The Function And Mechanism Of IGFBP5 On Fat Deposition In Intramuscular Adipocytes And Myocytes

The fat content in the muscle has an important effect on the quality of the livestock and poultry meat.Increasing the fat content in the muscle can improve the tenderness and flavor of meat and so on.With the deepening of the research,it has been confirmed that intramuscular adipocytes are the most important reservoir of fat in muscle.The intramuscular adipocytes accumulate gradually through proliferation and differentiation,and form the marbling pattern visible to the naked eye to a certain extent.In addition,some of the fat in the muscle is stored directly in the myocytes,and this part of the fat also accounts for a certain proportion of the muscle.IGFBP5 is a secretory protein that is highly expressed in muscle and fat and has been found to regulate the metabolic process of muscles,bones,and so on.Other studies have shown that IGFBP5 may affect lipid metabolism.So we want to study the effects of IGFBP5 on intramuscular fat formation.We isolated porcine intramuscular adipocytes and constructed a cellular model of intramyocellular lipid deposition in mice.We studied the role and mechanism of IGFBP5 in muscle fat storage from two aspects,through RT-qPCR,Western blot,Oil Red O staining,cellular immunofluorescence and other biochemical and molecular detection methods.The main results as follows:1.IGFBP5 was found to be highly expressed in muscle and fat by qPCR in tissues of 3-day-old and 180-day-old pigs;adding IGFBP5 recombinant protein to cultured pig intramuscular pre-adipocytes significantly increased the rate of EdU positive cells.(P<0.05)and the number of S phase cells(P<0.05),these indicators indicate that IGFBP5 can significantly promote the proliferation of porcine intramuscular precursor adipocytes.2.The addition of IGFBP5 recombinant protein inhibited the differentiation of porcine intramuscular pre-adipocytes,while the interference with the expression of IGFBP5 promoted the differentiation of porcine intramuscular pre-adipocytes.Since the expression of IGFBP5 is significantly decreased in the early stage of intramuscular pre-adipocytes differentiation,it is speculated that the reduction of IGFBP5 plays an important role in the early stage of differentiation.Three days after induction of differentiation,a series of related enzymes synthesized for de novo fatty acids,such as acetyl-CoA carboxylase(ACACA),fatty acid synthase(FASN),ultralong-chain fatty acid elongase(ELOVL),stearoyl-CoA desaturase(SCD),and the key enzymes for triglyceride synthesis,such as diacylglycerol acyltransferase(DGAT),were decreased in the IGFBP5-treated group(P<0.05).At the same time,it was found that the key enzymes responsible for lipolysis,such as hormone-sensitive lipase(HSL),and the genes involved in fatty acid oxidation,such as carnitine acyltransferase 1(CPT1)and mitochondrial transcription factor A(TFAM),were significantly decreased in the IGFBP5-treated group(P<0.05).So IGFBP5 does not enhance lipolysis and oxidation while inhibiting fat synthesis.3.Based on the mouse C2C12 myoblast cell line and induced by sodium oleate,a cellular model of intramyocellular lipid deposition was successfully constructed.In a certain concentration range,with the increase of sodium oleate concentration,the deposition of fat in myocytes increased;in the model construction,high glucose DMEM medium was better than low glucose medium in myocytes filling with fat(P<0.05);as the concentration of BSA increases,the deposition of fat in the myocytes decreases(P<0.05).At the same time,oleateinduced intramyocellular lipid deposition is accompanied by insulin resistance in myocytes.Furthermore,expression of IGFBP5 was inhibited in an oleate-induced intramyocellular lipid deposition cellular model.4.The addition of IGFBP5 recombinant protein inhibits lipid deposition in myocytes and improves insulin sensitivity in myocytes.In the process of IGFBP5 inhibiting lipid deposition in myocytes,IGFBP5 not only inhibits the expression of fatty acid transporter 1(FATP1)(P<0.05),which regulates the uptake of long-chain fatty acids,but also inhibits the mitochondrial glycerol-3 phosphoacyltransferase(GPAM)and diacylglycerol acyltransferase 2(DGAT2)(P<0.05),which regulate triglyceride synthesis.Thus,IGFBP5 inhibits fatty acid uptake and triglyceride synthesis.In addition,the addition of IGFBP5 promoted the reduction of lipids in myocytes and also improved the insulin sensitivity of myocytes.During this process,the expression of lipolysis gene triglyceride lipase(ATGL)and hormone-sensitive lipase(HSL)was enhanced(P<0.05),accompanied by an increase in the level of free fatty acids in the medium,so IGFBP5 promotes the lipolysis in myocytes and the transport of free fatty acids from the inside to the outside of the myocytes.In conclusion,this study investigated the role of IGFBP5 in intramuscular fat formation and storage from two aspects of porcine intramuscular adipocytes and constructed intramyocellular lipid deposition cellular models,and analyzed the mechanism of IGFBP5 on lipid metabolism.This experiment comprehensively studied the effect of IGFBP5 on intramuscular fat metabolism.The research methods and results have certain reference significance for further study of the formation mechanism of fat in muscle.