ISSN 20790570, Advances in Gerontology, 2013, Vol. 3, No. 3, pp. 155–172. © Pleiades Publishing, Ltd., 2013. Original Russian Text © A.G. Trubitsyn, 2012, published in Uspekhi Gerontologii, 2012, Vol. 25, No. 4, pp. 563–581.

The Joined Aging Theory A. G. Trubitsyn Institute of Biology and Soil Science, Far East Branch, Russian Academy of Sciences, pr. 100letiya Vladivostoka 159, Vladivostok, 690022 Russia email: [email protected] Abstract—In attempts to develop an instrument for prolonging life, mankind has created more than 300 the ories of aging, each of which offers its own original cause of the agedependent degradation. Among them, there are many logically perfect theories based on actual, repeatedly checked facts, but none of them have given a practically significant result. The theory presented here is based on the conception that life is a phe nomenon that represents many interrelated physicochemical processes propelled by the energy of the mito chondrial bioenergetic machine. The gradual agedependent degradation of all vital processes is caused by a programmed decrease in the bioenergetics level. This theory unites all existing aging theories that are built on authentic facts; the data accumulated in different fields of biology have served the basis to show that the fun damental phenomena that accompany the aging process, such as an increase in the level of reactive oxygen species (ROS), a decrease in the general level of protein synthesis, the limitation of cellular proliferation (Hayflick limit), a decrease in the efficiency of reparation mechanisms are caused by the attenuation of bioenergetics. Each of these phenomena in turn generates a number of harmful secondary processes. Almost all of the current theories are based on one of these destructive phenomena or their combination. Hence, each theory describes one side of the aging process that is initially caused by a programmed decrease in the bioen ergetics level. The united theory makes it possible to understand the nature of the aging clock and explains the phenomenon of lifespan extension under the conditions of food restriction. Failures of attempts to develop a remedy for senescence are explained by the fact that the currently used manipulations with the sep arate secondary phenomena of bioenergetics attenuation are not capable of advancing longevity beyond the bounds of its natural duration, although they can improve the quality of life in old age. The only way to achieve unlimited healthy life is to find a way of managing bioenergetics. Keywords: aging, genetic program, bioenergetics, reactive oxygen species, Hayflick limit, general level of pro tein synthesis, reparation mechanisms, aging clock, longevity DOI: 10.1134/S2079057013030120

INTRODUCTION The dream of unlimited and healthy life has stimu lated the search for the causes of aging. Aging and lifespan limitation was explained by Hippocrates (460–377 BCE) in his work On Diet as the gradual loss of the natural heat given to each organism in some amount upon birth. An analogous thought about the attenuation of natural heat was subsequently advanced by Aristotle (384–322 BCE) in his work On Youth and Old Age, Life and Death, and Respiration. The views of these ancient thinkers persisted in science until the 19th century; moreover, they were supported by accu rate experiments in the late 19th century. A German physiologist, M. Rubner (1854–1932) carried out cal orimetric investigations on people of different ages and came to the conclusion about the gradual decrease in heat reproduction in an organism. However, no ways of affecting this cause of aging were proposed at that time, and the interest in it faded away in the 20th century. This theory was replaced by theories that explained the cause of the agedependent degradation by manageable factors. Almost each large discovery in

biology and related fields of science was accompanied by the origin of a new aging theory, and they reached a number of more than 300 by the late 20th century [76]. They are subdivided into two alternative groups: sto chastic theories assert that the organism degradation is caused by the injury of biopolymers by accidental fac tors, and the adherents of programmed aging believe that aging is controlled by a genetic program. The theoretical basis of stochastic theories is very convincing: as early as the middle of the 20th century, the sign of “lifespan” was shown to be uncontrollable by the individual (Darwin) natural selection by R.W. Medawar [75]. He noted that animals almost never lived to old age in a natural habitat and perished from different external causes, even in young age. As a result, natural selection cannot differentiate based on the sign of lon gevity; consequently, specific aging genes that deter mine a natural lifespan may not exist either. This opin ion is still prevalent [65, 66]. The evidentiary basis of the stochastic theories is also strong; experiments give indisputable proof that the number of damaging agents (such as ROS) and polymers damaged by them

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increases with aging, and the number of functionally impaired biopolymers grows due to the stochastic errors in the reduplication, transcription, translation, and processing processes. This is supposed to decrease the efficiency of the reparation mechanisms, disturb the homeostasis of tissue regeneration, and yield many other destructive processes that accompany aging. The idea of the programmed character of aging that was advanced by A. Weisman as early as the late 19th cen tury [115] was not supported for almost 100 years. It only came to be reanimated in the late 20th century under the pressure of empirical data, which indisput ably indicated that lifespan was at least partially deter mined by a genetic program [9, 11, 12, 105]. However, since there is no convincing mechanism of pro grammed aging, this viewpoint has not yet been widely recognized, although lifespan has already been shown to be controlled by the natural selection, and longevity genes have been shown to exist [14, 28]. The adherents of this idea are searching for the longevity genes almost at random by trial and error [63]. Many genes have been found, the modification of which affects the lifespan of different organisms. This has given birth to the idea that the aging process is multifactorial, i.e., it is controlled by many genes [2, 60]. At present, the attention of gerontologists is focused on several theories based on the reliably ascer tained processes, which can actually result in age dependent degradation, including growth in the level of ROS, a decrease in the general level of protein syn thesis, the interruption of cellular dividing after a cer tain number of duplications (the Hayflick limit), a change in the hormonal status, the weakening of repa ration mechanisms, etc. [4]. However, none of these theories has yet yielded practically significant results, despite significant efforts. This paper substantiates the joint aging theory, which shows how the destructive processes that under lie all the modern aging theories are determined by the only programmed process—the decrease in the bioen ergetics level (the degradation of the value of the Gibbs energy, ΔG). The presented bioenergetic mechanism of pro grammed aging has been composed using the materi als of several of the author’s previously published arti cles [13–17, 109, 110]. In addition, a significant part of the presented material has been published in the book Bioenergetics issued by InTech Publishing House [111]. BIOENERGETICS General Notion Any energy system is quantitatively described as follows based on the value of the motive force and the effect generated by it: F = kA. In mechanics, these val ues are force and work (k is the friction); in electro technology, they are electromotive force (EMF) and

