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Calorie restriction (CR) induces a metabolic shift towards mitochondrial respiration; however, molecular mechanisms underlying CR remain unclear. Recent studies suggest that CR-induced mitochondrial activity is associated with nitric oxide (NO) production. To understand the role of mitochondria in CR, we identify and study Saccharomyces cerevisiae mutants with increased NO levels as potential CR mimics. Analysis of the top 17 mutants demonstrates a correlation between increased NO, mitochondrial respiration, and longevity. Interestingly, treating yeast with NO donors such as GSNO (S-nitrosoglutathione) is sufficient to partially mimic CR to extend lifespan. CR-increased NO is largely dependent on mitochondrial electron transport and cytochrome c oxidase (COX). Although COX normally produces NO under hypoxic conditions, CR-treated yeast cells are able to produce NO under normoxic conditions. Our results suggest that CR may derepress some hypoxic genes for mitochondrial proteins that function to promote the production of NO and the extension of lifespan.
Calorie restriction (CR) extends lifespan in a variety of organisms and has also been shown to ameliorate many age-associated diseases such as diabetes and cancers [1–3]. However, the molecular mechanisms underlying CR-induced beneficial effects are still not fully understood. Owing to a short lifespan and well-established molecular genetic techniques, the budding yeast Saccharomyces cerevisiae represents a powerful system to identify new components in CR signaling pathways and to study these factors at the molecular/genetic level. Yeast lifespan can be studied in two distinct ways: replicative lifespan (RLS) and chronological lifespan (CLS). RLS measures the number of cell divisions an individual yeast cell undergoes before senescence (division potential), whereas CLS measures the length of time cells remain viable at a nondividing state (postmitotic survival). RLS may serve as a model to understand the mechanisms of replicative senescence such as in stem cells, while CLS may be more relevant to postmitotic cell senescence in adult animals [4, 5].
Moderate CR can be imposed in yeast by reducing the glucose concentration from 2% to 0.5% in rich media [6–9], which extends both RLS and CLS. In yeast, CR is suggested to function through reducing the activities of conserved nutrient-sensing pathways. Decreasing the activities of the Ras-cAMP/PKA (cyclic AMP-activated protein kinase A) pathway, Sch9 (homolog of mammalian S6K kinases) and Tor1 kinases have been shown to mimic CR and extend lifespan [6, 10, 11]. The recent identification of additional CR-specific longevity genes provides further insight into the molecular mechanisms underlying CR and the resulting metabolic alterations [7, 12–17]. The Sir2 family proteins (sirtuins) are among identified CR downstream targets; they are conserved longevity factors that were originally discovered and studied in yeast . Sirtuins are NAD+-dependent protein deacetylases that are responsive to metabolic changes and stress and have been shown to play important roles in several CR models [3, 18, 19].
Mitochondria have also been shown to play important roles in CR. In yeast, CR induces a shunting of carbon metabolism from fermentation to the mitochondrial TCA cycle . This metabolic shift to respiration is necessary and sufficient for the activation of Sir2-mediated lifespan extension in certain yeast strains . A link between CR and increased mitochondrial metabolism has also been reported in higher eukaryotes including mammals [13, 14, 20–22]. Notably, the age-dependent decline in expression of genes encoding components of the mitochondrial respiratory chain has been reported in several species [22–24]. Since mitochondria are the major sites of energy production in eukaryotic cells, these findings highlight the crucial role of energy metabolism in CR. Since CR regimens involve the reduction of nutrient input, it is believed that a global change in nutrient sensing and regulatory pathways as well as changes in the mitochondrial respiratory chain are translated to physiological responses to counteract age-induced effects [25–27].
The role of the mitochondrial respiratory chain in CR is still unclear. In yeast, it has been suggested that CR activates mitochondrial respiration to prevent the accumulation of toxic metabolites [28–30]. Although the mitochondrial electron transport chain is also the primary site for reactive oxygen species (ROS) generation in the eukaryotic cell, increased mitochondrial electron flow during CR would be expected to reduce ROS levels [3, 31–33]. Recently, cytochrome c oxidase (COX) of the mitochondrial respiratory chain has been shown to catalyze the reduction of nitrite to nitric oxide (NO) . When considered with the findings that the respiratory chain is involved in CR and that NO has been implicated in CR, it is of interest to ask if the NO involved is produced by mitochondrial COX.
