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Національний університет фізичного виховання і спорту України (Київ)
Национальный университет физического воспитания и спорта Украины
ул. Физкультуры, 1, Киев, Украина, 03680
National University of Physical Education and Sports of Ukraine
Str. Fizkultury, 1, Kyiv, Ukraine, 03680
Inroduction. The protein product encoded by PPARG gene is peroxisome proliferator-activated receptor gamma 2 (PPARG2). This is an intracellular factor that plays a role in adipogenesis, glucose and fat homeostasis. The functions of this transcription factor lies in the regulation of genes associated with the accumulation of fat, differentiation of adipocytes and myoblasts, as well as insulin sensitivity [20, 21, 22]. PPARγ is expressed mainly in adipose tissue [9 21], to a lesser extent, in other types of cells, such as macrophages, smooth muscle fibers, endothelial cells, and cardiac myocytes [7, 9, 12, 15].
The analysis of gene networks regulating intracellular cholesterol in hepatocytes and lipid metabolism in adipocytes showed that the factor PPARγ belongs to the key regulators of gene expression of lipid metabolism . A large number of research papers that appeared recently indicate the interest in PPARγ, as a regulator of functions of the cardiorespiratory system [18, 25, 26].
PPARG gene, localized in chromosome 3 (3p25). Associations of gene polymorphisms with various metabolic disorders have been established. In particular, the analysis of polymorphisms of Pro12®Ala, C1431T, C-2821T and the recently discovered A-2819G polymorphism is often conducted [3, 4, 6, 7, 15]. The most studied polymorphism of PPARG gene is Ala®Pro12 polymorphism, which is a cytosine to guanine substitution at position 34 of exon 2 (with the replacement of proline to alanine at position 12 of protein isoform PPARγ2) (rs1801282). The following genotypes have been identified: Рго/Рго - homozygotes for the normal allele, Pro / Ala - heterozygotes, Ala/Ala – homozygotes for the rare allele. The experimental data indicate a reduced ability of PPARγ2 factor in binding with gene promoters which it activates while replacing proline to alanine [13, 24].
PPARγ2 reduced activity, which is associated with the Ala-allele carrier state leads to increased insulin sensitivity and increased glucose utilization . On this basis, the Ala-allele is considered protective against the development of type II diabetes.
Replacement of Pro> Ala at position 12 moderately reduces the receptor function, so Pro12Ala polymorphism is an indicator of reducing the risk of type 2 diabetes, hyperinsulinemia, insulin resistance and atherosclerosis, obesity [5, 10, 11,14, 17, 23]. It is believed that insulin sensitivity in patients with the Ala-allele is associated with less active lipolysis in adipose tissue and glycolysis in the liver, leading to a decrease in free fatty acids and activation of their consumption by muscle tissue.
The meta-analysis of the data from 30 studies with a total sample of 19,136 people showed that the Ala-allele carriers have a higher BMI than Pro / Pro homozygotes .
Based on these data, PPARG Ala-allele carriers are considered to be more prone to speed-power sports compared with Pro-allele carriers because their muscles mainly utilize glucose as well as increased insulin sensitivity . As you know, insulin has anabolic effects on skeletal muscle and improves strength performance.
Correlation of Pro12 → Ala polymorphism with the cross-sectional area (CSA) of muscle fibers allowed finding that the Ala allele is associated with an increase in the volume of both slow and fast muscle fibers .
Clinical data demonstrating the association of PPARG Ala-allele with increased sensitivity to insulin, suggest an increased anabolic action of insulin on muscle tissue and thus Ala-allele carrier state may give advantages to sprinters and weightlifters. The Ala-allele is associated with big CSA both of as slow (significantly) and fast (at trend level) muscle fibers. The mechanism that allows us to consider the Ala-allele as a marker of increased susceptibility to the development and manifestation of speed-power qualities, lies in reducing the Ala- allele transcriptional activity.
Therefore, many researchers demonstrated the influence of the polymorphism on the metabolic processes that influence the properties of muscle tissue and the physical quality which can be considered as a genetic marker of disposition to sports in which competitive exercises are provided mainly by anaerobic mechanisms of energy supply.
Connection of the work with academic programs, plans, themes. The work is performed under theme 2.22 “Development of a comprehensive system of identifying of individual-typological characteristics of sportsmen based on genome manifestations” of the consolidated plan of research work in the field of physical culture and sports for 2011 - 2015 (state registration number 0111U001729).
