The Effects Of Lycopene On Skeletal Muscle Fiber Type Conversion And Pork Quality And Its Underlying Mechanism

Based on the My HCs isoform,skeletal muscle fibers are divided into type I(slow-twitch,slow oxidative),type IIa(fast-twitch,fast oxidative),type IIx(fast-twitch,intermediate)and type IIb(fast-twitch,fast glycolytic).Different muscle fiber types have significant differences in metabolic characteristics and physicochemical properties,and are closely related to meat quality and fatigue resistance ability.Recently,investigation of the effects of nutrition and miRNAs on regulating skeletal muscle fiber type conversion is a research hotpot.Lycopene,mainly isolated from tomatoes and guavas,has strong antioxidant capacity.Lycopene is beneficial to maintain healthy body because of its anti-inflammatory and anti-cancer functions.However,the effects of lycopene on skeletal muscle fiber type conversion and meat quality are still unclear.Therefore,this study firstly investigated the effects of lycopene on the capacity of fatigue resistant and muscle fiber type conversion,and then focused on the role of lycopene in meat quality and explored the mechanism of lycopene on muscle fiber type conversion and meat quality using C2C12 myotubes or porcine myotubes as experimental objects.Exp.1 Effect of lycopene on muscle fiber type conversion of miceTo investigate the effects of lycopene on anti-fatigue capacity and skeletal muscle fiber type conversion of mice,a total of sixty mice were randomly divided into three groups of twenty animals according to their body weight which were fed with normal standard diets supplemented with 0,100 mg/kg or 300 mg/kg lycopene,respectively.All mice were housed in an individual cage.After 6 weeks,five mice were selected from every group performed the forced swimming test and obtained the time to exhaustion respectively.The remaining mice were anesthetized with CO₂and collected muscle samples.Our results showed that:(1)300 mg/kg lycopene significantly up-regulated the time to exhaustion of mice and ATP concentration in TA muscle;(2)300 mg/kg lycopene promoted the protein expressions of slow My HC and TNNI1,and decreased fast My HC and TNNI2 protein levels;300 mg/kg lycopene increased the number of slow-twitch fiber and decreased the number of fast-twitch fiber;300 mg/kg lycopene markedly increased the m RNA levels of My HC I,TNNI1and TNNC1,and decreased My HC IIa,My HC IIx and My HC IIb m RNA levels;(3)300 mg/kg lycopene markedly enhanced SDH and MDH enzyme activities,and decreased the enzyme activity of LDH;(4)300 mg/kg lycopene up-regulated the concentration of hemoglobin,and increased Complex III and Complex IV enzyme activities in TA muscle;300 mg/kg lycopene also up-regulated the m RNA levels of NRF1,TFAM,TFB1M,CS,ATP5G and Cytc in TA muscle;(5)lycopene up-regulated NRF1,Ca MKK?,P-AMPK,Sirt1,PGC-1?protein levels,and increased the expressions of AMPK?1,AMPK?2,Sirt1 and PGC-1?;(6)lycopene up-regulated the expression levels of P-Akt and P-Fox O1,and down-regulated Fox O1 protein level;(7)100 mg/kg and 300 mg/kg lycopene decreased miR-22-3p level in TA muscle of mice.These data indicated that lycopene enhanced anti-fatigue ability of mice,activated AMPK/Sirt1/PGC-1?signaling pathway and Akt/Fox O1 signaling pathway,and promoted skeletal muscle fiber type conversion from fast-twitch fiber to slow-twitch fiber.Lycopene also inhibited the expression of miR-22-3p.Exp.2 Effect of dietary lycopene supplementation on meat quality and muscle fiber type conversion of finishing pigsTo verify the effect of lycopene on meat quality and skeletal muscle fiber type conversion of finishing pigs,eighteen 63.89±1.15 kg barrows were randomly divided into three groups:control group,100 mg/kg lycopene group and 200 mg/kg lycopene group.There were 6 replicates in each group and 1 pig in each replicate.The experiment lasted 10 weeks.In vivo test results showed that:(1)supplementation with200 mg/kg lycopene significantly increased muscle a*_45minvalue,intramuscular fat and crude protein,and decreased muscle L*_45min value and muscle b*_45minvalue in longissimus dorsi of finishing pigs;(2)supplementation with 100 mg/kg and 200mg/kg lycopene significantly decreased the contents of MDA in liver and longissimus dorsi;100 mg/kg lycopene increased T-AOC and T-SOD enzyme activities in serum and up-regulated T-SOD,GSH-Px and CAT enzyme activities in liver;200 mg/kg lycopene increased GSH-Px and CAT levels in serum,increased T-AOC and GSH-Px enzyme activities in liver,and up-regulated T-SOD and CAT enzyme activities in longissimus dorsi;(3)200 mg/kg lycopene significantly increased SOD1,SOD2,CAT,GPX1,GST,GR and Nrf2 m RNA levels,and decreased the expression level of Keap1;(4)100 mg/kg and 200 mg/kg lycopene markedly promoted slow My HC expression and decreased fast My HC level,accompanied by increased the number of slow-twitch fiber and decreased the number of fast-twitch fiber;200 mg/kg lycopene up-regulated My HC I,My HC IIa,My HC IIx and TNNI1 m RNA levels and decreased the level of My HC IIb;(5)200 mg/kg lycopene markedly increased SDH and MDH enzyme activities and decreased LDH activity;(6)supplementation with lycopene