In order to begin to elucidate how CR modulates complex genetic and metabolic networks to alter stress resistance, genomic stability, and lifespan, it is essential to uncover additional factors in the CR pathway. Towards this end, we have explored the relationship between NO and CR and have identified new genes in CR.
Yeast strains BY4742 MATα his3Δ1 leu2Δ0 lys2Δ0 ura3Δ0 and the genome-wide gene deletion collections (nonessential genes) were acquired from Open Biosystems. Medium used for replicative lifespan (RLS) analysis was YEP (2% bacto peptone, 1% yeast extract, 1.5% agar) supplemented with filter-sterilized glucose at a final concentration of 2% or 0.5%. Medium used for chronological lifespan (CLS) analysis was minimal synthetic SD (6.7g/L yeast nitrogen base) supplemented with 4x auxotrophic amino acids (leucine, histine, uracil, and lysine) and glucose to a final concentration of 2% or 0.5%. Gene deletions were made by replacing the wild-type genes with the reusable Kanr marker as described in  and verified by Polymerase Chain Reaction (PCR) using oligonucleotides flanking the genes of interest.
Different concentrations of GSNO were added to yeast cells during chronological lifespan assays at different time points. GSNO was made as follow: 200μL of 0.5M GSH in 0.5M HCL was added to 200μL of 0.5M NaNO2 and then incubated at 4°C in the dark. After 30 minutes, 400μL of 0.25M NaOH was added to neutralize the solution. This reaction generated GSNO at final concentration of ~125mM. Inactivated GSNO was generated by exposing GSNO to UV illumination for 30min (photolysis) .
Diaminofluorescein-FM diacetate (Kamiya Biomedical) was diluted to 1μM in reaction buffer (0.2M phosphate buffer, pH 7, 1mM EDTA, 0.1% glucose) immediately prior to the assay. Cells at different growth stages were harvested, washed twice, and then resuspended into 120μL of reaction buffer (0.2M potassium phosphate buffer, pH 7, 1mM EDTA, 0.1% glucose). 100μL of cell suspension was added to 100μL reaction buffer with 1μM of DAF-FM DA in 96-well fluorescence assay plates (~5×106 cells were analyzed in each well). After 2 hours of incubation (30°C), fluorescence signals were detected using a plate reader with excitation at 485nm and emission monitored at emission at 535nm [36, 37].
Cells were grown in YPD media containing either 0.5% or 2% glucose on a shaker (200rpm) at 28°C and harvested in mid-exponential phase. Cells were washed twice with cold distilled water, pelleted, and kept on ice until use. Nitrite-dependent NO synthesis was measured essentially as described in [34, 38]. Briefly, cells were resuspended in 2mL PBS (pH 6.5) in an NO chamber and bubbled 5min with nitrogen. Then the chamber was closed, and NO production was measured at 28°C after the addition of 1mM sodium nitrite using an NO electrode (amiNO-700, Innovative Instrument) as described in [34, 38].
Cells were grown as above and harvested in mid-exponential phase. Mitochondria were prepared as described in . NO production was measured with an amino-700 nitric oxide sensor connected to an inNOII nitric oxide measuring system (Innovative Instruments, Inc). Simultaneous measurement of O2 concentration in the chamber was performed by a 2mm Clark-type electrode Oxy-2 (Innovative Instruments, Inc) connected to the inNOII. Except where noted, all solutions were nitrite-free. Measurements were performed at 28°C in a final reaction volume of 2mL, in a thermostated chamber with a close-fitting lid containing ports for the electrodes and a Hamilton syringe. Assays for NO production by the mitochondrial respiratory chain were performed in NO Assay Medium (6mM succinate, 650mM mannitol, 10mM K2HPO4 [pH 6.5], 0.1mM EDTA, and 10mM KCl). Except where noted, the NO assay medium was prebubbled with air in order to be saturated with oxygen (200μM) prior to addition of NaNO2, which was added to a final concentration of 1mM, and the measurement of NO production.
