Antioxidant Gene Expression in Active and Sedentary House Mice (Mus domesticus) Selected for High Voluntary Wheel-Running Behavior

Corresponding author: Anne M. Bronikowski, 339 Science 2, Iowa State University, Ames, IA 50011., abroniko{at}iastate.edu (E-mail)

Communicating editor: M. NOOR

	ABSTRACT

TOP ABSTRACT Quantitative assays for mRNA... Selection for increased... LITERATURE CITED

We present liver mRNA levels of the two antioxidant enzymes catalase (CAT) and Mn-superoxide dismutase (SOD2) in four treatment groups of house mice assayed by RNase protection at 20 months of age. These groups were mice from four replicate selection and four replicate control lines from the sixteenth generation of selective breeding for high voluntary wheel running, housed with or without running wheels from age 3 weeks through 20 months. Exercising control females had induced CAT expression; SOD2 exhibited a similar pattern in females from two of the four control lines. Exercising male mice had induced CAT expression, but not SOD2 expression, irrespective of genetic background. We discuss these results with respect to both evolutionary (genetic) and training (exercise-induced) adaptations and explore predictions of these results in relation to the oxidative-damage theory of senescence.

EVOLUTIONARY senescence theory argues that organisms senesce because the power of natural selection decreases with age (reviewed in ROSE 1991 ). Thus, selection is less effective against deleterious mutations expressed after reproductive peaks and, indeed, is virtually powerless against mutations expressed at postreproductive ages. This age-specific selection differential may result in the fixation of deleterious late-acting mutations ("mutation accumulation"; MEDAWAR 1952 ), particularly if the mutations are beneficial early in life ("antagonistic pleiotropy"; WILLIAMS 1957 ; reviewed in CHARLESWORTH 1994 ; MARTIN et al. 1996 ). Laboratory studies, primarily on Drosophila spp., support this life history theory tenet (e.g., PLETCHER et al. 1998 , PLETCHER et al. 1999 ) and furthermore point to the prevalence of pleiotropic genes involved in trade-offs between survival and reproduction (ROSE 1984 , ROSE 1990 ; SGRO and PARTRIDGE 1999 ; STEARNS et al. 2000 ) and survival and stress resistance (e.g., ARKING et al. 1991 ; TATAR et al. 1997 ).

The oxidative stress hypothesis of aging states that the senescent phenotype, the intrinsic physiological and biochemical decline with age resulting in decreasing survival, results from the accumulation of oxidative damage to cellular components (e.g., SOHAL et al. 1990 ; BARJA et al. 1994 ; reviewed in FINKEL and HOLBROOK 2000 ). Such damage is caused by an imperfect balance between the production of highly reactive oxygen species (ROS; e.g., in the mitochondrial electron transport pathway) and their breakdown by enzymatic and nonenzymatic antioxidants (SOHAL and WEINDRUCH 1996 ). Specifically, the antioxidant enzymes in this study are involved in the removal of two ROS: Mn-superoxide dismutase (SOD2), present within mitochondria, converts superoxide anions into H₂O₂, and catalase (CAT) converts H₂O₂ into water and oxygen in the cytosol. Genetic mutations or environmental interventions that increase an organism's ability to either break down ROS or decrease their production can have the consequence of prolonging both average and maximum life span (e.g., WEINDRUCH and WALFORD 1988 ; SOHAL et al. 1995 ; reviewed in BECKMAN and AMES 2000 ).