promoted Cytc protein expression and up-regulated the m RNA levels of Cytc,TFAM,TFB1M,CS,COX1;(7)lycopene markedly induced the expressions of P-AMPK,NRF1,Ca MKK?,Sirt1,PGC-1?and increased AMPK?1,AMPK?2,Sirt1,PGC-1?m RNA levels in longissimus dorsi;(8)100 mg/kg and 200 mg/kg lycopene significantly suppressed the expression of miR-22-3p.These data indicated that lycopene could improve meat quality of finishing pigs,and might regulate muscle fiber type conversion from fast-twitch fiber to slow-twitch fiber through AMPK signaling pathway.Exp.3 The mechanism study of lycopene regulates skeletal muscle fiber type conversion via AMPK signaling pathway and miR-22-3pThese results of Exp.1 and Exp.2 indicated that lycopene improved meat quality of finishing pigs,enhanced anti-fatigue capacity of mice,promoted muscle fiber type conversion from fast-twitch fiber to slow-twitch fiber,and inhibited the expression of miR-22-3p in skeletal muscle.Whereas,the mechanism of lycopene is not reported.Previous results found that the levels of miR-22-3p in different skeletal muscles were markedly different and its sequence was highly conserved,we could guess that miR-22-3p might involve in skeletal muscle fiber type transformation.This study firstly investigated the effect and mechanism of miR-22-3p on skeletal muscle fiber type conversion in C2C12 and porcine myotubes,results showed that:(1)the expressions of miR-22-3p in TA and EDL were higher than in SOL;(2)miR-22-3p mimics significantly inhibited My HC I and My HC IIa and promoted My HC IIb in C2C12 myotubes,while miR-22-3p inhibitor treatment got reverse results.Meanwhile,miR-22-3p mimics significantly decreased the number of slow-twitch fibers in C2C12myotubes,while miR-22-3p inhibitor increased the number of fast-twitch fibers;(3)miR-22-3p mimics effectively decreased MDH enzyme activity and increased LDH activity;(4)miR-22-3p mimics significantly suppressed P-AMPK,Sirt1 and PGC-1?protein levels and inhibited AMPK enzyme activity in C2C12 myotubes,while miR-22-3p inhibitor up-regulated P-AMPK and Sirt1 protein levels;(5)Compound C attenuated the promoting effect of miR-22-3p inhibitor on the formation of slow-twitch fiber,and AICAR eliminated the inhibitory effect of miR-22-3p mimics on the slow-twitch fiber formation.The experimental results of porcine myotubes showed that:(1)miR-22-3p mimics significantly inhibited the protein expression of My HC I and promoted My HC IIb expression;(2)miR-22-3p mimics markedly decreased the protein levels of P-AMPK,Sirt1 and PGC-1?;(3)AICAR eliminated the inhibitory effect of miR-22-3p mimics on My HC I protein expression.These results indicated that miR-22-3p promoted muscle fiber type conversion from oxidative fiber to glycolytic fiber.To investigate the mechanism by whether lycopene-induced skeletal muscle fiber type conversion through miR-22-3p and AMPK signaling pathway,C2C12 and porcine myotubes were considered as experimental objects.The results of lycopene-treated C2C12 myotubes showed that:(1)supplementation with 1?M,2.5?M and 5?M lycopene significantly up-regulated slow My HC protein and down-regulated fast My HC protein,accompanied by increased the percentage of slow-twitch fiber and decreased the number of fast-twitch fibers;the effect of 2.5?M lycopene got to the optimal point;2.5?M lycopene effectively increased My HC I,TNNI1,TNNC1,My HC IIa and My HC IIx m RNA levels,and decreased My HC IIb m RNA level;(2)2.5?M lycopene up-regulated SDH and MDH enzyme activities and decreased LDH activity;(3)2.5?M lycopene increased Cytc protein level,and upregulated Cytc,NRF1,TFAM and CS m RNA levels;(4)lycopene could activate AMPK/Sirt1/PGC-1?and Akt/Fox O1 signaling pathway;(5)lycopene regulated muscle fiber type conversion through AMPK/Sirt1/PGC-1?and Akt/Fox O1 signaling pathway;(6)lycopene significantly suppressed miR-22-3p expression;(7)C2C12myotubes treated with 2.5?M lycopene and miR-22-3p mimics,miR-22-3p mimics attenuated the promoting effect of lycopene on the formation of slow-twitch fiber.The results of porcine myotubes incubated with lycopene found that:(1)2.5?M lycopene increased My HC I,My HC IIa,My HC IIx m RNA levels and decreased the expression of My HC IIb;(2)2.5?M lycopene up-regulated the m RMA levels of AMPK?1,AMPK?2,Sirt1 and PGC-1?;(3)2.5?M lycopene effectively inhibited the m RNA level of miR-22-3p.These data indicated that lycopene regulated skeletal muscle fiber type conversion through AMPK/Sirt1/PGC-1?signaling pathway and miR-22-3p,and Akt/Fox O1 signaling pathway involved in lycopene-induced muscle fiber type conversion.In summary,this study investigated the effects of lycopene on skeletal muscle fiber type conversion and meat quality and its mechanism,and researched the relationship between lycopene and miR-22-3p in regulation of muscle fiber type conversion.This study verified that miR-22-3p promoted muscle fiber type transformation from oxidative fibers to glycolytic fibers.We also found that lycopene could improve meat quality of finishing pigs,enhance fatigue resistant of mice,and regulated skeletal muscle fiber type conversion from fast-twitch to slow-twitch through AMPK/Sirt1/PGC-1?signaling pathway and miR-22-3p.