Pancreatic β-Cell Membrane Fluidity and Toxicity Induced by Human Islet Amyloid Polypeptide Species.

Emily H Pilkington,Pu Chun Ke,William J Stanley,Esteban N Gurzov,Aleksandr Kakinen,Thomas P Davis,Sara A Litwak

doi:10.1038/srep21274

Abstract

Aggregation of human islet amyloid polypeptide (hIAPP) into fibrils and plaques is associated with pancreatic β-cell loss in type 2 diabetes (T2D). However, due to the rapidness of hIAPP conversion in aqueous phase, exactly which hIAPP species is responsible for the observed toxicity and through what mechanisms remains ambiguous. In light of the importance of understanding hIAPP toxicity for T2D here we show a biophysical scheme based on the use of a lipophilic Laurdan dye for examining MIN6 cell membranes upon exposure to fresh and oligomeric hIAPP as well as mature amyloid. It has been found that all three hIAPP species, especially fresh hIAPP, enhanced membrane fluidity and caused losses in cell viability. The cell generation of reactive oxygen species (ROS), however, was the most pronounced with mature amyloid hIAPP. The correlation between changes in membrane fluidity and cell viability and their lack of correlation with ROS production suggest hIAPP toxicity is elicited through both physical and biochemical means. This study offers a new insight into β-cell toxicity induced by controlled hIAPP species, as well as new biophysical methodologies that may prove beneficial for the studies of T2D as well as neurological disorders.

Highlights

(6-Dodecanoyl-2-dimethylaminonaphthalene) is a lipophilic dye capable of partitioning into cell phospholipid membranes (A)
We show our ratiometric imaging of MIN6 pancreatic cells resulting from their exposure to human islet amyloid polypeptide (hIAPP) of the three states, quantified by generalised polarisation (GP) of the cell membranes partitioned with a Laurdan dye
In order to investigate the specific properties of oligomeric hIAPP, polyphenol resveratrol was utilised as a fibrillation inhibitor

Summary

Introduction

(6-Dodecanoyl-2-dimethylaminonaphthalene) is a lipophilic dye capable of partitioning into cell phospholipid membranes (A). Research by Meng et al refuted this, revealing that rifampicin had no effect on hIAPP fibrillation and demonstrating that the ligand can interfere with ThT fluorescence[29]; illustrating limitations of current methodology to characterise hIAPP species, and proving that the exact science of hIAPP-mediated toxicity is still unclear. Ratiometric imaging, a dual-channel confocal fluorescence technique, was used to visualise cell membrane perturbation by each hIAPP species (Fig. 1A,B). This was achieved via the employment of a lipophilic Laurdan dye, utilised as a probe for membrane lipid order[33,34,35,36] and applied to a number of applications, including nanoparticle exocytosis[37], viral budding[38], and yeast reproduction[39]. In addition to its direct implication for research in T2D, this study demonstrates the use of ratiometric imaging as an effective tool for examining the biophysical and toxicological manifestations of hIAPP that remain a challenge due to the kinetic nature of this most aggregation prone polypeptide

Methods

Results

Conclusion