Each mutant strain from the deletion collection (Open Biosystems) was inoculated into SD in 96-well plates and allowed to grow for 2 days. 4μL saturated culture was then added into 400μL fresh SD and allowed to grow for 12hr. To prepare for NO measurement, cells were spun down in 96-well plates at 3000RCF, washed, and then resuspended into 120μL reaction buffer (0.2M phosphate buffer, pH 7, 1mM EDTA, 0.1% glucose). 100μL cell suspension was added to 100μL reaction buffer with 1μM of DAF-FM DA (Kamiya Biomedical) prealiquated in 96-well fluorescence assay plates. Multiple wild-type (WT) controls grown under both normal (2% glucose) and CR (0.5% glucose) conditions were included on each 96-well plate as internal controls. Average NO levels of WT controls grown under normal condition (after normalized to cell number) were set to 1. NO levels of the mutants were normalized to the WT controls on the same plate. We have determined the variations among different plates and different experiments to be ~20% (data not shown), which was used as the standard deviation for the initial screen.
Three to four single colonies derived from each strain were analyzed in each experiment as described in  with a few modifications. Cells were grown in 10mL SD (at a starting OD600 of 0.1) in 50mL tubes on a roller drum set at 250rpm to ensure proper aerations. Cell viability (~2×108 cells were analyzed in each sample) was monitored every 2-3 days by plating a fraction of the culture onto fresh media to determine the colony-forming units (CFU). Cell survival rates were calculated by normalizing the CFU of each time point to the CFU of day 0 (48hr after starting CLS, when cells just entered stationary phase). Numbers on the x-axes of CLS graphs denote the number of days after entering stationary phase (nonmitotic state). GSNO-treated cells were grown in the dark for the first 24hr.
All RLS analyses were carried out on YEP agar plates supplemented with glucose at a final concentration of 2% or 0.5% with 50 cells per strain per experiment  using a micromanipulator under a microscope. All RLS assays were repeated at least twice by two different individuals.
Oxygen consumption was monitored using a Clark-type electrode as previously described in .
About 300 OD600 units of yeast cells grown to different stages were harvested and homogenized in 1.5mL lysis buffer containing 20mM Tris-HCl, pH 8, 0.5mM EDTA, 0.1% NP-40, 1mM phenylmethylsulphonyl fluoride (PMSF) (Sigma), and protease inhibitor tablet (Roche) using a beads beater. The GSNO reductase (GSNOR) activity of Sfa1 was determined by measuring GSNO-dependent NADH consumption using fluorescence (340/460nm) as described in  in 200μL reaction buffer (200μM NADH, 20mM Tris-HCl, pH 8, 0.5mM EDTA) with 15μg (total proteins) cell lysate and 0 or 200μM GSNO at room temperature. The NO metabolizing activity of Yhb1 was determined by measuring NO-dependent NADH consumption using fluorescence (340/460nm) as described in  in a 200μL reaction mix containing 200μM NADH, 20mM Tris-HCl, pH 8, 0.5mM EDTA, 15μg lysate, and 0 or 300μM MAHMA NONOate (Cayman Chemical) at room temperature. The NADPH-dependent NO-metabolizing activity was measured by MAHMA NONOate-dependent consumption of NADPH using fluorescence (340/460nm) as described in  in a 200μL reaction mix containing 250μM NADPH, 20mM Tris-HCl, pH 8, 0.5mM EDTA, 15μg lysate, and 0 or 300μM MAHMA NONOate at room temperature.
Approximately 1kb of the CYC7 promoter was cloned into the URA3+, pYES2.1 vector (Invitrogen) using the BamHI site immediately upstream of the β-galactosidase gene. For the liquid β-galactosidase assay, 5 OD600 units of yeast cells grown in SD w/o uracil for 18hrs (starting OD600 = 0.1) under normoxic condition were washed, lysed by bead beating in breaking buffer (100mM Tris-HCl pH 8, 20% glycerol, 1mM DTT, 0.02% SDS, 8mM PMSF), and separated from debris by centrifugation. 50μL of lysate was then added to 450μL of Z buffer (0.1M sodium phosphate buffer pH 7, 10mM KCl, 1mM MgSO4, 50mM β-mercaptoethanol), preincubated at 30°C for 5min, and then 100μL 4mg/mL ONPG in Z buffer was added. After 1hr incubation at 30°C, the reaction was stopped with 250μL of 1M NaCO3 and read at 420nm. This reading was then normalized to protein concentration of the lysate and finally normalized to the WT 2% glucose sample.
