Characterization of the active components of the multimerized sTNFRII-adiponectin fusion protein showing both TNFα-antagonizing and glucose uptake-promoting activities

BackgroundThe sTNFRII-adiponectin fusion protein had been previously shown to have strong TNFalpha antagonistic activity. However, the protein exists as a mixture of different multimers. The aim of the present study was to characterize its active components. MethodsIn this study, the protein was isolated and purified by Ni-NTA affinity and gel exclusion chromatography, and further identified by Coomassie staining and western blotting. The TNFalpha antagonistic and hypoglycemic activities were determined in vitro, and glucose detection kit and 2-NBDG 2-deoxy-2-[(7-nitro-2,1,3-benzoxadiazol-4-yl)amino]-D-glucosewere used to measure their effects on glucose metabolism (including glucose consumption and glucose uptake in HepG2 and H9C2 cells). Effect of the protein on glucose uptake was also examined in a 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 TNFalpha 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 dose-dependent 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. ConclusionIn summary, the hexamer and trimer of sTNFRII-adiponectin fusion protein had both TNFalpha-antagonizing and glucose uptake-promoting activities, although neither could improve glucose uptake in the cell model of insulin resistance.