Sci. Aging Knowl. Environ., 17 July 2002
High-Octane Endurance--Yeast in the Metabolic Fast Lane Live Longer
Yeast don't shoot craps or snort cocaine, but a different kind of fast life might invigorate them, according to new research. Many scientists have wondered whether slowing metabolism could prolong life. The findings suggest instead that speeding it up might do the trick, at least in yeast. The results from this series of experiments also tie together the effects of known life extenders, showing that calorie restriction (CR) harnesses a protein that governs aging, Sir2p. Although many questions remain about how faithfully the findings will transfer to mammals, the work challenges some old ideas and hints that researchers should look at the relation between metabolism and longevity in a new way.
Like many animals, yeast live longer when food is scarce. For the microbes, cutting back calories means living on nutrient Jell-O that contains 0.5% glucose instead of the usual 2%. Under these conditions, a mother yeast cell buds to produce a daughter about 30 times on average instead of the usual 23 before she comes to her senses, claims old age, and becomes infertile; this eventual inability to reproduce provides a way to measure yeast longevity (see Kaeberlein Perspective). The benefits of CR apply to a vast range of creatures, from insects to worms to mammals, and scientists have hungered to dig into its mechanism. Some observations have hinted that metabolism sputters in calorie-restricted animals, leading to the supposition that this biochemical lethargy promotes long life.
For Saccharomyces cerevisiae, however, robust metabolic activity might stall rather than accelerate aging, report Leonard Guarente, a molecular biologist at the Massachusetts Institute of Technology in Cambridge, and colleagues in the 18 July issue of Nature. When yeast are swimming in sugar, they chew each glucose molecule a bit and then spit out the remains. When glucose supplies dwindle, however, they munch their food completely and survive longer. Yeast forced to adopt the second approach, through a change in cuisine or genetic tinkering, live longer, the team reports. The biochemical pathways that underlie these two metabolic schemes spew different amounts of a molecule that activates the life-span-extension protein Sir2p, which Guarente says might explain how low glucose concentrations delay aging.
"The notion has been that when animals have higher [metabolic rates], they have a shorter life-span, but there's never been a control," says Victor Darley-Usmar, a free-radical biochemist at the University of Alabama, Birmingham. The new study provides that control because "in the same organism, you've changed the degree of [metabolism]. Now, where you've got greater [metabolism], you've got extended life-span, so you've turned that concept upside down."
When glucose abounds, S. cerevisiae break down each molecule into ethanol and produce two ATP molecules in the process. When the sugar is scarce, they digest their food further, funneling its carbons into CO2 and squeezing out all of the energy stored in its bonds. The yield from one glucose molecule: 30 molecules of ATP. The team tested whether glucose concentrations that prolong yeast's life are low enough to prompt them to harness the second pathway. S. cerevisiae need oxygen only to devour their meals completely. The researchers measured oxygen consumption and found that the organisms depleted about twice as much oxygen in low compared to high glucose concentrations, suggesting that yeast shunt glucose into the oxygen-consuming pathway when the sugar is scarce.
To explore whether the lean-times pathway--called respiration--is required for yeast to extend life-span when glucose concentrations dive, Guarente and colleagues exploited a mutant that can't break glucose all the way down to CO2. Unlike normal yeast, organisms lacking a protein, cytochrome C1, that is required for respiration spawned the same number of progeny whether they were grown in low or high glucose concentrations. This result suggests that yeast must digest the sugar completely to derive the age-retarding benefits of glucose deprivation.
The researchers next wondered whether the metabolic shift toward respiration could by itself bestow extra staying power on the yeast. To test this idea, they tricked the cells into decimating glucose even when the sugar was plentiful. They engineered yeast that generate extra dollops of a protein, Hap4p, that activates several hundred genes and prods the microbe toward respiratory metabolism. Even when encountering a glucose feast, the organisms thoroughly gnaw every sugar morsel. This genetic intervention increased life-span by 35% in cells grown in plentiful glucose.
Together, the observations indicate that metabolic decisions dictate life-span. The researchers "could nicely manipulate the way that energy was processed," says Steven Austad, a biogerontologist at the University of Idaho in Moscow. "When they shunt energy through aerobic respiration, the yeast live longer. That effect goes away if they block aerobic respiration."
The team decided to probe deeper into the mechanism by which small amounts of glucose keep yeast mothers young. Other studies suggest that CR extends life-span by increasing resistance to oxidative stress. For example, calorie-restricted mammals suffer unusually small amounts of damage from reactive oxygen species (ROS), and adding enzymes or drugs that detoxify ROS in flies and worms lengthens life-span (see Genes/Interventions Database entries on the drug EUK-8, Drosophila melanogaster SOD2, and D. melanogaster SOD1). At first glance, these observations seem incompatible with the new finding that a shift toward respiration underlies long life. People often associate increased metabolic rate with increased ROS production, because mitochondria spit out these molecular miscreants as a byproduct of respiration. However, the correlation doesn't necessarily hold up. "How hard the mitochondrion is working is not the point," says Darley-Usmar. "What matters is efficiency." If the respiratory apparatus runs cleanly, it can churn out large amounts of ATP without gumming up the cell with ROS.
Exposing yeast to oxidative agents hikes the abundance of messenger RNAs (mRNAs) that encode protective enzymes, says Guarente, so measuring such mRNAs "might be a window into whether the cells are seeing a lot of ROS," he reasoned. The researchers used microarrays to assess the activity of all the genes in Hap4p-overproducing yeast and normal yeast grown in low glucose concentrations. Quantities of mRNAs rose for only a few antioxidant genes. To test whether long-lived yeast stave off oxidative assaults unusually well, the team then assessed resistance to two chemicals that generate ROS. The glucose-deprived and Hap4p-overproducing yeast succumbed more easily to these agents than did controls. The results show that shielding yeast from ROS is not central to the life-span extension provoked by low glucose concentrations, says Guarente. "Yeast live longer not because of a reduction in ROS and oxidative damage," he says. "They live longer because of the way carbon is utilized."
