In C. elegans, CR was recently shown to require two sensory neurons, termed ASI neurons, in the head of the roundworm (Bishop and Guarente, 2007). Signals emanating from these two cells trigger an increase in respiration, which, again, proved to be essential for CR-induced life span extension, as two different inhibitors of electron transport prevented this effect (see Figure 1). The response to CR in C. elegans requires two transcription factors, SKN-1 (Bishop and Guarente, 2007) and PHA-4 (Panowski et al., 2007), but so far sirtuins have not been implicated.
It appears that the degree and timing of energy limitation may influence the relationship between mitochondrial function and longevity. In yeast, a CR regimen using one tenth as much glucose as the moderate CR regimen, described above, was shown to extend yeast life span by a mechanism that did not require mitochondria or SIR2 (Kaeberlein et al., 2005b). In C. elegans, an RNAi-imposed reduction in mitochondrial function during development surprisingly extends life span, but in adult worms such a reduction in mitochondrial function has no effect (Dillin et al., 2002; Rea et al., 2007).
A priori, one cannot be certain which of the experimental conditions in the lower organisms is relevant to the standard CR protocol in mammals (30%-40% reduction of ad libitum feeding). However, recent findings have begun to link these mechanisms to mammalian CR. First, mice lacking SIRT1 are unable to mount the increase in foraging activity typical of this diet (Chen et al., 2005), suggesting a role of this sirtuin in the brain during CR. Moreover, transgenic mice that over-express SIRT1 resemble CR mice in physiological parameters (decreased blood glucose, insulin, fat, and cholesterol) and behavioral assays (improved rotarod performance and delayed mating) (Bordone et al., 2007).
Second, CR increases respiration as well as mitochondrial number per cell in mice (Nisoli et al., 2005) (see Figure 1). These increases require the endothelial nitric oxide synthase (eNOS); nitric oxide is known to be capable of activating the SIRT1 promoter in cultured cells. These findings fit with the fact that the NAD/NADH ratio and SIRT1 protein levels are increased in several rodent tissues during CR (Cohen et al., 2004; Guarente, 2006). Interestingly, SIRT1 also deacetylates and activates the eNOS enzyme (Mattagajasingh et al., 2007), indicating that a positive feedback mechanism between SIRT1 and eNOS may reset the levels of this sirtuin during CR.
Further molecular insight into the increase in mitochondrial function in CR is suggested by the relationship between SIRT1 and PGC-1α, a transcriptional coactivator of nuclear genes encoding mitochondrial proteins. SIRT1 was shown to deacetylate PGC-1α at several lysine residues and thereby increase its ability to transcriptionally activate target genes (Rodgers et al., 2005; Gerhart-Hines et al., 2007). Thus, the increase in SIRT1 during CR should result in mitochondrial biogenesis in tissues such as muscle and white fat. Indeed, feeding mice the putative SIRT1 activator resveratrol upregulates mitochondrial number in muscle (Lagouge et al., 2006). This compound also triggers numerous salutary effects to counteract a high caloric diet, including improved physical activity and longer average life span (Baur et al., 2006; Lagouge et al., 2006).
Finally, some of these effects found in mice also have been observed in a six month human trial in which subjects were calorie restricted to a degree that resulted in the expected reductions in body weight and blood insulin (Civitarese et al., 2007). Most strikingly, muscle punch biopsies from these calorie-restricted individuals showed upregulation of SIRT1, eNOS, the mitochondrial protein TFAM, and mitochondrial number, compared to biopsies from control individuals on a normal diet (Figure 1). These findings suggest that a conserved pathway may operate in mammals during CR involving the activation of SIRT1 and eNOS that triggers an increase in mitochondrial activity.
Why Is Activation of Mitochondria Good?
Increased respiration in yeast and worms is required for CR-induced longevity, but the situation in mammals is only correlative. However, it is still worth considering possible mechanisms to explain how salutary effects might be caused by or associated with the increase in number of mitochondria, if only to spur new experiments. In yeast, Sir2p lies downstream of (that is, it responds to) increased respiration in the CR longevity pathway (Figure 1). It has been suggested that this sirtuin is activated by the respiration-triggered increase in the NAD/NADH ratio (Lin et al., 2004) and the induction of the PNC1 nicotinamidase (Anderson et al., 2003). In mammals on CR, it seems likely that sirtuins both trigger (SIRT1) and respond to (SIRT3, 4, 5) an increase in mitochondrial number and activity (Figure 1). I consider below five possible ways in which increased mitochondrial number and activity may be an integral part of a mechanism by which CR exerts antiaging effects in mammals.
