01 October 2015

Slowing Down Aging: Are we ready? (4)

translated by Evgenia Ryabtseva
(The end, the beginning of the article is here.)

Activators of the signaling mechanism mediated by AMP-dependent kinase

AMP-dependent kinase (AMPK) is a conservative energy-sensitive serine-threonine kinase that is activated when the intracellular energy level decreases, which leads to an increase in AMP concentration (Ruderman & Prentki, 2004). Activation of AMPK increases the sensitivity of tissues to insulin, which leads to an increase in glucose consumption by skeletal muscles, as well as a decrease in glucose production in the liver and increased fatty acid oxidation in some tissues (Ruderman & Prentki, 2004; Ruderman et al., 2013). Scientists have developed several activators of this enzyme, including 5-aminoimidazole-4-carboxamidriboside (AICAR). In addition, a number of drugs approved by the US Food and Drug Administration (FDA), such as biguanides, thiazolidinediones, glucagon-like peptide-1 receptor agonists, salicylates and resveratrol, have the ability to activate AMPK (Coughlan et al., 2014).

The drug metformin belonging to the biguanide class, which is a first-line drug for type 2 diabetes, activates AMPK in the liver (Rena et al., 2013). Data obtained during experiments on animal models and in vitro indicate that metformin alters metabolic and cellular processes (Cabreiro et al., 2013) associated with the development of age-related diseases (Ruderman & Prentki, 2004). It is very important that metformin therapy increases the life expectancy of rats (Anisimov, 2010), mice (Martin-Montalvo et al., 2013) and nematodes (Cabreiro et al., 2013). Physical activity also stimulates AMPK and causes stimulation of glucose uptake and mitochondrial biogenesis during and after physical activity. The use of 5-aminoimidazole-4-carboxamidriboside has very similar effects (Hayashi et al., 1998; Song et al., 2002).

Currently, many studies are being conducted to clarify the possibility of demonstrating the effect of methomine on aging in patients with type 2 diabetes mellitus. It should be noted that when conducting a Prospective study of diabetes mellitus in the UK (UKPDS), in comparison with other antidiabetic drugs, the use of metformin reduced the risk of developing cardiovascular diseases (Group, 1998), the incidence of cancer and overall mortality (Wu et al., 2014), as well as, possibly, the extinction of cognitive functions (Ng et al., 2014). Proven safety and registered positive effect on longevity indicate the need to evaluate the effectiveness of metformin use to increase human life expectancy. However, in order to conduct clinical studies of the effect of metformin on aging, it is extremely important to analyze the known effects of its long-term use not only by relatively healthy people, but also by people with various diseases for whom the use of metformin can be detrimental. For example, according to recently obtained data, metformin stimulates the inhibition of mitochondrial glycerophosphate dehydrogenase and gluconeogenesis. This indicates the insufficiency of our understanding of the mechanisms of action of this powerful drug and the need for further research to study its effect on relatively healthy people (Madiraju et al., 2014). Gluconeogenesis is necessary during periods of fasting, but it also plays an important role in the metabolism of people who consume mainly ketogenic foods. This makes metformin potentially dangerous for people who follow fairly popular ketogenic diets.

Inhibitors of inflammatory mechanismsChronic mild inflammation is considered one of the characteristic signs of aging.

This phenomenon is so widespread that to focus on the immunogenic pathogenic components of many major age-related pathologies, including cancer, susceptibility to infections and dementia (Franceschi & Campisi, 2014), the term "inflamaging" (from the English. inflammaging) was coined (Franceschi et al., 2000; Franceschi & Campisi, 2014). Thus, inflammation is associated with a variety of age-related conditions, genes and mechanisms that regulate inflammatory processes and are potential targets for combating them (Franceschi & Campisi, 2014). Apparently, inflamaging is more complex than previously thought, with many different organs and tissues involved in the production of inflammatory stimuli (Franceschi et al., 2007; Cevenini et al., 2012). Their list includes the immune system, adipose tissue, skeletal muscles, liver and intestines. An extremely important role belongs to the intestine, which is the largest immune organ of the body, populated by billions of bacteria capable of releasing inflammatory signals both into the intestinal lumen and systemic blood flow (Biagi et al., 2010).

