Modulation of cytokine production by physical activity is of considerable interest, since it might be a promising strategy for correcting metabolic processes at both cellular and systemic levels. The ...content of IL-6, IL-8, and IL-15 in the plasma and the concentration of monovalent cations in the skeletal muscles of trained and untrained mice were studied at different periods after static and dynamic exercises. Dynamic loads caused an increase in the IL-6 content and decrease in the IL-15 content in the plasma of untrained mice, but produced no effect on the concentration of IL-8. In trained mice, the effect of a single load on the concentration of IL-6 and IL-15 in the plasma was enhanced, while the concentration of IL-8 decreased. Static loads produced a similar, but more pronounced effect on the plasma concentration of IL-6 and IL-15 compared the dynamic exercises; however, the concentration of IL-8 in response to the static exercise increased significantly. Prior training reinforced the described response for all the myokines studied. Dynamic load (swimming) increased the intracellular content of sodium but decreased the content of potassium in the mouse
musculus soleus
. Similar response was observed after the static load (grid hanging) in the
musculus biceps
; but no correlation of this response with the prior training was found. Possible mechanisms involved in the regulation of cytokine secretion after exercise are discussed, including triggering of gene transcription in response to changes in the Na
+
i
/K
+
I
ratio.
The cardiotonic steroids (CTS), such as ouabain and marinobufagenin, are thought to be adrenocortical hormones secreted during exercise and the stress response. The catalytic α-subunit of Na,K-ATPase ...(NKA) is a CTS receptor, whose largest pool is located in skeletal muscles, indicating that muscles are a major target for CTS. Skeletal muscles contribute to adaptations to exercise by secreting interleukin-6 (IL-6) and plethora of other cytokines, which exert paracrine and endocrine effects in muscles and non-muscle tissues. Here, we determined whether ouabain, a prototypical CTS, modulates IL-6 signaling and secretion in the cultured human skeletal muscle cells. Ouabain (2.5–50 nM) suppressed the abundance of STAT3, a key transcription factor downstream of the IL-6 receptor, as well as its basal and IL-6-stimulated phosphorylation. Conversely, ouabain (50 nM) increased the phosphorylation of ERK1/2, Akt, p70S6K, and S6 ribosomal protein, indicating activation of the ERK1/2 and the Akt-mTOR pathways. Proteasome inhibitor MG-132 blocked the ouabain-induced suppression of the total STAT3, but did not prevent the dephosphorylation of STAT3. Ouabain (50 nM) suppressed hypoxia-inducible factor-1α (HIF-1α), a modulator of STAT3 signaling, but gene silencing of HIF-1α and/or its partner protein HIF-1β did not mimic effects of ouabain on the phosphorylation of STAT3. Ouabain (50 nM) failed to suppress the phosphorylation of STAT3 and HIF-1α in rat L6 skeletal muscle cells, which express the ouabain-resistant α1-subunit of NKA. We also found that ouabain (100 nM) promoted the secretion of IL-6, IL-8, GM-CSF, and TNF-α from the skeletal muscle cells of healthy subjects, and the secretion of GM-CSF from cells of subjects with the type 2 diabetes. Marinobufagenin (10 nM), another important CTS, did not alter the secretion of these cytokines. In conclusion, our study shows that ouabain suppresses the IL-6 signaling via STAT3, but promotes the secretion of IL-6 and other cytokines, which might represent a negative feedback in the IL-6/STAT3 pathway. Collectively, our results implicate a role for CTS and NKA in regulation of the IL-6 signaling and secretion in skeletal muscle.
Type 2 diabetes mellitus accounts for about 90% of cases of diabetes and is considered one of the most important problems of our time. Despite a significant number of studies on glucose metabolism, ...the molecular mechanisms of its regulation in health and disease remain insufficiently studied. That is why non-drug treatment of metabolic disorders is of great relevance, including physical activity. Metabolic changes under the influence of physical activity are very complex and are still difficult to understand. This study aims to deepen the understanding of the effect of physical exercise on metabolic changes in mice with diabetes mellitus. We studied the effect of forced treadmill running on body weight and metabolic parameters in mice with metabolic disorders. We developed a high-fat-diet-induced diabetic model of metabolic disorders. We exposed mice to forced treadmill running for 4 weeks. We determined glucose and insulin levels in the blood plasma biochemically and analyzed Glut-4 and citrate synthase in M. gastrocnemius muscle tissue using Western blotting. The research results show that daily treadmill running has different effects on different age groups of mice with metabolic disorders. In young-age animals, forced running has a more pronounced effect on body weight. At week 12, young obese mice had a 17% decrease in body weight. Body weight did not change in old mice. Moreover, at weeks 14 and 16, the decrease in body weight was more significant in the young mice (by 17%) compared to the old mice (by 6%) (p < 0.05). In older animals, it influences the rate of glucose uptake. At 60 min, the blood glucose in the exercised older mice decreased to 14.46 mmol/L, while the glucose concentration in the non-exercised group remained at 17 mmol/L. By 120 min, in mice subjected to exercise, the blood glucose approached the initial value (6.92 mmol/L) and amounted to 8.35 mmol/L. In the non-exercised group, this difference was 45%. The effects of physical activity depend on the time of day. The greater effect is observed when performing shift training or exercise during the time when animals are passive (light phase). In young mice, light phase training had a significant effect on increasing the content of Glut-4 in muscle tissue (84.3 ± 11.3%, p < 0.05 with control group—59.3 ± 7.8%). In aged mice, shift training caused an increase in the level of Glut-4 in muscle tissue (71.3 ± 4.1%, p < 0.05 with control group—56.4 ± 10,9%). In the group of aged mice, a lower CS level was noticed in all groups in comparison with young mice. It should also be noted that we observed that CS increased during exercise in the group of young mice, especially during light phase training. The CS content in the light phase subgroup (135.8 ± 7.0%) was higher than in the dark phase subgroup (113.3 ± 7.7%) (p = 0.0006). The CS decreased in aged chow-fed mice and increased in the high-fat-fed group. The CS content in the chow diet group (58.2 ± 5.0%) was 38% lower than in the HFD group (94.9 ± 8.8%).
