Dietary Restriction: Critical Co-Factors to Separate Health Span from Life Span Benefits

Abstract

Dietary restriction (DR), typically a 20%–40% reduction in ad libitum or “normal” nutritional energy intake, has been reported to extend life span in diverse organisms, including yeast, nematodes, spiders, fruit flies, mice, rats, and rhesus monkeys. The magnitude of the life span enhancement appears to diminish with increasing organismal complexity. However, the extent of life span extension has been notoriously inconsistent, especially in mammals. Recently, Mattison et al. reported that DR does not extend life span in rhesus monkeys¹ in contrast to earlier work of Colman et al.² Examination of these papers identifies multiple potential confounding factors. Among these are the varied genetic backgrounds and composition of the “normal” and DR diets. In monkeys, the correlation of DR with increased health span is stronger than that seen with life span and indeed may be separable. Recent mechanistic studies in Drosophila ³ implicate non-genetic co-factors such as level of physical activity and muscular fatty acid metabolism in the benefits of DR. These results should be followed up in mammals. Perhaps levels of physical activity among the cohorts of rhesus monkeys contribute to inconsistent DR effects. To understand the maximum potential benefits from DR requires differentiating fundamental effects on aging at the cellular and molecular levels from suppression of age-associated diseases, such as cancer. To that end, it is important that investigators carefully evaluate the effects of DR on biomarkers of molecular aging, such as mutation rate and epigenomic alterations. Several short-term studies show that humans may benefit from DR in as little as 6 months, by achieving lowered fasting insulin levels and improved cardiovascular health. Optimized health span engineering will require a much deeper understanding of DR.

Introduction

C aloric or dietary restriction (DR), typically a 20%–40% reduction in ad libitum or “normal” nutritional energy intake, has been associated with increased life span in diverse organisms including yeast, nematodes, spiders, fruit flies, fish, mice, rats, and rhesus monkeys.⁴ (Note that we use the terms DR and calorie restriction [CR] synonymously. The literature sometimes refers to CR to mean decreased caloric intake and DR to mean restriction of one of more components of intake.) First observed in laboratory rats as long ago as 1935,⁵ the incredible diversity of organisms that respond to DR with increased life span has suggested that DR fundamentally slows the rate of aging, perhaps by nutritional stress-induced stimulation of pathways involved in survival. However, besides the obvious problems in consistently defining what constitutes aging beyond longevity, critics have pointed out that “normal” diet is a nebulous concept and that DR may merely define an optimal diet. Moreover, debate continues over whether DR actually slows the rate of aging versus only delaying the onset of age- associated diseases, at least in mammals. Additional relevant questions concern whether the effects of DR are truly universal and to what extent should results from invertebrates or even short-lived mammals correctly inform human health, given the large physiological differences.

The comparative maximum benefit of DR to life span appears to decrease with increasing organismal complexity. DR can increase life span of yeast three-fold, nematodes two- to three-fold, Drosophila two-fold, and mice 30%–50%.⁶ Maximal benefit is achieved when DR is begun early in life, and the later a DR regimen is initiated, the less the benefit for any particular species.