Cells were first inoculated in SD (staring OD600 = 0.1) and allowed to grow for 12hrs. The arginine analogue and NOS inhibitor L-NAME (Sigma) was then added to these cells at 200mM final concentration as previously described in . After 2hr incubation at 30°C, cells were washed and then incubated with the DAF-FM DA fluorescence dye to determine relative NO levels as described previously.
For hydrogen peroxide treatments, cells were pregrown in SD with 2% or 0.5% glucose (CR) for 20hrs (staring OD600 = 0.1) then were treated with 0.5% H2O2 for 1hr. Following this treatment, cells were plated on YPD in 5-fold serial dilutions. For GSNO toxicity test, cells were pregrown in SD with 2% or 0.5% glucose (CR) or pretreated with 25μM GSNO for 18hr. GSNO was then added to cells at a final concentration of 1.5mM. After 24hr treatment, cells were removed from the GSNO-containing SD, plated on YPD in 5-fold serial dilutions, and then allowed to grow for 2 days at 30°C.
Statistical analysis of CLS was carried out using the AUC method (area under the curve) [44, 45]. P values were calculated for each pair of lifespans as shown in Supplemental Table 2 (see Supplementary Material available online at doi: 10.4061/2011/673185). Statistical analysis of RLS was carried out using the JMP statistics software (SAS), and the Wilcoxon rank sums tests P values were calculated for each pair of lifespans as shown in Supplemental Table 3. All other P values were calculated using Student's t-test (two-tailed).
Moderate CR in yeast results in a metabolic shift from fermentation to mitochondrial respiration . In mice, CR-induced mitochondrial biogenesis was attributed to increased expression of endothelial nitric oxide synthase (eNOS) . To further understand the role of NO in CR, we determined whether CR increased NO levels in yeast using an NO-sensitive dye, diaminofluorescein-FM diacetate (DAF-FM DA). Although DAF-FM DA is fairly specific to NO and NO oxidation products [36, 37], we further confirmed that it did not cross-react with other reactive oxygen species (such as superoxide and hydrogen peroxide) in vivo under our assay conditions (Supplemental Figure 1). As shown in Figure 1(a), CR significantly increased NO-related signals during early growth stages, while cells were actively dividing. The long-lived tor1Δ and hxk2Δ mutants [6, 11] also showed increased NO signals (Figure 1(b)). In addition, CR, tor1Δ, and hxk2Δ also conferred resistance towards nitrosative stress (treatments of a physiological NO donor S-nitrosoglutathione, GSNO) (Figure 1(c)). We next determined whether the activities of major NO detoxification enzymes were increased by CR. Yeast flavohemoglobin (Yhb1), which catalyzes the conversion of NO to nitrite or nitrous oxide, and GSNO reductase (GSNOR, Sfa1) are the two major enzymes that protect yeast cells from nitrosative stress [40, 41, 46]. Interestingly, Yhb1 activity determined in the crude cell lysate  was increased by CR (Figure 1(d)), whereas Sfa1 activity  was not increased by CR, suggesting that Yhb1 may play an important role in CR. In addition, CR also slightly induced NADPH-dependent NO-metabolizing activity (Figure 1(d)), which has been reported in mammalian cells . Together, these results demonstrate that CR-induced increases in NO are not due to decreased NO degradation.
