Metformin supplementation and life span in Fischer-344 rats.

Calorie restriction (CR) has been known for more than 70 years to extend life span and delay disease in rodent models. Metformin administration in rodent disease models has been shown to delay cancer incidence and progression, reduce cardiovascular disease and extend life span. To more directly test the potential of metformin supplementation (300 mg/kg/day) as a CR mimetic, life-span studies were performed in Fischer-344 rats and compared with ad libitum feeding and CR (30%). The CR group had significantly reduced food intake and body weight throughout the study. Body weight was significantly reduced in the metformin group compared with control during the middle of the study, despite similar weekly food intake. Although CR significantly extended early life span (25th quantile), metformin supplementation did not significantly increase life span at any quantile (25th, 50th, 75th, or 90th), overall or maximum life span (p > .05) compared with control.

Caloric restriction mimetics: a novel approach for biogerontology.

Caloric restriction remains the only nongenetic intervention that has been consistently and reproducibly shown to extend both average and maximal lifespan in a wide variety of species. If shown to be applicable to human aging, it is unlikely that most people would be able to maintain the 30-40% reduction in food intake apparently required for this intervention. Therefore, an alternative approach is needed. We first proposed the concept of caloric restriction (CR) mimetics in 1998. Since its introduction, this research area has witnessed a significant expansion of interest in academic, government, and private sectors. CR mimetics target alteration of pathways of energy metabolism to potentially mimic the beneficial health-promoting and anti-aging effects of CR without the need to reduce food intake significantly. To date, a number of candidate CR mimetics including glycolytic inhibitors, antioxidants and specific gene-modulators have been investigated and appear to validate the potential of this approach.

Dietary restriction in aging nonhuman primates.

Dietary restriction (DR) has been shown to benefit health and longevity in a wide variety of species, although most have maximal life spans of only a few years. In 1987, the National Institute on Aging began the first well-controlled long-term study in a species with a considerably longer life span and a closer physiology to humans. Using rhesus monkeys (Macaca mulatta), an extensive array of physiological measures have been conducted in both males and females to evaluate the effects of DR. Monkeys benefit from DR with a lower body weight, body fat, blood glucose and thus are at lower risk for developing diabetes. Changes in several endocrine measures indicate an altered hormonal axis; however, circadian patterns and timing relative to the onset of DR can obscure the differences. Despite the caloric deficit, female monkeys are not reproductively compromised, and both males and females may benefit immunologically. There remains much to be learned from this model of DR including whether long-term DR will increase life span in a primate species.

Effects of calorie restriction on chromosomal stability in rhesus monkeys (Macaca mulatta).

The basic tenet of several theories on aging is increasing genomic instability resulting from interactions with the environment. Chromosomal aberrations have been used as classic examples of increasing genomic instability since they demonstrate an increase in numerical and structural abnormalities with age in many species including humans. This accumulating damage may augment many aging processes and initiate age-related diseases, such as neoplasias. Calorie restriction (CR) is one of the most robust interventions for reducing the frequency of age-related diseases and for extending life span in many short-lived organisms. However, the mechanisms for the anti-aging effects of CR are not yet well understood. A study of rhesus monkeys was begun in 1987 to determine if CR is also effective in reducing the frequency of age-related diseases and retarding aging in a long-lived mammal. Male monkeys were begun on the diet in 1987, and females were added in 1992 to examine a possible difference in response to CR by sex. The CR monkeys have been maintained for over 10 years on a low-fat nutritional diet that provides a 30% calorie reduction compared to a control (CON) group. Because of the greater similarity of nonhuman primates to humans in life span and environmental responses to diet compared with those of rodents, the rhesus monkey provides an excellent model for the effects of CR in humans. This study examined the effects of CR on chromosomal instability with aging. Significant age effects were found in both CR and CON groups for the number of cells with aneuploidy: old animals had a higher loss and a higher gain than young animals. However, there was no effect of age on chromosomal breakage or structural aberrations in either diet group. Diet had only one significant effect: the CR group had a higher frequency of chromatid gaps than did the CON group. CR, implemented in adult rhesus monkeys, does not have a major effect on the reduction of numerical or structural aberrations related to aging.

Delay of T cell senescence by caloric restriction in aged long-lived nonhuman primates.

