Bioengineering Against Fibrils and Hypoglycemia: Studies on Insulin, Glucagon and the Fusion of Both

Abstract

Insulin drugs are vital for blood glucose control in type 1 and late-stage type 2 diabetes mellitus. Unfortunately, however, they have two notable problems: (1) an intrinsic propensity to physical degradation (amyloid-like fibrillation), which reduces potency and can lead to occlusion of insulin pumps’ catheters, impairing timely drug administration; and (2) an ever-present risk for iatrogenic hypoglycemia with potential acute (or even fatal) consequences and chronic sequelae. The risk of hypoglycemia, its immediate and long-term complications, and associated anxiety can confound efforts to achieve effective glycemic control. Further, insulin’s physical instability impacts worldwide distribution by imposing a refrigeration requirement—often a barrier to global access. A combined solution to these two problems could benefit patients worldwide. To circumvent these limitations, glucose-responsive technologies have been sought to reduce hypoglycemic risk; diverse strategies have focused on novel devices, delivery modes, or protein engineering. In the present doctoral work, we describe an alternative glucose-responsive approach that exploits an endogenous glucose-dependent switch in hepatic physiology: preferential insulin signaling (under hyperglycemic conditions) versus preferential counter-regulatory glucagon signaling (under hypoglycemic conditions). Glucagon, traditionally regarded as a counter-regulatory hormone, has been underutilized in routine glucose control due to a marked propensity to fibrillation. Motivated by the pilot success of a counterintuitive strategy—co-infusion of insulin and glucagon—we have bioengineered and tested a fibrillation-resistant insulin-glucagon fusion protein with favorable relative hormonal activities. The N-terminal glucagon moiety was stabilized as a partial α-helix by Lys13-Glu17 lactam bridge and fused to a C-terminal insulin moiety stabilized as a single chain with a foreshortened C domain. Our in vitro studies demonstrated (a) marked resistance to fibrillation on prolonged agitation at 37 °C and (b) unaffected dual hormonal signaling activity. Glucodynamic responses were monitored in rats relative to control fusion proteins lacking one or the other hormonal activity. Results showed that insulin’s efficacy in hyperglycemia was unaffected, but enhanced endogenous glucose production was observed under hypoglycemic conditions. Together, these findings provide proof of principle for the translational application of a novel glucose-responsive insulin formulation with augmented physical stability, addressing two major problems of insulin replacement therapy in a single molecule.