Impact of caloric restriction on health and survival in rhesus monkeys from the NIA study.

Calorie restriction (CR), a reduction of 10­40% in intake of a nutritious diet, is often reported as the most robust non-genetic mechanism to extend lifespan and healthspan. CR is frequently used as a tool to understand mechanisms behind ageing and age-associated diseases. In addition to and independently of increasing lifespan, CR has been reported to delay or prevent the occurrence of many chronic diseases in a variety of animals. Beneficial effects of CR on outcomes such as immune function, motor coordination and resistance to sarcopenia in rhesus monkeys have recently been reported. We report here that a CR regimen implemented in young and older age rhesus monkeys at the National Institute on Aging (NIA) has not improved survival outcomes. Our findings contrast with an ongoing study at the Wisconsin National Primate Research Center (WNPRC), which reported improved survival associated with 30% CR initiated in adult rhesus monkeys (7­14 years) and a preliminary report with a small number of CR monkeys. Over the years, both NIA and WNPRC have extensively documented beneficial health effects of CR in these two apparently parallel studies. The implications of the WNPRC findings were important as they extended CR findings beyond the laboratory rodent and to a long-lived primate. Our study suggests a separation between health effects, morbidity and mortality, and similar to what has been shown in rodents, study design, husbandry and diet composition may strongly affect the life-prolonging effect of CR in a long-lived nonhuman primate.

Sex as a risk factor regarding presbyopia in the rhesus monkey.

PURPOSE: To determine the effect of sex as a risk factor regarding presbyopia. METHODS: Maximum accommodation was pharmacologically induced (40% cabachol corneal iontophoresis) in 97 rhesus monkeys (49 males and 48 females) ranging in age from 8 to 36 years old. Accommodation was measured by Hartinger coincidence refractometry. RESULTS: Accommodative amplitude measured refractometrically decreased with age, and the rate of change was not different between males and females (p = 0.827). CONCLUSIONS: Presbyopia is essentially sex neutral, and no one is spared. There may be modest variations between different populations for various reasons, but essentially it is monotonously predictable. At present there is no biological therapeutic.

Effects of an Unripe Avocado Extract on Glycaemic Control in Individuals with Obesity: A Double-Blinded, Parallel, Randomised Clinical Trial.

BACKGROUND: Unripe avocados (Persea americana) are naturally enriched in mannoheptulose (MH), which is a candidate caloric restriction mimetic. OBJECTIVES: To evaluate the effects of a diet supplement made from unripe avocado on glucose tolerance, and cardiometabolic risk factors in free-living nondiabetic adults with obesity. METHODS: In a double-blinded, randomised controlled trial, 60 adults (female n = 47, age 48 ± 13 years, BMI 34.0 ± 2.6 kg/m2) were stratified by sex and randomised to avocado extract (AvX, 10 g finely ground, freeze-dried unripe avocado) or placebo (10 g finely ground cornmeal plus 5% spinach powder) daily, for 12 weeks. The primary outcome was a change in glucose area under the curve (AUC) in response to a 75 g oral glucose tolerance test. A post-hoc analysis was subsequently performed in a subgroup with insulin AUC above the median of baseline values after removal of participants >2 SD from the mean. RESULTS: There were no between-group differences in glucose AUC (p = 0.678), insulin AUC (p = 0.091), or cardiovascular outcomes. In the subgroup analysis, insulin AUC was lower in AxV versus placebo (p = 0.024). CONCLUSIONS: Daily consumption of unripe avocado extract enriched in MH did not alter glucose tolerance or insulin sensitivity in nondiabetic adults with obesity, but the data provided preliminary evidence for a benefit in insulin AUC in a subgroup of participants with elevated baseline postprandial insulin levels.

Glycolytic inhibition: an effective strategy for developing calorie restriction mimetics.

Calorie restriction mimetics encompass a growing research field directed toward developing treatments that mimic the anti-aging effects of long-term calorie restriction without requiring a change in eating habits. A wide range of approaches have been identified that include (1) intestinal inhibitors of fat and carbohydrate metabolism; (2) inhibitors of intracellular glycolysis; (3) stimulators of the AMPK pathway; (4) sirtuin activators; (5) inhibitors of the mTOR pathway, and (6) polyamines. Several biotech companies have been formed to pursue several of these strategies. The objective of this review is to describe the approaches directed toward glycolytic inhibition. This upstream strategy is considered an effective means to invoke a wide range of anti-aging mechanisms induced by CR. Anti-cancer and anti-obesity effects are important considerations in early development efforts. Although many dozens of candidates could be discussed, the compounds selected to be reviewed are the following: 2-deoxyglucose, 3-bromopyruvate, chrysin, genistein, astragalin, resveratrol, glucosamine, mannoheptulose, and D-allulose. Some candidates have been investigated extensively with both positive and negative results, while others are only beginning to be studied.

