Abstract:

Objective To investigate the transcriptional regulation mechanism of the Furin gene in adipose tissue under obesity conditions and its effect on the secretion of the adipokine asprosin. Methods Twenty-four C57BL/6J mice were randomly divided into normal control diet （NCD） group and high-fat diet （HFD） group， with 12 mice in each group. The mice were fed for 12 consecutive weeks to establish the diet-induced obesity models. Real-time fluorescence quantitative PCR（RT-qPCR） method was used to detect the expression levels of Furin， fibrillin 1 （FBN1）， and the candidate transcription factor specificity protein 1 （SP1） mRNA in subcutaneous white adipose tissue（WAT） （sWAT） and epididymal WAT（eWAT） of the mice in two groups. Candidate transcription factors for the Furin gene promoter region were screened based on the JASPAR database. In the 293T cells， the SP1 over-expression plasmid （pcDNA-SP1 group） and its control plasmid containing green fluorescence protein （GFP） （pcDNA-GFP group） were transfected. Simultaneously， a dual-luciferase reporter vector containing the Furin promoter region was co-transfected with the aforementioned plasmids to verify the regulatory interaction between SP1 and the Furin promoter. The stromal vascular fraction （SVF） cells from sWAT were isolated and induced to differentiate into mature adipocytes. SP1 （Ad-SP1 group） or GFP control （Ad-GFP group） was overexpressed via adenoviral transduction. Western blotting method and enzyme-linked immunosorbent assay （ELISA） were used to detect the expression levels of SP1 and Furin proteins in cells in various groups and the levels of asprosin in the cell supernatant in Ad-GFP and Ad-SP1 groups， respectively. Results Compared with NCD group， the body weight， sWAT weight， and eWAT weight of the mice in HFD group were significantly increased （P<0.001）. Compared with NCD group， the expression levels of Furin and SP1 mRNA in sWAT and eWAT of the mice in HFD group were significantly increased （P<0.001）. JASPAR database prediction suggested that SP1 was a core candidate factor regulating Furin transcription. In the dual-luciferase experiment， compared with pcDNA-GFP group， the luciferase activity in 293T cells in pcDNA-SP1 group was significantly increased （P<0.001）. Compared with Ad-GFP group，the expression levels of SP1 and Furin proteins in SVF-derived mature adipocytes in Ad-SP1 group were significantly increased （P<0.001）， and the level of asprosin in the cell supernatant was significantly increased （P<0.001）. Conclusion The expression of SP1 mRNA in WAT and adipocytes of obese mice was up-regulated. SP1 can enhance the transcriptional activity of Furin by directly binding to its promoter region. SP1 overexpression may upregulate the expression level of Furin protein in adipocytes， promoting the secretion of asprosin.

Key words: Obesity, Furin, Specificity protein 1, Asprosin, Transcriptional regulation