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Mitochondria are a major intracellular source of reactive oxygen species (ROS) they are involved in aging and lifespan regulation. Mitohormesis involves a variety of ROS during several growth stages.
Mitochondria are a major intracellular source of reactive oxygen species (ROS) they are involved in aging and lifespan regulation. Mitohormesis involves a variety of ROS during several growth stages.
Mitochondria are a major intracellular source of reactive oxygen species (ROS) they are involved in aging and lifespan regulation. Mitohormesis involves a variety of ROS during several growth stages.
Mitochondria, reactive oxygen species, and chronological aging: A message
from yeast Yong Pan Gladstone Institute of Virology and Immunology, University of California, San Francisco, 1650 Owens Street, San Francisco, CA 94158, United States a b s t r a c t a r t i c l e i n f o Article history: Received 1 April 2011 Received in revised form 22 July 2011 Accepted 15 August 2011 Available online 22 August 2011 Section Editor: T.E. Johnson Keywords: Chronological lifespan Mitochondrial theory of aging Mitohormesis Hormetic ROS Oxidative phosphorylation Target of rapamycin Reactive oxygen species Saccharomyces cerevisiae As a major intracellular source of reactive oxygen species (ROS), mitochondria are involved in aging and life- span regulation. Using the yeast chronological aging model, researchers have identied conserved signaling pathways that affect lifespan by modulating mitochondrial functions. Caloric restriction and a genetic mimetic with reduced target of rapamycin signaling globally upregulate the mitochondrial proteome and respiratory functions. Recent discoveries support the notion that an altered mitochondrial proteome induces mitohormesis. Mitohormesis involves a variety of ROS during several growth stages and extends lifespan in yeast and other organisms. Here we recap recent advances in understanding of ROS as signals that decelerate chronological aging in yeast. We also discuss parallels between yeast and worm hypoxic signaling. In sum, this mini-review covers mitochondrial regulation by nutrient-sensing pathways and the complex underlying interactions of ROS, metabolic pathways, and chronological aging. 2011 Elsevier Inc. All rights reserved. 1. Introduction: Mitochondria and aging Mitochondria serve many essential functions, but their primary role is to generate ATP from nutrients. They also participate in signal transduction, metabolite conversion, and the biosynthesis of organic molecules. In addition, they are the major intracellular source of reac- tive oxygen species (ROS), which damage cellular components and accelerate aging of the organism. Since mitochondrial ATP production and ROS generation involve oxidative phosphorylation (OXPHOS), active regulation by conserved signaling pathways is warranted. Here, we reviewrecent advances in understanding howmitochondria contribute to aging, with an emphasis on the regulation of OXPHOS by the target of rapamycin (TOR) pathway and its effects on the chro- nological lifespan (CLS) of yeast. The OXPHOS system consists of protein complexes that transfer high-energy electrons from NADH and FADH2 to molecular oxygen. Potential energy is harvested to establish a proton gradient across the inner mitochondrial membrane. This so-called mitochondrial membrane potential is dissipated through ATP synthase (OXPHOS Complex V) to generate ATP. Normally, donation of electrons to oxygen is catalyzed by Complex IV, or cytochrome oxidase. However, electrons may react prematurely with oxygen in nonenzymatic reac- tions, yielding ROS. The mitochondrial theory of aging, rst proposed by Harman, has garnered support over the years (Harman, 1972). According to Harman, cellular damage caused by ROS generated in mitochondria is the major force driving the aging process. Subsequent research conrmed that mitochondria generate several types of ROS, including superoxide, hydrogen peroxide, and hydroxyl radicals. Because the chemical prop- erties of the ROS are different, their preferred cellular targets differ (D'Autreaux and Toledano, 2007). The highly reactive hydroxyl radical is indiscriminate. Negatively charged superoxide primarily targets iron-sulfur clusters, and neutrally charged hydrogen peroxide reacts preferably with cysteine thiols. ROS also collaborate with metals in multi-step reactions, yielding