Small Molecule Increases Lifespan and "Healthspan" of Obese Mice
Risk of Death Cut By 31 Percent for Obese Mice Treated with Compound, and Treated Mice Seen Living as Long as Lean Mice
In Obese Mice, Molecule Reversed Nearly All Pathways Activated In Mice By High Calorie Diets
Findings Suggest Broad Implications for the Treatment of Age-related Diseases, Including Diabetes and Heart Disease
BOSTON-November 1, 2006-Researchers have used a single compound to increase the lifespan of obese mice, and found that the drug reversed nearly all of the changes in gene expression patterns found in mice on high calorie diets--some of which are associated with diabetes, heart disease, and other significant diseases related to obesity. The research, led by investigators at Harvard Medical School and the National Institute on Aging, is the first time that the small molecule resveratrol has been shown to offer survival benefits in a mammal. The study is reported in the November 1 advanced online edition of Nature.
"Mice are much closer evolutionarily to humans than any previous model organism treated by this molecule, which offers hope that similar impacts might be seen in humans without negative side-effects," says co-senior author David Sinclair, HMS associate professor of pathology, and co-director of the Paul F. Glenn Labs for the Biological Mechanisms of Aging.
"After six months, resveratrol essentially prevented most of the negative effects of the high calorie diet in mice," said Rafael de Cabo, Ph.D., the study's other co-senior investigator from the National Institute on Aging's Laboratory of Experimental Gerontology, Aging, Metabolism, and Nutrition Unit. "There is a lot of work ahead that will help us better understand resveratrol's roles and the best applications for it."
Image of three 15 month old mice: (left) high calorie diet + resveratrol; (center) high calorie diet; (right) standard diet. Photo by Doug Hansen.
Resveratrol is found in red wines and produced by a variety of plants when put under stress. It was first discovered to have an anti-aging properties by Sinclair, other HMS researchers, and their colleagues in 2003 and reported in Nature. The 2003 study showed that yeast treated with resveratrol lived 60 percent longer. Since 2003, resveratrol has been shown to extend the lifespan of worms and flies by nearly 30 percent, and fish by almost 60 percent. It has also been shown to protect against Huntington's disease in two different animal models (worms and mice).
"The "healthspan" benefits we saw in the obese mice treated with resveratrol, such as increased insulin sensitivity, decreased glucose levels, healthier heart and liver tissues, are positive clinical indicators and may mean we can stave off in humans age-related diseases such as type 2 diabetes, heart disease, and cancer, but only time and more research will tell," says Sinclair, who is also a co-founder of Sirtris, a company with an author on this paper and which is currently in a phase 1b trial in humans with diabetes using an enhanced, proprietary formulation of resveratrol. [Harvard has license and equity interests with Sirtris, which is not a public company.]
Investigators identified resveratrol while looking for compounds that activate Sir2, an enzyme linked to lifespan extension in yeast and other lower organisms. For the last 70 years, scientists have been able to increase the lifespan of a variety of species by reducing their normal food consumption by 30 to 40 percent - a diet known as calorie restriction. Through this research, scientists identified Sir2 as a key contributor to life extension. Without Sir2, for example, fruit flies see none of the benefits from either calorie restriction or treatment by resveratrol. The mammalian version of the Sir2 gene is SIRT1, which has the same enzymatic activity as Sir2, but modifies a wider variety of molecules throughout cells. Indicators in this study show that resveratrol might also be activating SIRT1 in mice, as well as other known longevity pathways.
How the Study Was Done This study examined three groups of mice, one on a standard diet (SD), another on a high calorie diet (HC) with 60 percent of calories coming from fat, and a third group of mice on the same high calorie diet but also treated with resveratrol (HCR). At middle age, or roughly 52 weeks of life, the researchers put the mice on the different diets.
