**3. The complex role of molecular pathways in the aging process and DM**

Human body cells constantly receive signals from the body and the environment, causing processes such as damage, infection and stress. The modern field of research, called "epigenetics," explores how the environment and time affect the functioning of genes and the development, health and aging of humans. Some epigenetic changes are serious triggers for the development of DM or conditions that increase the risk of developing related DM to age. The response to internal and external signals from cells and the production of these signals occurs through biological pathways that are important for the development of aging and DM, including oxidative stress and/or cellular metabolism. Everyday millions of destructive events occur in the DNA structure, but in cells there are powerful mechanisms that protect DNA from damage, and these mechanisms remain active in old age.

Today, the goal is to sequester 100 genes in 1000 healthy elderly people, which can shed light on the inherited variability that underlies the protection of some people from aging diseases, including DM, enabling them to live a healthy life at their age. Sequencing allows researchers to determine whether the presence of mutation carrier makes the elderly more secure or more vulnerable to the effects of damaging factors. Topol et al. perform study compared genetic sequencing in healthy volunteers, aged 80 years, and persons whose death has been linked to diseases associated with aging, including type 2 DM [40]. Scientists are finding that healthy people have an extremely low probability of genetic variations associated with the development of the disease. This fact proves the idea that protective genes play major role in the successful aging people. Therefore, the identification of molecular bases of protective effect would develop similar medicines, including effective means of preventing type 2 DM. Barzilai et al. sequenced several candidate genes of centenarians, including a variant gene that modifies the mechanism of cholesterol metabolism [41]. Scientists have sequenced genes of IGF1, its receptor and have identified mutations that are unique to women aged 100 years [42]. Calorie restriction increases life span and reduces age-related deterioration of work systems and physiological responses of age-related diseases, including with the development of DM. Restriction of caloric intake in animals in the experiment leads to a decrease in the level of glucose and insulin in the blood plasma and reduces inflammatory responses and the intensity of oxidative stress.

can lead to insulin resistance, chronic inflammation with systemic consequences for the body. Many genes transcribed by TNF-α are activated by the transcription factor in NF-κB adipose

The peroxisome proliferator-activated receptors (PPARs) alter gene expression, affecting the metabolism of fats and carbohydrates in response to changes in lipid levels in food. Ligands of these transcription factors are fatty acids and their derivatives. PRAPs act on the nucleus of the cell by forming heterodimers with another nuclear receptor—retinoid X receptor (RXR) that binds to regulatory regions of DNA. PPARs include genes necessary for β-oxidation of fatty acids and the formation of ketone bodies during fasting and stimulate the expression of genes encoding proteins that provide β-oxidation and dissipation of energy due to the formation of mismatched mitochondria. In mice with non-functioning receptor, leptin-activated PPAR-γ prevents the development of obesity by stimulating the synthesis of proteins

Ghrelin is a peptide hormone consisting of 28 amino acids produced by P/D1 cells of mucous membrane of the fundus of the stomach. Ghrelin receptors are expressed by neurons in the arcuate nucleus and ventromedial hypothalamus, here the processes associated with the action of ghrelin are mediated: stimulation of the production of releasing hormones, increased appetite, changes in the level of glucose and lipid metabolism, regulation of secretion and contractions of walls of the gastrointestinal tract [38]. It stimulates the release of GH. The concentration of ghrelin in the blood increases before eating and falls immediately after its intake. The concentration of ghrelin in the blood plasma increases with age, which contributes

Consequently, numerous biomedical studies have proved the key role of neuroimmuneendocrine interactions in the human body, which trigger the universal molecular pathological

**3. The complex role of molecular pathways in the aging process and DM**

Human body cells constantly receive signals from the body and the environment, causing processes such as damage, infection and stress. The modern field of research, called "epigenetics," explores how the environment and time affect the functioning of genes and the development, health and aging of humans. Some epigenetic changes are serious triggers for the development of DM or conditions that increase the risk of developing related DM to age. The response to internal and external signals from cells and the production of these signals occurs through biological pathways that are important for the development of aging and DM, including oxidative stress and/or cellular metabolism. Everyday millions of destructive events occur in the DNA structure, but in cells there are powerful mechanisms that protect

Today, the goal is to sequester 100 genes in 1000 healthy elderly people, which can shed light on the inherited variability that underlies the protection of some people from aging diseases, including DM, enabling them to live a healthy life at their age. Sequencing allows

DNA from damage, and these mechanisms remain active in old age.

involved in the cleavage of fatty acids and thermogenesis [37].

to weight gain in people as they age [39].

pathways in the development of aging and DM.

tissue cells.

