Effects Of Dietary Leucine Levels On Proteome Of Skeletal Muscle In Early Weaning Piglets

Early weaning strategy can lead to early weaning stress and upregulate autophagy level in the skeletal muscle,liver and spleen of piglets.Autophagy is an evolutionarily conserved catabolic process that has been involved in several physiological and pathological cellular process,incluing growth,development,differentiation,metabolism,immunity,inflammation and apoptosis.Leucine is not only the substrate for protein synthesis,but also can regulate autophagy via mTORC1,Sestrin2 and glutamate dehydrogenase signaling pathways.Therefore,it is necessary to investigate the molecular mechanism underlying autophagy in response to leucine and provide valuable data for precision feeding of early weaning piglets.In this study,every three early weaning piglets that fed with 1.66% and 2.10%leucine level diets,were selected for slaughter at 21,22 and 26 days old.We applied iTRAQ to identify differentially expressed proteins and screen autophagy-related proteins regulated by leucine in longissimus muscle.A total of 1919 proteins and 402 differentially expressed proteins were identified and the unique peptide,length of pepdide,protein sequence coverage were in accordance with mass spectrometry standard.The result of GO analysis showed that differentially expressed proteins were mainly distributed in the intracellular part and organelles,and were involved in metabolic process of nutrients,and mainly played the role of binding and catalysis.KEGG pathway analysis showed that protein synthesis signaling pathway was significantly enhanced,including eIF2,eIF3 E,eIF5,eEF1 signaling and 40 S ribosomal proteins.Western blot analysis showed the activity of mTOR and its downstream protein S6K1 was significantly stimulated.Meanwhile,the contents of F0F1-ATP synthase,NADH dehydrogenase,iron-sulfur protein in oxidative phosphorylation pathway,cis-aconitate hydratase,isocitrate dehydrogenase,succinate dehydrogenase,2-oxoglutarate dehydrogenase,succinyl coenzyme A in citrate cycle pathway,pyruvate kinase in glycolysis,glucose phosphate isomerase in pentose phosphate pathway were significantly increased.In experimental group,we found that the contents of AMP and ADP were significantly decreased,the ATP content was significantly increased,and the ratio of AMP/ATP was significantly decreased via high performance liquid chromatography.Eventually,we screened three autophagy-related proteins of CAPNS1,RACK1,PRKAR1 A,and their expressions were upregulated by early weaning stress-induced autophagy.In addition,AMPK was activatedand mTOR was repressed.Thus,our data suggested that leucine could activate multiple energy synthesis pathways to stimulate mTOR by repressing AMPK,then inhibiting autophagy and the expression of RACK1.In summary,we obtain the following conclusions:(1)protein synthesis is enhanced in skeletal muscle of weaning piglets fed with 2.10% leucine level diet,including mTOR and eIF2 signaling pathways;(2)energy synthesis is promoted in skeletal muscle of weaning piglets fed with 2.10% leucine level diet,with decreasing AMP content and increasing ATP content;(3)the expressions of CAPNS1,RACK1 and PRKAR1 A are upregulated by early weaning stress-induced autophagy;(4)leucine is likely to inhibit autophagy by repressing the expression of RACK1.