Protein Synthesis In Soleus Muscle Research Articles

ВКЛАД mTORC1 И GSK-3 В РЕГУЛЯЦИЮ БИОГЕНЕЗА РИБОСОМ В ПОСТУРАЛЬНОЙ МЫШЦЕ КРЫСЫ В УСЛОВИЯХ МОДЕЛИРУЕМОЙ МИКРОГРАВИТАЦИИ

The investigation was aimed to reveal a potential role of mechanistic target of rapamycin (mTORC1) and glycogen synthase kinase (GSK-3) in the regulation of ribosome biogenesis and protein synthesis in rat soleus muscle during simulated gravitational unloading. Wistar rats were subject to 7-day tail-suspension (hindlimb unloading) and treated with rapamycin (mTORC1 inhibitor) or AR-A014418 (GSK-3 inhibitor). Protein synthesis was measured with SUnSET. Phosphorylation of glycogen synthase-1 (GS-1) and ribosomal protein S6 (rpS6) was measured with the use of Western-blotting. Levels of 28S and 18S rRNA were determined using agarose gel electrophoresis. Expression of 47S pre-rRNA and c-Myc was assessed with RT-PCR. Gravitational unloading led to an increase in phosphorylation of GS-1 (a marker of GSK-3 activity) and inhibited phosphorylation of rpS6 (a marker of mTORC1 activity), expression of the ribosome biogenesis markers (total RNA РНК, 28S and 18S rRNA, 47S pre-rRNA, с-Myc) and protein synthesis in soleus muscle. GSK-3 inhibition prevented an increase in GS-1 phosphorylation and, partly, a reduction in the markers of both ribosomal biogenesis and protein synthesis. The mTORC1 inhibitor contributed to the phospho-rpS6 reduction but did not substantially influence the parameters of translational capacity and protein synthesis. To sum up, unlike mTORC1, an elevated GSK-3 activity can contribute to the inhibition of ribosome biogenesis and protein synthesis in rat soleus muscle under 7-day gravitational unloading.

Signaling Pathways Regulating Protein Synthesis in the Rat Soleus Muscle in the Early Period of Gravitational Unloading

The effects of different durations of gravitational unloading (antiorthostatic suspension of rats for 1, 3, or 7 days) on the intensity protein synthesis, ribosomal RNA contents, and intracellular signal pathways involved in controlling protein biosynthesis were studied in rat soleus muscle. The contents of key markers of the anabolic signal pathways (p-AKT, p-GSK-3β, p-p70s6k, p-4E-BP1, p-90RSK1) were determined by gel electrophoresis followed by immunoblotting. The intensity of protein synthesis in soleus muscle was evaluated by puromycin labeling, i.e., the SUnSET method. A significant decrease in protein synthesis intensity in the soleus muscle was seen after 3 and 7 days of gravitational unloading. Gravitational unloading for 3 and 7 days led to a significant decrease in the content of 28S rRNA. Functional unloading for 24 h led to a significant increase in the content of p-p70s6k and a decrease in the content of p-4E-BP1 in the soleus muscle (p < 0.05). By day 3 of suspension, there were decreases in the contents of markers such as phospho-AKT and phospho-p90RSK1 as compared with controls (p < 0.05). After 7 days of gravitational unloading, there were significant decreases in the levels of phosphorylated AKT and GSK-3β compared with the control group (p < 0.05). These results lead to the conclusion that decreases in the intensity of protein synthesis in rat soleus muscle at the early stages of gravitational unloading may be associated with suppression of ribosome biogenesis and decreases in the activity of mTORC1-dependent signal pathways.

