During yeast chronological aging resveratrol supplementation results in a short-lived phenotype Sir2-dependent

Abstract

Resveratrol (RSV) is a naturally occurring polyphenolic compound endowed with interesting biological properties/functions amongst which are its activity as an antioxidant and as Sirtuin activating compound towards SIRT1 in mammals. Sirtuins comprise a family of NAD⁺-dependent protein deacetylases that are involved in many physiological and pathological processes including aging and age-related diseases. These enzymes are conserved across species and SIRT1 is the closest mammalian orthologue of Sir2 of Saccharomyces cerevisiae. In the field of aging researches, it is well known that Sir2 is a positive regulator of replicative lifespan and, in this context, the RSV effects have been already examined. Here, we analyzed RSV effects during chronological aging, in which Sir2 acts as a negative regulator of chronological lifespan (CLS). Chronological aging refers to quiescent cells in stationary phase; these cells display a survival-based metabolism characterized by an increase in oxidative stress. We found that RSV supplementation at the onset of chronological aging, namely at the diauxic shift, increases oxidative stress and significantly reduces CLS. CLS reduction is dependent on Sir2 presence both in expired medium and in extreme Calorie Restriction. In addition, all data point to an enhancement of Sir2 activity, in particular Sir2-mediated deacetylation of the key gluconeogenic enzyme phosphoenolpyruvate carboxykinase (Pck1). This leads to a reduction in the amount of the acetylated active form of Pck1, whose enzymatic activity is essential for gluconeogenesis and CLS extension.

Keywords: Chronological aging; Oxidative stress; Resveratrol; Saccharomyces cerevisiae; Sir2.

Graphical abstract

Fig. 1

RSV supplementation at the diauxic shift determines a short-lived phenotype. Wild type (wt) cells were grown in minimal medium/2% glucose and the required supplements in excess (see Materials and methods). At the diauxic shift (Day 0), resveratrol (RSV, 100 μM) was added to the expired media and (A) survival over time of treated and untreated cultures was assessed by colony-forming capacity on YEPD plates. 72 h after the diauxic shift (Day 3) was considered the first age-point, corresponding to 100% survival. Data referring to the time points where chronological aging cultures showed 50% (Mean CLS) and 10% (Maximum CLS) of survival are reported in the Table. In parallel, for the same cultures the concentrations of extracellular ethanol (B) and acetate (C) together with Icl1 and Pck1 enzymatic activities (D) and intracellular trehalose content (E) were measured. All data refer to mean values of three independent experiments with three technical replicates each. Standard deviations (SD) are indicated. Statistical significance as assessed by one-way ANOVA test is indicated (*P≤0.05 and **P≤0.01).

Fig. 2

RSV supplementation at the diauxic shift triggers oxidative stress. Cultures of Fig. 1 were analyzed for the presence of intracellular superoxide by the conversion of non-fluorescent dihydroethidium into fluorescent ethidium (Eth). (A) Summary graphs of the percentage of fluorescent/superoxide positive cells (% Eth) are reported. About 1000 cells for each sample (three technical replicates) in three independent experiments were examined. At the indicated time points, catalases/Sod enzymatic activities (B) and the levels of intracellular malondialdehyde (MDA) (C) were measured. All the measurements were normalized to the protein content. Day 0, diauxic shift. SD is indicated. Statistical significance as in Fig. 1 (*P≤0.05 and **P≤0.01).