Mutations and drugs that confer longevity benefits have helped identify the key targets of DR. Especially in invertebrates, such studies have given credence to DR's possible universality and provide the strongest evidence that DR actually affects the rate of aging. Evolutionarily conserved molecular mechanisms for dietary restriction result in inhibition of nutrient-sensing pathways and alter organismal and cellular metabolism in yeast, worms, flies and mammals (Fig. 1). Specifically, inhibition of the target of rapamycin (TOR)/ribosomal S6 protein kinase (S6K) pathways by genetic knockout or by the TOR/mammalian (m)TOR inhibitor rapamycin results in increased life span in yeast, worms, flies, and mice. In worms and flies, rapamycin-mediated life span increase requires the conserved “metabolic brake” eukaryotic initiation factor 4E binding protein (4E-BP), autophagy (which increases with decreased mTOR/S6K activity), and reduced (S6K) activity. Low levels of nutrients associated with DR similarly inhibit the TOR pathway. Interestingly, rapamycin treatment of adult mice results in a 10% increase in life span in older adult mice,⁷ which may even exceed the benefit of DR in that model. In multi-cellular animals, DR inhibits insulin/insulin-like growth factor-1 (IGF-1) (IIS) pathways, which leads to increased increased life span via inhibition of phosphoinositide 3-kinase (PI3K) and Akt, and stimulation of master transcription factor DAF-16 (worms)/Foxo (flies, mammals?), which in turn activates multiple protective downstream pathways. Ultimately, the combination of IIS and TOR/S6K inhibition results in increased levels of enzymes that protect cells from oxidative damage such as superoxide dismutase (SOD) and catalase (except flies), proteins that stabilize protein conformation such as heat shock proteins/chaperones, autophagy, endoplasmic reticulum (ER) stress response, xenobiotic metabolism, and reduced translation and fat accumulation.⁶ Reduced levels of amino acids are thought to predominately inhibit the mTOR pathway, whereas glucose restriction targets the IIS pathway (Fig. 1). However, in humans, unlike other studied multicellular animals including rodents, DR by itself does not lower the IIS growth factor IGF-1; instead reducing protein intake lowers IGF-1,⁸ suggesting that humans may have altered mTOR/IIS regulation.

FIG. 1.

Evolutionarily conserved molecular mechanisms for dietary restriction (DR). Inhibition of nutrient-sensing pathways alter organismal and cellular metabolism in yeast, worms, flies, and mammals. DR down-regulates the target of rapamycin (TOR)/mammalian (m)TOR pathway and the insulin/insulin-like growth factor-I (IGF-1) (IIS) pathways leading to increased levels of superoxide dismutase (SOD), catalase (except files), heat shock proteins/chaperones, autophagy, endoplasmic reticulum (ER) stress response, and reduced translation and fat accumulation. Reduced sugar/insulin/IGF-1 eventually leads to increased levels of Daf16 (worms)/Foxo (mammals, flies), which acts on multiple downstream pathways to effect increased longevity. Reduced levels of amino acids, especially methionine, are thought to predominately inhibit the mTOR pathway, whereas glucose restriction not only targets the Daf16/IGF-1 pathway, but also activates master nutrient sensor adenosine monophosphate (AMP) kinase. Rapamycin, an inhibitor of TOR, extends life span in adult mice, at least as well as DR does. ATP, Adenosine triphophate; PI3K, phosphoinositide 3-kinase; AMPK, AMP-activated protein kinase. (Color image is available online at www.liebertpub.com/rej).

Increasing organismal complexity not only diminishes maximum benefit of DR on life span, but perhaps also decreases the consistency of the life span enhancement, which has created controversy as to the value of DR to extending human life span. However, even in invertebrates, DR has not always resulted in life span enhancement. Reported exceptions include Drosophila melanogaster (which more often than not, actually do respond to DR), the nematode Caenorhabditis remanei (unlike its cousin C. elegans), medflies, butterflies, the housefly, and the spider Latrodectus hasselti. In mammals, specifically rodents, the results are even more problematic. A meta-review of the literature by Swindell reveals that DR works more consistently in rats (median of experiments support a 14%–45% increase) than in mice (median of experiments support a 8% decrease in life span to a 20% increase).⁹ Especially problematic are mouse studies in which life span decreases or remains unaffected and those in which many animals die from non-aging effects. It is not reassuring that non-inbred mouse strains newly derived from wild animals tend to not respond to DR.

Unfortunately, technical differences in DR protocols, including diet composition and varied genetic backgrounds of the animals may account for much of the inconsistency, which makes interpretation of the data more difficult.⁹ Interestingly, even in cases where life span enhancement is not seen, often health span benefits are seen, including reduction of age-associated pathologies such as cancer, heart disease, and kidney disease, suggesting DR may have medical value independent of increasing life span.