To identify additional components in the CR pathway, we screened the yeast deletion collection (~4500 strains) for mutants with elevated NO levels similar to CR. About 157 high-NO mutants were identified and ranked by NO levels, growth rate, and resistance to GSNO (Supplemental Table 1). CR increases both replicative and chronological lifespan (RLS and CLS). Therefore, we used these criteria to classify potential “CR genetic mimics.” Among the top 15 hits, 5 mutants (hxt17Δ, pkh2Δ, gup1Δ, hhf1Δ, and soy1Δ) showed extended CLS and RLS (Table 1). Hxt17 is a high-affinity hexose transporter, which may mimic CR by decreasing the activity of the glucose sensing pathways . Pkh2 is a Ser/Thr protein kinase that activates Sch9 , and pkh2Δ is likely to behave similar to sch9Δ, which has been shown to mimic CR . Recently, pkh2Δ was identified as an RLS-extending mutant , providing further validation for our screen. Gup1 is an ER membrane-localized O-acyltransferase involved in remodeling the fatty acid moiety of the GPI anchor . HHF1 encodes one of two identical histone H4 proteins. Depleting histone H4 has been shown to alter the expression levels of specific yeast genes including many genes that regulate energy homeostasis [50, 51]. Soy1/Aim4 is suggested to participate in mitochondrial genome maintenance ; however, its molecular function remains unclear.
We next determined whether these long-lived mutants function in the same pathway as CR to extend lifespan. As expected, CR did not significantly alter CLS (Figures 2(a)–2(d)) or RLS (Figures 3(a)–3(d)) of most of these long-lived mutants, suggesting that these mutations might be in genes that function in the same (or overlapping) pathway to extend CLS and/or RLS (detailed statistical analysis of CLS and RLS is shown in Supplemental Tables 2 and 3, resp.). Two notable exceptions were observed in our studies: the gup1Δ (Figures 2(c) and 3(c)) and the soy1Δ (Figure 3(b)) mutants. CR further increased gup1Δ RLS (gup1Δ versus gup1Δ, CR; P = .038) and vice versa (CR versus gup1Δ, CR; P = .004) (Figure 3(c)), suggesting a positive interaction between gup1Δ and CR in RLS extension. In addition, although CR-induced CLS was not further increased by gup1Δ (CR versus gup1Δ, CR; P = .073), the gup1Δ mutant exhibited longer CLS under CR (gup1Δ versus gup1Δ, CR; P = .035). Interestingly, CR appeared to decrease soy1Δ-induced RLS (soy1Δ versus soy1Δ, CR; P = .008) (Figure 3(b)), suggesting a negative interaction between soy1Δ and CR. However, soy1Δ did not significantly change CR-induced RLS (CR versus soy1Δ, CR; P = .49). A potential positive interaction was also observed between hhf1Δ and CR. CR appeared to extend RLS more significantly when combined with hhf1Δ (CR versus hhf1Δ, CR; P = .001). However, CR did not significantly change hhf1Δ-induced RLS (Figure 3(d)) (hhf1Δ versus hhf1Δ, CR; P = .19).
We next determined whether these long-lived, high-NO mutants displayed an increase in mitochondrial respiration similar to CR cells. As shown in Figure 4(a), all of these mutants showed increased oxygen consumption, demonstrating a correlation between increased mitochondrial respiration and NO production. In addition, a functional electron transport chain was required for CR-induced lifespan. CR failed to extend RLS [7, 12] and CLS (Figure 4(b)) in the respiratory-deficient cyt1Δ mutant lacking cytochrome c1. Deletion of CYT1 also eliminated the long RLS (Figure 4(c)) and CLS (B. Li and S.-J. Lin, unpublished) of potential CR mimics. We then further examined how CR increased NO production. Although there is biochemical evidence that NOS homologs are present in yeast , the existence of nitric oxide synthases in yeast remains controversial since no NOS sequence homolog has been identified. In contrast, cytochrome c oxidase (COX) has been shown to produce NO in a nitrite- (NO2−-) dependent manner under hypoxic conditions . Therefore, the mitochondrial electron transport chain is a potential source of NO production in CR cells. Our results (Figure 4(d)) showed that CR- and CR mimics-induced NO increases were largely abolished in cells lacking CYT1. In addition, CR-induced NO production was not inhibited by the NOS inhibitor (arginine analog) L-NAME (Figure 4(e)).
