Nonhuman IAPP Variants Inhibit Human IAPP Aggregation

Abstract

Aim: To identify naturally occurring variants of IAPP capable of inhibiting the aggregation of human IAPP and protecting living cells from the toxic effects of human IAPP.

Background: The loss of insulin-producing β-cells and the overall progression of type 2 diabetes appears to be linked to the formation of toxic human IAPP (hIAPP, Islet Amyloid Polypeptide, amylin) amyloid in the pancreas. Inhibiting the initial aggregation of hIAPP has the potential to slow, if not stop entirely, the loss of β-cells and halt the progression of the disease.

Objective: To identify and characterize naturally occurring variants of IAPP capable of inhibiting human IAPP aggregation.

Methods: Synthetic human IAPP was incubated with synthetic IAPP variants identified from natural sources under conditions known to promote amyloid-based aggregation. To identify IAPP variants capable of inhibiting human IAPP aggregation, Thioflavin T-binding fluorescence, atomic force microscopy, and cell-rescue assays were performed.

Results: While most IAPP variants showed little to no ability to inhibit human IAPP aggregation, several variants showed some ability to inhibit aggregation, with two variants showing substantial inhibitory potential.

Conclusion: Several naturally occurring IAPP variants capable of inhibiting human IAPP aggregation were identified and characterized.

Keywords: Islet amyloid polypeptide; amylin; amyloid; pancreas.; protein aggregation; type 2 diabetes.

Figure 1.

Sequence comparison between human IAPP and animal IAPP variants in this study. Only amino acids that differ from the sequence of hIAPP are indicated. IAPP variants found to inhibit hIAPP aggregation are indicated in the top half of the figure, with variants showing no ability to inhibit hIAPP aggregation listed in the bottom half of the figure.

Figure 2.

37 μM Human IAPP mixed with 75 μM of each indicated animal IAPP. Samples were incubated at 37°C for 40 minutes with shaking. Fluorescence at 488 nm was recorded at the indicated time points. The average of at least three trials is shown with error bars indicating the standard deviation among trials.

Figure 3.

IAPP variants showing no inhibition of hIAPP aggregation. 37 uM Human IAPP mixed with 75 uM of each animal IAPP. Samples were incubated at 37 degrees C for 40 minutes with shaking. All scans are shown as 10 μM × 10 μM.

Figure 4.

IAPP variants showing inhibitory potential against hIAPP aggregation. 37 uM Human IAPP mixed with 75 uM of each animal IAPP. Samples were incubated at 37 degrees C for 40 minutes with shaking. All scans are shown as 10 μM x 10 μM.

Figure 5.

AFM images of hIAPP with dilutions of raccoon IAPP. Inhibition of hIAPP aggregation with diluted concentrations of raccoon of raccoon IAPP. All samples contained 37 μM hIAPP. 1:1 sample contained 37 μM hIAPP mixed with 37 μM raccoon IAPP. 1:0.5 sample contained 37 μM hIAPP mixed with 18.5 μM raccoon IAPP. Samples were mixed at 37°C for 40 minutes with shaking at 200 rpm prior to depositing onto freshly cleaved mica.

Figure 6.

MTT viability assay showing the average of four separate experiments performed in triplicate. The black bar shows the average viability of cells alone. The blue bars indicate the average cell viability upon the addition of 12.8 μM hIAPP or animal IAPP variants individually (animals indicated on the horizontal axis). The red bars display cell viability of hIAPP mixed with each animal IAPP variant (1:1 ratio of each IAPP with 12.8 μM of individual IAPP). Asterisks show a significant increase (p<0.05) in cell viability between hIAPP alone and hIAPP mixed with raccoon or chicken IAPP variants.

Figure 7.

LDH cytotoxicity assay. The first bar shows the average cytotoxicity of cells alone. The blue bars indicate the average cell cytotoxicity upon addition of 12.8 μM hIAPP or animal IAPP variants individually (animals indicated on the horizontal axis). The red bars display cell cytotoxicity of hIAPP mixed with each animal IAPP variant (1:1 ratio of each IAPP with 12.8 μM of individual IAPP).

Figure 8.

Cell morphology. The top panel shows cells alone, cells treated with 12.8 μM hIAPP, and cells treated with 12.8 μM of each IAPP animal variant. The bottom panel shows cells treated with 12.8 μM of hIAPP mixed with each IAPP animal variant (1:1 ratio of each IAPP).