The University of Wisconsin Study

Dietary Restriction and Aging in Rhesus Monkeys

Life Extension Magazine® presented last month (July 1998 LEF Magazine) the preliminary findings of a ground-breaking study at the National Institute on Aging on how calorie restriction might extend and improve the lives of rhesus monkeys-the closest relative to mankind yet studied. In part two of the series, here are details from a complementary study that also shows promising results.

As longevity increases in most parts of the world and baby boomers (among those being, for example, the authors of this article) begin to grow old, the number of older persons in our society is increasing rapidly. This demographic reality has forced health care providers and scientists into a race with time to deal with higher rates of disease and disabilities, rising long-term care needs, and the high medical costs associated with these increases.

Can interventions be discovered by basic and clinical researchers that delay the onset of age-related diseases? Can the results of promising avenues of research be translated efficiently into medical practice so as to increase the number of people who age in a way that is minimally influenced by diseases? And, as is the dominant focus of Life Extension magazine, can strategies be developed to truly retard the aging process and thereby increase the maximum human life span?

Dietary restriction (often called calorie restriction, or calorie restriction with adequate nutrition) is well known among gerontologists and, increasingly, the general populace because it is the only intervention that repeatedly and strikingly increases maximum life span and retards the rate of aging in warm blooded animals (with laboratory rodents being most often studied). Life span extension by dietary restriction depends specifically on a reduction of caloric intake and this must occur without deficiencies of any essential nutrient. Thus, the bottom line of dietary restriction is that it is a state of very healthful "undernutrition without malnutrition."

There are two major issues about dietary restriction and aging that are being investigated. First, we know that caloric intake reduction can slow the aging process in animals such as mice and rats, but how does it actually do so? This is an extremely important question because, when it is answered, researchers will be quite well positioned to develop drugs aimed at triggering the most important actions of dietary restriction and, hopefully, its benefits as well. Note that the goal would be for this to occur in people eating normal caloric intakes.

The second issue concerns whether dietary restriction will be able to retard the rate of aging in animals closely related to humans...for example in nonhuman primates such as rhesus monkeys.

Our group at the University of Wisconsin-Madison is conducting a long-term, longitudinal study called "Dietary Restriction and Aging in Rhesus Monkeys." This project is directed by one of the authors (Weindruch) and is funded by the National Institute on Aging, which is one of the components of the National Institutes of Health. Our study tests the hypothesis that dietary restriction will influence the aging process in a primate species in a manner similar to that observed in rodents. We further hypothesize that dietary restriction's influence will be reflected by an altered rate of change of certain measurable markers of biological aging and, eventually, by increased longevity.

Our monkey dietary restriction study focuses on two major topics in the biology of aging. One is the development of nonhuman primates (in this case, the rhesus monkey) as a model for the study of aging. This species, whose real name is Macaca mulatta, is genetically very closely related to humans. A long-lived species, these monkeys have a maximum life span of about 40 years, roughly one-third that of humans. The other major issue we are trying to address is whether or not dietary restriction retards the rate of aging in a primate species.

To achieve this latter goal, the caloric intake of adult female and male rhesus monkeys has been restricted by 30 percent below that previously consumed by each animal. These monkeys are being compared with control animals that are being fed in a conventional fashion; that is, allowed free access to food for about eight hours per day.

The only other major, highly controlled study of dietary restriction's influence on aging in rhesus monkeys is being carried out by NIA scientists Drs. George Roth, Donald Ingram, Mark Lane and other collaborators in Bethesda, Md. (See Life Extension Magazine®, July 1998). These two studies should eventually-perhaps in 15 years-provide important data on longevity and disease patterns in a primate species subjected to a dietary restriction regimen.

Our rhesus monkey dietary restriction study began in 1989 when the NIA provided funds to study 30 young adult males, ranging in age from 8 to 14 years. All animals were previously fed the same standard monkey diet in the manner that monkeys are conventionally maintained in most primate facilities. This is done by giving each monkey free access to the food for about eight hours per day, typically from about 8 a.m. to 4 p.m., as well as being given daily treats such as fresh fruits (yes, including bananas), peanuts or raisins.

