Effect Of Porcine Intramuscular Adipocyte Exosomes On The Transformation Of Muscle Fiber Type

Skeletal muscle is the main source of pork products and muscle fiber is the basic unit of muscle tissue.According to metabolic and contractile characteristics,muscle fibers can be divided into four subtypes: slow oxidation type(Ⅰ),fast oxidation type(Ⅱa),intermediate type(Ⅱx type),fast glycolysis type(Ⅱb type).Skeletal muscle is a highly heterogeneous tissue containing a variety of cells,such as skeletal muscle satellite cells,intramuscular adipocytes,fibroblasts,and macrophages.In addition to their own energy storage function,intramuscular adipocytes can also function as secretory organs to secrete leptin,adiponectin and lipid droplets to affect the physiological functions of other tissue cells.Exosomes are nanoscale extracellular vesicles(30-160 nm in diameter)with lipid bilayer that can be secreted by a variety of cells.They contain a variety of functional substances such as non-coding RNAs,m RNAs,proteins and lipids that mediate communication between cells and tissues.However,porcine intramuscular adipocytes are an important cell group in skeletal muscle.The composition of secreted exosomes and their role in the transformation of muscle fiber types remain unclear.Therefore,this study successfully isolated porcine intramuscular adipocyte exosomes.The source,internalization,function and composition differences of exosomes from porcine adipocytes were investigated.mi RNA and lipid libraries of exosomes from two sources were constructed by transcriptome and lipid sequencing analysis.The differentially expressed mirnas and lipids were screened and the potential functions of differentially expressed mirnas and lipids were predicted.Based on the above studies,further through the co-culture experiment of porcine intramuscular precursor adipocytes and mature intramuscular adipocytes exosomes and porcine skeletal muscle satellite cells,as well as differential mi RNA treatment of porcine skeletal muscle satellite cells,the influence of the exosome components from porcine intramuscular adipocytes and mature intramuscular adipocytes on the transformation of muscle fiber types was explored.The main findings are as follows:1.Exosomes can be secreted by porcine intramuscular precursors and mature intramuscular adipocytes.Both porcine intramuscular preadipocytes and mature intramuscular adipocytes can secrete exosomes,which contain adiponectin and leptin.2.Biological characteristics of exosomes of porcine intramuscular preadipocytes and mature intramuscular adipocytes.Identification of intramuscular preadipocytes and mature intramuscular adipocytes exosomes by transmission electron microscopy,atomic force microscopy,nano-particle size and exosome labeled protein WB.3.Transcriptome sequencing analysis of exosomes from porcine intramuscular precursor adipocytes and mature intramuscular adipocytes.The composition of exosomes of intramuscular preadipocytes and mature intramuscular adipocytes was preliminally detected by Raman spectroscopy.Nucleic acid was the largest difference,followed by lipid,and protein content was the lowest.A total of 13 differentially expressed mirnas were identified by transcriptome sequencing,and the differentially expressed mirnas were mainly involved in AMPK signaling pathway.Among them,mi R-1285 is highly expressed in mature intramuscular adipocyte derived exosomes,which can increase the proportion of glycolytic muscle fibers in skeletal muscle satellite cells.4.Sequencing analysis of exosomal lipids of porcine muscle precursor adipocytes and mature intramuscular adipocytes.A total of 1246 differentially expressed lipids were detected.Compared with exosomes derived from intramusculus precursor fat cells,777 lipids were up-regulated and 469 lipids were down-regulated,and these differentially expressed lipids were mainly related to AMPK and other signaling pathways.5.Extracellular uptake of intramuscular adipocytes and its effect on the transformation of muscle fiber types.The exosomes of intramuscular adipocytes can be taken up by satellite cells of skeletal muscle,most of the exosomes are internalized by endosomes and endoplasmic reticulum,and a small part is absorbed by lysosomes.Mature intramuscular adipocyte derived exosomes can increase the proportion of glycolytic muscle fibers in porcine skeletal muscle satellite cells.In conclusion,this study successfully isolated and identified porcine intramuscular adipocyte exosomes,and mature intramuscular adipocyte exosomes can improve the proportion of glycolytic muscle fibers in porcine skeletal muscle satellite cells.Transcriptome and lipid sequencing were further used to detect differentially expressed mirnas and lipids in exosomes,and it was found that differentially expressed mirnas and lipids may be enriched in AMPK signaling pathway.This study has reference significance for exploring the interaction between pig fat and muscle to regulate muscle fiber type transformation,and also provides a new idea for improving pork quality.