Caloric Restriction Diet Induces Specific Epigenotypes Associated with Life Span Extension

The lifespan of Wistar rats on caloric restriction diet (CRD) is correlated with the changes in prooxidantantioxidant balance, in the contents of triiodothyronine, and thyroxin (epigenotype characteristics). It has been shown that in a month after the moment of one-month-old rats began to receive CRD the part (up to 15 %) of experimental animals died without any apparent cause, irrespective of the degree of calorie restriction (40 %, 57 % and 60 % weight-loss). The rest of animals with 40 %, 57 % and 60 % weight-loss had longer life span in comparison with control group. The CRD-induced life-span prolongation in animals was accompanied by the induction of specific epigenotypes featured by acceleration of the electron transfer rate in electron transport chain and subsequent reduced production of reactive oxygen species and increased antioxidant activity. The activity of glutathione reductase, glutathione transferase, NADHcytochrome C reductase, isocitrate dehydrogenase was elevated. The activity of Se-dependent glutathione peroxidase was higher more than 30 times as compared with control. Likewise, the epigenotypes of animals with 40 %, 57 % and 60 % weight-loss CR, were characterized by 37, 43 and 56 % decrease in triiodothyronine and 50, 25, 39 % decrease in tyrosine, respectively. The observed induction of specific epigenotypes is associated with higher life-span and is related to the multivariant metabolic strategies of adaptation to CRD.


INTRODUCTION
The first study of life-span gain possibility in experiments by caloric restriction diet (CRD) was published more than 70 years ago by C.M. McCay [1].In 60-th of the last century in Biology research institute of the Kharkov National University in laboratory of professor V.N.Nikitin the complex research of CRD influence on the life-span of Wistar rats was conducted [2,3].The conversion of one-month-old animals to CRD was shown to be accompanied by decrease of metabolism rate [4], rate of physiological systems becoming [5] and extension of average and maximal life-span [6].
For the last years there the numerous studies on CRD influence on average and maximal life-span of different objects from yeast cells [7] to monkeys [8] are conducted.And now one may say about formed conception of direct relation of metabolism rate and lifespan.The free-radical theory of aging conformed to this conception [9,10] It was shown that calorie-restricted animals have decreased rate of reactive oxygen species generation and as a consequence number of products of free-radical damages of macromolecules in organism [11,12].As known in case of "excess" of It would seem there is a logic direct relation between CRD and free-radical hypothesis.However along with destructive processes reactive oxygen species induce the synthesis of antioxidants able to neutralize their cytotoxic action [15][16][17].
Despite of vast numbers of researches on CDR model some specialists prejudiced verity of life-span extension and supposed that it could be only effect of animal selection [18,19].
One may assume that CRD executes some interrelated functions: selection of genotypes with potentially high life-span, induction of antioxidant system that can provide resistance of organism both to reactive oxygen species, and to others stress-factors.
Earlier we assumed that conversion of animals to CRD induced the formation of specific epigenotypes providing extension of average and even maximal lifespan [18].For epigenotypes providing potentially high life-span the cooperative changes in set of metabolic systems and first of all in prooxidant-antioxidant, hormonal and also nucleic-protein and lipid metabolism can be typical [20][21][22].
On this basis, the aim of this work was to research mitochondrion functional activity, content of some products of free radical processes, activity of glutathione-dependent and NAD + -dependent enzymes of mitochondrion of rat liver and also content of thyroid hormones in blood serum of rats maintained during 2 months (from one month age) on CRD, providing different degree of animal growth inhibition.