A Mitochondrial Buffer to Aging
There is ample evidence that damage to mitochondria increases progressively with age (Wallace, 2005). This has been observed in the form of the accumulation of mutations in mitochondrial DNA and a decline in the activity of mitochondrial enzymes and components of the electron transport chain. In the case of aging skeletal muscle syncitia, for example, zones of metabolically inactive tissue have been observed, due to expansion of mitochondria that become damaged during aging (Bua et al., 2006). A large body of evidence links mitochondrial dysfunction with diabetes (Lowell and Shulman, 2005), although a recent report surprisingly showed that deleting the apoptosis inducing factor AIF1 in muscle or liver reduced mitochondrial function and had salutary effects on metabolic disease (Pospisilik et al., 2007).
A higher pool of functional mitochondria may ameliorate tissue damage simply by buffering cells (or the syncitia in muscle) against the gradual decline in the ability to produce energy as mitochondria become damaged during aging. One may well wonder why mitochondrial number is not normally set to a higher level during ad libitum feeding to forestall this decline. It is important to remember that aging occurs postreproductively and is nonadaptive. Thus, under normal conditions, mitochondrial number and function will only fall sway to selective pressure until the reproductive period has been completed. During subsequent aging, there is minimal selective pressure to maintain mitochondrial (or any other) robustness. By this logic, CR and perhaps other stressors may impose a new selective landscape in which robust somatic maintenance, rather than reproduction, is now at a premium and mitochondrial biogenesis favored.
Mitochondrial Regulation of Sirtuins
A second possibility is that the increase in mitochondrial function serves a regulatory function by changing the activity of regulatory proteins, exemplified by CR and Sir2p in yeast. Proteins capable of responding to metabolic changes in mitochondria that alter the NAD/NADH ratio include the three sirtuins located in that cellular compartment (SIRT3, 4, and 5). Both SIRT3 and SIRT4 have been shown to have important metabolic functions in mitochondria (Guarente, 2006). Importantly, these two sirtuins were also recently shown to be involved in a stress-resistance pathway (Yang et al., 2007). Cells that sustain DNA damage will acutely deplete their nuclear NAD pool because of the activation of the NAD-cleaving enzyme poly-ADP-ribose polymerase (PARP), which is involved in DNA repair. However, the mitochondrial pool of NAD is preserved in part through the translocation of the NAD synthetic enzyme Nampt to mitochondria, where reinforced synthesis of NAD, SIRT3, and SIRT4 appears to be required to prevent apoptosis.
In the steady-state condition of CR, any changes in the NAD/NADH ratio due to altered metabolic activity in mitochondria are likely to equilibrate to the other cellular compartments by shuttle systems that operate across the mitochondrial membrane. Thus, the non-mitochondrial sirtuins and indeed any other cellular proteins that bind to NAD may also be affected. In principle, it is also possible that changes in other small molecules due to altered mitochondrial activity may play important regulatory roles during CR.
Mitochondrial Regulation of ROS
ROS remain a likely cause of the decline in cellular and organismal vitality during aging. There is good evidence that species that are long-lived produce high levels of antioxidant enzymes and, moreover, that CR leads to a reduction in the production of ROS in a given organism (Weindruch and Walford, 1988). Furthermore, mice with a transgene targeting the antioxidant enzyme catalase to the mitochondrial matrix have a longer life span (Schriner et al., 2005). Although ROS can be generated by a variety of enzymes of oxidative metabolism, the electron transport chain is their most significant source. How might the biogenesis of mitochondria, described above, relate to the reduction of ROS observed during CR? It was first assumed that ROS production by the electron transport chain would be proportional to the respiration rate; however, recent evidence suggests that this view is too simplistic (Lopez-Lluch et al., 2006). In fact, most ROS are produced by complexes I (NADH dehydrogenase) and III (cytochrome b-c1 complex) in the electron transport chain (Barja, 2007; Pamplona and Barja, 2007), and this production actually appears to be enhanced by the stalling of electrons at these complexes (Kushnareva et al., 2002; Barros et al., 2004) (see Figure 2). For example, ischemia will stall electron transport due to lack of a terminal electron acceptor and cause ROS production upon oxygen reperfusion resulting in severe tissue damage.