high-molecular-weight products, such as carbonylated protein aggregates (Nystrm, 2005). ROS-induced damage is complex and frequently irreversible, and it further impairs mitochondrial function, rendering them prone to further ROS genera- tion. This positive feed-forward relationship between mitochondrial ROS generation and damage was hypothesized to be responsible for oxidative stress and accelerated aging (Harman, 1972). Because ROS damage cellular components, eukaryotes have evolved detoxifying enzymes, such as superoxide dismutases and catalases. Consistent with the mitochondrial theory of aging, transgenic mice over-expressing catalase in various cellular compartments have a longer lifespan than controls (Schriner et al., 2005). Interestingly, most lifespan gain is achieved by catalase over-expressed in mitochon- dria, conrming that ROS generated in mitochondria are a major Experimental Gerontology 46 (2011) 847852 E-mail address: yong.pan@gladstone.ucsf.edu. 0531-5565/$ see front matter 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.exger.2011.08.007 Contents lists available at SciVerse ScienceDirect Experimental Gerontology j our nal homepage: www. el sevi er . com/ l ocat e/ expger o lifespan-limiting factor. However, the relationship between detoxica- tion enzymes and lifespan is non-linear. For example, neither global reduction nor over-expression of manganese superoxide dismutase (Sod2) alters lifespan even though such variation of the enzyme levels signicantly affects resistance against paraquat (Jang et al., 2009; Van Remmen et al., 2003). These observations highlight a complex relation- ship between ROS, detoxication enzyme and lifespan in higher organ- isms. Nevertheless, the reductionist approach to use simpler model organisms has shed new light on the roles of ROS in aging. 2. Yeast models for the mitochondrial theory of aging The budding yeast, Saccharomyces cerevisiae, is a well-established model for studying mitochondrial function and aging. Despite evolu- tionary divergence from metazoans, multiple molecular pathways that regulate mitochondria and aging are conserved in yeast and higher organisms. Unlike many higher organisms, budding yeast can endure lack of oxygen and do not require mitochondria for ATP production. Thus, it is an ideal system for exploring mitochondria's important roles besides energy production. Inglucose-richenvironments, yeast fa- vors sugar fermentationover respiration, resulting indistinct features of upregulation of respiratory gene expression upon sugar depletion. Genomic tools to better understand changes in expression revealed patterns of yeast mitochondrial proteins during the switch to respira- tion (i.e., the diauxic shift). By probing yeast expression proles at different growth stages, Brown and colleagues revealed a major change in yeast gene expression during the diauxic shift in glucose-based YPD media (DeRisi et al., 1997). Specically, the tricarboxylic acid (TCA) cycle was globally upregulated, and glycolysis was globally downregu- lated, consistent with metabolite data. This expression pattern sug- gested that the direction of metabolic ow during the post-diauxic phase is reversed from that during log growth. Consistent with increased expression of OXPHOS, the respiratory chain regulator HAP4 was one of the two genes encoding transcription factors that are upregu- lated during diauxic shift. Two aging models, replicative aging and chronological aging, have been developed to model different aspects of yeast aging. Replicative lifespan measures the number of times a yeast mother cell divides before reaching senescence (Steinkraus et al., 2008). In contrast, CLS registers the percent viability of yeast culture in the stationary phase (Fabrizio and Longo, 2007). Using the yeast CLS system, researchers conrmed the importance of mitochondrial ROS in aging. As we shall discuss in depth in the following sections, several genetic mutations inuence ROS levels in various yeast growth stages and subsequently affect CLS. Like other aging models, ROS detoxifying enzymes have important but complicated roles in CLS. [Mitochondrial ROS have equally critical effects on replicative aging, a topic reviewed in this journal (Ugidos et al., 2010)]. 