144 Diabetes and Its Complications

Genetic analysis identified several genes that affect life span and associated with damage to the pituitary development, the decrease of the secretion of GH, food intake and apoptosis. The work of these genes converges in the region of the IGF-1 receptor pathway and reproduces many effects of limiting calorie intake. Although *dwarf* mice having the defect in the synthesis of GH or the IGF-1 signaling pathway are also characterized by an increase of life expectancy, people with signaling defects associated with GH are prone to the development of diseases associated with aging. One of the targets of IGF-1, the signal pathway within the cell, is the repression of proteins responsible for stress resistance, including SOD and heat shock proteins, as well as a decrease in IGF signaling can increase life expectancy by increasing the expression of genes responsible for stress resistance. The mutation of the receptor to IGF-1, a phosphorylation target (p66 Shc), also increases the life span without affecting other organs and systems. When Shc is activated, the levels of intracellular oxygen radicals increase, suppressing the factor FKHRL1, which is involved in apoptosis (**Figure 1**).

Let us consider the scheme of molecular pathological pathway insulin/IGF-1 in humans, where mechanisms of aging and the appearance of type 2 DM are converging.

IGF-1 and IGF-1R provide the activity of proliferative signaling system that stimulates growth in many cell types and blocks apoptosis. In vivo, IGF-1 acts as an immediate response to effects of many growth factors and GH. One of the components of IGF-1, mitogenic signaling, is associated with the tyrosine kinase receptor via Shc, Grb2 and Sos-1, activating the RAS and MAP kinase cascade (raf, Mek, Erk). The end point of the MAP kinase pathway is the modification of the activity of transcription factors, such as the activation of ELK transcription factors. The serum response factor (SRF) and AP-1 provide mitogenic activity of many growth factors. IGF-1R signals for cellular survival and growth in response to IGF-1 and IGF-2. IGF-1R activates three signaling pathways that converge on the phosphorylation process of the BAD protein and block apoptosis. The first pathological pathway is activated by the

molecular pathways in the cell: insulin/IGF-1, sirtuins and mTOR. In the 1980s, scientists discovered the first gene that limited the lifespan of *Caenorhabditis elegans* and called it *Age-1.* The effects of the gene *Age-1* are realized through the molecular pathway of insulin/IGF-1: when the activity of the gene *Age-1* decreases, the molecular pathway of insulin/IGF-1 decreases and the life of *C. elegans* lengthens. Recent studies have shown that in people with mutations in the pathway of insulin/IGF-1, the risk of developing DM may be reduced.

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The sirtuin pathological pathway in the cell regulates the metabolism of the cell. In the 1990s, scientists from the Massachusetts Institute of Technology discovered an extrocopy of the equivalent of sirtuin, sirtuin 2, which increases the life span of yeast. mTOR pathway (mammalian target of rapamycin) plays a role in the aging processes of various organisms, controlling the rate of protein synthesis, which is important for the functioning of the cell. The inhibition of this pathway in rapamycin mice leads to an increase in life expectancy [45].

The development of DM is associated with hyperlipidemia III and IV types. An example of a gene associated with healthy aging and long life expectancy is the cholesteryl ester transfer protein gene. The homozygous variant 405VV of the *CETP* gene is associated with low concentrations of the CETP protein in the blood, high concentrations of HDL cholesterol and large HDL particles, which determines the protection of the human body from the develop-

The work of biological clock is determined by oscillatory genes and genes responsible for unidirectional movement of time (telomere activity). Oscillatory genes synchronize behavioral and biochemical processes with a day/night cycle. Telomeres, which are repetitive series of DNA sequences that form terminal regions of chromosomes, are shortened during each subsequent division of the cell. The shortened telomeres are registered in various pathological conditions associated with aging. The activity of all processes in human cells is supported by NADH and ATP, synthesized from nutrients. Limiting the intake of calories increases the level of AMP and NAD and healthy life span of animals. Silent Information Controller T1 (SIRT1), NAD-dependent deacetylase, reduces the telomere reduction process, whereas the 1α receptor activator γ, activated by the peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α), is phosphorylated by kinase AMP and deacetylated SIRT1. Thus, PGC-1α is a key component of circadian oscillator that combines human biological clock and energy metabolism. Reactive forms of oxygen, formed in conditions of genetic mutation of biological clocks, lead to an accelerated reduction of telomeres. The above-mentioned processes are

The *Timeless* gene (Tim) was found in Drosophila and encodes a protein that regulates the circadian rhythm [47]. Oscillations of mRNA and protein rhythmically occur in time as part of the work of negative coupling in the transcription-translation system involved in the work

The timeless protein connects the cell cycle with the circadian rhythm in humans. In the model of "direct coupling," two cycles are separated by key protein, the expression of which is determined by the circadian pattern [51]. The special role of the Tim gene in creating circadian rhythm is realized with the help of the *Cry* gene in humans. The transcription of the *Cry* and *Per* genes is activated by the *CLOCK/BMAL1* complex and suppressed by the PER/CRY complex.

ment of DM, cardiovascular diseases and Alzheimer's disease [46].

of the period (per) gene of the periodic oscillatory protein [48–50].

described in patients with DM.

