The search for anti-aging interventions: From elixirs to fasting regimens

Abstract

Introduction

An organism’s lifespan is inevitably accompanied by the aging process, which involves functional decline, steady increase of a plethora of chronic diseases and ultimately death. Thus, it has been an ongoing dream of mankind to improve health span and extend life. In the last century, developed countries have profited from medical advances and improvements in public healthcare systems and better living conditions derived from their socioeconomic power to achieve a remarkable increase in life expectancy. The current US population reports estimate that the percentage of people aged 65 years and older in the USA will increase from 13% in 2010 to 19.3% in 2030. However, according to the WHO, age itself remains the greatest risk factor for all major life-threatening disorders, and the number of people suffering from age-related diseases is anticipated to almost double over the next two decades. The fact that healthspan has not increased at the same pace as lifespan is a source of grave concern (Mercken et al., 2012). Indeed, in 2009–2010 almost 70% of the adult population in the USA was obese or overweight and one in every three adults suffered from hypertension (NCHS, 2012). Both, obesity and hypertension represent major risk factors for stroke and cardiovascular disease. Although weight loss and increase in physical activity are generally prescribed to avoid such age-associated diseases, only a small percentage of people have the discipline to change their lifestyle accordingly (Wing and Phelan, 2005). The prevalence of age-related pathologies represents major psychological and social impediments as well as an economic burden that urgently needs appropriate interventions.

Dietary regimens and drugs that can slow the aging process continue to raise interest among the general public as well as the scientific and medical communities. Besides the direct applicability of such interventions to meet the need for improving health and longevity, they help provide valuable insights for basic research. Indeed, the molecular mechanisms underlying aging-associated defects appear to be interconnected and affect the same pathways as those responsible for diseases such as cancer, cardiovascular and neurodegenerative disorders. Therefore, efforts aimed at identifying extrinsic solutions to slow aging require a two-pronged approach: greater understanding of the molecular mechanisms involved in the aging process and application of this knowledge in a practical manner. Unfortunately, only a few proposed aging interventions have received good press, the reasons ranging from their intrinsic complexities to the multifactorial causes underlying aging. One of the problems in aging research is the relatively low reproducibility of experimental findings due to the simple fact that aging is a long-lasting process. A long experiment is more prone to develop artifacts that are difficult to control, hence increasing the possibilities and time windows for experimental discrepancies. Furthermore, it is our opinion that some inconsistencies in the field arise from over-interpretation of lifespan-shortening scenarios associated with aging, where, in many cases, the reduced lifespan may have rather resulted from collateral damage.

To consolidate the core of reliable advances in anti-aging research, the present review considers only pharmacological interventions, regimens and genetic manipulations that meet three highly selective criteria: (i) promotion of healthspan and/or life extension; (ii) validation in at least three model organisms; and (iii) confirmation by at least three different laboratories. There are only a few proposed aging interventions that have met such stringent criteria: fasting regimens, caloric restriction, exercise, and the use of low molecular weight compounds, including spermidine, metformin, resveratrol and rapamycin (Table 1). From a molecular point of view, these interventions may act through epigenetic mechanisms (histone acetylation/methylation), the insulin/TOR pathway, the Ras signaling pathway, mitochondrial function, proteostasis, autophagy, and stress resistance. This review summarizes the current knowledge on these promising approaches and critically evaluates their applicability as anti-aging interventions.

Why do we age?

