| The deposition of fat in muscle,recognized by the intramuscular fat(IMF),is an important meat quality.In recent decades,extensive selection for increasing carcass lean-meat percentage had led to a dramatic decrease in IMF content,which had a passive effect on the pork quality.To improve the IMF content,we should initially understand the unique deposition mechanism of IMF.Due to the particular location in close vicinity to skeletal muscle fibers,the development of intramuscular adipocytes is exclusively affected by the paracrine activity of skeletal muscle fibers.Therefore,the mechanism that skeletal muscle affects the IMF development should be clarified.Insulin signaling plays an important role in the regulation of fat deposition,which can induce the adipocyte differentiation,promote the synthesis of triglycerides in mature adipocytes and inhibit their lipolysis.In this study we primarily investigate the difference of insulin signaling pathway between the intramuscular and subcutaneous adipocytes.Additionally,the mechanism of insulin signaling and lipid metabolism in intramuscular adipocytes was explored by treating adipocytes with muscle conditioned cultured mediLun(MCM).Furthermore,the mechanism that the selected gene and myokine affected insulin signaling patknvay and lipid metabolism in adipocytes were validated.All above will illustrate the impacts which influencing the regulatory development mechanism in intramuscular adipocytes.The main results achieved were as follows:1.Differences in insulin signal between intramuscular and subcutaneous adipocytes.Dedifferentiated i.m.and s.c.preadipocytes were isolated by the ceiling culture method.The isolated preadipocytes were verified by immunofluorescence staining of the preadipocyte marker,transmembrane protein Pref-1,which is expecially expressed in preadipocytes.The result showed that the two types of preadipocytes with positive Pref-1 reactions had a good proliferation and differentiation ability.These analyses confirmed that the isolated preadipocytes with high purity had the ability to re-differentiate.The effects of insulin signaling on the adipocyte differentiation was examined via culturing the s.c.preadipocytes and s.c.adipocyte with insulin,knocking down the INSR and IGF-1R in preadipocytes.The results showed that insulin could not induce the differentiation of precursor adipocytes exclusively,but the effect of dexamethasone(Dex)and 3-methyl-1-butylxanthine(IBMX)on cell differentiation was improved(p<0.05).Since treated the adipocytes with insulin,the expression levels of FABP4 and PPARG in adipocytes was increased(p<0.05).The content of triglyceride and the differentiation level was significantly deereased(p<0.01).Furthermore,the adipocye differentiation was inhibited by downregulating the expression of INSR or IGF-IR.The content of glycerides and the Oil red O positive lipid drops were decreased significantly(p<0.01).To clairifiy the difference of insulin signaling and lipid metabolism between i.m.and s.c.adipocytes,the expression levels of INSR and IGF-1R,the adipocyte differentiation,the expression pattern of glucose metabolism and fatty acid metabolism related genes,and also the function of mitochondrial were detected.The results shoxvn that the mRNA expression levels of INSR and IGF-1R in i.m.preadipocytes were both significantly higher than those in s.c.preadipocytes(p<0.01),whereas the content of glycerides and the expression levels of adipocytes marker,FABP4 and PPARG,were significantly lower in i.m.adipocytes than that in s.c.adipocytes(p<0.01).The mRNA levels of Glutl and Glut4 were higher i.m.preadipocytes during the early stage of adipocyte differentiation(p<0.05),meanwhile the expression levels of PKM2 and PDK4,the major genes of glycolysis,in i.m.preadipocytes were higher than those in s.c.preadipocytes at the late stage of differentiation(C,D)(p<0.05).However,the expression pattern of fatty acid transport genes,FAT and LPL,in i.m.preadipocytes and s.c.preadipocytes were different from that of glucose metabolism genes.The expression levels of FAT and LPL in s.c.preadipocytes were significantly higher than that in i.m.preadipocytes at the late stage of differentiation.To elucidate the difference of adipogenic ability between intramuscular and subcutaneous adipocytes,the fatty acid capacity and mitochondrial function of the two types o’f adipocytes were verified.The results showed that the expression of genes involves in fatty acid oxidations,including cpt-1α、ept-1α、PPARa,was higher in i.m.adipocytes than that in s.c.adipocytes(p<0.05).Moreover,the mitochodondrial content was also higher in i.m.adipocytes(p<0.05).Consistent with the mitochondrial DNA copy number,the mRNA levels of genes implicated on mitochondrial biogenesis,including PGC-1α,NRF-1,TFAM were higher in i.m.adipocytes(p<0.01).2.MCM enllances intramuscular adipocytes insulin signaling and inhibits adipocyte differentiationCell proliferation,apoptosis and differentiation phenotype were measured by treating s.c.adipocytes with different concentrations of MCM.The results showed that MCM inllibited cell proliferation with the most significant influence at the protein concentration of 120 μg/ml(p<0.01).Flow cytometry showed that MCM induced the apoptosis of preadipocytes with protein concentration of 120μg/ml,the early and late apoptotic rates increased from 6.5%to 15.1%(p<0.01)and 2.8%to 6.4%(p<0.05),respectively.The content of Oil Red O positive lipid drops was significantly lower in MCM-treated adipocytes with the most significant effects at protein concentration of 120 μg/ml.The Oil Red O staining indicated MCM inhibited adipocyte differentiation.In order to detect the effect of MCM on the insulin signal in adipocytes,the expression levels of insulin signaling related genes and NCOA3 were detected by treating s.c.adipocytes with MCM at protein concentration of 120 μg/ml.The results