One such environmental intervention that remains equivocal with respect to its effects on ROS production, aging, and life span is exercise. Moderate exercise unequivocally improves overall health (e.g., increased cardiovascular performance, BLAIR et al. 1995 ; decreased collagen degradation, THOMAS et al. 1992 ; balanced neuroendocrine function, TUMER et al. 1997 ), offsets the risk of age-related disease (ASTRAND 1992 ), and increases median life span (HOLLOSZY 1988 , HOLLOSZY 1993 ; reviewed in MCCARTER 2000 ). But through increased oxygen consumption and metabolism, exercise can increase the production of highly damaging ROS (e.g., JI et al. 1998 ; BEJMA and JI 1999 ) and is associated with increased antioxidant enzyme activity and upregulation of these enzymes in skeletal muscle, heart, and liver (HOLLANDER et al. 1999 ; BEJMA et al. 2000 ; JI 2000 ). However, because the balance between ROS production and cellular antioxidant defenses is imperfect, increased ROS production may result in increased cellular oxidative damage and thus limit maximum life span (SOHAL and WEINDRUCH 1996 ).

To test proximate and evolutionary mechanisms of aging related to exercise and oxidative damage, we measured antioxidant gene expression in active and sedentary middle-aged mice from the sixteenth generation of a replicated artificial selection experiment for high voluntary wheel running behavior (Fig 1; details of the selection experiment in SWALLOW et al. 1998 ). By generation 15, selection lines were running on average 150% more than control lines (11.3 km/day vs. 4.5 km/day; BRONIKOWSKI et al. 2001 ), primarily by increasing running speed (rpm; GARLAND 2002 ). We hypothesize that selectively bred mice will have levels of antioxidant gene expression lower than those of control mice, but that within each selection group, active mice will have levels of antioxidant gene expression higher than those of sedentary mice. We discuss these results in the context of evolutionary theories of senescence, oxidative stress mechanisms of aging, and an ongoing aging experiment involving full siblings of the individuals used herein.

View larger version (18K): In this window In a new window Download PPT slide   Figure 1. The nested, hierarchical colony design. Activity group and genetic background are crossed, line is nested with genetic background (four lines per background), and family is nested within line (5 families per line). Eight full sibs (four males and four females) from each of 40 families were used to construct the colony. Parental mice were from the fifteenth generation of a selective-breeding experiment for increased voluntary wheel-running exercise (SWALLOW et al. 1998 ). Pups were weaned at 21 days of age and placed in treatment groups at 28 ± 3 days of age in the mouse facility at Washington State University. Four males and four females from each of the 5 families in each line were used, with one-half placed in the active group and one-half placed in the sedentary group. Each activity group thus contained two females and two males from each of the 5 families within each of the eight lines, for a total of 160 individuals per activity group and 320 individuals in the new study colony. Mice in the active group were placed individually in cages with a 10-cm-radius running wheel and electronic wheel-revolution counter built into the cage top. Mice in the sedentary group were placed individually in standard rodent cages. Extra sibs were placed in similar housing and were used as sentinel mice to monitor the colony for the presence of specific-pathogen exposure; monthly panels were all negative for exposure during this study. Mice were checked daily; food and water were available ad libitum. Cage bottoms were cleaned once every 2 weeks, wheels were cleaned once every 4 weeks, and clean wheels were randomly assigned to active mice. At 20 months of age, one active male, one sedentary male, one active female, and one sedentary female from each of the 40 families were decapitated, exsanguinated, and dissected. Tissues and organs were placed in a -80° freezer.

	Quantitative assays for mRNA expression

TOP ABSTRACT Quantitative assays for mRNA... Selection for increased... LITERATURE CITED

Gene expression levels (CAT, SOD2, ß-actin), body mass, liver mass, age, and wheel-running revolutions are in Table 1. Mice from selection lines with access to running wheels averaged higher daily revolutions over the 2 weeks prior to measuring antioxidant expression than did active mice from control lines (Table 2). Daily revolutions translate to selection and control mice running, on average, 4.0 and 2.7 km per day, respectively, for females, and 3.5 and 2.5 km per day, respectively, for males in the 2 weeks prior to sacrifice. These values compare to 7.7 and 4.5 km per day (selection and control, respectively) for the same individual females at 2 months of age and 6.2 and 4.8 per day for these same individual males at age 2 months. Thus, both females and males of selection lines continued running more than those of control lines through middle age, but the differences and the magnitudes were considerably smaller.