MATERIALS AND METHODS
The research was conducted on males Wistar rats maintained at standard vivarium conditions in accordance with the guidelines of the European Convention for the Protection of the Vertebrata using for the experimental and scientific aims (Strasburg, 1986).The animals of control groups were fed ad libitum.The rats of experimental groups at one-month age were conversed from standard diet to one of calorie restricted diets resulted in growth inhibition of different degree: diet by McCay-Nikitin [2], resulted in growth inhibition by 63 % («severe»), and two diets resulted in less growth inhibition, -by 57 % («moderate») and 40 % («mild»).At 3-and 24-months age the part of rats was decapitated at ether narcosis; the blood serum, and mitochondrion and postmitochondrion fractions from liver were collected.In each group for assessment of life span were used 30-40 animals [23].The thyroxin ( 4 ) and triiodothyronine ( 3 ) contents in blood serum were estimated using the standard radioimmunological kits ("Total T4 RIA" and "Total T3 RIA", IMMUNOTECH, Czech Republik).The rate of respiration and oxidative phosphorylation at FAD + -(succinate) and NAD + -(glutamate+malate,oxibutirate)-dependent substrate oxidation in mitochondrion was estimated polarographically: the rate of oxygen consumption was registered in nonphosphorylating (V 2 ), phosphorylating (V 3 ) and disengaged (V 3 ) states; the efficiency of phosphorylation was estimated (ADP/O; the proportion of ADP quantity transformed in ATP, to oxygen quantity consumed by mitochondrion in phosphorylating state 3) [24].The rate of rotenone-resistant NADH-cytochrome c-reductase and NADH-oxidase activity of mitochondrion was determined by NADH oxidation fluorimetrically in hypotonic medium with cytochrome c [25].
The protein content was determined by Lowry assay in Miller modification [32].In each group for assessment of biochemical parameters were used 7 animals.
The data obtained were analyzed statistically by non-parametric (Wilcoxon-Mann-Whitney test) and parametric (t-test) methods using "Statistika V.6".The compliance of feature distribution to normal one was analyzed by Shapiro-Wilk test.The correlation analysis was conducted by Pearson.The animal survival rate was estimated by Kaplan-Meier method and comparison of curves of survival rate was drawn by Gehan with Yates correction.Differences between treatment groups were considered significant if P was less than 0.05 (p<0.05).
In the study were used the reagents produced by Applichem.

The Life-Span of Experimental Animals Maintained on Calorie Restricted Diets
One-month rats were divided on 4 experimental groups: the 1 st group -control -remained on standard diet; the 2 nd group was restricted in food quantity by such way that after 2 months this group animals weight was 40 % less than control group -conditionally "mild" diet; the 3 rd group was restricted by 57 % at "moderate" diet and the 4 th group was restricted by 63 % at "severe" diet (Figure 1).(1) -tacking the standard diet.
(2) -tacking the "mild" calorie-restricted diet since the second month of their life.
(3) -tacking the "moderate" calorie-restricted diet since the second month of their life.
(4) -tacking the "severe" calorie-restricted diet since the second month of their life.* p <0.05 as compare to control.
Thus, in the control group, fed by standard diet, individual animals died after 10 months of life and mass death began with 18 months.Last animal in this cohort died at the age of 33 months.It should be noted that the curve of the control group mortality was not linear, but S-shaped (Figure 2).Importantly that immediately after the transfer of experimental animals on the CRD some part of animals (10-15%) perished with no pathological manifestations.
The marked difference in the survival of animals on different CRD was shown in later stages of ontogeny (Figure 2).
It should be paid attention on the time of the death of the last animal in each experimental group.So, in the control group the last animal died at the age of 33 months, in the 2 nd -35 months, in the 3 rd group -39 months and in the 4 th group at 46 months (Figure 2).
We have suggested, that transfer of animals on CRD performs a dual function: 1. provides the selection of animals already having a specific pattern of metabolic rate -epigenotype 2. "selects" animals with epigenotypes able to induce those metabolic chains that can enhance resistance to a limited diet and as a result, gain life-span.To check this suggestion the pattern of metabolic values was determinedepigenotype after 2 months of the transfer of animals on different diets.Unlike epigenome characterizing the pattern of expressing genes under epigenotype we understand already implemented metabolic pattern, i.e. implemented epigenome.It should be noted that epigenotype gives more information to understand the biological functions than the epigenome does.In epigenotype characteristic we included the content of thyroid hormone (thyroxine, triiodothyronine) and values of prooxidant-antioxidant systems of experimental animals.