Despite its importance, the mechanistic details of the most important inflammatory stimuli are unclear and require further careful study. Inflammatory stimuli may be endogenous (e.g., chronic cytomegalovirus infection) (Sansoni et al., 2014), but they are highly likely to have an endogenous origin, possibly related to decay products formed as a result of continuous cell and tissue renewal (Franceschi & Campisi, 2014). For example, circulating mitochondrial DNA (mtDNA), recognized by immune sensors as a foreign nucleic acid, is a powerful inflammatory stimulus and its level in the blood increases as the body ages (Pinti et al., 2014). Blood concentrations of pro-inflammatory galactosylated N-glycans, which are also one of the most powerful biomarkers of human biological age (Dall'Olio et al., 2012), and pro-inflammatory circulating microRNAs ("inflammoMIR") (Olivieri et al., 2013) also increase with age, which may contribute to the development of inflamaging.

The most important triggers of age-related inflammation seem to be localized at the cellular and molecular levels. Physiological aging of cells is associated with a pro-inflammatory secretory phenotype associated with physiological aging (SASP, from the English Senescence-Associated Secretory Phenotype), the development of which is triggered by damaging agents (radioactive radiation, viruses) and, possibly, prolonged exposure to cellular decay products (Coppe et al., 2010). Physiological aging of cells can also spread to surrounding cells (Jurk et al., 2014). Secondly, DNA and telomere damage caused by reactive oxygen species and other agents can trigger an inflammatory response to DNA damage (Vitale et al., 2013). Thirdly, the activation of the NF-kB-mediated signaling pathway by inflammasomes may be caused by exposure to reactive oxygen species and cellular decay products (Youm et al., 2013). These mechanisms provide many targets for therapy aimed at suppressing inflamaging both locally and at the systemic level. Possible strategies for influencing these targets include the elimination of cells that have entered the phase of physiological aging (for example, by activating natural killers) (Tchkonia et al., 2013), deactivation of inflammasomes (Youm et al., 2013), adherence to diets rich in omega-3 fatty acids ("Mediterranean diet") (Berendsen et al., 2013), and other dietary strategies, such as the low-calorie diet discussed above.

In experiments on mice, anti-inflammatory drugs have demonstrated their potential as a means to increase life expectancy, but their effects are relatively poorly expressed (Strong et al., 2008). For example, nordihydroguyretoic acid (NDHA) and aspirin increased survival, but did not increase the maximum life expectancy. Other anti-inflammatory drugs had no effect on life expectancy, which indicates the need for further larger studies.

Modulators of epigenetic mechanismsThe term "epigenetics" describes inherited phenotypic changes caused by post-replicative chromatin modifications, rather than classical genetic changes based on mutations.

Such covalent and non-covalent modifications of DNA and proteins (e.g. histones) alter the state of chromatin conformation and entail corresponding changes in transcriptional activity (Jaenisch & Bird, 2003; Goldberg et al., 2007; Baker et al., 2008). Epigenetic effects can be mediated by three fundamentally different mechanisms: (i) methylation, (ii) posttranslational modifications of histones, and (iii) interference of non-coding RNA (Goldberg et al., 2007; Baker et al., 2008). The results of studies involving twins indicate that the genetic profile at birth determines life expectancy by only 25%, on the basis of which it is suggested that epigenetic factors also contribute to aging. Such epigenetic factors are likely to be influenced by lifestyle, diet, and external stressful influences. This indicates the potential for developing strategies to alleviate age-related cellular function disorders (Imai et al., 2000; Longo, 2009).

Despite the fact that manipulations of enzymes (sirtuins, histone acetyltransferases, histone deacetylases) regulating the status (de) of chromatin acetylation (and other targets) can increase the lifespan of yeast, fruit flies and roundworms, the role of histone modifications in regulating lifespan is very poorly studied. Recently, a model line of drosophila flies has been developed to assess the effect of such histone modifications on aging (Pengelly et al., 2013).

