The molecular basis by which dietary restricted feeding reduces mitochondrial reactive oxygen species generation.

Restricted feeding regimes in rodents that extend longevity lower the rate of mitochondrial reactive oxygen species generation. This effect is not dependent upon the depression of the state III and IV mitochondrial respiration rates. Mitochondria from liver, skeletal muscle and brown adipose tissue adapt to DR feeding with a lowered membrane potential that results from an enhanced proton leak across the inner membrane. Mitochondrial ROS generation is very sensitive to the magnitude of the membrane potential and the enhanced proton leak and ROS generation rates are reversible by exogenous insulin in liver and heart mitochondria. The adenine nucleotide translocase (ANT) was shown to be the dominant proton leak channel induced under DR feeding regimes in these tissues, while in brown fat mitochondria, UCP1 was activated, but this was not sensitive to exogenous insulin treatment. The effect of DR to modify the proton leak, membrane potential and ROS generation rate can be simulated by a range of non-esterified free fatty acids (NEFA) acting on the ANT to enhance its protonophoric activity. Mobilisation of NEFA under DR feeding, when insulin plasma concentrations are extremely low, explains the indirect action of insulin to counteract the effects of DR feeding on mitochondrial ROS generation.

Dietary restriction in rodents--delayed or retarded ageing?

Dietary restriction (DR) feeding increases survival significantly in strains of rats and mice. There remains however, the question as to whether these two species are always responding in an identical manner to the feeding regime. Enhanced survival can be achieved either through a set-point effect, where there is a change in the elevation of the Ln age-specific mortality rate or, by a decrease in the slope of the Ln age-specific mortality rate that results in a significant increase in the time to double the rate of mortality. It is only the second response that is evidence of a slower rate of ageing. These two possible responses to DR feeding may confound attempts to identify the biochemical mechanisms underlying the effect of DR on survival. A general lack of consistency is evident in the data and this is apparent when evaluating the free radical hypothesis of ageing in this model. Further, this hypothesis as currently viewed may be too simplistic to explain the variety and complexity of the ageing phenotype. What may be more important is not oxidative macromolecular damage but the slow transition to this cellular endpoint through the slow development of oxidative stress and the role it plays in modifying cell gene expression profiles.

The effect of aging and caloric restriction on mitochondrial protein density and oxygen consumption.

It has been proposed that part of the anti-aging mechanism of caloric restriction (CR) involves changes in mitochondrial function. To investigate this hypothesis, mitochondria from various tissues of male Brown Norway rats (fully fed and CR) were isolated and respiration rates determined. In mitochondria from liver, heart, brain and kidney, there were no significant effects of CR on state 4 mitochondrial respiration rate. Further experiments using liver mitochondria under a variety of incubation conditions confirmed that CR does not alter mitochondrial respiration rate in this tissue. However, the respiration rate of mitochondria from brown adipose tissue (BAT) of CR animals was approximately three-fold higher compared to mitochondria from fully fed controls. Mitochondrial protein density was significantly higher in liver tissue of CR animals; it was significantly lower in heart and unchanged in BAT. It is concluded that whilst CR results in tissue-specific changes in mitochondrial respiration rate, these effects do not explain the CR-induced changes in free radical production reported previously for these organelles.

Oxidative stress and mitochondrial function with aging--the effects of calorie restriction.

Accumulated oxidative stress resulting from a gradual shift in the redox status of tissues is now considered to be a key mechanism underlying the aging process. Calorie-restricted (CR) feeding, an experimental protocol to extend survival and delay aging in rodents, is recognized to slow the rate of accrual of age-related oxidative stress. This conclusion is based on the increase in tissues with age of the oxidation products of proteins, lipids and DNA. The functional consequence, however, of the accumulation of these non-specific oxidative markers is more difficult to determine. A shift in the redox status of tissues with age and calorie restriction feeding may have a greater impact on cell function through activation of redox sensitive transcription factors than through the accumulation of these non-specific oxidative markers. Activation of such transcription factors will stimulate signalling pathways that will lead to a change in the gene expression profile and cell functioning. Little research has been conducted in this area. It has been proposed that CR feeding slows the rate of accrual of oxidative damage because mitochondria in these animals have a lower rate of superoxide generation when compared with mitochondria from control animals. This proposal is based on in vitro observations using isolated mitochondria and clearly requires further confirmation in isolated cells or using an in vivo approach. The application of metabolic control analysis to identify in isolated mitochondria the mechanism underlying this response has suggested one possible explanation for the lower superoxide production rates observed.