The Content of Thyroid Hormones (Thyroxine and Triiodothyronine) in Animals Maintained for Two Months on Different Calorie Restricted Diets
The triiodothyronine content in animals maintained for 2 months on different CRD was significantly reduced compared to control animals (Figure 3A).There was correlation between the weight loss caused by the "mild", "moderate" and "severe" CRD and reduction of serum triiodothyronine in animals (r=0,983).Thus, if the body weight was reduced by 40, 57 and 63%, the content of triiodothyronine was decreased by 37, 43 and 56%, respectively (Figure 3A).
The content of thyroxine in the blood serum of animals maintained on the CRD was significantly lower than in control animals (Figure 3B).However, this reduction was not dependent on the type of CRD.Thus, in the case of "mild" CRD thyroxine content was 50% lower compared to the control and at a "moderate" and "severe" CRD only for 25 and 39%, respectively (Figure 3B).
The tiriyodtironin and thyroxine are known to perform similar metabolic functions, however, the activity of triiodothyronine is 4-5 times higher than that of thyroxine.Perhaps this explains the absence of a direct correlation between the decrease in the amount of thyroxine in the blood and weight loss of the animals on the CRD.
Consequently, the content of thyroid hormones is reduced in the animals on CRD.This result suggests that after 1 month on CRD animals survived such only those who have low thyroid function.
The triiodothyronine and thyroxine are known to be involved in the regulation of a large number of metabolic processes, and in particular, the oxidative breakdown of fats, carbohydrates, protein synthesis, the synthesis of sex hormones, mineral metabolism, which results in the regulation of growth and development.
Such a wide range of metabolic activity of thyroid hormones is accompanied by many cooperative (1) -animals in "control" group.
(3) -animals tacking the "moderate" calorie restricted diet.(4) -animals tacking the "severe" calorie restricted diet.* p <0.05 as compare to control.changes of metabolic chains in the body.Such cooperative changes can lead to the induction of specific epigenotype, which provides a potentially high life-span of animals with altered epigenotype.
We hypothesized that epigenotype of potentially high life-span may be associated with changes in the activity of mitochondrial oxidative phosphorylation, the activity of pro-and antioxidant systems.

Values of Oxidative Phosphorylation of Rat Liver Mitochondria Treated with 2 Months Different CRD
To determine the efficiency of oxidative phosphorylation in mitochondria of liver 3 different substrates were used: succinate-FAD-dependent, glutamate + malate -NAD-dependent and hydroxybutyrate -NAD +-dependent substrates.
The maintenance of animals on CRD for 2 months was accompanied by a reduction of oxidative phosphorylation in all three states: V2 (nonphosphorylating), V3 (phosphorylating) and V3r (fragmented) in Chansu (Table 1).
However, the greatest reduction was shown for V3, and it depended on the degree of diet restriction.So for the "mild" CRD it was 21 % and for the "moderate" and "severe" -29 and 30 % respectively.Reduction in the intensity of phosphorylation for V2 and V3 did not depend on the degree of calorie restriction and was manifested only for "moderate" and "severe" diets (Table 1).
Respiratory control was reduced insignificantly only in the case of "moderate" and "severe" CRD compared with the respiratory control of animals on the standard diet.However, the efficacy of phosphorylation (ratio of ADRs / O) remained the same for all the groups of animals (Table 1).
Therefore, the maintenance of experimental animals on CRD with different degrees of caloric restriction and varying degrees of growth retardation for 2 months was accompanied by a slight decrease in the oxygen consumption rate and the degree of coupling of oxidative phosphorylation in the mitochondria of the liver when used glutamate + malate and hydroxybutyrate as oxidation substrates.Thus, V2 did not change compared to control animals (Table 2, 3).Phosphorylating oxidation speed (V3) decreased only in the case of "moderate" and "severe" CRD by 21 and 22 % respectively when using glutamate + malate, and by 21 and 32 % respectively when using -hydroxybutyrate (Table 2, 3).
In the case of the glutamate + malate oxidation respiratory control decreased in animals maintained on a "mild", "moderate" and "severe" diets, and in the case of -hydroxybutyrate the decrease is shown only for animals on "moderate" and "severe" CRD (Table 2, 3).However, the efficiency of phosphorylation (ADP / 0) remained unchanged in all groups of experimental animals (Table 2,

