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Научни трудове на Съюза на учените в България-Пловдив. Серия Г. Медицина, фармация и дентална медицина т. XXII. ISSN 1311-9427 (Print), ISSN 2534-9392 (On-line). 2017. Scientific works of the Union of Scientists in Bulgaria-Plovdiv, series G. Medicine, Pharmacy and Dental medicine, Vol.XXII. ISSN 13119427 (Print), ISSN 2534-9392 (On-line). 2018.
ГЛИКОГЕНОВ МЕТАБОЛИЗЪМ В СКЕЛЕТНИ МУСКУЛИ НА ТРЕНИРАНИ ЗА ИЗДРЪЖЛИВОСТ ПЛЪХОВЕ СЛЕД ПРИЛАГАНЕ
НА АНТИАНДРОГЕН Фанка Гергинска1, Слави Делчев1, Михаела Шишманова-Досева2, Георги ВасилевЗ, Катерина Георгиева4 1Катедра по анатомия, хистология и ембриология, Медицински факултет, Медицински университет - Пловдив,2Катедра по фармакология и лекарствена токсикология,Фармацевтичен факултет, Медицински университет - Пловдив, 3 Медицински университет - Пловдив, 4Катедра по физиология, Медицински факултет, Медицински университмт - Пловдив
GLYCOGEN METABOLISM IN- HIND LIMB MUSCLES OF ENDURANCE TRAINED RATS AFTER ANTIANDROGEN ADMINISTRATION Fanka Gerginskal, Slav1 Delchevl, Michaela Shishmanova-Doseva2, Geo rgi Vasilev3, Katerina Georgieva4 lDepartment of Anatomy, Histology and Embryology, F acuity of Medicine, Medical Un iversity - Plovdiv, 2Department of Pharmacology and Drug» Toxicology,Faculty of Pharmacy, Medical University - Plovdiv, 3Medical University - Plovdiv, 4Department of Physiology, Faculty of Medicine, Medical University - Plovdiv
Abstract
Depletion of muscle glycogen is an accurate marker for the onset of exhaustion in aerobic physical exercise. Glycogen Synthase (GS) catalyses the processes of addition of a-1,4 glucose units to the growing glycogen molecule. The type and utilization of energy substrates, used by muscles, depend on the hormonal status and physical activity. There is a lack of data about the influence of androgens via their androgen receptor (AR) on glycogen content and GS in aerobic exercise of skeletal muscles. The aim of the study was to investigate the effect of endurance training and ARblocker on glycogen content and expression of GS in skeletal muscles. Male Wistar rats were allocated in trained and untrained groups. The trained rats were subjected to submaximal training. Half of the trained and untrained rats received Flutamide for 8 weeks. An immunohistochemical study for GS and PAS staining for glycogen were carried out, followed by morphometrical and statistical analyses. Training increased glycogen content and immunoexpression of GS in soleus (Sol) and extensor digitorum longus (EDL). AR blockade decreased glycogen in Sol and immunoexpression of GS in EDL of trained animals. The increased expression of GS in trained
rats reveals the role of the enzyme in adaptation processes to endurance training. The lowered glycogen content in Sol after AR blockade in trained rats proves the participation of androgens in adaptation to exercise via AR. The lowered expression of GS after antiandrogen administration in EDL of endurance trained animals can be explained by differences in AR content in diverse types of skeletal muscles, which defines their response to androgens.
