The effect of aging and dietary restriction on DNA repair

DNA repair was studied as a function of age in cells isolated from both the liver and the kidney of male Fischer F344 rats. DNA repair was measured by quantifying unscheduled DNA synthesis induced by UV irradiation. Unscheduled DNA synthesis decreased approximately 50% between the ages of 5 and 30 months in both hepatocytes and kidney cells. The age-related decline in unscheduled DNA synthesis in cells isolated from the liver and kidney was compared in rats fed ad libitum and rats fed a calorie-restricted diet; calorie restriction has been shown to increase the survival of rodents. The level of unscheduled DNA synthesis was significantly higher in hepatocytes and kidney cells isolated from the rats fed the restricted diet. Thus, calorie restriction appears to retard the age-related decline in DNA repair.     

Effect of age and dietary restriction on protein synthesis by isolated kidney cells

The influence of age and food restriction on kidney protein synthesis was studied in Fischer F344 rats. The rate of total protein synthesis by suspensions of kidney cells declined 60% between 4 and 31 months of age. The rate of protein synthesis by kidney cells isolated from 19-month old rats fed a restricted diet (60% of diet consumed by rats fed ad libitum) was 45% higher than the rate of protein synthesis by kidney cells isolated from 19-month old rats fed ad libitum. The excretion of protein in the urine was measured to assess the effect of the age related decline in protein synthesis on kidney function. A dramatic increase in proteinuria was observed with increasing age, and rats fed the restricted diet excreted significantly less protein in the urine than rats fed ad libitum.     

Influence of aging and caloric restriction on activation of Ras/MAPK, calcineurin, and CaMK-IV activities in rat T cells

The signaling cascade mediated by Ras (p21ras) and MAPK (mitogen-activated protein kinase) and calcium/calmodulin regulating enzymes, calcineurin (CaN) and CaMK-IV, are considered to be essential for T-cell growth and function. In the present study, the effect of aging and caloric restriction (CR) on the induction of Ras and MAPK activation by concanavalin A (ConA) was studied. Splenic T cells were isolated from young (4-6 months) and old (22-24 months) rats that had free access to food (control group), and from caloric restricted old (22-24 months) rats that beginning at 6 weeks of age were fed 60%(40% caloric restriction) of the diet consumed by the control rats. We found that the induction of Ras activity in T cells isolated from control old rats was lower (P<0.001) than that in control young rats. However, the levels of Ras activity in T cells isolated from CR old rats were similar to the levels in the age-matched control rats. The induction of MAPK activity in T cells isolated from control old rats and CR old rats was significantly less than in T cells isolated from control young rats, and caloric restriction significantly (P<0.05) reduced the age-related decline in MAPK activation. We also measured the induction of CaN and CaMK-IV activities by ConA in T cells from control young and old and CR old rats. The induction of both CaN and CaMK-IV activity decreased with age. Caloric restriction significantly (P<0.05) reduced the age-related decline in CaN activity, but had no significant effect on CaMK-IV activity. The changes in Ras/MAPK activation and in CaN and CaMK-IV activity with age or with CR were not associated with alterations in their corresponding protein levels. Thus, caloric restriction has a differential effect on the activation of the upstream signaling molecules that are altered with age.     

Effect of dietary restriction on the degradation of proteins in senescent mouse liver parenchymal cells in culture

We previously reported that the half-life of protein degradation in cells from old mice is about 50% longer than that in cells from young or middle-aged ones. In the present study we investigated the degradation rate of microinjected proteins (horseradish peroxidase (HRP) and ovalbumin (OVA] and pulse-labeled proteins in hepatocytes from dietary-restricted old mice. Dietary restriction was initiated when mice were 23 months of age and performed in two steps (first 80% and then 60% of the ad libitum intake), the total period being 70 days. Hepatocytes were isolated from mice fed a restricted diet and fed ad libitum. The half-lives of HRP, OVA, and pulse-labelled proteins in the hepatocytes from mice fed a restricted diet were about 40% shorter than those in the cells from mice fed ad libitum. These values were close to those in the cells of young animals. These results are discussed in relation to our previous findings that a similar regimen reduces the percentage of heat-labile enzymes accumulated in tissues of aged animals.     