Since the COX complex was reported to produce NO under hypoxic conditions , we examined whether calorie-restricted yeast cells could synthesize NO under normal (normoxic) conditions. Using an NO-specific electrode, we verified that calorie-restricted BY4742 WT (ρ+) cells could produce NO when grown under normoxic conditions (Figure 4(f)). In contrast, calorie restriction failed to induce NO production either in the BY4742 (ρ0), a rho0 strain lacking mitochondrial DNA, or in the respiratory-deficient cyt1Δ mutant under normoxic conditions. These findings further support the conclusion that the NO synthesis in calorie-restricted yeast cells requires a functional mitochondrial respiratory chain. To explore this further, we examined the oxygen sensitivity of nitrite-dependent NO synthesis by mitochondria isolated from WT cells. As shown in Figure 4(g), oxygen sensitivity reported previously for nitrite-dependent mitochondrial NO synthesis  was reduced in mitochondria isolated from WT cells grown under CR. These results demonstrated that yeast mitochondria are capable of NO synthesis at oxygen concentrations that are in the normoxic range experienced by yeast cells grown in colonies or in liquid culture.
Eukaryotic cells harbor both aerobic and hypoxic isoforms for several components of the electron transport chain to ensure proper electron transfer in response to different oxygen concentrations [32, 55–57]. It has been reported that, although COX-specific NO synthesis is strongly inhibited by oxygen when COX contains the aerobic isoform of subunit V (Va), the oxygen inhibition is relieved when COX contains subunit Vb, the hypoxic subunit V isoform . Interestingly, CR increased the expression of the hypoxia inducible cytochrome c CYC7 (Figure 4(h)) as well as COX5b, which encodes COX hypoxic subunit Vb (P. Castello and R. Poyton, unpublished). Therefore, CR might induce NO production in part by derepressing expression of the hypoxic isoforms of mitochondrial electron transport complexes and perhaps additional hypoxia responsive genes.
Next, we examined the roles of NO detoxification enzymes Yhb1 and Sfa1 in CR. CR failed to extend RLS in the sfa1Δyhb1Δ mutant (Figure 5(a)), indicating that Yhb1 and Sfa1 are required for optimal NO homeostasis during CR-induced RLS. Deleting YHB1 partially abolished CR-induced RLS (P = .03, CR versus CR, yhb1Δ), whereas deleting SFA1 alone had no significant effect. Therefore, it appeared that Yhb1 and Sfa1 play redundant roles under our CR/RLS assay condition. The lifespan of potential CR mimics hxt17Δ, pkh2Δ, and hhf1Δ was also reduced to WT levels in the yhb1Δsfa1Δ mutant (Figure 5(b)). Deleting YHB1/SFA1 was not likely to prevent CR-induced RLS solely by reducing mitochondrial activity. As shown in Figure 5(c), CR-induced increases in oxygen consumption and NO production were only partially reduced in the yhb1Δsfa1Δ mutant. Interestingly, yhb1Δsfa1Δ did not abolish CR-induced CLS (Figure 5(d)), suggesting that additional NO-detoxification enzymes and stress response factors might compensate for the loss of Yhb1/Sfa1 for CR-induced CLS. In line with our results, it has been reported that the stress response factors such as Rim15 and superoxide dismutases play important roles in CR-induced CLS [9, 58].
We then examined whether treating cells with NO donors was sufficient to mimic CR to extend lifespan. Interestingly, GSNO appeared to be the most effective NO donor for lifespan extension among the different NO donors tested (B. Li and S.-J. Lin, unpublished). As shown in Figures 6(a) and 6(b), both a single treatment of 25μM S-nitrosoglutathione (GSNO) (which caused mild growth inhibition, Figure 1(c)) and multiple treatments of 5μM GSNO (no growth inhibition observed) extended CLS to a level similar to CR. GSNO did not further increase CR-induced (Figures 6(a) and 6(b)) or CR mimics-induced (Figure 6(c)) CLS suggesting that GSNO and CR may function in the same pathway to extend CLS. Interestingly, GSNO treatments partially rescued the short CLS of the respiratory-deficient cyt1Δ mutant (Figure 6(d)), suggesting that it may partially compensate for the loss of respiration or may also confer some respiration-independent beneficial effects. For example, GSNO may extend lifespan by promoting specific protein modifications . However, GSNO treatment (25μM) did not appear to have an impact upon RLS (B. Li and S.-J. Lin, unpublished); this could be an effect of GSNO instability in the plate-based RLS assay. It is also likely that GSNO only confers partial beneficial effects of CR. Since CR confers resistance to various stresses, we examined whether GSNO might mimic CR by increasing stress resistance. As shown in Figure 6(e), cells pretreated with a low dose of GSNO were more resistant to GSNO-induced toxicity. Pretreatment of GSNO also slightly increased the resistance to hydrogen peroxide compared to calorie-restricted cells (Figure 6(f)). Therefore, GSNO may extend CLS in part by increasing stress responses, possibly via inducing hormesis effect. Interestingly, not all of the high-respiration activity CR genetic mimics examined showed increased resistance towards hydrogen peroxide (Figure 6(f)), although these mutants all showed increased resistance towards GSNO (Supplemental Table 1). It is possible that different mutants only partially mimic CR by activating a different subset of stress response pathway. It is also very likely that additional mechanisms play important roles in these mutants.