3).
These results allow us to conclude that the CRD reduces the rate of oxidative phosphorylation in liver mitochondria.
The degree of reduction of phosphorylation depends on the caloric restriction.It was shown in most for the "severe" CRD that provides the greatest effect of increasing life span.At that the rate of phosphorylating oxidation (V3) and the degree of coupling of oxidative phosphorylation (respiratorycontrol) decreased mostly.This indicates a certain decrease in the flux density of electrons in electric transport chain of the mitochondria of liver cells of animals primarily on "severe" CRD and, consequently, a decrease in the rate of generation of free radicals and reduce cross-processes in the cells of these animals.

Some Values of Lipid Peroxidation in Liver of Rats Maintained for 2 Months on "Severe" CRD
The activity of free radical processes in the liver of animals was assessed by the rate of accumulation of TBA-active products and the number of Schiff basesthe end products of peroxidation.
It was found that in the animals maintained for 2 months on "severe" CRD the content of TBA-active products was lower by almost 30% than in the liver of control animals (Figure 4).Therefore, maintenance of the animals on the "severe" CRD was accompanied by reduction of products of free radical reactions, which correlated well with a reduction of oxidative phosphorylation and a decrease in the density of electron transport in electric transport chain of the mitochondria.However, the reduction products of free radical processes in animals maintained on the CRD can be also connected with the high activity of their antioxidant enzymes.

Activity of Antioxidant Enzymes in Liver of Rats Maintained for 2 Months on a Different CRD
To clarify the role of antioxidant enzymes in lifespan increase of rats the activity of glutathionetransferase (GT), isocitratedehydrogenase (ICDH), Se-dependent glutathioneperoxidase (Se-GP) and glutathionreductase (GR) was determined.
The activity of all the enzymes varied differently against different CRD.If the activity of GR remained unchanged, the GT activity slightly (11%) increased compared to control, and only on the "severe" CRD (Table 4).
At the same time the activity of ICDH and the Se-GP increased dramatically.Thus, the activity of ICDH increased 1.7-fold, 1.9 and 1.8 times compared with  the control, respectively, for the "mild", "moderate" and "severe" diets (Table 4).The activity of Se-GP increased even more.Thus, it was higher 19, 32 and 37 times in the animals maintained for 2 months on the "mild", "moderate" and "severe" CRD respectively compared to the control (Table 4).
Consequently, the response of the studied antioxidant enzymes on CRD varied.The activity of Se-GP increased the worst (very strongly) compared to the control level.The degree of increase in the activity depended on the degree and extent of calorie restriction and growth retardation (r=-0,991).It should be noted that high correlation was revealed between the changes of the Se-GP activity of and the content of T3 in serum of animals maintained for two months at various CRD (r = -0,967).
It is known that the outer mitochondrial membrane is rotenone-sensitive chain NADH oxidation, which is involved in the reduction of dehydroascorbic acid, and thus may be involved in improving the reliability of the antioxidant system of the cell.The activity of the redoxchain was judged by the change of the activity of NADH-oxidase and NADH -cytochrome c reductase in animals maintained for 2 months on different CRD.(1) -animals in "control" group.
(2) -animals tacking the "severe" calorie restricted diet.It was found that the maintenance of experimental animals on different CRD was accompanied by significant increase of NADH-oxidase activity and the degree activity increase is strongly correlated with the level of calorie restriction.Since in the animals maintained on a "mild", "moderate" and "severe" diets NADH-oxidase activity increased by 2, 2.9, and 3.5 times, respectively, compared to the control (Figure 5).
Activity of NADH-cytochrome-c-reductase also increased, depending on the degree of calorie restriction, but much less compared to the NADHoxidase (Figure 5).
It should be noted that there is a pronounced correlation (r=0,972 and r=0,970) between the increase of activity of NADH-oxidase and NADH-cytochrome-creductase and growth retardation of the animals maintained on the CRD.