current strength; etc. In chemical thermodynamics, in particular bioenergetics, these are the change in free energy (the Gibbs energy, ΔG) and flow (the rate of a reaction). The terms “bioenergetics level” and “level of energy production” are also used to designate the value of a motive force in bioenergetics. To specify these notions, let us note that the value ΔG is deter mined for macroergic coenzymes that function in the bioenergetic machine (ATP, NAD, NADPH, FAD, CSH, etc.) based on the ratio of the concentrations of their reduced forms to oxidized coenzymes and tem perature. For example, for ATP, ΔG = DG0 – RTln[ATP]/[ADP][Pi], where DG0 is the standard Gibbs energy (the value ΔG under the following condi tion: [ATP] = [ADP] = [Pi] = 1 M at 298 K), R is the gas constant, and T is the absolute temperature. The greater the negative value of the Gibbs energy, the larger the energy potential produced by the bioener getic machine. As follows from the abovepresented expression for ΔG, the only variable that determines the bioenergetics level of the cell in warmblooded animals is the ratio between the reduced and oxidized forms of macroergic coenzymes ([ATP]/[ADP], [NADH/NAD+], etc.). Here, following H. Westerhoff and K. van Dam [7], for convenience, we will call the value ΔG voltage and designate it using these variables. The value of the mitochondrial membrane potential (Δψ) expressed in volts is also voltage. AgeDependent Dynamics of Bioenergetics As we have mentioned, the decrease in the bioener getics level with aging was first shown according to the decrease in the heat production in a organism as early as the late 19th century and has been proven many times since then at a higher experimental level [46, 84, 119]. For example, J. Hayashi et al. [46] have mea sured the level of mitochondrial energy production according to the activity of cytochrome oxidase in fibro blasts taken from 16 people at an age of 0–97 years. Fig ure 1 shows the curve for the age dependence of the bioenergetics level, which was constructed using the results of their experiments. The curve shows that the average level of mitochondrial energy production gradually decreases with aging against the background of significant individual distinctions. Scheme of the Bioenergetic Machine In any eukaryotic cell, mitochondria produce about 90% of the energy; therefore, here, we will only consider the scheme of the mitochondrial bioener getic machine (Fig. 2). In many respects, the scheme of the operation of this machine is analogous to that of the operation of a heat power plant, which is more familiar to us. In both cases, the output voltage is gen erated at three stages. At an electric power plant, these stages are as follows (1) the energy of fuel combusted in a firebox (coal, oil, gas) produces the pressure of ADVANCES IN GERONTOLOGY

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steam; (2) the steam rotates a turbine; (3) the turbine rotates an electric generator that produces the output voltage. In a bioenergetic machine: (1) the energy of fuel combusted in the Krebs cycle (carbohydrates, proteins, and fats, which come from food) produces the primary energy potential [NAD(P)H/NAD(P)+]; (2) the transmembranous electrochemical proton gra dient (Δψ) is formed at the expense of its energy; (3) (Δψ) is the motive force for ATPsynthase that produces the output voltage [ATP]/[ADP]. If there are no excessive loads (at state 4 or a similar state), the change in the value [NADH]/[NAD+] entails the pro portional change in the values (Δψ) and [ATP]/[ADP] (just as at an electric power plant, the decrease in steam pressure will entail the deceleration of turbine rotation and decrease in output voltage). THE CAUSE OF THE AGEDEPENDENT GROWTH IN THE LEVEL OF ROS The agedependent growth in the level of ROS is the basis of the most popular freeradical (mitochon drial) aging theory. D. Harman laid the foundation of this theory as early as the middle of the 20th century [44], but its modified variant is still prevalent among the aging theories. Adherents to this theory assert that there are no specific aging genes, since longevity can not be controlled by the natural selection [65, 66, 75, 108]. They believe that agedependent degradation is mainly due to the injury of cellular structures by ROS produced mitochondria [108]. This theory charms researchers with its simplicity and clarity. Indeed, the production of ROS has been reliably ascertained to increase with aging; consequently, the number of inju ries in cellular structures also grows. The conclusion is evident. The mode for prevention of aging is also obvi ous, i.e., the neutralization of ROS by antioxidants. The supposed cause for agedependent growth in the production of ROS is that they provoke mutations in mitochondrial DNA (mtDNA), which leads to growth in defects in the respiratory chain that cause the subse quent growth in the production of ROS, an increase in the number of injuries, and a reduction in the bioener getics level. This produces a to vicious cycle that deter mines the progressive degradation of all organism functions. The origin of the freeradical theory immediately stirred up disputes that still continue. Many experi mental data have been obtained that contradict the assertions of this theory. The aging process has not been shown to cause a significant loss in the activity of the respiratory chain [22, 85]. These data are proved by the experiments, which do not directly deal with the electrontransport chain. Thus, the experiments on the intracellular organelle transport have shown that mitochondria from aged donors being transferred into ρ0 HeLa cells (HeLa cells without mtDNA) com ADVANCES IN GERONTOLOGY

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Fig. 1. Age dependence of level of mitochondrial energy in human fibroblast cells (according to the data of J. Hayashi et al., 1994 [46]. Bioenergetics level is determined in vivo according to activity of cytochrome oxidase.

pletely restore the functional activity [46, 57]. The transfer of the HeLa cell nuclei into cells of aged donors also removes mitochondrial dysfunctions [58]. The authors have come to the conclusion that mito chondrial dysfunctions are caused by nuclear factors, rather than mtDNA mutations. The conclusion that the agedependent accumulation of mtDNA muta tions is controlled by the nuclear genome was also recently drawn by Y. Yao et al. [117]. In the last decade, the tension of debates has reached its apogee. On one hand, in this theory, aging has already been asserted as no longer being an unsolved problem of biology [47, 51]. On the other hand, R. Howes has stated that the freeradical theory has lead gerontology to a deadlock; half a century of experiments with antioxidants have given no result in both the prolongation of life and the suppression of oldage cancer, diabetes, or cardiovas cular pandemics [53]. He is echoed by G. Bjelakovic et al. [24], who, based on vast factual material, have shown that antioxidants do not have a positive effect and, at best, do not increase lifespan. The following question is posed by D. Gems et al. [39] in the conclu sion of his recent review on this subject: is the theory actually dead, or does it only need modifying? The accumulated experimental data show that this theory is correct with regard to the harmful effect of ROS on an organism, but the cause of their agedependent growth due to the formation of the vicious cycle must be rejected. The leading role of the nuclear genome has been convincingly shown, and it is necessary to

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Krebs cycle NAD(P)H/NAD(P)+

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Fig. 2. Bioenergetic machine and ROS detoxification mechanism. Bioenergetic machine. Primary energy potential NAD(P)H/(NAD)(P)+ generated in the Krebs cycle is transformed by the electrontransport chain (ETC) into the electrochem ical proton gradient Δψ. ATP synthase reduces ADP to ATP at the expense of its energy. Mechanism of ROS detoxification. The •–

superoxide radical ( O 2 ) produced in the ETC is converted into hydrogen peroxide (H2O2) by SOD (MnSOD). H2O2 is then converted into water and molecular oxygen in the reaction catalyzed by glutathione peroxidase (GP) and partially in the Fenton reaction. The latter produces an extremely aggressive hydroxyl radical. Rate of detoxification is predetermined mainly by the activity of glutathione peroxidase (GP), which is supported at the expense of the energy coming from the bioenergetic machine through the chain of oxidationreduction reactions, which are sequentially catalyzed by nicotinamide transhydrogenase (NT) and glutathione reductase (GR). The mechanism of the growth in the ROS level. The programmed decrease in the bioenergetics level entails the proportional reduction in the activity of glutathione peroxidase, which increases the level of H2O2 in the mito chondrial matrix. Since hydrogen peroxide is also a substrate for the Fenton reaction, this augments the flow of H2O2 through this reaction, which increases the content of aggressive free radicals. As a result, the programmed attenuation of bioenergetics entails growth in the total level of ROS and their aggression.