Calorie restriction (CR) extends longevity and ameliorates age-related diseases in many organisms. Understanding how CR induces these beneficial effects may therefore provide insights into the molecular mechanisms underlying age-associated diseases. Considerable progress has been made in the discovery of genes/proteins and metabolites that affect longevity. However, many molecular aspects of CR-induced events remain unclear. Uncovering additional long-lived CR-mimicking mutants will likely contribute to the understanding of the molecular mechanisms of CR. To identify new components in the CR pathway, we developed a nitric oxide- (NO-) based screen for mutants that showed higher intracellular levels of NO as potential CR mimics.
Nine of the top 17 high-NO mutants summarized in Table 1 show a positive correlation between increased mitochondrial respiration and NO production. There is also a positive correlation between increased respiration and longevity. These nine mutants showed increased oxygen consumption (9 out of 17) and increased CLS and/or RLS. In addition, mutants that showed both extended RLS and CLS (5 out of 17) all showed increased oxygen consumption as well as NO levels (Table 1). Two mutants (avl9Δ and tps1Δ) that did not show either extended CLS or RLS also did not show increased oxygen consumption. However, it is noteworthy that 5 long-lived mutants (with extended RLS or CLS) did not possess increased oxygen consumption.
Although CR cells have increased NO levels, a higher intracellular NO concentration is not sufficient to extend lifespan under all growth conditions and/or genetic backgrounds. For example, the avl9Δ and tps1Δ mutants have higher NO levels but do not show increased lifespan (Table 1). It is possible that increases in the stress response as well as functional mitochondria are also required for NO-induced lifespan extension. Supporting this model, our results showed that treating the respiratory deficient cyt1Δ mutant with GSNO only partially restored its CLS (Figure 6(d)). In addition, it has been shown that several stress response factors play important roles in CLS and CR-induced CLS in S. cerevisiae [9, 33]. In Schizosaccharomyces pombe, the stress-responsive MAP kinase Sty1 was demonstrated to be essential for CR-mediated CLS extension .
NO detoxification enzymes Yhb1 and Sfa1 appear to play an important role in CR-induced RLS (Figure 5(a)) but not CLS (Figure 5(d)). One possible explanation is that perhaps actively dividing cells are more susceptible to CR-induced NO stress. In line with this model, CR increases NO more significantly in early growth stages during which most cells were still actively dividing (Figure 1(a)). Yhb1 and Sfa1 are dispensable for CR-induced CLS, which may be due to the fact that other NO metabolizing factors and/or stress response factors play more important roles in CR-induced CLS. Interestingly, the sfa1Δyhb1Δ double mutant (under non-CR conditions) showed decreased NO levels (Figure 5(c)) suggesting that functional NO detoxification is required to support NO production in yeast cells. These results highlight the differences in sensitivity towards NO production and detoxification activities for CR-induced RLS and CLS. It is possible that yeast RLS and CLS have different sensitivities towards certain NO metabolites such as peroxynitrite. Peroxynitrite is formed by the reaction of NO with superoxide, both of which can be produced by the electron transport chain . It is possible that during replicative aging, superoxide detoxification is not as efficient, making NO scavenging more important. During chronological aging, the high activity of superoxide dismutases can maintain consistently low levels of superoxide, compensating for the lack of NO scavenging. Supporting this possibility, several stress response factors including superoxide dismutase activities have been reported to be upregulated during CLS [9, 33, 58]. In addition, lower levels of superoxide have also been observed in calorie-restricted yeast cells during CLS . Transcription factors associated with respiration (Hap4), stress response (Msn2), and a MAP kinase important in osmoregulation (Hog1) can drive the expression of Yhb1 [61, 62], Sfa1 [63, 64], or both and may regulate increased RLS and resistance to nitrosative stress during CR.
