Examining the effects of vitamin B12 conjugation on the biological activity of insulin: a molecular dynamic and in vivo oral uptake investigation

Abstract

The practical use of the vitamin B12 uptake pathway to orally deliver peptides and proteins is much debated. To understand the full potential of the pathway however, a deeper understanding of the impact B12 conjugation has on peptides and proteins is needed. We previously reported an orally active B12 based insulin conjugate attached at LysB29 with hypoglycemic properties in STZ diabetic rats. We are exploring an alternative attachment for B12 on insulin in an attempt to determine the effect B12 has on the protein biological activity. We describe herein the synthesis, characterization, and purification of a new B12–insulin conjugate, which is attached between the B12 ribose hydroxyl group and insulin PheB1. The hypoglycemic properties resulting from oral administration (gavage) of such a conjugate in STZ diabetic rats was similar to that noted in a conjugate covalently linked at insulin LysB29, demonstrating the availability of both positions on insulin for B12 attachment. A possible rationale for this result is put forward from MD simulations. We also conclude that there is a dose dependent response that can be observed for B12–insulin conjugates, with doses of conjugate greater than 10−9 M necessary to observe even low levels of glucose drop.