The blots shown are representative tracings of an experiment done eight times. Graphs are mean ± S.E 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. (C) Immunodetection for poly(ADP-ribose) in the liver, heart, kidney and lung from 3 month, 12 month and 24 month old rats. Poly(ADP-ribose) (green) and DAPI (blue). Higher immunoreactivity for poly(ADP-ribose) was observed in 12 and 24 month old rats compared to 3-month old rats.

Figure 6. Increased PARP activation alters pyridine nucleotide metabolism with aging.

(A) NAD⁺ content and (B) NADH content were determined in the rat liver, heart, kidney and lung with aging using a spectrophotometric assay. All values are means ± S.E from tissue obtained from eight different rats for each age group. Significance ^*p<0.01 compared to 3 month old rats. (C) NAD⁺∶NADH ratio was determined as the total NAD⁺ content divided by total NADH levels. All values are means ± S.E from tissue obtained from eight different rats for each age group. Significance ^*p<0.01 compared to 3 month old rats.

Figure 7. Reduced NAD⁺ levels contributes to reduced Sirt1 activity in the liver, heart, kidney and lung with aging.

(A) Reduced Sirt1 activity was observed in the rat liver, heart, kidney and lung after 12 months of age using a flourometry. All values are means ± S.E from tissue obtained from eight different rats for each age group. Significance ^*p<0.01 compared to 3 month old rats. (B) Western blotting for Sirt1 in (i) liver, (ii) heart, (iii) kidney, and (iv) lung with aging using anti-Sirt1 antibody. The blots shown are representative tracings of an experiment done eight times. Graphs are mean ± 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. (C) Immunodetection for Sirt1 in the liver, heart, kidney and lung from 3 month, 12 month and 24 month old rats. Sirt1 (red) and DAPI (blue). Higher immunoreactivity for Sirt1 was observed in 12 and 24 month old rats compared to 3-month old rats.

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 ± 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.

Figure 9. Oxidative stress-mediated reduction in mitochondrial respiratory chain activity in the liver, heart, kidney and lung with age.

Reduced (A) complex I, (B) complex II, (C) complex III, and (D) complex IV, activities at 24 months of age. All values are means ± S.E from tissue obtained from eight different rats for each age group. Significance ^*p<0.01 compared to 3 month old rats.

Figure 10. Schematic representation for the association between oxidative stress, PARP-mediated and decline in NAD⁺ content, and NAD⁺ dependent functions with aging.

Oxidative stress to DNA activates PARP leading to poly(ADP-ribosylation) of proteins in a reaction which consumes NAD⁺. Depletion of cellular NAD⁺ stores attenuates the activity of Sirt1 deacetylase leading to hyperacetylation of p53, and consequently tilting the balance to cell death via an apoptotic mechanism.