(2007) Vaanholt, Lobke Maria
In evolutionary biology, ageing is usually defined as a persistent decline in the age-specific fitness components of an organism due to internal physiological deterioration. This definition integrates effects on reproduction and survival. Gerontologists simply define ageing as an increase in the likelihood that an individual will die in a certain time interval. As we age, intracellular processes degenerate and ultimately fail. This can lead to age-related diseases, such as cardiovascular disease, Parkinson’s disease etc., and ultimately to death. There has been much speculation on the role of energy metabolism in the causation of these processes. This has led to the formation of several intriguing theories which attribute the causation of death ultimately to the very motor of life itself; the rate of living theory (Pearl, 1928; Rubner, 1908) and free radical theory of ageing (Harman, 1956).
This idea was summarized by Murray (1926) in the statement:
‘If aliveness is measured by the velocity of chemical activity (heat production) an organism may in this sense be said to dig its own grave. The more abundant its manifestations of life, the greater will be its rate of senescence’.
The primary aim of this thesis was to investigate the relationship between ageing and metabolic rate. In two large-scale experiments I manipulated the energy expenditure of a group of animals by either increasing their physical activity or exposing them to cold. Survival curves were created for different experimental groups and I looked at changes that occurred in several physiological parameters that might be involved in ageing to explain differences that occurred in life span (Part II: Metabolism & Ageing). In addition, I explored the behavioural and physiological consequences of changes in energy balance in mice that had been selectively bred for high levels of spontaneous physical activity (Part I: Activity & Metabolism).
PART I: Activity & Metabolism
Life history theory
The evolution of life histories has been explained by the presence of limited resources that results in trade-offs between survival (maintenance of the body) and reproduction (Stearns, 2000). In times of plenty, resources can be allocated to growth and reproduction, but when resources are scarce, energy has to be allocated to enable survival of the individual and future success. In many species the reproductive season is tuned to coincide with the peak in food availability. When food is scarce, reproduction ceases and energy is allocated to increase the chance of survival into the next year. There is a large variation in the way animals deal with such trade-offs. When there is a genetic basis for these decisions, natural selection favours life-history traits that result in a higher fitness. The main environmental factors influencing the available resources for endothermic animals are environmental temperature and food availability.
In part I of this thesis we investigated the effects of low ambient temperatures or food availability on metabolism and the amount of voluntary activity mice were willing to engage. We used mice that had been selected by T. Garland Jr. for high wheel-running activity and their random-bred controls. Detailed description of the selection protocol and the main characteristics of these mice is provided in box 1.1. The amount of wheel-running activity was the selection criteria. After 31 generations of selection the mice ran approximately 2,7 times as much as control animals (Rhodes et al., 2005). With the selection for wheel-running activity other traits have been co-selected (i.e. small body size) and much research has been undertaken to uncover these co-selected traits.
In chapter 2 we investigate mice exposed to various ambient temperatures. We measured wheel-running activity and metabolic rate simultaneously to determine whether high-activity mice have evolved to have a lower running economy and whether they would more likely use heat generated by activity to substitute for thermoregulatory heat at low ambient temperatures than control mice do. In chapter 3 we manipulated food availability using a system in which animals had to run in a running wheel for a set number of revolutions to obtain a food pellets. This approach was used to study effects of food availability on physiological and behavioural responses in control and selected mice. Previous studies in rats by T. Adage showed that rats with low spontaneous levels of wheel-running activity have more difficulties to cope with a workload schedule than rats with high spontaneous levels of wheel-running activity. Similar effects were expected between control and high-activity mice.