find the mechanism by which the genetic program controls the level of ROS. Mechanism of ROS Scavenging In the process of aerobic metabolism, a small part of the electrons that flow from NADH through the respiratory chain directly react with oxygen, reducing •– it to anion superoxide ( O 2 ) [36, 96], which is able to injure cellular biopolymers. To prevent these injuries, the cell disposes of an echelon protection system, in which three functional lines of defense can be condi tionally distinguished, i.e., prophylactic mechanisms, ROSscavenging mechanism, and emergency mecha nisms. The prophylactic mechanisms either prevent •– the formation of O 2 , or oxidize the superoxide inversely into O2 in the place of its formation [27, 30, 95]. The

emergency mechanisms are activated in the case of the catastrophic growth in ROS, when the joint action of other mechanisms can no longer improve the situation [104]. However, ROS not only injure biopolymers, but also play an important role in regulating the activity of transcription factors, growth factors, and other intrac ellular signaling systems [29, 30, 91, 107]. Cells need ROS, but their concentration must be supported at a safe level. A specialized ROSscavenging mechanism exists in the mitochondrial matrix to support the •– homeostasis of ROS. The process of O 2 detoxifica tion by this mechanism is implemented at two stages (Fig. 2): at the first stage, the manganesecontaining mitochondrial SOD (MnSOD) transforms the super oxide produced by the respiratory chain into hydrogen peroxide [56, 95]. Then, after penetrating the cytosol, H2O2 is decomposed into water and molecular oxygen ADVANCES IN GERONTOLOGY

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by catalase; in the mitochondrial matrix, it is decom posed by the glutathione and thioredoxin systems (there is no catalase in the mitochondrial matrix) [21, 114]. The glutathione system consists of glutathione peroxidase (GP) and glutathione reductase (GR). The activity of GP is supported thanks to the energy of the oxidation of glutathione (GSH), which is concur rently converted in its oxidized sulfide form (GSSG). Glutathione reductase then catalyzes the reduction of the oxidized glutathione at the expense of the oxida tion of NADPH [21, 55]. NADP+ that is formed in the reaction is reduced again to NADPH in the isocitrate dehydrogenase reaction of the Krebs cycle [21] and in the reaction catalyzed by nicotinamide transhydrogenase (NT), which supports the dynamic balance between NADP/NAD+ and NADPH/NADP+ [113]. An analogous thioredoxin system that also consists of thioredoxin peroxidase (TP) and thioredoxin reductase functions in parallel with the glutathione system [81]. The activity of TP is analogously sup ported by the oxidation of thioredoxin, which is reduced by thioredoxin reductase at the expense of the oxidation of NADPH [70] (Fig. 2 does not show this parallel system in order to simplify the scheme). The reaction catalyzed by these peroxidases is simple. H2O2 takes two electrons from glutathione (thiore doxin) and two protons from the medium, and is decomposed into two water molecules as follows: H2O2 + 2e– + 2H+ = 2H2O. In this system, the direct detoxification of H2O2 is performed by GP and TP, and the remaining reactions are energy drives, through which energy in the form of highenergy electrons is transmitted from the bioener getic machine (from the Krebs cycle) to glutathione peroxidase and thioredoxin peroxidase, which sup ports their activity. The activity of any energydepen dent chemical reaction depends on energy supply [7]. Thanks to this, the activity of the ROSscavenging mechanism depends on the voltage of cellular bioen ergetics; the greater the value of NAD(P)H/NAD(P)+ generated by the Krebs cycle, the higher the activity of GP and TP and, vice versa, the attenuation of bioen ergetics leads to a decrease in their activity. Fenton Reaction In parallel to the two mentioned decomposition modes, there is also the third H2O2 decomposition mode catalyzed by biovalent iron ions, which is called the Fenton reaction. Being presented most simply, the Fenton reaction represents the chain of reactions, in which H2O2 is decomposed into water and molecular oxygen, and Fe2+ is regenerated as follows [37, 40]: Fe2+ + H2O2 → Fe3+ + •OH + OH–, Fe2+ + •OH → Fe3+ + OH–, •OH

•

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H O 2 + Fe2+ → Fe3+ + H O 2 , •

H O 2 + Fe3+ → Fe2+ + O2 + H+. In contrast to GP and TP, iron decomposes H2O2 due to its ability to be cyclically oxidized and reduced [73]. However, secondary free radicals and a number of highly active compounds that can cause a wide range of biological injuries are generated by this redox activity as byproducts [37, 72]. Among these com pounds, the hydroxyl radical (•OH) is an actual chem ical predator. The reaction capability is so high that, upon being formed, it immediately reacts with any biological molecule in its environs, which produce secondary radicals with different activity [43, 122]. •– Among O 2 , H2O2, and •OH, only hydroxyl radical is able to cause the rupture of a double DNA chain. Mechanism of AgeDependent Growth in Level of ROS Since the Fenton reaction takes place in parallel with the glutathione and thioredoxin peroxidase reac tions, hydrogen peroxide can be transformed into neu tral products in two ways, one of which is harmless, while another involves the additional production of highly aggressive ROS. According to the theorem about the partition of metabolic flows, the share of the total H2O2 flow (Qf) that passes through the Fenton reaction depends on the total activity of both peroxi dases (Ap), as well as the activity of the Fenton reaction (Af), as follows: Qf = Af/(Af + Ap). This entails an important consequence; the lower the activity of GP and TP, the greater the H2O2 flow through the Fenton reaction. The programmed agedependent decrease in the bioenergetics level lowers the activity of GP and TP, which increases the concentration of their sub strate, i.e., H2O2. Since hydrogen peroxide is also a substrate for the Fenton reaction, this strengthens the flow of H2O2 through this reaction with all of the abovementioned consequences. This leads to growth in both the total content of ROS and their aggression. Consequently, the agedependent growth in the level of ROS and their aggression is caused by the pro grammed decrease in cellular bioenergetics, and the freeradical theory itself passes from the class of sto chastic theories into the class of programmed theories. NATURE OF THE DECREASE IN GENERAL LEVEL OF PROTEIN SYNTHESIS Modern View on the Problem The rate of synthesis of each specific protein is finely regulated by transcription factors depending on the demand for it in the current situation. However, the proportional decrease in the synthesis rate of all proteins is also observed in the aging process. This phenomenon should be ranked as the second most important harmful process, which contributes to the

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progressive weakening of the support and reparation mechanisms [87]. Meanwhile, there is already a sig nificant amount of empirical data that make it possible to explain this phenomenon by the attenuation of cel lular bioenergetics, i.e., to transfer it from the class of stochastic phenomena into the class of programmed phenomena.

40S mRNA

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Fig. 3. Initiation of translation and its relationship with bioenergetics. The initiation process is divided into three stages. (1) The preceding double eIF2–GTP complex is bound with the initiatory mettRNA and then joint to the small 40S ribosomal subunit. (2) The formed complex is bound with the matrix RNA. Concurrently, GTP is hydro lyzed to GDP in the mettRN–eIF2–GTP–mRNA com plex: the energy is spent to form the bond, and inorganic phosphor is released in the environment. (3) After the 60S ribosomal subunit is joined, the preinitiation complex decomposes into components that enter the next initiation cycle, but eIF2 remains bound with GDP; moreover, the eIF2–GDP complex is stable and inactive. The substitu tion of GDP for GTP is catalyzed by the eIF2B nucleotide exchange factor, and the released GDP is reduced in the reaction GDP + ATP ↔ GTP + ADP, which is catalyzed by nucleotide diphosphate kinase (NDK). The formed ADP is reduced by the bioenergetic machine. The pro grammed decrease in the energy potential produced by the bioenergetic machine ([ATP]/[ADP]) decelerates the reinactivation of the eIF2–GDP complex and the entire process of translation initiation.

agedependent degradation [94]. S. Rattan, who has comprehensively studied the effect of the agedepen dent decrease in the protein synthesis rate, writes that it causes the insufficiency of enzymes for the support of reparation mechanisms and normal functioning of cellular metabolism; the inefficiency of the intracellu lar and intercellular signaling pathways; and a decrease in the production and secretion of hormones, antibod ies, neuromediators, and components of the extracel lular matrix [86]. It is believed that the decrease in the rate of protein synthesis is caused by the stochastic accumulation of molecular injuries during their expression and by DNA reduplication, transcription, processing, and translation processes, as well as by the