Fig. 3

RSV supplementation at the diauxic shift increases Pck1 deacetylation without affecting Sir2 levels. Wt cells expressing Pck1-3HA were grown and supplied at the diauxic shift (Day 0) with RSV as in Fig. 1. At different time points, total protein extracts from both treated and untreated cultures were prepared and immunoprecipitated with anti-HA antibody. Afterwards, Western analyses were performed with anti-HA and anti-Ac-K antibodies followed by densitometric quantification of signal intensity of the bands relative to the total Pck1 (Pck1-3HA) and the acetylated form (Ac-K). The ratios of Ac-K to correspondent Pck1-3HA values are reported (A). (B) Wt cells expressing Sir2-3HA were grown and supplied at Day 0 with RSV as in Fig. 1. At different time points, total protein extracts from both treated and untreated cultures were prepared and subjected to Western analysis using anti-HA antibody. Protein extracts from exponentially growing cells were also analyzed (Exp). Pgk1 was used as loading control. The filter stained with Ponceau S Red is also shown. One representative filter is shown. (C) Bar chart of the relative levels of Sir2-3HA expressed in arbitrary units. For each time point, band intensities relative to Sir2-HA and Pgk1 detected by Western analysis were quantified and the values of Sir2-3HA signals were normalized against Pgk1 ones. Then, all values obtained were further normalized against the normalized Sir2-3HA level measured in exponential phase, which was arbitrary set to 1. (D) Total protein extracts prepared from cultures in (B) were subjected to Western analysis using antibodies anti-H4K16ac and anti-H4. The filter stained with Ponceau S Red is also shown. (E) Bar chart of the relative levels of H4K16ac expressed in arbitrary units. Densitometric quantification of signal intensity of the bands relative to the total H4 and the acetylated form (K16ac) was performed. The ratios of H4K16ac to correspondent H4 values are reported. All data refer to mean values determined in three independent experiments with three technical replicates each. SD is indicated. Statistical significance as in Fig. 1 (**P≤0.01).

Fig. 4

RSV supplementation at the diauxic shift tosir2Δ cells does not determine a short-lived phenotype. Sir2Δ cells were grown and supplied at the diauxic shift (Day 0) with RSV as in Fig. 1. (A) Survival over time of treated and untreated cultures was assessed. In the Table are shown Mean CLS and Maximum CLS values. Bar charts report Pck1 enzymatic activity (B), the ratio between Ac-K and Pck1-3HA values (C), the percentage of intracellular superoxide-accumulating cells (D), catalases/Sod enzymatic activities (E) and the levels of intracellular malondialdehyde (MDA) (F) determined for both cultures. All data refer to mean values of three independent experiments with three technical replicates each. SD is indicated.

Fig. 5

RSV reduces the beneficial effects induced bySCH9deletion and severe CR on CLS. (A) CLS of sch9Δ and sch9Δsir2Δ cells supplemented with RSV at the diauxic shift (Day 0) as in Fig. 1. Survival of wt and sir2Δ strains is also shown for comparison. (B) The indicated strains grown as in Fig. 1, at Day 1 were transferred to water containing 100 μM RSV. Every 48 h, cultures were resuspended in fresh water and each time RSV was added. Survival of both treated and untreated cells was evaluated. In parallel, Pck1 enzymatic activity (C) and the ratio of Ac-K to Pck1-3HA values (D) were determined. All data presented are the mean values±SD of three biological replicates. Statistical significance as in Fig. 1 (**: P≤0.01).

Fig. 6

NAM supplementation during chronological aging rescues the negative effects of RSV on CLS and Pck1 activity. (A) Wt cells were transferred to water as in Fig. 5B supplemented with 100 μM RSV or 5 mM nicotinamide (NAM). Every 48 h, cultures were resuspended in fresh water containing RSV or NAM and survival of the cells over time was monitored. In addition, at the time point corresponding to 50% of survival RSV-treated cells were switched to water supplemented with both NAM and RSV. Survival of wt cells in water is also shown for comparison. During the treatments in (A), at the indicated time points, Pck1 enzymatic activity (B) and the ratio of Ac-K to Pck1-3HA values (C) were determined. All data refer to mean values of three independent experiments with three technical replicates each. SD is indicated. Statistical significance as in Fig. 1 (**: P≤0.01).

Fig. S1

Cells lacking Sir2 are irresponsive to RSV supplementation at the diauxic shift. Bar charts of Icl1 enzymatic activity (A) and intracellular threalose content (B) determined for RSV-treated and untreated sir2Δ cultures of Fig. 4. Data refer to mean values determined in three independent experiments with three technical replicates each. SD is indicated.