Although our selection regime did not influence CAT expression in sedentary animals, running did induce enhanced transcription in control, but not in selected, female mice and in both control and selected male mice (Table 2). Posthoc analysis of least-squares means from the full-model ANCOVA indicated that active control females had CAT expression significantly higher than that of the other female groups (Fig 2A). The magnitude of the effect of running in males was similar in both selection and control genetic backgrounds (Fig 2B).

View larger version (17K): In this window In a new window Download PPT slide   Figure 2. Least-squares mean expression levels (±SE) of CAT by activity group and genetic background from the two-way nested ANCOVA for females (a) and males (b).

In contrast to CAT, neither selection regime nor activity group affected SOD2 expression in either males or females (Table 2). For females, running was positively associated with SOD2 expression in two control lines (Table 2 and Fig 3A). For males, SOD2 expression varied among lines within both the selection and control groups (Table 2 and Fig 3B). Finally, ß-actin was affected neither by genetic background nor by activity group (Table 2), but the a priori contrast between active selection and control females indicated higher ß-actin expression in active control females.

View larger version (24K): In this window In a new window Download PPT slide   Figure 3. Least-squares mean expression levels (±SE) of manganese-superoxide-dismutase (SOD2) by activity group and genetic background from the two-way nested ANCOVA for (a) females, where active control females had higher SOD2 in lines two and four, and for (b) males.

	Selection for increased voluntary wheel running and long-term exercise training and antioxidant expression

TOP ABSTRACT Quantitative assays for mRNA... Selection for increased... LITERATURE CITED

An understanding of the relationship among selective breeding for exercise, exercise level, and antioxidant expression is fundamental for revealing pleiotropic effects of genes involved in running behavior on oxidative damage and life span and hence may shed light on both proximal and evolutionary mechanisms of senescence (MARTIN et al. 1996 ). In females the selection group by activity group interaction on CAT expression was significant and was driven by control females with wheel access having CAT expression higher than that of selected females with wheel access, despite significantly higher levels of exercise in females from selected lines. In males, CAT expression was higher in active mice irrespective of selection group, despite significantly higher levels of running in selected males with wheel access. Finally, no significant effects of selection group or activity group were identified for SOD2 expression.

The results of this study bear on several questions relating exercise to aging. First, wheel running was significantly higher in selection mice than in control mice, both at the normal testing age (2 months) and at sacrifice (20 months, Table 1), although the amount of running declined significantly between ages 2 and 20 months. This result from analyses of "landmark age" data is not surprising in light of identified differences in both the position and shape of the entire wheel-running ontogeny between these same selected and control individuals; selected mice run more throughout ontogeny but also have a greater rate of decline in running as they age (T. J. MORGAN and P. A. CARTER, unpublished data). Recent comparative reviews of diverse taxa (insects, rodents, and primates) suggest that age-related decline in physical activity (including rodent wheel revolutions; e.g., HOLLOSZY 1993 ) is widespread in various animal taxa and has a definite physiological basis (SALLIS 2000 ). Additionally, INGRAM 2000 summarized diverse evidence that changes in the dopaminergic system correlated with decreased activity with age. Thus we hypothesize that early-age selection on voluntary exercise and persistence of this differential through (at least) midlife reflect a persistent underlying difference in the dopamine/motivation system between genetic backgrounds (see RHODES et al. 2001 ).