Effect of caloric restriction on mitochondrial reactive oxygen species production and bioenergetics: reversal by insulin.

To gain insight into the antiaging mechanisms of caloric restriction (CR), mitochondria from liver tissue of male Brown Norway rats were used to study the effects of CR and insulin on mitochondrial reactive oxygen species production and bioenergetics. As assessed by hydrogen peroxide measurement, CR resulted in a decrease in the production rate of reactive oxygen species. This decrease was attributed to a decrease in protonmotive force in mitochondria from the CR animals. The decrease in protonmotive force resulted from an increase in proton leak activity and a concomitant decrease in substrate oxidation activity. Each of these effects of CR was reversed by subjecting CR animals to 2 wk of insulin treatment. To achieve continuous and stable insulin delivery, animals were placed under temporary halothane anesthesia and miniosmotic pumps were implanted subcutaneously. To gain further insight into how CR and insulin exerted its effects on mitochondrial bioenergetics, the effects of CR and insulin were quantified using modular metabolic control analysis. This analysis revealed that the effects of CR were transmitted through different reaction branches of the bioenergetic system, and insulin reversed the effects of CR by acting through the same branches. These results provide a plausible mechanism by which mitochondrial reactive oxygen species production is lowered by CR and a complete description of the effects of CR on mitochondrial bioenergetics. They also indicate that these changes may be due to lowered insulin concentrations and altered insulin signaling in the CR animal.

Molecular mechanisms linking calorie restriction and longevity.

Calorie-restricted feeding retards the rate of ageing in mammalian and invertebrate species. The molecular mechanisms underlying this effect include a lower rate of accrual of tissue oxidative damage that is associated with a significantly lower rate of mitochondrial free radical generation in rodent species. To identify the important sites of control and regulation for mitochondrial free radical generation during ageing and calorie-restricted feeding, metabolic control analysis is being applied to the study of mitochondrial bioenergetics. With ageing an increase in the mitochondrial proton leak is observed in mouse hepatocytes and in rat skeletal muscle. Limited data suggest that calorie-restricted feeding lowers the inner mitochondrial membrane potential and this may explain the reduced rate of free radical generation. A lowered unsaturation/saturation index is observed for mitochondrial membrane lipids in calorie-restricted rodents resulting in an altered membrane structure and function. Plasma concentrations of insulin and triiodothyronine are significantly lower under calorie-restricted feeding conditions and these hormones exert transcriptional control over desaturase enzymes that are important in the control of membrane lipid unsaturation. A loss of double bonds should make the mitochondrial membranes more resistant to peroxidation damage and would also reduce the proton conductance of the membrane, raising the membrane potential at a given respiration rate. This effect however, appears to be offset by other membrane changes that may include increased activity of uncoupling proteins. These unidentified adaptations increase the proton leak in calorie-restricted animals resulting in a lowering of the membrane potential and ROS generation.

Use of primary cultures of rat hepatocytes for the study of ageing and caloric restriction.

Primary cultures of hepatocytes are widely used to investigate liver function, but this technology has not been exploited fully in the study of ageing and caloric restriction (CR). Hepatocytes were isolated from adult and aged, fully fed, and calorie restricted male Sprague-Dawley rats and their viability and biochemical status assessed over 48h in primary culture. The in vivo differences in cellular protein and DNA content due to age and CR were maintained over the 48h experimental period. The results of this study confirm earlier reports that protein synthesis and degradation rates decline with age in liver tissue, and this decline is retarded by CR. Rates of protein synthesis and degradation in the first year of life were depressed in response to CR feeding and were only significantly higher than recorded for control animals during the second year of life. Cells from rats of both ages and diets maintained linear rates of extracellular protein synthesis, intracellular protein synthesis, protein degradation and albumin secretion between 24 and 48h in culture. These findings indicate that hepatocytes from CR rats did not respond adversely to the relatively rich culture medium and cells from CR animals did not immediately revert to the fully fed phenotype.

Calorie restriction and age-related oxidative stress.