Survival Benefit At 60 weeks of age, the survival rates of HC and HCR fed mice groups began to diverge and remained separated by a three to four month span. At 114 weeks of age, 58 percent of the HC fed mice had died, compared to 42 percent of the HCR and SD groups. Presently, the team has found resveratrol to reduce the risk of death from the HC diet by 31 percent, to a point where it is not significantly increased over the SD group.(Note: Given that mice are still living, final calculations can't be made.) "The median lifespan increase we are seeing is about 15 percent at this point," says Sinclair. "We won't have final lifespan numbers until all of the mice pass away, and this particular strain of mouse generally lives for two-and-a-half-years. So we are around five months from having final numbers, but there is no question that we are seeing increased longevity.
The team also found that the HCR fed mice had a much higher quality of life, outperforming the HC fed mice on motor skill tests. "The mice on resveratrol have not been just living longer," says Sinclair. "They are also living more active, better lives. Their motor skills actually show improvement as they grow older." The resveratrol fed mice also showed improved motor function with age over its HC fed counterparts. Researchers watched how well the mice did walking on a rotarod, similar to walking on a log in the water, a common measure of balance and motor coordination. At 24 months of age, the HC fed group would fall off the rotarod after 60 seconds, while the HCR group would stay on for nearly 120 seconds. The HCR group steadily improved their motor skills as they aged to the point where they were indistinguishable from the SD fed group.
Reversing Genetic Pathways Triggered by High Calorie Diet The research team also wanted to see if resveratrol could reverse the changes in gene expression patterns triggered by high calorie diets. Using liver tissue of five mice at 18 months of age from each group, the team performed a whole-genome microarray and identified which genes were turned on or off. The researchers then used a database generated by the Broad Institute that groups individual genes into common functional pathways to see where there were major differences.
"We made a striking observation," says Sinclair. "Resveratrol opposed the effects of high caloric intake in 144 out of 153 significantly altered pathways. In terms of gene expression and pathway comparison, the resveratrol fed group was more similar to the standard diet fed group than the high calorie group."
Improved Health Biomarkers: Glucose and Insulin In humans, high calorie diets can increase glucose and insulin levels leading to diabetes, cardiovascular disease, and non-alcoholic fatty liver disease. In the HC fed mice, researchers found biomarkers that might predict diabetes, including increased levels of insulin, glucose and insulin-like growth factor-1 (IGF-1). Conversely, the HCR fed group had significantly lower levels of these markers, paralleling the SD group. For example, a standard diabetes glucose test on the HCR fed group found considerably higher insulin sensitivity, meaning the HCR group had a lower disposition toward diabetes than the HC fed group. Lower insulin levels also predict increased lifespan in mice.
Organ Protection: Heart and Liver Three pathologists examined heart tissue from the SD, HC, and HCR mice, and while not knowing which organ belonged to which mouse group, they looked for subtle changes in the abundance of fatty lesions, degeneration and inflammation. On a relative scale of 0-4, the assessment produced mean scores of 1.6 for the SD group, 3.2 for the HC group, and 1.2 for the HCR group.
The researchers also found that the livers of mice at 18 months of age on the HC diet were greatly increased in size and weight. Liver tissue studies of these mice showed a loss of cellular integrity, and a build-up of lipids, which is common to high fat diets. In contrast, the HCR group had normal, healthy livers. Livers of mice at 18 months old: (left) normal diet; (center) high calorie diet; (right) high calorie diet + resveratrol.
Links to Calorie Restriction Lifespan Model The researchers also looked for metabolic ties to resveratrol's impact: pathway changes that mimicked those caused by calorie restriction. They examined AMP-activated kinase (AMPK), a metabolic regulator that promotes insulin sensitivity and fatty acid oxidation. It's been shown in previous work that the lifespan of worms has been extended by the addition of copies the AMPK gene, and chronic activation of AMPK is seen on calorie-restricted diets. The researchers examined the livers of the HCR fed group and found a strong tendency for AMPK activation, as well as two downstream indicators of its activity.