Mechanism of Controlling Rate of General Protein Synthesis The cellular bioenergetics was first discovered to affect the level of protein synthesis by D. Young [120]. He noted that the rate of the introduction of amino acids into a growing polypeptide chain depended on the income of the major fuel of the bioenergetic machine (glucose, pyruvate, lactate) into cells. The observed effect was supposed to be related to the level of ATP production. The rate of this process was later ascertained to actually be determined by the value of the GTP/GDP ratio, which in turn depends on the value of the ATP/ADP ratio [77, 121]. The dynamic balance between the values of these ratios, which is catalyzed by nucleotide diphosphate kinase (NDK), is supported in the cell. Subsequently, the quantitative parameters of the effect of the ATP/ADP and GDP/GTP ratios on the protein synthesis rate was investigated in detail by J. Hucull et al. [54]. By relying on the results of their investigations, the authors came to the conclusion that the change in the rate of general protein synthesis, which is caused by the changes in the ATP/ADP ratio and/or GTP/GDP ratio and takes place in the range that is compatible with the normal energetic level, was a physiological regulatory mecha nism. At the present moment, the molecular mechanism of protein synthesis has been well studied and expounded in detail in a number of reviews [78, 82, 87]. The general protein synthesis rate has been shown to be controlled at the level of translation [54, 64, 82]. Among three translation stages (initiation, elongation, and termina tion), initiation is a regulatory link [35, 78, 82]. The task of this stage is to sequentially join first 40S, then 60S ribosomal subunits to the molecule of matrix RNA (mRNA) and to assemble a polyfunctional 80S ribosome. This stage involves not less than 12 recycling eukaryotic initiation factors (eIF). The initiation starts with the junction of an initiation methionine transfer RNA (mettRNA) to a preexisting double eIF2– GTP complex (Fig. 3). The formed triple eIF2– GTP–mettRNA complex binds the mRNA with the 40S ribosomal subunit. Concurrently, GTP in its com position is hydrolyzed to the GDP, and the oxidation energy is spent to form the bond. After the 60S subunit is joined, this preinitiation complex decomposes into subunits, which then enter a new initiation cycle. However, eIF2 continues to be bound with GDP; moreover, a double eIF2–GDP complex is stable and functionally inactive, i.e., it is not able to enter a new ADVANCES IN GERONTOLOGY

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initiation cycle (to be bound with a new mettRNA). Its activation requires the replacement of GDP by GTP, which is catalyzed by the factor of eIF2B nucle otide exchange. The released GDP is reduced in the reaction GDP + ATP ↔ GTP + ADP, which is cata lyzed by nucleotide diphosphate kinase (NDK). The formed ADP is reduced in the mitochondrial bioener getic machine. The decrease in the energy potential produced by the bioenergetic machine (the ATP/ADP ratio) results in a decrease in the GTP/GDP ratio, which decelerates the reduction of the eIF2–GDP complex and entire process of translation initiation. Consequently, the general level of protein synthesis is regulated by the eIF2 recycling rate, which is in turn determined by the state of cellular bioenergetics. If the reaction of GDP reduction in the eIF2– GDP complex is interrupted, then protein synthesis is completely blocked in the cell [31, 34]. This is the basis for a series of natural mechanisms that protect an organism from every possible stress situation, i.e., the phosphorylation of the eIF2 αsubunit by different specific protein kinases blocks the reaction of the exchange of GDP for GTP, which causes the complete interruption of protein synthesis in a cell and subse quent apoptosis [31, 32, 35]. These specific protein kinases are expressed in a cell in the case of extreme situations, such as the occurrence of doublehelical replicative viral RNA [79, 93], irreparable injuries of the genetic apparatus [93, 117], catastrophic amino acid shortage [32], and malignant cell transformation [33]. Under normal physiological conditions, when there are no specialized protein kinases, the guanine nucleotide exchange, together with the general level of protein synthesis, is proportional to the existing level of cellular bioenergetics [54]. Consequently, the age dependent decrease in the general level of protein syn thesis is caused by the programmed decrease in the bioenergetics level. CAUSE OF LIMITED CELLULAR PROLIFERATION Modern Views on the Nature of the Phenomenon The decay of tissues is the most noticeable change in the aging process that enables the age of a person to be determined fairly accurately based on his or her external appearance. The cause of this phenomenon was revealed as early as half a century ago [48], i.e., higher eukaryotic cells do not divide infinitely; rather, they enter a nondividing but viable state after a certain number of duplications. For example, embryonic human fibroblasts can divide 53 ± 6 times during 302 ± 27 days and remain in the stationary state for 305 ± 41 more days [23]. This limitation of division called the Hayflick limit is the basis of the replicative aging theory, which has been recognized as one of the brightest modern theories of aging [3]. ADVANCES IN GERONTOLOGY

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The basic postulate of this theory is that the accu mulation of all nondividing cells disturbs the homeo stasis of tissue regeneration, which leads to tissue deg radation [52, 59, 118]. A convincing mechanism by which the division of senescent cells ceases was theo retically forecasted by A.M. Olovnikov as early as 1973 and then proved experimentally [42]. The eukaryotic chromosome ends from the 3'DNA end have multiply repeating TTAGGG nucleotide sequences called telomeres. They prevent the endtoend chromosome fusion, protect DNA from nuclease digestion, and play a role in the disjunction of duplicated chromo somes in the mitosis. Telomeres are synthesized in embryonic cells by a special telomerase enzyme, which is absent in most somatic cells. Due to the RNA primer, which is required during the initiation of DNA reduplication, the telomeric chromosome ends of somatic cells become 50–200 nucleotides shorter with each division. As a result, after a certain number of duplications, the telomeric end is exhausted, and divi sions cease due to chromosome erosion [59, 68]. The mechanism has been confirmed by many empirical facts, i.e., 90–95% of potentially immortal tumor cells have the telomerase activity, and their telo meric chromosome ends do not become shorter. The suppression of the telomerase activity in these cells results in the shortening of a telomeric end and termi nation of divisions, i.e., aging. The restoration of the telomerase activity turns them into immortal cells again. Meanwhile, the facts that contradict this expla nation were accumulated. The aging cells that have the telomerase activity have been discovered, and, vice versa, there are potentially immortal tumor cells that lack telomerase activity. However, the most convinc ing data were obtained by the group of researchers headed by M. Blasco [25]. They managed to obtain the mice zygotes that lacked the telomerase gene, but had fullsized initial telomeric chromosome ends. The mice that developed from these zygotes proved to be not only viable, but also fertile. This initial length of telomeres was sufficient to support the normal viability of six generations of mice. For example, in the first generation, the mice normally passed the stages of youth and maturity and died from old age, with 80% of the telomeres in the reserve. The abnormalities caused by the exhaustion of telomeric chromosome ends only appeared in the fifth and sixth generations. These data were confirmed by another group of researchers headed by E. Herrera [49]. They obtained an analogous mice line, but with a shortened initial telomeric end, and repeated the experiments of M. Blasco and the group of researchers. These mice were only viable for four generations. Abnormalities in late generations are related to the exhaustion of telom eres in the tissue cells with the most intensive division [59]. At present, the researchers of the telomeric mechanism are inclined to the following conclusion: the loss of a telomeric end actually results in chromo some erosion and death of a cell, but the termination

