Study On Leucine Regulating Porcine Skeletal Muscle Fiber Type Transformation By AdipoQ Signaling Pathway

Muscle fiber type transformation plays an important role in improving meat quality,which is regulated by nutrients.Numerous studies indicated that Leu promoted the formation of slow-twitch muscle fibers and promoted porcine skeletal muscle fiber type transformation from fast-twitch to slow-twitch in vitro,but how this effect is achieved in vivo remain poorly understood.Furthermore,an our previous study indicated that Leu regulated the expression of slow-twitch muscle fibers expression through AMPK signaling pathway,and the secretion of adiponectin(AdipoQ)was regulated by Leu and located upstream of AMPK.Therefore,whether Leu affects porcine skeletal muscle fiber type transformation through AdipoQ signaling pathway remains to be further studied.On this basis,we investigated the effect of Leu on porcine skeletal muscle fiber transformation and AdipoQ and its receptor expression in weaning piglets,and then further explored whether Leu regulates the transformation of porcine skeletal muscle fibers through AdipoQ signaling pathway in porcine skeletal muscle satellite cells by adding Leu and AdipoR1-siRNA.The main research contents and results are as follows:1.Effect of dietary Leu supplementation on skeletal muscle fiber type transformation and AdipoQ and its receptors expression in weaning pigletsIn this experiment,a total of 54 21-day-old crossbred Duroc × Landrace ×Yorkshire male piglets were randomly divided into 3 treatments,18 piglets in each group.Three treatments were obtained by supplementing the basal diet with 0%,0.25%,0.5% of Leu.The experiment lasted for 42 days.The results showed that dietary Leu supplementation had no significantly difference in growth performance,however,dietery supplementation 0.25% Leu increased the growth performance of pigs to a certain extent.Compared with the control group,dietary 0.25% Leu supplementation significantly decreased serum inflammatory factors(IL-1β,IL-6 and TNF-α)concentrations(P < 0.01).At the same time,the protein expression levels of slow MyHC,Myoglobin,Troponin I-SS,p-AMPK and PGC-1α were significantly increased in Longissimus dorsi(LD)muscle by 0.25% Leu supplementation(P <0.05),while fast MyHC protein expression level was significantly reduced(P < 0.01).Moreover,the mRNA expression levels of MyHC I,Tnnc1,Tnnt1,Myoglobin,AMPKα2 and PGC-1α were significantly increased(P < 0.05),and the mRNA expression level of MyHC IIb was significantly decreased(P < 0.05).The ATPase staining results indicated that compared with control group,0.25% Leu supplementation significantly increased the number of slow muscle fibers and decreased the number of fast muscle fibers in LD muscle(P < 0.05).Furthermore,0.5%Leu treatment group significantly increased succinate dehydrogenase(SDH)activity and significantly decreased lactate dehydrogenase(LDH)activity(P < 0.05).Serum AdipoQ concentration was measured by ELISA kit.The results showed that 0.25%treatment Leu group significantly increased serum AdipoQ content when compared with the control group.And 0.25% Leu treatment group significantly increased the protein and mRNA expression levels of AdipoQ and adiponectin receptor 1(AdipoR1)in LD muscle by Western Blot and real-time quantitative PCR analysis(P < 0.05),but there was no significant difference in adiponectin receptor 2(AdipoR2)expression level between different treatment groups(P = 0.75).2.Study on the mechanism of Leu in regulating porcine skeletal muscle fiber transformationTo explore the mechanism of Leu in regulating porcine skeletal muscle fiber transformation,the porcine skeletal muscle satellite cells were treated with Leu(0,1,2 and 4 mM)for 24 h after 3 day of differentiation.On this basis,the effect of Leu on porcine skeletal muscle fiber type transformation and the expression of AdipoQ and its receptors were further investigated.Subsequently,AdipoR1-siRNA mediated down-regulation AdipoQ signaling was used to demonstrate whether Leu regulates porcine skeletal muscle fiber type transformation through AdipoQ signaling pathway.The results showed that 4 mM Leu extremely significantly enhanced the protein expression levels of slow MyHC(P < 0.001),and extremely significantly decreased fast MyHC protein expression level(P < 0.001).Then the optimum concentration of Leu was set to be 4 mM.Compared with control group,4 mM Leu group significantly increased the protein expression levels of Myoglobin,Troponin I-SS,AdipoQ,AdipoR1,p-AMPK and PGC-1α and the mRNA expression levels of AMPKα2,PGC-1α,AdipoQ and AdipoR1(P < 0.001),but there was no significant difference in protein expression and mRNA expression of AdipoR2(P > 0.05).At the same time,4mM Leu significantly increased the SDH activity(P < 0.05),and significantly decreased the LDH activity(P < 0.01).However,knockdown of AdipoR1 expression by AdipoR1-siRNA abolished Leu-induced upregulation of the protein expressioin of AdipoR1,slow MyHC,p-AMPK,PGC-1α and mRNA expression of AdipoR1,MyHC I,AMPKα2 and PGC-1α,and downregulation the protein expression of fast MyHC and mRNA expression of MyHC IIb.In summary,supplementation of 0.25% Leu to a pig diet can increase the expression of slow twitch muscle fiber,decrease the expression of fast twitch muscle fiber,and increase the expression of AdipoQ and its receptor.And then we further demonstrated that Leu regulates the transformation of porcine skeletal muscle fibers through AdipoQ signaling pathway in porcine skeletal muscle satellite cells by adding Leu and AdipoR1-siRNA.This study confirmed that Leu increased AdipoR1 through AdipoQ signaling pathway,activiated AMPK in the downstream,and then promoted porcine skeletal muscle fiber type transformation in both in vivo and in vitro studies.