What have we learned from animal models of aging about caloric restriction?

Caloric restriction describes a scientific intervention in which total daily calories provided to an animal or organism are limited to about 70% of those of the animal's freely fed counterparts. Sometimes described as "undernutrition without malnutrition," caloric restriction is the only intervention actually documented to extend life span in laboratory animals. Its effects in humans are, of course, unknown. Scientists would have great difficulty designing experiments that would involve enough participants to be statistically valid. They would also be faced with the challenge of having to study them long enough to know the effects on longevity.

But scientists have studied caloric restriction in laboratory animals, with promising results. They have looked at the effects of caloric restriction in each of the major categories of experimental animals listed below. For additional information, see the Caloric Restriction Information Center.

• Yeast
• Roundworms
• Fruit flies
• Rodents
• Nonhuman primates

Yeast
Researchers at Louisiana State University have recently published an article detailing the extension of life span in yeast that they achieved through caloric restriction. They reduced the amount of glucose (sugar) in the medium in which their yeast was cultured and found that life span was increased and the appearance of certain characteristic signs of aging was delayed. They found similar results when they decreased the concentration of amino acids in the yeast culture medium, which suggests that a reduction in total available calories was the important factor in extending life span, not the reduction of one particular nutrient.

Caloric restriction in yeast has also been shown to have an important relationship to sirtuins, a family of enzymes that play critical roles in a wide array of vital life processes. SIR2 is an important sirtuin found in yeast, while its homolog in humans is SIRT1. In September 2003, a team of investigators found that resveratol, a sirtuin-activating compound found in red wine, increased the life span of yeast cells by more than two-thirds. And structural biologists at The Wistar Institute, led by Ronen Marmorstein, have been using yeast models to study the role of sirtuins in gene expression. Using X-ray crystallography and other techniques, Marmorstein and his group examined how sirtuin molecules bind to NAD, an important compound in glucose metabolism. Sirtuins rely on NAD to properly facilitate gene expression. If levels of NAD are sufficient, then sirtuins can effectively shut down certain genes that, left unchecked, would expend needless energy, promote the production of genetic abnormalities, and contribute to the aging process. If NAD molecules are consumed in glucose metabolism, as they would be in a high-calorie diet, then sirtuins do not function effectively.

Interestingly, other recent research seems to suggest the opposite. Rozalyn M. Anderson and others from the Harvard Medical School have studied caloric restriction in yeast quite extensively, and they have found that the activity of SIR2 and SIRT1 are not affected by physiological alterations in NAD levels. While such contradictory findings seem confusing, they simply illustrate the complicated nature of aging research, as well as the need to have multiple investigators conduct similar experiments to verify results.

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Roundworms
In studying roundworms and longevity, scientists have identified several groups of genes that seem to promote longer life spans. One group produces a protein called PABP, another group produces heat shock proteins (proteins that protect the metabolism from the stress of high environmental temperature) and the third group produces proteins that are similar to certain human proteins, although their function is yet undetermined. The genes in the third group were all stimulated to produce their proteins in the face of starvation, suggesting that caloric restriction can induce changes that increase life span in roundworms.

Interesting research by scientists at the Washington University School of Medicine in St. Louis revealed that anti-seizure drugs extended the lifespan of roundworms considerably. The anticonvulsant ethosuximide extended roundworm life from an average of 17 days to an average of 20 days, and the drug trimethadione had a bigger effect, extending lifespan by 47%. When the researchers looked into why the epilepsy medicines increased longevity, they discovered that the drugs affect aging by influencing the neural system involved in the insulin-signaling pathway. In addition to metabolism, the C. elegans insulin pathway regulates free radical protection and may be related to the longevity increase caused by caloric restriction in mammals.

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Fruit flies
Demonstrating that even the most serious scientists can have a sense of humor, researchers studying the fruit fly have identified a gene that they have labeled Indy (for "I'm Not Dead Yet"). Indy is analogous to a gene in humans that produces a protein in the kidneys that might be important in the Krebs cycle, a biochemical cascade involved in energy generation. Mutations in the Indy gene in fruit flies result in a doubling of the life span without a reduction in either fertility or physical activity. The researchers speculate that these mutations, which reduce the reutilization of certain substances, produce a physical state that mimics caloric restriction. This indicates that caloric restriction might be effective in increasing longevity in fruit flies.

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Rodents
It was research with rodents that first established the link between caloric restriction and aging. Indeed, scientists first recognized the value of caloric restriction more than 60 years ago, when they found that rats fed a low-calorie diet lived longer on average than free-feeding rats. What's more, some of the treated animals survived longer than the oldest-living animals in the control group, which means that the maximum life span (the oldest attainable age), not merely the average life span, increased.

Since those classic experiments were conducted, the findings have been replicated in many other organisms, from fruit flies to roundworms. But rats and mice continue to be a favorite model to study the effects of caloric restriction on aging. Hundreds of papers each year present findings from research conducted on rats and mice in laboratories all over the world-a body of work that would be difficult to summarize adequately on this web site. However, to provide a glimpse into the varied and numerous ways rodents can be used in aging research, two recent studies are highlighted below:

In 2003, researchers at the University of Florida measured the levels of cytochrome c and other indicators of apoptosis in the brain cells of rats to better understand the mechanisms of memory loss and mental decline in Alzheimer's and Parkinson's disease. They studied three groups of rats: 12- and 26-month-old animals given unlimited food and water, and a group of 26-month-old rats given 40 percent fewer calories than those in the unrestricted groups. The researchers found that cytochrome c increased with age in the normally nourished animals. In the 26-month-old, calorie-restricted rats, however, the protein did not increase; instead, it remained at the level of the young rats.

Researchers at the University of North Carolina at Chapel Hill Lineberger Comprehensive Cancer Center studied the expression of two proteins called p16INK4a and ARF in a rodent model of aging. They discovered that, as cells and tissues age, expression of p16INK4a and ARF dramatically increases. The researchers also found that the increase in the two proteins can be substantially inhibited by decreasing caloric intake, suggesting that decreased expression of p16INK4a and ARF could mediate the known anti-aging effects of caloric restriction.

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Nonhuman primates
The National Institute on Aging is conducting long-term experiments on nonhuman primates, such as rhesus and squirrel monkeys, in the hopes that their results will increase our understanding of human aging. Among the studies being conducted are several involving caloric restriction. Study animals are given diets with about 30% fewer calories than the diets of the control animals, and they receive nutritional supplements to guard against vitamin deficiencies in order to achieve the goal of undernutrition without malnutrition.

Early results with these primates demonstrate that the calorie-restricted monkeys weigh less than their freely fed counterparts, and they have both less fat and less lean body mass. They also have lower body temperatures. The calorie-restricted monkeys metabolize glucose (sugar) more efficiently, with better glucose tolerance and greater insulin sensitivity. The research suggests that they do not develop insulin resistance as often as their freely fed counterparts. This improved glucose metabolism suggests that they are less likely to develop diabetes as they age. They also show signs that they are less predisposed to heart disease and cancer, two other diseases of aging. Further, caloric restriction seems to slow the age-related decline in melatonin secretion that monkeys share with humans. These results are still preliminary. Primates have a rather long life span, and years of study must be conducted before it is known whether these changes seen in the calorie-restricted monkeys will translate into longer average life spans.

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