Characterization and Mechanisms of Action of Avocado Extract Enriched in Mannoheptulose as a Candidate Calorie Restriction Mimetic.

Increased consumer interest in the avocado (Persea americana or Persea gratissima) has been attributed to established health benefits of this fruit associated with a wide range of ingredients. In search of effective calorie restriction mimetics (CRM), we present herein a consideration of possible health benefits of the rare sugar, mannoheptulose (MH), which acts as an intracellular glycolytic inhibitor and presents the highest concentration of this inhibitor in unripe avocados. A method for producing an extract of unripe avocado (AvX) to enrich concentrations of MH is described. Experiments using myocyte cultures demonstrated a pattern of CRM-like responses when treated with AvX. In vivo experiments confirmed that orally consumed AvX is bioavailable in both mice and dogs, as observed in urine and blood samples. Additional experiments in both these species demonstrated CRM-like improvements in glucose and insulin responses. In sum, the MH-enriched AvX exhibits promise as a CRM.

An Avocado Extract Enriched in Mannoheptulose Prevents the Negative Effects of a High-Fat Diet in Mice.

Beginning at 16 weeks of age and continuing for 44 weeks, male C57BL/6J were fed either a control (CON) diet; a high-fat (HF) diet (60% unsaturated); or the HF diet containing an extract of unripe avocados (AvX) enriched in the 7-carbon sugar mannoheptulose (MH), designed to act as a glycolytic inhibitor (HF + MH). Compared to the CON diet, mice on the HF diet exhibited higher body weights; body fat; blood lipids; and leptin with reduced adiponectin levels, insulin sensitivity, VO2max, and falls from a rotarod. Mice on the HF + MH diet were completely protected against these changes in the absence of significant diet effects on food intake. Compared to the CON diet, oxidative stress was also increased by the HF diet indicated by higher levels of total reactive oxygen species, superoxide, and peroxynitrite measured in liver samples by electron paramagnetic resonance spectroscopy, whereas the HF + MH diet attenuated these changes. Compared to the CON, the HF diet increased signaling in the mechanistic target of the rapamycin (mTOR) pathway, and the addition of the MH-enriched AvX to this diet attenuated these changes. Beyond generating further interest in the health benefits of avocados, these results draw further new attention to the effects of this rare sugar, MH, as a botanical intervention for preventing obesity.

Effects of moderate calorie restriction on testosterone production and semen characteristics in young rhesus macaques (Macaca mulatta).

We have previously reported a modest influence of moderate calorie restriction (CR) on testicular gene expression in young adult rhesus macaques (Macaca mulatta); however, it is unclear if these modifications correspond to subsequent changes in testicular function or sperm physiology. This study extends our earlier findings to examine potential physiological differences due to this differential gene expression. Animals were subjected to 30% CR (CR, n = 5) or were fed a standard control diet (CON, n = 5) starting during their peripubertal period. Circulating testosterone (T) levels were measured across a 24-h period after 7 yr of dietary treatment and were found to be similar in CR and CON males; however, maintenance of daily minimum T levels was significantly higher in the CR animals. Semen collection was performed on the same cohort of animals three times per male (CR, n = 4; CON, n = 4) after 8 yr of treatment, and samples were assessed by a variety of measures. Parameters, including semen quality and sperm cell viability and function, showed less variability in semen samples taken from CR males, but overall testicular function and sperm quality were comparable regardless of diet. There is mounting evidence that CR may promote health and longevity in a wide range of organisms, including nonhuman primates. Importantly, our data suggest that moderate CR has no obvious lasting detrimental effect on testicular function and sperm parameters in young adult primates and may in fact help maintain higher levels of circulating T.

Chronic ingestion of 2-deoxy-D-glucose induces cardiac vacuolization and increases mortality in rats.

