Enhanced insulin receptor interaction by a bifunctional insulin-transferrin fusion protein: an approach to overcome insulin resistance.

Bifunctional fusion protein design has been widely utilized as a strategy to increase the efficacy of protein therapeutics. Previously, we proposed a novel application of the bifunctional fusion protein design through the introduction of proinsulin-transferrin (ProINS-Tf) fusion protein as a liver-specific protein prodrug to achieve a glucose-lowering effect in type 1 diabetic mice. In this report, we studied the binding characteristics of this activated fusion protein to the insulin receptor to elucidate its mechanism in eliciting insulin receptor-mediated signaling. We found that, with the assistance of the transferrin moiety binding to the transferrin receptor, the activated ProINS-Tf exhibited significantly higher binding affinity to the insulin receptor compared with the native insulin, resulting in a prolonged and stronger Akt phosphorylation. This enhanced induction by activated ProINS-Tf overcame insulin resistance in palmitate-treated HepG2 cells. ProINS-Tf also demonstrated a better glucose-lowering effect than native insulin, even with a much lower dose and less frequent injections, in non-obese diabetic mice with insulin resistance symptoms. The activated ProINS-Tf, serving as a bivalent protein molecule, could be a new insulin analog to overcome insulin resistance, which is associated with several diseases, including type 2 diabetes and non-alcoholic fatty liver disease.