Second, young selected mice from generation 14 had liver SOD2 enzyme activity lower than that of control mice (THOMSON et al. 2002 ); herein, no significant effects of selection were measured for SOD2 gene expression. The difference between the results of these two studies may be caused by post-translational modification in SOD2 enzymes or other molecular events that are not reflected in SOD2 expression (e.g., mRNA stability). Alternatively, SOD2 expression and/or activity may change with age, duration of exercise, and selection generation. The THOMSON et al. 2002 mice from selection generation 14 were housed with running wheels for 8 weeks beginning at 3 weeks of age and thus were assayed at 11 weeks of age. The mice herein were from generation 16, were housed with running wheels for almost 20 months beginning at 3 weeks of age, and were assayed at 20 months of age. Further investigation into the age specificity and effect of exercise duration on antioxidant expression and activity is warranted to address these hypotheses.

Third, active males had higher CAT expression, regardless of selection group. The effects of long-term exercise on the steady-state dynamics of the enzymatic antioxidant defense system are not clear. For example, JI 1993 found significant increases in antioxidant enzyme activity in heart, liver, and skeletal muscle after a single (acute) exhaustive exercise bout, but not after 12 weeks of training. On the other hand, in a separate study employing 10 weeks of endurance training HOLLANDER et al. 1999 found increased SOD2 and CAT expression in skeletal muscle. In addition, Holloszy and colleagues (e.g., HOLLOSZY et al. 1985 ; HOLLOSZY and SCHECHTMAN 1991 ; HOLLOSZY 1993 ) documented an extension of average life span, but not maximum life span, in exercising rodents (see also MLEKUSCH et al. 1996 ). These latter studies suggest that the upregulation of antioxidant defenses in response to increased ROS production is not costly or at least is less costly than the assorted beneficial effects of exercise on overall health. Thus we predict that within a given selection group, active mice may have longer average life spans.

Finally, active-selected females had CAT expression lower than that of active-control females, which is similar to the SOD2 enzyme activity patterns in young mice reported in THOMSON et al. 2002 . In general, lower antioxidant expression rates are thought to correlate with lower ROS production (reviewed in FINKEL and HOLBROOK 2000 ). Some studies have shown an increase in ROS production with exercise (e.g., JACKSON et al. 1985 ; ALESSIO 1993 ; BEJMA and JI 1999 ), and many others have shown that short-term exercise training increases antioxidant enzyme activity and/or expression in skeletal muscle and other organs in rats and mice (e.g., LEEUWENBURGH et al. 1994 ; NAKAO et al. 2000 ). Indeed, in studies that have measured all components of the oxidative system (oxidants, antioxidants, and actual damage to cellular components), increased levels of oxidants tend to predict levels of induction of the antioxidant defense system (e.g., AGARWAL and SOHAL 1993 ; SOHAL et al. 1994 ); thus lower CAT expression in exercising selection females may reflect lower ROS production and hence may reflect a beneficial pleiotropic effect of selection on voluntary exercise. However, an imperfect match between ROS production and antioxidant induction can result in increased oxidative damage; thus if lower CAT expression does not reflect lower ROS production, then active selected females should be experiencing more oxidative damage. This suggests a costly pleiotropic effect of early-age exercise selection on antioxidant defenses, which is consistent with the antagonistic pleiotropy theory of aging: genes with beneficial early effects having detrimental effects on life span through the physiological trade-off between exercise and oxidative damage. The ongoing study of whether early-age selection for voluntary exercise has positive or negative genetic correlations with average and maximal life span will aid in distinguishing between these two predictions.

	ACKNOWLEDGMENTS

For colony care and data collection, we thank S. Hall, S. Kane, S. Thomson, L. Jenkins, M. Baze, B. Irwin, M. Schmit, A. Poopatanapong, J. Robertson, E. Leber, F. Muller, and D. Baker; and for technical assistance, the veterinarians of Laboratory Animal Resources at Washington State University (WSU). We thank K. B. Kreiger and K. A. Vonnhame for laboratory assistance and S. P. Ford for the use of his molecular laboratory. This colony and research were supported by grants from the WSU School of Biological Sciences (P.A.C.), the National Science Foundation (DEB-0083638 to P.A.C., DEB-0105079 to P.A.C. and T.J.M., and IBN-9728434 to T.G.), and the National Institutes of Health (AG05784 to A.M.B.).

