Life‐long caloric restriction reduces oxidative stress and preserves nitric oxide bioavailability and function in arteries of old mice

Aging impairs arterial function through oxidative stress and diminished nitric oxide (NO) bioavailability. Life‐long caloric restriction (CR) reduces oxidative stress, but its impact on arterial aging is incompletely understood. We tested the hypothesis that life‐long CR attenuates key features of arterial aging. Blood pressure, pulse wave velocity (PWV, arterial stiffness), carotid artery wall thickness and endothelium‐dependent dilation (EDD; endothelial function) were assessed in young (Y: 5–7 month), old ad libitum (Old AL: 30–31 month) and life‐long 40% CR old (30–31 month) B6D2F1 mice. Blood pressure was elevated with aging (P < 0.05) and was blunted by CR (P < 0.05 vs. Old AL). PWV was 27% greater in old vs. young AL‐fed mice (P < 0.05), and CR prevented this increase (P < 0.05 vs. Old AL). Carotid wall thickness was greater with age (P < 0.05), and CR reduced this by 30%. CR effects were associated with amelioration of age‐related changes in aortic collagen and elastin. Nitrotyrosine, a marker of cellular oxidative stress, and superoxide production were greater in old AL vs. young (P < 0.05) and CR attenuated these increase. Carotid artery EDD was impaired with age (P < 0.05); CR prevented this by enhancing NO and reducing superoxide‐dependent suppression of EDD (Both P < 0.05 vs. Old AL). This was associated with a blunted age‐related increase in NADPH oxidase activity and p67 expression, with increases in superoxide dismutase (SOD), total SOD, and catalase activities (All P < 0.05 Old CR vs. Old AL). Lastly, CR normalized age‐related changes in the critical nutrient‐sensing pathways SIRT‐1 and mTOR (P < 0.05 vs. Old AL). Our findings demonstrate that CR is an effective strategy for attenuation of arterial aging.     

Reduced food consumption increases water intake and modulates renal aquaporin-1 and -2 expression in autoimmune prone mice

Aquaporin-1(AQP1) and AQP2 are members of the aquaporin family of cell membrane water channel transport proteins and have been implicated in the regulation of renal water excretion. We have previously shown that calorie restriction (CR) relative to ad libitum (AL) feeding extends lifespan and delays the onset of autoimmune kidney disease in lupus-prone (NZBxNZW)F1 (B/W) mice. To determine if AQP1 and/or AQP2 expression is influenced by CR, mice were fed an AL or CR (40% less food) diet until 4 (young) or 9 (old) months of age when mice were sacrificed. Kidneys were removed and the expression of AQP1 and AQP2 was determined at the protein and mRNA levels using western blotting and RT-PCR respectively. While age did not significantly increase AQP1 expression in the AL groups, CR did increase both the protein (1.4-fold) and mRNA (2.4-fold) levels. In old mice, AQP1 expression was higher (1.8-fold) in CR compared to the AL group while CR had no effect in young mice. In contrast, AQP2 showed an age related decrease (55%) in the AL groups and an increase in the protein (8.4-fold) and mRNA (1.7-fold) levels in the CR groups. Relative to AL, CR decreased AQP2 expression at the protein (90%) and mRNA (50%) levels in the young mice while an increase at the protein (2.9-fold) and mRNA (1.9-fold) levels was evident in the old mice. Interestingly, a significant increase in water intake per gram body weight was found in both young and old CR fed mice when compared to their AL counterparts which may contribute to the prevention of autoimmune disease with age and differences in longevity. These data show, for the first time, significant age and diet influences in renal AQP1 and AQP2 expression at both protein and mRNA levels in lupus-prone mice.     

Response of Lens Epithelial Cells to Hydrogen Peroxide Stress and the Protective Effect of Caloric Restriction