3. Caloric restriction and its genetic mimetics Among a handful of manipulations that extend lifespan, caloric restriction (CR) shows high levels of evolutionary conservation. CR, typically in the forms of less frequent feeding or a reduction in the quantity or quality of food, aims at inducing under-nutrition without malnutrition (Mair and Dillin, 2008). In mammals, typical experi- ments decrease calorie intake by 2050%, which extends median life- span by 3080%. In yeast, a common CR protocol uses 0.5% glucose upon inoculation (a 75% reduction in sugar content from standard media) without alterations in concentrations of other nutrients. Compared to the standard 2% glucose medium, CR medium strongly stimulates oxygen consumption (Lin et al., 2002). The increase in oxygen consumption is recapitulated by HAP4 over-expression. Com- parison of gene expression proles further revealed that 55 of 124 genes differentially regulated by CR are similarly regulated by HAP4 over-expression. In addition to glucose, restriction of several other easily fermentable sugars strongly affects CLS (Smith et al., 2007). Intriguingly, yeast in complex sugar- or non-fermentable carbon source-based media are longer-lived than that in a glucose-based medium, but restrictionof these carbonsources leads to more attenuated or entirely absent CLS extension (Smith et al., 2007). This observation is consistent with the fact that metabolizing complex sugars or non- fermentable carbon sources already extensively involves respiration. Complex sugars and non-fermentable carbon sources, like restriction of easily fermentable sugars, extend CLS via enhancing mitochondrial functions. Manipulation of genetic mimetics of CR produces CLS extension similar to that of CR. Such mimetics typically reduce evolutionarily conserved, nutrient-sensing pro-growth signals. Ras and TOR are two well-studied signaling pathways (Table 1). Reduced activity of ei- ther pathway by knockout or loss-of-function mutation in Ras or TOR renders strains longer-lived than wildtype controls (Powers et al., 2006; Wei et al., 2008). Like yeast under CR, ras2 and tor1 strains have enhanced resistance to oxidative stress as they enter the station- ary phase (Wei et al., 2008). Despite different signaling circuitries, both Ras and TOR inuence key anti-stress regulators, such as Rim15, Msn2/4, and Gis1. These factors directly or indirectly increase the levels of detoxication enzymes (e.g., Sod2) and protein chaper- ones (e.g., heat shock proteins). Besides regulating stress-resistance genes, Ras directly regulates mitochondrial respiration and ROS production. Strains over- expressing constitutively active Ras2 val19 have reduced respiration (Hlavata et al., 2003). Furthermore, Ras2 val19 engages yeast in an altered respiratory mode with elevated mitochondrial membrane potential and failed ATP production. Interestingly, supplementation with a chemical uncoupler or expression of uncoupling protein restores respiratory function and reduces ROS accumulation. Evidently, blocking potential dissipation through ATP synthase increases ROS production. Not surprisingly, Ras2 val19 -expressing yeast accumulates excessive oxidative damage products. In agreement with the mitochon- drial theory of aging, this yeast strain has a much shorter CLS than wild- type (Fabrizio et al., 2003). 4. TOR signaling and mitochondria In budding yeast, nutrient-sensing TOR signaling involves TORC1 (Loewith et al., 2002). TORC1 is a well-conserved complex in evolu- tion, with yeast and mammals sharing homology in its constituents. In yeast or mammalian systems, TORC1 is specically inhibited by rapamycin. Furthermore, yeast TORC1 directly phosphorylates Sch9p, a yeast homolog of ribosomal S6 kinase 1 (S6K1), the target of mammalian TORC1 (Urban et al., 2007). Two kinases, Tor1p and Tor2p, constitute TORC1 in yeast; Tor1p is not essential for viability, although it is present in the complex. For this reason, deletions of TOR1 (tor1) have been generated to study the effects of reduced TORC1 signaling. Like the Ras pathway, the TOR pathway regulates mitochondria and controls CLS. Tor1 was identied in a genetic screen as a long- lived mutant (Powers et al., 2006). CLS extension by deletion of TOR1 was recapitulated by treatment of wildtype yeast withrapamycin. Dele- tion of TOR1 increases respiratory activity and expression of OXPHOS proteins (Bonawitz et al., 2007). Enhanced respiration is required for CLS extension in tor1yeast, since CLS in wildtype and tor1is indistin- guishable in respiration-incompetent cells. The necessity of respiration in CLS extension is conrmed using strains with reduced