Effect of DR on Non-Human Primates

If the maximum DR effect on life span does diminish with organismal complexity, perhaps it is because more complex organisms already make better use of the stress signaling pathways to maintain homeostasis. More complex animals have a greater need to coordinate responses efficiently among a large number of cells when subjected to stresses such as starvation, to reduce cell division/reproduction, and conserve energy. To assess the relevance of DR to humans requires study of animals more closely related to humans, such as other primates. Life span studies on humans are not practical, but two studies on rhesus monkeys have been ongoing since the 1980s at the National Institute of Aging (NIA) and the Wisconsin National Primate Research Center (WNPRC). Initial results from the WNPRC, reported in 2009, suggested that young animals (7–14 years old) undergoing DR had a trend toward a longer life span. This trend becomes statistically significant if animals that died from non-aging causes are not considered. Most importantly, they observed that DR significantly reduced the incidence of age-associated diseases such as diabetes, cancer, cardiovascular disease, and brain atrophy.² In an important recent study, Mattison et al. at the NIA report that contrary to the WNPRC study, DR had no effect on the survival of rhesus monkeys, regardless of whether DR was initiated in young monkeys (1–14 years old) or older animals (16–23 years old). Although 50% of young animals remain alive, statistical projection suggests that less than 0.1% of the DR animals will outlive the controls. Some health span–related parameters did improve: DR-treated older animals had lower triglycerides and glucose levels and DR-treated males had significantly lower cholesterol levels than untreated males. However, cholesterol levels in DR-treated females were unaffected, and in young DR animals, glucose levels were unaffected, whereas triglycerides were slightly lower only in males.¹

To explain the contradictory results between the NIA and WNPRC results, differences in diet have been invoked: The untreated WNPRC animals were allowed to feed ad libitum and as a consequence weighed 10% more than the NIA control animals, suggesting that the control animals in the NIA study may already be subject to a weak DR effect. Interestingly, the weight of rhesus monkey in both studies exceeds that of wild rhesus monkeys, raising again the problem of what constitutes a “normal” diet. Also, the NIA diet may have been healthier: Sucrose made up 28.5% of the WNPRC animal diet, but only 3.9% of the NIA diet, which may explain an increase in diabetes incidence (40% in the WNPRC vs. 12.5% in the NIA).¹⁰ The NIA diet also included more plant-derived micro-nutrients and phytochemicals than the WNPRC diet. It is also unclear if differences in the physical activity levels of the animals in the two studies may have played a role (see below). However, it is clear that DR results in at least modest increased health span in these non-human primate studies.

Physical Activity Levels and Fatty Acid Metabolism in Muscle May Play Critical Roles in DR

Katewa et al. show that DR induces an increase in fatty acid synthesis and breakdown in D. melanogaster. Inhibition of fatty acid synthesis or oxidation genes inhibited DR-mediated life span extension. Furthermore, this increase in fatty acid metabolic rate correlated with an increase in physical activity that was at least partially required for DR to extend longevity in flies. Increased fatty acid metabolism was monitored by feeding the flies with 14C-labeled glucose for 24 hr. Triglyceride synthesis rates increased 2.8-fold in DR animals, whereas chasing with unlabeled food for 60 hr resulted in a 63% decrease in triglycerides in DR animals. Triglyceride synthesis was unaffected in control animals. Together these results demonstrate that both lipogenesis and lipolysis are increased during DR. Using a strain that expresses an inducible RNA interference (RNAi) that targets acetyl coenzyme A carboxylase (dACC), DR-mediated life span increase was reduced (113% increase in controls to 52% in dACC-inhibited females and from a 22% increase to 5% in dACC-inhibited males). Knockdown of dACC also reduced starvation and cold stress resistance in DR treated flies.³