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G 2

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Fig. 4. Phases of the proliferation cycle. A cell sequentially passes four phases in the cycle. G1 phase (gap 1) is the preparation of everything needed for DNA reduplication; S phase is reduplication; G2 phase is preparation for mito sis. The cycle is completed by the M phase (mitosis) with the formation of a daughter cell. The rate of proliferation rate is caused by the duration of the G1 phase.

of cellular proliferation in the process of normal phys iological aging takes place earlier than this critical moment and the cell, which has exhausted its entire proliferation potential, still contains a large reserve of telomeres. The telomeric mechanism serves as an additional barrier on the path to the multiplication of malignantly transformed cells [59]. It could be con cluded that the telomeric apparatus was not initially involved in the mechanism responsible for the cessa tion of the division of senescent cells. Indeed, the results of the initial experiments carried out by L. Hay flick and P. Moorhead showed that a cell entered a nondividing but viable state after a certain number of duplications. A cell cannot remain viable if it has ceased to divide due to the chromosome erosion. Meanwhile, the question about the nature of the Hay flick limit has remained unanswered. It is likely that the alternative cause of this phenomenon must be sought in the mechanism of cell division. Cause for Termination of the Proliferation of Senescent Cells The cycle of cellular division (proliferation cycle) is subdivided into the following four phases [1, 99]: G1, S, G2, and M (Fig. 4). The G1 phase (gap 1) involves the synthesis of the precursor molecules that are needed for the DNA reduplication and duplication of all cellular structures in the forthcoming division. The next S phase (synthesis) involves the duplication of the amount of DNA, and, after a short G2 phase, a cell enters the M phase (mitosis). Many studies have shown that nondividing cells are all at the G1 phase. If a cell has passed this phase, it passes the others auto matically with an almost equal rate. Since senescent cells enter irreversible proliferation rest, from here on, we will only be interested in events that take place in the G1 phase.

The rate of cell division is controlled by regulatory factors, which are endogenous and exogenous for a cell; they represent the stimulators and inhibitors of proliferation. Let us exemplify this regulation based on the data from one of the first works in this field [69], which precisely reflect the essence of the phenome non. Different growth factors were observed to affect a cell culture of mice fibroblasts grown in a medium impoverished in exogenous regulatory factors. A cell was shown to enter the state of proliferation rest at the boundary of M/G1 phases (the G0 phase) immedi ately after mitosis ended. The exogenous platelet derived growth factor (PDGF) was required to bring the cell out of this state. Without this factor, no struc tural or biochemical changes took place in the resting cell, and it remained insensitive to other proliferation stimuli. This primary stimulus was called the compe tence factor. After receiving this signal, the cell started to perform the biochemical reactions for preparing a new division cycle, but they ceased after a while. The PDGF was no longer needed for the subsequent devel opment of biochemical events, but the epidermal growth factor (EGF) came to be required. Its income caused the prolongation of biochemical and structural changes; however, after a while, there was a new inter ruption at the boundary of G1/S phases, which was called the restriction point R. The last several hours of the G1 phase only passed through the stimulation with the somatomedin C (SmC). The latter two factors were called the first and second progression factors. Cells of each tissue are stimulated by their own growth factors. In addition to the growth factors, the passage of the cell cycle is regulated by a large group of inhibi tors [101, 102]. Cells can go out of the cycle and pass into the state of proliferation rest. Three types of rest are differenti ated: (1) Irreversible rest or a state of terminal differenti ation, in which cells lose the receptors to the growth factors and become incapable of returning to the pro liferation cycle (for example, nervous cells, secretary cells, cells of the cardiac muscle). (2) temporal rest is needed for cells to perform their working functions in the composition of some tissues. This takes place when a cell does not get the necessary proliferation stimuli by the growth factors or if a medium contains exogenous inhibitors that cancel their proliferation signal. These cells retain the integ rity of their receptor apparatus and are able to return to the cycle under the corresponding conditions (for example, liver cells, fibroblasts, etc.). (3) The proliferation rest of senescent cells that have entirely exhausted their proliferation potential is similar to temporal rest, in that cells retain their recep tor apparatus and the integrity of all the structures needed for division. Nevertheless, division itself does not take place. The first experiments on determining the causes for the termination of the proliferation of senescent cells ADVANCES IN GERONTOLOGY

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were carried out by the group of researchers headed by S. Rittling [92]. They observed over 11 biochemical reactions, which sequentially took place at the G1 phase in young and senescent cells. It was ascertained that the reactions all took place in senescent cells, just like in young ones, although at a lesser rate; however, senescent cells stopped at the restriction point and, with no reaction to the proliferation stimulus by the second progression factor, deepened into the state of rest. If these cells are again stimulated by proliferation factors during some time interval, they will again pass through all stages of preparation for the transition to the S phase and return to the state of proliferation rest again. Hence, the authors made the conclusion that the restriction point became impassable for senescent cells that had exhausted their entire proliferation potential. The events that take place at the restriction point are intensively studied mainly by the researchers of cancer genesis. Their interest is aroused by the fact that malig nantly transformed cells pass this point without stopping; meanwhile, the cycle is obligatorily retarded at this point in healthy dividing postembryonic cells and, as we have mentioned, this point becomes an insurmountable bar rier for senescent cells. At present moment, much success has been achieved in studying the biochemical events at this point. Cyclindependent kinases (Cdk) are major reg ulators of the reactions that take place in the division cycle. They are controllers for all events, i.e., they determine the sequence of reactions, as well as their duration and intensity [99]. The function of the cyclindependent kinases is simple. Figuratively speaking, newly synthesized gene regulatory proteins of the division cycle, which are designated as E2F, come off the translation line in a package. The retino blastoma protein (Rb) serves as this package. While these proteins are bound with Rb, they are not active. The cyclindependent kinases phosphorylate the Rb protein, after which the gene regulatory proteins are released and activate the protein genes, the work of which is necessary in the division cycle [101]. Cdk can exist in the active and inactive states. The activity of the cyclindependent kinases is regulated fairly com plexly [80], but one must know two principal aspects in order to consider the theme discussed here, i.e., (1) the Cdk is activated when joined with cyclin, which is specific to it (which follows from the designa tion) and (2) the formed active Cdk–cyclin complex can be inactivated again if the specific inhibitor of the cyclindependent kinases is joined with it. At present, mammals have been discovered to have eight types of the cyclindependent kinases, which designated by ciphers (Cdk1, Cdk2, etc.); ten types of cyclins designated by the Latin alphabet letters (cyclin A, cyclin B, etc.); and a large group of Cdk inhibitors, which have individual cipher designations (p1, p2, etc.). The passage of the G1 phase is regulated by three ADVANCES IN GERONTOLOGY