Caloric restriction (CR) has long been known to increase median and maximal lifespans and to decreases mortality and morbidity in short-lived animal models, likely by altering fundamental biological processes that regulate aging and longevity. In rodents, CR was reported to delay the aging of the immune system (immune senescence), which is believed to be largely responsible for a dramatic increase in age-related susceptibility to infectious diseases. However, it is unclear whether CR can exert similar effects in long-lived organisms. Previous studies involving 2- to 4-year CR treatment of long-lived primates failed to find a CR effect or reported effects on the immune system opposite to those seen in CR-treated rodents. Here we show that long-term CR delays the adverse effects of aging on nonhuman primate T cells. CR effected a marked improvement in the maintenance and/or production of naïve T cells and the consequent preservation of T cell receptor repertoire diversity. Furthermore, CR also improved T cell function and reduced production of inflammatory cytokines by memory T cells. Our results provide evidence that CR can delay immune senescence in nonhuman primates, potentially contributing to an extended lifespan by reducing susceptibility to infectious disease.

The potential for dietary restriction to increase longevity in humans: extrapolation from monkey studies.

Based on results emerging from long-term studies of dietary restriction in rhesus monkeys, we offer our views regarding whether dietary restriction can increase longevity in humans. Because lifespan data in monkeys remain inconclusive currently, we respond that "we do not for sure". Based on the vast literature regarding the effects of healthy, low calorie diets on health and longevity in a wide range of species, including humans, and based on data emerging from monkey studies suggesting that dietary restriction improves markers of disease risk and health, we respond that "we think so." Because it is unlikely that an experimental study will ever be designed to address this question in humans, we respond that "we think we will never know for sure." We suggest that debate of this question is clearly an academic exercise; thus, we would suggest that the more compelling discussion should focus on whether basic mechanisms of DR can be discovered and if such discoveries can lead to the development of effective DR mimetics. Even if proof that DR or DR mimetics can increase longevity in humans will likely never emerge, we would suggest that endpoints regarding disease risk and disease incidence as well as maintenance of function can be examined in human clinical trials, and that these will be highly relevant for evaluating the effectiveness of such treatments.

Calorie restriction mimetics: an emerging research field.

When considering all possible aging interventions evaluated to date, it is clear that calorie restriction (CR) remains the most robust. Studies in numerous species have demonstrated that reduction of calories 30-50% below ad libitum levels of a nutritious diet can increase lifespan, reduce the incidence and delay the onset of age-related diseases, improve stress resistance, and decelerate functional decline. A current major focus of this research area is whether this nutritional intervention is relevant to human aging. Evidence emerging from studies in rhesus monkeys suggests that their response to CR parallels that observed in rodents. To assess CR effects in humans, clinical trials have been initiated. However, even if results from these studies could eventually substantiate CR as an effective pro-longevity strategy for humans, the utility of this intervention would be hampered because of the degree and length of restriction required. As an alternative strategy, new research has focused on the development of 'CR mimetics'. The objective of this strategy is to identify compounds that mimic CR effects by targeting metabolic and stress response pathways affected by CR, but without actually restricting caloric intake. For example, drugs that inhibit glycolysis (2-deoxyglucose), enhance insulin action (metformin), or affect stress signaling pathways (resveratrol), are being assessed as CR mimetics (CRM). Promising results have emerged from initial studies regarding physiological responses which resemble those observed in CR (e.g. reduced body temperature and plasma insulin) as well as protection against neurotoxicity (e.g. enhanced dopamine action and up-regulated neurotrophic factors). Ultimately, lifespan analyses in addition to expanded toxicity studies must be accomplished to fully assess the potential of any CRM. Nonetheless, this strategy clearly offers a very promising and expanding research endeavor.

Caloric restriction modulates insulin receptor signaling in liver and skeletal muscle of rat.

OBJECTIVE: We investigated how the insulin/insulin-like growth factor-1 signaling pathway is involved in the robust antiaging effects produced by caloric restriction. METHODS: We subjected male rats to feeding ad libitum or calorie restriction, i.e., 60% of the ad libitum amount, for 2 and 25 mo and then assessed the effects of calorie restriction on insulin receptor (IR) signaling in liver and skeletal muscle. RESULTS: The results indicated that aging was accompanied by a significant decrease in IR tyrosine phosphorylation after insulin stimulation in live and skeletal muscle, which was associated with a significant increase in the activity of protein tyrosine phosphatase-1B. However, these age-related alterations were attenuated by long-term calorie restriction. Expression profile of mRNA showed an increased expression of mRNAs for IR and insulin-like growth factor-1 receptor in both tissues of calorie-restricted rats, but increased expression of IR mRNA was dissociated with the IR gene product in rats maintained on long-term calorie-restricted diet. CONCLUSION: IR signaling may play an important role in aging and its retardation by calorie restriction, and normal function of IR in liver and skeletal muscle is required for healthy aging and extending lifespan in mammals.

Age-related decline in caloric intake and motivation for food in rhesus monkeys.

