Insulin As a Model System for Beta-Amyloid Protein Aggregation Reversibility

The appearance of long, insoluble beta-amyloid protein deposits on brain tissue is a characteristic of Alzheimer’s disease, the most common form of adult dementia that affects over 5.4 million elderly Americans.  The role of these amyloid plaques remains unknown, although it is presumed that the plaques and/or small pieces of beta-amyloid fibrils block cell communication and disrupt processes that normal cells need to survive.  In order to prevent or cure Alzheimer’s disease, it is important to understand the formation mechanism of the beta-amyloid fibers as they play a key role in the progression of the disease.  Human insulin is a classic amyloidogenic protein that aggregates at high temperature (> 60ºC) in acidic solution (pH = 1.6) to form long, insoluble fibers.  It is known that the formation of amyloid protein fibrils is irreversible, although it is unclear at what point irreversibility is attained.  Since insulin is relatively inexpensive, readily available, exhibits a similar molecular and fibril structure to beta-amyloids, and is relatively unaffected by heat treatment, it is an ideal model system to study beta-amyloid aggregation.  This study aims to assess the time dependence of heat treatment on aggregation and the effects of thermal quenching after heat treatment; quenching refers to transferring the samples from heat to room temperature in order to not only stop but potentially reverse aggregation. 

In order to achieve this goal, solutions of insulin are heat-treated to induce aggregation and analyzed using dynamic light scattering (DLS).  Samples of human insulin are prepared at concentrations of 8 mg/ml and pH = 1.6.  These solutions are heated to 65˚C for variable intervals and then monitored with DLS after removal from heat.  Samples heated for 5 minutes show no aggregation; scattering intensity remains constant at ~35kHz.  Samples heated for two five-minute intervals (total 10 min on heat) yield small aggregation visible in the form of intensity spikes and changes in size distribution that disappears after sufficient thermal quenching.  Samples heated for three five-minute intervals (total 15 min on heat) yield further aggregation which also disappears after thermal quenching.  Samples heated for four five-minute intervals (total 20 min on heat) display aggregation that is irreversible, even after days spent at room temperature; in fact, the aggregation seems to progress during that time.  Work on continuous sample heating is in progress and will be included in the final report.  The work performed indicates that the early aggregation of insulin is reversible by thermal quenching to room temperature.