Keywords: Glycogen, Glycogen Synthase, skeletal muscles, Flutamide, endurance training
The glycogen depots in the skeletal muscles are a source of energy at rest and during physical exercise. The type and utilization of energy substrates used by the muscles depend on the hormonal status and physical activity (Cunha et al., 2005). Depletion of the muscle glycogen is an accurate marker of the onset of exhaustion during aerobic physical exercises. Glycogen synthase (GS) catalyses the processes of addition of a-1,4 glucose units to the growing glycogen molecule, which is a key stage in the polysaccharide biosynthesis (Cid et al., 2005; Adeva-Adany et al., 2016). Studies indicate the influence of the glycogen content in skeletal muscles and muscle contraction on the GS activity (Nielsen et al., 2001; Lai et al., 2009). Higher levels of glycogen have a strong inhibitory effect on the GS activity (Nielsen et al., 2001; Adeva-Adany et al., 2016). During exercise, inhibitory factors are released which, together with the stimulating factors, determines the final effect on the GS activity (Nielsen and Richter, 2003). The amount of glycogen in the depots is also associated with the role of the protein glycogenin, which forms the basis on which the processes of polysaccharide growth take place (Alonso et al., 1995). It has been found that glycogenin is actually the 38-kDa subunit of the muscle GS (Pitcher et al., 1987). Furthermore, this protein is related to the GS activity and is subject to hormonal control (Alonso et al., 1995). Testosterone (Ts) has important physiological significance for the maintenance of the skeletal muscle functions (Manttari et al., 2008; Salehzadeh et al., 2011) and regulation of glycogen metabolism (Van Breda et al., 1993; Ramamani et al. 1999). Data on the androgen influence via their androgen receptor (AR) on glycogen and GS content in aerobic physical exercise in skeletal muscles are scarce.
The aim of this study was to investigate the effect of an AR blocker (BAR) on the glycogen content and the expression of GS in skeletal muscles during endurance training. Material and methods
Male Wistar rats (180-200g, n=24) were allocated into two groups: trained (T) and non-trained (NT). The trained rats were subjected to 8-week training on treadmill (EXER-3R-Treadmill, Columbus Instruments, Columbus, OHIO, USA) with submaximal loading (70-75% VO2max) 5 days a week. The duration of the training increased gradually during the first week. During the second week it reached up to 40 minutes a day and remained such to the end of the experiment. Half of the trained (T+F) and untrained (NT+F) rats were treated with the AR blocker Flutamide (15 mg-kg-1) dissolved in sesame oil and administered subcutaneously for 8 weeks, the rest animals received sesame oil for the same time period. Two days after the last training the rats were decapitated under Thiopental anaesthesia (30 mg-kg"1). The weight of m. soleus (Sol) and m. extensor digitorum longus (EDL) was measured up to 3 minutes after the decapitation (laboratory scale TP512A). Parts of the muscles were fixed in Bouin's fixative for 24 hours and embedded in paraffin. Immunohistochemical reaction was applied on thin sections (5 ^m). Primary antibody Anti-glycogen synthase CT (04-357 Chemicon, Millipore, Germany, 1:250) and ImmunoCruz ABC Staining System (Santa Cruz Biotechnology, USA) were used. Other parts of the muscles were frozen immediately in liquid nitrogen and stored in -800C until the moment of analysis. The PAS staining was applied (McManus, 1948). The average saturation of the reaction for glycogen and the intensity of the immune expression of GS in myocytes of six animals from each group were determined by software "DP-Soft" (Olympus, Japan) in arbitrary units (AU). To test for the two main effects of exercise training and BAR administration and for the interaction between them the results were assessed by two-way ANOVA. When the F-criterion was significant, depending on the homogeneity of the dispersions, Tuckey or Games-Howell post hoc tests were applied. A P<0.05 value was accepted as statistically significant. The results are presented as x±SEM.
Results
A tendency of lowering the muscle mass of Sol was found in rats treated with Flutamide, which had lower values compared to placebo treated groups (0.103±0.005 g v/s 0.117±0.005 g; P=0.078). The weight of EDL also had a tendency of lowering after BAR administration compared to placebo (0.131±0.006 g v/s 0.148±0.006 g; P=0.056). Training had no main effect (P>0.05). No interaction between two factors was found (P>0.05) (Figure 1).
0,2 0,18 0,16 0,14 0,12 3 0,1 (0,08 0,06 0,04 0,02 0
Figure 1. *P=0.078
##
_ so -1=|-1::|| I
< ;.
40 -rin-;:■--
:tt Ut
NT NT+F T T+F
Figure 2. Glycogen content in Soleus and EDL (AU). ##P<0.01 trained v/s untrained.
*P<0.001, compared to NT; **P<0.05, compared to NT; #P<0.001, compared to T.
□ Sol II EDL
Weight of Soleus and EDL (g) at the
end of the experiment. , **P=0.056 Flutamide treated groups v/s placebo.