respiration. Over-expression of Puf3, a suppressor of mitochondrial ribosomal bio- genesis, reduces but does not eliminate respiration (Chatenay-Lapointe and Shadel, 2011). The over-expression partially abolishes lifespan extension by deletion of TOR1. Similar to tor1, increased respiration and extended CLS were found in yeast devoid of SCH9, the major medi- ator of the regulation of expression of OXPHOS proteins and CLS by TORC1 (Lavoie and Whiteway, 2008; Pan and Shadel, 2009). Although 848 Y. Pan / Experimental Gerontology 46 (2011) 847852 enhanced respiration is required for CLS extension, it alone is insuf- cient. Deletion of PUF3 increases mitochondrial translation and oxygen consumption in both wildtype and tor1 without changes in CLS (Chatenay-Lapointe and Shadel, 2011). Genomic approaches have been used to study TOR-regulated tran- scriptional change. For instance, in rapamycin-treated yeast cultures, glycolytic enzymes are downregulated and TCA cycle proteins are upre- gulated, as during the diauxic shift (Hardwick et al., 1999). Similarly, reduced TOR signaling increases respiration during exponential growth and extends CLS, and microarray analyses of sch9 strains during expo- nential growth in SD medium conrmed that many mitochondrial electron transport components are upregulated (Lavoie and Whiteway, 2008). However, another microarray analysis of sch9strains at various growth stages showed downregulation of many mitochondria- associated genes (Wei et al., 2008). These discrepancies suggest that growth stage and strain specicity contribute to gene expression and indicate that other complementary methods are needed to better understand how TOR regulates the OXPHOS system. Since mRNA abundance does not always reect protein abundance, proteomic studies have been conducted. The Grifn group treated yeast with rapamycin during the robust growth phase (Bandhakavi et al., 2008). Among more than 100 differentially expressed proteins, the OXPHOS units Atp15p, Inh1p, Qcr2p, and Qcr7p were upregulated. Putative regulators of mitochondrial gene expression and DNA replica- tion, including Mnp1p, Abf2p, and Pet10p, were also upregulated. Similarly, Schreiber and colleagues combined chemical genetics and genomic and proteomic tools to study growth inhibition by rapamycin (Huang et al., 2004). They identied small molecules that reverse the effects of rapamycin on cells and discovered a new TOR signaling com- ponent, Nir1p. To compare the mitochondrial proteomes of wildtype and tor1, two-dimensional difference gel electrophoresis was used (Pan and Shadel, 2009). The result conrmed the global upregulation of OXPHOS components in tor1 yeast. The regulation of mitochondrial function and lifespan by the TOR pathway is remarkable (Table 1), as knockout of a single gene, such as TOR1 or SCH9, leads to a well-orchestrated reconguration of metabolism and extension of CLS. The global upregulation of OXPHOS is a complex process involving both the mitochondrial and nuclear genomes. Coordinating the two genomes is paramount, as imbalanced OXPHOS is prone to extreme ROS generation. A master regulator, in the form of transcription or translation factors, may act downstream of TOR1/SCH9 to ensure the proper global upregulation. Sch9 may directly phosphorylate transcription factors such as Hap4, which acti- vates OXPHOS biogenesis. Knockout of Hap4 reduces the increase in cytochrome c expression in sch9 strains (Lavoie and Whiteway, 2008). However, evidence of a direct physical interaction between Hap4 and Sch9 will be required to establish Hap4 modication by Sch9. An intriguing alternative to an activator is the preferential translation of mRNAs. With reduced TOR signaling, translation rates do not decrease equally for all mRNAs. A well-characterized example is the regulation of translation by translation initiation factor eIF4E (Koromilas et al., 1992). Under physiological conditions, the supply of eIF4E is limited, and mRNAs must compete for it. Transcripts with complex 5 UTRs rely heavily on eIF4E for effective translation. Nutrient scarcity alters the translation prole, favoring mRNAs with simple 5 structures. Nucleus-encoded mitochondrial proteins in Drosophila share a simple 5 UTR, and their expression is increased upon TOR inactivation (Zid et al., 2009). The same mechanism may apply to regulation of mito- chondrial biogenesis by TOR in yeast. Reduced protein translation was recently identied to extend yeast replicative lifespan (Steffen et al., 2008). Deletion of several 60S subunit-encoding genes extends lifespan in a Gcn4-dependent manner. Whether disruption of the same array of genes changes expression of OXPHOS proteins and CLS remains an un- answered but intriguing question. 