The natural occurrence of insulin receptors in groups on adipocyte plasma membranes as demonstrated with monomeric ferritin‐insulin

Abstract This study was designed to document whether the reported distribution of insulin receptors in small groups of receptor sites randomly distributed in the glycocalyx of adipocytes and isolated adipocyte plasma membranes was a naturally occurring phenomena or due to artifacts. Possible artifacts include: (1) oligomeric forms of ferritin in the ferritin‐insulin preparation, (2) an uneven distribution of the glycocalyx on the plasma membrane, or (3) ligand‐induced aggregation of occupied receptor complexes. Biogel A 1.5m chromatography of the ferritin‐insulin conjugate revealed the ferritin in the ferritin‐insulin complex to consist of 55% monomers, 15% dimers, and 30% oligomers. The monomer peak was purified (> 95%) for use in these studies. Cationic ferritin, a glycocalyx marker, when incubated with paraformaldehyde‐fixed plasma membranes, was found to be uniformly distributed on the surface of the plasma membrane indicative of uniformly distributed glycocalyx. The ability to demonstrate and inhibit ligand‐induced aggregation on the isolated plasma membrane was established with a multivalent ligand, ferritin‐concanavalin A. More than 66% of the ferritin‐concanavalin A receptors were found in large clusters of 5 or more and 34% as singletons or clusters of up to 4 when incubated at 24°C with fresh membranes. Only 38% of the ferritin‐concanavalin A receptors were in large clusters; 62% were singletons or clusters up to 4 on membranes prefixed with paraformaldehyde before incubation. The distribution of the monomeric ferritin‐insulin was similar on both adipocytes and purified adipocyte plasma membranes and was consistent with earlier reports with ferritin‐insulin. The quantitative distribution of the monomeric ferritin‐insulin as singletons or in groups of 2–6 was comparable between the intact cells and isolated membranes incubated at 24°C. The binding of 500 μUnits monomeric ferritin‐insulin per ml to the isolated plasma membranes was studied under incubation conditions similar to those used with ferritin‐concanavalin A. Under all three conditions, fresh membranes at 24°C and 0–4°C and prefixed membranes at 24°C, the pattern of distribution of the monomeric ferritin‐insulin as singletons or groups of 2–6 was identical, indicating that the ligand was not causing aggregation into clusters as did the concanavalin A. Thus, the occurrence of insulin receptors in small groups appears to be a natural phenomenon in the plasma membrane structure of adipocytes.