Electrical pulse stimulation (EPS)-treated cultured myotubes are widely employed as an in vitro model of muscle contraction. Here we examined time-dependent EPS action and dose-dependent ouabain ...action on Na+i and K+i in C2C12 myotubes. After 2 h of EPS (40 V, 1 Hz, 10 ms) Na+i increased by ∼150% whereas K+i declined by ∼20%. 3 μM ouabain had a negligible impact on Na+i and K+i in control cells but increased the Na+i/K+i ratio in EPS-treated myotubes by 85%. Thus, our results show for the first time that EPS results in dissipation of Na+ and K+ gradients in cultured myotubes and suggest that the augmented production of endogenous cardiotonic steroids may contribute to elevation of the Na+i/K+i ratio in exercising muscle.
•Electrical pulse stimulation (EPS) increases Na+ content in C2C12 myotubes.•EPS of C2C12 myotubes resultes in the loss of intracellular K+.•EPS sharply increases sensitivity of C2C12 myotubes to low doses of ouabain.•EPS-treated myotubes might be used as an in vitro model of exercising muscle.
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•In C2C12 myotubes, electrical pulse stimulation (EPS) resulted in Ca2+i oscillations and elevation of the Na+i/K+i ratio.•Additions of nicardipine abolished Ca2+i oscillations but ...did not affect elevation of the Na+i/K+i ratio.•EPS resulted in differential expression of 3215 transcripts.•In EPS-treated myotubes, differential expression of 1018 transcripts was preserved in the presence of nicardipine.•Elevation of the Na+i/K+i ratio contributes to Ca2+i-independent mechanism of transcription regulation triggered by EPS.
Elevation of Ca2+i and AMP-activated protein kinase (AMPK) are considered as major signals triggering transcriptomic changes in exercising skeletal muscle. Electrical pulse stimulation (EPS) of cultured myotubes is widely employed as an in vitro model of muscle contraction. This study examines the impact of Ca2+i-mediated and Ca2+i-independent signaling in transcriptomic changes in EPS-treated C2C12 myotubes. Electrical pulse stimulation (40 V, 1 Hz, 10 ms, 2 h) resulted in Ca2+i oscillations, gain of Na+i, loss of K+i, and differential expression of 3215 transcripts. Additions of 10 μM nicardipine abolished Ca2+i oscillations but did not affect elevation of the Na+i/K+i ratio seen in EPS-treated myotubes. Differential expression of 1018 transcripts was preserved in the presence of nicardipine, indicating a Ca2+i-independent mechanism of excitation–transcription coupling. Among nicardipine-resistant transcripts, we noted 113 transcripts whose expression was also affected by partial Na+,K+-ATPase inhibition with 30 μM ouabain providing the same elevation of the Na+i/K+i ratio as in EPS-treated cells. Electrical pulse stimulation increased phosphorylation of CREB, ATF-1, Akt, ERK, and p38 MAPK without any impact on phosphorylation of acetyl-CoA carboxylase and Unc-51 like autophagy activating kinase-1, i.e. downstream markers of AMPK activation. Unlike CREB, ATF-1, and MAPKs, an increment in Akt phosphorylation was abolished by nicardipine. Thus, our results show that Ca2+i-independent signaling plays a key role in altered expression of 30% of studied genes in EPS-treated myotubes. This signaling pathway is at least partially triggered by dissipation of transmembrane gradients of monovalent cations.
Elevation of Ca
and AMP-activated protein kinase (AMPK) are considered as major signals triggering transcriptomic changes in exercising skeletal muscle. Electrical pulse stimulation (EPS) of cultured ...myotubes is widely employed as an in vitro model of muscle contraction. This study examines the impact of Ca
-mediated and Ca
-independent signaling in transcriptomic changes in EPS-treated C2C12 myotubes. Electrical pulse stimulation (40 V, 1 Hz, 10 ms, 2 h) resulted in Ca
oscillations, gain of Na
, loss of K
, and differential expression of 3215 transcripts. Additions of 10 μM nicardipine abolished Ca
oscillations but did not affect elevation of the Na
/K
ratio seen in EPS-treated myotubes. Differential expression of 1018 transcripts was preserved in the presence of nicardipine, indicating a Ca
-independent mechanism of excitation-transcription coupling. Among nicardipine-resistant transcripts, we noted 113 transcripts whose expression was also affected by partial Na
,K
-ATPase inhibition with 30 μM ouabain providing the same elevation of the Na
/K
ratio as in EPS-treated cells. Electrical pulse stimulation increased phosphorylation of CREB, ATF-1, Akt, ERK, and p38 MAPK without any impact on phosphorylation of acetyl-CoA carboxylase and Unc-51 like autophagy activating kinase-1, i.e. downstream markers of AMPK activation. Unlike CREB, ATF-1, and MAPKs, an increment in Akt phosphorylation was abolished by nicardipine. Thus, our results show that Ca
-independent signaling plays a key role in altered expression of 30% of studied genes in EPS-treated myotubes. This signaling pathway is at least partially triggered by dissipation of transmembrane gradients of monovalent cations.
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