The Differential Function And Mechanism Of PPAR Signaling In Regulation Of Porcine Subcutaneous And Intramuscular Fat Depositon

Intramuscular fat content is closely related to meat tenderness,flavor and juiciness.In a long period,the breeding goal of high lean meat percentage has resulted in a significant decrease in the intramuscular fat content of the muscle,which leads to the decline in pork quality.With the continuous improvement of people’s living standard,there are increased demands for high quality pork.Thus,in order to improve the falling pork quality,moderate improvement of pork intramuscular fat content without affecting backfat thickness becomes a major goal of the new phase pig breedings.Previous studies have shown that there are distinct mechanisms underlying the fat deposition in different parts of the body,but the difference of molecular mechanism between intramuscular and subcutaneous fat are not fully understood.Therefore,comparing gene profiles of subcutaneous and intramuscular fat to figure out the differentially expressed genes,signaling pathways and the potential targets for regulating intramuscular fat deposition,is of great significance to improve the depot-specific regulation network of intramuscular fat.It will lay a solid theoretical foundation for improving the meat quality of pigs.Peroxisome proliferator-activated receptors(PPARs)are members of the superfamily of the nuclear receptor transcription factors that regulate the expression of the target genes,and it can be divided into three types: PPARα,β/δ and γ.PPARγ is mainly expressed in adipose tissue,acting as the master regulator of adipogenesis and critical for maintaining mature adipocyte function.Previous studies have found that PPARγ activator significantly increased intramuscular fat content in pigs,mice and humans,but had no significant effect on total fat content.We also found significant differences of PPAR signaling pathways through transcriptome sequencing between subcutaneous and intramuscular adipocytes.According to the above results,it is speculated that PPAR signaling may play a different role in the proliferation or differentiation between the porcine intramuscular adipocytes and subcutaneous adipocytes.To verify this hypothesis,we used the molecular techniques like flow cytometry,Oil Red O staining,Real-time PCR and Western Blot to test the change of morphology or gene expression during the proliferation and differentiation of porcine intramuscular and subcutaneous adipocytes after PPARγ agonist treatment,the related signaling pathways of proliferation and differentiation were also examined.In addition,high throughput transcriptome sequencing technology was used to determine the different mechanism of PPARγ activation on differentiation of intramuscular adipocytes and subcutaneous adipocytes.The main results obtained in this study are as follows:1.Activation of PPARγ can increase the thermogenic genes(Ucp1,etc.)and mitochondrial function genes(Cidea,etc.)expression of the in vitro cultured mouse subcutaneous adipocytes.2.Using RNA-seq technology,1051 differentially expressed genes were identified between porcine subcutaneous and intramuscular adipocytes.The expression level of PPARγ in porcine subcutaneous adipocytes is significantly higher than in intramuscular adipocytes,and KEGG pathway enrichment analysis aslo shows that PPAR signaling pathways is highly enriched.3.Treating porcine subcutaneous and intramuscular preadipocytes with PPARγ agonist,the results of CCK8,flow cytometry,Ed U and the level of cell cycle genes(Cyclin B and Cyclin D)and the key signaling pathway of proliferation Erk all had no significant difference compared with control group(P≥0.05).It suggested that activation of PPARγ had no influence on proliferation of subcutaneous and intramuscular adipocytes.4.In the adipocytes differentiation process,activation of PPARγ can significantly promote the porcine intramuscular adipocytes differentiation.Oil Red O Staining showed a 1.62-fold than that of the untreated group(P<0.01),although there also had a promoting effect to subcutaneous adipocytes differentiation(1.27 folds),the Oil Red O staining results showed no statistical difference compared with control group(P≥0.05).5.Using high throughput transcriptome sequencing technology,PPARγ activation in porcine subcutaneous adipocytes caused 165 genes significantly changed during differentiation,while the total number of genes regulated by PPARγ agonist in porcine intramuscular adipocytes were just 83.These results suggest that activating PPARγ has a different effect on the gene expression profile between porcine subcutaneous and intramuscular adipocytes.6.Further KEGG pathway enrichment analysis of differentially expressed genes revealed that activation of PPARγ during differentiation significantly changed PPAR signaling pathways,however,the enrichment factor of the differentially expressed genes in PPAR pathway was higher in the intramuscular adipocytes than that of subcutaneous adipocytes.It was notable that PPARγ agonist significantly activated AMPK signalling pathway of intramuscular adipocytes,but had no effect on the AMPK signaling pathway of subcutaneous adipocytes.Western blot result is consistent with the transcriptome sequencing results,further confirming that activation of PPARγ may affect porcine intramuscular adipocytes differentiation by activation AMPK signaling pathways.7.Matured muscle cells(myotubes)were treated with PPARγ agonist,and then the culture medium was collected to treat adipocyte cells.The results showed that the treating group and control group had no significant difference in the differentiation degree,which revealed that PPARγ agonist may not indirectly affect the intramuscular fat cell differentiation by influencing the muscle cell secretion.In summary,PPARγ and PPAR signaling pathways were firstly indentified differentially expressed between porcine subcutaneous and intramuscular adipocytes by using RNA-seq technology,Therefore,it is speculated that PPARγ may play different roles in the proliferation and differentiation of subcutaneous and intramuscular adipocytes.It was found that activation of PPARγ did not affect the proliferation of both subcutaneous and intramuscular adipocytes,but it promoted the intramuscular adipocyte differentiation.Using high throughput transcriptome sequencing technology to screen differentially expressed genes,followed by KEGG pathway enrichment analysis showed that PPARγ agonist promoted porcine intramuscular adipocytes differentiation by specifically activating AMPK signaling pathways,while had no significant effect on AMPK pathway of subcutaneous adipocytes.In addition,treating muscle cells with PPARγ agonist not indirectly affected the intramuscular adipocyte differentiation by influencing the myocytes secretion.This study suggests that there is a significant difference in PPAR signaling between subcutaneous and intramuscular adipocytes,and this intrinsic difference may result in the different response to PPARγ agonist.This provides a theoretical basis for targeting PPAR signaling to differentially regulate porcine subcutaneous and intramuscular fat deposition.