Jane S. McLaren

Uncoupled and surviving: individual mice with high metabolism have greater mitochondrial uncoupling and live longer

Two theories of how energy metabolism should be associated with longevity, both mediated via free‐radical production, make completely contrary predictions. The ‘rate of living‐free‐radical theory’ ( Pearl, 1928 ; Harman, 1956 ; Sohal, 2002 ) suggests a negative association, the ‘uncoupling to survive’ hypothesis ( Brand, 2000 ) suggests the correlation should be positive. Existing empirical data on this issue is contradictory and extremely confused ( Rubner, 1908 ; Yan & Sohal, 2000 ; Ragland & Sohal, 1975 ; Daan et al., 1996 ; Wolf & Schmid‐Hempel, 1989 ]. We sought associations between longevity and individual variations in energy metabolism in a cohort of outbred mice. We found a positive association between metabolic intensity (kJ daily food assimilation expressed as g/body mass) and lifespan, but no relationships of lifespan to body mass, fat mass or lean body mass. Mice in the upper quartile of metabolic intensities had greater resting oxygen consumption by 17% and lived 36% longer than mice in the lowest intensity quartile. Mitochondria isolated from the skeletal muscle of mice in the upper quartile had higher proton conductance than mitochondria from mice from the lowest quartile. The higher conductance was caused by higher levels of endogenous activators of proton leak through the adenine nucleotide translocase and uncoupling protein‐3. Individuals with high metabolism were therefore more uncoupled, had greater resting and total daily energy expenditures and survived longest – supporting the ‘uncoupling to survive’ hypothesis.

EFFECT OF LONG-TERM COLD EXPOSURE ON ANTIOXIDANT ENZYME ACTIVITIES IN A SMALL MAMMAL

Aerobic organisms continually face exposure to reactive oxygen species (ROS) and many have evolved sophisticated antioxidant systems to effectively remove them. Any increase in ROS production or weakening in this defense system may ultimately lead to oxidative stress and cellular damage. We investigated whether long-term cold exposure, which is known to lead to an elevation in metabolic rate, increased the activities of the ROS-scavenging enzymes, catalase (CAT), selenium-dependent glutathione peroxidase (GPx), and total superoxide dismutase (Total-SOD) in liver, cardiac muscle, kidney, skeletal muscle (vastus lateralis), and duodenum of short-tailed field voles (Microtus agrestis), born and maintained at either 8 +/- 3 degrees C or 22 +/- 3 degrees C. CAT, GPx, and Total-SOD activities were determined at age 61 +/- 1.9 days. An increase in CAT activity in voles maintained at 8 +/- 3 degrees C was observed in skeletal muscle (71%) and kidney (20%), with both CAT and GPx activities significantly elevated (by 40 and 43%, respectively) in cardiac muscle, when compared to voles at 22 +/- 3 degrees C. Total-SOD activity and protein content did not differ significantly between groups in any tissue. We suggest that the compensatory increases in CAT (skeletal muscle, cardiac muscle, kidney) and GPx (cardiac muscle), but not Total-SOD activities, resulting from long-term cold exposure may reflect the elevated metabolic rate, and possibly also increased ROS production, at this time.

The impact of experimentally elevated energy expenditure on oxidative stress and lifespan in the short-tailed field vole Microtus agrestis

Life-history theory assumes that animal life histories are a consequence of trade-offs between current activities and future reproductive performance or survival, because resource supply is limited. Empirical evidence for such trade-offs in the wild are common, yet investigations of the underlying mechanisms are rare. Life-history trade-offs may have both physiological and ecological mediated costs. One hypothesized physiological mechanism is that elevated energy metabolism may increase reactive oxygen species production, leading to somatic damage and thus compromising future survival. We investigated the impact of experimentally elevated energy expenditure on oxidative damage, protection and lifespan in short-tailed field voles (Microtus agrestis) maintained in captivity to remove any confounding ecological factor effects. Energy expenditure was elevated via lifelong cold exposure (7±2°C), relative to siblings in the warm (22±2°C). No treatment effect on cumulative mortality risk was observed, with negligible effects on oxidative stress and antioxidant protection. These data suggest that in captive animals physiologically mediated costs on life history do not result from increased energy expenditure and consequent elevations in oxidative stress and reduced survival.

