Novel Gene Screen And Their Function In Adipocyte Biology

Obesity is resulted from increasing of number and size of adipocytes. Development of adipocyte tissue is regulated by a lot of transcription regulator. Many genes go up or down during adipocytes differentiation regulating adipocytes differentiation and function. Although a lots of regulator were discovered, there are still many novel genes’function in adipocytes need to be uncovered, Their expressions were dynamically changed during adipocytes differentiation, which implied they may involved in the regulation of adipocytes differentiation.We found a list of novel genes that have not been reported yet in adipocytes from microarray and gene expression data base(GEO Datasets2659,2660and GEO Dataset2818). Ogn and Prrxl were selected and their expression profile during timecourse of3T3-L1adipocytes differentiation were analyzed by Real-time qPCR. Then pSuper retrovirus shRNA RNAi vector for Ogn and Prrx1were constructed and transfected to HEK293T cells. The virous were collected and filtered before infecting3T3-L1preadipocytes. After selection with antibiotic, their function in adipocytes differentiation and function were studied. Oil red O staining and BODIPY staining were used to show lipid accumulation in adipocytes after shRNA knockdown. Realtime PCR and Western blot were used to study change of gene expression or protein expression of makers for adipocytes differentiation. Furthermore, their functions in glucose uptake and lipids lipolysis were also studied. Results are shown as follows:1Ogn regulate3T3-L1adipocytes differentiation through Wnt/β-catenin signaling pathway1) According to microarray data and gene expression data base, Ogn mRNA expression is decreased during adipocytes differentiation. Our results showed similar results in3T3-L1adipocytes. Ogn mRNA expression was higher in SVF than adipocytes fraction in WAT of mice.2) Retrovirus mediated shRNA infection of3T3-L1preadipocytes showed that knockdown Ogn could promote adipogenesis, increase lipids accumulation as well as PPARy, FABP4mRNA and protein expression.3) Over-expression of Ogn in3T3-L1preadipocytes could not effect adipocytes differentiation. PPARy mRNA was decreased in Ogn-overexpression adipocytes but not significantly.4) Knockdown of Ogn in3T3-L1preadipocytes could decrease basal level of glucose uptake but not insulin stimulates glucose uptake.5) Wnt3a could inhibit adipogenesis by stabling β-catenin in cytoplasm and promoting nuclear translocation. Knockdown of Ogn in3T3-L1preadipocytes could partially block this inhibition by decreasing β-catenin nuclear translocation. Thus Ogn could regulate adipocyte differentiation through wnt/β-catenin signaling pathway.2Prrxl Inhibits Adipogenesis by Activating TGFβ Signaling1) Prrx1a, Prrx1b and Prrx2are down-regulated during adipogenesis in consistence with microarray data and gene expression data base. Prrx1a or Prrx1b mRNA expression is about80%lower in adipocytes fraction than that of in SVF part in WAT of mice. Prrx2is hardly detected in adipocytes in WAT of mice.2) Knockdown of Prrxl in3T3-L1preadipocytes could promote lipids accumulation shown by Oil red O staning or BODIPY staning, stimulate PPARγ、C/EBPa and FABP4mRNA or protein expression in adipocytes.3) Over-expression of Prrx1a or Prrx1b in3T3-L1preadipocytes can suppression Γ promoter activity, but could not effect lipids accumulation or gene expression in adipocytes.4) Real-time qPCR results showed that Tgfb2and Tgfb3mRNA expression was decreased in Prrx1knockdown in3T3-L1preadipocytes, which suggests Prrx1may regulating TGFP signaling pathway. In deed, using SB431542, a specific inhibitor of TGFβ signaling pathway, treats3T3-L1preadipocytes could phenocopy the knockdown of Prrxl. These data suggest that Prrxl could inhibit adipogenesis by activating TGFβ signaling pathway.5) Wnt3a and TNFα can inhibit adipogenesis dose dependently in adipocytes. However, Knockdown of Prrxl in3T3-L1preadipocytes barely blocks this effect. This indicates that Prrxl regulate dipogenesis did not affect Wnt/β-catenin and TNFa signaling pathway.6) By Feeding BL6and C3H mice with high fat diet from3weeks to15weeks, we constructed obese mouse model. In this model, qPCR data showed that Prrxla and Prrxlb were corelated with Tgfb3mRNA expression in WAT of mice.