The aim of the work is to establish differences in the distribution of genotypes according to PPARG Ala®Pro12 gene polymorphism in groups of athletes specializing in sports with different nature of energy supply of the muscular work.
Materials and methods of the research.
In the course of the study 567 people DNAs were studied, including 249 athletes of different sports and 318 people who had no experience in regular exercise. All athletes were divided into 3 groups according to the nature of the mechanisms of muscle activity energy supply during training and competition load: 1) athletes who specialize in events with predominant manifestation of endurance (n = 100), 2) athletes who specialize in events with speed and power manifestation (n = 87), 3) athletes who specialize in events with combined requirements for endurance and power manifestation (n = 62).
The examination of athletes was performed on the basis of the laboratories of theory of sports training methodology and reserve capacities of athletes of the Research Institute of National University of Physical Education and Sports of Ukraine. Molecular genetic analysis was performed on the basis of the Laboratory of General and Molecular Pathophysiology Department of O.O.Bogomoletz Institute of Physiology of the National Academy of Science of Ukraine.
DNA was extracted from buccal epithelium using a set of reagents DiatomTM. Pro12Ala [CCG (Pro) → GCG (Ala)] gene polymorphism (rs1801282) at position 34 of exon 2 of PPARG gene was determined by amplification followed by restriction. Amplification was performed with direct 5'-GCC AAT TCA AGC CCA GTC-3 'and reverse - 5'-GAT ATG TTT GCA GAC AGT GTA TCA GTG AAG GAA TCG CTT TCC G-3' primers synthesized by (“Metabion”, Germany). For amplification 50-100 ng of DNA were added to a mixture containing 5 μL of PCR-buffer (“Amplisense”, Russia), 2.5 μL of dNTP, 25 pmol/L of each primer and 0.1 U of Tag-polymerase (“AmpliSense”, Russia), the volume was adjusted to 25 ml with deionized water. Polymerase chain reaction (PCR) was performed in the thermocycler “Applied Biosystems 2700” (USA). For amplification of the gene, the following PCR conditions are necessary: pre-denaturation - 94°C (5 min), 38 cycles of amplification: denaturation - 94°C (30 sec), annealing of primers - 64°C (30 sec), DNA synthesis - 72°C (60 sec); the final synthesis – 72°C (10 min). The products of PCR amplification are DNA fragments 270 base pairs in length. The presence of nucleotide substitution C to G ay 34 position 34of B exon of PPARG gene creates restriction site (CG ↓ CG) for endonuclease Bsh1236I. The structure of the restriction mixture was composed of deionized water, 10X buffer R («Fermentas»), restrictase Bsh1236I («Fermentas», Lithuania).
incubation of the restrictive mixture (8 μL) with PCR products (6 μL) was performed in a separate tube in the thermostat at 37ºC (for 24 h). The reaction products were separated using horizontal electrophoresis in 1.5% agarose gel (160V within 45 min) and identified under ultraviolet light after ethidium bromide staining by the transilluminator (“Biokom”, Russia). The presence of the restriction site determines the distribution of amplicons into two fragments of 227 and 43 base pairs in length. Thus, the genotype Pro / Pro was associated with non-restricted fragments 270 bp in length, Pro/Ala genotype– with three fragments of 270, 227, and 43 bp in length, and Ala/Ala genotype– with two fragments of 227 and 43 bp in length.
probability of differences in the distribution of samples was determined by c2 criterion. P value <0.05 was considered significant.
Findings and their discussion. Usage of PCR allowed us to set the frequency distribution of allelic variants Pro12®Ala polymorphism of PPARG gene in the Ukrainian population. The distribution of allelic variants for this polymorphism is: Pro/Pro – 64.2%; Pro/Ala – 34.0%; Ala/Ala – 1.9%; frequency of occurrence of the rare Ala allele - 18.9%. This distribution corresponds to the Hardy-Weinberg equilibrium (χ2 = 0.15). The frequency of occurrence of the minor Ala-allele, set in our studies, is slightly higher than the frequency in Asian and European countries [16, 19, 27], but is close to the frequency of the Eastern European countries  (see Table 1). In most populations homozygous Pro/Pro genotype is dominant, and the frequency of Ala/Ala genotype is very low.