Calorie restriction (CR), the purposeful reduction of energy intake with maintenance of adequate micronutrient intake, is well known to extend the lifespan of laboratory animals. Compounds like 2-deoxy-D-glucose (2DG) that can recapitulate the metabolic effects of CR are of great interest for their potential to extend lifespan. 2DG treatment has been shown to have potential therapeutic benefits for treating cancer and seizures. 2DG has also recapitulated some hallmarks of the CR phenotype including reduced body temperature and circulating insulin in short-term rodent trials, but one chronic feeding study in rats found toxic effects. The present studies were performed to further explore the long-term effects of 2DG in vivo. First we demonstrate that 2DG increases mortality of male Fischer-344 rats. Increased incidence of pheochromocytoma in the adrenal medulla was also noted in the 2DG treated rats. We reconfirm the cardiotoxicity of 2DG in a 6-week follow-up study evaluating male Brown Norway rats and a natural form of 2DG in addition to again examining effects in Fischer-344 rats and the original synthetic 2DG. High levels of both 2DG sources reduced weight gain secondary to reduced food intake in both strains. Histopathological analysis of the hearts revealed increasing vacuolization of cardiac myocytes with dose, and tissue staining revealed the vacuoles were free of both glycogen and lipid. We did, however, observe higher expression of both cathepsin D and LC3 in the hearts of 2DG-treated rats which indicates an increase in autophagic flux. Although a remarkable CR-like phenotype can be reproduced with 2DG treatment, the ultimate toxicity of 2DG seriously challenges 2DG as a potential CR mimetic in mammals and also raises concerns about other therapeutic applications of the compound.

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.

Nutritional interventions in aging and age-associated diseases.

Dietary interventions have proven to be the most robust and consistent means of ameliorating the diseases and dysfunctions of aging. A large literature includes both quantitative (caloric restriction [CR]) and qualitative (micronutrients, antioxidants, etc.) alterations of nutrient and caloric intake and the resulting positive outcomes for age-related disease protection, maintenance of vitality, and quantitative and qualitative life-span extension. In recognition of the importance of this research area, two sessions concerning nutritional interventions in aging and age-associated diseases were organized for this symposium, one dealing with diet and the other with dietary supplements. This brief report covers both, providing selected examples of the most recent research into both these types of interventions and their mechanisms of action.

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.

Caloric restriction improves health and survival of rhesus monkeys.

Caloric restriction (CR) without malnutrition extends lifespan and delays the onset of age-related disorders in most species but its impact in nonhuman primates has been controversial. In the late 1980s two parallel studies were initiated to determine the effect of CR in rhesus monkeys. The University of Wisconsin study reported a significant positive impact of CR on survival, but the National Institute on Aging study detected no significant survival effect. Here we present a direct comparison of longitudinal data from both studies including survival, bodyweight, food intake, fasting glucose levels and age-related morbidity. We describe differences in study design that could contribute to differences in outcomes, and we report species specificity in the impact of CR in terms of optimal onset and diet. Taken together these data confirm that health benefits of CR are conserved in monkeys and suggest that CR mechanisms are likely translatable to human health.

Functional recovery of the striatal cholinergic system in aged rats by adenoviral vector-mediated gene transfer of dopamine D2 receptor.

We previously reported that the striatal dopamine-acetylcholine (ACh) interaction was affected by the aging process, possibly via a decrease of the striatal D(2)R expression. In the current study, the ACh responses to the infusion of 0.1microM of the D(2)R agonist quinpirole were measured with microdialysis techniques after adenoviral vector-mediated gene transfer of D(2)R into the striatum of 25-month-old rats. The D(2)R gene-transferred rats showed significantly stronger responses of the striatal cholinergic neurons to the infusion of the D(2)R agonist than did control vector-transferred rats. The current study suggests that age-associated functional decline with decreased gene expression of the receptor may be restored by intervention.

Manipulation of health span and function by dietary caloric restriction mimetics.

After nearly a century of rigorous investigation and testing, dietary caloric restriction (CR) remains the most robust and reproducible method for slowing aging and maintaining health, function, and vitality. This intervention has been applied to species across the evolutionary spectrum, but for a number of reasons, practical applicability to humans has been questioned. To overcome these issues, we initiated the field of CR mimetics in 1998 and have observed its development into a full-fledged antiaging industry. Basically, strategies that enable individuals to obtain the biological benefits of CR without reducing actual food intake can be considered CR mimetics, whether functional, pharmaceutical, nutraceutical, or other. Some of the best known candidates include resveratrol and related agents, the antidiabetic drug metformin, and rapamycin and other mTOR regulators. While the mechanisms of action vary, these and essentially all CR mimetic candidates work through at least some of the same pathways as actual CR. While the entire field continues to evolve rapidly, the current status will be reviewed here, with particular focus on recent developments, the most practical relevance and applicability for potential consumers, and new strategies for the future.

