| Skeletal muscle is an important component of animal body and also the main meat producing organ of livestock and poultry.However,the mechanism of skeletal muscle growth,especially the regulation of nutrition,remains unclear.Lysine(Lys),the first limiting amino acid in cereal-based diets,has been thought to promote muscle fiber hypertrophy by increasing protein synthesis.Stimulated by the external environment,the satellite cells(SCs)distributed in the basal lamina and sarcolemma of skeletal muscle fibers are the dynamic source for myofiber hypertrophy via proliferation,differentiation and fuse into myofiber to form new nuclei.Despite that,whether dietary Lys activates SCs to promote skeletal muscle growth has not been reported,and the specific mechanisms still unknown.Thus,this study was designed to explore the relationship between Lys,SCs and skeletal muscle growth and its intensive mechanism,so as to provide theoretical basis for the precise regulation of livestock production by nutrition.The main research contents and results are as follows:Experiment 1: Effects of dietary Lys supply on growth performance and meat quality of weaned piglets.A total of 30 Duroc × Landrace × Large White weaned piglets with similar weights were randomly divided into 2 groups for 14 d.The control group was fed a basic diet containing 1.31 % Lys(n = 12).The Lys deficiency group was fed a restricted diet containing 0.83 % Lys(n = 18).On day 15,six piglets were selected from each group to evaluate meat quality.Moreover,six piglets from the Lys deficiency group were assigned to the Lys rescue group(Lys = 1.31 %;n = 6)for an additional 14 d.The remaining piglets in the control group and Lys deficiency group were kept on the same diets as those administered from 0 to 14 d.That is,from 15 to 28 d,the piglets were divided into a control group(n = 6),a Lys deficiency group(n = 6)and a Lys rescue group(n = 6).Results are as follows:(1)Compared with the control group,the average daily weight gain and feed conversion rate of weaned piglets in the Lys deficiency group were significantly reduced in the first(0-14 d),the later(15-28 d)and the whole experiment(0-28 d)period(P < 0.05).Compared with Lys deficiency for 28 d,the average daily feed intake in the Lys rescue group was significantly increased from 15 to 28 d(P < 0.05).In addition,the average daily weight gain and feed conversion rate in the Lys rescue group were significantly increased from 15 to 28 d and 0 to 28 d(P < 0.05).(2)In longissimus dorsi muscle,compared with the control group,dietary Lys deficiency for 14 d significantly increased cooking loss(P < 0.05).Compared with the control group and Lys deficiency group,we found that the a* detected at 45 min and 24 h was significantly increased after Lys deficiency for 14 d and then re-supplementation for another 14 d(P < 0.05).Moreover,dietary Lys deficiency for 14 d and then re-supplementation for another 14 d significantly increased the a* value at 45 min compared to dietary Lys deficiency for 28 d in the semimembranosus(P < 0.05),and the cooking loss also tended to be decreased in the control group(P = 0.051).Experiment 2: Effects of dietary Lys supply on skeletal muscle growth of weaned piglets.The longissimus dorsi muscle,psoas major muscle and others completed separated to analysis the effects of dietary Lys supply on skeletal muscle growth.Results are as follows:(1)Compared with the control group,the relative weight of the longissimus dorsi muscle was significantly decreased after dietary Lys deficiency for 14 d(P < 0.05).Compared with the Lys deficiency group at 28 d,the relative weight of the longissimus dorsi muscle was significantly increased after Lys deficiency for 14 d and then re-supplementation for another 14 d(P < 0.05).(2)Compared with the control group,the relative weight of the extensor carpi radialis muscle and total forequarters muscle were significantly decreased after dietary Lys deficiency for 14 d(P < 0.05).Compared to dietary Lys deficiency for 28 d,the relative weight of the lateral head of triceps of brachii muscle and extensor carpi radialis muscle were significantly increased in the control group and Lys rescue group(P < 0.05).(3)Compared with the control group,the relative weight of the semimembranosus muscle and total hindquarters muscle were significantly decreased after dietary Lys deficiency for 14 d(P < 0.05).Compared to dietary Lys deficiency for 28 d,the relative weight of the biceps femoris muscle,cranial tibial muscle,soleus muscle and total hindquarters muscle were significantly increased in the control group and Lys rescue group(P < 0.05),and the relative weight of semimembranosus muscle and semitendinosus muscle were significantly increased in the Lys rescue group(P < 0.05),and the relative weight of gemelli muscle was significantly increased in the control group(P < 0.05).Experiment 3: Effects of dietary Lys supply on proteomic profile of longissimus dorsi muscle of weaned piglets.Considering above findings,we collected longissimus dorsi muscle samples for isobaric tag for relative and absolute quantitation(i TRAQ)analysis to learn more about the role of Lys in governing skeletal muscle growth.Results are as follows:(1)The Kyoto Encyclopedia of Genes and Genomes(KEGG)functional annotation analysis of total proteins showed that the focal adhesion,mammalian target of rapamycin(mTOR)signaling pathway,lysosome,cell cycle,Lys biosynthesis were required for Lys promoted longissimus dorsi muscle growth,and can be used for further research.