The restricted monkeys are healthier than the controls, are leaner, have lower levels of circulation glucose and insulin, and have greater insulin sensitivity.

Each animal's normal food intake was determined over a six-month period so that we would have the data needed to establish the level of food restriction on an individual basis. In our view, this is an important consideration because rhesus monkeys, like people, show great individual-to-individual variation in food intake. Also like people, some skinny monkeys are fairly big eaters, whereas some fatter ones are light eaters.

A significant difference between our study and that being conducted by the NIA scientists is that we have based food intakes of the calorically restricted monkeys on the basis of each animal's past intakes, whereas our colleagues at NIA have relied on charts that provide recommended food intakes for animals on the basis of age and body weight.

To initiate the study, 15 of the animals continued to "dine" in the conventional fashion. The caloric intake of the other 15 was gradually restricted, accomplished by reducing the food intake by 10 percent per month for three months. These 30 monkeys are referred to as "Group 1."

The aims of the initial study, from 1989 to 1994, with the Group 1 monkeys were to assess the effects of dietary restriction on potential biomarkers of aging: the function of the immune system, the visual system and the regulation of glucose, and later body composition and metabolic rate.

Group 2 was made up of young females (15 on the control diet and 15 subjected to dietary restriction) at ages very similar to those of the Group 1 males at the onset of dietary restriction in 1989. Dietary restriction was carried out in the same way as Group 1.

Another cohort, Group 3, also was initiated, and included 16 male rhesus monkeys, eight on a calorie-restricted diet and eight controls, all of similar ages as Group 1 at the onset of restriction. Group 3 is distinct in that these monkeys are undergoing surgical biopsies so that we can study liver, muscle, spleen and other tissues in alternate years. This has provided us with the opportunity to conduct biochemical studies of tissues well studied in diet-restricted rodents, but not yet studied in a primate species.

To date, it is clear that a 30-percent reduction in calorie intake can be safely imposed on rhesus monkeys. In fact, in agreement with the findings from the NIA study, several findings indicate that the restricted monkeys are healthier than the conventionally fed controls, are much leaner, have lower levels of circulating glucose and insulin, and have greater insulin sensitivity. This means that the insulin in the blood actually works better to remove glucose in the restricted monkeys.

Also, leptin, a hormone involved in appetite control made by fat cells, is much reduced in the diet-restricted monkeys. This makes sense, because they have much less body fat. We also find that blood lipids are beneficially altered by dietary restriction.

There is a very strong rationale for investigating mitochondria in the context of dietary restriction. Mitochondria are the minute structures within cells that serve as the power plants. As a consequence of their normal metabolic activity, mitochondria produce free radicals, highly reactive molecules often derived from oxygen. Free radicals carry an unpaired electron on their surface that makes them prone to causing damage to other molecules they may encounter. Denham Harman first proposed in 1956 that free radicals may be involved in aging, although it was unclear at that time where they may be coming from. It was not until more than 20 years later that scientists discovered that mitochondria are important sources of free radical production.

NORMAL DIET Food intake daily: 662 calories Body weight: 31.5 pounds Body fat: 26% MEASURES OF HEALTH Abdominal measure: 24.4 inches Body Mass: 47.6 (kg/m2) Basal gucose: 74 (milligrams per deciliter of blood) Basal insulin: 44 (micromoles/milliliter) Insulin Sensitivity: 1.8 (x 10-4) Leptin: 5.8 (ng/milliliter)

REDUCED DIET Food intake daily: 488 calories Body weight: 20.5 pounds Body fat: 8.6% MEASURES OF HEALTH Abdominal measure: 16.5 inches Body Mass: 34.2 (kg/m2) Basal gucose: 53 (milligrams per deciliter of blood) Basal insulin: 10 (micromoles/milliliter) Insulin Sensitivity: 9.1 (x 10-4) Leptin: 1.0 (ng/milliliter)

The current mitochondrial free radical explanation of aging partly derives from an understanding of how mitochondria produce a molecule known as ATP (adenosine triphosphate), the molecule that provides energy for many essential cellular activities such as the making of proteins, pumping of ions across cell membranes and muscle contraction, to name a few. The synthesis of ATP takes place by a complex sequence of reactions known as the electron transport system and oxidative phosphorylation, which occurs in the inner membrane of mitochondria. Using oxygen, these reactions extract energy from nutrients and use it to manufacture ATP. However, free radicals are also produced as a consequence of this process. Thus, the aging process may be a result of our cells carrying out very basic function: producing the energy required for life.