Hydrogen peroxide (H₂O₂) has been reported to be present at significant levels in the lens and aqueous humor in some cataract patients and suggested as a possible source of chronically inflicted damage to lens epithelial (LE) cells. We measured H₂O₂effects on bovine and mouse LE cells and determined whether LE cells from old calorically restricted mice were more resistant to H₂O₂-induced cellular damage than those of same age ad libitum fed (AL) mice. Bovine lens epithelial cells were exposed to H₂O₂at 40 or 400 μM for 2 h and then allowed to recover from the stress. The cells were assayed for DNA damage, DNA synthesis, cell viability, cell morphology, response to growth stimuli, and proliferation potential. Hydrogen peroxide-treated cells showed an increased DNA unwinding 50% greater than that for untreated controls. These DNA strand breaks appeared to be almost completely rejoined by 30 min following removal of the cells from a 2-h exposure. The 40 μM exposure did not produce a significantly lower DNA synthesis rate than the control, it responded to growth factor stimuli, and it replicated as did the control cells after removal of H₂O₂. The 400 μM H₂O₂severely affected DNA synthesis and replication, as shown by increased cell size and by markedly reduced clonal cell growth. The cells did not respond to growth stimulation by serum or growth factors and lost irreversibly the capacity to proliferate. The responses of LE cells from old adlib diet (AL) and calorically restricted (CR) mice to H₂O₂were significantly different. Exposure of LE cells to 20, 40, or 100 μM H₂O₂for 1 h induces a significant loss of cellular proliferation in cells from old AL mice. LE cells from long-term CR mice of the same strain and age were more resistant to oxidative damage at all three concentrations of H₂O₂than those of both old and young AL mice and showed a significantly higher proliferation potential following treatment. It is concluded that CR results in superior resistance to reactive oxygen radicals in the lens epithelium.     

Association between life-span extension by caloric restriction and thiol redox state in two different strains of mice

The hypothesis that the life-extending effect of caloric restriction (CR) is associated with an attenuation of the age-related pro-oxidant shift in the thiol redox state was tested employing a novel experimental design. Amounts of GSH, GSSG, and protein mixed disulfides (Pr-SSG) in the skeletal muscle and liver were compared between two strains of mice that have similar life spans when fed ad libitum (AL), but different life spans under the standard CR regimen. The life span of one strain, C57BL/6, is extended under CR, whereas it remains unaffected in the other strain, DBA/2. Mice were fed AL or 40% less food starting at 4 months and compared at 6 and 24 months of age. The amounts of GSSG and Pr-SSG increased and the GSH:GSSG ratios decreased with age in both strains of AL-fed mice. CR prevented these age-related changes in the C57BL/6, whose life span is extended by CR, but not in the DBA/2 mice, in which it remains unaffected. CR enhanced the activity of glutamate-cysteine ligase in the C57BL/6, but not in the DBA/2 mice. The results suggest that longevity extension by CR may be associated with the attenuation of age-related pro-oxidizing shifts in the thiol redox state.     

Cellular proliferation potential during aging and caloric restriction in rhesus monkeys (Macaca mulatta)

Caloric restriction (CR) is the most successful method of extending both median and maximal lifespans in rodents and other short-lived species. It is not yet clear whether this method of life extension will be successful in longer-lived species, possibly including humans; however, trials in rhesus monkeys are underway. We have examined the cellular proliferative potential of cells from CR and AL (ad libitum fed) monkey skin cells using two different bioassays: colony size analysis (CSA) of dermal fibroblasts isolated and cloned directly from the skin and beta-galactosidase staining at pH 6.0 (BG-6.0) of epidermal cells in frozen sections of skin. Decreases in both proliferative markers occurred with age, but no differences were observed between CR and AL animals. Skin biopsies were obtained from AL and CR rhesus monkeys from two different aging colonies, one at the National Institute on Aging (NIA) and one at the University of Maryland-Baltimore (UMB). These biopsies were used as a source of tissue sections and cells for two biomarkers of aging assays. The CR monkeys had been maintained for 9–12 years on approximately 70% of the caloric intake of control AL animals. In the CSA studies, the fraction of small clones increased significantly and the fraction of large clones decreased significantly with increasing age in AL monkeys. The frequency of epidermal BG-6.0 staining cells increased with age in older (>22 years) AL monkeys, but most predominately in those of the UMB colony, which were somewhat heavier than the NIH AL controls. Old monkeys on CR tended to have fewer BG-6.0-positive cells relative to old AL-derived epidermis, but this effect was not significant. These results indicate that cellular proliferative potential declined with age in Macaca mulatta, but was not significantly altered by CR under these conditions. Although these experiments are consistent with an absence of effect of CR on monkey skin cell proliferative potential, we have found in previous experiments with mice that a longer duration of CR (as a fraction of total lifespan) was needed to demonstrate CR-related improvement in clone size in mice. Further studies on the now mid-aged monkeys will be needed as their age exceeds 20 years to conclusively rule out an effect of CR on proliferative potential of skin cells from these primates. J. Cell. Physiol. 180:123–130, 1999. © 1999 Wiley-Liss, Inc.     

