Characterization of the receptor binding properties of monoiodoinsulin isomers and the identification of different insulin receptor specificities in hepatic and extrahepatic tissues.

Abstract

Insulin was iodinated with 125I using a modification of the lactoperoxidase method. This iodinated insulin mixture was then separated by HPLC, allowing studies to be performed on the pure monoiodoinsulin isomers labeled at the tyrosyl residuesat A14, B16, or B26. Binding studies in adipocytes revealed the relative affinity for these isomers to be 1.00:0.99:1.30 (A14, B16, and B26, respectively). The increase in binding affinity was shown to be due to a slower dissociation rate rather than an accelerated association rate. Receptor mediated internalization of the different iodinated isomers was assessed and these results showed that the isomers were internalized and degraded in proportion to their relative binding affinities. The biologic activity of the B26 isomer to stimulate adipocyte glucose oxidation was measured and showed that the B26 isomer had 30% greater biologic potency compared with native insulin, consistent with its 30% increase in binding potency. Thus, the B26 monoiodoinsulin isomer behaves as a superagonist. Binding studies using extrahepatic tissue (i.e., adipocytes, cultured human fibroblasts, IM-9 lymphocytes, and human placental membranes) consistently revealed that the relative binding affinity for the B26 isomer was always greater than the A14 isomer (1.29, 2.11,1.22, and 1.35, respectively) while the binding affinity of the B16 isomer was generally comparable to the A14 isomer. In hepatic tissue (whole cell, liver plasma membranes, and partially purified, solubilized liver receptors) binding studies illustrated that the B26 isomer consistently had a lower relative affinity when compared to the A14 isomer (0.75 : 0.69 : 0.77). In conclusion, (1) the B26 monoiodoinsulin isomer exhibits higher binding affinity and greater biologic activity in adipocytes than native insulin, and (2) the B26 monoiodoinsulin isomer has a higher binding affinity in extrahepatic tissues but exhibits a lower binding affinity in hepatic tissues when compared with the A14 monoiodoinsulin isomer, indicating a striking difference in functional specificity between the hepatic and the extrahepatic insulin receptor.