Using genome-wide transcription analysis, Katewa et al. observed that dACC inhibition reversed DR-mediated changes in a cluster of genes involved in muscle structure and function. To confirm that the effect was muscle specific, Katewa repeated experiments using RNAi targeting dACC in the fat body, neurons, and muscle using inducible promoters specific for each tissue type. Only muscle-specific dACC-targeted RNAi reduced DR-mediated life span enhancement. To dissect this effect, Katewa and colleagues used RNAi knockdown to target CG4389 and CG7834, which encode for mitochondrial long-chain-3-hydroxyacyl-coenzyme A dehydrogenase and electron transport flavoprotein β-subunit proteins, respectively. Similar to inhibition of dACC, muscle-specific knockdown of CG4389 or CG7834 significantly reduced the DR-dependent life span extension, demonstrating that increased fatty acid metabolism in muscle is required for DR life span increase. Interestingly, treatment with muscle-specific RNAi targeting dACC, CG7834, or CG4389 also resulted in a significant reduction in DR- associated physical movement.³

DR is reported to enhance spontaneous movement-related activity in diverse species, including flies, rodents, and primates, probably due to selection for increased foraging under conditions of nutritional scarcity. Katewa et al. confirmed that their DR flies had higher levels of spontaneous activity, but inhibition of dACC lowered spontaneous activity in DR-treated flies at all ages, showing dACC is required for the increased physical activity. To test whether spontaneous activity is necessary for DR, wing defects were induced genetically by expressing the cell death–inducing gene reaper in wings. In flies with defective wings, DR only induced a very modest 14% life span extension versus 61% for appropriate controls. As an alternate strategy, when flies wings were mechanically clipped, DR-mediated life span was modestly reduced from 97% to 33%. Although the effects of limiting physical activity are less than those seen with RNAi knockdown of dACC, increased physical activity is at least partially required for DR-mediated life span increase.

Finally, consistent with an important role for fatty acid metabolism and physical activity in DR, over-expression of AKH, a glucagon ortholog that maintains glucose and triglyceride homeostasis in flies and mammals, increased fatty acid metabolism, spontaneous physical activity, and life span by 33% in ad libitum–fed flies. As expected, over-expression of AKH did not further increase life span in DR flies.³

These results suggest that modulation of fatty acid metabolism and conditions that allow or limit physical activity may prove important to interpreting DR experiments in other systems, such as the rhesus monkey studies. There is a cautionary note here. In Katewa's study, DR was implemented by reducing the total amount of food available to the flies. When DR in flies involves reducing calories by only lowering sugar, fatty acid metabolism is probably unaffected, which may eliminate a physical activity effect on DR.¹¹

Medical Implications

Human data

First, it should be clear that invertebrate, rodent, and perhaps even rhesus monkey DR results may not translate well to humans. There are two types of studies that evaluate the potential impact of DR on humans. The first connects genetic mutations in the DR pathways to longevity and health span. Ecuadorians with growth hormone receptor (GHR) mutations that produce severe GHR and IGF-1 deficiencies do not seem to live longer, but are remarkably free of diabetes and cancer and have reduced incidence of stroke¹² Interestingly, insulin and mTOR expression are also reduced (Fig. 1). Alcohol abuse and accidents leading to premature death appear to limit any definitive conclusions regarding the effects this mutation may have on longevity, although it is curious that no exemplar of increased maximum longevity emerged.¹²

Although life span studies on humans may be impractical, shorter-term DR human health span studies have been reported, although many are small studies that require replication. The best-controlled human study is the NIA's CALERIE research program. The Phase I CALERIE clinical trial determined the short-term (6 or 12 months) effects of 20% or 25% CR in non-obese humans. DR subjects had decreased whole body and visceral fat, reduced body weight, reduced energy expenditure, improved fasting insulin levels, and improved fatty acid and inflammatory biomarkers (low-density lipoprotein [LDL], total cholesterol-to-high-density lipoprotein [HDL] ratio, and C-reactive protein [CRP]). Reduced bone density, a potential problem, was not observed. A longer 2-year Phase II trial is in progress. Regarding life span, the authors of the CALERIE study estimated that on the basis of comparison with rodent data, a 55-year-old male beginning a DR regimen would gain an average of 2 months of additional life span.^13
–15