Exercise & obesity
Obesity is becoming an increasingly prevalent health problem in affluent societies. It is often associated with metabolic derangements such as impaired glucose tolerance, insulin resistance, high blood pressure, dyslipidemia, and abdominal obesity. When these metabolic abnormalities are displayed in concert (often referred to as the “metabolic syndrome”), they entail a high risk of developing into life-threatening conditions such as cardiovascular disease and diabetes mellitus type 2 (for review see (Carroll and Dudfield, 2004; Moller and Kaufman, 2005)). Increased dietary fat intake in combination with a sedentary existence are factors precipitating the development of obesity and the associated metabolic syndrome. Adipose tissue produces several hormones, such as leptin and adiponectin, that are important for energy homeostasis. Levels of these hormones are associated with metabolic risk factors. Adiponectin levels are decreased and leptin levels increased in obese compared with lean subjects (Park et al., 2004).
Mice that have been selected for high wheel-running activity (for detailed description see box 1.1.) have decreased levels of leptin even when correcting for fat mass (Girard et al., 2006). Leptin is produced by adipose tissue and informs the body about its available fat stores and is involved in regulating food intake. Selected mice have a high food intake to cover the increased costs of wheel-running activity (Swallow et al., 2001a), and lowering levels of leptin may be an adaptation to increase food intake and maintain energy balance.
High-activity mice have a lean phenotype (Dumke et al., 2001; Swallow et al., 1999a) and adiponectin levels are thus expected to be increased in high-activity mice. This together with low levels of leptin might make high-activity mice less prone to develop metabolic derangements on a high-fat diet and would make these mice a suitable model to study the metabolic syndrome.
In chapter 4 we measured hormone levels of leptin, adiponectin and corticosterone in aging male mice selected for high wheel-running activity and their random-bred controls. We studied correlations between the hormones and body composition. In chapter 5 we describe a study in which we exposed selected males and females to a high fat diet. Body composition, food efficiency, energy metabolism and glucose tolerance were tested to determine whether high-activity mice responded differently to a high-fat diet than controls.
Part II: Metabolism & Ageing
“Rate of living” and “free radical” theory of ageing
Instinctively we know that things (cars, machines) break down faster if you use them more often and more intensively. The same might be applicable to animal (and human) life. This notion that the rate of energy turnover determines the rate of breakdown is known as the “rate of living” theory (Pearl, 1928). In 1908, Rubner noted that food intake per gram decreased with increasing life span among five domestic animals (guinea pig, cat, dog, cow and horse). He calculated the energy intake per gram per life span (life-time energy potential, LEP) and found that the variation in LEP between species was small (1,5 fold), although the variation in body mass was very large. Including data for men the variation in life-time energy expenditure was slightly larger, but still only 5-fold. He concluded that mass-specific energy metabolism times the maximal lifespan was a constant (Rubner, 1908). Energy metabolism might thus be the factor that determines our life span. In 1928 Pearl postulated the “rate of living theory” that states that there is an inverse relationship between energy expenditure and life span (Pearl, 1928). An extensive body of evidence exists that is consistent with this theory. Comparative studies have shown that energy expenditure tends to show an inverse relationship with body size and longevity when compared across mammalian or bird species (Ku et al., 1993; Speakman, 2005a). Also evidence from intra-specific studies show evidence for the rate of living theory. Increasing ambient temperature (and thereby energy expenditure) decreased life span of nematodes proportionally (Van Voorhies and Ward, 1999). Honey bees that were forced to fly with extra loads had decreased life spans (Wolf and Schmid-Hempel, 1989), and flies prohibited to fly (thereby decreasing metabolic rate) had increased life spans (Yan and Sohal, 2000). Brood size increases in kestrels resulted in increased energy turnover and a subsequent decrease in the survival of parents that had enlarged broods (Daan et al., 1996). In hibernating hamsters survival was higher than in hamsters that did not hibernate (Lyman et al., 1981). A moderate increase of the level of basal metabolism of young adult rats adapted to hypergravity compared to controls in normal gravity was accompanied by a roughly similar increase in the rate of organ aging and reduction of survival (Economos et al., 1982). In contrast there are also numerous studies that showed no relationship or a positive relationship between energy expenditure and life span (in