Other researchers, however, question that conclusion. Quantifying protective enzymes doesn't directly assess oxidative injury. "I wish they had measured oxidative damage to some component of the cells," says Austad. "It would have been very interesting if they had gotten longer life despite having greater oxidative damage." Guarente says that such a phenomenon would be difficult to show even if it were occurring. Although it's trivial to separate old from young mammals, he points out, one can't easily isolate elderly yeast from the newly budded youngsters crowding a culture. Finally, Darley-Usmar and Bruce Kristal, a biochemist at Weill Medical College of Cornell University in New York City and the Burke Medical Research Institute in White Plains, New York, propose mechanisms by which cells could increase their resistance to oxidative stress without producing more mRNA for the antioxidant enzymes--by altering protein stability, for instance--and they argue that the study does not address whether ROS plays a central role in governing life-span.
Some experts agree with Guarente that oxidative stress is not crucial to the life-span extension derived from low glucose conditions, but they underscore an additional message. The finding "points out the difference between yeast and mammalian systems," says Roger McCarter, a physiologist at the University of Texas Health Science Center in San Antonio. "There's a lot of indirect evidence that reduced oxidative damage plays a role in CR [in mammals]," says McCarter. "In yeast, that doesn't seem to be true."
To tie their new findings to a life-extending protein that they've been studying for years, the researchers performed a battery of tests designed to probe the involvement of Sir2p in yeast's response to low glucose. Extra Sir2p ups the number of times a yeast cell divides. It confers this perseverance by its ability to "silence" chromosomal regions, a process that shuts off genes and prevents cellular machinery from cutting and pasting DNA together in new and often troublesome ways. The long life granted by low glucose conditions depends on Sir2p: Strains engineered to lack the protein don't benefit from CR or from boosting Hap4p output. And additional experiments showed that silencing intensifies in low glucose concentrations and in Hap4p-overproducing strains--but only if Sir2p is present. These results show that silencing grows stronger under the same conditions that prolong life-span and that both processes depend on Sir2p.
The protein's quirky biochemical needs suggest how these findings might fit together. Sir2p requires a molecule called NAD+ to function--and respiratory activity generates NAD+. Depending on what other biochemical events are occurring in the cell, this phenomenon could cause NAD+ to accumulate. According to Guarente's model, a glucose shortage promotes oxidative metabolism, which emits NAD+ that can then activate Sir2p. He says he'd like to measure NAD+ in live cells so he can test his proposal that its quantities mount when glucose inventory is meager. The work, however, leaves open the possibility that Sir2p is activated by an NAD+-independent mechanism, he adds.
Whether yeast metabolism resembles that of any multicellular organism is debatable, says McCarter. Unlike the microbe, mammals rarely use anaerobic metabolism. Sprinting down the street generates a burst of ATP production through such a pathway, but in general, we need oxygen-based respiration to meet our energetic needs. The research, however, might help deflate a contentious, century-old notion of aging that some researchers have invoked to explain how CR works in warm-blooded creatures. In one form, that idea--called the rate-of-living hypothesis--asserts that oxygen-breathing creatures are born with the capacity to process a defined allotment of calories. Once that share vanishes, so does the organism's life (see "The Two Faces of Oxygen"). The theory gained support from nutritional studies. Cutting back calories initially makes metabolic rate per unit weight drop. However, that's because dividing metabolic activity by body weight right after starting a diet--before a person has shed pounds--gives an artificially low number. With prolonged food restriction, the body shrinks, and metabolic rate scaled to body weight climbs back to where it was before the sundae-skimping began--in both humans and lab animals. "The 'rate-of-living' idea has been loved by all for more than 100 years, and it'd be nice if it were true," says McCarter, "but available evidence in warm-blooded animals doesn't support it."
Others, however, say that the idea remains viable. They hypothesize that the metabolic rate of a key organ or tissue falls in dieting mammals and that this decline cultivates long life. "Whole-animal studies are simply not the correct way to answer this question," says Jon Ramsey, who studies energy metabolism at the University of California, Davis.
Although debate on this issue continues, scientists on both sides agree that CR does not deliver its goods to mammals by stepping up metabolism, as it does in yeast. Perhaps the discrepancy means that wizened yeast, but no one else, should care about the new work. Some scientists, however, are cautiously extrapolating the findings, seizing upon the idea that metabolic strategy rather than rate is the shared theme in CR-related longevity. "Under CR [in yeast], you see this shift in [metabolic pathway] utilization that seems to be important for life-span," says Arlan Richardson, a molecular gerontologist at the University of Texas Health Science Center in San Antonio. "That's the big thing I'm excited about." Even if metabolic rate remains steady in mammals, "the utilization of fuels [might be] different--and maybe that's what's important in CR," he says.
The work should prod scientists to focus their attention on this issue. Says McCarter, "The emphasis needs to shift away from metabolic rate to the type of metabolism that's taking place." Although fast living doesn't enliven everyone, different strategies might rejuvenate other creatures. Researchers' hearts are already racing at the challenge of uncovering those rousing alternative lifestyles.
July 17, 2002
Evelyn Strauss is senior news editor of SAGE KE. She is attempting to live an alternative and long life.
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Citation: E. Strauss, High-Octane Endurance--Yeast in the Metabolic Fast Lane Live Longer. Science's SAGE KE (17 July 2002), http://sageke.sciencemag.org/cgi/content/full/sageke;2002/28/nf9
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