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Cdk (2, 4, and 6); cyclins A, D, E; and inhibitors p15, p16, p18, p19, p21, p27, and p57 [101, 102]. The studies on events that take place in the G1 phase have been increasing greatly in recent years; more and more new biochemical subjects have been discovered, and the ways that they interact have been revealed. The information on them can be found in a series of reviews and original papers [65, 88, 101]. In this paper, we will only touch upon the major events that are minimally sufficient to understand the causes for the interruption of divisions of senescent cells. Omitting needless details, the scheme of the pas sage of the G1 phase, which was expounded in [100, 102], can be presented as follows (Fig. 5). The level of the p27 inhibitor in a resting cell is high, which inhibits the reactions of the preparation for division. In response to the stimulation by the competence fac tor, cyclin D starts to be expressed in a cell and the active cyclin D–Cdk4 complex is formed, which phosphorylates the Rb. As a result, the gene regulatory E2F proteins are released, and the phosphorylated Rb degrades. Subsequently, the E2F2 proteins activate the protein genes needed for DNA reduplication in the S phase and genes of the cyclin E, Cdk2, and E2F. The resulting cyclin E and Cdk2, when joined, form the active cyclin E–Cdk2 complex, which starts to interact with p27, as well as to additionally activate the genes of the E2F regulatory proteins, which phospho rylate the Rb. It is important to note that the cyclin E– Cdk2 complex activates the genes of its own compo nents, i.e., it reproduces itself. This results in the for mation of a positive feedback loop that promotes the rapid removal of p27 and avalanchelike growth in the number of E2F proteins and Sphase proteins, which permits a cell to pass the restriction point. Moreover, the outburst of the E2F superexpression induces the synthesis of the p53 inhibitor that interrupts the E2F expression, which has become unnecessary in the S phase. However, this and subsequent reactions of the cycle are already beyond the interests of the discussed theme. The character of the interaction between the p27 and active cyclin E–Cdk2 complex was simulta neously and independently given a significant specifi cation by two groups of researchers [98, 112]. Before these works, the interaction between the p27 and active cyclin E–Cdk2 complex was believed to have the only consequence, i.e., the inactivation of the complex. Their experiments studied the kinetics of the molecular interactions between these compounds and showed that not only did the inhibitor inactivate the complex, but the complex was also able to attack the inhibitor, phosphorylating it by threonine 187. Figura tively speaking, the p27 inhibitor and cyclin E–Cdk2 complex compete for survival. The outcome of this competition is determined by the energy supply of the reaction. If the level of ATP is high, the cyclin E– Cdk2 complex has an advantage, i.e., it phosphory lates the p27, after which this inhibitor becomes a tar

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Cdk4

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(b) p27 Cyclin E

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Fig. 5. Simplified scheme of the control over the passage of the restriction point. In response to the stimulation by mitogens, the active cyclin D–Cdk4 complex that phosphorylates the retinoblastoma proteins (Rb) is synthesized in a cell. As a result, the E2F gene regulatory proteins are released, and the phosphorylated Rb degrades. The E2F proteins activate the genes of the pro teins needed for the DNA reduplication in the S phase, genes of cyclin E, and cyclindependent kinase 2 (Cdk2), as well as E2F. When joined with each other, cyclin E and Cdk2 form an active complex that interacts with the p27 inhibitor of cyclindependent kinases. There are two possible outcomes of this interaction. (a) In young cells, the bioenergetics level is within the physiological norm, and the cyclin E–Cdk2 complex phosphorylates p27. As a result, the inhibitor degrades, and cyclin E–Cdk2 phosphory lates Rb, which additionally releases the gene regulatory proteins. Since E2F activates the genes of cyclin E, Cdk2, and E2F, the positive feedback chain is formed, which promotes the rapid removal of p27 and avalanchelike growth in the number of the Sphase proteins, which permits a cell to pass the restriction point. (b) During the agedependent decrease in the bioenergetics level lower than some critical level, p27 forms a strong bond with the cyclin E–Cdk2 complex, and the latter loses its activity. As a result, the superexpression of the Sphase proteins does not take place, the level of p27 remains high, and the entrance to the S phase becomes impossible.

get for the ubiquitindependent proteolytic machine and is destroyed. If the bioenergetics level becomes lower than a certain value, then the p27 already inac tivates the cyclin E–Cdk2. As a result, the positive feedback loop of the E2F synthesis is blocked and the pathway to the S phase becomes impossible. The capa bility to inactivate its inhibitor is only immanent to the cyclin E–Cdk2 complex and has not yet been discov ered in other analogous complexes [112]. Thus, the central event at the restriction point of the G1 phase in the cellular cycle is the start of the self accelerating reaction cascade controlled by the

cyclin E–Cdk2 complex. This is a necessary condition for the removal of the p27 inhibitor and accumulation of all the precursors for DNA reduplication and cell division. This condition is only fulfilled at the normal physiological bioenergetics level. When bioenergetics decreases in aging cells to some threshold level, the cyclin E–Cdk2 loses its capability to inactive p27 and becomes its own target. As a result, the evacuation of the inhibitor is interrupted, and it becomes impossible to enter the S phase. All of this can be summed up briefly and somewhat exaggeratedly as follows: the passage of the restriction ADVANCES IN GERONTOLOGY

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point is hindered by the p27 inhibitor of the cyclin dependent kinases. A cell has a special pump for its evacuation. The efficiency of its works depends on energy supply. In case of the programmed decrease in the cellular bioenergetics lower than some threshold level, it ceases to pump out the inhibitor, and cell divi sions become impossible. DECREASE IN EFFICIENCY OF REPARATION MECHANISMS The agedependent decrease in the efficiency of reparation mechanisms is mentioned as one of the causes of the agedependent degradation by almost each stochastic aging theory, usually without explain ing the nature of the decrease itself. From the standpoint of the idea of the programmed aging mechanism, which is developed here, the reason for the weakening of reparation mechanisms is on the surface. The key role in restoring the injured DNA structures is played by the excisional reparation, dur ing which the injured sites are recognized and cut off in one of chains, and the formed gaps are then built up complementarily [71, 103]. This process, which fol lows the cutandpatch principle, is has multiple stages and is catalyzed by a large group of enzymes; moreover, each stage requires energy expenses in the form of ATP oxidation. As has been mentioned, the activity of any energydependent chemical reaction depends on energy supply [7]. Consequently, the pro grammed decrease in the bioenergetics level with aging will inevitably cause a decrease in the efficiency of reparation. Nature of the Aging Clock The answer to the question about the aging clock is among the key conditions for understanding the nature of aging itself. In the opinion of V.N. Anisimov, “the entire history of ideas and concepts in gerontol ogy can be briefly characterized as the history of the search for an aging clock” [4]. It is evident that the term “aging clock” should be understood as the mech anism that predetermines lifespan. At present, the mechanism of the biological clock that coordinates the physiological processes of an organism over time has been studied fairly completely [4, 90, 106]. It is also believed that the biological clock is related to the aging clock [8, 106]. Just as in a technical clock, the biological mechanism of time reckoning is based on the oscillation rhythms assigned by a special oscilla tory circuit. Time genes are contained in each cell of all eukaryotic organisms, which vary from onecelled organisms to higher animals and plants. In vertebrates, they are only active in the suprachiasmatic hypotha lamic nucleus. These genes express the proteins that inhibit their own transcription. These systems with negative feedback inevitably produce oscillations due to each link of a system that reacts to the signals of ADVANCES IN GERONTOLOGY