Manuscript received February 28, 2002; Accepted for publication May 20, 2002.

	LITERATURE CITED

TOP ABSTRACT Quantitative assays for mRNA... Selection for increased... LITERATURE CITED

AGARWAL, S. and R. S. SOHAL, 1993  Relationship between aging and susceptibility to protein oxidative damage. Biochem. Biophys. Res. Commun. 194:1203-1206.[Medline]

ALESSIO, H. M., 1993  Exercise-induced oxidative stress. Med. Sci. Sports Exerc. 25:218-224.[Medline]

ARKING, R., S. BUCK, A. BERRIOS, S. DWYER, and G. T. BAKER, 1991  Elevated paraquat resistance can be used as a bioassay for longevity in a genetically based long-lived strain of Drosophila.. Dev. Genet. 12:362-370.[Medline]

ASTRAND, P. O., 1992  Physical activity and fitness. Am. J. Clin. Nutr. 55:1231S-1236S.[Abstract/Free Full Text]

BARJA, G., S. CADENAS, C. ROJAS, R. PEREZ-CAMPO, and M. LOPEZ-TORRES, 1994  Low mitochondrial free radical production per unit O₂ consumption can explain the simultaneous presence of high longevity and high aerobic metabolic rate in birds. Free Radical Res. 21:317-327.[Medline]

BECKMAN, K. B., and B. N. AMES, 2000 Oxidants and aging, pp. 755–796 in Handbook of Oxidants and Antioxidants in Exercise, edited by C. K. SEN, L. PACKER and O. O. P. HÄNNINEN. Elsevier, Amsterdam.

BEJMA, J. and L. L. JI, 1999  Aging and acute exercise enhance free radical generation in rat skeletal muscle. J. Appl. Physiol. 87:465-470.[Abstract/Free Full Text]

BEJMA, J., P. RAMIRES, and L. L. JI, 2000  Free radical generation and oxidative stress with ageing and exercise: differential effects in the myocardium and liver. Acta Physiol. Scand. 169:343-351.[Medline]

BLAIR, S. N., H. W. KOHL, III, C. E. BARLOW, R. S. PAFFENBARGER, JR., and L. W. GIBBONS et al., 1995  Changes in physical fitness and all-cause mortality. A prospective study of healthy and unhealthy men. J. Am. Med. Assoc. 273:1093-1098.[Abstract]

BRONIKOWSKI, A. M., P. A. CARTER, J. G. SWALLOW, I. A. GIRARD, and J. S. RHODES et al., 2001  Open-field behavior of house mice selectively bred for high voluntary wheel running. Behav. Genet. 31:309-316.[Medline]

CHARLESWORTH, B., 1994 Evolution in Age-Structured Populations, Ed. 2. Cambridge University Press, Cambridge, UK.

EL MOUATASSIM, S., P. GUERIN, and Y. MENEZO, 1999  Expression of genes encoding antioxidant enzymes in human and mouse oocytes during the final stages of maturation. Mol. Hum. Reprod. 5:720-725.[Abstract/Free Full Text]

FINKEL, T. and N. J. HOLBROOK, 2000  Oxidants, oxidative stress and the biology of ageing. Nature 408:239-247.[Medline]

GARLAND, T., JR., 2002 Selection experiments: an underutilized tool in biomechanics and organismal biology, pp. 25–56 in Vertebrate Biomechanics and Evolution I: Theory and Aquatic Animals, edited by V. L. BELS, J.-P. GASC and A. CASINOS. Bios Scientific Publishers, Oxford.