Mitochondria are also special structures because they contain their own unique genetic material, known as the mitochondrial DNA. Although mitochondrial DNA makes up only a very tiny fraction of the total DNA in a cell (more than 99.9 percent of the DNA is in the cell's nucleus), it codes for some essential molecules involved in the mitochondrial processes that make ATP. An emerging theme is that mitochondrial DNA mutations, accumulating over a life span, contribute to aging, cancer and other degenerative diseases. As investigators have discussed, it is well known that the assembly of functional mitochondria requires the joint expression of both mitochondrial and nuclear genes.

Further, the mutation rate for mitochondrial DNA is much higher than for nuclear genes, leading to the view that aging and certain major degenerative diseases (for example, Parkinson's and Alzheimer's diseases, ischemic heart disease and diabetes) may be partly due to the accumulation of mitochondrial DNA mutations leading to deficits in ATP production.

A discovery of great potential importance is the correlation of the accumulation of mitochondrial DNA deletions with aging; that is, mitochondrial genomes with big pieces missing. We are investigating the role that mitochondrial DNA deletions may play in the loss of skeletal muscle mass with aging, known as "sarcopenia." This age-associated loss of skeletal muscle contributes to a loss of strength and an overall increase in physical frailty. Sarcopenia is an important biological component in the age-associated increase in the occurrence of injurious falls, immobility, and the need for hospitalization or nursing home placement.

Much of this work has not yet involved the monkeys from the three cohorts because these animals are not yet old enough to display sarcopenia. Instead, we have studied normally fed, old rhesus monkeys, which are losing muscle mass, as well as old rats and mice subjected to dietary restriction, so we can better know what to study when our dietary restriction monkeys reach appropriate ages.

Thigh muscle samples from rhesus monkeys were examined for the presence of age-associated mitochondrial DNA deletions. Several normally fed animals from 6 to 34 years of age were examined for mitochondrial DNA deletions in a region occupying about one-half of the mitochondrial genome. All samples from animals over 13 years of age contained mitochondrial DNA deletion products, whereas the presence of deletions was greatly reduced or absent in younger animals. The specific deletion patterns varied from individual to individual. Some deletions were common to several animals while others appeared to be unique to a particular animal. Later work showed that the deleted mitochondrial genomes are distributed in a mosaic manner, with most cells (the muscle fibers) having no or low levels of deletions, while a subset of the cells had high levels.

These data demonstrate the importance of studying aging skeletal muscle using microscopic techniques, instead of grinding up the whole tissue for biochemical assays. Accordingly, we have used microscopy to study thigh muscle samples from rhesus monkeys (ages 2 to 39 years) processed for study of mitochondrial electron transport system activities. We analyzed 1,000 to 7,000 fibers per animal. In the animals up to 26 years of age, which approximates late middle age, the enzymatic activities displayed a typical "checkerboard" appearance, with those fibers containing high levels of mitochondria staining more intensely while fibers with fewer mitochondria stained less intensely.

In the two monkeys over 30 years, however, no such pattern was present in the muscle sections, and staining intensity was much lower than in the younger animals. In the older rhesus monkeys, 20 to 39 years of age, individual fibers were found which had no detectable activity of one of the enzymes, but were overly reactive for another. The levels of these abnormal fibers increased with the age of the animal. None of these abnormal enzymatic activities was detected in the younger rhesus monkeys, ages 2 to 16 years.