Calorie restriction (CR) in mammals has been recognized as the best characterized and most reproducible strategy for extending maximum survival, retarding physiological aging, and delaying the onset of age-related pathologic conditions in mammals. The overwhelming majority of studies using CR have used short-lived rodent species, although current work using rhesus and squirrel monkeys will determine whether this paradigm is also relevant to manipulating the rate of primate aging. The mechanism by which restricted calorie intake modifies the rate of aging and pathology has been the subject of much controversy, although an attenuation in the lifetime accumulation of oxidative damage appears to be a central feature. Although the majority of studies have focused on the ability of cells from calorie-restricted animals to scavenge free radicals to explain the slower accrual of oxidative damage with age, it is not established that CR has a consistent effect to upregulate the activity of these enzymes in all tissues. A major effect of calorie-restricted feeding now appears to be on the rate of production or leak of free radicals from the mitochondria. The details of the adaptation and the signaling pathway that induces this effect are currently unknown.

Differential display analysis can reveal patterns of gene expression in immortalised hepatoma cells which are similar to those observed in young adult but not old adult liver cells.

We used the differential display technique to examine whether there were any patterns of gene expression which were characteristic of both young adult rat liver and of immortalised rat hepatoma cell lines, but not of old adult rat liver. No genes were detected which appeared to be clearly expressed in young liver and immortalised cell lines, but not in old liver. However, 14 genes were detected in old liver which were down-regulated in young liver and the hepatoma cell lines. This observation lends support to the idea that immortalisation of malignant cells may involve, at least in some aspects, a reversal of the ageing process in these cells and that the genes involved have a recessive action.

Fluorescence in situ hybridization analysis of the fos/jun ratio in the ageing brain.

We have examined the expression of the fos and jun genes in the cerebellum of the rat brain during ageing, by use of a semi-quantitative fluorescence in situ hybridization (FISH) method. In these experiments we have utilised the digital imaging capabilities of a cooled CCD camera system to store the fluorescence intensities of individual cells and to compare the data from each target (fos or jun) gene with that of a control (beta-actin) gene. In this way we have been able to obtain a relative quantitation of fos and jun mRNA levels. Purkinje cells were analysed in brain from Sprague-Dawley rats of 6, 13 and 23 months of age. Data obtained in this way demonstrated that the level of fos expression decreased significantly during ageing but, in contrast, that of jun increased between 6 and 13 months and thereafter remained constant. We subsequently carried out a further comparison of fos/jun ratios in purkinje cells in Wistar rats and also observed a highly significant fall in the ratio between 6 and 23 months. This change in the fos/jun ratio has important implications for the composition of the AP-1 transcription factor and for the expression of genes that it regulates.

Differential display analysis of gene expression indicates that age-related changes are restricted to a small cohort of genes.

It is clear that there is a genetic component associated with the ageing process. Although evolutionary theory has suggested that the activity of certain genes may facilitate ageing by favouring resource utilisation by the germ cells at the expense of somatic cells, there is reason to believe that the senescent phenotype, which is the endpoint of the ageing process, may be due to alterations in the levels of expression of other genes. To investigate this situation we have used the differential display technique to survey gene expression during ageing of the rat brain, heart and liver. By optimising this technique it is possible to identify up to 10000-14000 PCR products, which represent genes expressed in the tissue under study. Interestingly, only a relatively small cohort (approximately 2%) of these genes appear to show significant changes in their levels of expression during ageing. Characterisation of the latter has so far revealed certain genes, such as glial fibrillary acidic protein, which are associated with the senescent phenotype. It has also revealed that the level of fos, a component of the AP-1 transcription factor, decreases with age, which has implications for AP-1 regulated genes. The differential display technique has also revealed an increase in mitochondrial RNA during ageing of the heart, which may be due to a gene dosage effect caused by the presence of increased numbers of mitochondrial genomes in myocytes in old age. The differential display technique therefore appears to offer a powerful tool for identifying genes which contribute to the emergence of a senescent phenotype.

Age-related changes in gene expression in the rat brain revealed by differential display.