5. Mitohormesis: That which does not kill yeast makes it long-lived How do alterations in expression of OXPHOS proteins translate into extended lifespan? Increasing evidence suggests that mitohormesis is a contributor, pointing the mitochondrial theory of aging in a new direc- tion. Hormesis, a phenomenon by which a low dose of toxic stimulus induces resistance against ensuing stress of similar nature, has a well- known role in aging. Worms exhibit extended lifespan in response to a wide variety of environmental stress including heat, hyperbaric oxygen and a ROS generator (Cypser and Johnson, 2002). Built on hor- metic extension of lifespan, the mitohormesis theory posits that ROS production by mitochondria under glucose restriction enhances stress resistance, thereby extending lifespan (Schulz et al., 2007). The authors established that lifespan extension by CR in worms requires oxidative hormetic signals. The respiratory inhibitor azide and the superoxide inducer paraquat both generate CR-like lifespan extension, which is eliminated by antioxidants. Similarly, disruption of Complex I subunit NUO-1 and Complex III subunit ISP-1 induces production of ROS (Yang and Hekimi, 2010). In particular, both nuo-1 and isp-1 mutations induce mitochondrial superoxide without elevating overall ROS, indi- cating that superoxide serves as a hormetic ROS signal in these mutant strains (Yang and Hekimi, 2010). In agreement with superoxide's signal- ing role, pro-oxidant paraquat mimics lifespanextensioninnuo-1 andisp- 1 worms while anti-oxidant n-acetyl cystein (NAC) abolishes lifespan ex- tension in these mutants. Mitohormesis is conserved as a lifespan-extending mechanism in yeast. CR in yeast increases hydrogen peroxide in the stationary phase (Mesquita et al., 2010). A high peroxide level stimulates Sod activity and thus extends CLS. In agreement with mitohormesis, CLS is extended by deletion of peroxisomal or cytosolic catalase and shortened by its over-expression (Mesquita et al., 2010). In tor1 yeast by comparison, superoxide generated during growth serves as the hormetic signal (Pan et al., 2011). Reduction of TOR signaling during growth promotes coupled respiration and boosts OXPHOS elec- tron ow, thereby temporarily elevating mitochondrial membrane Table 1 Cited yeast genes that alter cellular ROS levels and chronological lifespan. Genes Mammalian orthologue Manipulation Phenotype Reference TOR1 mTOR Knock-out Mild increase in superoxide during growth; lower ROS in stationary phase. Extended lifespan Bonawitz et al., 2007; Pan et al., 2011 SCH9 S6K Knock-out Lower ROS in stationary phase. Extended lifespan Lavoie and Whiteway, 2008; Pan and Shadel, 2009 RAS2 RAS Knock-out Reduced damage to mitochondrial aconitase; enhanced resistance to paraquat. Extended lifespan Fabrizio et al., 2003 RAS2 RAS Expression of constitutive val19 allele Reduced respiration; failed mitochondrial ATP production; high ROS. Shortened lifespan Hlavata et al., 2003; Fabrizio et al., 2003 SOD2 SOD2 Knock-out No major impact on lifespan of wildtype; reverted lifespan extension by SCH9 deletion. Fabrizio et al., 2003 HAP4 n.a. Knock-out Reduced oxygen consumption; Reverted lifespan extension by SCH9 deletion Lavoie and Whiteway, 2008 849 Y. Pan / Experimental Gerontology 46 (2011) 847852 potential and enhancing superoxide generation. The superoxide peak suppresses ROS production in stationary phase and prolongs CLS (Pan et al., 2011). In agreement with superoxide's hormetic effects, superoxide inducer menadione during growth effectively prolongs CLS while expression of SOD2 prevents a full lifespan extension. The apparent negative roles of ROS detoxication enzymes in the last two examples are noteworthy. These observations, although super- cially contradicting Harman's mitochondrial theory of aging, provide an explanation on non-linearity between levels of anti-oxidant en- zymes and lifespan. In yeast, Longo and colleagues observed little life- span change by either over-expression or knock-out of Sod2 (Fabrizio et al., 2003). Only in combination with over-expression of Sod1 do high levels of Sod2 extend CLS. Perhaps, the benets of low-level super- oxide dismutase