HOLLANDER, J., R. RIEBIG, M. GORE, J. BEJMA, and T. OOKAWARA et al., 1999  Superoxide dismutase gene expression in skeletal muscle: fiber-specific adaptation to endurance training. Am. J. Physiol. 277:R856-R862.[Abstract/Free Full Text]

HOLLOSZY, J. O., 1988  Exercise and longevity: studies on rats. J. Gerontol. 43:B149-B151.[Medline]

HOLLOSZY, J. O., 1993  Exercise increases average longevity of female rats despite increased food intake and no growth retardation. J. Gerontol. 48:B97-B100.[Medline]

HOLLOSZY, J. O. and K. B. SCHECHTMAN, 1991  Interaction between exercise and food restriction: effects on longevity of male rats. J. Appl. Physiol. 70:1529-1535.[Abstract/Free Full Text]

HOLLOSZY, J. O., E. K. SMITH, M. VINING, and S. ADAMS, 1985  Effect of voluntary exercise on longevity of rats. J. Appl. Physiol. 59:826-831.[Abstract/Free Full Text]

INGRAM, D. K., 2000  Age-related decline in physical activity: generalization to nonhumans. Med. Sci. Sports Exerc. 32:1623-1629.[Medline]

JACKSON, J. J., R. H. EDWARDS, and M. C. SYMONS, 1985  Electron spin resonance studies of intact mammalian skeletal muscle. Biochim. Biophys. Acta 847:185-190.[Medline]

JI, L. L., 1993  Antioxidant enzyme response to exercise and aging. Med. Sci. Sports Exerc. 25:225-231.[Medline]

JI, L. L., 2000 Exercise-induced oxidative stress in the heart, pp. 689–712 in Handbook of Oxidants and Antioxidants in Exercise, edited by C. K. SEN, L. PACKER and O. O. P. HÄNNINEN. Elsevier, Amsterdam.

JI, L. L., C. LEEUWENBURGH, S. LEICHTWEIS, R. FIEBIG, and J. HOLLANDER et al., 1998  Oxidative stress and aging: role of exercise and its influences on antioxidant systems. Ann. NY Acad. Sci. 845:102-117.

LEEUWENBURGH, C., F. RUSSEL, R. CHANDWANEY, and L. L. JI, 1994  Aging and exercise training in skeletal muscle: responses of glutathione and antioxidant enzyme systems. Am. J. Physiol. 267:R439-R445.[Abstract/Free Full Text]

MARTIN, G. M., S. N. AUSTAD, and T. E. JOHNSON, 1996  Genetic analysis of aging: role of oxidative damage and environmental stresses. Nat. Genet. 13:25-34.[Medline]

MCCARTER, R. J. M., 2000 Caloric restriction, exercise and aging, pp. 797–829 in Handbook of Oxidants and Antioxidants in Exercise, edited by C. K. SEN, L. PACKER and O. O. P. HÄNNINEN. Elsevier, Amsterdam.

MEDAWAR, P. B., 1952 An Unsolved Problem of Biology. H. K. Lewis, London.

MLEKUSCH, W., H. TILLIAN, M. LAMPRECHT, H. TRUTNOVSKY, and R. HOREJSI et al., 1996  The effect of reduced physical activity on longevity of mice. Mech. Ageing Dev. 88:159-168.[Medline]

NAKAO, C., T. OOKAWARA, T. KIZAKI, S. OHISHI, and H. MIYAZAKI et al., 2000  Effects of swimming training on extracellular superoxide dismutase, Cu,Zn-Superoxide dismutase and Mn-superoxide dismutase in mouse tissues. J. Appl. Physiol. 88:649-654.[Abstract/Free Full Text]

PLETCHER, S. D., D. HOULE, and J. W. CURTSINGER, 1998  Age-specific properties of spontaneous mutations affecting mortality in Drosophila melanogaster. Genetics 148:287-303.[Abstract/Free Full Text]

PLETCHER, S. D., D. HOULE, and J. W. CURTSINGER, 1999  The evolution of age-specific mortality rates in Drosophila melanogaster: genetic divergence among unselected lines. Genetics 153:813-823.[Abstract/Free Full Text]

RHODES, J. S., G. R. HOSACK, I. A. GIRARD, A. E. KELLEY, and G. S. MITCHELL et al., 2001  Differential sensitivity to acute administration of cocaine, GBR 12909, and fluoxetine in mice selectively bred for hyperactive wheel-running behavior. Psychopharmacology 158:120-131.[Medline]

ROSE, M. R., 1984  Laboratory evolution of postponed senescence in Drosophila melanogaster.. Evolution 38:1004-1010.