As noted, rodent models (mice and rats) have been used to guide the studies of primate mitochondrial function. The influence of dietary restriction initiated in late middle-age rats (at 17 months) on muscle fibers showing mitochondrial abnormalities was analyzed, as was mitochondrial DNA deletion accumulation in discrete skeletal muscles. Tissues from three groups of rats were studied: Young (3-4 months) fed without restriction; old (30-32 months) restricted at 17 months; and old controls.

We found that dietary restriction, started in late middle-age, can retard age-associated increases in the number of skeletal muscle fibers having mitochondrial enzyme abnormalities and decrease the accumulation of mitochondrial DNA deletions. In about 10 years, we should know if similar outcomes occur in monkeys subjected to adult-onset dietary restriction.

We have also studied the changes in body composition (a big decrease in fat and a much milder drop in lean mass), lower circulating levels of insulin and glucose, and greater insulin sensitivity. All of these changes argue that the restricted monkeys are healthier than the controls. This statement is supported by the observation that currently three of the Group 1 controls are either diabetic or pre-diabetic, whereas none of the Group 1 restricted animals are showing any signs of developing the disease, a common one for conventionally fed rhesus monkeys.

There also are several experiments being conducted to learn the nature of dietary restriction's influence (or non-influence) on several possible indicators of biological age. For example, we reported several lowered immune responses in dietary restriction animals. The capacity of white blood cells to undergo cell division when stimulated with an appropriate chemical was reduced in restricted monkeys, as compared with controls, during the interval after two to four years of dietary restriction.

Natural killer cell activity and antibody responses to influenza vaccine were also reduced during this interval in restricted monkeys. Neither cell surface antigens nor peripheral blood lymphocyte counts appear to be affected by dietary restriction thus far. These results, suggesting lowered immune responses in restricted animals, are not those predicted based on work in rodents.

In another aspect of our studies, we initiated a collaboration with the laboratories of Dr. Rajindar Sohal at Southern Methodist University and Dr. William Cefalu (Bowman Grey School of Medicine) to investigate oxidative stress and atherogenesis. Higher total sulfhydryl content of the plasma was found; data show a greater reducing capacity in the plasma in the restricted monkeys, which suggests a greater ability to remove free radicals from the blood.

Also, triglyceride levels have been found to be reduced in the restricted animals, and the lipoproteins extracted from dietary restriction plasma are less prone to being oxidized in an in vitro system than are lipoproteins from controls. Another quite interesting (and potentially important) observation is that the lipoproteins extracted from the plasma of restricted monkeys are less adherent to blood vessel walls than are lipoproteins from controls.

As we prepare the renewal application for the Program Project to fund this work from 1999 to 2004, some new directions and opportunities are apparent. For example, a new project will be proposed dealing with the immune response to influenza in these animals. This will be spearheaded by Dr. David Watkins, a world leader in the very specialized field of studying immunity in monkeys. Other work will measure levels of hormones and metabolites in urine to assess the status of the hypothalamo-pituitary-adrenal axis. Another new approach will be the use of a special type of water (called "doubly-labeled water") to measure metabolic rate.

There is widespread agreement among gerontologists about the importance of determining dietary restriction's influences on aging in nonhuman primates. This topic, which might appear to be a simple one to pursue, is not so straightforward. Good markers of biologic age in rhesus monkeys have not been established. The animal-to-animal variation inherent in genetically different animals (versus inbred stains of mice) makes it essential to study adequate numbers of animals in order to gather meaningful data.

In addition, most of what is known about the biologic effects of dietary restriction in rodents is the result of cross-sectional studies of tissues from killed animals, while studies of dietary restriction in monkeys have been minimally invasive.

Despite these chronic challenges, we hope that our study is providing a better understanding of the biology of aging in primates and that it contributes significantly to the body of knowledge which can be used by the scientific and medical communities to attenuate the development of the undesirable biological expressions of aging.

Co-authors Richard Weindruch, Ph.D. (lead scientist) and Jennifer Christensen are associated with the Wisconsin Regional Primate Research Center, located at the University of Wisconsin-Madison, one of seven Regional Primate Research Centers funded by the National Institutes of Health. These centers serve as regional and national resources for solving human health problems through research on nonhuman primate models.