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other links with some lag. The greater this internal lag of the system, the larger the oscillation periods. The oscillatory circuit of the suprachiasmatic nucleus is adjusted so that its rhythms are close to the day illumi nation rhythms. The signals from these circadian rhythms are transmitted into the pineal gland (epiphy sis), which secretes melatonin in time with oscilla tions, with the maximum in at dark times of day. The basic destination of the suprachiasmatic oscillatory system is to change the melatonin production rate. The rhythmic fluctuations in the concentration of melatonin in the blood change the daily activity of the almost entire endocrine system, and, subsequently, the activity of all organism systems. There is a special mechanism for adjusting this clock to the daily rhythm, i.e., light information is transmitted from the eye retina photoreceptors into the suprachiasmatic nucleus and, as a result, the oscillatory rhythms are adjusted to the daily illumination rhythms. As age increases, the production of melatonin by the epiphysis decreases, and daily biorhythms are mis matched. This aggravates the diseases that accompany aging or is a direct cause of their origin, including sleep rhythm disorders, hypertension, sugar diabetes, immunity suppression, cardiovascular diseases, risk of malignant cell transformation, and many others [2, 4, 6, 19, 60, 83]. The introduction of exogenous melato nin at a suitable time of day significantly normalizes the work of the circadian mechanism and improves health, thus increasing lifespan [2, 5, 8, 20, 61, 90]. The entire assembly of empirical data indicates that the biological clock serves to coordinate the functions of different organism organs and tissues, but there is no information on its role in the aging clock [61]. This clock generates cycles one after another, but does not sum up their assembly. As for the programmed aging clock, it must contain an integrator of events that take place over time. The example of a primitive, but prin cipally correct organization of such a clock can be found in the philosophic doctrine of yogis. They believe that a person is given a certain number of res pirations at birth. Respirations take place in time, and some unknown executive mechanism aggravates the state of an organism after each respiration, i.e., the results of previous respirations (the course of the clock) are summed up and weaken the organism down to the zero level. This respiration aging clock can be slowed down or sped up; the less frequent the respira tion, the longer the life, and vice versa. Proliferation Clock of Programmed Aging The theory developed here makes it possible to out line the actual mechanism of the aging clock. Let us note that aging is immanent not only to organisms, but also to the cells of mammals that are cultivated in vitro and are not affected by the biological clock. L. Hay flick and P. Moorhead [48] noted in the conclusive part of their historical work on cell cultivation that the

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amount of cell divisions in a culture was only deter mined by internal factors, i.e., the number of duplica tions was programmed in each cell. This conclusion was drawn based on experiments in which the growth of a culture of fibroblast cells taken from human embryos was interrupted by freezing to –70°C for dif ferent periods. Independently of the duration of these periods and number of congelations, the irreversible termination of proliferation took place after the sum mary passage of about 50 divisions (passages). As has been shown above, the termination of cell prolifera tion is caused by a decrease in the bioenergetics level. With allowance for the experiments made by L. Hay flick and P. Moorhead, this means that the rate of bioenergetics attenuation is strictly related to the number of duplications. This gives us grounds to con clude that the genetic program switches the bioener getics level of a cell to a new lower value in the process of each regular division. Consequently, the lifespan of cells is counted based on the proliferation clock, i.e., the course of time is predetermined by the rate of divi sions and the executive mechanism (aging mecha nism) sums up the results of events that decrease the bioenergetics level after each duplication. According to the generally accepted definition, lifespan is the interval from birth to death. According to the calendar clock, the lifespan of fibroblast cells of a human embryo in a permanent culture is little more than 600 days (with allowance for the postproliferation period), and part of the cells of the same culture, which has undergone congelation, live longer by the congelation period. According to the proliferation clock, their lifespan is equal (50 divisions), i.e., the aging clock is not synchronized with the calendar clock. Are these conclusions applicable to the organism as a whole? The later works by L. Hayflick give an affir mative answer. The number of cell duplications in a culture in vitro was shown to be inversely proportional to the donor’s age, and the number of divisions of embryonic cells in animals of different species was shown to correlate directly with their specific lifespan. However, if cells in a culture are usually descendants of one cell and grow old almost synchronously, all is much more complex in an organism. First, the rate of cell divisions varies in different tissues: from the zero rate (e.g., nervous cells and cardiac hystiocytes) to an almost permanent one (e.g., epithelial cells). Second, each tissue represents a conglomerate of differentage cells. This heterogeneity arises as a result of tissues being constantly regenerated due to tissuespecific stem cells being involved in the process of division and differentiation in proportion to the death of senescent cells. It was shown that, as stem cells divided both in vitro [26] and in vivo [50, 62, 97], their proliferation potential decreased and they reached the Hayflick limit, i.e., stem cells also grew old. Relying on these and other data, A. Ho et al. [50] came to the conclu sion that a living organism was as old as its stem cells.

These and many other data indicate that the prolifera tion clock predetermines the aging rate of not only a cellular culture, but also an organism as a whole; moreover, the leading role in an organism is played by the aging clock of stem cells. Consequently, from the standpoint of the theory developed here, the aging clock counts out the number of past divisions rather than a calendar time. This clock assigns a degradation rate to an organism and, consequently, longevity. Specific lifespan is strictly controlled by natural selection [14, 110, 111]. It can be neither greater nor smaller than the optimal value predetermined by the environmental conditions. If these conditions change in the evolution process, lifespan in individuals of a species must also change; otherwise, the death of the species is inevitable. According to the proliferation aging clock, this can be achieved by the change in the initial value of bioenergetics and/or value of its decrease during each cell division. CAUSE OF GROWTH IN LIFESPAN IN CASE OF FOOD RESTRICTION The proliferation aging clock makes it possible to suggest the solution of one more inveterate problem of gerontology. As early as the 1930s, a paradoxical phe nomenon was discovered, i.e., strict food restriction (calorie restriction, dietary restriction), which seems to be harmful to an organism, increases the maximum specific lifespan. The interest in this phenomenon has not lessened; this manipulation remains the only method that reliably increases the lifespan of mam mals while maintaining their health. The mechanism of this phenomenon has not been revealed until now, although it is intensively searched for at all levels vary ing from evolutionary to molecular. The comprehen sive analytical review of the literature on this subject is presented in the book by V.N. Anisimov [4, 5]. Here, we will only briefly touch upon the aspects needed to understand the suggested explanation of the essence of this phenomenon. Absorbed food is known to fuel two processes in an organism, i.e., it is combusted in the bioenergetic machine and used as construction materials to built proper structures. At present, lifespan extension in the case of starvation is believed to be mainly caused by a decrease in ATP production due to the shortage of fuel that enters the bioenergetic machine. The suggestion is logically perfect and confirmed by the experiments, which show that ATP production decreases in the case of food restriction [74]. Moreover, facts have been obtained that cast doubt on this most widespread standpoint. First, calorie restriction causes the expres sion of mitochondrial genes and stimulates the pro duction of new mitochondria in cells [74], which makes it possible to support the ATP/ADP value (the output voltage of the bioenergetic machine) at a level that is immanent to animals with complete diets. The recently obtained data [38] show that these mitochon ADVANCES IN GERONTOLOGY

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Genetic program

Decrease in the bioenergetics level in proportion to cell division Growth in ROS Decrease in the protein synthesis rate production

Termination of cell proliferation

Decrease in the efficiency of reparation

Other energy dependent processes

Secondary destructive processes Aging (degradation of all organism functions) Fig. 6. Mechanism of aging. The genetic program decreases the bioenergetics level as cells divide, which increases the content of ATP in tissues, decreases the total level of protein synthesis, rate of tissue regeneration, efficiency of the reparation mechanisms, and generates a number of other harmful processes. Each of these phenomena in turn entails a series of destructive processes. Their growing destructive effect leads to the progressive degradation of an organism up to the level incompatible with life.

