Amyloid Fibrillation of Insulin: Amelioration Strategies and Implications for Translation

Abstract

Insulin is a therapeutically relevant molecule with use in treating diabetes patients. Unfortunately, it undergoes a range of untoward and often unpredictable physical transformations due to alterations in its biochemical environment, including pH, ionic strength, temperature, agitation, and exposure to hydrophobic surfaces. The transformations are prevalent in its physiologically active monomeric form, while the zinc cation-coordinated hexamer, although physiologically inactive, is stable and less susceptible to fibrillation. The resultant molecular reconfiguration, including unfolding, misfolding, and hydrophobic interactions, often results in agglomeration, amyloid fibrillogenesis, and precipitation. As a result, a part of the dose is lost, causing a compromised therapeutic efficacy. Besides, the amyloid fibrils form insoluble deposits, trigger immunologic reactions, and harbor cytotoxic potential. The physical transformations also hold back a successful translation of non-parenteral insulin formulations, in addition to challenges related to encapsulation, chemical modification, purification, storage, and dosing. This review revisits the mechanisms and challenges that drive such physical transformations in insulin, with an emphasis on the observed amyloid fibrillation, and presents a critique of the current amelioration strategies before prioritizing some future research objectives.

Figure 1

Overview of the insulin molecule, its delivery platforms, and complications associated with diabetes. (A) Insulin is released into the bloodstream from the β-cells of the pancreatic islets of Langerhans. (B) Lateral and anteroposterior ocular view in a case of diabetic retinopathy. (C) Foot ulcer found in diabetic patients. (D) Capillary blood glucose measurement (glycosimetry). (E, F) Insulin administration by (E) insulin pen and (F) subcutaneous injection.

Figure 2

Human insulin molecule with its two polypeptide chains (A and B). The two interchain disulfide bonds (A7–B7, A20–B19) and one intrachain disulfide bond (A6–A11) are also shown. The darkened residues remain conserved across all the species. In porcine insulin, alanine replaces threonine (B30), whereas in bovine insulin, in addition to the substitution in porcine insulin, two additional substitutions are noted: alanine for threonine (A8) and valine for isoleucine (A10). The residues that help dimerization and hexamerization are marked with “D” and “H”, respectively.

Figure 3

Thermoenergetic profile of insulin oligomers. Although physiologically active, the monomer (PDB: 3I40) is most unstable and quickly forms relatively stable dimers (PDB: 6S34). In the presence of Zn²⁺, the monomers gradually form the T6 hexamer (PDB: 1MSO), while adding both Zn²⁺ and m-cresol produces an even more stable R6 hexamer (PDB: 1EV6) bearing two extra chloride ions.

Figure 4

Sigmoidal (black) and double-sigmoidal (red) kinetics of insulin fibrillation passing through the lag, growth, and equilibrium phases—as determined by Th-T fluorescence.

Figure 5

Successive stages of development in insulin oligomers: protofilaments, protofibrils, and fibrils. (A) Side and top views of an intertwined conglomeration of eight helical insulin oligomers (color scale: purple → red → light yellow) that form a protofilament. The gray segments at the open ends mark additional precursors. (B) Two intertwined protofilaments form a protofibril of 100 Å diameter. The orange ellipse shows the boundaries of an assembled protofibril. (C) Three protofibrils (orange, red, and yellow) interweave to form a mature insulin amyloid fibril. Both the side and frontal views are shown here. Reproduced from ref (62) under an open access Creative Commons License.

Figure 6

Chemical structures of phenol and phenolic compounds used as additives and preservatives in insulin formulations to impart stability.

Figure 7

Chemical structure of the heptapeptide LVEALYL.

Figure 8

Chemical structures of the flavonoid compounds used to inhibit insulin fibrillation.

Figure 9

Chemical structures of polyphenolic compounds used to inhibit insulin fibrillation.