The more complex a problem, the more important it may be to ask simple questions. So, why do we age? Actually, we do hold the capacity for immortality. The molecular clock in our germ line stem cells, which sustain gamete production, is kept at zero as evidenced by the fact that our offspring are not born with the father’s or mother’s age. Then, why do somatic cells age? Firstly, the maintenance of repair activity in all our cells represents a vast energetic demand and, secondly, the pressure of recombination and dying of generations allows organisms to adapt to changing environments (e.g., ice ages). This implies that aging may be an atavistic, adaptive and altruistic program, by which single cells or organisms eventually die for the benefit of the whole population in a highly coordinated (programmatic) fashion. In that case, groups harboring individual cells or organisms capable of dying in times of stress (e.g., nutrient deprivation, starvation) would have a selective evolutionary advantage over groups in which every single cell or organism is selfish. In fact, one of Darwin’s most famous theories is that selection can work at the group level. However, most evolutionary biologists reject this idea. For instance, Maynard Smith argued that if groups would altruistically inhibit the generation of offspring, an egoist would infect and take advantage. August Weismann maintained that an individual’s demise would depend solely on the fulfillment of its contribution to the species (sufficient reproductive activity) and not to the group. Further, aging as an adaptive program has been questioned because this process seldom occurs in nature, where most individuals die at relatively young age due to predators or infection. However, aging may start early in life. For instance, several lines of evidence indicate that the general physical fitness already of a 30-year old person is less than that of a 20-year old.

The last decade has provided evidence for the existence of group selection. Within chronologically aged yeast cultures, single yeast cells induce programmed cell death (PCD) as a result of the accumulation of oxygen radicals and metacaspase activation (Fabrizio et al., 2004; Herker et al., 2004). Genetic disruption of PCD-mediated cell removal improves short-term survival of aged cultures; however, the surviving cells lose their ability to re-grow, indicating an accumulation of damaged cells in the absence of PCD (Fabrizio et al., 2004; Herker et al., 2004). Hence, the demise of single individuals in an aged population has a cleaning function that allows only the fittest to survive. This evolutionary advantage can be simulated in a competition experiment carried out between PCD-functional and –defective yeast populations that mimic the encounter of a group containing altruistic individuals with one comprising only the egoistic kind. Strains with dysfunctional PCD have an initial advantage but are eventually overgrown and outcompeted by wild type cells (Fabrizio et al., 2004; Herker et al., 2004). Features like superoxide levels and regrowth frequency are connected to PCD, an adaptive mechanism required for programmed aging through selective advantage at least in yeast (Fabrizio et al., 2004; Herker et al., 2004). Many observations argue that programmed aging (sometimes referred to as a pseudo-program) also occurs in higher eukaryotes, for example, the rapid aging after the flowering of annual plants or the accelerated aging (progeria) of Pacific salmon after spawning. As to its possibility as a selective advantage in human populations, Darwin stated in his famous work “The Descent of Man”, “There can be no doubt that a tribe including many members who… were always ready to aid one another, and to sacrifice themselves for the common good would be victorious over most other tribes; and this would be natural selection.” The existence of some programmatic features during aging is a conditio sine qua non for a therapeutic approach to combat aging.

Behavioral anti-aging interventions

Healthy aging to combat diseases

Pharmacological anti-aging interventions

Although virtually all obese people know that stable weight loss would reduce their elevated risk for metabolic diseases and enhance their overall odds for survival, only 20% of overweight individuals are able to lose 10% of their weight for a period of at least 1 year (Wing and Phelan, 2005). A similar behavior can be found in most of the population with regard to food choice and exercise, possibly because the acute satisfaction of comfort overrides the potential long-term benefits that lifestyle changes bring. Thus, drugs that may work as CR mimetics have drawn general attention in recent years, with some of them yielding promising results in current anti-aging trials (Cloughesy et al., 2008; Patel et al., 2011).

