Arginine Promotes Skeletal Muscle Slow-Twitch Fibers Formation By Improving Mitochondrial Function

Arginine(Arg)is a kind of conditional essential amino acid,which plays an important role in promoting the growth and development of piglets.Arginine may also play an important regulatory role in the formation of porcine muscle fiber types.Therefore,the effects of Arg on the formation of porcine slow-twitch muscle fibers and its mechanism were investigated in vitro and in vivo.Experiment 1.Effects of dietary Arg supplementation on slow-twitch muscle fibers formation and muscle mitochondrial function of weaned pigletsA total of sixty healthy DLY(Duroc× Landrace× Yorkshire)piglets(6.55±0.36 kg)were randomly assigned into four groups,including control group(basal diet),0.5% Arg(basal diet+ 0.5% Arg),1% Arg(basal diet+ 1% Arg)and 1.5% Arg(basal diet+ 1.5% Arg).Each treatment consisted of five replicates with three piglets per replicate.The trial lasted 28 d.The results were as follows:1)Dietary supplementation Arg had no significant effect on average daily feed intake(ADFI),average daily gain(ADG)and Feed/gain(F/G)of weaned piglets.2)Compared with the control group,the addition of 1% Arg significantly enhanced the serum SOD,CAT and T-AOC activities(P < 0.05),but decreased the serum MDA content.1% Arg markedly up-regulated the SOD1,CAT,GR and Nrf2 mRNA levels in longissimus dorsi muscle and psoas major muscle(P < 0.05).3)Compared with the control group,1% Arg significantly enhanced the NO content and NOS activity(P < 0.05)in longissimus dorsi muscle.1% Arg increased the proportion of type I muscle fiber and reduced the proportion of type II muscle fiber in the longissimus dorsi muscle.Meanwhile,the addition of 1% Arg improved the SDH activity in longissimus dorsi muscle and psoas major muscle(P < 0.05).The addition of 1% Arg significantly up-regulated the Slow-MyHC and Fast-MyHC protein expression levels in psoas major muscle and up-regulated the Slow-MyHC protein expression level in longissimus dorsi muscle(P < 0.05).The addition of 1% Arg improved the MyHC I,Tnni1,Tnnc1 and Tnnt1 mRNA levels in longissimus dorsi muscle and psoas major muscle(P < 0.05).4)Compared with the control group,the addition of 1% Arg increased the Cytc protein level in longissimus dorsi muscle and psoas major muscle(P < 0.05).What's more,the addition of 1% Arg had significantly increased Cytc?Nrf1?ND1?TFB1M and ATP5 G gene expression in longissimus dorsi muscle and psoas major muscle(P < 0.05).5)Compared with control group,the addition of 1% Arg up-regulated the phosphorylation of AMPK and ACC protein,and up-regulated SIRT1 and PGC-1? protein expression in both longissimus dorsi muscle and psoas major muscle of piglets(P < 0.05).Meanwhile,1% Arg had significantly increased AMPK?1,AMPK?2 and PGC-1? mRNA levels in longissimus dorsi muscle and psoas major muscle(P < 0.05).Experiment 2 Mechanism of Arg promoting the formation of slow-twitch fibers expression in skeletal musclePorcine muscle satellite cells were isolated from the longissimus dorsi muscle of a newborn piglet.Three days after differentiation,the cells were treated with different concentrations of Arg(0,50,100 and 200 ?g/mL)and Rot(0,0.1,0.5 and 1?M),an inhibitor of respiratory chain,for 72 h.Western Blot and qPCR analysis were used to detect the mRNA level and protein expression to find out the best concentration of Arg and Rot.On this basis,Arg and Rot were added to explore whether Arg affected the formation of slow muscle fibers in porcine muscle satellite cells by improving the function of mitochondria.The results showed that 100 ?g/mL Arg significantly increased the mRNA expression of Slow MyHC protein and MyHC I gene expression,significantly increased the expression of SIRT1,PGC-1? and Cytc protein,and significantly decreased the protein expression of MyHC IIb.Western Blot results showed that 0.1 ?M rotenone and 1 ?M Rot significantly down-regulated the protein expression of Slow MyHC,PGC-1? and Cytc.Finally,after treated with 100 ?g/mL Arg and 0.1?M Rot,100 ?g/mL Arg mediated up-regulation of AMPK ?1,AMPK ?2,SIRT1,PGC-1?,Cytc,TFB1 M,ATP5G mRNA expression and the Slow-MyHC,SIRT1,PGC-1? and Cytc protein expression were weakened by 0.1 ?M Rot.In conclusion,in vivo and in vitro experiments shows that the Arg affected the formation of slow muscle fibers in pigs by improving the function of mitochondria.