DISCUSSION
The increase of average and maximum life-span of rats maintained on CRD is well known [2,6,7] and our results confirm this.In addition, we noticed that the transfer of 1 month rats on CRD was followed by the death of 10 to 15% of the animals for a month and it didn't depend on the degree of caloric restriction.The superiority of the experimental animals maintained on CRD in life-span compared with the control group was clearly manifested in the later stages of ontogeny (Figure 2).
The results on the dynamics of mortality and life span leads to the following assumptions: 1. Initial (selected for the experiment) cohort of animals is highly heterogeneous in their ability to respond to caloric restriction.Thus, 10 -15% of the animals are not able to survive in such conditions and die.In some animals, about 15-20%, a significant increase of life-span compared to control animals is observed (Figure 2).

2.
There is no direct correlation between the degree of caloric restriction and life-span increase.
Thus, the difference in life-span between the groups maintained on the "mild" and "moderate" diets were little, while on the "severe" CRD there was significant increase of life-span compared to others groups of animals (Figure 2).
These results can be explained on the basis of the concept of epigenotype [33].The essence of the concept is that the metabolic pattern is interoperable sequence of biochemical cycles, which provides a coordinated response to various stimuli (factors).Since epigenotype consists of a large number of on the one (1) -animals in "control" group.
(3) -animals tacking the "moderate" calorie restricted diet.(4) -animals tacking the "severe" calorie restricted diet.* p <0.05 as compare to control.hand related, and on the other relatively autonomous elements (biogeochemical cycles), then the cell can generate an infinite number of combinationsepigenotype. Epigenotype diversity can be repeatedly extended by the presence of alternative metabolic pathways in biological systems.
These results suggest that the transition from one to another epigenotype occurs in salutatory manner.Thus, life-span varies quite dramatically on CRD providing 60% of body weight restriction, while at the restriction by 40 and 50% there were almost no differences in life-span.
We believe that the transfer of 1 month animals on CRD, which induces metabolic shift from anabolic to catabolic type, results in the death of the animals whose epigenotype for unclear reasons can not "do" a saltatory transition to a epigenotype providing catabolic pathway.Therefore on the first stage CRD selects epigenotype.
Later in the "selected" epigenotype cooperative changes of all the elements of the metabolic system occur.But since there is an infinite variety of metabolic variants of epigenotypes, in the population there will be individuals with different types of epigenotype among which there will epigenotypes providing high life-span.Consequently, on the conditional second stage CRD performs the function of specific induction of epigenotype.
Increase of life-span of individuals with specific epigenotypes is realized as hormesis effect [33].
The data obtained in this study suggested the epigenotype concept.Thus, the transfer of animals on CRD is characterized not mere by saltatory increase of the Se-dependent glutathione peroxidase (30 times) or isocitrate dehydrogenase activity, but also by changes in relation of the various antioxidant enzymes, hormonal status, and other values of metabolism.Presenting the epigenotype measures as the spatial portrait, the differences of epigenotypes of control animals and animals maintained for 2 months on "severe" CRD is rather obvious and compelling (Figure 6).
These results allow us to hypothesize that the CRD performs a dual function: 1 -"selection" of animals with specific epigenotype; 2 -induction of hormesis response on the life-span.It is important that forming of specific epigenotypes will influence not only on lifespan, but also on the response of the organism to other factors, susceptibility to infectious agents or pharmaceuticals.

Figure 1 :
Figure 1: Weight of 3-month old animals (g).(1) -tacking the standard diet.(2)-tacking the "mild" calorie-restricted diet since the second month of their life.(3)-tacking the "moderate" calorie-restricted diet since the second month of their life.(4) -tacking the "severe" calorie-restricted diet since the second month of their life.* p <0.05 as compare to control.