Resveratrol is a polyphenol that is found in grapes and in red wine. Its potential to promote lifespan was first identified in yeast (Howitz et al., 2003) and has since gained fame because it was suggested to be responsible for the so-called French paradox (French winemakers do not suffer from cardiovascular diseases though enjoying a high-fat diet). In monkeys fed a diet high in sugar and fat, resveratrol attenuated peripheral inflammation in adipose tissue (Jimenez-Gomez et al., 2013), maintained pancreatic homeostasis by preventing β-cell dedifferentiation (Fiori et al., 2013) and improved vascular function, particularly pulse wave velocity (Mattison et al., 2014). Resveratrol supplementation prolongs the lifespan of mice fed a high-fat diet or fed every other day (Baur et al., 2006), but not of mice on regular chow. Resveratrol prolongs the life of flies (Bauer et al., 2004), worms (Baur and Sinclair, 2006), and the replicative lifespan of yeast (Howitz et al., 2003), but not yeast’s chronological lifespan (our unpublished observations). However, recent data claim that the extent of lifespan extension in worms and flies on a resveratrol-supplemented diet may be shorter than previously reported (Burnett et al., 2011). Nevertheless, resveratrol offers protection in models of stress- or age-associated diseases, including chronic overfeeding, insulin resistance, type 2 diabetes, or cardiovascular dysfunction (Baur et al., 2006; Lagouge et al., 2006). The mechanisms behind these effects may rely on the fact that resveratrol mimics some of the metabolic actions of CR, as a series of studies on humans have suggested (Timmers et al., 2012). Resveratrol interacts with many stress-related targets in the cell, including the mammalian NAD⁺-dependent deacetylase SIRT1 (Baur and Sinclair, 2006; Lagouge et al., 2006), although the resveratrol-SIRT1 interaction may be indirect (Beher et al., 2009). SIRT1 is a member of a family of proteins (sirtuins) that have been linked to longevity in yeast, flies and worms (Haigis and Guarente, 2006; Kaeberlein et al., 1999; Mouchiroud et al., 2013), but the effects and extent in lifespan extension have been challenged (Burnett et al., 2011). Still, recent evidence in mammals suggests a connection between sirtuins and a longevity-promoting function; for example, a study indicates that brain-specific overexpression of SIRT1 is sufficient to promote longevity in mice (Satoh et al., 2013). Overall, it is clear that resveratrol or SIRT1 overexpression prevents several age-associated diseases and pathogenic conditions in mice, including oxidative stress in the aging heart, neurodegeneration, or diabetes (Haigis and Guarente, 2006). Importantly, activation of autophagy by resveratrol is required for lifespan extension in C. elegans (Morselli et al., 2010).

Rapamycin, an inhibitor of the TOR kinase, is a natural product secreted by a soil bacterium originally discovered on Easter Island (also known as Rapa Nui, hence the name rapamycin). Before its introduction in the anti-aging field, rapamycin was already well known as an immunosuppressive drug for the treatment of allograft rejection. Rapamycin is a strong inducer of autophagy and it extends lifespan in all organisms tested so far, including yeast, flies, worms and mice (Bjedov and Partridge, 2011; Kapahi et al., 2004). Middle-aged mice on a diet supplemented with rapamycin survived significantly longer (Harrison et al., 2009); however, reports of the potent immunosuppressive properties of rapamycin suggest that the drug is unsuitable for lifespan extension in humans. Moreover, long-term administration of rapamycin has been found to cause a variety of adverse health effects in patients, including impaired wound healing, anemia, proteinuria, pneumonitis, and hypercholesterolemia. Chronic inhibition of mammalian TOR (mTOR) function by rapamycin promotes insulin resistance and diabetes in laboratory mice (Lamming et al., 2012). It is interesting that intermittent rapamycin feeding, which was recently shown to also increase lifespan in mice, opens up new opportunities to avoid immunosuppression and its potentially detrimental effects on lifespan extension (Anisimov et al., 2011a). Because of its anti-proliferative properties, rapamycin or its analogs (rapalogs) are used as anticancer drugs (Benjamin et al., 2011; Guba et al., 2002).

Spermidine is a naturally occurring polyamine that triggers autophagy and, thereby, lifespan extension when supplemented in the food supply of yeast, flies and worms (Eisenberg et al., 2009). Endogenous spermidine concentrations decrease as the organism age, including in humans with the exception of centenarians (Pucciarelli et al., 2012). Acute injections of spermidine in mice activate autophagy in multiple organs (Morselli et al., 2011) while chronic spermidine feeding promotes increase in healthspan (Eisenberg et al., 2009). The impact of long-term effects of spermidine on autophagy has not been investigated. Spermidine has been found to impede a number of neurological pathologies and induce neuronal autophagy. It inhibits neurodegeneration and alleviates pathogenesis and motor dysfunction in a mouse model for TDP-43 aggregation-induced frontotemporal dementia and amyotrophic lateral sclerosis (Wang et al., 2012). Moreover, age-associated memory loss in flies is reversed by spermidine in an autophagy-dependent fashion (Gupta et al., 2013). In yeast cells, spermidine controls transcription of autophagy-relevant genes through hypoacetylation of several promoter regions (Eisenberg et al., 2009). Intriguingly, the promoter region of ATG7, an essential autophagy gene, remains selectively acetylated, which allows for its transcription in a state of general gene silencing (Eisenberg et al., 2009). Lifespan extension in two short-lived mouse models has been reported after either oral administration of spermidine or the upregulation of bacterial polyamine production in the gut (Matsumoto et al., 2011; Soda et al., 2009). It is possible, however, that factors other than polyamines are secreted by gut bacteria to influence longevity.

