Characterization of the Active Components of the Multimerized sTNFRIIAdiponectin Fusion Protein Showing Both TNFα-Antagonizing and Glucose Uptake-Promoting Activities.

ABSTRACT

BACKGROUND: The sTNFRII-adiponectin fusion protein previously showed strong TNFα antagonistic activity. However, the fusion protein exists as mixture of different multimers. The aim of the present study was to characterize its active components. METHODS: In this study, the fusion protein was isolated and purified by Ni-NTA affinity and gel exclusion chromatography, and further identified by Coomassie staining and western blotting. The TNFα antagonistic and glucose uptake-promoting activities were determined in vitro. The glucose detection kit and 2- NBDG (2-deoxy-2-[(7-nitro-2,1,3-benzoxadiazol-4-yl)amino]-D-glucose) were used to measure their effects on glucose metabolism (including glucose consumption and glucose uptake in HepG2 and H9C2 cells). The effect of the fusion protein on glucose uptake was also examined in free fatty acid (FFA)- induced insulin resistance cell model. RESULTS: The sTNFRII-adiponectin fusion protein was found to exist in three forms 250 kDa (hexamer), 130 kDa (trimer), and 60 kDa (monomer), with the final purity of 90.2%, 60.1%, and 81.6%, respectively. The fusion protein could effectively antagonize the killing effect of TNFα in L929 cells, and the multimer was found to be superior to the monomer. In addition, the fusion protein could increase glucose consumption without impacting the number of cells (HepG2, H9C2 cells) in a dosedependent manner. Mechanistically, glucose uptake was found to be enhanced by the translocation of GLUT4. However, it could not improve glucose uptake in the cell model of insulin resistance. CONCLUSION: In summary, the active components of the fusion protein are hexamers and trimers. The hexamer and trimer of sTNFRII-adiponectin fusion protein had both TNFα-antagonizing and glucose uptake-promoting activities, although neither of them could improve glucose uptake in the cell model of insulin resistance.