Calorie restriction and exercise have also been previously shown to increase the number of mitochondria in the liver. Mitochondria generate energy in cells. Through electron microscopy, investigators showed that the livers of the HCR fed mice had considerably more mitochondria than the HC group, and were not significantly different from those of the SD group.
Links to SIRT1 The team also asked if SIRT1 was activated by resveratrol in mice, as Sir2 is in lower organisms. To determine this, they looked at the amount of a specific chemical modification (acetylation) on the molecule PGC-1alpha. Removal of the "acetyl" chemical groups on PGC-1alpha activates this protein so that it can turn on certain genes that generate mitochondria and turn muscle into the type suited for endurance. The only enzyme known to remove the acetyl chemical groups on PGC-1alpha is SIRT1, and therefore the activity of PGC-1alpha is one of the most reliable and specific markers of SIRT1 activity in mammals. The research team found that levels of PGC-1alpha were three-fold lower in the HCR fed mice than in the HC mice, consistent with what would be expected when SIRT1 was being activated by resveratrol.
"This work demonstrates that there may be tremendous medical benefits to unlocking the secrets behind the genes that control our longevity," says Sinclair, "No doubt many more remain to be discovered in coming years."
This study was supported by Paul F. Glenn and the Glenn Foundation for Medical Research, the U.S. National Institutes of Health and the National Institute on Aging, the Ellison Medical Research Foundation, the American Heart Foundation, and the American Diabetes Association.
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Red Wine Component Inhibits Ill Effects of Excess Weight
A compound that increases the life span of yeast, worms, and fruit flies now has been shown to improve health and survival in mice fed a high-calorie diet. Treatment with resveratrol, a plant-derived molecule found in red wine, prevented many of the health consequences of obesity, even as mice gained weight. David Sinclair, who led the research, said that resveratrol “reduced the deleterious effects of a high-calorie diet. The mice were statistically similar to mice fed a lean diet.”
Photo by Graham Ramsay
David Sinclair (left) and Joseph Baur found that resveratrol, which can extend the lives of lower organisms, also keeps overfed mice healthy.
An HMS associate professor of pathology, Sinclair first identified resveratrol in a screen of molecules that enhance the activity of Sirt1, the mammalian version of the protein Sir2 that has been shown to affect life span in yeast and other lower organisms. Since then, treatment with resveratrol has been demonstrated to increase life span in worms, flies, and fish. This study, published online in Nature on Nov. 1, is the first in a series of studies by Sinclair’s group to determine whether resveratrol has the same impact on mammals.
Living Lean So far, the most reliable way to extend life span of an organism is through caloric restriction. Since Sir2 seems to mediate the effects of caloric restriction in some lower organisms, Sinclair’s team has been investigating whether manipulating the equivalent pathway in mammals can achieve the same benefits without cutting back on food. Joseph Baur, a postdoctoral fellow in Sinclair’s lab and first author on the paper, said that studying the compound’s effects in high-fat diets also makes sense given the prevalence of overweight and its known health risks. “Obesity tends to accelerate a lot of aging-related diseases,” he said.
The study compared three groups of mice: one fed a normal diet; another that began a high-calorie, high-fat diet at middle age; and a group that began the same high-calorie diet but were simultaneously treated with resveratrol. The researchers do not yet have final numbers on the total life span of the animals, since some are still alive. But after six months of treatment, mice that received resveratrol had a significantly higher survival rate. At 114 weeks of age, 42 percent of treated mice had died, compared with 58 percent of their untreated peers fed a high-calorie diet
Resveratrol did not prevent mice from gaining weight on a richer diet, but it did prevent health problems that arise with obesity. Most noticeable were differences in the livers of the two sets of mice. Untreated mice who followed the high-calorie diet had swollen livers filled with fat deposits—the livers of the treated mice, however, looked normal. Pathologists who examined the hearts of the mice also found that the group treated with resveratrol had healthier hearts, comparable to those of normal mice. Treated mice also had lower levels of glucose and insulin in their blood and higher insulin sensitivity. In a test of motor skills, the -resveratrol-treated mice outperformed untreated overweight peers and even showed improvement with age.