Human studies have documented age-related declines in caloric intake that are pronounced at advanced ages. We examined caloric intake from a longitudinal study of aging in 60 male and 60 female rhesus monkeys (Macaca mulatta) collected for up to 10 years. Monkeys were provided a standardized, nutritionally fortified diet during two daily meals, and intake was measured quarterly. About half of the monkeys were on a regimen of caloric restriction (CR) representing about a 30% reduction in caloric intake compared to controls (CON) of comparable age and body weight. CR was applied to determine if this nutritional intervention retards the rate of aging in monkeys similar to observations in other mammalian studies. Following reproductive maturity at 6 years of age, there was a consistent age-related decline in caloric intake in these monkeys. Although males had higher intake than females, and CON had higher intake compared to CR, the sex and diet differences converged at older ages (>20 years); thus, older CR monkeys were no longer consuming 30% less than the CON. When adjusted for body weight, an age-related decline in caloric intake was still evident; however, females had higher intake compared to males while CR monkeys still consumed less food, and again differences converged at older ages. Motivation for food was assessed in 65 of the monkeys following at least 8 years in their respective diet groups. Using an apparatus attached to the home cage, following an overnight fast, monkeys were trained to reach out of their cage to retrieve a biscuit of their diet by pushing open a clear plastic door on the apparatus. The door was then locked, and thus the biscuit was irretrievable. The time spent trying to retrieve the biscuit was recorded as a measure of motivation for food. We observed an age-related decline in this measure, but found no consistent differences in retrieval time between CR and CON groups of comparable age and time on diet. The results demonstrate an age-related decline in food intake and motivation for food in rhesus monkeys paralleling findings in humans; however, we found no evidence that monkeys on a long-term CR regimen were more motivated for food compared to CON. Examining the relationship of selected blood proteins to food intake following 7-11 years on the study, we found a negative correlation between globulin and intake among males and females after accounting for differences in age. In addition, a positive correlation was observed between leptin and intake in males.

Caloric restriction mimetics: the next phase.

Calorie restriction (CR) mimetics are agents or strategies that can mimic the beneficial health-promoting and anti-aging effects of CR, the only intervention conclusively shown to slow aging and maintain health and vitality across the phylogenetic spectrum. Our lead compound, developed at the National Institute on Aging, was 2-deoxyglucose, an analogue of the native sugar, that acted as a glycolytic inhibitor, having limited metabolism and actually reducing overall energy flow--analogous to CR. This agent reduced insulin levels and body temperature of rats, similar to the physiological effects of CR, but toxicity was noted in long-term studies, which apparently prevented life-span extension. We previously demonstrated that lower insulin and body temperature (as well as maintenance of dehydroepiandrosterone levels) correlate with longevity in non-CR humans. The recent work of other investigators shows that humans subjected to short-term CR also have lower insulin and body temperature. Obviously, longer-term CR is extremely difficult to maintain; hence, the need for CR mimetics. The next phase of calorie restriction studies includes basic investigations as well as possible clinical trials of a number of candidate CR mimetics, ranging from glycolytic inhibitors to lipid-regulating agents to antioxidants and specific gene modulators. The scope of these ongoing studies in various laboratories, as well as their practical implications, are reviewed and analyzed here.

Effects of phentermine and phenformin on biomarkers of aging in rats.