We have used the polymerase chain reaction (PCR)-based technique of differential display to analyse changes in gene expression during ageing of the rat brain. In this approach we have compared three young adult (6 months) with three old adult (20 months) animals. RNA preparations from the homogenised brains were subjected to reverse transcriptase (RT)-PCR using 36 different combinations of primer pairs. Any PCR product which was consistently found to be more prominent in the three young brains compared to the three old brains, and vice versa, was scored as potentially representing a gene which was differentially expressed during the ageing of this tissue. Out of a possible 2000+ PCR products we identified 44 that might represent genes that exhibit differential expression during ageing of the rat brain. An initial screen of these fragments, by Southern-blotting the PCR products and hybridising them with cDNA probes derived from either young or old brain RNA preparations, indicated that 40% of them represented genes that were differentially expressed. This approach is likely to prove invaluable for identifying cohorts of genes that show differential expression during the ageing process.

Retardation by restricted feeding of age-related changes in steroidogenic activity of rat pre- and post-ovulatory follicles.

Reproductive ageing in female rodents is accompanied by changes in circulating peptide and steroid hormones leading to irregular, lengthened oestrous cycles prior to loss of fertility. In this study, the effect of ageing is reported on steroid hormone synthesis within individual ovarian follicles and its retardation by restricted feeding for two groups of ad libitum fed animals (114 and 350 days) and two groups of diet-restricted animals (350 and 600 days). Follicles from ad libitum fed animals of 350 days showed a transition in follicular steroid hormone synthesis to release elevated amounts of oestradiol-17beta on all days of the cycle. This age-related change in follicle steroid release was significantly delayed by maintaining animals on a restricted feeding regime, and was not complete even by 600 days of age. This effect of diet as a means to manipulate ageing of the follicular steroidogenic pathways provides a useful system for investigating the control of reproductive ageing in rodents.

Lymphoproliferative responses in diet-restricted and aging Sprague-Dawley rats.

Immunological effects of aging and dietary restriction (i.e., chronic underfeeding without malnutrition) were investigated in male CFY Sprague-Dawley rats. From weaning, diet-restricted animals were given the amount of food that maintained their body weights at approximately 50% of age-matched ad libitum controls. Cells from the spleen, Peyer's patches, brachial/axillary lymph nodes, and mesenteric nodes of 12- and 20-month-old rats were tested in vitro against the T-cell mitogens phytohaemagglutinin and concanavalin A, and the B-cell mitogen Salmonella minnesota lipopolysaccharide (with dextran sulphate). No clear boosting effect of dietary restriction nor age-related decline in T- or B-cell response was observed when a standard foetal bovine serum supplement was used. In general however, serum from diet-restricted rats supported better proliferative responses than serum from age-matched controls, suggesting that dietary restriction may promote lymphocyte proliferation by an indirect mechanism. A feature of this study was the variation in phenotype and immune responsiveness among closely related animals. Thus in an outbred population, any beneficial effects of dietary restriction upon immune responses could be outweighed by variation among individuals.

The influence of age and chronic restricted feeding on protein synthesis in the small intestine of the rat.

Rates of protein synthesis (measured in vivo) and growth of the small intestine were studied as a function of age in ad libitum fed (control) and chronic dietary-restricted rats. At weaning, the fractional rates of synthesis in the mucosal and muscularis externa and serosal layers of the small intestine of control animals were similarly high (90-100% per day). Although these rates subsequently declined with age in the muscularis externa and serosa, they remained constant in the mucosa. Restricted feeding (50% reduced intake), when imposed from weaning onwards, significantly extends the maximum life span of rodents. However, the change in nutritional status slows the accumulation of protein, RNA, and DNA in both layers of the small intestine. Although underfeeding did not prevent the age-related fall in muscularis externa and serosal protein synthesis, significantly higher rates (both fractional and per ribosome) were found when compared age for age with controls. Mucosal fractional synthetic rates were similarly increased by the reduced food intake. These changes in protein turnover in the small intestine are consistent with the higher rates of whole body turnover previously observed in chronically underfed rats.

Failure of dietary restriction to enhance cytolytic and chemiluminescent activity of peritoneal exudate cells from ageing rats.

Investigations into the cytolytic activity of peritoneal macrophages from male Sprague-Dawley rats (activated in vivo with Corynebacterium parvum) utilised SV40-3T3 and L-929 target cells in a 72 h [3H]thymidine release assay. The dietary restricted rats under test were given a measured amount of food from weaning sufficient to maintain their body weights at approximately 50% of age-matched controls fed ad libitum. Cytolytic activity was similar in the two dietary groups at both 13 and 21 months of age, nor were chemiluminescent responses of peritoneal cells to latex or zymosan particles affected by the dietary restriction. No differences were seen in the effectiveness of zymosan particles opsonised with serum pooled from dietary restricted versus control rats in stimulating chemiluminescent responses by standard preparations of peritoneal cells, and concentrations of serum C3 and IgG appeared to be unchanged by the dietary regime.