Metformin, a biguanide first isolated from the French liliac, is the most prescribed drug for the treatment of type-2 diabetes. Metformin decreases hepatic gluconeogenesis and increases insulin sensitivity (Berstein, 2012). It is a potent, indirect activator of adenosine monophosphate-activated protein kinase (AMPK) (Scarpello and Howlett, 2008), an enzyme involved in cellular and whole-organism energy balance, as well as in glucose and fat metabolism. Interestingly, AMPK is also induced by CR. Upon metformin exposure, AMPK is activated by an increase of the AMP/ATP ratio, thereby inhibiting mTOR, a protein kinase involved in the control of cellular proliferation and tumor growth (Dazert and Hall, 2011). Although the direct target of metformin is not known, it indirectly inhibits complex I of the electron transport chain (El-Mir et al., 2000; Viollet et al., 2011). Therefore, metformin compromises ATP production in mitochondria, leading to an increase of the AMP/ATP ratio without ROS accumulation. The transcription factor SKN-1/Nrf2 is activated upon metformin treatment, resulting in increased expression of antioxidant genes and subsequent protection from oxidative damage (Berstein, 2012). Metformin increases mean and maximum lifespan in different female mouse strains predisposed to high incidence of mammary tumors (Anisimov et al., 2011b; Martin-Montalvo et al., 2013). Recently, we and others have been testing pharmacological interventions that mimic the effects of CR and can delay aging and the incidence of age-related diseases (Anisimov et al., 2011b; Baur et al., 2006; Harrison et al., 2009; Martin-Montalvo et al., 2013). Some metabolic effects of metformin resemble those of CR, even though food intake was increased (Anisimov et al., 2011b; Martin-Montalvo et al., 2013). At a biochemical level, metformin supplementation is associated with inhibition of chronic inflammation and reduction in oxidative damage, two well-known factors that compromise health and lifespan (Martin-Montalvo et al., 2013). There are now several reports demonstrating that the effects of metformin on transcription mimic the gene expression profile of animals on CR through the transcriptional regulatory pathways controlling lifespan extension (Anisimov et al., 2011b; Martin-Montalvo et al., 2013).

Common molecular denominators

Conclusion

The purpose of aging research is the identification of interventions that may avoid or ameliorate the ravages of time. In other words, the quest is for healthy aging, where improved longevity is coupled to a corresponding healthspan extension. Unfortunately, numerous claims about substances or behaviors predicted to hold such benefits have been proven erroneous. In recent years, however, fasting and exercise regimens, and a number of small molecules have been shown to prolong life and/or sustain health late in life in model organisms as different as yeast and monkeys. All these interventions are compatible with the concept of programmed aging and, thus, amenable to interventions aimed at modulating molecular components and pathways responsible for the aging process. Autophagy up-regulation, improved stress resistance and mitochondrial efficiency are among the cellular functions needed to hold off features associated with accelerated aging, such as free radical generation, excess caloric intake, chronic hyperglycemia, and fat accumulation. More research is needed to gain further insights into the long-term effects of these interventions alone and in combination, and clarify their applicability to humans. The current epidemics of obesity, diabetes and related disorders constitute major impediments for healthy aging. It is only by extending the healthy human lifespan that we will truly meet the premise of the Roman poet Cicero: “No one is so old as to think that he may not live a year.”

Acknowledgments

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