Electron stalling occurs when the rate of entry of electrons into complex I of the electron transport chain exceeds their rate of transit through the slowest step of the chain. For example, if electron transit through complex IV were limiting, rapid entry of electrons into the electron transport chain could trigger electron stalling at complexes I and III (Figure 2). Ironically, the old idea that CR reduces carbohydrate use as fuel for the TCA cycle could explain a reduction in ROS production because NADH production and the rate of entry of electrons into the electron transport chain would be reduced. But this idea has been discredited because respiration does not decrease during CR.
The biogenesis of mitochondria during CR offers another possible explanation. The increase in electron transport chain components should restrain ROS production because it will increase the number of entry points for electrons into the electron transport chain, thereby lowering the rate of entry into the chain and reducing the probability of electrons becoming stalled at complexes I and III (Figure 2). Further studies on mitochondrial bioenergetics should resolve whether such an effect is observed in tissues from animals on CR. The model discussed in this section does not require any additional regulatory components to translate the upregulation of mitochondria into beneficial effects.
Mitochondrial Reprogramming to Fat Oxidation
CR changes the way in which different available energy sources in food are handled by the body. In caloric excess, carbohydrates are used for energy and fat is stored, presumably because it has a higher energy content per gram. However, during CR all energy sources are used, notably fat and amino acids, and the animal minimizes carbohydrate use in peripheral tissues to maintain adequate blood glucose levels for the brain (Weindruch and Walford, 1988). Therefore, in energy-producing tissues like muscle, there is a shift toward the β-oxidation of fatty acids to derive energy during CR, a process that occurs in mitochondria.
Unlike carbohydrate catabolism, in which most of the reducing power generated for electron transport is in the form of NADH, β-oxidation of fatty acids produces FADH at a molar equivalence of NADH. FADH must enter the electron transport chain not via complex I but via the electron transfer flavoprotein dehydrogenase (ETF) (coupled to acyl-CoA dehydrogenase) (Figure 2). ETF passes electrons to ubiquinone, which then donates them to complex III and thence onward down the chain. Therefore, use of fat as an energy source increases the frequency with which electrons enter the electron transport chain by bypassing complex I. Given that complex I is one of the primary stations of ROS production, this simple metabolic shift from carbohydrate to fat utilization may reduce ROS production.
But what about the increase in mitochondrial biogenesis observed during CR? When electrons enter the electron transport chain at complex I, their transport results in the synthesis of three molecules of ATP by oxidative phosphorylation. However, when electrons enter at ETF only two ATP molecules are made because the proton-pumping complex I has been bypassed. Thus, it is possible that the observed mitochondrial biogenesis during CR compensates for the reduction in ATP production per reducing equivalent when fat is used as an energy source. This model would suggest that it is not an increase in respiration or even mitochondrial biogenesis per se that is beneficial during CR but rather the partial bypass of complex I in the production of ATP. In this regard, it is interesting to note that the human CR trial did not record any increase in respiration but actually showed a decrease in whole body energy expenditure (Civitarese et al., 2007), even though there was an increase in mitochondrial components.
As for model three, this idea is appealing for its simplicity but bears the caveat of placing great emphasis on the importance of ROS in aging. It is interesting to note that one of the most effective drugs for diabetes, metformin, can act as a partial inhibitor of complex I (Owen et al., 2000). Much of the benefit of this drug in treating metabolic disease has been attributed to the resulting activation of AMP-dependent protein kinase (AMPK) (Zhou et al., 2001), which helps to reduce gluconeogenesis in liver and to drive the oxidation of fat in muscle. However, it is clearly possible that the inhibition of complex I per se can help to reduce cellular damage and to foster long-term health benefits in metformin-treated individuals. The activation of AMPK may still serve an important regulatory role by inducing fatty-acid oxidation to ensure the maintenance of energy production.
Recycling of Damaged Mitochondria
Cells have the cytosolic ubiquitin/proteasome system and mitochondrial proteases to degrade damaged or unwanted proteins and to replace them with newly synthesized ones (Varshavsky, 2005). Cells can also degrade damaged structures as large as organelles by a process called autophagy (Scherz-Shouval and Elazar, 2007). This process transports damaged organelles, for example mitochondria, to lysosomes where they are degraded and their contents recycled. Obviously, if mitochondria were degraded in this fashion, new synthesis would be required to maintain energy homeostasis. In this regard, it is possible that the increase in mitochondrial biogenesis induced by CR is a consequence of increased organelle turnover, and any increase in net mitochondrial synthesis is but a corollary. An increase in mitochondrial turnover might be beneficial for cells, given that they are among the most damaged structures during aging. It would not matter according to this model whether ROS or other agents generate the damage; the important outcome would be better maintenance of young mitochondria in aging cells. Consistent with this idea, autophagy has been reported to be required for the CR extension of life span in C. elegans (Jia and Levine, 2007).
Importantly, a recent paper showed that SIRT1 deacetylates and activates several proteins of the autophagic machinery (Lee et al., 2007). Cells from SIRT1-deficient mice are defective in autophagy, and SIRT1-deficient mice have damaged organelles and display early postnatal lethality like mice defective in autophagy. Thus, the idea that salutary effects of CR spring from the activation of autophagy and the clearance of defective mitochondria is attractive.
However, several questions arise. First, can the autophagic machinery selectively recognize and destroy damaged mitochondria? Second, does CR actually induce autophagy in mammals? One might expect the answer to be yes, given that one consequence of autophagy is to provide a source of carbon and energy, and induction of autophagy during acute starvation is clearly observed. However, long-term CR is a steady-state process and does not impose the same degree of energy limitation as starvation, so whether it triggers autophagy is still an open question. In conclusion, the interesting possibility that autophagy of damaged mitochondria plays an important role in mammalian CR awaits further experimentation.
Conclusion
Mitochondria have long been proposed to play an important role in aging. Recent genetic findings in lower organisms have pinpointed sirtuins as antiaging genes, and at least four of the seven mammalian sirtuin homologs have mitochondria-associated functions. CR is perhaps the most robust intervention that extends mammalian life span and has been associated with an increase in SIRT1 levels in several tissues and a corresponding increase in mitochondrial components. Here, I have presented several models for how this increase in mitochondria may have the effect of slowing aging and disease. Some of the models rely on an important role of ROS in the aging process, whereas others do not. It is hoped that the next few years will see a further convergence of genetic pathways with mitochondrial function, which will provide a comprehensive view of aging and antiaging mechanisms and will also explain how CR works. It seems likely that we are on the right track of acquiring this understanding, and that it will involve mechanisms rich in new and old ideas about aging and how to counteract it.
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