Members of the Caloric Restriction Society (CRS) voluntarily limit total energy intake in the hopes of retarding aging. CRS members (predominately males with an average age 50±10 years who have undergone DR for an average of 6 years) when compared to age-matched controls consuming typical American diets, had a lower body mass index (BMI), reduced body fat, less total serum cholesterol, LDL cholesterol, total cholesterol/LDL, and higher HDL cholesterol. Fasting plasma insulin and glucose levels also were decreased for CRS members.¹⁶ Lower levels of plasma CRP and tumor necrosis factor-α (TNF-α) reflected decreased inflammation in CRS members. Circadian heart rate variability (HRV), a measure of autonomic function, was higher in CRS members, equivalent to people 20 years younger, while baseline average heart rate was lower. These data suggest re-balancing of the sympathetic/parasympathetic axis toward the parasympathetic in CRS members.¹⁷ Left ventricular diastolic function in CRS members was similar to that found in people about 16 years younger.¹⁸ Taken together, CRS members appear to have substantially less risk for cardiovascular disease and diabetes. Although these data derive from a small cohort, they suggest that DR could have substantial health span benefit in humans.

Composition of diet

Does composition of diet matter in DR? Although the early dogma was that only total calories were important in DR, subsequent data suggest that this may not be true and that the composition of the DR diet does indeed matter. The variation in results in rhesus monkey DR studies may be partially due to differences in the amount of sucrose in their diet (see above). The type of DR may impact the effect of physical activity on DR (see above). Furthermore, in humans, unlike rodents, severe CR does not alter IGF-1 and IGF-1:IGF1 binding protein-3 (IGFBP-3) levels. In contrast, moderately reducing protein intake from 1.67 grams/kg to 0.95 gram/kg reduces IGF-1 levels 27%.⁸ In humans, these data suggest that caloric-based DR may preferentially involve the mTOR/S6K pathway (Fig. 1). Interestingly, DR via protein restriction extends life span in rodents, although not quite as much as restricting total calories, and in particular restriction of methionine appears to be sufficient to induce much of the benefit of protein restriction, perhaps by reducing oxidative damage to proteins and lowering mitochondrial reactive oxygen species (ROS).^19,20 It has been hypothesized that about 50% of the life extension effects of DR in rodents may be due to methionine restriction.¹⁹

Health span benefits versus potential problems

Benefits

On the basis of clinical trials, potential health span benefits from DR in humans minimally include protection from diabetes and increased cardiovascular health. Moreover, data from the rodent and rhesus monkey studies suggest that DR may decrease the incidence of cancer as well, if these results translate to humans.

Potential drawbacks

DR reduces BMI and muscle mass.²¹ Although high BMI is considered unhealthy, low BMI is associated with increased mortality from all causes in middle-aged and elderly humans.²² Reduced muscle mass has been observed in DR humans. Loss of strength associated with reduced muscle mass may lower quality of life and conflict with the benefits of increased muscle mitochondrial biogenesis. Wound healing is diminished in DR-treated rats,²³ which suggests that wound healing be carefully investigated in humans undergoing DR. Genetic background may be of great importance in humans as well as in the model systems. For example, there is some evidence that DR accelerates the course of neurodegeneration in a mouse model of amyotrophic lateral sclerosis (ALS),²⁴ which may contraindicate DR for people with a genetic predilection for ALS. DR may have paradoxical effects on immune function. On the one hand, DR has been reported to increase the number of naïve T cells and the diversity of the T cell repertoire (which decrease in aging) as well as slowing the decline in antibody production, T cell proliferation, and antigen presentation in a variety of mammals (for review, see Spindler⁴). On the other hand, DR-treated mice have higher mortality after influenza infection, perhaps due to reduced reserves.^20,25 Also, DR in old rhesus monkeys decreases T cell proliferation and can produce lymphopenia.²⁶

Alternatives

Should DR prove safe and effective in humans, adherence to a low-calorie diet may prove difficult for many. Therefore, it is useful to find alternate ways to achieve similar health span benefits. There are two approaches: The discovery of drugs that mimic DR and the possibility of modifed DR which includes alternate day fasting (ADF), protein or methionine restriction, and combining a modest reduced-calorie diet with exercise.