of RT-PCR showed that IRS-1 mRNA level was significantly increased in MCM-treated group(p<0.01),and the protein expression of IGF-1R,IRS-1 and p-IRS-1 were significantly increased(p<0.05).While it was found that MCM inhibited the mRNA and protein expression of NCOA3(p<0.05).To further validate the effect of MCM on adipocyte insulin signaling,MCM was used to treat adipocytes in different stages of differentiation.The results showed that the expression levels of INSR,IGF-1R,IRS-1 and p-IRS-1 were significantly increased in early to middle differentiation stage(p<0.05).To detect the effect of MCM on adipocyte differentiation when the insulin signaling change,the s.c.adipocytes in different stages of differentiation were treated with MCM.The results showed that MCM inhibited the adipocyte differentiation and played the main role in the pre-differentiation to middle stage.The content of glycerides and the expression levels of adipocyte marker,FABP4 and PPARG,were significantly decreased(p<0.05).The effect of MCM on cell lipolysis,lipid synthesis and fatty acid oxidation-related phenotype were further investigated to elucidate the inhibition effects of MCM on lipid deposition.The results showed that the fatty acid oxidase related genes(Cpt1α,Cpt1β and PPARa),the genes related to heat production(PGC-1α,PRDM16 and Cidea),the number of mitochondrial DNA,the mitochondrial hyperplasia-related genes,including PGC-1α,NRF-1 And TFAM,were significantly higher than those in the control group(p<0.05).3.MCM affects intramuscular adipocytes insulin signal and differentiation level tlrough the regulation on NCOA3.The study aboved showed MCM inhibited the expression of NCOA3.It has been reported that the insulin sensitivity of adipose tissue was inproved when NCOA3 expression was inllibited.We speculated that the differential expression of NCOA3 in intramuscular and subcutaneous adipose tissue may result the differential insulin signal and fat deposition in the tavo tissues.The expression of NCOA3 in the tAvo adipose tissues was detected and the miRNAs regulating NCOA3 was screened.The mechanism that miRNA regulation on NCOA3 expression was explored.Finally,miRNA mimic and inhibitor were used to detect the effect of the miRNA on adipocyte differentiation.The expression of NCO A3 in intramuscular adipose tissue was significantly lower than that in subcutaneous adipose tissue(p<0.01).The expression ratio of NCOA3 in intramuscular and subcutaneous adipose tissue(NCOA31/NCOA3s)was significantly higher in IMAT/SCAT high group(p<0.05).The expression of miR-17-5p in intramuscular adipose tissue was significantly higher than that in subcutaneous adipose tissue(p<0.01).The expression ratio of miR-17-5p(miR-17-5pI/miR-17-5ps)in intramuscular and subcutaneous adipose tissue was lower in the IMAT/SCAT high group(p<0.05).RT-qPCR and Western blotting showed that miR-17-5p was able to target NCOA3 by dual luciferase activity assay.To investigate the regulation of MCM on miR-17-5p,the expression level of miR-17-5p was determined by treatment of subcutaneous adipocytes with MCM.RT-qPCR results showed that the expression of miR-17-5p in MCM-treated group was significantly higher than that in control group(p<0.05).To investigate the action of miR-17-5p on adipocyte differentiation,miR-17-5p mimics and inhibitors were transfected into intramuscular preadipocytes,respectively.The results showed that the content of glycerides and the expression of PPARG and FABP4,the adipogenic differentiation makers,were significantly reduced in miR-17-5p mimics groups(p<0.05).However,the content of glycerides and the expression of PPARG and FABP4,were significantly increased in miR-17-5p inhibitors groups(p<0.05).4.Myokine FGF21 induced browning of white adipocytes.The IMF shows the characteristic of brown adipose tissue.FGF21 xvas able to regulate the insulin sensitivity and induce the browning of white adipocytes.We examined the effect of myokine FGF21 on the lipid metabolism and insulin signal of porcine adipocytes.The expression level of FGF21 in muscle and adipose tissue was detected,and the expression level of FGF21 in muscle tissue vas significantly higher than that in subcutaneous adipose tissue ip<0.01).FGF21 was used to treat differentiating adipocyte.After 9 days,the levels of triglyceride in FABP4 and PPARG were significantly decreased(p<0.01),but the adipocyte differentiation efficiency did not change significantly.FGF21 was used to treat mature adipocytes for 3 days,and the effect of FGF21 on the expression of insulin sensitivity related genes and lipolysis was examined.The results showed that the expression levels of INSR,IGF-IR and IRS-1,ATGL and HSL were significantly increased and the release of glycerol was increased(p<0.05)in FGF21 treated group.To elucidate the effect of FGF21 on adipocyte insulin sensitivity,the consumption of glucose in adipocytes under FGF21 was examined.The results showed that the consumption of glucose in FGF21 treated cells was significantly higher than that in the control group(p<0.05).Under the combined action of insulin and FGF21,the consumption of glucose was sig1ificantly higher than that of insulin alone.The expression of brown adipocyte marker gene was measured after 6 days of incubation with FGF21 cells.RT-qPCR results showed that the expression levels of PPARα;Cidea,PRDM16,Elovl3,PGC-1α,CPT－1α and CPT-1β in the FGFZ1-treated cells were significantly higher than that in the control group(p<0.05)In summary,the differences of insulin signaling and adipogenic differentiation between intramuscular and subcutaneous adipocytes were compared.Furthemore,the regulation effects of MCM on insxxlin signaling and adipogenic differentiation was examined.The research provides the theoretical basis for exploring the regulation of muscle tissue on the developluent of IMF and also increasing the IMF content to improve the pork quality... |