Further Reading

Aspnes LE, Lee CM, Weindruch R, Chung SS, Roecker EB, Aiken JM: Caloric restriction reduces fiber loss and mitochondrial abnormalities in aged rat muscle. Faseb J. 11:573-581, 1997.
Bandy B, Davison AJ: Mitochondrial mutations may increase oxidative stress: implications for carcinogenesis and aging? Free Rad. Biol. Med. 8:523-539, 1990.
Chance B, Sies H, Boveris A: Hydroperoxide metabolism in mammalian organs. Pysiol. Rev. 59:527-603, 1979.
Cortopassi GA, Arnheim N: Detection of a specific mitochondrial DNA deletion in tissues of older humans. Nucl. Acids Res. 18:6927-6933, 1990.
Fishbein L: "Biological effects of dietary restriction." New York: Springer-Verlag; 1991.
Harman D: Aging: a theory based on free radical and radiation chemistry. J. Gerontol. 11:298-300, 1956.
Hart RW, Neuman DA, Robinson RT: "Dietary Restriction: Implications for the Design and Interpretation of Toxicity and Carcinogenicity Studies." Washington, DC: ILSI Press; 1995.
Kemnitz JW, Roecker EB, Weindruch R, Elson DF, Baum ST, Bergman RN: Dietary restriction increases insulin sensitivity and lowers blood glucose in rhesus monkeys. Am. J. Physiol. 266:E540- E547, 1994.
Kim M-J, Roecker EB, Ershler WB, Aiken JM, Weindruch R: Oxidative stress-induced interleukin-6 production by peripheral mononuclear cells of rhesus monkeys: Influences of age and dietary restriction (in press).
Lee CM, Chung SS, Kaczkowski JM, Weindruch R, Aiken JM: Multiple mitochondrial DNA deletions associated with age in skeletal muscle of rhesus monkeys. J. Gerontol. Biol. Sci. 48:B201-B205, 1993.
Lee CM, Weindruch R, Aiken JM: Age-s associated alterations of the mitochondrial genome. Free Rad. Biol. Med. 22:1259-1269, 1997.
Miquel J: An integrated theory of aging as the result of mitochondrial-DNA mutation in differentiated cells. Arch. Gerontol. Geriatr. 12:99-117, 1991.
Ramsey JJ, Roecker EB, Weindruch R, Kemnitz JW: Energy expenditure in adult male rhesus monkeys during the first 30 months of dietary restriction. Am. J. Physiol. 272:E901- E907, 1997.
Roecker EB, Kemnitz JW, Ershler WB, Weindruch R: Reduced immune responses in rhesus monkeys subjected to dietary restriction. J. Gerontol. Biol. Sci. 51:B276-B279, 1996.
Schwarze S, Lee CM, Chung SS, Roecker EB, Weindruch R, Aiken JM: High levels of mitochondrial DNA deletions in skeletal muscle of old rhesus monkeys. Mech. Ageing Dev. 83:91-101, 1995.
Sohal RS, Weindruch R: Oxidative stress, caloric restriction, and aging. Science 273:59-63, 1996.
Wallace DC: Mitochondrial genetics: a paradigm for aging and degenerative diseases? Science 256:628-632, 1992.
Weindruch R: Caloric restriction and aging. Sci. Am. 274(1):46-52, 1996.
Weindruch R, Sohal RS: Caloric intake and aging. New Engl. J. Med. 337:986-94, 1997.
Weindruch R, Walford RL: Dietary restriction in mice beginning at one year of age: Effects on life-span and spontaneous cancer incidence. Science 215:1415-1418, 1982.
Weindruch R, Walford RL. "The Retardation of Aging and Disease by Dietary Restriction." Springfield, IL: C.C. Thomas; 1988.
Yen T-C, Su J-H, King K-L, Wei Y-H: Ageing-associated 5 kb deletion in human liver mitochondrial DNA. Biochem. Biophys. Res. Comm. 178:124-131, 1991.