Most of the studies undertaken to understand the problem of aging have been based on the premise that it is attributable to a decline in cellular functions and impaired stress resistance due to oxidative damage to cellular macromolecules (Rattan 2006
). The longevity-promoting effects of CR are hypothesized primarily because of reduced oxidative damage due to decreased oxidative stress (Sohal and Weindruch 1996
) in the CR-induced hypometabolic state. Besides the observation of enhanced respiration in yeast cells by Guarente laboratory (Lin et al. 2002
), our laboratory has reported enhanced ROS and concomitant stronger antioxidant defense system upon calorie restriction (Agarwal et al. 2005
). Though it has been speculated that functional mitochondria are vital for the beneficial effects of CR in yeast (Lin et al. 2004
), no experimental evidence is cited, indicating a role of this organelle in CR. Our results give evidence in favor of an interesting role for this organelle in the benefits accrued from calorie restriction in yeast cells.
The mitochondrial content of the cells growing in the normal and calorie-restricted condition was determined to ascertain if the enhanced respiratory activity upon calorie restriction as reported by our and Guarente laboratory was due to increased mitochondrial biogenesis. Apparently, the mitochondrial content remained same in either condition as determined by the fluorescence experiments (Fig. ).This observation is in contrast to the results obtained in mammalian system where the authors associate calorie restriction with increased mitochondrial content (Civitarese et al. 2007
; Nisoli et al. 2005
). Our next set of experiments was aimed to see if calorie restriction affected mitochondrial functionality. Oxygen consumption in isolated mitochondria (Fig. ) show more than twofold increase upon calorie restriction, which corroborated the previous reported results. H2
production was increased in CR mitochondria, ascertaining CR association with hypermetabolic cellular status rather then hypometabolic status. Moreover, this increased ROS generation in CR correlated well with the oxygen consumption (an approximately twofold increase, Fig. ). Further, blockage of the electron transport chain with antimycin further increased the H2
levels, whereas coincubation with myxothiazol reduced this increase. These findings can be explained on the basis of electron transfer in the complex III and the site of action of these two inhibitors. According to the Q cycle mechanism, two ubiquinone-binding sites are present in the cytochrome bc1
complex. The Qo
site or the ubiquinol-oxidizing site is present on the P side of the inner mitochondrial membrane whereas the Qi
site, the ubiquinone-reducing site, is located on the N side of the membrane. Antimycin inhibits the reduction of ubiquinone at the Qi
site, resulting in the accumulation of unstable semiquinone, which in turn transfers a single electron to molecular oxygen to produce superoxide. Myxothiazol prevents the oxidation of ubiquinol at the Qo
site, preventing the formation of ubisemiquinone at this site and having no transfer of electrons to the molecular oxygen (Raha and Robinson 2000
). Thus, it can be concluded that complex III is the source of H2
production in the CR mitochondria.
Lower ROS production in CR mitochondria has been reported (Barros et al. 2004
)—a result contrary to our observation. Closer examination of the data revealed that choice of substrate set (ethanol, malate, and glutamate) for mitochondrial respiration might be the cause of low H2
production because of the following reasons. Mitochondrial inner membrane is virtually impermeable to NADH coenzymes. Metabolism of a mixture of ethanol, malate, and glutamate generates intramitochondrial NADH, which can be oxidized by internal NADH dehydrogenase only; it cannot be utilized by external dehydrogenases (Nde1 and Nde2). In this study, we have measured internal dehydrogenase activity and found that the marginal increase in activity is also reflected in slightly increased H2
levels, whereas the higher external dehydrogenase activity in CR correlates very well with the H2
levels detected with the corresponding substrate for these dehydrogenases (Fig. , ). All these data together indicate that substrates specific to internal dehydrogenase might generate low ROS in mitochondria, which however may not be a true reflection of mitochondrial status in CR.
Indirect evidences have accumulated, conferring a vital role to complex I of respiratory chain in stress and life span regulation of yeast and worms. In contrast to many eukaryotic cells, S. cerevisiae
lacks the multi-subunit complex I-type NADH dehydrogenase. Instead, it contains an “internal mitochondrial NADH dehydrogenase,” encoded by NDI1
and an “external NADH dehydrogenase,” encoded by NDE1
, which oxidizes mitochondrial and cytosolic NADH, respectively (Horne et al. 2001
). Jazwinski’s group has suggested the necessity of mitochondria for life span extension by transient heat stress (Shama et al. 1998
). Later, it was established that transient heat stress actually induces oxidative stress, and Nde1 and Nde2 are vital for this induction (Davidson and Schiestl 2001
). In CR, NAD levels remain the same, but NADH levels drop down finally, leading to an increase in the NAD/NADH ratio, which mediates Sir2-dependant life span extension. The exact cause of the depletion of NADH levels has however not been documented. As an indirect evidence for overactivity of NADH dehydrogenase, it has been cited that overexpression of Nde1 and Nde2 extends its life span in yeast (Lin et al. 2004
) while subsequent mutation in nematode (gas-1
) reduces complex I activity and shortens life span (Kayser et al. 2004
). Our results suggest that CR specifically upregulate external NADH dehydrogenase activity (Fig. ).