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 and caloric restriction mimetics: current status and promise for the future.

Dietary caloric restriction is the most reproducible means of extending longevity and maintaining health and vitality. It has been shown to be relevant to a wide rage of species, including primates. Examination of key markers of the calorically restricted phenotype, such as plasma insulin, dehydroepiandrosterone sulfate, and body temperature, suggest that they may predict longevity in humans as well. However, most people would be unwilling or unable to adopt the 30% to 40% reduction in food intake necessary to achieve optimal health and longevity benefits. For this reason, a number of laboratories are pursuing caloric restriction mimetics: ways to achieve the benefits of restriction without eating less. This approach will undoubtedly remain a major focus of biogerontology for the foreseeable future.

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.

Caloric restriction versus drug therapy to delay the onset of aging diseases and extend life.

There are two firmly established methods of prolonging life. Calorie restriction (CR) using nutrient-rich diets to prolong life in lower animals, and life saving medications in humans to delay the development of the major diseases of middle and old age. These two approaches have different mechanisms of action. In rats, CR at 40% below ad libitum intake begun soon after weaning and continued until death, reduces body weight by about 40% and increases lifespan. There have been no lifelong CR studies performed on humans. However, in healthy adult human subjects about 20% CR over a period of 2-15 years, lowers body weight by about 20% and decreases body mass index (BMI) to about 19. This CR treatment in humans reduces blood pressure and blood cholesterol to a similar extent as the specific drugs used to delay the onset of vascular disease and so extend human life. These same drugs may act by mechanisms that overlap with some of the mechanisms of CR in retarding these pathologies and thus may have similar antiaging and life prolonging actions. Such drugs may be regarded as CR mimetics which inhibit the development of certain life shortening diseases, without the need to lower calorie intake. In developed countries, better medical care, drug therapy, vaccinations, and other public health measures have extended human life by about 30 years during the 20th century without recourse to CR, which is so effective in the rat. The percentage gain in human life expectancy during the 20th century is twice that achieved by CR in rat survival. However, rat longevity studies now use specific pathogen-free animals and start CR after weaning or later, thereby excluding deaths from infectious diseases and those associated with birth and early life. There is a need to develop CR mimetics which can delay the development of life-threatening diseases in humans. In the 21st century due to the human epidemic of overeating with a sedentary lifestyle, it may necessary to utilize CR to counter the aging effects of overweight. Since the greatest life-extending effects of CR in the rodent occur when started early in life, long-term antiaging therapy in humans should be initiated soon after maturity, when physiological systems have developed optimally.

Calorie restriction mimetics: can you have your cake and eat it, too?

Strong consensus exists regarding the most robust environmental intervention for attenuating aging processes and increasing healthspan and lifespan: calorie restriction (CR). Over several decades, this paradigm has been replicated in numerous nonhuman models, and has been expanded over the last decade to formal, controlled human studies of CR. Given that long-term CR can create heavy challenges to compliance in human diets, the concept of a calorie restriction mimetic (CRM) has emerged as an active research area within gerontology. In past presentations on this subject, we have proposed that a CRM is a compound that mimics metabolic, hormonal, and physiological effects of CR, activates stress response pathways observed in CR and enhances stress protection, produces CR-like effects on longevity, reduces age-related disease, and maintains more youthful function, all without significantly reducing food intake, at least initially. Over 16 years ago, we proposed that glycolytic inhibition could be an effective strategy for developing CRM. The main argument here is that inhibiting energy utilization as far upstream as possible provides the highest chance of generating a broad spectrum of CR-like effects when compared to targeting a singular molecular target downstream. As an initial candidate CRM, 2-deoxyglucose, a known anti-glycolytic, was shown to produce a remarkable phenotype of CR, but further investigation found that this compound produced cardiotoxicity in rats at the doses we had been using. There remains interest in 2DG as a CRM but at lower doses. Beyond the proposal of 2DG as a candidate CRM, the field has grown steadily with many investigators proposing other strategies, including novel anti-glycolytics. Within the realm of upstream targeting at the level of the digestive system, research has included bariatric surgery, inhibitors of fat digestion/absorption, and inhibitors of carbohydrate digestion. Research focused on downstream sites has included insulin receptors, IGF-1 receptors, sirtuin activators, inhibitors of mTOR, and polyamines. In the current review we discuss progress made involving these various strategies and comment on the status and future for each within this exciting research field.

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.