Of the many reported therapeutics that increase longevity in rodents, rapamycin represents a rational choice (Fig. 1) that inhibits key regulator mTOR and extended the life span of both male (10%) and female adult mice (13%).⁷ However, unless and until a safe rapamycin analog is approved for human use that does not decrease glucose tolerance through inhibition of complex mTORC2²⁷ and lacks immunosuppressive activity, it can not be recommended for enhancing health span. Unfortunately, rapamycin's immunosuppressive capability is linked to its ability to bind mTOR, so a simple analog that elicits health span enhancement and is not immunosuppressive may be difficult.

The adenosine monophosphate-activated protein kinase (AMPK) agonist metformin (Fig. 1) is an agent that appears capable of inducing some of the effects of DR; specifically it may prevent the onset of diabetes and reduce the incidence of cancer. Metformin is used in the treatment of type 2 diabetes; it increases glucose uptake in muscle and peripheral insulin sensitivity and decreases gluconeogenesis in the liver. It reproduces up to 75% of the genome-wide gene expression changes seen with DR in old mice²⁸ and extends life span in some mouse strains (between 8 and 38%)^29,30 but not rats.³¹ However, it has been hypothesized that metformin induces a mild DR effect by suppressing appetite that may explain the increased murine life span.⁴

ADF, in which subjects alternate fasting, with ad libitum feeding represents a more attractive way to achieve DR, because it imposes less stringent requirements on an individual. In rodents, ADF has been reported to achieve comparable results to conventional DR both in terms of life span extension and increased health span without body weight loss, although more well-controlled studies are needed (for review, see Varady³²). Unfortunately, human ADF data is inadequate and contradictory reports about potential benefits do not allow for useful conclusions.³³

Even more intriguing is the possibility of using methionine restriction (MR) to achieve some of the effects of DR in humans, similar to those seen in rodents. Diets focused on fruits and vegetables can significantly lower methionine.³⁴ However, human studies are necessary to confirm the validity of MR.

Combining a caloric-restricted diet with exercise (CE) would appear to be a particularly attractive way to achieve the health span benefits of DR, if the energy lost in exercise is balanced by restricting fewer calories. In animals and humans, CE generally achieves similar results to DR,^14,35

–38 although in animals there appear to be few additional benefits beyond decreased loss of cardiac and skeletal muscle function in old age,^39,40 whereas in humans increased reduction in diastolic blood pressure, LDL cholesterol, and insulin sensitivity have been observed.^38,41

In humans, epidemiological studies indicate that continued exercise correlates with an average of 2 years of additional life span in the elderly.⁴² At the same time, inactivity plays a major role in the secondary aging of essential physiological functions, suggesting exercise alone may be of benefit to human longevity.⁴³

Biomarkers

Finally, it would be of great value in evaluating the potential benefits of DR in humans to have better biomarkers for aging at the molecular and cellular levels to assess how much fundamental alteration of life span is really possible without SENS. Some biomarkers to consider in future DR studies include evaluation of the somatic cell mutation rate genome wide, changes in telomere length, epigenomic changes, and perhaps the development of new biomarkers, for example, protein aggregation.

Conclusion

To understand the maximum potential benefits from DR requires differentiating fundamental effects on aging at the cellular and molecular levels from suppression of age-associated diseases, such as cancer. To that end, it is important that the investigators carefully target the effects of DR on molecular aging biomarkers, such as mutation rate and epigenomic alterations. Furthermore, large, well-controlled human studies are needed to confirm the benefits of DR. A combination of modest balanced diet and exercise may be a good compromise for improved health span while waiting for specific therapeutics to slow aging and/or to promote regeneration/rejuvenation.

Footnotes

Author Disclosure Statement

There are no competing financial interest.

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