mammals (Holloszy and Smith, 1987; Holloszy and Smith, 1986; Navarro et al., 2004; 2003; Speakman et al., 2004)), and comparative studies show that for a certain body mass birds expend up to 4 times more energy than a mammal, and live longer (Speakman, 2005b). Another line of evidence comes from experiments on calorically restricted animals. Caloric restriction (CR; decreasing energy intake) is widely recognized as the only (non-genetic) manipulation that increases mean and maximum life span in mammals (first shown by (McCay et al., 1935)). In 1977 Sacher proposed that CR extended life span by decreasing metabolic rate. A study by Masoro et al. found that following the initiation of CR there was a brief period of reduced food intake per gram body mass, but this was followed by a lifetime where the intake per gram body mass was higher in CR rats than ad-libitum fed rats (Masoro et al., 1982). In a study where mass-specific 24-h metabolic rates were measured mass-specific (based on lean mass) metabolic rates were reduced upon the initiation of CR, but increased to levels higher than ad-libitum fed animals later on (McCarter and Palmer, 1992). Similar results were shown in rhesus monkeys (Ramsey et al., 1996). These studies disagree with a role for metabolic rate in the life extending effect of CR. Interpretation of the results is confounded because metabolic rate is usually normalized for body mass or lean mass, whereas the relative sizes of organs are not the same for animals that are CR or fed ad libitum (Greenberg, 1999b; Greenberg and Boozer, 2000).
A related theory of ageing was suggested by Harman in 1956 known as the “free radical theory” (Harman, 1956). This theory specifies the reason why there should be a direct link between energy metabolism and the rate of ageing. Free radicals or radical oxygen species (ROS) are produced as by-products of normal oxidative phosphorylation, and can cause damage to macromolecules which may result in malfunction and eventually cell death (for review see (Beckman and Ames, 1998)). The body has evolved defense systems against these radicals in the form of antioxidant enzymes (e.g. superoxide dismutase, catalase and gluthione peroxidase) that scavenge ROS and transform them into less toxic products. A small amount of ROS escape conversion. If damage to macromolecules has occurred the processes of DNA repair and protein synthesis can repair most of this damage. Despite these defense systems a small amount of damage still occurs and this accumulates with age resulting in malfunction of cells and eventually death (see Figure 1.1. for a graphical representation of the process). When energy expenditure (and oxidative phosphorylation) increases, the production of ROS will also increase. This would explain the relationship between ageing and metabolism proposed by the rate of living theory.
The relationship between oxidative phosphorylation and ROS production is not linear. Oxidative phosphorylation takes place on the inner membrane of mitochondria as a result of the transport of electrons over the membrane (electron transport chain; ETC). The ETC consists of 4 complexes. NADH and FADH2 that have been formed in the tricarboxylic acid cycle (TCA) donate their electrons to subsequently complex I or II which are then passed on to ubiquinone (Q). Q moves across the membrane to complex III and the electrons are passed on to cytochrome C that moves on to complex IV where the electrons are accepted by molecular oxygen and combined with protons to form water (for a more detailed description see (Brand, 2000a)). During this process protons are pumped across the membrane into the inner membrane space and a proton motive force builds up. When oxidative phosphorylation is coupled these protons are pumped back to the matrix via an ATP-ase pump resulting in the phosphorylation of ADP to ATP (ATP synthesis).
Free radicals are generated during oxidative phosphorylation when an oxygen molecule promiscuously reacts with one of the transported electrons before it reaches complex IV. This can for instance occur when the supply of ADP is limited thereby blocking up the system. Agents that increase respiration rate and thereby lower proton motive force (i.e., ATP synthesis) thus lower the rate of ROS production. ROS production is thus not linearly related to the rate of electron transport. The flow of electrons in the ETC is usually tightly coupled to the production of ATP, and it does not occur unless the phosphorylation of ADP can proceed. This prevents a waste of energy, because high-energy electrons do not flow unless ATP can be produced. If electron flow is uncoupled from the phosphorylation of ADP there would be no production of ATP, and the energy of the electrons would be wasted as heat. Uncoupling agents abolish the link between oxidation and phosphoryalation, allowing electron transport to proceed without coupled ATP synthesis, thereby increasing the respiration rate and lowering ROS production (Brand, 2000b). Therefore, metabolic rate and free radical production are not necessarily linearly related.
