| The aim of this study was to focus on the protein metabolic responses and its molecular mechanisms induced by casein (CAS), soy protein isolate (SPI) and ZEIN dietary protein.Experiment 1. Tissue protein synthesis rates in growing rats were determined by the flooding-dose method in order to study protein metabolic responses induced by casein, SPI and zein protein diets. Protein synthesis rate and proteolysis rate were increased in gastrocnemius muscle of growing rats fed zein diet (P＜0.05), in which group, mRNA levels of mTOR, Rheb and atroginl (MAFbx), MuRF1, pUb and FoxO, and protein level of phosphorylated mTOR (Ser2448) were increased in gastrocnemius muscle compared with control group (P＜0.05 or P＜0.01). However, responses of protein metabolism showed the opposite effects in liver. Accumulated protein (mg/d) in liver and gastrocnemius muscle was 15.88,1.19; 16.41,0.92; 0.97,0.14 and 7.39,0.69 in casein, SPI, zein and mix dietary group, respectively. Plasma concentrations of Lys, Thr and Val in zein fed group were lower than those in casein group, however, Leu concentration in plasma was higher than casein group (P＜0.05). Transcriptional levels of InR (P＜0.01), IGF-I (P＜0.01) and IGF-I R (P＜0.05) were up-regulated in gastrocnemius muscle. But mRNA level of IGF-I R in liver was down-regulated (P＜0.05) with no significant changes to be observed in InR and IGF-I mRNA levels (P>0.05).Experiment 2. Based on Experiment 1., this experiment was conducted to compare the effect of supplementation with essential amino acids to different protein diets on activity of digestive enzymes, endocrine state and expression of genes related to amino acid transporters and protein metabolism in early-weaned piglets. Activities of SDH and XOD in liver andγyGT in jejunal brush border membrane were normalized to control levels by adding amino acids except to activities of APN, CPA, DPP-Ⅳ. Plasma concentrations of insulin in zein fed group was decreased, and IGF-1 level in SPI fed group was increased (P＜0.05). mRNA levels of InR, IGF-I and IGF-I R in skeletal muscles were higher in zein and SPI group (P<0.05), and values of which were normalized to control levels with amino acids supplementation (P>0.05). In SPI fed group, mRNA levels of PepTl (P＜0.05) in jejunal, EAAC1 (P<0.05) and ASCT1 (P<0.01) in liver, and PAT1 and ATF4 in skeletal muscles were up-regulated (P<0.05), which mostly normalized to control levels with amino acid addition except to PepT1 (P>0.05). In zein fed group, mRNA levels of ASCT1 (P＜0.01) in liver, CD98, PAT1 and ATF4 in skeletal muscles were up-regulated (P＜0.05), which mostly normalized to control levels with amino acid addition (P>0.05). mRNA levels of EAAC1 and PepT1 of piglets fed SBM were higher than control group (P＜0.05).The values related to mTOR pathway and Ubiquitin (Ub)-proteasome pathway (UPP) were mostly down-regulated to normal levels with amino acids supplementation (P>0.05). With amino acids supplementation, mRNA levels of MyoD and MyoG (Myogenin) were down-regulated and gene expression level of MSTN (Myostatin) was up-regulated to that in corn-soybean meal based diet.Experiment 3. Amino acids, especially branched chain amino acids (leucine) were the main factors for regulating protein metabolism. Thus, This experiment was to study the effects of BCAA on molecular mechanisms of protein metabolism in L6 myotubes. Phosphorylation of S6K1 (Thr 389), ribosomal efficiency and total protein concentration in L6 myotube were increased by BCAA (P<0.05), which was dependent on mTOR pathway. Transcriptional levels of LAT1 and CD98 were induced by BCAA dependent of mTOR pathway (P<0.05). mRNA level of SNAT2 were up-regulated by BCAA dependent of insulin involved, which was mTOR-dependent (P<0.05). BCAA did not affect mRNA levels of GCN2 and ATF4, but translational level of ATF4 was up-regulated by BCAA, dependent on mTOR signaling pathway (P<0.05). No significant changes were observed in mRNA levels of MuRF1 and MAFbx without insulin involved. However, mRNA level of MAFbx was reduced by BCAA in insulin involved (P<0.05), which could not be completely blocked by rapamycin. And expressions of MuRF1 and MAFbx were higher without insulin involved than those in insulin involved (P<0.05).In summary, protein metabolism and its molecular mechanisms were changed by different protein sources. Reasons for such metabolic responses induced by protein sources may be partly related to amino acid profiles in protein diets, which were reflected in transport ability of amino acid in small intestine, liver and skeletal muscles, and endocrine state. Protein synthesis and proteolysis may be induced by amino acids (BCAA) and hormones dependent of mTOR and Ubiquitin (Ub)-proteasome signal transduction pathways. |
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