BACKGROUND: Caloric restriction (CR) is the only treatment known to substantially prolong both average and maximal life span in experimental animals. Interventions that mimic certain effects of CR could be potential anti-aging treatments in humans. Drugs which reduce appetite (anorexiants) represent one class of candidate treatments. Agents that reduce the glucose utilization by the organism could also represent another class of candidate CR mimetics. OBJECTIVE: In our study, we addressed the following questions: (1) Does treatment with an anorexiant reduce caloric intake and body weight of experimental animals comparable to that caused by CR? (2) Does treatment with an antidiabetic agent influence caloric intake and body weight? (3) Does treatment with any of these drugs affect metabolic parameters of an organism in the way similar to that observed with CR? METHODS: One hundred and twenty 6-month-old female Wistar-derived LIO rats were randomly subdivided into four groups and exposed to: (1) ad libitum feeding with placebo (controls); (2) the antidiabetic drug phenformin (2 mg/kg); (3) the anorectic drug phentermine (1 mg/kg), and (4) the same amount of food as the group with the least food intake during the previous week (pair-fed controls). Food and water intake, body weight, and rectal temperature were measured weekly during 16 weeks. At the end of the 16th week of the experiment, serum levels of glucose, total beta-lipoprotein and pre-beta-lipoprotein fractions, cholesterol, triglycerides, insulin, total triiodothyronine, and free thyroxine were estimated. The contents of diene conjugates and Schiff's bases, total antioxidant activity, the activities of Cu/Zn superoxide dismutase, glutathione S-transferase, and glutathione peroxidase, and the generation of reactive oxygen species (ROS) were studied in brain and liver homogenates and in the serum. RESULTS: The controls exposed to pair feeding had a significantly reduced food consumption (about 20%) as compared with the ad libitum fed controls and thus exhibited a moderate CR. Treatment with phentermine reduced the caloric intake by about 12% as compared with the ad libitum fed controls. Body weight and water intake in this group were only slightly decreased (by about 2 and 5%, respectively) as compared with the controls. The mean rectal temperature in the phentermine group (38 degrees C) was significantly higher than in the ad libitum fed (37.8 degrees C) and pair-fed (37.6 degrees C) controls. Treatment with phentermine also resulted in significantly reduced ROS levels in all tissues studied, while the highest ROS production was found in ad libitum (blood serum) and pair-fed (brain) controls. Treatment with phenformin did not significantly influence food and water consumption, body weight, and temperature when compared with the ad libitum fed controls. Rats treated with this antidiabetic drug showed intermediate values of ROS generation. Differences among the groups in total antioxidant activity were not obvious. CONCLUSIONS: Treatment with phentermine reduces caloric intake slightly less than is commonly observed in CR studies. CR due to forcibly reduced feeding and CR due to substance-suppressed appetite appear to act through different metabolic mechanisms and thus may affect aging and longevity in different ways.

Caloric restriction increases neurotrophic factor levels and attenuates neurochemical and behavioral deficits in a primate model of Parkinson's disease.

We report that a low-calorie diet can lessen the severity of neurochemical deficits and motor dysfunction in a primate model of Parkinson's disease. Adult male rhesus monkeys were maintained for 6 months on a reduced-calorie diet [30% caloric restriction (CR)] or an ad libitum control diet after which they were subjected to treatment with a neurotoxin to produce a hemiparkinson condition. After neurotoxin treatment, CR monkeys exhibited significantly higher levels of locomotor activity compared with control monkeys as well as higher levels of dopamine (DA) and DA metabolites in the striatal region. Increased survival of DA neurons in the substantia nigra and improved manual dexterity were noted but did not reach statistical significance. Levels of glial cell line-derived neurotrophic factor, which is known to promote the survival of DA neurons, were increased significantly in the caudate nucleus of CR monkeys, suggesting a role for glial cell line-derived neurotrophic factor in the anti-Parkinson's disease effect of the low-calorie diet.

Aging in rhesus monkeys: relevance to human health interventions.

Progress in gerontological research has been promoted through the use of numerous animal models, which have helped identify possible mechanisms of aging and age-related chronic diseases and evaluate possible interventions with potential relevance to human aging and disease. Further development of nonhuman primate models, particularly rhesus monkeys, could accelerate this progress, because their closer genetic relationship to humans produces a highly similar aging phenotype. Because the relatively long lives of primates increase the administrative and economic demands on research involving them, new emphasis has emerged on increasing the efficient use of these valuable resources through cooperative, interdisciplinary research.

Development of calorie restriction mimetics as a prolongevity strategy.

By applying calorie restriction (CR) at 30-50% below ad libitum levels, studies in numerous species have reported increased life span, reduced incidence and delayed onset of age-related diseases, improved stress resistance, and decelerated functional decline. Whether this nutritional intervention is relevant to human aging remains to be determined; however, evidence emerging from CR studies in nonhuman primates suggests that response to CR in primates parallels that observed in rodents. To evaluate CR effects in humans, clinical trials have been initiated. Even if evidence could substantiate CR as an effective antiaging strategy for humans, application of this intervention would be problematic due to the degree and length of restriction required. To meet this challenge for potential application of CR, new research to create "caloric restriction mimetics" has emerged. This strategy focuses on identifying compounds that mimic CR effects by targeting metabolic and stress response pathways affected by CR, but without actually restricting caloric intake. Microarray studies show that gene expression profiles of key enzymes in glucose (energy) handling pathways are modified by CR. Drugs that inhibit glycolysis (2-deoxyglucose) or enhance insulin action (metformin) are being assessed as CR mimetics. Promising results have emerged from initial studies regarding physiological responses indicative of CR (reduced body temperature and plasma insulin) as well as protection against neurotoxicity, enhanced dopamine action, and upregulated brain-derived neurotrophic factor. Further life span analyses in addition to expanded toxicity studies must be completed to assess the potential of any CR mimetic, but this strategy now appears to offer a very promising and expanding research field.

