Effects Of Glucocorticoids On The Protein Metabolism Of Skeletal Muscles In Broiler Chickens

In order to study the effects of glucocorticoids on the protein metabolism of skeletal muscles in broiler chickens (Gallus gallus domesticus), we did the experiments in vivo and in vitro. In vivo, we used exogenous glucocorticoids to induce stress on the later stage of broilers'development, and in vitro, we treated the myoblasts of broiler chickens with dexamethasone. The mRNA level of several genes which were important for the protein metabolism were measured looking forward to constructing genetic regulatory networks of skeletal muscle protein metabolism as well as finding out its'detailed molecular mechanism.The experiments in vivo were separated into two parts. In trial 1, 144 male AA broilers of 28 day were allocated into 12 treatments according to body weigh, 12 broilers per treatment. At the age of 29d, chickens were randomly separated into two groups: the basal diet group and the high protein ration, of which the latter was derived from a basal diet group through a 3d adjustment. At the age of 32d, chickens were randomly subjected to one of the following three treatments for 3 days: subcutaneous injection of DEX (2mg/kg BM), sham-treated (2 mg/kg BM of saline, control), sham-treated and limited-fed treatment to keep the feed consumption as that of the DEX chickens in the previous day. BM was recorded daily. At the age of 35d, half chickens of each treatment were randomly exposed to fasting for 12h or fasting 12h and refeeding 1h before samples were obtained. A blood sample was drawn from a wing vein by using a heparinized syringe within 30s and collected in iced tubes. Plasma was obtained after centrifugation at 400g for 10 min at 4°C and was stored at ?20°C for further analysis. Immediately after the blood sample had been obtained, the chicks were selected and sacrificed by exsanguination, and then harvested and weighed the breast and thigh muscle respectively. A 1 to 2g sample was respectively obtained from left PM, cooled down in liquid nitrogen and stored at -80℃for further analysis. The muscle of right leg and whole breast were harvested and weighed respectively, the breast and thigh mass were expressed as percentage of body mass (%). After three days'treatment, DEX had a significantly decrease (P<0.01) on daily gain of broiler chickens, in contrast, DEX had no significant (P>0.05) effect on food intake. The concentration of plasma uric acid (UA), total amino acid (T-AA) and insulin (INS) were higher at both sampling ages. The mRNA level of several genes that are important for protein proteolysis were significantly higher after three days exposure. The effect of glucocorticoid was not significantly inhibited by the high protein ration. In trial 2, 72 male AA broilers of 35 day were allocated into 12 treatments according to body weigh, 6 broilers per treatment. At the age of 36d, chickens were randomly subjected to one of the following two treatments for 3 days: subcutaneous injection of DEX (2mg/kg BM), sham-treated (2mg/kg BM of saline, control). BM was recorded daily. At the age of 38d, chickens of each treatment were exposed to fasting for 12h before blood samples were obtained. A blood sample was drawn from a wing vein by using a heparinized syringe within 30s and collected in iced tubes. After obtained the blood samples, every treatment was randomly separated into two perfusing treatments, leucine (500mg/kg BM) and salt (5ml). After perfusing 1h, another blood sample was drawn from a wing vein by using a heparinized syringe within 30 s and collected in iced tubes. Plasma was obtained after centrifugation at 400g for 10 min at 4°C and was stored at ?20°C for further analysis. Immediately after the blood sample had been obtained, the chicks were selected and sacrificed by exsanguination, and then harvested and weighed the breast and thigh muscle respectively. A 1 to 2g sample was respectively obtained from left PM, cooled down in liquid nitrogen and stored at -80℃for further analysis. The muscle of right leg and whole breast were harvested and weighed respectively, the breast and thigh mass were expressed as percentage of body mass (%). After three days treatment, DEX had a significantly decrease (P<0.01) on daily gain of broiler chickens, in contrast, DEX had no significant effect on food intake. The mRNA level of several genes that are important for protein proteolysis were significantly higher after three days exposure, while the mRNA level of genes that are important for protein synthesis were not significantly affected. The effect of glucocorticoid was not significantly inhibited by the perfusing of leucine. The experiments in vitro were separated into two parts also. In trial 3, cells were cultured in DMEM medium containing 10% heat-inactivated FBS, when cells were 70% confluent, the proliferation medium was replaced by a differentiation medium containing 2% horse serum. After 48h of differentiation, cells were incubated for 1h with serum-free DMEM and subjected to different treatments for three hours including serum-free DMEM, 1uM Dexamethasone, 100nM Insulin, 1uM Dexamethasone and 100nM Insulin, four random fields of each of the six replicates were selected. The mRNA level of IGF-I was significantly inhibited by the treatment of dexamethasone after three hours'treatment, and the mRNA level of MyoG was significantly increased by the treatment of dexamethasone. The mRNA level of MyoG was significantly increased by the treatment of insulin, and the mRNA levels of several genes that are important for protein proteolysis were significantly inhibited by the treatment of insulin after three hours'treatment. When insulin was in the medium, the mRNA level of MyoD and Atrogin-1 was significantly increased by the treatment of glucocorticoid, which indicated that glucocorticoids had taken part in the metabolism of myoblasts. In trial 4, after 48h of differentiation, cells were incubated for 1h with serum-free DMEM and subjected to different treatments for three hours including serum-free DMEM, 100nm and RU486 1uM Dexamethasone (after the treatment of RU486 alone for 1h, the cells were treated with RU486 and dexamethosone for 3h), two random fields of each of the six replicates were selected. The mRNA levels of IGF-I,MyoD,MSTN,MuRF1 were not significantly affected by the treatment, which indicated that the effects of dexamethasone could be inhibited by the RU486.