ATP levels were determined in isolated mitochondria to see if the increased respiration is reflected in ATP generation. Surprisingly, increased oxygen consumption upon calorie restriction did not entail in higher levels of ATP (Fig. ). It could be because of uncoupling of ATP synthase complex with respiratory chain or lack of internal ADP. However, addition of succinate enhanced ATP production, and addition of ADP enhanced it further (Fig. ), which clearly indicates that isolated mitochondria were well coupled and lack of ATP production is not simply due to lack of ADP or reduced efficiency of ATP synthase complex. Unaltered ATP levels in spite of higher respiratory activity of calorie-restricted mitochondria can be explained on the basis of observed ROS generation (a measure of the electron leak) in the mitochondria. As per the expectation, the increased oxygen consumption should translate into the higher ATP generation. But, instead of that, it is being translated into observed higher H2O2 levels.
Higher levels of H2
do not seem to be in accordance with life span extension benefit of calorie restriction, at least in the face value. But this observation seems to make sense when seen in the light of mitohormesis theory (Tapia 2006
). According to this theory, ROS from mitochondrial metabolism upon calorie restriction are the key element that initiates a cascade of events that culminates in the life span extension. A higher H2
level as detected in calorie-restricted mitochondria provides evidence of stress that the cells are subjected to, when put on that particular diet regimen. If the calorie restriction is a form of hormesis, then a stronger defense mechanism would be expected. This was indeed observed in augmented antioxidant system as evaluated in terms of SOD and GPx activity (Fig. ) in CR mitochondria. SOD (Cu–Zn SOD and Mn SOD) activity was found to be elevated in the mitochondria of CR cells. Expression of GPX1
is induced on glucose starvation, expression of GPX2
is induced by oxidative stress in a Yap1p-dependent manner, and expression of GPX3
is constitutive (Inoue et al. 1999
). Augmentation of SOD and GPx in response to CR has been reported previously at the cellular level (Agarwal et al. 2005
), and probably the same mechanism is applicable to annul the effect of mitochondrial ROS. Life span extension upon calorie restriction has been described in C. elegans
, owing to increased stress resistance subsequent to increased respiration and ROS levels (Schulz et al. 2007
). Thus, our results suggest that oxidative stress generated from mitochondria activates the scavenging pathway to exert beneficial effects of CR. The result of the Halo assay (Fig. ) further provides evidence of calorie restriction eliciting a hormetic response. Such a response should bestow protective effects towards subsequent stress conditions. The calorie-restricted cells were more resistant to the stress in the form of hydrogen peroxide as compared to normal cells. Though the role of an improved defense system in calorie-restriction-mediated life span extension is central, other players cannot be ruled out. It could be that the increased NADH dehydrogenase activity is a part of the hormetic responses and responsible for the lowering of NADH, which in turn augments parallel Sir2-mediated life span extension effects.
In the various reports regarding cellular response to oxidative stress, a role for a functional mitochondrial respiratory chain has been underscored. It has been known that respiratory-deficient yeast strains are sensitive to ROS (Grant et al. 1997
) and a fully functional electron transport chain is necessary for maintaining resistance to H2
(Thorpe et al. 2004
). It is also reported that daughter cells lose age asymmetry in the absence of Atp2 and show clonal-senescence phenotype at higher temperature (Borghouts et al. 2004
). The reduction in the mitochondrial ROS generation in respiratory-deficient atp2
cells points out the need of the functional respiratory chain for the generation of ROS. The markedly reduced life span in atp2
in CR and NR as compared to wild type clearly illustrates the role of intact electron transport chain in life span regulation.
To conclude, our study sheds light on the mechanism of life span extension upon calorie restriction in yeast. Evidence is provided supporting a key role for mitochondria in the string of events culminating in the longer life span in CR yeast. Our results buttress the notion that calorie restriction is indeed a form of hormesis—a multipronged response (Rattan 2008
) as is evident by higher H2
levels, augmented antioxidant defense system, resistance to subsequent stress, and increased NADH dehydrogenase activity. ROS have been traditionally held as damage-causing entities, but they are equally important for normal cellular functioning, and they have been appreciated as mediators of cellular metabolism (Allen and Tresini 2000
; Cadenas 2004
). It is highly likely that the increased ROS levels activate an array of sensors and mediators, which in turn orchestrate the multipronged response to calorie restriction, ultimately leading to life span extension.