in tackling stress resistance are canceled out by the enzymes' negative roles in initiating hormesis. The dichotomy of detox- icationenzymes ininuencing lifespanmay alsoexplainwhy inhigher organisms Sod2 levels can vary by fourfold without dramatic change in lifespan (Jang et al., 2009; Van Remmen et al., 2003). Intriguingly, the aforementioned yeast studies entertain the idea that different hormetic ROS affect CLS through effects on distinct phases of the yeast life cycle (Fig. 1). While superoxide induces hormesis dur- ing growth, hydrogen peroxide does so in stationary phase. A critical next question to ask is whether and where the two hormetic ROS con- verge upon the same signaling pathway to promote CLS. In the hormetic superoxide example, the adaptive phase (when mitochondrial ROS sig- nals are initiated) and the effective phases (when death occurs) appear to be separable (Fig. 1). Early alterations of mitochondrial functions in yeast and worms inuence survival in stationary phase and adulthood respectively. Respiratory carbon sources, such as glycerol, during growth pre-adapt yeast and extend its lifespan (Piper et al., 2006). Even though during the stationary phase the culture is switched to water, effects on lifespan extension persist. Similarly in worms, a reduction in expression of OXPHOS proteins during robust growth can signicantly extend lifespan (Dillin et al., 2002). In contrast, a similar alteration in adulthood has no effect on lifespan. This work, along with Hekimi's study (Yang and Hekimi, 2010), implies that superoxide has an adaptive window during growth to affect worm lifespan. The lifespan extension by enhanced respiration in yeast and by reduced respiration in worm highlights that hormetic superoxide, not oxygen consumption per se, is responsible for lifespan extension. The examples also illustrate that separation between the adaptive and effective phases may be evolutionarily conserved. Possibly during growth, organisms have abundant resources available to ration between pro-growth and pro-longevity pathways. Hormetic ROS signals may account for the lifespan extension asso- ciated with hypoxia. As hypoxia promotes ROS production, one would predict that hypoxia has the same effect on CLS as ROS. Indeed, during log growth and early stationary phase, hypoxia extends lifespan, and hypoxic treatment deep into the stationary phase shortens lifespan (Bonawitz et al., 2007). The effects of hypoxia highly resemble that of superoxide inducer menadione with similar treatment windows (Pan et al., 2011). Furthermore, hypoxia signicantly extends the CLS of wildtype, but has limited effects on already long-lived tor1. These observations can be explained by shared hormetic superoxide during growth in both TOR1 deletion and hypoxia. Hypoxia extension of yeast CLS nds a mechanistic parallel in Caenorhabditis elegans. As discussed in preceding paragraphs, nuo-1 and isp-1 produce hormetic superoxide, thereby extending wormlife- span (Yang and Hekimi, 2010). Kenyon and colleagues further identify the hypoxia-inducible factor (HIF-1) as the convergence point of hormetic ROS and hypoxic signals (Lee et al., 2010). A loss-of-function mutant allele or RNAi knock-down of HIF-1 decreases the lifespan of the long lived isp-1 mutant, but has no inuence over that of wildtype. Likewise, superoxide inducer paraquat extends wormlifespan, which is partially abolished by HIF-1 mutation. Although RNAi against respirato- ry chain prolongs lifespan only if knockdown is initiated at the larval stages (Dillin et al., 2002), HIF-1 function is required through adulthood to ensure lifespan extension (Lee et al., 2010; Mehta et al., 2009). While consistent with the proposed model, the worm data suggest the Fig. 1. Conceptual model of yeast ROS signaling and CLS. The blue solid line in the middle designates the articial boundary of adaptive and effective phases. Three representative cellular ROS scenarios of the adaptive phase are depicted in different colors on the left. Block arrows point to corresponding cellular ROS levels and their impacts on oxidative dam- age and CLS in the effective phase on the right. Flow charts depict signaling events responsible for respective ROS scenarios and changes of cellular ROS levels between the adaptive and effective stages: constitutively active nutrient sensors or loss of key stress-resistant pathways cause high ROS in adaptive phase, thereby initiating a vicious cycle of ROS pro- duction (top left); nutrient sensor with reduced activity, non-fermentable media or probably hypoxia promotes a hormetic response (middle left); wildtype, grown in fermentable medium, lacks hormetic response (bottom left). 