Life-long vitamin C supplementation in combination with cold exposure does not affect oxidative damage or lifespan in mice, but decreases expression of antioxidant protection genes

Oxidative stress is suggested to be central to the ageing process, with endogenous antioxidant defence and repair mechanisms in place to minimize damage. Theoretically, supplementation with exogenous antioxidants might support the endogenous antioxidant system, thereby reducing oxidative damage, ageing-related functional decline and prolonging life- and health-span. Yet supplementation trials with antioxidants in animal models have had minimal success. Human epidemiological data are similarly unimpressive, leading some to question whether vitamin C, for example, might have pro-oxidant properties in vivo. We supplemented cold exposed (7+/-2 degrees C) female C57BL/6 mice over their lifespan with vitamin C (ascorbyl-2-polyphosphate), widely advocated and self administered to reduce oxidative stress, retard ageing and increase healthy lifespan. No effect on mean or maximum lifespan following vitamin C treatment or any significant impact on body mass, or on parameters of energy metabolism was observed. Moreover, no differences in hepatocyte and lymphocyte DNA oxidative damage or hepatic lipid peroxidation was seen between supplemented and control mice. Using a DNA macroarray specific for oxidative stress-related genes, we found that after 18 months of supplementation, mice exhibited a significantly reduced expression of several genes in the liver linked to free-radical scavenging, including Mn-superoxide dismutase. We confirmed these effects by Northern blotting and found additional down-regulation of glutathione peroxidase (not present on macroarray) in the vitamin C treated group. We suggest that high dietary doses of vitamin C are ineffective at prolonging lifespan in mice because any positive benefits derived as an antioxidant are offset by compensatory reductions in endogenous protection mechanisms, leading to no net reduction in accumulated oxidative damage.

The consequences of acute cold exposure on protein oxidation and proteasome activity in short-tailed field voles, microtus agrestis

During cold exposure, animals upregulate their metabolism and food intake, potentially exposing them to elevated reactive oxygen species (ROS) production and oxidative damage. We investigated whether acute cold (7 +/- 3 degrees C) exposure (1, 10, or 100 h duration) affected protein oxidation and proteasome activity, when compared to warm controls (22 +/- 3 degrees C), in a small mammal model, the short-tailed field vole Microtus agrestis. Protein carbonyls and the chymotrypsin-like proteasome activity were measured in plasma, heart, liver, kidney, small intestine (duodenum), skeletal muscle (gastrocnemius), and brown adipose tissue (BAT). Trypsin-like and peptidyl-glutamyl-like proteasome activities were determined in BAT, liver, and skeletal muscle. Resting metabolic rate increased significantly with duration of cold exposure. In skeletal muscle (SM) and liver, protein carbonyl levels also increased with duration of cold exposure, but this pattern was not repeated in BAT where protein carbonyls were not significantly elevated. Chymotrpsin-like proteasome activity did not differ significantly in any tissue. However, trypsin-like activity in SM and peptidyl-glutamyl-like activity in both skeletal muscle and liver, were reduced during the early phase of cold exposure (1-10 h), correlated with the increased carbonyl levels in these tissues. In contrast there was no reduction in proteasome activity in BAT during the early phase of cold exposure and peptidyl-glutamyl-like activity was significantly increased, correlated with the lack of accumulation of protein carbonyls in this tissue. The upregulation of proteasome activity in BAT may protect this tissue from accumulated oxidative damage to proteins. This protection may be a very important factor in sustaining uncoupled respiration, which underpins nonshivering thermogenesis at cold temperatures.

Antioxidant enzyme activities, lipid peroxidation, and DNA oxidative damage: the effects of short-term voluntary wheel running

We examined the effect of voluntary exercise on antioxidant enzyme activities (catalase, glutathione peroxidase, superoxide dismutase) in skeletal muscle (hind- and forelimb) and heart of a model small mammal species: short-tailed field vole Microtus agrestis. In addition, DNA oxidation was determined in lymphocytes and hepatocytes using the comet assay and lipid peroxidation estimated in hindlimb muscle by measurement of thiobarbituric-acid-reactive substances. Voles (approximately 6 weeks old), exposed to a 16L:8D photoperiod (lights on 0500 h), ran almost continuously during darkness. We studied the effects of voluntary running over 1 or 7 days duration, with or without an 8-h rest period, on various biomarkers of oxidative stress compared to nonrunning controls. No differences were observed in antioxidant enzyme activities, except in heart total superoxide dismutase activity (P=0.037), with the lowest levels in 1- and 7-day runners at 0500 h. DNA oxidative damage, in lymphocytes or hepatocytes, and lipid peroxidation did not differ between groups. There was no evidence of any significant increase in any oxidative stress parameter in running individuals, despite having significantly elevated energy expenditures compared to sedentary controls.

