Cloning, Tissue Expression Analysis Of GPR43 In Swine And Study On The Role Of Fatty Acid To GPR43 In Mouse

Adipose tissue is an energy store tissue of organism, it is very important in energy metabolism. But excess of fat deposition leads to human obesity and related metabolism disease, also causes the decrease in quality of domestic product. The decrease of fat deposition are becoming the common issue which scholars at home and abroad is concerned. GPR43 is one of the GPCRs, has a important role in obesity and diabetes. In this study, the porcine GPR43 cDNA has been cloned. Then, we detected the transcriptional expression of GPR43 gene in adipose tissue and primary cultured adipocytes of pig, and analysised the regulatory role of fatty acid to GPR43 in mouse adipose tissue and adipocyte.1. The length of GPR43 cDNA sequence cloned by PCR technology was 486 bp and GenBank accession number was EU122439. Homologous alignment analysis using Clustal W software showed that pig GPR43 gene had 89%, 83% and 84% homology to that of human, mouse and rat respectively. Further CDD analysis indicated that pig GPR43 protein contained the conserved 7 transmembrane receptor 1 domain of GPCR family.2. we used real-time quantitative PCR and SQ RT-PCR to determine the expression level of GPR43 in different types, different growth-stage and various regions adipose tissue of pig. The results showed that the expression level of GPR43 mRNA was significantly higher in adipose tissue of obese pigs than that of lean pigs, and also the expression level gradually increased as age increased, we further found that the abundance of GPR43 mRNA level increased more in subcutaneous fat than visceral fat. All of those results suggested that the expression level of GPR43 has correlation with the obese degree, age and fat depot. The increase of GPR43 mRNA level may be the reason of obese pigs adipocyte proliferation and fat deposition rate than lean pigs. In the lipid metabolism process, the increase of GPR43 mRNA may inhibited lipolysis. In addition, to investigate the involvement of GPR43 in pig primary cultured adipocytes differentiation in vitro, GPR43 and PPARγmRNA levels were analyzed at different cultured time. The results showed that the levels of GPR43 mRNA increased after adipocyte differentiation. This suggests that GPR43 might be involved in the regulation of pig adipocytes differentiation. 3. To support the hypothesis that GPR43 is involved in adipose tissue development, we next investigated the relationship between GRP43 and the other lipolysis genes(HSL and TGH) in adipose tissue by semi-quantitative PCR. Applying Pearson's correlation analysis to the data obtained from the adipose tissue samples, we revealed that the expression level of GPR43 gene had a negative correlation with HSL gene and THG gene expression. The result suggested that GPR43 has a important role in lipolysis.4. Docosahexaenoic acid(DHA) is one of the polyunsaturated fatty acids which is necessary for the growth and development of the body and have an important role in regulation of fat deposition. In this experiment, we fed with different concentrations of DHA to mice, and analyzed expression level changes of GPR43, HSL and TGH in adipose. The result showed that DHA down-regulated GPR43 and up-regulated HSL and TGH mRNA levels. These results clearly demonstrate that DHA promote adipose lipolysis through inhibiting the expression of GPR43.5. The effects of acetate, a potent activator of GPR43, and SB203580, a strong inhibitor of p38MAPK, on differentiation of mouse preadipocyte were tested by SQ RT-PCR. It showed that acetate had the remarkable activation role on differentiation of mouse preadipocyte, and up-regulated GPR43, PPARγand C/EBPαmRNA expression. Conversely, SB203580 significantly inhibited the differentiation of mouse preadipocyte, and down-regulated GPR43 and PPARγmRNA expression. These findings indicated that acetate through GPR43 positively modulated the differentiation of preadipocytes, and closely related with p38MAPK.