Influence of caloric restriction and exercise on tumorigenesis in rats

Underfeeding or caloric restriction have been shown to inhibit the growth of spontaneous, transplanted, or chemically induced tumors in rats and mice. At 40% caloric restriction, growth of 7,12-dimethylbenz(a)anthracene-induced mammary and 1,2-dimethylhydrazine-induced colonic tumors is inhibited significantly even when the restricted diet contains twice as much fat as the control diet. Some inhibitory effects become evident even at 10% caloric restriction. In studies involving high fat diets, we find that rats receiving 20% fat ad libitum exhibit significantly higher 7,12-dimethylbenz(a)anthracene-induced mammary tumor incidence, multiplicity, and weight than rats ingesting the same amount of fat daily, but in a diet containing 25% fewer calories. In a study of intermittent ad libitum and restrictive feedings, chemically induced tumorigenicity varies inversely with feed efficiency. Exercise has also been shown to inhibit tumor growth. Sedentary rats fed ad libitum have a 108% higher incidence of 1,2-dimethylhydrazine-induced colon tumors than rats fed ad libitum but subjected to vigorous treadmill exercise. Caloric flux (either reduced intake or increased outflow) appears to reduce tumorigenicity in rodents.     

Evidence that DNA repair may not be modified by age or chronic caloric restriction

Pretreatment of animals with mixed-function oxidase inducers has been shown to increase the metabolic activation capacity of isolated hepatocytes resulting in an apparent increase in DNA repair. We recently reported decreases in chemically-induced DNA repair, measured as unscheduled DNA synthesis (UDS), in hepatocyte cultures isolated from aging ad libitum (AL) and caloric restricted (CR) diet-fed animals. In the present study, we evaluated the effects of pretreatment with Aroclor 1254 (ARO) on the genotoxicity of 4 carcinogens, from different chemical classes, in primary hepatocytes isolated from male Fischer 344 rats. ARO-induced old AL- and CR-derived cultures, treated with 2-acetylaminofluorene (2-AAF), aflatoxin B₁ (AFB₁), 7,12-dimethylbenz[a]anthracene (DMBA), and dimethylnitrosamine (DMN), exhibited significant induction-related increases in DNA repair in comparison to uninduced old AL and CR animals. These data indicate that the constitutive levels of specific cytochrome P450 decline with age and chronic caloric restriction, while the ability to respond to exogenous inducers is retained, and suggest that DNA repair may not be modified with age or diet restriction.     

Decreased fidelity of DNA polymerases and decreased DNA excision repair in aging mice: effects of caloric restriction.

Hepatic DNA polymerases from calorie restricted and ad libitum 26 month old C57BL/6 mice showed a decline in fidelity of nucleotide incorporation compared with weanling animals. Both alpha and beta polymerases from calorie restricted aged mice exhibited a higher level of fidelity than polymerases from ad libitum aged mice. UV-initiated unscheduled DNA synthesis was significantly higher in hepatocytes from weanling and 18 month old calorie restricted animals compared with cells from 18 month old ad libitum animals, while MMS-initiated unscheduled DNA synthesis did not differ significantly between cells from young and old or ad libitum and calorie restricted animals. These data suggest that calorie restriction could play a significant role in decreasing the age-related decline of cellular mechanisms expected to reduce the rate at which mutations accumulate during aging, and could potentially prolong the onset age of mutation-associated diseases of the elderly.     

Dietary restriction retards age-related decrease in cell population of rat corneal endothelium

The surface areas of corneal endothelial cells from 12- and 18-month-old male Fischer 344 rats fed ad libitum or a calorie-restricted diet were compared. The rats fed the restricted diet in both age groups showed a statistically significant reduction in the mean cell area of the corneal endothelium. The data indicate that dietary restriction retarded the age-related endothelial cell loss and the subsequent enlargement that takes place to compensate for cell loss. This is the first report to suggest that dietary restriction retards age-related cell loss.     

Tissue-specific effect of refeeding after short- and long-term caloric restriction on malic enzyme gene expression in rat tissues

Restricting food intake to a level below that consumed voluntarily (85%, 70% and 50% of the ad libitum energy intake for 3 or 30 days) and re-feeding ad libitum for 48 h results in an increase of malic enzyme (ME) gene expression in rat white adipose tissue. The increase of ME gene expression was much more pronounced in rats maintained on restricted diet for 30 days than for 3 days. The changes in ME gene expression resembled the changes in the content of SREBP-1 in white adipose tissue. A similar increase of serum insulin concentration was observed in all groups at different degrees of caloric restriction and refed ad libitum for 48 h. Caloric restriction and refeeding caused on increase of ME activity also in brown adipose tissue (BAT) and liver. However, in liver a significant increase of ME activity was found only in rats maintained on the restricted diet for 30 days. No significant changes after caloric restriction and refeeding were found in heart, skeletal muscle, kidney cortex, and brain. These data indicate that the increase of ME gene expression after caloric restriction/refeeding occurs only in lipogenic tissues. Thus, one can conclude that caloric restriction/refeeding increases the enzymatic capacity for fatty acid biosynthesis.     

