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

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.

Figure 1. Laurdan dye as an indicator of cell membrane lipid order and ratiometric imaging.

Laurdan (6-Dodecanoyl-2-dimethylaminonaphthalene) is a lipophilic dye capable of partitioning into cell phospholipid membranes (A). When excited at the 405 nm laser line, Laurdan emits fluorescence at 450 nm when the cell membrane is in the gel/liquid ordered phase (l_o; left panel), and redshifts to 500 nm when the cell membrane is in the liquid disordered phase (l_d; right panel). hIAPP monomers, oligomers and amyloid fibrils are predicted to cause lipid disorder (A). Intensity shifts between the ordered and disordered channels can be quantified as generalised polarisation (GP) values. A flowchart outlining the calculation of GP values from raw ratiometric confocal images in the ordered and disordered channels is represented by (B).

Figure 2. Characterisation of hIAPP species.

(A) Dynamic light scattering shows that monomeric hIAPP (0 min) readily aggregated to form large fibrils in aqueous solution over time (450 min), but was stabilised in oligomeric form by resveratrol. (B) TEM images of mature amyloid fibrils and plaques (2 weeks old). The characteristic helical structure of the fibrils can be seen in the lower image.

Figure 3. Ratiometric imaging of MIN6 cells treated for 2 h with fresh and stabilised oligomeric hIAPP, as well as mature hIAPP amyloid.

The shift in GP values for MIN6 cells labelled with Laurdan dye was recorded over 2 h (B). Blue channel = l_o phase; green channel = l_d phase; greyscale channel = live/bright-field; blue-green channel = merge. Inset panels represent merged channels. Arrows indicate signs of unhealthy cells, including shrinkage, blebbing/cytoplasm leakage, and membrane disorder. Scale bars: 40 μm.

Figure 4. Viability and ROS production in MIN6 cells treated with fresh, stabilised oligomeric and mature amyloid hIAPP.

MIN6 cells were left untreated (control) or incubated with fresh hIAPP, stable oligomeric hIAPP, mature amyloid or resveratrol alone. (A) Cell viability after 24 h of treatment was evaluated by Hoechst-33342 (blue) as a live stain, and propidium iodide (red) as a dead stain. White arrows indicate propidium iodide positive cells. Data shown are means and SEM of 3 independent experiments. *P < 0.05. Scale bar: 100 μm. (B) ROS positive cells were identified by DCFDA staining after treatment for 2 and 4 h. Representative FACS profiles after 2 h treatment are shown. H₂O₂ (1.96 mM) was used as positive control. Data shown are means and SEM of 4–6 independent experiments, *P < 0.05, ***P < 0.001.