Short‐term calorie restriction reverses vascular endothelial dysfunction in old mice by increasing nitric oxide and reducing oxidative stress

To determine if short‐term calorie restriction reverses vascular endothelial dysfunction in old mice, old (O, n = 30) and young (Y, n = 10) male B6D2F1 mice were fed ad libitum (AL) or calorie restricted (CR, approximately 30%) for 8 weeks. Ex vivo carotid artery endothelium‐dependent dilation (EDD) was impaired in old ad libitum (OAL) vs. young ad libitum (YAL) (74 ± 5 vs. 95 ± 2% of maximum dilation, P < 0.05), whereas old calorie‐restricted (OCR) and YCR did not differ (96 ± 1 vs. 94 ± 3%). Impaired EDD in OAL was mediated by reduced nitric oxide (NO) bioavailability associated with decreased endothelial NO synthase expression (aorta) (P < 0.05), both of which were restored in OCR. Nitrotyrosine, a cellular marker of oxidant modification, was markedly elevated in OAL (P < 0.05), whereas OCR was similar to Y. Aortic superoxide production was 150% greater in OAL vs. YAL (P < 0.05), but normalized in OCR, and TEMPOL, a superoxide dismutase (SOD) mimetic that restored EDD in OAL (to 97 ± 2%), had no effect in Y or OCR. OAL had increased expression and activity of the oxidant enzyme, NADPH oxidase, and its inhibition (apocynin) improved EDD, whereas NADPH oxidase in OCR was similar to Y. Manganese SOD activity and sirtuin1 expression were reduced in OAL (P < 0.05), but restored to Y in OCR. Inflammatory cytokines were greater in OAL vs. YAL (P < 0.05), but unaffected by CR. Carotid artery endothelium‐independent dilation did not differ among groups. Short‐term CR initiated in old age reverses age‐associated vascular endothelial dysfunction by restoring NO bioavailability, reducing oxidative stress (via reduced NADPH oxidase–mediated superoxide production and stimulation of anti‐oxidant enzyme activity), and upregulation of sirtuin‐1.     

Maternal caloric restriction partially rescues the deleterious effects of advanced maternal age on offspring

While many studies have focused on the detrimental effects of advanced maternal age and harmful prenatal environments on progeny, little is known about the role of beneficial non‐Mendelian maternal inheritance on aging. Here, we report the effects of maternal age and maternal caloric restriction (CR) on the life span and health span of offspring for a clonal culture of the monogonont rotifer Brachionus manjavacas. Mothers on regimens of chronic CR (CCR) or intermittent fasting (IF) had increased life span compared with mothers fed ad libitum (AL). With increasing maternal age, life span and fecundity of female offspring of AL‐fed mothers decreased significantly and life span of male offspring was unchanged, whereas body size of both male and female offspring increased. Maternal CR partially rescued these effects, increasing the mean life span of AL‐fed female offspring but not male offspring and increasing the fecundity of AL‐fed female offspring compared with offspring of mothers of the same age. Both maternal CR regimens decreased male offspring body size, but only maternal IF decreased body size of female offspring, whereas maternal CCR caused a slight increase. Understanding the genetic and biochemical basis of these different maternal effects on aging may guide effective interventions to improve health span and life span.     

Mild Calorie Restriction Induces Fat Accumulation in Female C57BL/6J Mice

This study investigated the effects of mild calorie restriction (CR) (5%) on body weight, body composition, energy expenditure, feeding behavior, and locomotor activity in female C57BL/6J mice. Mice were subjected to a 5% reduction of food intake relative to baseline intake of ad libitum (AL) mice for 3 or 4 weeks. In experiment 1, body weight was monitored weekly and body composition (fat and lean mass) was determined at weeks 0, 2, and 4 by dual energy X‐ray absorptiometry. In experiment 2, body weight was measured every 3 days and body composition was determined by quantitative magnetic resonance weekly, and energy expenditure, feeding behavior, and locomotor activity were determined over 3 weeks in a metabolic chamber. At the end of both experiments, CR mice had greater fat mass (P < 0.01) and less lean mass (P < 0.01) compared with AL mice. Total energy expenditure (P < 0.05) and resting energy expenditure (P < 0.05) were significantly decreased in CR mice compared with AL mice over 3 weeks. CR mice ate significantly more food than AL mice immediately following daily food provisioning at 1600 hours (P < 0.01). These findings showed that mild CR caused increased fat mass, decreased lean mass and energy expenditure, and altered feeding behavior in female C57BL/6J mice. Locomotor activity or brown adipose tissue (BAT) thermogenic capacity did not appear to contribute to the decrease in energy expenditure. The increase in fat mass and decrease in lean mass may be a stress response to the uncertainty of food availability.     

