The Pathway Of Glucocotricoids In Regulating Protein Synthesis Of Chicken Skeletal Muscle

The experiments were conducted to study the pathway of glucocorticoids in regulating proteinsynthesis of chicken （Gallus gallus domesticus） skeletal muscle. The underlying mechanismsregulating protein synthesis were further investigated in vivo and in vitro.We carried out three animal experiments. In trial1, the male AA broilers（36-d old）wererandomly subjected to one of the following two treatments for3days: subcutaneous injection ofdexamethasone （DEX,1mg/kg BM, twice per day）, sham-treated （1mg/kg BM of saline, control）. BMand feed intake were recorded daily. At the age of38d, chickens of each treatment were exposed tofasting for12h before blood and molecular samples were obtained. After obtained the samples, everytreatment was randomly divided into four perfusing treatments: leucine （500mg/Kg BM）, glucose（2.5ml/Kg BM）, leucine and glucose mixture, salt （5ml）. Glucocorticoids retard the growth of skeletalmuscle and decreased the growing performance of broiler chickens. Compared with the control, themRNA levels of AMPKα2and IGF-I were significantly down-regulated by DEX, while mRNAexpression of mTOR、Akt1、4E-BP1and S6K1were obviously increased. Perfusing leucinesignificantly decreased the mRNA levels of IGF-1and4E-BP1on DEX condition. Moreover,perfusing leucine significantly increased IGF-IR mRNA expression on DEX condition. The resultssuggested the mTOR and AMPK pathways were involved in protein synthesis by glucocorticoids.Perfusing leucine and glucose had no significant effects on protein synthesis by DEX treatment. Intrial2, the male AA broilers（29-d old）were assigned randomly into two groups （64chickens/group）according to the body mass （BM）, representing supplementation with1.0%L-leucine or0.68%L-alanine （isonitrogenous control） to the basal diet. Each group was divided into2treatments. Theexperimental chickens were randomly subjected to one of the following2treatments for4days:subcutaneous injection of DEX （1mg/kg BM, twice per day）, sham-treated （1mg/kg BM of saline,control）. BM and feed intake were recorded daily. At the end of the experiment, half of all theexperimental chickens were sampled at fasting state after12h feed withdrawal, the other half ofchickens were sampled at feeding state after3h re-feeding. The results suggested the glucocorticoids retarded the growth of skeletal muscle and decreased the growing performance of broiler chickens andregulated the protein synthesis by mTOR and AMPK pathways. In addition, leucine can remit theeffects on protein synthesis by DEX treatment. In trial3, the male AA broilers（34-d old）wereassigned randomly into two groups （64chickens/group） according to the body mass （BM）,representing supplementation with basal diet （3112MJ/Kg）or high energy diet （3612MJ/Kg）. Eachgroup was divided into2treatments. The experimental chickens were randomly subjected to one ofthe following2treatments for4days: subcutaneous injection of DEX （1.0mg/kg BM, twice per day）,sham-treated （1mL saline, control）. BM and feed intake were recorded daily. At the end of theexperiment, half of all the experimental chickens were sampled at fasting state after12h feedwithdrawal, the other half of chickens were sampled at feeding state after3h re-feeding. Underdietary high energy diet, the phosphorylation of AMPKα2Thr¹⁷²、mTOR Ser²⁴⁴⁸and p70S6K Thr³⁸⁹were significantly actived at fasting state. Under dietary basal diet, the phosphorylation of mTORSer²⁴⁴⁸was significantly actived, while the phosphorylation of p70S6K Thr³⁸⁹and Akt Ser⁴⁷³weresignificantly inhibited at fasting state. At re-feeding state, DEX treatment significantly inhibited thephosphorylation of mTOR Ser²⁴⁴⁸and p70S6K Thr³⁸⁹under dietary high energy diet, whilesignificantly actived he phosphorylation of AMPKα2Thr¹⁷²and p70S6K Thr³⁸⁹under dietary basaldiet. The results suggested that the mTOR and AMPK pathways were involved in protein synthesis byglucocorticoids. Furthermore, glucocorticoids regulated the phosphorylation of p70S6K that may getthrough mTOR pathway or other pathway.We carried out three experiments in vitro. Myoblasts were prepared from SPF chicken embryos（16-d old）. In trial4, myoblasts subjected to following treatments:40nM DEX,40nM DEX+10μMLY294002,40nM DEX+10μM Akt inhibitor,40nM DEX+25μM Rapamycin or control for3hours.The expression of GR、mTOR、S6K1、AMPKα2and Akt1were significantly inhibited by DEX. ThemRNA levels of Akt1、S6K1and AMPKα2were significantly decreased by cooperation of DEX andPI3K/Akt inhibitor. In trial5, myoblasts subjected to following treatments:1μM DEX,1μMDEX+100nM RU486,1μM DEX+25μM Rapamycin or control for3hours. The phosphorylation ofAMPKα Thr¹⁷²and mTOR Ser²⁴⁴⁸were significantly actived by DEX. The phosphorylation of mTORSer²⁴⁴⁸was significantly inhibited by DEX and RU486, while p70S6K Thr³⁸⁹phosphorylation wasactived and the same results were got by DEX and Rapamycin. The results of trial4and5showed thatglucocorticoids may affect protein synthesis pathways by GR and which regulated protein synthesis by the mTOR and AMPK pathway, dependent of Akt. Furthermore, glucocorticoids regulate thephosphorylation of p70S6K that may get through mTOR pathway or other pathway. In trial6,myoblasts subjected to following treatments:1μM DEX,10mM BCAA,1μM DEX+10mM BCAA orcontrol for0.5h,1h,3h,12h. The results showed that DEX and BCAA regulated protein synthesiswhich may have time effect. The expression of S6K1was significantly decreased by DEX for3hours.The expression of AMPKα2、mTOR、4E-BP1and S6K1were significantly inhibited by BCAA for1hour. The results suggested DEX and BCAA may regulat protein synthesis by AMPK and mTORpathways.