The tale of visfatin – or: About knowing what to measure ..

Visfatin has been described as an adipocytokine expressed in visceral adipose tissue that exerts insulin-like effects through direct interaction with the insulin receptor. Subsequent studies raised controversy on the association of visfatin with obesity and diabetes. This may be attributed to variances in the detection systems. We evaluated visfatin preparations of various sources, their molecular features and their detection in immunoassays.

Visfatin mRNA and protein was endogenously expressed in human preadipocytes and adipocytes, while secretion was seen only from mature adipocytes. Overexpression and secretion of visfatin was achieved by transfection of COS-7 cells with human PBEF full length cDNA. Visfatin protein was readily detectable in the cell lysates and supernatants by Western blot in both cell systems. Commercial immunoassays with distinct polyclonal anti-peptide antibodies (Elisa, Ria) both detected visfatin recombinant protein in dilution series and in the cell supernatants. Direct comparison of serum samples did, however, not show any correlation between both assays. We subjected serum to size exclusion chromatography (SEC) with subsequent immunoassay to search for specific peaks in human serum. A single peak was detected by Elisa at app. 800kDa. The Ria detected a lower signal between 50 to 100 kDa that corresponded with detection of visfatin in identical serum fractions by Western blot, while there was no signal at 800kDa in Western blot. I125-labeled visfatin subjected to SEC also eluted with peaks at 100kDa and a smaller peak at 50 kDa, equivalent to the MW of the dimer and monomer, respectively.

In conclusion, there are major differences in the qualitative and quantitative detection of visfatin by distinct immunoassays that need to be considered in clinical association studies, which is the focus of ongoing research.