Caloric Restriction Shortens Lifespan through an Increase in Lipid Peroxidation,Inflammation and Apoptosis in the G93A Mouse,an Animal Model of ALS

Caloric restriction (CR) extends lifespan through a reduction in oxidative stress, delays the onset of morbidity and prolongs lifespan. We previously reported that long-term CR hastened clinical onset, disease progression and shortened lifespan, while transiently improving motor performance in G93A mice, a model of amyotrophic lateral sclerosis (ALS) that shows increased free radical production. To investigate the long-term CR-induced pathology in G93A mice, we assessed the mitochondrial bioenergetic efficiency and oxidative capacity (CS – citrate synthase content and activity, cytochrome c oxidase - COX activity and protein content of COX subunit- I and IV and UCP3- uncoupling protein 3), oxidative damage (MDA – malondialdehyde and PC – protein carbonyls), antioxidant enzyme capacity (Mn-SOD, Cu/Zn-SOD and catalase), inflammation (TNF-α), stress response (Hsp70) and markers of apoptosis (Bax, Bcl-2, caspase 9, cleaved caspase 9) in their skeletal muscle. At age 40 days, G93A mice were divided into two groups: Ad libitum (AL; n = 14; 7 females) or CR (n = 13; 6 females), with a diet equal to 60% of AL. COX/CS enzyme activity was lower in CR vs. AL male quadriceps (35%), despite a 2.3-fold higher COX-IV/CS protein content. UCP3 was higher in CR vs. AL females only. MnSOD and Cu/Zn-SOD were higher in CR vs. AL mice and CR vs. AL females. MDA was higher (83%) in CR vs. AL red gastrocnemius. Conversely, PC was lower in CR vs. AL red (62%) and white (30%) gastrocnemius. TNF-α was higher (52%) in CR vs. AL mice and Hsp70 was lower (62%) in CR vs. AL quadriceps. Bax was higher in CR vs. AL mice (41%) and CR vs. AL females (52%). Catalase, Bcl-2 and caspases did not differ. We conclude that CR increases lipid peroxidation, inflammation and apoptosis, while decreasing mitochondrial bioenergetic efficiency, protein oxidation and stress response in G93A mice.     

Opposing effects on cardiac function by calorie restriction in different‐aged mice

Calorie restriction (CR) increases average and maximum lifespan and exhibits an apparent beneficial impact on age‐related diseases. Several studies have shown that CR initiated either in middle or old age could improve ischemic tolerance and rejuvenate the aging heart; however, the data are not uniform when initiated in young. The accurate time to initiate CR providing maximum benefits for cardiac remodeling and function during aging remains unclear. Thus, whether a similar degree of CR initiated in mice of different ages could exert a similar effect on myocardial protection was investigated in this study. C57BL/6 mice were subjected to a calorically restricted diet (40% less than the ad libitum diet) for 3 months initiated in 3, 12, and 19 months. It was found that CR significantly reversed the aging phenotypes of middle‐aged and old mice including cardiac remodeling (cardiomyocyte hypertrophy and cardiac fibrosis), inflammation, mitochondrial damage, telomere shortening, as well as senescence‐associated markers but accelerated in young mice. Furthermore, whole‐genome microarray demonstrated that the AMP‐activated protein kinase (AMPK)–Forkhead box subgroup ‘O’ (FOXO) pathway might be a major contributor to contrasting regulation by CR initiated in different ages; thus, increased autophagy was seen in middle‐aged and old mice but decreased in young mice. Together, the findings demonstrated promising myocardial protection by 40% CR should be initiated in middle or old age that may have vital implications for the practical nutritional regimen.     