The animals subjected to submaximal training had higher glycogen content in Sol compared to the sedentary groups (56.00±1.75 v/s 37.92±1.75 AU; P<0.001). The BAR application also had a main effect on glycogen, as the ones receiving Flutamide had lower content in comparison with placebo treated rats (32.52±1.75 v/s 61.41±1.75 AU; P<0.001). There was a significant interaction between the two factors (P<0.05). The highest glycogen content was established in muscle fibres of Sol in group T. The statistical analysis showed a significant difference to the NT group (P<0.001), as well as to the T+F group (P<0.001).
Training had a significant main effect on glycogen content of EDL. Trained animals had higher glycogen compared to untrained (30.91±1.63 v/s 22.54±1.63 AU; P<0.01). Application of BAR had no main effect (P>0.05). There was no significant two-way interaction (Figure 2).
The immunoexpression of GS was stronger in Sol of trained animals compared to the untrained ones (105.06±0.97 v/s 91.65±0.97 AU; P<0.001). Treatment with Flutamide had no significant main effect (P>0.05). No two-way interaction was found (P>0.05) (Figure 3, 5).
T
T+F
Figure 3. Immunoreaction for glycogen synthase in soleus (Magnification x400)
Figure 4. Immunoreaction for glycogen synthase in EDL (Magnification x400)
NT
T
100 90 80 70 60
100 90 80 70 60
1 *
n **
NT NT+F T T+F
Figure 5. Intensity of the immunoexpression of glycogen synthase in soleus (AU). *P<0.001 trained v/s untrained.
Figure 6. Intensity of the immunoexpression of glycogen synthase in EDL (AU). *P<0.001 compared to NT;
**P<0.001 compared to T.
In EDL the immunoexpression of GS was stronger in trained animals in comparison with untrained (101.39±U4 v/s 94.94±1.14 AU; P<0.01). Application of BAR had a significant main effect, as the animals receiving Flutamide had weaker expression (93.11±1.14 v/s 103.22±1.14 AU; P<0.001). Two-factor interaction was also established (P<0.05). The strongest immunoexpression was found in EDL of the T group, as the differences were significant to the NT group (P<0.001), as well as to the T+F (P<0.001), (Figures 4, 6). Discussion
The tendency to decrease of the muscle mass of Sol and EDL after BAR treatment for period of 8 weeks is also confirmed by other studies (Allan et al., 2008). Our results correspond also to the data of other researches that found effect of the decreased endogenous Ts levels on the muscle mass of EDL and Sol in people and animals after orchidectomy (Axell et al., 2006). The obtained data suggests that both types of muscle fibres (fast and slow) are characterized by androgen dependent type of their mass and the effect of the androgens is accomplished mainly through genomic mechanism via binding to AR.
Our results show that the glycogen content and the expression of GS in Sol and EDL in trained animals during rest is higher than that of untrained animals which is also confirmed by other researches. The activation of GS after a single exhausting training is important for the so called "glycogen overcompensation" following an initial depletion of the glycogen content (Gomes et al., 2009). The glycogen depletion itself appears to be a stimulus for the activation of GS and respectively of a glycogen synthesis. The increased expression of GS in trained rats proves the participation of the enzyme in adaptation processes to endurance training.
Blocking of AR in trained rats decreased glycogen content in the muscles studied which proves the participation of AR and androgens in glycogen maintenance. Those changes are expressed mainly in Sol, which participates actively in endurance training and this fact even more certainly highlights the androgen role in process of adaptation. The training also increased the expression of GS enzyme in EDL. According to its fibre content EDL is not resistant to continuous contractions, typical for aerobic physical exercise. The increased expression of GS enzyme corresponds to the increased glycogen content in the same muscle. However, the application of BAR decreased the expression of GS while there wasn't any effect on Sol. This could be explained by the differences of AR content in skeletal muscles which determine their response to the androgens. Glycogen in EDL of animals of the same group is reduced without statistical significance. This fact indicates preservation of muscle glycogen in type II muscle fibres.
CONCLUSIONS
Androgens influence glycogen content in the skeletal muscles of endurance trained rats as the blocking of AR leads to depletion of its amount. The expression of the GS in Sol remains higher in comparison with the results observed in untrained animals, which shows a protective effect of the training to some extent in respect to the blockade of androgen action.
120
120
110
110
NT
T
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