Comparative analysis of the distribution of Pro12®Ala polymorphism of PPARG gene in different populations [1, 15, 16, 19, 27].
Genotyping of athletes of different sports has allowed us to establish differences in the distribution of allelic variants according to Pro/Ala polymorphism. The overall distribution of allelic variants of Pro/Ala polymorphism of PPARG gene in a group of athletes (n = 249) (65.1% Pro/Pro, 31.3% Pro/Ala and 3.6% Ala/Ala) vs. the same distribution in the control group statistically did not differ (rχ2 = 0.4), while in the group of sportsmen both the frequency of occurrence of Ala/Ala genotype (1.7%), and the rare Ala-allele was somewhat higher.
When dividing the sampled population of sportsmen into subgroups by nature of energy supply of competitive exercises it was found that the prevalence of allelic forms of PPARG gene is different in these subgroups (Table 2).
Frequency of occurrence of allelic variants of Pro/Ala polymorphism of PPARG gene among athletes of different sports, % (n=567)
p1 - statistical probability of differences in the distribution of genotypes compared with the control group, P2 - statistical probability of differences in the distribution of genotypes compared with endurance athletes, P3 - statistical probability of differences in the distribution of alleles compared with the control group, * - probable differences per c2 criterion.
All examined groups had the highest frequency of Pro/Pro- genotype, although in the group of athletes who specialize in endurance types, this value was higher than that in the control group by 12.5% (p <0.05), and in the group of speed-power sports and sports that require the combined development of power and endurance, it was lower by 9.3% and 4.8%. By using a comparative analysis of the distribution of allelic variants we found that the probability of differences between sampled population of athletes who specialize in speed and power types and sports with predominant development of endurance. Thus, the frequency of Pro/Pro genotype differed in these groups by 21.8% and frequency of the minor allele was higher in the group of sportsmen of speed and power types by 11.2% (p = 0.005). These results suggest that the Pro-allele may promote high physical performance in sports with predominant manifestation of endurance, and the Ala-allele - in speed and power sports.
In addition, the athletes who specialize in sports with predominant development of endurance are likely to differ in the distribution of genotypes from the athletes who specialize in sports with the combination of power and endurance (mixed group). Thus, the frequency of occurrence of Pro/Pro genotype in the group of mixed sports was lower by 17.3% (rχ2 = 0.02) than in the group of endurance sports.
No fundamental differences have been established between endurance sports, studied by us, as for the distribution of genotypes according to this polymorphism, although among athletes involved in rowing the Ala-allele percentage is higher than among athletes of other types in this subgroup (by 3.9% than in athletes engaged in cross-country skiing).
The distribution of genotypes and alleles according to this polymorphism among athletes of speed and power sports differed only in the group of athletes involved in jumping (Fig. 1). Among the athletes who specialize in speed and power sports the greatest frequency of the Ala-allele (31%) was associated with the athletes involved in sprint running for (p <0.05), but among the athletes involved in jumping (14.7% ), this allele is less common than in the control group (3.3%). It is obvious that this polymorphism is not important for athletes who specialize in field-and-track jumps.
Thus, a group of athletes of sports with predominant development of endurance according to the distribution of genotypes based on Pro/Ala polymorphism of PPARG gene is significantly different from athletes of speed-power sports and athletes with the combination of power and endurance. Thus, Pro/Ala polymorphism of PPARG gene can be considered a molecular genetic marker for selection in endurance sports, where the Pro allele may be considered favorable for the development of endurance, and Ala - speed and power.
Conclusions. 1. The distribution of allelic variants of PPARG gene according to Pro/Ala polymorphism in the groups of athletes in different sports has probable differences. The frequency of Pro/Pro genotype in the group of athletes specializing in sports with predominant development of endurance is higher than the frequency in the control group and group of athletes of speed and power sports by 12.5% (p χ2 = 0.01) and 21.8% (p χ2 = 0.005).
2. Pro/Ala polymorphism of PPARG gene may be used as a molecular marker of genetic predisposition to sports. Pro-allele promotes high physical performance in sports with the predominant manifestation of endurance, and Ala-allele - in speed and power sports.
3. In further studies it is planned to study the information value of this molecular genetic marker and its contribution to the individual variation in data in the overall assessment of genetic predisposition to the development of high physical performance in various sports, based on the definition of polymorphisms of several dozens of genes.
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