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1SPORT PHYSIOLOGY
Electrical stimulation of c2c12 cell culture as a model of human physical activity
UDC 577.3:612.74
Dr. Med., Associate Professor E.Yu. Dyakova1 Postgraduate student Yun Yuyfen1 Postgraduate student E.A. Yugankina1' 2 A.E. Chernykh1
1National Research Tomsk State University, Tomsk 2Tomsk State Pedagogical College, Tomsk
Corresponding author: [email protected] Received by the editorial office on 22.05.2024
Abstract
Objective of the study was to using a physical exercise model, evaluate the effect of electrical stimulation on the glucose transport mechanism of the C2C12 cell culture.
Methods and structure of the study. For the study, the cells were randomly divided into 4 groups: 2 control groups and 2 experimental groups (with insulin resistance). Insulin resistance was formed using a high-glucose medium (25 mM). In each group, half of the cells were exposed to electrical stimulation, the other half remained intact. Half of the cells in each subgroup were treated with insulin to trigger the glucose transport mechanism. The content of the phosphorylated form of the Akt protein (pAkt) in the cells served as a marker for the operation of this mechanism.
Results and conclusions. It was found that in cells with insulin resistance, pAkt was lower compared to the control group. The addition of insulin increased pAkt content, but its level in the experimental group remained reduced. Electrical stimulation increased the concentration of pAkt in all groups, bringing the level of this protein in the experimental groups to values comparable to the control group. Physical activity is one of the methods for treating various diseases and improving both the psychological and functional state of the body. It has been proven that physical activity has a positive effect on the body with insulin resistance. Modeling physical activity on cell cultures using electrical stimulation makes it possible to elucidate the mechanisms of such positive effects.
Keywords: electrical stimulation, exercise model, insulin resistance, cell culture.
Introduction. To achieve the goals of scientific research, the most popular research method is the modeling of certain conditions, processes and systems. For example, running loads are used to study the effect of physical activity on a metabolic disorder such as insulin resistance. Aerobic exercise is considered a core component of any regimen for the prevention/treatment of insulin resistance [2]. Experiments were conducted on mice to determine the effect of running loads during the formation of insulin resistance on the content of protein markers in insulin-sensitive tissues: muscles, liver, brown and white fat [1, 4, 5]. Exercise has been found to have a positive effect on the physical condition of laboratory animals. However, the mechanism associated with the glucose transport system is difficult to trace in the
whole organism. Cell culture experiments are suitable for this purpose. Imitation of physical activity is carried out using the method of electrical stimulation on muscle cells. In this case, either muscle cultures from cell banks or primary cultures of myoblasts isolated from animal muscle tissue are used. This approach allows us to minimize the influence of unaccounted factors and study individual mechanisms. The key factor in the implementation of the glucose transporting function of the cell is the Akt protein, the phosphorylated form of which (pAkt) indicates the activation of this pathway.
Objective of the study was to using a physical exercise model, evaluate the effect of electrical stimulation on the glucose transport mechanism of the C2C12 cell culture.
SPORT PHYSIOLOGY
Methods and structure of the study. The study was carried out on a cell culture of mouse myoblasts C2C12 (collection of cell cultures of the Institute of Cytology of the Russian Academy of Sciences, St. Petersburg). Cells were seeded at a density of 3 10 cells/well in 8 six-well plates containing DMEM supplemented with 5 mM glucose, 10% heat-inactivated fetal bovine serum (FBS), 100 units/ml penicillin, and 100 Mg/ml streptomycin. Cells were stored in a CO2 incubator at 37°C in a humidified atmosphere. Five days after seeding, cells were differentiated in DMEM containing 5 mM glucose, antibiotics, 2% calf serum, and 1 nM insulin. Cell morphology was assessed using phase-contrast microscopy at 400 magnification without prior fixation.
Next, two groups were formed - experimental (EG, 24 wells) and control (CG, 24 wells). In the EG plates, the differentiation medium with 5.5 mM glucose was replaced with a medium containing 25 mM glucose. Insulin resistance developed in these cells. In CG plates, mannitol was added to the medium with 5.5 mM glucose to equalize osmolarity. The cells were placed in a CO2 incubator for two days. Next, the cells were subjected to the serumstarvation procedure - serum starvation to bring them to a state of basal activity [3]. To do this, the cells were placed in a serum-free medium for 1 hour, after which the medium was replaced with serum.
The next stage was electrical stimulation (in two tablets from the EG and in two tablets from the CG) for 6 hours. Electrical stimulation was performed using a C-Pace pulse generator (C-Pace EP, IonOptix, USA) with a voltage of 40 V, a stimulus duration of 10 ms, and a frequency of 1 Hz. At the same time, from each group CG and EG, two tab-
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Content of pAkt in myocyte samples during electrical stimulation (1 - CG without the addition of insulin, 2 - CG with the addition of insulin, 3 - EC without the addition of insulin, 4 - EG with the addition of insulin)
lets remained intact (were not subjected to electrical stimulation).
After electrical stimulation, 10 nM insulin was added to half the wells of each subgroup, and the corresponding amount of serum was added to the second half of the wells; the plates were placed in an incubator for 30 minutes. The cells were then washed with PBS and frozen in liquid nitrogen. Next, samples were prepared in which the protein concentration was determined by the Lowry method. Electrophoresis was carried out in SDS-polyacrylamide gel in accordance with the Laemmli method. Proteins were transferred from the gel to a nitrocellulose membrane (BioRad, USA). Incubation with HRP-conjugated secondary antibodies was carried out for 1 h at room temperature in 5% dry milk in PBST. Visualization of antigen-antibody complexes was carried out using the ECL kit and ChemiDoc XRS + Molecular Imager (BioRad, USA).
Statistical processing of data was carried out using the statistical analysis package STATISTICA 12.0. The Mann-Whitney U test was used to perform statistical analysis.
Results of the study and discussion. The phos-phorylated form of the Akt protein was detected in all samples studied. The concentration of pAkt in samples that were placed in a medium with increased glucose content was lower than in the control group, indicating partially formed insulin resistance in the experimental group. When insulin was added, the pAkt content increased; this increase was higher in the control group (see figure).
The figure shows that electrical stimulation led to an increase in the phosphorylated form of Akt in all groups. Moreover, its content in the experimental group was almost the same as in the control group when exposed to electrical stimulation.
Conclusions. Electrical stimulation of cell culture as a model of human physical activity causes positive changes at the cellular level, affecting, in particular, the glucose transport system of the cell. An increase in the level of the phosphorylated form of the Akt protein after stimulation indicates that physical activity during a metabolic disorder such as insulin resistance increases the cell's availability for glucose, improving the state of carbohydrate metabolism in the body as a whole.
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SPORT PHYSIOLOGY
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