The polyamine spermidine contained in the body directly inhibits histone acetyltransferases, thus maintaining histone H3 in a hypoacetylated state (Eisenberg et al., 2009). Functionally, this leads to increased resistance to temperature and oxidative stress, as well as a noticeable decrease in the number of human and yeast cells dying as a result of necrosis. Surprisingly, this mechanism increases the chronological life expectancy of representatives of different species, including fruit flies, roundworms and human cells. These data are consistent with the existing knowledge base on the role of histone acetylation in maintaining longevity, including observations according to which deletion of the gene encoding histone acetyltransferase sas2 prolongs the replicative lifespan of yeast (Dang et al., 2009). Sas2 is an antagonist of Sir2 (sirtuin-2), an important histone deacetylase involved in the aging process, and its deletion stabilizes the levels of Sir2 in aging cells, thus providing a low basal level of acetylation of specific histone residues associated with the regulation of longevity (Raisner & Madhani, 2008). Finally, a simple way to change the age-associated acetylation of histones is dietary strategies that deplete the reserves of acetyl coenzyme–A, the only donor of acyl residues for acetylation reactions. Indeed, it has recently been demonstrated that the elimination of acetyl coenzyme-A is sufficient to induce autophagy and increase life expectancy. However, it is unknown whether these effects depend on epigenetic changes (Eisenberg et al., 2014; Marino et al., 2014).

Only non-toxic natural compounds such as spermidine and resveratrol, which provide chromatin deacetylation, can be considered for clinical studies (Morselli et al., 2011). However, it should be borne in mind that a mechanistic understanding of this strategy is a very complex issue, since drugs can have many unintended side effects and, even at the epigenetic level, an integrated reaction of many histone regions may be necessary for the manifestation of anti-aging effects. However, the data obtained in experiments on mice and with the participation of humans indicate that spermidine is potentially safe for testing its effects on human life expectancy, both dependent and independent of epigenetics. In one clinical study, a traditional Japanese cuisine product rich in polyamine (fermented soybeans) demonstrated a significant increase in the concentration of polyamine in the blood of participants, which was not accompanied by recorded undesirable side effects (Soda et al., 2009).

Other promising potential drugs and their targetsSignaling mechanism mediated by beta-2-adrenergic receptor (beta-2-adrenergic receptor)

Chronic use of beta-2-adrenergic receptor agonists increases mortality and morbidity (Ho et al., 2010).

Conversely, beta-2-adrenergic receptor antagonists or beta blockers reduce mortality after myocardial infarction and improve the health of patients with heart failure (Bristow, 2000; Ellison & Gandhi, 2005). Oral administration of beta-blockers metoprolol and nebivolol, initiated at 12 months of age, increased the median and average life expectancy of C3B6F1 male mice on an isocaloric diet by 6.4% and 10%, respectively (Spindler et al., 2013). None of the drugs had an effect on body weight and the amount of food consumed, excluding a low-calorie diet and a change in energy consumption as possible explanations for the described effects. The drugs also increased the lifespan of fruit flies without affecting the amount of food they consumed. The potential effect of prolonged use of beta-blockers on the duration of a healthy human life needs further study in mice and in clinical studies. Only after that it can be considered as an anti-aging intervention suitable for influencing healthy people.

Meso-nordihydroguyretoic acidNordihydroguyretoic acid is a lignin contained in high concentrations in rubber plants (V.E.Tyler, 1994).

Oral administration of nordihydrogwayaretic acid increases the life expectancy of fruit flies and mice (Spindler et al., 2014). In vitro studies have demonstrated the ability of nordihydroguairetoic acid to suppress intracellular inflammatory signals, proliferation of tumor cells, activation of receptors for insulin-like growth factor-1 and insulin-like growth factor-2, as well as oxidative phospholyriation (Pardini et al., 1970; Lu et al., 2010). Nordihydroguyretoic acid dose-dependently reduces body weight without changing the amount of food consumed, which indicates its ability to reduce nutrient absorption or increase the degree of calorie utilization (Spindler et al., 2014). Nordihydroguyretoic acid did not have an obvious toxic effect on mice, but its use was associated with an increase in the incidence of liver, lung and thymus tumors, as well as bleeding into the abdominal cavity (Spindler et al., 2014). When conducting preclinical studies of nordihydrogvayaretic acid as an anti-aging drug, less toxic derivatives of this compound should be used (Meyers et al., 2009; Castro-Gamero et al., 2013), although associations with manifestations of toxicity make it an unlikely candidate for drugs to increase the duration of a healthy human life.

Statins and angiotensin converting enzyme inhibitorsStatins (inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A reductase)

reduce the frequency of age-related rhythm disturbances (Ludman et al., 2009; Spindler et al., 2012) and mortality from many types of cancer (Zeichner et al., 2012; Nielsen et al., 2013). The positive effect of statins on health is due to a decrease in the level of protein isoprenylation (Spindler et al., 2012) and suppression of cholesterol biosynthesis (Ludman et al., 2009). Statins increase the lifespan and healthy lifespan of fruit flies by reducing protein isoprenylation (Spindler et al., 2012).

