Resistin has different molecular isoforms in human circulating blood

Aim: Resistin, a novel 10 kD cystein-rich protein, causes insulin resistance and glucose intolerance in mice and may thus, connect the molecular link between obesity and type 2 diabetes. In humans, however, the role of resistin remains unclear. As there are no clear data on the molecular properties of resistin in human blood so far, we established biochemical and immunological methods for the detection and characterization of potential isoforms of this protein.

Methods: Anti-human resistin antibodies were raised in rabbits against resistin peptides as well as recombinant resistin proteins. Using these antibodies we established a Western blotting method as well as an „in-house“ immunoassay for the detection of resistin in serum. For comparison, levels of resistin were also measured by a commercially available ELISA. The molecular distribution of the resistin protein was analysed by size exclusion chromatography (SEC). Blood samples were withdrawn from children and adolescents (age range 8–18 years).

Results: Western Blot analysis revealed bands for resistin at 10 kD under reducing and at 20 kD under non-reducing conditions, respectively. After SEC we detected two major isoforms of this protein at 48–66 kD and in the exclusion volume >600 kD. Spiking of a serum pool with recombinant resistin led to increased levels at these observed peaks. Moreover, a direct comparison between both assays revealed the accordance of resistin levels (r=0.53, p<0.001, n=129).

Conclusions: Resistin circulates in human blood in different isoforms. This fact may be explained by the presence of specific or unspecific resistin binding proteins and / or by the aggregation of the monomeric resistin molecule. Furthermore, methodological differences in the recognition of these molecular variants by different antisera may lead to conflicting results in the interpretation of serum resistin levels of clinical studies. The question whether or not individual isoforms of the circulating resistin protein may have biological relevance remains unclear and should be a goal for further studies.