(2)Quantitative analysis of differential proteins showed that compared with the control group,10 proteins were down-regulated and 62 proteins were up-regulated in the Lys deficiency group;meanwhile,13 proteins were down-regulated and 31 proteins were upregulated in the Lys rescue group.Compared with the Lys deficiency group,21 proteins were down-regulated and 3 proteins were up-regulated in the Lys rescue group.(3)Gene Ontology(GO)enrichment analysis of differential proteins showed that compared with the control group,the protein serine/threonine kinase inhibitor activity was down-regulated in the Lys rescue group,whereas the sarcolemma and basal plasma membrane related proteins were up-regulated.Furthermore,compared with the Lys deficiency group,the negative regulation of fat cell differentiation,muscle hypertrophy,positive regulation of myoblast differentiation related proteins were up-regulated in the Lys rescue group.Experiment 4: Effects of dietary Lys supply on the status of SCs in longissimus dorsi muscle of weaned piglets.In order to further study the function of Lys on skeletal muscle growth,the longissimus dorsi muscles were selected for next work.The status and number of SCs were determined by immunohistochemical fluorescence staining assay,the level of key proteins in myogenic regulatory factors(MRFs)and mammalian target of rapamycin complex 1(mTORC1)pathway were detected by western blotting assay.Results are as follows:(1)Compared with the control group,dietary Lys deficiency for 14 and 28 d significantly reduced the ratio of total SCs,proliferative SCs and differentiated SCs to total cells in the longissimus dorsi muscle(P < 0.05).Compared with Lys deficiency for 28 d,the ratio of total SCs,proliferative SCs and differentiated SCs to total cells in the longissimus dorsi muscle were significantly increased in the Lys rescue group(P < 0.05).Meanwhile,the ratio of proliferated or differentiated SCs to total SCs were significantly increased(P < 0.05).In addition,Lys re-supplementation resulted in a significantly higher number of proliferative SCs in the longissimus dorsi muscle than in the control group(P < 0.05).(2)Compared with the control group,dietary Lys deficiency for 14 and 28 d significantly reduced the expression levels of myosin heavy chain(My HC)and myogenic regulatory factor 4(MRF4)in the longissimus dorsi muscle(P < 0.05).Compared with Lys deficiency for 28 d,dietary Lys re-supplementation significantly increased My HC and myogenin protein expression levels in the longissimus dorsi muscle(P < 0.05).(3)Compared with the control group,dietary Lys deficiency for 14 and 28 d significantly reduced the key protein levels of mTORC1 pathway in the longissimus dorsi muscle of weaned piglets(P < 0.05).Compared with Lys deficiency for 28 days,dietary Lys re-supplementation reactivated the key proteins in the mTORC1 pathway(P < 0.05).Experiment 5: Effects of medium Lys supply on the proliferation ability of SCs.SCs in the longissimus dorsi muscle of 5 days old piglets were isolated and cultured in vitro to detect whether the mTORC1 pathway mediates Lys induced SC proliferation and protein synthesis.Results are as follows:(1)Compared with the control group,the Lys deficiency for 48 h resulted in significant inhibition of the viability,proliferation and protein synthesis of SCs and the key protein levels in the mTORC1 pathway(P < 0.05).(2)Compared with Lys deficiency for 96 h,Lys deficiency for 48 h and then resupplemented for another 48 h significantly up-regulated the viability,proliferation ability and protein synthesis of SCs(P < 0.05).Meanwhile.the level of key proteins in mTORC1 pathway was also significantly up-regulated by Lys rescue(P < 0.05).(3)Compared with the control and Lys rescue conditions,we found Lys resupplemented with rapamycin significantly decreased the protein levels of the mTORC1 pathway(P < 0.05).Meanwhile.the rescued cell viability by Lys re-supplementation was inhibited by the simultaneous addition of rapamycin and reduced to the Lys deficiency level(P < 0.05).Moreover,Lys re-supplementation with rapamycin suppressed SC proliferation and protein synthesis of SCs(P < 0.05).Experiment 6: Effects of medium Lys supply on the differentiation ability of SCs.The SCs isolated from the longissimus dorsi muscle of 5 days old piglets were selected to measure the influence of Lys supply on the differentiation ability and the mechanism of mTORC1 pathway was also detected.Results are as follows:(1)Compared with the control group,Lys deficiency for 48 h significantly reduced the fusion index of SCs(P < 0.05),and also inhibited the key protein levels of My HC,myogenin and MRF4(P < 0.05).In addition,the key protein levels in the mTORC1 pathway of SCs during differentiation were significantly down-regulated(P < 0.05).(2)Compared with Lys deficiency for 96 h,the fusion index of SCs,and the key protein levels of My HC,myogenin and MRF4 were also significantly increased in the Lys rescue group(P < 0.05).Furthermore,compared with Lys deficiency for 96 h,the mTORC1 pathway was re-activated in the Lys rescue group(P < 0.05).(3)After medium Lys re-supplemented with rapamycin for another 48 h,we found the key proteins in the mTORC1 pathway,which were up-regulated by re-supplemented Lys was inhibited rapamycin(P < 0.05).Furthermore,the results of western blotting also showed that the increased My HC,myogenin,MRF4 and SC differentiation with re-supplemented Lys were suppressed by rapamycin(P < 0.05).In conclusion,we found Lys is not only a material for protein synthesis but also a signaling molecule to activate mTORC1 pathway to control SC proliferation and differentiation to promote skeletal muscle growth of pigs. |
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