Angiotensin converting enzyme inhibitors are antihypertensive agents (Crowley et al., 2012) that suppress the activity of angiotensin receptor-1, thereby reducing secondary mortality and morbidity in myocardial infarction (Corvol et al., 2004; Gradman, 2009; Hoogwerf, 2010; Crowley et al., 2012), as well as mitigating the severity of adverse events in patients with congestive heart failure (Corvol et al., 2004; Gradman, 2009; Hoogwerf, 2010; Crowley et al., 2012). Combined oral administration of statins and angiotensin converting enzyme inhibitors increases the lifespan of mice by about 19% without changing the level of cholesterol in the blood serum and the amount of food consumed. However, monotherapy with each of the drugs is not effective. Statins and angitensin-converting enzyme inhibitors are generally well tolerated and, when used together, can increase the life expectancy of people with normal blood pressure and blood cholesterol. However, before their recognition as anti-aging drugs, it is necessary to conduct additional studies on the mechanisms of action of these drugs and their potential impact on the aging process. Given the widespread use of statins in many countries, it is quite possible to begin studying their effect on healthy life expectancy in a broader context on populations of relatively healthy people receiving therapy for a moderate increase in blood cholesterol levels.

Hexosamine cascade and glycobiologyThe result of the activity of the hexosamine cascade is the production of a metabolite of uridine diphosphate-N-acetylglucosamine (UDP-GlcNAc), which is a precursor of N-bound glycosylation in the endoplasmic reticulum and O-bound glycosylation in the cytosol and other compartments of the intracellular space.

Activation of this cascade by mutations leading to the appearance of a new function in the gene of the enzyme glutamine-fructose-6-phosphate-aminotransferase-1 (GFAT-1) leads to the appearance of an excess of UDP-GlcNAc and an increase in the lifespan of C.elegans roundworms (Denzel et al., 2014). Activation of the hexosamine cascade or an increase in the activity of GlcNAc enhances some aspects of protein quality control, increasing the activity of proteasomes, autophagy and degradation mechanisms associated with the endoplasmic reticulum, which together leads to a decrease in the severity of characteristic phenotypes in models of proteotoxic disease. In mice, ischemia/reperfusion of the heart triggers a stress reaction in the endoplasmic reticulum, one of the consequences of which is an increase in GFAT activity. There is evidence that an increase in GFAT activity or GlcNAc therapy protects against the harmful effects of ischemia (Wang et al., 2014b). In addition, the metabolite glucosamine associated with these mechanisms has been shown to increase the lifespan of rodents (Weimer et al., 2014). The results of these studies indicate the potential importance of the hexosamine cascade in regulating protein quality control and longevity. Earlier work in this area also suggests that excessive activation of the hexosamine cascade can lead to symptoms similar to those of diabetes mellitus (Hawkins et al., 1996). Thus, in order to obtain positive effects, it is necessary to optimize the activity of this cascade and the tissue reactions caused by it. An interesting fact is that the body of a long-lived rodent known as a naked digger contains a large number of hyaluronic acid glycoconjugates (consisting of GlcNAc and glucuronic acid subunits), presumably having protective properties against cancer and possibly other age-related diseases (Tian et al., 2013). There is also an assumption that glycoconjugates act as predictive biomarkers of human aging (Dall'Olio et al., 2013). It is quite obvious that the hexosamine cascade can provide new targets for the treatment of age-related diseases and further work in this direction deserves close attention.

Signaling mechanisms triggered by DNA damageStudies in mice have provided confirmation of the principle according to which the removal of the "upper" elements of the cascade of reactions developing in response to DNA damage (Exo1-dependent removal of the terminal site) and the "lower" control points of DNA damage (p21-dependent cell cycle arrest, Puma-dependent apoptosis) can prolong the period of tissue viability maintenance. the lifespan of aging mice without telomerase (Choudhury et al., 2007; Schaetzlein et al., 2007; Sperka et al., 2011; Wang et al., 2012).

