Comparison Of Erhualian Porcine Gene Expression Profiles Between Subcutaneous And Intramuscular Fat Tissues And Preliminary Exploration Of The Regulatory Mechanisms Underlying Differential Fat Deposition Between The Tissues

The intramuscular fat (IMF) depot is a complex biological process. It is well documented that IMF content was decreased significantly due to long-term intensive selection for increased leanness and thin back fat. Increasing marbling IMF content while not influence other fat content is the aim of modern pig breeding. The fat depot is restricted to certain areas in the body and depot-specific adipocytes have different physiological function and lipid metabolism. Preadipocytes in different adipose tissues have various capacities of differentiation, and mature adipocytes of various depot origins have distinct metabolic behaviors. Biological differences among various depots might give a clue to depot-specific regulation of fat tissues. In order to understand the differential mechanisms regulating the fat deposition between IM and SC tissues, we compared the whole gene expression of SC and IM adipose tissue using Gene-chip microarray technology. From the above result, the expression level of PPARy, which is a critical factor in adipocyte differentiation, was different between the two fat tissues. Then we chose PPARy as the research object, and investigated the molecular mechanism of different expression between IM and SC fat tissues from miRNA, myokines and unclear reporter co-activator regulation.The main results achieved were as follows:1. In this study, whole gene expressions of dorsal subcutaneous (SC) adipose tissue and intramuscular (IM) adipose tissue, which isolated from Longissimus Dorsi (LD) muscle tissue of7months Erhualian pig were compared using Affymetrix Gene-Chip microarray technology. The result revealed that1228genes expressed higher in SC adipose tissue, while965genes expressed higher in IM adipose tissue. We found that the SC adipose tissue had a stronger capacity of lipid metabolism and fatty acid metabolism compared to IM adipose tissue. The different expressions of PPARγ and C/EBPa, which are the critical factors for adipocyte differentiation and lipid accumulation, are the main reasons for different metabolism and lipid depots between SC and IM fat tissues. And ANGPTL4, NNAT, NOR-1and CLIC5may also play important roles in the regulation of fat deposition between IM and SC adipose tissues.2. In this study, pure mature adipocytes were isolated from longissimus muscle by collagenase Type I and allowed to dedifferentiate into fibroblast-like cells in ceiling culture. These fibroblast-like cells exhibited the ability of redifferentiate into mature adipocytes when were adipogenically induced in vitro. The redifferentiation process was supported by lipid accumulation in the cytoplasm and high expression adipogenesis genes. The dedifferentiatie adipocyte (DFAT) can be stably obtained by this thechnology, and the DFAT cells can be used to study the mechanisms of intramuscular adipocytes differentiation and metabolism.3. To investigate the molecular mechanisms that regulate different lipid deposition ability between intramuscular (IM) fat and subcutaneous (SC) fat tissue, PPARy mRNA and protein expressions were evaluated between the two tissues. The potential microRNAs (miRNAs) targeting PPARy were also determined and verified. Besides, the functions of potential miRNA were detected in DFAT cells differentiation to adipocyte. The mRNA and protein levels of PPARy significantly increased in the SC fat tissue. Furthermore, the expression levels of miR-128and miR-130a, which potentially target PPARy3’-untranslated region (3’-UTR), were dramatically different between IM fat and SC fat tissue. The dual luciferase activity assay result showed only miR-130a suppressed PPARy expression. After over expression of miR-130a in DFAT cells, the differentiation ability to adipocyte was markedly inhibited by suppressing PPARy expression. Hence, the expression level of miR-130a is significantly different between IM and SC fat tissues, the tissue-variance of miR-130a levels result in the differential levels of PPARy, and might be the reason for differential fat deposition between IM and SC fat tissues.4. The intramuscular DFAT cells differentiation to adipocyte was significantly inhibited by adding muscle tissue culture fluid, so the result implied that there was myokine inhibited adipocyte differentiation in the culture fluid. Previous study found that Myostatin (MSTN) can influence adipocyte differentiation, so we do more research about MSTN on intramuscular DFAT cells. In this report, intramuscular DFAT cell was treated by different dose MSTN to test its role in cell proliferation, differentiation and lipolysis. The MSTN mRNA expression levels of muscle and the MSTN protein levels in blood serum were detected, and their relationship with intramuscular fat content were also measured. The results showed that altered dose of MSTN had different influences in intramuscular DFAT cells proliferation,100ng/ml of MSTN promoted cells proliferation, while the dose below100ng/ml inhibited cells proliferation. In differentiation process, MSTN treatment markedly inhibited intramuscular DFAT cells differentiation to adipocyte and had dose-dependent by oil red O staining. MSTN inhibited IM DFAT cells differentiation to adipocyte by decreasing C/EBPβ, C/EBPa, PPARy and SREBPlc expressions. After differentiation, effects of MSTN on lipolysis were detected though the releasing of glycerol and the expressing levels of adipose triglyceride lipase (ATGL) and hormone sensitive lipase (HSL). The releasing of glycerol was drastically reduced by MSTN treatment as well as the expressions of ATGL and HSL were drastically reduced by MSTN treatment. In this study, we found there was no correlation between MSTN mRNA expression levels and IMF content, and MSTN protein levels in blood serum can’t influence the IMF content.5. SRC-2, which is a coactivitor of PPARy, can promoter aipocyte differentiation by enhancing transcriptional activity of PPARy protein.In order to verify the ability of SRC-2in IM adipocyte differentiation, the SRC-2was knockdown by siRNA, and IM DFAT cells differentiation ability was observed by RT-qPCR and red oil O staining. The result showed that the differentiation was inhibited by knockdown SRC-2expression, and the gene expressions of PPARy, FABP4, LPL and ADIPOQ were significantly reduced.