Dietary restriction influence bile formation in aging rats

Food restriction is one of the most effective interventions which increases the survival of rodents and influences a variety of physiologic and pathologic processes. Thus, we examined whether life-long caloric restriction would influence bile formation, one of the important hepatic functions. Female Sprague Dawley rats were subjected soon after weaning to a restricted diet (60% of the diet consumed by the rats fed ad libitum) and bile formation determined at 3.5, 12 and 24 months of age. Rats had their bile ducts cannulated under nembutal anesthesia and bile collected at 10 min. intervals. Bile flow rate decreased 35% between 3.5 and 24 months of age. This decrease was associated with a reduction of the bile acid dependent fraction of bile flow (BADF) up to 12 months of age, thereafter the bile acid independent fraction (BAIF) also decreased. Phospholipid and cholesterol secretion rates increased with age, but did not correlate with bile acid secretion. In rats fed the restricted diet, bile flow was about 20% higher at 3-5 months of age when compared with the ad libitum fed group. This bile flow rate remained constant until 24 months of age. The increased bile flow was attributed to higher BADF and BAIF. The phospholipid and cholesterol secretion followed that of bile acids. It thus appears that dietary restriction exerts a beneficial effect on the age related decline in bile formation.     

Ultrastructural morphometry of matrical changes induced by exercise and food restriction in the rat calcaneal tendon.

The ultrastructural morphometry of collagen fibril populations in 24 calcaneal tendons obtained from 12 Fischer 344 rats were studied to elucidate matrical changes induced by food restriction and/or endurance exercise. Rats were randomly assigned to four equal groups: ad libitum control (AC), ad libitum exercise (AE), restricted diet control (RC) and restricted diet exercise (RE) groups. Beginning from 6 weeks of age, animals in the two food restriction groups were fed 60% of the mean food consumption of ad libitum fed rats. Then, starting from 6-7 months of age, the rats in the two exercise groups performed 40-50 min of treadmill running at 1.2-1.6 miles h-1 every day for a total of 10 weeks. Endurance training did not significantly alter body weight, but food restriction with or without exercise resulted in a significant loss of body weight. In ad libitum fed controls, food restriction alone did not significantly alter the mean collagen fibril CSA, but predisposed a preponderance of small-sized collagen fibrils. Endurance training per se induced a significant (32%) increase in mean fibril CSA (P less than 0.05), but this adaptive response to exercise was prevented by food restriction, as indicated by a 33% decline in fibril CSA (P less than 0.05). These findings demonstrate that dietary restriction modifies the adaptation of tendon collagen morphometry in response to endurance training, and that weight loss is better achieved with food restriction than endurance exercise.     

Attenuation of plasma dyslipidemia and oxidative damage by dietary caloric restriction in streptozotocin-induced diabetic rats

Oxidative stress has been proposed as the pathogenic mechanism linking insulin resistance with endothelial dysfunction during diabetes. The present study investigated the attenuation of plasma dyslipidemia and oxidative damage by caloric restriction in experimental diabetes. Forty male Wistar rats were divided into ad libitum and calorie-restricted groups. The calorie-restricted group was subjected to 30% caloric restriction for 63 days before induction of diabetes to 50% of both groups. Caloric restriction significantly (p<0.01) reduced the body weights, reactive oxygen species (ROS), catalase, total cholesterol levels and non-significantly reduced SOD activities in non-diabetic and diabetic rats. Caloric restriction was also found to improve blood glucose levels, glycated hemoglobin, malondialdehyde, triglyceride, oxidized glutathione and reduced glutathione levels and significantly (p<0.05) increased GPx and GR activities in the experimental animals. The non-diabetic rats fed ad libitum had the most significant increases in body weight which could be due to dyslipidemia. These results indicate that dietary caloric restriction attenuates the oxidative damage and dyslipidemia exacerbated during diabetes as evidenced by the significant reduction in their body weights, ROS, total cholesterol levels and the increases in GPx activity and redox status.     

Heat Shock and Caloric Restriction Have a Synergistic Effect on the Heat Shock Response in a sir2.1-dependent Manner in Caenorhabditis elegans

The heat shock response (HSR) is responsible for maintaining cellular and organismal health through the regulation of proteostasis. Recent data demonstrating that the mammalian HSR is regulated by SIRT1 suggest that this response may be under metabolic control. To test this hypothesis, we have determined the effect of caloric restriction in Caenorhabditis elegans on activation of the HSR and have found a synergistic effect on the induction of hsp70 gene expression. The homolog of mammalian SIRT1 in C. elegans is Sir2.1. Using a mutated C. elegans strain with a sir2.1 deletion, we show that heat shock and caloric restriction cooperate to promote increased survivability and fitness in a sir2.1-dependent manner. Finally, we show that caloric restriction increases the ability of heat shock to preserve movement in a polyglutamine toxicity neurodegenerative disease model and that this effect is dependent on sir2.1.