Isolation And Identification Of Porcine Fast And Slow Muscle Exosomes And Their Effect On Lipid Formation Of Intramuscular Preadipocytes

Pork contains an appropriate amount of intramuscular fat（marbling）to make the meat quality excellent,and it is generally believed that 2.5～3%intramuscular fat content in pork is the best.However,the enthusiastic pursuit of lean meat rate has led to the development of pig selection to lean pigs,and the content of intramuscular fat in meat is getting lower and lower,making the meat quality more"chai"not in line with the eating habits of Chinese consumers.Skeletal muscle in animal organisms is a highly heterogeneous secretory tissue composed mainly of muscle fibers with different metabolic and contractile properties.Because of the anatomical proximity between muscle fibers and intramuscular fat,exosomes may mediate cell-to-cell communication between them.However,the effect of porcine fast and slow muscle exosomes on intramuscular fat deposition remains unclear.Based on this,this study isolates and identifies exosomes representing Extensor digitorum longus（EDL）and Soleus muscle（SOL）,representing the extensor digitorum longus（EDL）muscle of porcine fast and slow muscle,and explores the effect of porcine fast and slow muscle exosomes on adipogenesis of intramuscular preadipocytes.Therefore,in this study,SOL and EDL-derived exosomes（denoted as SOL-EXO and EDL-EXO）were identified by scanning electron microscopy（SEM）,transmission electron microscopy（TEM）,nanoparticle tracking analysis（NTA）and WB separation,and their content components were analyzed by Raman spectroscopy.Secondly,the effects of SOL-EXO and EDL-EXO on lipid formation in intramuscular preadipocytes were clarified by Bodipy and Adipored immunofluorescence staining,oil red O staining extraction and WB.Finally,proteomic sequencing analysis of SOL-EXO and EDL-EXO predicted the differentially expressed proteins that may have an effect on adipogenic preadipocytes in porcine muscles and the signaling pathways they are enriched to.The main findings were obtained as follows:1.Slow twitch fat deposits in pigs are higher than fast troughs.The separation of porcine soleus muscle（SOL）,gastrocnemius muscle（GAS）,tibialis anterior（TA）and extensor toe longus（EDL）for fast and slow muscle fluorescence double standard staining and oil red O frozen section staining showed that the amount of intramuscular fat deposition of slow-twitch SOL was higher than that of fast-twitch EDL and that the lipid-forming ability of SOL-derived intramuscular precursor adipocytes was stronger than that of EDL.2.The average particle size of pig SOL-EXO and EDL-EXO is about 100 nm,and it is double-layered in the shape of a membrane dish.Under TEM,both SOL and EDL myosatellite cells secrete exosomes.Both SOL-EXO and EDL-EXO are double-layered disc balls under SEM.NTA analysis showed that the average particle size of SOL-EXO was84.61 nm and that of EDL-EXO was 84.92 nm.WB can detect the expression of membrane surface proteins CD9,CD63,CD81 and TSG101.3.Porcine SOL-EXO protein content is higher than EDL-EXO.NTA analysis showed that the concentration of SOL-EXO was 7.43×10¹⁰ cells/m L,and the DEL-EXO was5.85×10¹⁰ cells/m L.At the same time,Raman spectroscopy content composition analysis showed that the lipid,nucleic acid and protein content of SOL-EXO were higher than those of EDL-EXO,and the protein content was the most significant difference between them.4.Porcine SOL-EXO and EDL-EXO proteomic sequencing assays.In this study,a total of 33262 peptides and 4173 proteins were identified.A significant difference in expression of 72 proteins was found between SOL-EXO and EDL-EXO,of which 31 were downregulated in SOL-EXO and 41 upregulated in SOL-EXO.GO enrichment analysis showed that differentially expressed proteins were involved in the localization of cytoplasmic membrane proteins,the formation of secreted granule cavities,and nitrogen metabolism in biological processes.KEGG pathway enrichment analysis showed that differentially expressed proteins were related to fatty acid metabolism and amino acid biosynthesis.5.Porcine SOL-EXO and EDL-EXO promote and inhibit the adipogenic differentiation of intramuscular preadipocytes.The quantitative results of Bodipy and Adipored immunofluorescence staining,oil red O staining extraction and TG content showed that compared with the NC group,the content of lipid droplets was significantly increased after SOL-EXO treatment of porcine intramuscular preadipocytes,while the content of lipid droplets after EDL-EXO treatment was significantly reduced.The WB results showed that compared with the NC group,SOL-EXO increased the expression of the key adipogenic genes PPARγ,FABP4 and C/EBPβand decreased the expression of the lipolytic gene ATGL.After EDL-EXO treatment of intramuscular preadipocytes,the expression of key adipogenic genes PPARγ,FABP4 and C/EBPβdecreased,and the expression of the lipolytic gene ATGL increased.In summary,this study was the first to isolate and identify pig SOL-EXO and EDL-EXO,both of which had an average particle size of about 80 nm and were double-membrane disc-shaped.The protein content of SOL-EXO is higher than that of EDL-EXO;It was found that SOL-EXO promotes the adipogenic differentiation of intramuscular preadipocytes,but EDL-EXO has the opposite effect.The results of this study clarify the effect of porcine fast and slow muscle exosomes on lipid formation of intramuscular preadipocytes,which provides a new idea for exploring intramuscular fat deposition in pigs from the perspective of muscle-lipid interaction.