Glycosylated Insulin Complexed to Concanavalin A: Biochemical Basis for a Closed-Loop Insulin Delivery System

Abstract

The oligosaccharides maltose, maltotriose, mannotriose, and mannotetrose have been chemically attached to insulin molecules. Incubation of oligosaccharide and insulin at different molar ratios, with or without addition of cyanoborohydride, showed a nearly linear increase in carbohydrate attachment over time. The intravenous t½ of 125I-labeled sugar-insulinderivatives was identical to that of unmodified insulin (3.0 min). Biologic activity of these derivatives, assessed in rats by use of a blood glucose depression assay, did not differ significantly from control. These glycosylated insulin molecules are reversibly bound to the glucose-binding lectin Concanavalin A (Con A). Such sugar-insulin/lectin complexes serve as an insulin reservoir from which sugar-insulin molecules are displaced by glucose. Release of sugar-insulin molecules is a function of the particular sugar-insulin and of the ambient glucose concentration. Glucose displacement of glycosylated insulin complexed to Con A is in direct proportion to the amount of glucose present in the surrounding fluid. At each glucose concentration, the relative binding affinity of the maltotriose derivative is less than that of the mannotriose derivative, while the relative binding affinity of both maltotriose and mannotriose are less than that of the mannotetrose derivative. Prolonged incubation at 37°C causes sugar-insulin, like unmodified insulin, to spontaneously aggregate into high-mol-wt, nondiffusable complexes. This aggregation phenomenon was found to be markedly inhibited when glycosylated insulins were synthesized utilizing partially sulfated insulin. Results from the studies described in this report provide the biochemical basis for a closed-loop, glucose-controlled insulin delivery system, utilizing glyco-sylated insulin complexed to Con A.