**Figure 1.** Scheme of participation of IGF-1-signaling in the regulation of life expectancy (database of pathways of BioCarta).

IGF-1R PI3 kinase, and the AKT signaling pathway phosphorylates BAD and blocks apoptosis. The second pathological pathway is activated by IGF-1R involving the Ras-Map-kinase pathway with the blockade of apoptosis. The third pathological pathway involves the interaction of Raf with mitochondria in the response to the activation of IGF-1R. The similarity of these pathological ways blocks apoptosis and increases the response to IGF-1R stimulation. The function of proapoptotic BAD molecule is regulated by the phosphorylation of three sites (ser 112, 136 and 155), which reduces the possibility of BAD heterodimerization by survival proteins of BCL-XL or BCL-2 cells. Phosphorylated BAA binds to 14-3-3 and is sequestered in the cytoplasm. Phosphorylation of ser-136 is associated with activation of Akt and phosphorylation of Ser-112 is due to the activation of the Ras-MAPK pathway. BAD Ser 155 is a large phosphorylation site that induces the formation of growth factors and is protected by inhibitors of protein kinase A.

It is known that FOXO3A gene prevails among long livers and probably determines longer life span, being one of the members of the family of transcription factors that mediate insulin action and resistance to stress. The relationship between polymorphisms of the FOXO3A gene and human life expectancy is presented in eight independent cohorts of centenarians [43]. Cell resistance to stress and cell survival in aging and DM may increase with high expression of the protein of the FOXO3A gene due to its effect on the activation of several members of the family of serum glucocorticoid-regulated kinases.

Genome-wide association studies (GWAS) have identified genes associated with DM and aging [44]. Most of the detected longevity genes have distant effect on one of the three molecular pathways in the cell: insulin/IGF-1, sirtuins and mTOR. In the 1980s, scientists discovered the first gene that limited the lifespan of *Caenorhabditis elegans* and called it *Age-1.* The effects of the gene *Age-1* are realized through the molecular pathway of insulin/IGF-1: when the activity of the gene *Age-1* decreases, the molecular pathway of insulin/IGF-1 decreases and the life of *C. elegans* lengthens. Recent studies have shown that in people with mutations in the pathway of insulin/IGF-1, the risk of developing DM may be reduced.

The sirtuin pathological pathway in the cell regulates the metabolism of the cell. In the 1990s, scientists from the Massachusetts Institute of Technology discovered an extrocopy of the equivalent of sirtuin, sirtuin 2, which increases the life span of yeast. mTOR pathway (mammalian target of rapamycin) plays a role in the aging processes of various organisms, controlling the rate of protein synthesis, which is important for the functioning of the cell. The inhibition of this pathway in rapamycin mice leads to an increase in life expectancy [45].

The development of DM is associated with hyperlipidemia III and IV types. An example of a gene associated with healthy aging and long life expectancy is the cholesteryl ester transfer protein gene. The homozygous variant 405VV of the *CETP* gene is associated with low concentrations of the CETP protein in the blood, high concentrations of HDL cholesterol and large HDL particles, which determines the protection of the human body from the development of DM, cardiovascular diseases and Alzheimer's disease [46].

The work of biological clock is determined by oscillatory genes and genes responsible for unidirectional movement of time (telomere activity). Oscillatory genes synchronize behavioral and biochemical processes with a day/night cycle. Telomeres, which are repetitive series of DNA sequences that form terminal regions of chromosomes, are shortened during each subsequent division of the cell. The shortened telomeres are registered in various pathological conditions associated with aging. The activity of all processes in human cells is supported by NADH and ATP, synthesized from nutrients. Limiting the intake of calories increases the level of AMP and NAD and healthy life span of animals. Silent Information Controller T1 (SIRT1), NAD-dependent deacetylase, reduces the telomere reduction process, whereas the 1α receptor activator γ, activated by the peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α), is phosphorylated by kinase AMP and deacetylated SIRT1. Thus, PGC-1α is a key component of circadian oscillator that combines human biological clock and energy metabolism. Reactive forms of oxygen, formed in conditions of genetic mutation of biological clocks, lead to an accelerated reduction of telomeres. The above-mentioned processes are described in patients with DM.

