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August 1998


The University of Wisconsin Study


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.

The Value of Animal Studies

Views on the use of animals in research engender huge and highly charged differences among people, rivaling those triggered by the topic of abortion. However, as loving pet owners (four cats, one dog, and Elmo the hamster, all of which provide sources of great affection in our two households) the authors feel very strongly that many of the country's biggest medical breakthroughs would not have been possible without animal research.

The list of such breakthroughs is far from trivial, including a vaccine for polio, insulin treatments for diabetics, medication for high blood pressure, kidney dialysis, radiation and chemotherapy treatments for cancer. Animal research is necessary to help understand basic biology and pathology, and also to test new diagnostic tools, surgical techniques and the latest drug therapies.

Animal research has been absolutely essential for the bulk of the progress made to date in understanding the aging process. By studying rhesus monkeys, the University of Wisconsin Primate Center's Aging Research Group and their collaborators have made progress in learning more about the aging process and associated pathological and physiological changes in this valuable animal model.

It is quite likely that the success of future efforts to attain a better understanding of the aging process and how to retard its progression will depend, in very large part, on the appropriate use of animal models.

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.