Many studies support the importance of antioxidants, oxidative stress and repair of oxidative damage for the ageing process. For instance, the importance of antioxidants enzymes is clear from studies with over-expression or knocking out of these enzymes. Overexpression of catalase and superoxide dismutase in Drosophila melanogaster increased median and maximum lifespan up to 30% (Orr and Sohal, 1994; Sohal et al., 1995), and mice lacking manganese superoxide dismutase died within 10 days (Li et al., 1995), whereas administration of superoxide dismutase-catalase mimetics increased lifespan up to three times in mice (Melov et al., 2001). In CR animals life span extending effects have also been attributed to differences in oxidative stress. Increased antioxidant enzyme activity, DNA repair and protein synthesis, and decreased numbers of oxidatively damaged molecules have been shown in CR animals (for reviews see (Gredilla and Barja, 2005; Tavernarakis and Driscoll, 2002; Yu, 1996)).
Whereas the “free radical” theory has gained much support in recent years, the rate of living theory has been discarded as invalid by many researchers based on inter-specific comparisons and the lack of effects on (or increases in) energy metabolism in CR animals. This is remarkable since the free radical theory of ageing is itself the main theory postulating the mechanism connecting energy turnover and ageing. As argued by Speakman (2002; 2005c) the reasons to dispute the theory may not always be valid, because the arguments that are used to test the theory are fraught with problems. Firstly, maximum life span is not a good measure of ageing. Maximal life span is determined by a single point in every data base and is highly affected by the sample size used and also by the conditions in which animals are housed (i.e. laboratory or natural conditions). Secondly, basal metabolic rates have been used in most studies to estimate life-time energy potential. Basal metabolic rate is the metabolism of an animal when fasting and resting at thermo-neutral temperatures and contributes only 40% to the total daily energy expenditure. The latter is a better measure of metabolism. Using a single measure of metabolic rate in the life time of an animal might not be sufficient to make an accurate estimate of life-time energy potential. Thirdly, testing for consistency in life-time energy expenditure per gram of tissue by inter-specific comparisons between birds and mammals is not the best way to test the rate of living theory and inter-specific comparisons are complicated by the fact that animals from different species may reflect adaptive or genetic differences in free-radical production or differences in defence and repair mechanisms. Therefore, intra-specific comparisons are more convincing when looking at associations between energy expenditure and ageing. A fourth argument relates to the scaling of energy expenditure to body mass. Greenberg has shown that in cases where no relation was found between life-time energy expenditure per gram body mass and life span, a relationship does exist when one calculates the energy expenditure for certain metabolically active organs and relate this to life span (Greenberg, 1999a). Life-time energy expenditure per gram dry lean body mass instead of total body mass might be a better measure to test the rate of living theory since this contains the tissue that is metabolically most active. A stronger correlation between energy metabolism and dry lean mass is usually found then between body mass and energy expenditure.
In studies on energy expenditure and life span almost never the body composition and energy turnover are followed throughout life. In order to resolve some of the confusion in this area we carried out two large scale experiments. We manipulated energy metabolism by either increasing activity through selection (chapter 6) or by decreasing environmental temperature (chapter 8) and looked at the relationship between energy metabolism and survival in intra-specific comparison. Mice selectively bred for high wheel-running activity were used to investigate the effects of increased voluntarily exercise. In the cold experiment, c57Bl/6J mice were used that were subjected to 10C compared to 22C in control mice. An additional group that was exposed to cold early in life was added. This for the first time tests one basic implicit proposition in the rate of living and free radical theories: that the effects of energy turnover are cumulative. Energy turnover increase in youth should still have and effect in old age. In both experiments we paid specific attention to the effects of age and experimental manipulation (i.e. cold or activity) on two systems that are involved in defending the body against ROS, the antioxidant defence system and protein turnover (chapter 7 and chapter 9).
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