IGF-1R PI3 kinase, and the AKT signaling pathway phosphorylates BAD and blocks apoptosis. The second pathological pathway is activated by IGF-1R involving the Ras-Map-kinase pathway with the blockade of apoptosis. The third pathological pathway involves the interaction of Raf with mitochondria in the response to the activation of IGF-1R. The similarity of these pathological ways blocks apoptosis and increases the response to IGF-1R stimulation. The function of proapoptotic BAD molecule is regulated by the phosphorylation of three sites (ser 112, 136 and 155), which reduces the possibility of BAD heterodimerization by survival proteins of BCL-XL or BCL-2 cells. Phosphorylated BAA binds to 14-3-3 and is sequestered in the cytoplasm. Phosphorylation of ser-136 is associated with activation of Akt and phosphorylation of Ser-112 is due to the activation of the Ras-MAPK pathway. BAD Ser 155 is a large phosphorylation site that induces the formation of growth factors and is protected by

**Figure 1.** Scheme of participation of IGF-1-signaling in the regulation of life expectancy (database of pathways of

It is known that FOXO3A gene prevails among long livers and probably determines longer life span, being one of the members of the family of transcription factors that mediate insulin action and resistance to stress. The relationship between polymorphisms of the FOXO3A gene and human life expectancy is presented in eight independent cohorts of centenarians [43]. Cell resistance to stress and cell survival in aging and DM may increase with high expression of the protein of the FOXO3A gene due to its effect on the activation of several members of the

Genome-wide association studies (GWAS) have identified genes associated with DM and aging [44]. Most of the detected longevity genes have distant effect on one of the three

inhibitors of protein kinase A.

BioCarta).

146 Diabetes and Its Complications

family of serum glucocorticoid-regulated kinases.

The *Timeless* gene (Tim) was found in Drosophila and encodes a protein that regulates the circadian rhythm [47]. Oscillations of mRNA and protein rhythmically occur in time as part of the work of negative coupling in the transcription-translation system involved in the work of the period (per) gene of the periodic oscillatory protein [48–50].

The timeless protein connects the cell cycle with the circadian rhythm in humans. In the model of "direct coupling," two cycles are separated by key protein, the expression of which is determined by the circadian pattern [51]. The special role of the Tim gene in creating circadian rhythm is realized with the help of the *Cry* gene in humans. The transcription of the *Cry* and *Per* genes is activated by the *CLOCK/BMAL1* complex and suppressed by the PER/CRY complex. Timeless protein in humans (hTIM) is responsible for the production of electrical oscillations emanating from the suprachiasmatic nucleus of the hypothalamus (SCN) and determining for all circadian rhythms in the human body. This protein interacts with the key products of the activity of the oscillation genes *CLOCK, BMAL, PER1, PER2* and *PER3.* Sancar et al. investigated the role of hTIM in the work of cell cycle in humans [52]. It plays integral role in phases of G2/M and the intra-S cell cycle. In the G2/M phase of the cell cycle, hTIM binds the ATRIP subunit to the ATR protein kinase responsible for DNA damage. Binding of hTIM and ATR subsequently leads to phosphorylation of Chk1, resulting in cell cycle arrest or apoptosis. The *Timeless* gene influences to the development of human diseases. DNA damage associated with telomeres is increased in cells with reduced replication of the *Timeless* gene, along with disruption of telomere replication. Swi1 is a protein associated with the Timeless protein, which is responsible for DNA replication in the telomere region [53]. Single nucleotide polymorphism in the *Timeless* gene, which leads to the replacement of glutamine by arginine in the amino acid sequence of the protein, has not demonstrated an association with changes in morning or evening diurnal rhythms in humans [54]. The Timeless protein can be responsible for circadian rhythms in pancreatic β-cells [55]. It is believed that the Timeless protein can be identified as a kinase suppressor with Ras-1-like activity [56].

