Model organisms such as yeast, worms, and flies have been instrumental in the discovery of life span regulatory pathways that have a common evolutionary origin. Among these, the insulin/IGF-I-like pathways control longevity in organisms as phylogenetically distant as yeast and mice. Akt, Tor, and Ras function in the mammalian IGF-I signaling pathway and have been implicated in life span regulation in different model organisms 
. In this study, we show that longevity regulatory pathways control the shift from respiration to glycolysis and glycerol biosynthesis. This metabolic switch, which leads to the removal of pro-aging carbon sources and glycerol accumulation, creates an environment in the sch9
Δ culture that mimics calorie restriction ().
Inhibition of the Tor1/Sch9 and Ras pathways activates stress response transcription factors and glycerol biosynthesis.
The genetic and genomic data revealed two parallel longevity signaling pathways controlled by Tor1/Sch9 and Ras, in agreement with our previous work 
. The beneficial effects of reduced activities of both pathways is additive (), and the sch9
Δ double mutant is one of the longest lived genetic mutants 
. In agreement with the genetic data, the gene expression profile of the day 2.5-old ras2
Δ mutant shows that approximately 67% of the genes differentially expressed are not significantly changed in the other two mutants (). Our genetic analysis of the interactions between the Tor, Sch9 and Ras2 indicates a stronger overlap between the Tor1 and Sch9 pathways in the regulation of stress resistance, longevity, and age-dependent genomic instability. It also suggests that TORC1 functions upstream of Sch9 in the regulation of these readouts in agreement with what has been proposed by others 
and with the demonstration of the direct phosphorylation of Sch9 by TORC1 
. Our microarray analysis indicates similarities but also differences between the set of genes controlled by Tor and Ras. On the one hand, TOR1
deletion further increased the heat-shock resistance of ras2
Δ mutants, and on the other hand no additional life span extension was observed. Furthermore, the overexpression of constitutively active Ras2 abolished CLS extension associated with deficiency of TOR1
, suggesting an overlapping of the two pathways and possibly an upstream role of TORC1.
Despite the higher degree of differential expression profile observed in ras2
Δ mutants, there are remarkable similarities in the expression pattern of genes involved in key metabolic pathways in all three long-lived mutants. The genome-wide association (transcription factor binding motif enrichment test) and the genetic analyses indicate that longevity modulation by the Tor/Sch9 and Ras signaling depends on the protein kinase Rim15 and its downstream stress response transcription factors, Msn2/4 and Gis1 
. The most striking result is that genes involved in glycolysis/fermentation are consistently upregulated, while mitochondrial related genes are down-regulated, in all three long-lived mutants, suggesting a cellular state that favors glycolysis and diminished mitochondrial functions including TCA cycle and oxidative phosphorylation. Part of our results may appear to contradict recent results showing that respiration is upregulated in the tor1
Δ mutant 
. This discrepancy may be explained by the difference in the time point of observation. Bonawitz and colleagues measured higher respiration rates in exponentially growing or day 1 tor1
Δ cultures relative to wild type yeast. By day 2 this difference was no longer observed 
. The role of respiration in replicative life span regulation is still unclear. On the one hand, increased respiration has been shown to mediate the beneficial effect of CR (0.5% glucose) 
; on the other hand, growth on lower glucose-containing medium (0.05% glucose) can extend the replicative life span of respiratory-deficient yeast 
. Moreover, a study from the Jazwinski's group indicated that respiration does not directly affect replicative longevity 
. The different effect of respiration on life span may also be contributed to the experimental systems used for life span studies. The replicative life span analysis is mostly carried out on the solid rich YPD medium, where cells are constantly exposed to glucose and other nutrients. The energy required for growth is mainly derived from fermentation. In contrast, our chronological longevity studies are performed by monitoring population survival in a non-dividing phase in which fermentation is minimized 
The gene expression profiles of long-lived mutants showed the induction of key genes required for glycerol biosynthesis. High levels of extracellular and intracellular glycerol were detected in the sch9Δ culture and triglyceride catabolism appeared to contribute to glycerol generation (). This shift towards the production of glycerol represents a fundamental metabolic change in the physiology of the long-lived mutants.
Genetic analysis performed by deleting genes required for glycerol biosynthesis in the sch9
Δ mutant indicates that glycerol production is required for life span regulation (). Increased glycerol biosynthesis may contribute to life span regulation through several distinct mechanisms. First, cells lacking Sch9 utilize glucose and ethanol and accumulate glycerol, a carbon source that does not promote aging and cell death. This metabolic change creates an environment that mimics calorie restriction. CR, achieved by either lowering glucose in growth medium or by removing ethanol, extends the yeast CLS 
. Conversely, addition of low concentration of ethanol reverses life span extension induced by CR 
. Here we show that cells lacking Sch9 deplete pro-aging carbon sources and activate glycerol biosynthesis. Whereas glucose and, to a lesser extent, ethanol promotes aging, glycerol acts as a “phantom carbon source” and does not inhibit the transactivation of stress response transcription factors Msn2/4 and Gis1, which play important roles in stress resistance and longevity modulation in both long-lived mutants and cells under calorie restriction () 
. Since glycerol was taken up by the cells and caused a minor enhancement of survival under starvation conditions, it is likely that its uptake provides nutritional support for long term survival (). Second, production and accumulation of glycerol may contribute to cellular protection since glycerol enhances resistance to osmotic stress and functions as molecular chaperone stabilizing/renaturing the newly synthesized or heat-inactivated proteins. However, our present and past results indicate that Sch9 also down-regulates stress resistance systems independently of the generation of glycerol. For example, in the BY4741 background Sch9 deficiency increased the resistance to multiple stresses in mutants with defects in glycerol biosynthesis (). Third, glycerol production may affect aging through the modulation of the redox balance of the cell, since its production contributes to the maintenance of NAD:NADH ratio 
. Easlon et al
. have recently shown that overexpression of the malate-aspartate NADH shuttle components extends yeast replicative life span 
. The latter two mechanisms, however, are less likely to contribute significantly to chronological life span extension, as addition of exogenous glycerol to the culture had little or no effect on heat-induced protein inactivation () or chronological survival in wild type cells (). Additionally, we overexpressed in wild type cells the bacterial NADH oxidase (NOX) or alternative oxidase (AOX), both of which increase NADH oxidation in yeast 
, did not significant alter the life span of the wild type cells (unpublished data).
In summary, we presented data showing enhanced expression of glycerol biosynthetic genes in three long-lived yeast mutants lacking SCH9, TOR1, or RAS2, whose homologs also play important roles in life span modulation in organisms ranging from worms, flies, to mammals. Our data also suggest that the switch to glycerol biosynthesis is required for life span extension in the sch9Δ mutants. We argue that the genetically induced carbon source substitution in the long-lived tor1Δ and sch9Δ cells creates a beneficial environment that mimics calorie restriction which, together with the intracellular regulation of stress resistance via transcription factors Gis1 and Msn2/4, results in life span extension and stress resistance (). In light of the conservation of the longevity regulatory pathways and the role of calorie restriction in extending life span of a wide range of species, it will be important to investigate further the possibility of an anti-aging role for glycerol in higher eukaryotes.