Insulin Fibril Formation Caused by Mechanical Shock and Cavitation

Abstract

Cavitation can occur when liquids are exposed to pressure waves of sufficient amplitude, producing rapidly expanding and collapsing gas bubbles that generate localized regions of high energy dissipation. When vials containing insulin were subjected to mechanical shock or when ultrasound was applied to the vials, the resulting cavitation events induced formation of insulin amyloid fibril nuclei that were detected by transmission electron microscopy and quantified by fluorescence spectroscopy following staining with the amyloid-sensitive dye thioflavin-T. Dropping insulin solutions in glass vials produced only minute amounts of insulin fibril nuclei, which could be detected by allowing the nuclei to grow. Cavitation-induced formation of amyloid aggregates may be relevant for iatrogenic insulin deposition disease, where insulin fibrils formed in vitro prior to administration to patients could serve as nuclei for growing fibril deposits in vivo.

Insulin aggregation starts at dynamic triple interfaces, originating from solution agitation

“…Besides dehydration of adsorbed proteins, other effects of agitation resulting from a dynamic airliquid interface have been frequently suggested to trigger protein aggregation: for instance, foaming is known for its detrimental effects on the stability of proteins [42,43]. Moreover the formation of cavitation bubbles by ultra-sonication has been proposed as a primary nucleationtriggering mechanism [26,44,45]. In our case however, neither of the experimental setups resulted in the formation of foaming nor bubbles.…”

“…Its stability has been extensively studied in various conditions (low pH and high temperature [22], physiological conditions [23]). It has also been known for a long time for its aggregation into amyloid fibrils [24] and its instability in medical devices [25,26]. At physiological pH and temperature, it has been shown by Sluzky and colleagues [23] and then by Ballet and coworkers [27] that a solution of human insulin can aggregate in the presence of a hydrophobic surface only when the solution is agitated.…”

Peptide Inhibitors of Insulin Fibrillation: Current and Future Challenges

“…The mechanism which leads insulin to form fibrils is a physical process that follows a sigmoidal curve, with a lag phase, rapid growth, and a plateau ( Figure 2 ). The mechanism of insulin’s fibril formation has been proposed to consist of three main reactions: nucleation, growth, and precipitation [ 43 ]. Nucleation consists of the formation of stable nuclei, and it is viewed as the assembly process toward an organized structure.…”

“…In these conditions, the meniscus’s continuous movement during agitation leads to the generation of transiently dry areas, causing the partial dehydration of adsorbed proteins that may then aggregate more easily [ 52 ]. Similarly, fibrillation is promoted by mechanical shocks and cavitation, which can occur when pressure waves of sufficient amplitude produce gas bubbles within liquids that rapidly expand and collapse, generating localized regions of high energy dissipation [ 43 ]. Finally, a recent study showed that increasing interfacial shear rate produces a monotonic increase in the fibrillation rate, with a monotonic decrease in fibrillation time [ 53 ].…”