Effects And Molecular Mechanism Of RXRα On Porcine Preadipocytes Differentiation

Adipose tissue stores energy passively, and also acts as an endocrine organ secreting multiple hormonal materials. The over development of the adipose tissue, also expressed as excessive body fat deposition, leads to human obesity and related complications, such as diabetes, coronary artery disease, atherosclerosis, hypertension and hyperlipoidemia, and the decrease of meat quality in animal products. The key loop of fat deposition is the over proliferation and differentiation of adipocytes. Thus, it is very important to study the molecular mechanism of adipocytes differentiation. Compared with rodent animals, the anatomic and physiological characteristics, and the mode of fat deposition of pig are much similar to those of human. Additionally, pig is the main cultivated animal. Therefore, to study the mechanism of porcine adipocytes differentiation is significant not only for human medicine, but also for animal production industry.The main marker genes during adipocytes differentiation include RXRα, C/EBPα, PPARγ, ADD-1/ SREBP-1, LPL and aP2. RXR belongs to super family of nuclear hormone receptor, which contains three subtypes (α,βandγ), and regulates many aspects of metazoan life as ligand activated transcriptional factor. However, it is still uncertain about the role of RXR in the adipocytes differentiating process. In addition to the pathway of RXR acting as a nuclear receptor, whether there is other independent signal pathway regulated by RXR or not still needs to be further studied. It has been identified that heterodimer of RXRα/PPARγinfluence adipocytes differentiation by modulating the expression of adipocyte specific genes. However, whether RXRαhas the direct role in adipocytes differentiation or not remains unclear. Furthermore, the role and mechanism of RXRαin porcine preadipocytes differentiation has not been reported.In this study, healthy, 1-3-old-day Landrace piglets were used as experimental animal to construct in vitro culture system of porcine preadipocytes. The time-spatial expression of RXRαduring porcine preadipocytes differentiating process was investigated with SQ RT-PCR and indirect immunofluorescence technique. Porcine preadipocytes were treated with 9-cisRA, a ligand of RXRα. Subsequently, the effects of 9-cisRA on porcine preadipocytes differentiation were detected by Oil red O staining, Oil red O extraction, GPDH activity analysis and SQ RT-PCR. The change of porcine preadipocytes differentiation and underlying molecular mechanism were checked by vector construction, chemical synthesis, liposome transfection, Oil red O staining, Oil red O extraction, GPDH activity analysis, SQ RT-PCR, indirect immunofluorescence, and Western Blotting while the overexpression or silencing of RXRαgene. The obtained results provided the information and basis for consummating the RXRαrelated adipocytes differentiation regulation theory. The influence of RXRαon the adipocyts apoptosis was studied as well in the present work.The main results were as follows:1. RXRαcontinuously expressed at 2-8 d during porcine preadipocytes differentiation and its expressing levels were no significant difference during the differentiating process.2. 9-cisRA, a ligand of RXRα, plays an important regulatory role in porcine preadipocytes differentiation. 9-cisRA at lower concentration(0-10 nmol/L)promoted preadipocytes differentiation, proved to be the evidences that the number of adipocytes, fat content in adipocytes, GPDH activity and the expression of RXRα,PPARγmRNA increased. On the other hand, 9-cisRA at higher concentration(100 nmol/L -10μmol/L)repressed preadipocytes differentiation, shown as the phenomena that the number of adipocytes, fat content in adipocytes, GPDH activity and the expression of RXRα, PPARγmRNA decreased.3. Overexpression of RXRαgene could promote porcine preadipocytes differentiation. It was shown that the number of adipocytes, fat content in adipocytes, GPDH activity, and the abundance of PPARγand C/EBPαmRNA increased.4. Silencing of RXRαgene could inhibit porcine preadipocytes differentiation. It was shown that the number of adipocytes, fat content in adipocytes, GPDH activity, and the abundance of PPARγand C/EBPαmRNA decreased.5. RXRαcould inhibit the apoptosis of porcine adipocytes.6. Lipofectamine 2000 was used to transfect pSUPER-RXRαRNAi and chemically synthesized siRNA. It was shown that the transfection efficiency was lower with the big pSUPER-RXRαRNAi, while the chemically synthesized siRNA was effectively transfected into porcine preadipocytes by using Lipofectamine 2000. On the other hand, siRNA with fluorescence marker could be used to check transfection efficiency and optimize the transfection method.Taken together, RXRαexpresses in the duration of porcine preadipocyte differentiation and enhances this process. Its ligand 9-cisRA influences the differentiation via RXR in different manners with different treating dosage. The effects of RXRαon the porcine preadipocyte differentiation closely relate to its influence on the expression of PPARγand C/EBPαgenes, and these effects might be due to its formation to heterodimer with PPARγor its directly regulation of the expression of C/EBPαgene. It is also concluded that RXRαmight influence the apoptosis of porcine preadipocytes, and this influence appears to be an inhibition effect.