Photoperiodic effects on body mass, energy balance and hypothalamic gene expression in the bank vole

SUMMARY We examined the effect of increasing photoperiod, at a constant low temperature, on the body mass and energy budget of the bank vole Clethrionomys glareolus. Simultaneously, we determined the hypothalamic gene expression of neuropeptides and receptors known to be involved in short-term energy balance. Despite an increase in body mass (approximately 10% of initial mass), we found no significant changes in any energetic parameters (food intake, energy assimilation rate, resting metabolic rate and total daily energy expenditure by doubly-labelled water). Apparent energy assimilation efficiency was higher in voles exposed to long-days (LD) compared to short-days (SD). Surprisingly, gene expression of corticotrophin releasing factor (CRF; in the paraventricular nucleus), and the melanocortin-3 receptor (in the arcuate nucleus), both known to be involved in appetite suppression and elevation of energy expenditure in short-term energy balance, were higher in voles kept in LD compared to SD. CRF expression was also elevated in females compared to males. These paradoxical data suggest an alternative mechanism for the control of seasonal body mass changes compared to short-term body mass changes, and between male and female voles. Furthermore, they highlight the need for studies to perform simultaneous measurements at both the molecular and whole animal levels.

Deleterious consequences of antioxidant supplementation on lifespan in a wild-derived mammal

While oxidative damage owing to reactive oxygen species (ROS) often increases with advancing age and is associated with many age-related diseases, its causative role in ageing is controversial. In particular, studies that have attempted to modulate ROS-induced damage, either upwards or downwards, using antioxidant or genetic approaches, generally do not show a predictable effect on lifespan. Here, we investigated whether dietary supplementation with either vitamin E (α-tocopherol) or vitamin C (ascorbic acid) affected oxidative damage and lifespan in short-tailed field voles, Microtus agrestis. We predicted that antioxidant supplementation would reduce ROS-induced oxidative damage and increase lifespan relative to unsupplemented controls. Antioxidant supplementation for nine months reduced hepatic lipid peroxidation, but DNA oxidative damage to hepatocytes and lymphocytes was unaffected. Surprisingly, antioxidant supplementation significantly shortened lifespan in voles maintained under both cold (7 ± 2°C) and warm (22 ± 2°C) conditions. These data further question the predictions of free-radical theory of ageing and critically, given our previous research in mice, indicate that similar levels of antioxidants can induce widely different interspecific effects on lifespan.

Lifelongα-tocopherol supplementation increases the median life span of C57BL/6 mice in the cold but has only minor effects on oxidative damage

The effects of dietary antioxidant supplementation on oxidative stress and life span are confused. We maintained C57BL/6 mice at 7 +/- 2 degrees C and supplemented their diet with alpha-tocopherol from 4 months of age. Supplementation significantly increased (p = 0.042) median life span by 15% (785 days, n = 44) relative to unsupplemented controls (682 days, n = 43) and also increased maximum life span (oldest 10%, p = 0.028). No sex or sex by treatment interaction effects were observed on life span, with treatment having no effect on resting or daily metabolic rate. Lymphocyte and hepatocyte oxidative DNA damage and hepatic lipid peroxidation were unaffected by supplementation, but hepatic oxidative DNA damage increased with age. Using a cDNA macroarray, genes associated with xenobiotic metabolism were significantly upregulated in the livers of female mice at 6 months of age (2 months supplementation). At 22 months of age (18 months supplementation) this response had largely abated, but various genes linked to the p21 signaling pathway were upregulated at this time. We suggest that alpha-tocopherol may initially be metabolized as a xenobiotic, potentially explaining why previous studies observe a life span extension generally when lifelong supplementation is initiated early in life. The absence of any significant effect on oxidative damage suggests that the life span extension observed was not mediated via any antioxidant properties of alpha-tocopherol. We propose that the life span extension observed following alpha-tocopherol supplementation may be mediated via upregulation of cytochrome p450 genes after 2 months of supplementation and/or upregulation of p21 signaling genes after 18 months of supplementation. However, these signaling pathways now require further investigation to establish their exact role in life span extension following alpha-tocopherol supplementation.