Our results indicate that COX is a major source of NO production under CR (Figure 7) because deleting components of the electron transport chain in the rho0 (Figure 4(f)), cyt1Δ (Figures 4(d) and 4(f)), and COX (B. Li and S.-J. Lin, unpublished) mutants largely abolishes CR-induced NO production and increased longevity (Figures 4(b) and 4(c)). Moreover, GSNO treatments could only partially compensate the loss of respiratory capacity (Figure 6(d)), indicating that both mitochondrial respiration and mitochondrial NO synthesis are required for optimal NO-induced longevity. Interestingly, the hypoxic isoforms of cytochrome c and COX subunit V, which are normally repressed by oxygen, are activated by CR (Figure 4(h)) (P. Castello and R. Poyton, unpublished). It is therefore possible that CR activates/de-represses factors that are normally induced by hypoxia to further augment NO production and respiration capacity. The upregulation of Cox5b in CR cells could explain both the increased rate of NO synthesis and the relaxation of oxygen sensitivity because a COX isozyme containing subunit Vb has been shown to support a higher rate of NO synthesis at high oxygen concentrations . In addition, increased NO has also been suggested to function in the induction of hypoxic genes (hypoxic signaling) [32, 34, 65]. It would be interesting to determine the roles of CR-induced NO production and hypoxic signaling in mitochondrial hormesis (mitohormesis), which also plays an important role increasing stress responses and lifespan . It remains unknown whether COX also plays important roles in CR-induced NO production in mammals, although rat liver mitochondrial COX and human endothelial cell COX also exhibit NO producing activity [34, 65]. In mammals, mitochondrial NO could also come from eNOS, which has been reported to physically interact with the outer mitochondrial membrane . eNOS has been suggested as a mediator of CR in mice, and induction of eNOS caused both mitochondrial biogenesis and enhanced expression of the NAD-dependent protein deacetylase SIRT1 . Together, these studies suggest that NO-mediated signaling and mitochondrial respiration work in concert to adapt cells to metabolic changes induced by CR leading to enhanced stress response and lifespan extension (Figure 7).
In summary, we have demonstrated that calorie-restricted yeast cells produce NO under normoxic conditions perhaps by derepressing the hypoxic subunit isoforms of COX and cytochrome c. CR-induced NO production may extend lifespan by increasing the stress response (mitohormesis). Our study may have uncovered potential novel components in the CR pathway and provided tools to analyze the interconnections between NO, mitochondrial respiration, CR, and longevity. Although the CR mimics identified in this study share similar NO levels, lifespan, and oxygen consumption phenotypes with CR, they may activate NO production and regulate mitochondrial respiration and lifespan via different mechanisms. It will be enlightening to examine these differences in future studies. Finally, we propose that CR likely confers its beneficial effects via a mitochondria-NO-mediated adaptive metabolic shift, which optimizes metabolism and at the same time improves cellular defense system against the oxidative stress that accumulates with age.
The authors thank Dr. Limin Liu, Dr. Moran Benhar, and Dr. Alfred Hausladen for discussions and suggestions on nitric oxide metabolism. They also thank members of the Parales laboratory for assistance with oxygen consumption assays and the screen for high NO mutants and Dr. Xiaowei David Yang for assistance with statistical analysis. This work was supported by grants from National Institute on Aging (AG24351) (to S.-J. Lin), the Ellison Medical Foundation (to S.-J. Lin), and NIH (GM30228) (to R. O. Poyton). B. Li and C. Skinner contributed equally to this work.
The authors have declared that no conflicts of interest exist.