Caloric restriction modulates early events in insulin signaling in liver and skeletal muscle of rat.

Mutations that extend life span in C. elegans suggest that the insulin/IGF-1 signaling (IS) pathway may play a key role in retarding aging and extending life span by caloric restriction (CR). To evaluate this hypothesis, male rats were subjected to either AL (ad libitum) or CR (40% from AL) for 2 and 25 months, and then the effects of CR on the early events in the IS pathway in liver and muscle were assessed. The results indicated that aging was accompanied by a significant decline in insulin receptor tyrosine phosphorylation (pY-IR) upon insulin stimulation in both tissues, which was correlated with a significant increase in the activity of protein tyrosine phosphatase 1B (PTP-1B). However, these alterations with age were attenuated by 25CR. Parallel changes observed in liver mRNA of CR rats were upregulated insulin receptor (IR), IGF-1R and IRS-1, but increased expression of IR mRNA was dissociated with the IR protein in 25CR rats. The expression of liver mRNAs involved in lipid metabolism was also analyzed. In contrast to 25AL rats, the expression of mRNAs for PPARs (alpha, delta, and gamma) was significantly increased in 25CR rats. SREBP-1c and fatty acid synthase were reduced, and other genes were increased, including hormone-sensitive lipase and PGC-1 by CR. The data suggest that the normal function of insulin receptor in liver and muscle is required for successful aging. An altered expression of transcription of a number of genes involved in lipid metabolism may also contribute to modulation of the IS pathway by CR.

Circulating adiponectin levels increase in rats on caloric restriction: the potential for insulin sensitization.

Caloric restriction (CR) has a well-known insulin sensitizing effect in vivo. Although this effect has been confirmed in rodents and primates for many years, its precise molecular mechanisms remain unknown. Here we show a significant increase in plasma adiponectin and a decrease in blood glucose, plasma triglyceride and insulin levels in rats maintained on CR diet for 2, 10, 15, and 20 months. Long-term CR rats exhibited significantly higher insulin-stimulated insulin receptor tyrosine phosphorylation and lower PTP-1B activity both in liver and skeletal muscle than those observed in rats fed ad libitum (AL). In addition, the triglyceride levels in these tissues were significantly lower in long-term CR animals. Interestingly, concentrations of plasma adiponectin in long-term CR rats were associated with increased expression of the transcription factor mRNAs for the peroxisome proliferator-activated receptor (PPAR)alpha, gamma and delta, but decreased expression for SREBP-1c, resulting in a concerted modulation in the expression of key transcription target genes involved in fatty acid oxidation and energy combustion in liver. Taken together, our findings suggest an important role for adiponectin in the beneficial effects of long-term CR.

Assessment of auditory function in rhesus monkeys (Macaca mulatta): effects of age and calorie restriction.

Age-related alterations in auditory function were evaluated in adult male rhesus monkeys (Macaca mulatta) involved in a long-term study evaluating the effects of caloric restriction (CR) on aging. We assessed 26 monkeys in a control group fed a low fat, high fiber diet at approximately ad libitum levels and 24 monkeys in a CR group that were fed the same diet reduced in amount by 30% compared to age- and weight-matched controls. The following measures of auditory function were obtained while monkeys were maintained under anesthesia: (1) distortion product otoacoustic emissions (DPOAEs); (2) auditory brainstem responses (ABRs); and (3) middle latency responses (MLRs). All DPOAE measures and peak II amplitude significantly decreased with age, while peak IV latency and ABR threshold significantly increased with age. We found no significant effects of CR on any auditory parameters examined.

Age-related decline in striatal volume in rhesus monkeys: assessment of long-term calorie restriction.

Using magnetic resonance imaging (MRI), we measured striatal volume in 22 male rhesus monkeys undergoing calorie restriction (CR) for 11-13 years and 38 monkeys who were fed ad libitum (CON). CR delays the onset of many age-related processes, and this study tested whether it would alter the age-related decline in striatal volume. The CON and CR groups were sub-divided into middle age (less than 24 years old) and old age groups. Contrary to expectation, volumes of the putamen (not the caudate nucleus) were larger bilaterally in the CON than in the CR group both at middle age and senescence. Regression analysis (region volume versus age) indicated bilateral age-related declines in putamen and caudate nucleus volumes in the old CON monkeys, but only for the putamen in the old CR monkeys. Because tests for slopes found no differences between the groups, the data do not establish an effect of CR. Further study, involving sequential imaging, is warranted in order to clarify the possible effects of CR on age-related changes in striatal volume.