Voluntary Exercise Has Only Limited Effects on Activity of Antioxidant Enzymes and Does Not Cause Oxidative Damage in a Small Mammal

Regular physical exercise has a number of beneficial effects, including the reduction in the risk of cancer, osteoporosis, obesity and cardiovascular disease (1,2). Voluntary exercise through wheel running has also been shown to increase average life expectancy in rats by nearly 10% (3). However, physical exercise also considerably increases total oxygen consumption relative to resting levels, which may, in turn, increase the generation of reactive oxygen species (ROS). If ROS generation exceeds the antioxidant protection and repair mechanisms, oxidative stress will occur and this process is thought to be intimately involved in the aging process (4) and as a causative factor in lipid, protein and DNA damage (5–8). Exercise-induced oxidative stress is also implicated in muscle contractile dysfunction possibly by impaired Ca metabolism and apoptosis of muscle cells (7,9). Considerable debate exists in the literature [e.g., Powers & Sen (10)] on how exercise affects the balance between ROS production, antioxidant protection and repair. During endurance training, in particular, increases occur in both enzymatic and nonenzymatic antioxidants (11,12), although these responses appear highly tissue specific (13,14). Repeated bouts of exercise may also increase resistance to oxidative stress (15), primarily through the induction of various stress proteins (16). Strenuous exercise has been shown to elevate urinary 8-hydroxy-deoxyguanosine (8oxodGuo) excretion, which may be interpreted as an increase in oxidative DNA damage or in DNA repair (6,17). Endurance exercise in dogs decreased the level of 8-oxodGuo in the DNA of colonocytes and lymphocytes, indicating an increase in DNA repair capacity (17), or in antioxidant activity. However, the response of the various antioxidant and repair mechanisms to exercise appears dependent on many factors, including exercise bout duration, exercise intensity, previous exercise exposure, subject species, subject age and assay technique employed (11–13). The majority of studies examining the relationship between exercise, ROS production and oxidative stress have exposed subjects to bouts of acute and/or exhaustive exercise, or examined the effects of endurance training (10), with fewer studies investigating the effects of voluntary exercise on oxidative stress parameters [e.g., Leeuwenburgh et al. (18)]. In the following study, we used a small (15–30 g) mammalian model, the short-tailed field vole (Microtus agrestis) to examine whether short-term (i.e., 1or 7-d) voluntary wheel running, with or without an 8-h recovery period, had any effect on the activities of the antioxidant enzymes catalase (Cat), glutathione peroxidase (Gpx) and total superoxide dismutase (totalSOD) or DNA oxidation. We measured oxidative DNA damage in lymphocytes and hepatocytes, employing the comet assay and lesion-specific enzymes endonuclease III (endo III) and formamidopyrimidine DNA glycosylase (FPG) (19–21). Antioxidant enzyme activities were measured in skeletal muscle (hindand forelimb) and heart because these tissues experience a large increase in oxygen consumption during exercise (18) and are thought to be relatively susceptible to oxidative stress (22). Using the doubly labeled water technique (23), we previously showed that the daily energy expenditure, and hence oxygen consumption, is over 40% higher in voles with access to running wheels compared to that in nonrunning sibling-matched controls (24). 1 Presented as part of the Waltham International Symposium: Pet Nutrition Coming of Age held at Vancouver, Canada, August 6–7, 2001. This symposium and the publication of symposium proceedings were sponsored by the Waltham Centre for Pet Nutrition. Guest editors for this supplement were James G. Morris, University of California, Davis, Ivan H. Burger, consultant to Mars UK Limited, Carl L. Keen, University of California, Davis, and D’Ann Finley, University of California, Davis. 2 Supported, in part, by a grant to J.R.S. from the Biotechnology and Biological Sciences Research Council (SAGE-1 initiative). 3 To whom correspondence should be addressed. E-mail: c.selman@abdn.ac.uk. 4 Abbreviations used: cat, catalase; endo III, endonuclease III; FPG, formamidopyrimidine DNA glycosylase; Gpx, glutathione peroxidase; ROS, reactive oxygen species; total-SOD, total superoxide dismutase.