Key Markers of mTORC1-Dependent and mTORC1-Independent Signaling Pathways Regulating Protein Synthesis in Rat Soleus Muscle During Early Stages of Hindlimb Unloading

Background/Aims: The purpose of the study was to assess the amount of rRNA and phosphorylation status of the key markers of mTORC1-dependent (70s6k, 4E-BP1) and mTORC1-independent (GSK-3β, AMPK) signaling pathways controlling protein synthesis in rat soleus during early stages of mechanical unloading (hindlimb suspension (HS) for 1-, 3- and 7 days). Methods: The content of the key signaling molecules of various anabolic signaling pathways was determined by Western-blotting. The amount of 28S rRNA was evaluated by RT-PCR. The rate of protein synthesis was assessed using in-vivo SUnSET technique. Results: HS for 3 and 7 days induced a significant (p<0.05) decrease in the rate of global protein synthesis in soleus muscle in comparison with control. HS within 24 hours resulted in a significant (p<0.05) decrease in p-4E-BP1 content, p-AMPK content and increase in p-p70s6k content in rat soleus muscle. Following three days of HS the content of p-AKT was decreased (p<0.05). After 7 days of HS the phosphorylation level of AKT and GSK-3beta was significantly reduced (p<0.05) compared to control. We also observed a significant decrease in the amount of 28S rRNA in rat soleus following 1, 3 and 7 days of HS. Conclusion: Taken together, the results of our study suggest that a decline in the global rate of protein synthesis in rat soleus during early stages of simulated microgravity is associated with impaired ribosome biogenesis as well as reduced activity of mTORC1-independent signaling pathways.

Effect of sympathetic denervation on the rate of protein synthesis in rat skeletal muscle.

Rates of protein synthesis were investigated in skeletal muscles from rats submitted to chemical and surgical sympathectomy. Three models of sympathetic denervation were used: 1) treatment with guanethidine (100 mg.kg(-1).day(-1) sc); 2) lumbar sympathetic denervation (surgical excision of the second and third lumbar ganglia of the sympathetic chain, from which arises the postganglionic fibers to the skeletal muscles of rat hindlimb); and 3) adrenodemedullation. Protein synthesis was estimated in isolated soleus muscle by the rate of incorporation of [(14)C]tyrosine (0.1 mM, 0.05 microCi/ml) into total protein. Soleus isolated after 2 and 4 days of chemical sympathectomy or after 3 days of lumbar denervation showed a 17-20% statistically significant decrease in in vitro rates of protein synthesis. These effects were reverted by addition of 10(-5) M isoproterenol or epinephrine in vitro. Neither clenbuterol nor isoproterenol (10(-7), 10(-6), or 10(-5) M) in vitro affected the rate of protein synthesis in soleus from normal rats. On the other hand, clenbuterol or epinephrine (10(-5) M) increased by 20% the rate of protein synthesis in soleus muscles from adrenodemedullated rats and prevented its decrease in muscles from fasted rats. The data suggest that the sympathetic nervous system stimulates protein synthesis in oxidative muscles, probably through the activation of beta(2)-adrenoceptors, especially in situations of hormonal or nutritional deficiency.

Effects of insulin on protein synthesis in muscles from normal and dystrophic mice.

Protein synthesis in soleus and extensor digitorum longus (EDL) muscles was measured in vitro to test the hypothesis that the lack of muscle protein accumulation in dystrophic conditions could be caused by a reduced sensitivity to insulin. We demonstrate that physiological insulin concentrations stimulate protein synthesis in soleus muscles from normal mice but not from muscles obtained from dystrophic (dy) animals. The difference is lost at very high insulin concentrations (1 microM) and could not be shown at any concentration with EDL muscles. These results, together with the reported reduced inhibitory effect of insulin on protein synthesis in dystrophic hamsters and on protein breakdown in dystrophic mice, suggest that protein metabolism in certain muscles from dystrophic animals may be less responsive to the anabolic effects of insulin.

Insulin affects only initiation and not elongation in protein synthesis in soleus muscles of lean and obese mice

The effect of insulin on polypeptide chain initiation and elongation has been studied in soleus muscles isolated from lean and goldthioglucose-obese mice. Insulin increased the amount of radioactivity present in nascent chains by ∼30% in muscles from both lean and obese mice, indicating that it stimulates peptide chain initiation. In contrast, elongation rates, estimated by measurement of half transit time, were similar in basal conditions and insulin-treated muscles of lean and obese animals. Thus, insulin increased the initiation without modifying the elongation rates. Obesity did not affect either basal rates of initiation and elongation or the effect of insulin.