Low-calorie diets extend lifespan in almost every model tested, but scientists can't yet agree on what controls this phenomenon. The histone deacetylase Sir2 provides a seductive link between gene silencing and calorie restriction (CR),1 but many debate the mechanism. Now researchers are turning to a new question: Does Sir2 have a role in CR-mediated longevity at all? While some still believe that Sir2 is the lynchpin of the CR-longevity pathway, others are more skeptical and propose alternative mechanisms. Recent findings haven't clarified the issue.

Some researchers, including biologist Leonard Guarente at Massachusetts Institute of Technology, contend that Sir2 is dependent on nicotinamide adenine di-nucleotide (NAD) and that CR activates Sir2 by reducing glucose metabolism, which increases the ratio of NAD to its reduced version, NADH.2 Others, such as Harvard Medical School pathologist David Sinclair, hold that nicotinamide, not NAD, is the control switch for Sir2, and that the...


In September, a group of researchers from Stan Fields' lab at the University of Washington (UW), Seattle, published a paper offering evidence that CR may work through a pathway not involving Sir2. In yeast lacking Sir2, CR did not extend lifespan.4 But when the scientists eliminated both Sir2 and Fob1, a DNA-binding protein known to induce recombination and promote production of the extrachromosomal rDNA circles that cause aging in yeast, CR increased lifespan to the same extent that it does in wild-type cells.

"As long as rDNA-circle levels are kept low, Sir2 is sort of irrelevant for CR," says UW postdoc Matt Kaeberlein. One interpretation, he says, is that two pathways exist, both affected by CR. "We can't rule that out," he notes, but seeing the full CR-mediated effect without Sir2 gives his group at UW reason to think otherwise.

"If you get rid of Sir2 and get rid of the deleterious effects of not having Sir2," says Sinclair, "calorie restriction has a way of extending lifespan. I would think of this alternative pathway as a backup to Sir2." He speculates that in the sir2/fob1 double mutant, CR could be acting through one of yeast's four Sir2 homologs.

Kaeberlein points out that his yeast strain is different from those used by most others who research aging, and Stephen Helfand, developmental biologist at the University of Connecticut Health Center in Farmington, says that might explain his results: "People are thinking that there are some strain differences here that are not accounted for."

Guarente agrees. "Different yeast strains can be very divergent." He adds that the researchers have yet to fully describe their pathway and determine how similar it is to the previously proposed Sir2 pathway.

But Kaeberlein says that his team's data are more consistent with genetic data from Caenorhabditis elegans, which show that CR functions independently of the Daf-16 forkhead transcription factor pathway,5 while Sir-2.1, the worm ortholog of Sir2, is directly involved in that pathway.6 This indicates that Sir-2.1 is not in the same pathway as CR in C. elegans.

Further studies have shown that when researchers calorie-restrict a worm that is long-lived due to mutations in the Daf-16 pathway, the effect on lifespan is additive,7 "just like we see with CR and Sir2 overexpression in yeast," says Kaeberlein. "It's not a perfect correlation," he admits, "but at least all of the genetic pieces appear to be the same." Unfortunately, this issue gets more complicated in mammals, where CR works on the insulin pathway, which is analogous to the Daf-16 mechanism.


Sinclair says, however, that C. elegans may be an exception, as other yeast data are consistent with findings in flies and mice. A recent study on aging in Drosophila melanogaster by Helfand's group provides support for this position: They suggested a Sir2-dependent model for CR-mediated lifespan extension in Drosophila.8 The team showed that calorie restriction in flies reduces expression of Rpd3, a histone deacetylase that normally inhibits Sir2. Lowering inhibition of Sir2 causes an increase in its activity, which lengthens the flies' lifespan. They also showed that blocking Sir2 activation eliminates the normal response to CR. According to these results, CR can't increase lifespan without Sir2.

"What we tried to do was provide the data to begin to bring together these already suspected parts of a pathway," says Helfand. "We think we have a reasonably good pathway for going from CR ... through Rpd3 and then through Sir2 and then other effectors."


A. Leonard Guarente's model of CR-mediated longevity in Saccharomyces cerevisiae indicates that CR regulates the NAD/NADH ratio, which controls Sir2 activation and extends lifespan. B. David Sinclair's longevity model suggests that CR increases Pnc1 activity, which eliminates nicotinamide, thus activating Sir2 and slowing aging. C. Matt Kaeberlein's yeast model places Sir2 and CR in separate life-extension pathways. D. In Caenorhabditis elegans, some genetic data indicate that Sir-2.1 works to slow aging through the Daf-16 pathway, while CR extends life independent of that pathway. E. Stephen Helfand's most recent model using Drosophila melanogaster shows CR blocking Rpd3, a Sir2 inhibitor, thus allowing CR to upregulate Sir2 and extend aging. F. In mammals, how CR lengthens lifespan is still unclear, but CR might activate Sirt1, which has several known targets downstream

Kaeberlein suggests another explanation: "Maybe something kills Sir2 mutants that doesn't kill wild-type flies. If that's the case, the inability of CR to extend lifespan is meaningless." Referring to his group's sir2/fob1 double mutants that still respond to CR, he asks, "How do we know that something similar isn't happening in flies?"

Matt Piper, a biologist at University College London, says he sees a role for Sir2 in lifespan extension by CR but notes that an organism's diet influences its physiology in multiple ways that might affect its lifespan. "Because diet is so complex, you have many different signaling pathways in the organism determining many different physiological processes, which result in lifespan extension or shortening," says Piper. "To put it all down to one gene in one pathway is a very big call." He explains that both the insulin-signaling pathway, which responds to sugar, and the TOR-signaling pathway, which is affected by dietary protein, probably play a role in CR-mediated longevity. Having at least two different signaling pathways for lifespan extension in flies and worms, Piper adds, "would suggest that there's probably a number of different feedins to get lifespan extension."

Helfand says he has little doubt that Sir2 plays a major role, but admits the need for clarification: "At the moment it's reasonably muddy."


Helfand points out that measuring yeast replicative lifespan, or the number of times a mother cell can divide before dying, might not be completely analogous to metazoan lifespan. "There are many important common features, but some of the differences may speak a little bit about the fact that yeast are different than flies, which are different than people," he says.

Regardless of what's going on in lower organisms, says Guarente, "the larger issue becomes which pathway is conserved in mediating CR in higher organisms." Both Guarente and Sinclair have started companies – Elixir Pharmaceuticals and Sirtris Pharmaceuticals, respectively – that aim to produce drugs that mimic the benefits of CR in humans. While scientists are unsure about components upstream of Sirt1 (the mammalian homologue of Sir2), he adds, they have identified some of its downstream targets, including fat metabolism and mobilization, glucose/ insulin metabolism, the FOXO family of transcription factors, and several cell-survival mediators, such as tumor suppressor p53 and DNA repair factor Ku70.9 He says that the experiments needed to clarify the mammalian pathway are underway now. "The way we're going about it is to look at various physiological changes that are triggered by CR and seeing if they're really mediated by Sirt1. ... I think we'll have a pretty good idea in a year or so of how this shakes up, but I would say that Sirt is looking good right now as being the center."

From the evidence in lower organisms, Kaeberlein remains unconvinced. "There are a lot of people ... trying to connect Sir2 to CR," says Kaeberlein. "They are finding interesting tidbits here and there. ... The problem is that there are also a lot of inconsistencies, [and] these inconsistencies need to be explained before anyone can say with any level of certainty that CR acts through Sir2."

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