March 7, 2013 | 1
What is it about sirtuins? Few research topics have engendered fiercer controversy in recent years than these enzymes, channels through which the famous red-wine ingredient resveratrol appears to exert effects like those of calorie restriction, a reduction of food intake known to slow aging in many species.
The most basic bone of contention is whether resveratrol really activates sirtuins. The idea that it revs up the enzymes dates from the first study that suggested it has anti-aging power—a 2003 investigation led by Harvard University’s David Sinclair. Two years later, other researchers published studies suggesting that resveratrol only stimulates sirtuins in misleading test-tube assays—and probably not in living cells. The skeptics’ findings put a cloud over Sinclair’s research and raised questions about work at Sirtris Pharmaceuticals, a biotech he cofounded.
But now Sinclair’s group has marshaled evidence that sirtuin activators really do stimulate the enzymes as originally proposed—at least in certain situations. Their new findings are reported in the March 8 issue of Science in a study coauthored by Sirtris researchers. A second study led by Clemens Steegborn at the University of Bayreuth in Germany, published this week in Aging, supports the Harvard group’s analysis.
Together the studies “appear to elegantly resolve” the mechanism controversy, according to a commentary in Aging by Sita Kugel and Raul Mostoslavsky, researchers at Massachusetts General Hospital in Boston. Mostoslavsky is a member of Sirtris’s scientific advisory board.
The new data won’t end the sirtuins debate, which concerns issues besides the mechanism question—the most hotly contested one is whether sirtuins are centrally involved in calorie restriction’s anti-aging effects. But the studies should restore lost luster to Sinclair’s work on sirtuins. They’re also good news for GlaxoSmithKline, which bought Sirtris in 2008 for a hefty $720 million—the skeptics’ reports had suggested that Glaxo’s effort to develop sirtuin activators as drugs was based on a cracked foundation.
The mechanism debate began after Sinclair and colleagues discovered in 2003 that resveratrol sped up the action of SIR2, a yeast sirtuin thought to mediate calorie restriction’s effects, and of SIRT1, a mammalian version of SIR2, in test-tube assays. Using a fluorescent molecule called Fluor-de-Lys to flag the enzyme’s activity, their experiments indicated that resveratrol interacted with the sirtuins in a way that accelerated their normal function. (SIR2 and SIRT1 regulate the activity of various “substrate” molecules in cells by removing pieces from them called acetyl groups.) The sirtuin stimulation extended yeast cells’ lifespans by up to 60%, according to the report.
But in 2005, skeptics reported that resveratrol failed to boost sirtuins’ action without the presence of Fluor-de-Lys. (Attached to substrates, the fluorescent molecules generated a telltale glow when acetyl groups were removed by sirtuins.) That suggested Fluor-de-Lys inadvertently had played a central role in producing the sirtuin-boosting effect—and that the effect didn’t occur outside the test tube. In 2009 and 2010, respectively, researchers at Amgen and Pfizer reported similar findings.
Meanwhile, many studies in living organisms were reported that indicated resveratrol and other sirtuin activators induce calorie-restriction-like effects by stimulating the enzymes. But many observers found them suspect. Indeed, soon after the Pfizer report, a scientist tracking the debate emailed me that it seemed “sirtuins and resveratrol and the entire Sirtris enterprise are a house of cards that is in the process of crumbling badly.”
It appeared to the skeptics that purported sirtuin activators, at most, might indirectly boost the enzymes via unknown knock-on effects in cells. That led many to conclude that Glaxo had made a costly blunder when it bought Sirtris in hopes of turning its compounds into drugs targeting SIRT1 and other sirtuins.
But Sinclair theorized that the fluorescent molecules may have acted a lot like naturally-occurring parts of sirtuin substrates in the test-tube assay. If so, his team’s apparently misleading experiments with Fluor-de-Lys could have pointed, fortuitiously, to a correct conclusion: That resveratrol can rev up sirtuins in living cells.
The new studies support this theory. Both indicated that the fluorescent molecules, which are hydrophobic (repelling water molecules), mimic hydrophobic amino acids found at two locations in certain SIRT1 substrate molecules. In fact, sirtuin activators appear to rev up SIRT1 only when it is interacting with a limited number of substrates that contain hydrophobic molecules at one or both of the two locations.
By a twist of fate, the substrate used by both Sinclair and his critics in their early test-tube experiments lacked the critical hydrophobic amino acids. That’s why resveratrol’s sirtuin-revving effect only occurred, as the skeptics reported, when that substrate was gussied up with Fluor-de-Lys—the fluorescent molecules substituted for the missing hydrophobic amino acids.
Importantly, according to the Sinclair group’s new data, substrates with such amino acids include ones thought to help induce some of calorie restriction’s key health-promoting effects, such as the “stress response,” which hardens cells against damage by DNA-mangling molecules.
In its latest study, Sinclair’s group also went beyond test-tube experiments to probe SIRT1’s action in living cells. This step was based on their discovery that the SIRT1 protein includes a single amino acid that’s critical for the boosting of the enzyme’s activity by resveratrol and other activators—when another amino acid is substituted for it, SIRT1 does its normal enzymatic thing but can’t be artificially revved up. That enabled a revealing experiment: Sirtuin activators added to cells containing normal SIRT1 were found to enhance the cells’ mitochondrial function in a way reminiscent of calorie restriction’s effect. (Mitochondria, cells’ energy dynamos, are spruced up by calorie restriction.) But when the activators were added to cells whose SIRT1 lacked the critical amino acid, the mitochondrial boost didn’t occur. The results imply that SIRT1 serves as a key channel for inducing the mitochondrial effect, which the Sirtris compounds can amplify in cells.
Besides clearing up the mechanism issue, said Mostoslavsky, the new findings should aid drug development since they’ve illuminated how different sirtuin activators exert selective effects on SIRT1 substrates. That promises to lead to sirtuin-boosting medicines that confer specific therapeutic benefits, with few side effects, by targeting certain SIRT1 substrates and not others.