850 Y. Pan / Experimental Gerontology 46 (2011) 847852 requirement of active signaling beyond the adaptive phase to maintain hormesis. In yeast, HIF-1-like factors that mediate hypoxic and hormetic ROS signaling remain to be identied. In the media pre-adaption example coveredpreviously, Piper andcolleagues showedthe de-repressionof res- piration and subsequent lifespan extension can be recapitulated by over- expression of Hap4 (Piper et al., 2006). The facts that HAP4 resides down- stream of TOR1 and SCH9 in regulating respiration and lifespan and that Hap4 contains an oxygen-sensing heme prosthetic group also make it an appealing candidate of HIF-1 functional analog in yeast. 6. Metabolism and lifespan: Toward a comprehensive picture Considerable evidence indicates that TOR has pleiotropic effects on metabolism. In addition to regulating mitochondria, TOR signaling like- ly regulates other energy production pathways that share metabolic in- termediates with mitochondrial OXPHOS. Proteomic experiments suggest that rapamycin represses glycolysis (Hardwick et al., 1999). The identication of acetic acid as a major contributor to chronological aging points to the fact that metabolites produced by glycolytic and other pathways directly affect CLS (Burtner et al., 2009). Medium acid- ity, as a result of acetic acid production, decreases CLS. Under CR, yeast cultures produce substantially less acetic acid and are thus exposed a higher media pH. Long-lived strains, such as sch9, have greater resis- tance to acetic acid. Elevated acidity increases intracellular ROS levels in stationary phase, suggesting a close link between metabolites and mitochondrial functions (Pan et al., 2011). Despite the shared feature of low ROS in stationary, reduction of acetic acid likely acts indepen- dently of mitohormesis in promoting CLS. Neutralization or swapping of media has minimal impact on oxygen consumption during growth and early stationary phase (Pan et al., 2011). Still, toxic metabolites likely contribute to a vicious cycle of ROS production in late stationary phase. The development of metabolomics adds newtools to achieve a com- prehensive understanding of metabolism and lifespan. By analyzing long-lived genetic mutants and longevity-promoting culture condi- tions, Fukusaki and colleagues identied 87 yeast metabolites associat- ed with longer replicative lifespan (Yoshida et al., 2010). Enrichment in the glycolytic intermediates 3-phosphoglycerate and phosphoenol py- ruvate correlates negatively with lifespan, whereas abundance of the TCA cycle intermediates malate, succinate, citrate, and fumarate corre- lates positively with lifespan. The effects of these metabolites on CLS re- main to be determined. Given the fundamentally different nutritional environments replicative lifespan and chronological lifespan assays provide, one should be cautious about jumping to conclusion on a metabolite's effects on aging (Steinkraus et al., 2008). Nonetheless, the availability of metabolomic tools allows examination of new unknowns in CLS. For example, howdo environmental and intracellular stresses inuence each other and how do they affect various signaling pathways? Do alterations of other metabolic pathways also possess adaptive windows as does TOR regulation of mitochondria? The answers to these questions will help better understand how events early in life inuence the aging process. 7. Summary The principles of mitochondrial theory of aging are supported by decades of experiments, and great progress has been made in reveal- ing the roles of mitochondria. Past studies established detoxication of ROS as a major regulatory mechanism by mitochondria regulate the aging process. Using yeast CLS and other models, researchers found that several molecular events, such as hypoxia and hormetic ROS, have an evolutionarily conserved effects on aging. Long before damage by ROS becomes lethal, cellular signaling is affected either ensuring better detoxication or initiating events that exacerbate ROS damage (Fig. 1). Unlike Harman's initial model, genetic factors play an active role in modulating the production and dissipation of ROS, and thus aging. 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