Johnson showed that life expectancy increased to 65% due to the mutation of the *Age-1* gene to greater extent than caloric restriction [66]. The *Age-1* gene encodes catalytic subunit of class I phosphatidylinositol 3-kinase (PI3K). Ten years after Johnson's research, the analysis of the *daf-2* gene was performed, and Cynthia Kenyon demonstrated an increase in the half-

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Despite the fact that long livers can be characterized by unique set of genes, future biomedical research based on methods of high-performance screening of a pool of target molecules will lead to great advances in the diagnosis of aging stages and DM, as well as the development of methods for the prevention and therapy of accelerated aging of the human body and various

Research in the field of aging and diabetology is related to recent discoveries in genomics and proteomics, new analytical equipment allows identifying biomarkers of aging and DM, the development of new drugs occurs through high-throughput screening of target molecules in

At present, we have created unified technology platform for diagnostics and predictive pharmacology of aging and DM, taking into account interdisciplinary approach, including complex solution of problems of genomics, proteomics and metabolomics in the range of

The technological platform for diagnostics and predictive pharmacology of DM includes

• *Scientific component*: Search for target biomarkers (α-β-subunit of insulin receptor, tyrosine kinase, MEK1/2-MAPK-cascade, Shc-Grb2-SOS-Ras–Raf mitogen, atypical isoforms of protein kinases, molecular cascade of GH/IGF-1, etc.) with aging and DM as the basis for a new level of diagnosis; the development of cellular technologies in the treatment of DM in combination with the development of new drugs based on targeted biomarkers; *in vitro* and *in vivo* studies of individual PK processes of drugs for the prevention of aging and treatment of DM; combining information on phenotypic manifestations of drug effects on the basis of pharmacoproteomic profile with the results of PK studies; PK/PD modeling;

• *Technological component*: New methods of genomic, proteomic, pharmacokinetic, pharmacoprotein, pharmacogenomic studies; biomodeling; software on bioinformatics and for the registration of ADR; methods of visualization of molecular PD effects of drugs in biological

• *Medical component*: The introduction of technological platform for molecular diagnostics and drug monitoring in aging and DM, diagnostic of ADR, new biomolecular methods

**4. New technological platform for diagnostic and predictive** 

**pharmacology of aging stages and diabetes mellitus**

three components taking into account a unique instrument base:

personalization of therapy for stages of aging and each type of DM.

fluids and body tissues; creation of bank of biosamples.

life of *C. elegans* [67].

human body [68].

violations of carbohydrate metabolism.

universal molecular pathways [69].

The telomeric zinc finger-associated protein (TZAP) associated with long telomeres that have low concentration of protective complex competing with TRF1 and TRF2 factors linking telomeric repeats. In telomeres, TZAP causes a purification process that leads to rapid removal of telomere repeats. The regulation of the length of telomeres in human cells has been proposed: reduced concentration of protective complex in long telomeres leads to binding of TZAP protein and initiation of telomeres purification and sets an upper limit of telomere length [57]. Telomere shortening was previously associated with the development of DM in several pilot studies and in two large studies. Zee et al. showed that telomere length was less in the study group of patients with type 2 DM than in the control group (adjusted odds ratio = 1.748) [58].

Salpea et al. performed a study in which it was found that telomere length was less in type 2 DM and this fact corresponded to a high level of oxidative stress in these patients. Short telomeres are an independent predictor of the progression of diabetic nephropathy (DN) in patients with type 1 DM in the early onset of the disease [59, 60]. Astrup et al. showed that short telomere length is the predictor of all causes of death in type 1 DM [61]. Short telomeres were detected in arterial wall cells in patients with type 1 and type 2 DM [62]. Patients with IGT demonstrated shorter telomere length compared to healthy controls, and patients with DM and severe atherosclerosis showed the presence of shorter telomeres compared with patients with DM without atherosclerosis. The presence of obesity and insulin resistance was associated with the length of telomere leukocytes in the adult population [63]. The study found direct causal relationship between telomerase activity and insulin secretion, as well as glucose tolerance: the TERC-/- mutation showed ITG, which was caused by impaired glucosestimulated insulin secretion from pancreatic islet cells due to a decrease in pancreatic cell size and replication damage of producing insulin β-cells [64].

Klass showed that the life expectancy of *C. elegans* could vary with the presence of the mutation of the *Age-1* gene, and this effect is associated with caloric restriction [65]. Later, Johnson showed that life expectancy increased to 65% due to the mutation of the *Age-1* gene to greater extent than caloric restriction [66]. The *Age-1* gene encodes catalytic subunit of class I phosphatidylinositol 3-kinase (PI3K). Ten years after Johnson's research, the analysis of the *daf-2* gene was performed, and Cynthia Kenyon demonstrated an increase in the halflife of *C. elegans* [67].

Despite the fact that long livers can be characterized by unique set of genes, future biomedical research based on methods of high-performance screening of a pool of target molecules will lead to great advances in the diagnosis of aging stages and DM, as well as the development of methods for the prevention and therapy of accelerated aging of the human body and various violations of carbohydrate metabolism.