ROSE, M. R., 1990 Evolutionary genetics of aging in Drosophila, pp. 43–58 in Genetic Effects on Aging II, edited by D. E. HARRISON. Telford Press, Caldwell, NJ.

ROSE, M. R., 1991 The Evolutionary Biology of Aging. Oxford University Press, New York.

SALLIS, J. F., 2000  Age-related decline in physical activity: a synthesis of human and animal studies. Med. Sci. Sports Exerc. 32:1598-1600.[Medline]

SGRO, C. M. and L. PARTRIDGE, 1999  A delayed wave of death from reproduction in Drosophila.. Science 286:2521-2524.[Abstract/Free Full Text]

SOHAL, R. S. and R. WEINDRUCH, 1996  Oxidative stress, caloric restriction, and aging. Science 273:59-63.[Abstract]

SOHAL, R. S., B. H. SOHAL, and U. T. BRUNK, 1990  Relationship between antioxidant defenses and longevity in different mammalian species. Mech. Ageing Dev. 53:217-227.[Medline]

SOHAL, R. S., H. KU, S. AGARWAL, M. J. FORSTER, and H. LAL, 1994  Oxidative damage, mitochondrial oxidant generation and antioxidant defenses during aging and in response to food restriction in the mouse. Mech. Ageing Dev. 74:121-133.[Medline]

SOHAL, R. S., A. AGARWAL, S. AGARWAL, and W. C. ORR, 1995  Simultaneous overexpression of copper- and zinc-containing superoxide dismutase and catalase retards age-related oxidative damage and increases metabolic potential in Drosophila melanogaster.. J. Biol. Chem. 270:15671-15674.[Abstract/Free Full Text]

STEARNS, S. C., M. ACKERMANN, M. DOEBELI, and M. KAISER, 2000  Experimental evolution of aging, growth, and reproduction in fruitflies. Proc. Natl. Acad. Sci. USA 97:3309-3313.[Abstract/Free Full Text]

SWALLOW, J. G., P. A. CARTER, and T. GARLAND, JR., 1998  Artificial selection for increased wheel-running behavior in house mice. Behav. Genet. 28:227-237.[Medline]

TATAR, M., A. A. KHAZAELI, and J. W. CURTSINGER, 1997  Chaperoning extended life. Nature 390:30.[Medline]

THOMAS, D. P., R. J. MCCORMICK, S. D. ZIMMERMAN, R. K. VADLAMUDI, and L. E. GOSSELIN, 1992  Aging- and training-induced alterations in collagen characteristics of rat left ventricle and papillary muscle. Am. J. Physiol. 263:H778-783.[Abstract/Free Full Text]

THOMSON, S. L., T. GARLAND, JR., J. G. SWALLOW, and P. A. CARTER, 2002  Response of Sod-2 enzyme activity to selection for high voluntary wheel running. Heredity 88:52-61.[Medline]

TUMER, N. J., S. LAROCHELLE, and M. YUREKLI, 1997  Exercise training reverses the age-related decline in tyrosine hydroxylase expression in rat hypothalamus. J. Gerontol. A 52:B255-B259.

WEINDRUCH, R., and R. L. WALFORD, 1988 The Retardation of Aging and Disease by Dietary Restriction. Charles C. Thomas, Springfield, IL.

WILLIAMS, G. C., 1957  Pleiotropy, natural selection and the evolution of senescence. Evolution 11:398-411.

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