Effect of age and restricted feeding on polypeptide chain assembly kinetics in liver protein synthesis in vivo.

Polypeptide assembly rates during in vivo hepatic protein synthesis were studied as a function of age and restricted feeding in male rats. With ageing the time to assemble the average peptide in the liver of fully-fed rats significantly increased. In young rats maintained on a restricted feeding regime known to retard ageing, the time to assemble the average polypeptide was increased 2.5 times. With ageing the rate of peptide elongation increased so that at 2 years of age the underfed animals assembled peptides at a significantly faster rate than their age-matched controls. The rate of elongation of peptides during hepatic protein synthesis was shown to be directly dependent upon circulating T3 levels rather than the dietary status of the animal. On refeeding young diet restricted rats, polypeptide assembly kinetics did not immediately return to control values although the rate of protein synthesis was significantly increased. Total liver RNA content increased significantly in refed animals allowing for a greater rate of chain initiation to offset the slow rate of chain elongation. A period of 28 days of ad libitum feeding was required before assembly kinetics returned to control values and is probably indicative of a persistent impaired monodeiodination of T4 to T3.

The effects of age and chronic restricted feeding on protein synthesis and growth of the large intestine of the rat.

1. In vivo rates of protein synthesis and growth of the large intestine were studied in ad libitum fed control and chronic diet restricted rats between 3 and 149 weeks post partum. 2. Restricted feeding (50% reduced intake) when imposed from weaning significantly extends the life span of rodents through an unknown biochemical mechanism. 3. The change in nutritional status slows the accumulation of RNA, DNA and protein in the large intestine but does not modify the fractional rate of protein synthesis. 4. It was therefore deduced, that intracellular protein degradation, or the rate of mucosal cell extrusion into the gut lumen, is accelerated by chronic restricted feeding.

Failure of dietary restriction to influence natural killer activity in old rats.

Natural killer activity of Sprague-Dawley rats maintained on an ad libitum versus restricted diet was compared using an 18 hour 51Cr-release assay, against the K562 erythroleukemic line, Yac-1 lymphoma cells and SV40-3T3 cells. The results indicated that no enhancement of natural killer function was induced by dietary restriction of 10.5-month-old rats from weaning. Prolongation of the restricted diet into late life (24 months) similarly did not enhance basal natural killer activity over levels observed in the ad libitum controls. This suggests that the improved resistance to some tumours seen after prolonged dietary restriction depends on another defensive mechanism, reduced metabolic activity and/or a reduction of available nutrients at cancerous foci.

Effects of ageing and chronic dietary restriction on the morphology of fast and slow muscles of the rat.

The soleus and extensor digitorum longus muscles of the hindlimb and the flexor digitorum profundus muscle of the forelimb were studied in ad libitum-fed control and age-matched diet-restricted male rats at various ages from weaning to senescence. Growth of individual muscles was accomplished by fibre hypertrophy and not hyperplasia. Between weaning and one year, fibre numbers remained constant in the soleus but fell by 50% in the extensor digitorum longus. Both muscles displayed increasingly oxidative fibre type profiles with advancing age, irrespective of dietary status. This was particularly noticeable in the soleus, which transformed its fibre population from one containing 35% fast fibres at weaning to one with no fast fibres at 91 weeks. In senility, however, the fibre type population again displayed 25% fast fibres. The capillary: fibre ratio and the capillary density were correlated with muscle fibre size in both hindlimb muscles. Although capillarity increased with age, expected differences between fast and slow muscles were probably minimised by the high proportion of FOG fibres in the extensor digitorum longus. Both hindlimb muscles displayed significant increases in the ratio of connective: muscle tissue with increasing age. The soleus invariably contained more connective tissue than the extensor digitorum longus. Dietary restriction reduced the rate of increase, so that the connective tissue content was approximately one half that found in control muscles at one year. Various pathological features associated with old age were delayed considerably in the muscles of the diet-restricted rats. It is concluded that chronic dietary restriction imposed directly after weaning has a dramatic effect on the normal growth and ageing of skeletal muscle.