drial reconstructions by no means affect the phenom enon of lifespan extension caused by starvation. Sec ond, the extension of lifespan in animals on restricted diets is affected not only by the amount of calories consumed, but also by the content of food. Thus, the increased content of essential amino acids in the ratio of starving animals, such as methionine, tryptophan, cystine, and cysteine, decreases the lifespan extension effect, and, in the case of moderate starvation, even reduces it to zero [41, 123]. On the contrary, even if rations are complete, the shortage of some amino acids in food increases the lifespan and imitates the effect of food restriction [41]. The proliferation aging clock permits one to explain these facts and to advance a new mechanism of lifespan extension in case of food restriction. Starva tion results in a decrease in the rate of cell division due to the shortage of construction materials, in particular the shortage of irreplaceable components (essential amino acids and, as we can suppose, essential fatty acids). The decrease in the proliferation rate in the case of starvation is proven by direct experiments in vivo [116], as well as by the fact that injuries inflicted on animals on restriction diets heal more slowly due to the decelerated cell division [89]. The decrease in the rate of cell division in an organism is nothing but a decrease in the speed of the proliferation aging clock, which is what causes the extension of the lifespan counted out according to the calendar clock. In principle, the cause of lifespan extension is the same as in case of the temporal congelation of a cell culture; albeit, without the period when vital processes are completely switched off. It is natural that the effect of the decrease in the speed of the aging clock mani fests itself most strongly in the period of intense cell division, i.e., in the period of organism growth. ADVANCES IN GERONTOLOGY

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Organisms that survived a period of a sharp food short age are actually younger than organisms fed ad libi tum, which is what causes all of the observed positive effects, including lifespan extension. The changes in the range of expressed genes caused by starvation and reconstruction of a series of physiological processes [5] are likely determined by the mechanism by which scanty resources are redistributed in favor of the func tions that are the most significant for an organism’s survival. This mechanism is necessary to any organ ism, since the population of a species constantly varies under natural habitat conditions [28], and its periodic decrease is mainly caused by extinction due to starva tion. THE AGING MECHANISM According to all what has been stated above, the aging mechanism, which is a constituent part of the proliferation aging clock, is presented as follows (Fig. 6). The genetic program controls only one function in an organism, i.e., it decreases the bioenergetics level (the amount of natural heat) during each cell division. This raises the content of ATP, decreases the general level of protein synthesis, causes tissue aging, lowers the effi ciency of the reparation mechanisms, and yields a number of other harmful processes that directly depend on bioenergetics. These phenomena each pro duce a whole series of harmful destructive processes. Their destructive effect, which grows in proportion to the increase in the number of past divisions, leads to the progressive degradation of an organism to the level incompatible with life.

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CONCLUSIONS Immortality is an eternal dream that mankind has been ardently trying to make reality throughout its entire history. Even the scientifictechnical revolution of the 20th century has not approached us to the desired goal. Moreover, the theoretical base of modern gerontology deprives us even of the hope to achieve it in the foreseeable future. Actually, the stochastic theo ries consider aging to be due to the inevitable accumu lation of errors and, consequently, attempts at radi cally increasing the lifespan are doomed to fail. The programmed aging theories are also not optimistic, since they assert that aging is multifactorial, and almost every process that contributes to the degrada tion of the organism is controlled by its own genes; it is almost impossible to modify all of them. The presently expounded generalized theory shows that the entire diversity of the agingdependent destructive processes is caused by the only pro grammed process, which can only be controlled by several genes. According to the results of the research by S.V. Myl’nikov, these genes are not more than three in number in the D. melanogaster [10]. At first glance, this assertion is incompatible with the indisputable fact that there are many genes whose modifications slow down or speed up the aging process. However, this is apparently contradictory; the modification of any physiologically important gene disturbs the har monious ensemble of physicochemical processes of life, which have been polished up by the evolution for many million years. Changing the functional state of an organism (simply speaking, inflicting genetic wounds on it), these modifications must inevitably affect lifespan. If in this case modified genes hamper cell divisions without doing too much damage to the remaining functions, then they increase lifespan due to the decrease they caused in the speed of the prolif eration aging clock. An example of this is Ames dwarf mice and Snell dwarf mice, which live 50% longer than wildtype mice [5], which contain a mutation that restricts the production of growth hormone and other regulatory compounds that affect the celldivision process. If the modifications worsen health and affect proliferation insignificantly, then the effect will be the opposite. Let us note that the overwhelming majority of organisms with these modified genes (including the effect of lifespan extension) have deviations from the norm that are incompatible with the possibility that they live in a natural habitat. Apparently, none of these genes can be suggested to play the role of a natural aging gene without additional substantiation. The genes that actually implement the aging pro cess are yet unknown, but the prime cause of aging suggested here, in combination with the empirical material accumulated in different fields of science, makes it possible to indicate the directions of search ing for them; i.e., they are among the genes controlling the energy homeostasis of cells. These genes support

the constant bioenergetics level in intervals between cell divisions and decrease it during each duplication. One of the major conclusions of the abovestated theory is that no manipulations with separate second ary processes caused by the decrease in the bioenerget ics level can lead to the growth in the maximum lifespan. Some of them (e.g., the augmentation of melatonin production) can improve health, thus increasing lifespan, but only within the limits of a spe ciesspecific lifespan. This is evinced by the entire his tory of applied works in gerontology. The only possible way to achieve healthy and unlimited life is to develop methods for bioenergetics management, i.e., to mod ify the expression of aging genes so that their bioener getics level can either remain at a previous level during cell division, which will stop aging, or grow, which will result in the rejuvenation of an organism. The problem is complex but solvable in the near future; the bioener getic machine has already been studied fairly well, the regulator of energy homeostasis has become visible, and a mighty arsenal of experimental methods has been created. The time when mankind’s eternal dream comes true mainly depends on whether the conditions for studies in this field are created. REFERENCES 1. Alberts, B., Bray, D., Lewis, J., Raff, M., Roberts, K., and Watson, J.D., Molecular Biology of the Cell, New York: Garland, 1994. 2. Anisimov, V.N., Molekulyarnye i fiziologicheskie mekh anizmy stareniya (Molecular and Physiological Mech anisms of Aging), St. Petersburg: Nauka, 2003. 3. Anisimov, V.N., Priority fundamental gerontological studies: input of Russia, Usp. Gerontol., 2003, no. 12, pp. 9–27. 4. Anisimov, V.N., Molekulyarnye i fiziologicheskie mekh anizmy stareniya (Molecular and Physiological Mech anisms of Aging), St. Petersburg: Nauka, 2008, vol. 1. 5. Anisimov, V.N., Molekulyarnye i fiziologicheskie mekh anizmy stareniya (Molecular and Physiological Mech anisms of Aging), St. Petersburg: Nauka, 2008, vol. 2. 6. Anisimov, V.N. and Vinogradova, I.A., Lightdark conditions, melatonin and risk of cancer, Vopr. Onkol., 2006, vol. 52, no. 5, pp. 491–498. 7. Westerhoff H.V. and van Dam, K., Thermodynamics and Control of Biological Free Energy Transduction, Amsterdam: Elsevier, 1987. 8. Golub, V.V., Hypothalamus as a possible modulator of the rates of development and aging of mammals, Russ. J. General Chem., 2009, vol. 80, no. 7, pp. 1425–1433. 9. Goldsmith, T.C., The case for programmed mammal aging, Russ. J. Gen. Chem., 2009, vol. 80, no. 7, pp. 1434–1446. 10. Myl’nikov, S.V., Genetic determination of aging rate in some lines of Drosophila melanogaster, Usp. Geron tol., 1997, no. 1, pp. 50–56. ADVANCES IN GERONTOLOGY

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Translated by L. Solov’eva

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