This mechanism is a potential target for anti-aging intervention, as telomere dysfunction and DNA damage accumulate in aging stem cells and tissues (Jiang et al., 2008). Another point of contact discussed at the meeting between signaling mechanisms triggered by DNA damage, telomeres and aging was the paradoxical activation of DNA damage mechanisms under the action of mitochondrial reactive oxygen species (Schroeder et al., 2013), which increases the chronological lifespan of yeast by epigenetic suppression of subtelomeric DNA transcription. This indicates the possibility of influencing mitochondrial adaptive signaling mechanisms mediated by active oxygen forms to increase the duration of a healthy life.

Stem cellsIn the process of aging, stem cells of adult body tissues demonstrate functional disorders and an exponential increase in the number of precancerous mutations, the appearance of which is caused by internal cell defects and changes in the niche of stem cells, as well as the environment formed by circulating blood (Ju et al., 2007; Behrens et al., 2014).

Recent data suggest that reversing age-related disorders of stem cell stability and function may improve tissue maintenance and prevent age-related oncogenesis caused by stem cells (Patel & Demontis, 2014). 

Both stem cell-based interventions and other dietary and pharmacological approaches that induce regeneration and rejuvenation provided by stem cells are likely to become more important in the future to ensure a healthy life expectancy, but currently their testing as modulators of the life expectancy of model organisms is just beginning.

Retrotransposons or mobile genetic elementsThe results of studies on a variety of model systems, including yeast, fruit flies, and later experiments on mice and human cell cultures demonstrate that retrotransposons are activated when cells enter the phase of physiological aging, as well as during the aging of the body (De Cecco et al., 2013a,b; Sedivy et al., 2013).

Active retrotransposition is mutagenic and has a strong destabilizing effect on genomes. Given the importance of genome integrity in relation to aging and cancer risk, this indicates an interesting possibility of involvement of retrotransposition in the development of certain age-related pathologies that has not been studied. A number of nucleoside reverse transcriptase inhibitors currently used in HIV therapy, such as lamivudine and adefovir, block the retrotransposition of several endogenous elements, including elements of the LINE1 family, which is the only known active retrotransposon family in the human genome (Dai et al., 2011). Modern reverse transcriptase inhibitors have side effects, which prevents their long-term use as anti-aging agents. However, if studies on mice demonstrate their positive effect, new drugs can be developed to target elements of the LINE1 family in the human genome.

ConclusionAccumulating scientific data obtained in studies on various organisms and species indicate that exposure to aging will not only delay the development of chronic diseases, but also prevent many age-related changes in metabolism, while at the same time increasing the duration of a healthy life.

Scientists have identified a number of mechanisms involved in metabolism, growth, inflammation and epigenetic modifications that change the rate of aging and the likelihood of developing age-related diseases. Interventions that are potentially able to safely affect these mechanisms and trigger protective anti-aging reactions that increase life expectancy are getting closer to reality. These include periodic or prolonged fasting, adherence to a moderate low-calorie diet combined with a diet with a low glycemic index and restriction of protein intake, inhibition of the growth hormone/insulin–like growth factor-1 signaling axis, inhibition of the signaling mechanism mediated by TOR-S6K and activation of sirtuins or AMPK. It is also necessary to evaluate the effectiveness of additional pharmacological interventions, such as therapy with metformin, acarbose, spermidin, statins and beta-blockers. New strategies that are not yet ready for clinical trials, including the use of drugs that affect epigenetic modifications or inhibit retrotransposition, deserve close attention and additional research. Taking into account the material and technical aspects of conducting clinical trials, mainly aimed at increasing life expectancy or healthy life expectancy, the seminar participants came to the conclusion that primary studies should be planned in such a way that they allow assessing the therapeutic effect on age-related diseases and conditions (not on aging as such). At the same time, they must first be carried out on small cohorts for relatively short periods of time. Their main task should be to assess safety and portability. This approach is highly likely to provide early data on the most promising candidates, who will subsequently undergo longer and more thorough studies on their impact on aging.

In accordance with the title of the seminar "Interventions aimed at slowing down human aging: are we ready?", its participants believe that the time has come when it is necessary not only to consider certain therapeutic techniques as means for the treatment of age-related combined diseases, but also to begin conducting clinical trials, the ultimate goal of which is to increase the duration of a healthy life (and possibly longevity) of the human population, in compliance with the guiding principle of medicine "do no harm".

For references to the cited sources, see the original article.

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