Preformed Seeds Modulate Native Insulin Aggregation Kinetics.

Area of Science:

• Biochemistry
• Materials Science
• Pharmaceutical Science

Background:

• Insulin aggregation occurs during storage and at injection sites, leading to injection amyloidosis in diabetes patients.
• Understanding insulin stability and aggregation is crucial for the pharmaceutical and biotechnology industries.
• Research focuses on factors modulating insulin aggregation under physiological conditions.

Purpose of the Study:

• To investigate factors influencing insulin aggregation propensity near physiological conditions.
• To quantify the effect of pre-formed insulin "seeds" on aggregation kinetics.
• To elucidate the role of seed "maturity" in insulin nucleation-dependent aggregation.

Main Methods:

• Insulin aggregation kinetics studied at pH 7.2 and 37 °C with dithiothreitol (DTT).
• Techniques included UV-visible, fluorescence, and Fourier transform infrared spectroscopy.
• Microscopy methods: scanning electron microscopy (SEM) and atomic force microscopy (AFM).
• Insulin seeds prepared by incubating reduced insulin for varied durations (10 min to 12 h).

Main Results:

• Aggregation kinetics were fastest with 10-minute-old seeds, indicating nascent seeds accelerate nucleation.
• Intermediate seeds (30 min to 4 h) promoted transient fibril formation within 4 hours.
• Longer incubation (24 h) with intermediate seeds led to amorphous aggregate formation.
• Disulfide-reduced insulin favors amorphous aggregate formation under near-physiological conditions.

Conclusions:

• Insulin aggregation under reducing conditions at physiological pH and temperature primarily yields amorphous aggregates.
• The "maturity" of insulin seeds significantly impacts the nucleation-dependent aggregation pathway.
• Nascent seeds accelerate aggregation, while intermediate seeds can lead to transient fibril intermediates.