Hyperadrenocorticism of calorie restriction contributes to its anti‐inflammatory action in mice

Calorie restriction (CR), which lengthens lifespan in many species, is associated with moderate hyperadrenocorticism and attenuated inflammation. Given the anti‐inflammatory action of glucocorticoids, we tested the hypothesis that the hyperadrenocorticism of CR contributes to its attenuated inflammatory response. We used a corticotropin‐releasing‐hormone knockout (CRHKO) mouse, which is glucocorticoid insufficient. There were four controls groups: CRHKO mice and wild‐type (WT) littermates fed either ad libitum (AL) or CR (60% of AL food intake), and three experimental groups: (a) AL‐fed CRHKO mice given corticosterone (CORT) in their drinking water titrated to match the integrated 24‐hr plasma CORT levels of AL‐fed WT mice, (b) CR‐fed CRHKO mice given CORT to match the 24‐hr CORT levels of AL‐fed WT mice, and (c) CR‐fed CHRKO mice given CORT to match the 24‐hr CORT levels of CR‐fed WT mice. Inflammation was measured volumetrically as footpad edema induced by carrageenan injection. As previously observed, CR attenuated footpad edema in WT mice. This attenuation was significantly blocked in CORT‐deficient CR‐fed CRHKO mice. Replacement of CORT in CR‐fed CRHKO mice to the elevated levels observed in CR‐fed WT mice, but not to the levels observed in AL‐fed WT mice, restored the anti‐inflammatory effect of CR. These results indicate that the hyperadrenocorticism of CR contributes to the anti‐inflammatory action of CR, which may in turn contribute to its life‐extending actions.     

Lifelong Caloric Restriction Increases Working Memory in Mice

Caloric restriction (CR) is argued to positively affect general health, longevity and the normally occurring age-related reduction of cognition. This issue is well examined, but most studies investigated the effect of short-term periods of CR. Herein, 4 weeks old female mice were fed caloric restricted for 4, 20 and especially for 74 weeks. CR mice received 60% of food eaten by their ad libitum (AL) fed littermates, and all age-matched groups were behaviorally analyzed. The motor coordination, which was tested by rotarod/accelerod, decreased age-related, but was not influenced by the different periods of CR. In contrast, the age-related impairment of spontaneous locomotor activity and anxiety, both being evaluated by open field and by elevated plus maze test, was found aggravated by a lifelong CR. Measurement of cognitive performance with morris water maze showed that the working memory decreased age-related in AL mice, while a lifelong CR caused a better cognitive performance and resulted in a significantly better spatial memory upon 74 weeks CR feeding. However, a late-onset CR feeding in 66 weeks old mice did not ameliorate the working memory. Therefore, a lifelong CR seems to be necessary to improve working memory.     

Role of calorie restriction in alleviation of age-related morphological and biochemical changes in sciatic nerve

BackgroundAging is associated with structural, functional and biochemical alterations in the nervous system. Calorie restriction (CR) was found to retard most physiological indices of aging.ObjectivesThis work aimed to investigate the effect of CR on age-related changes in sciatic nerves.Materials and methodsThirty male albino rats aged 1 month were equally divided into three groups; Group I [control adult-ad libitum AL]: fed a regular diet and sacrificed at the age of 6 months, group II (aged-AL group): fed a regular diet AL and sacrificed at the age of 18 months, and group III (aged CR) fed a 40% calorie restricted diet and sacrificed at the age of 18 months. Rats were anesthetized and sciatic nerves were processed for light, electron microscope and morphometric studies. Oxidative stress in sciatic nerves was investigated by estimation of lipid perioxidation by product malondialdehyde (MDA) tissue level and antioxidant enzyme; superoxide dismutase activity (SOD).ResultsThe aged (AL) sciatic nerves appeared disorganized, with thick perineurium and increased collagen fibers associated with decreased g-ratio. Abnormal myelin forms were seen as outfolded myelin loops, thin denuded myelin, splitting of myelin into myelin figures and interlamellar vacuoles. Schwann cells revealed vacuolated cytoplasm. There was also significant increase in MDA level and a significant decrease in SOD activity in comparison to control adult (AL). Apparent structural and histomorphological improvement were noticed after CR in aged rats.ConclusionAging caused structural and biochemical alterations in sciatic nerves with alleviating effect of calorie restriction on such effects.     

