Effect And Mechanisms Of Phytol And Its Metabolites On Skeletal Muscle Fiber Type Transform

Skeletal muscle is the most important tissue of movement and energy homeostasis in the animal body,the function of skeletal muscle is affected by the fiber type in the skeletal muscle,the exercise ability and metabolic features are distinguishing in different muscle fiber types.At the same time,skeletal muscle fiber type can conversion between different types under the influence of a variety of internal and external factors.Phytol is a side chain of chlorophyll as a long chain adipose alcohols,it is widey exist in the grean plants in nature environment,when human and animals consume food rich in chlorophyll,phytol can be dissociation from chlorophyll and absorbed by small intestine,and broken down to phytanic acid and pristanic acid in liver.At present,the studies on phytol are focus on pathology such as refsum disease and the effect of green fodder on milk and meat quality in ruminant,however there is no researches between phytol and its effect on skeletal muscle fiber type transform.In this study we found for the first time that phytol and its metabolites pristanic acid has great influence on the skeletal muscle fiber type,and this influence is working through long chain fatty acid transporter CD36.The study includes the following five parts.Part 1:Effect of phytol on muscle fiber type in C2C12 cell line and its mechanismWe here used C2C12 cell line as experiment model,found that phytol significantly enhanced the expression of MHC I and MHC IIa,and also increased the expression of CD36and FABP3.Investigate the intracellular signaling,we found that the expression of PPARδand PGC-1αin PGC-1α/PPARs pathway and expression of Ca MKII and the level of p MEF2A/MEF2A in Ca²⁺pathway were all up-regulated by phytol treatment in C2C12 cell line,furthermore the two slow-fiber-type-related mi RNAs mi R-499 and mi R-208b were also up-regulated.The seahores x Fp cell mitochondria stress test show that the basal metabolism,spare respiratory capacity and ATP preduction were up-regulated by phytol treatment in C2C12 cell line.The above results suggested that phytol could promote the expression of MHC I and MHC IIa though signal pathway PPARδ/PGC-1αand Ca MKII/MEF2A,and the LCFA transporters CD36 and FABP3 were involved in this process,meanwhile phytol could also activate the mitochondrial oxidation,enhanced the respiratory capacity of cell.Part 2:Effect of phytol on muscle fiber type in mice and its mechanismWe next used C57BL/6J mice as as experiment model,found that used 0.5%phytol as feed additive had no effect on growth and muscle sampling and adipose tissue sampling characteristics,however the muscle grip and excise endurance of phytol feeded mice were significiently increased,enhanced the aerobic capacity of phytol feeded mice.The histochemistry staining of GAS and TA sections showed that the proportion of MHC I was significantly increased,and the proportion of MHC IIb was significantly decreased,the expression of protein level of MHC I also show an upward tendency.Investigate the intracellular signaling in phytol feeded mice GAS,we found that the protein expression of PPARδ,PGC-1α,Calcineurin,MEF2A,CD36 and FABP3 were all significantly up-regulated.It was reported that LCFA could not noly used as energy substance but also nuclear transcription factor ligand and activate the transcriptional activity,the IP（Immunoprecipitation）result showed that phytol could increased the molecular interactions of FABP3 and PPARδ.The above results suggested that phytol could promote the fast-slow and glycolytic-oxidative fiber type transformation,manifested in the transform of muscle fiber type constitute in phytol feeded mice GAS and TA,and the enhanced of muscle grip and excise endurance of mice.PPARδ/PGC-1αand Calcineurin/MEF2A signal pathway and the transport of LCFA participate in the process,meanwhile phytol could activated the transcriptional activity of PPARδas the ligand of PPARδ.Part 3:Effect of the two metabolites phytanic acd and pritanic acid on muscle fiber type was different with the effect of phytol in C2C12 cell lineThe metabolites of phytol in culding phytanic acid（PA）and pristanic acid（Pr A）,witch reported metabolized in liver,and there is no researche between PA and Pr A and their effect on skeletal muscle fiber type transform yet.So we used C2C12 cell line as the experiment model to study the effect of these two phytol metabolites on muscle fiber type transform.The result showed that PA had no influence on muscle fiber type in C2C12 cell line,the expression level of MHCs and signal pathway moleculers had no changed compared with control.After treatment of Pr A,the protein expression level of MHC I,PPARδ,Calcineurin,MEF2A and the level of p MEF2A significantly increased,but there is no changed in the expression level of MHC IIa,CD36,FABP3 and the level of p MEF2A/MEF2A.The intracellular location of FABP3 was detected by ICC（Immunocytochemistry）,it showed that the intracellular location of FABP3 after Pr A treatment had no migration.The seahores x Fp cell mitochondria stress test show that the spare respiratory capacity were up-regulated by Pr A treatment in C2C12 cell line,but the level of basal metabolism and ATP preduction had no changed compared with control.The above results suggested that PA had no influence on muscle fiber type and relative signal pathway,Pr A could significantly increased the protein expression of MHC I though Calcineurin/MEF2A signal pathway,and the up-regulation of PPARδmay also affected by Ca²⁺signal pathway.But Pr A had no effect on LCFA transport,the intracellular location of FABP3 after Pr A treatment had no migration means the same possibility.Meanwhile the Pr A could not activate the mitochondrial oxidation and enhanced the respiratory capacity of cell as phytol.Part 4:The RNA-seq showed the different mechanisms between phytol and Pr A treatment on C2C12 cell lineThe study in this research above showed that phytol and Pr A a huge different appearance in effect on muscle fiber type transform,because of there is no existing study in these field,so we used RNA-seq technology to lay the foundation for the further study.DEGs（Different expression genes）analysis showed that there were 3896 DEGs in Phy vs Con compare group and 197 DEGs in Pr A vs Con compare group.GO biological functional annotation showed that there were 192 annotations in Phy vs Con compare group and 62 annotations in Pr A vs Con compare group.KEGG pathway enrichment showd that Phy vs Con and Pr A vs Con compare groups had 8 and 1 signifcantly pathway enreched respectively.KOBAS pathway enrichment showd that Phy vs Con and Pr A vs Con compare groups had 86 and 7 highly signifcantly pathway enreched respectively.The post-transcriptional control of DEGs analysis showed that,the mechanism of phytol influence on muscle fiber type mainly though LCFA transport process,PPARδ/PGC-1αsignal pathway and Ca MKII/MEF2A signal pathway,meanwhile the mechanism of Pr A influence on muscle fiber type mainly though PPARδand cell metabolism.The above results suggested that there was a huge difference between the mechanisms of phytol and Pr A on the muscle fiber type transform,and the influence level of the phytol seems greater than Pr A.Part 5:Effect of phytol on muscle fuber type transform is induced by CD36We used LV-sh RNA-CD36 to interference the expression level of CD36 in C2C12 cell line and in GAS of C57BL/6J mice as experiment model.The results in C2C12 cell line showed that CD36 RNA interference could remove the effect of phytol on muscle fiber type such as the increased expression of MHC I,PPARδand CD36.Further interferes with CD36 though injected LV-sh RNA-CD36 in mice GAS,the enhanced of muscle grip and excise endurance by phytol feeded could be remove to the level of control group,the proportion of MHC I and MHC IIa in GAS,and the expression level of muscle fiber type transformation related signal pathway moleculers also had the same elimination situation.However because of TA had not injected the LV-sh RNA-CD36,the proportion of MHC I and MHC IIa in TA keeped at the same level as phytol feeded group.The above result suggested that the effect of phytol on muscle fuber type transform is surely induced by CD36.