Molecular Characteristics of Serum Visfatin and Differential Detection by Immunoassays

Abstract

CONTEXT: There are controversial results concerning the association of visfatin with obesity and diabetes. We aimed to characterize molecular features of visfatin, to assess visfatin detection by different immunoassays, and evaluate the association with human obesity and glucose metabolism. RESULTS: Distinct preparations of human visfatin (recombinant, endogenously expressed from human adipocytes, and overexpressed in COS-7 cells) were readily identified by three currently available immunoassays. However, direct comparison of native human serum samples did reveal great discrepancies between these assays and complete lack of correlation. To specify putative molecular isoforms of visfatin, we fractionated iodine-125-labeled recombinant visfatin spiked into human serum and supernatants of visfatin-overexpressing COS-7 cells by size exclusion chromatography. We obtained a distinct peak at approximately 100 kDa that was confirmed by subsequent Western blotting of the fractions and is equivalent to the molecular mass of the dimer. Only one of the immunoassays detected a similar peak in native human size exclusion chromatography serum fractions, whereas the others detected a peak at more than 500 kDa or did not show any distinct peak. We did not observe any differences in visfatin serum levels between lean or obese patients. In addition, there was no correlation between visfatin serum levels with visfatin mRNA expression in sc or visceral fat and with parameters of glucose metabolism. CONCLUSION: Differences in the qualitative and quantitative detection of visfatin by immunoassays need to be considered in clinical association studies and may explain the conflicting observations with respect to a putative relation of circulating visfatin to human obesity or insulin resistance.