“We need to find out what the exact targets are that resveratrol is hitting within fat tissue and organs.”
The team also examined gene expression changes in the liver tissue of the mice. They ran the data through a database from the Broad Institute that groups genes into functional pathways. Through that, the team identified 153 pathways that were significantly changed by either the high-calorie diet alone or the high-calorie diet plus resveratrol. In 144 of them, resveratrol produced an effect opposite that of a high-calorie diet; in other words, said Baur, “If a pathway went up in comparison to a standard diet, resveratrol made it go down,” and vice versa.
Missing Pieces One of the study’s surprises is that resveratrol seems to have uncoupled the health consequences of being overweight from the fat itself. “By looking at the physiology of these mice, you would think they are lean healthy mice, but they’re fat healthy mice,” Sinclair said. The implication, he said, is that “fat isn’t necessarily bad if you can block its effects.”
Matt Kaeberlein, assistant professor of pathology at the University of Washington, said the study suggests that taking resveratrol or a similar compound could be beneficial for people who are obese or are eating a high-fat diet—which unfortunately includes a majority of people in the United States and other developed countries. But the study leaves open many questions, such as how resveratrol works and whether it can mimic the effects of calorie restriction in lean animals, which Sinclair’s lab is currently testing. The attempt to translate the pathways controlling life span in lower organisms into mammals has generated a great deal of debate. “Because the biology is so complicated, many of these pathways tie together,” said Kaeberlein. “It’s difficult to untangle.”
Sinclair believes that the compound is keeping mice healthy by triggering the same life-extending response that a strict diet does. “We’ve been going up the tree of life from yeast to worms to flies, showing this molecule extends their life and mimics calorie restriction,” he said. However, Sinclair added, “There’s a lot to figure out about how resveratrol is working.” His team used a proxy measure of Sirt1 activity to show that the enzyme was more active in mice treated with resveratrol. He doesn’t rule out that other pathways may be involved, and the team is currently testing Sirt1’s role by treating Sirt1--knockout mice.
Photo courtesy of Joseph Baur
When mice were fed a high-calorie, high-fat diet, their livers were swollen and filled with fat deposits (center) compared with those of mice fed a normal diet (left). Treating mice with resveratrol while they were on a high-calorie diet, however, prevented many of the liver abnormalities (right).
Sinclair believes that because resveratrol is produced when plants are stressed, it may be one of many plant compounds that can trigger a “survival response” in animals that consume them. “It could be that we’ve evolved to sense molecules from the plant world.” He said that certain components of plants are not just beneficial as antioxidants or anti-inflammatories but are actually sending signals that change physiology. However, the daily dose of resveratrol given to mice in this study is the equivalent in humans of 100 glasses of wine, so its role at normal dietary levels is unknown.
Rafael de Cabo, the study’s co-senior author and an investigator at the National Institute on Aging’s Laboratory of Experimental Gerontology, said that he hopes this study will spur new methods for testing the effects of aging in the body. Using longevity as an endpoint is onerous because studies on mammals can take years or, in the case of primates, decades. “We need to find out what the exact targets are that resveratrol is hitting within fat tissue and organs,” de Cabo said, as well as understand how caloric restriction affects the entire body of an animal. Only then can scientists start to unravel the puzzle of how these interventions extend life.
Image of three 15 month old mice: (left) high calorie diet + resveratrol; (center) high calorie diet; (right) standard diet. Photo by Doug Hansen. 
The resveratrol fed mice also showed improved motor function with age over its HC fed counterparts. Researchers watched how well the mice did walking on a rotarod, similar to walking on a log in the water, a common measure of balance and motor coordination. At 24 months of age, the HC fed group would fall off the rotarod after 60 seconds, while the HCR group would stay on for nearly 120 seconds. The HCR group steadily improved their motor skills as they aged to the point where they were indistinguishable from the SD fed group. 