The effect of long-term caloric restriction on function of T-cell subsets in old mice

The effect of caloric restriction (from weaning to old age) on CD3-stimulated CD4+ and CD8+ lymphocyte proliferation and calcium mobilization was examined. Young ad libitum (ad lib) fed, old ad lib fed, old calorically restricted, and old calorically restricted mice which were fed ad lib during the last 6 weeks of their life (restricted/refed) were compared in both BDF1 [(C57BL/6 x DBA/2)F1] and C57BL/6 mice. Proliferation of CD4+ cells was lower in old ad lib animals than in young animals; this difference was not seen in CD8+ cells. Those CD4+ cells which did proliferate in old ad lib animals underwent similar cell cycle progression as young cells. In calorically restricted and calorically restricted/refed animals, CD4+ cell proliferation was similar to the young animals, and CD8+ cells showed a higher proliferative capacity than cells from either young or old ad lib mice. Differences in proliferative capacity were not correlated with alterations in transmembrane signaling efficiency as peak [Ca2+]i was reduced in both T-cell subsets in all groups of old mice relative to young mice. Additionally, reduced [Ca2+]i was observed in the CD8+ subset for which there was no deficit in proliferation, and the enhanced proliferation seen in old restricted and old restricted/refed mice did not manifest as increased [Ca2+]i mobilization. The percentage of CD4+ cells from both mouse strains was reduced in all groups of old mice compared with young mice, while the percentage of CD8+ cells was generally similar in young and all groups of old mice. Our studies would suggest that lifelong caloric restriction of mice prevents the age-associated decrease in T-cell proliferative capacity but that the enhanced proliferation of these cells is not due to increased efficiency of transmembrane signaling.     

Longitudinal determination of skin collagen glycation and glycoxidation rates predicts early death in C57BL/6NNIA mice.

In 1988, the National Institute on Aging launched a 10-year program aimed at identification of biomarkers of aging. Previous results from our laboratory showed that pentosidine, an advanced glycation product, formed in skin collagen at a rate inversely related to maximum life span across several mammalian species. As part of the Biomarkers Program, we investigated the hypothesis that longitudinal determination of glycation and glycoxidation rates in skin collagen could predict longevities in ad libitum-fed (AL) and caloric restricted (CR) mice. C57BL/6NNia male mice were biopsied at age 20 months and at natural death. Glycation (furosine method) was assessed by gas chromatography/mass spectrometry (GC/MS) and the glycoxidation products carboxymethyllysine (CML) and pentosidine were determined by GC/MS and HPLC, respectively. CR vs. AL significantly (P<0.0001) increased both mean (34 vs. 27 months) and maximum (47 vs. 31 months) life spans. Skin collagen levels of furosine (pmol/micromol lysine) were approximately 2.5-fold greater than CML levels and 100-fold greater than pentosidine. Individual accumulation rates modeled as linear equations were significantly (P<0.001) inhibited by CR vs. AL for all parameters and in all cases varied inversely with longevity (P<0.1 to <0.0001). The incidence of three tissue pathologies (lymphoma, dermatitis, and seminal vesiculitis) was found to be attenuated by CR and the latter pathology correlated significantly with longevities (r=0.54, P=0. 002). The finding that markers of skin collagen glycation and glycoxidation rates can predict early deaths in AL and CR C57BL/6NNia mice strongly suggests that an age-related deterioration in glucose tolerance is a life span-determining process.     

Effect of Chronic Caloric Restriction on the Circadian Regulation of Physiological and Behavioral Variables in Old Male B6C3F1 Mice

The circadian rhythms of food and water consumption, the number of feeding and drinking episodes, oxygen consumption, carbon dioxide production, respiratory quotient, gross motor activity, and body temperature were measured in male B6C3F, mice that were fed ad libitum (AL) or fed a caloric-restricted diet (CR). The CR regimen (60% of the normal AL consumption) was fed to mice during the daytime (5 hr after lights on). CR animals exhibited fewer feeding episodes but consumed more food per feeding bout and spent more total time feeding than AL mice. It appears that CR caused mice to change from their normal “nibbling behavior” to meal feeding. Compared to AL animals, the mean body temperature was reduced in CR animals, while the amplitude of the body temperature rhythm was increased. Spans of reduced activity, metabolism, and body temperature (torpor) occurred in CR mice for several hours immediately before feeding, during times of high fatty acid metabolism (low RQ). The acute availability of exogenous substrates (energy supplies) seemed to modulate metabolism shifting metabolic pathways to promote energy efficiency. CR was also associated with lower DNA damage, higher DNA repair, and decreased proto-oncogene expression. Most of the circadian rhythms studied seemed to be synchronized primarily to the feeding rather than the photoperiod cycle. Night-time CR feeding was found to be better than daytime feeding because the circadian rhythms for AL and CR animals were highly synchronized when this regimen was used.