The Impact of Wnt Inhibitory Factor-1 on Preadipocytes Differentiation
Wnt Inhibitory Factor-1 (WIF-1) is a secreted modular protein that inhibits the activities of Wnt proteins. The latter are known as extracellular signaling molecules involved in many aspects of embryonic development. During juvenile development, WIF-1 is expressed in adipose tissue in chickens. We hypothesize that WIF-1 regulates pre-adipocyte differentiation. This study was undertaken to examine the WIF-1 effect on the differentiation of mouse pre-adipocyte 3T3-L1 and chicken primary pre-adipocyte through the expression of transcriptional factors CCAAT/enhancer binding protein α (C/EBP α), and peroxisome proliferator-activated receptor γ (PPARγ). 3T3-L1 cells were treated with 0, 1, 5 and 20ng/ml of recombinant mouse WIF-1 protein and chicken pre-adipocytes cells were treated with 0, 2, 10µg/ml of recombinant mouse WIF-1 protein. Total RNA was isolated 24-48hours posttreatment. Levels of C/EBPα and PPARγ mRNA were analyzed by reverse transcription quantitative polymerase chain reaction (RT-qPCR). Results showed that there is no significant effect on PPARγ mRNA level, however, there is a difference in the expression of C/EBPα mRNA with 20ng/ml WIF-1 treatment in 3T3 cells comparing to the control. ELISA assay was used to detect β-catenin levels after treating 3T3-L1 cells with WIF-1 at concentrations 10 and 100ng/ml and with Wnt at concentrations 3 and 30ng/ml, and the combination of WIF-1 (100ng/ml) and Wnt (30ng/ml). Results showed that in both WIF-1 treatment concentrations, there was a reduction in β-catenin levels. Wnt treatments did not affect β-catenin levels. WIF-1 effect on β-catenin level could not be replicated in a replicated test. Thus, the results were not conclusive. In vitro cell culture system was done for chicken pre-adipocytes and used to examine WIF-1 activity. WIF-1 expression was examined after chicken pre-adipocytes transfection with CMV6-ENTRY-WIF-1 vector and results indicated that WIF-1 was expressed in the cytoplasm of WIF-1 plasmid transfected cells whereas no WIF-1 expression was detected in the control. Western blot was done after transfection chicken pre-adipocytes, and the protein expression signal was not shown due to the low transfection efficiency.
"The Impact of Wnt Inhibitory Factor-1 on Preadipocytes Differentiation"
ETD Collection for Tennessee State University.