Chitosan Oligosaccharides Attenuate Amyloid Formation of hIAPP and Protect Pancreatic β-Cells from Cytotoxicity

Abstract

The deposition of aggregated human islet amyloid polypeptide (hIAPP) in the pancreas, that has been associated with β-cell dysfunction, is one of the common pathological features of patients with type 2 diabetes (T2D). Therefore, hIAPP aggregation inhibitors hold a promising therapeutic schedule for T2D. Chitosan oligosaccharides (COS) have been reported to exhibit a potential antidiabetic effect, but the function of COS on hIAPP amyloid formation remains elusive. Here, we show that COS inhibited the aggregation of hIAPP and disassembled preformed hIAPP fibrils in a dose-dependent manner by thioflavin T fluorescence assay, circular dichroism spectroscopy, and transmission electron microscope. Furthermore, COS protected mouse β-cells from cytotoxicity of amyloidogenic hIAPP, as well as apoptosis and cycle arrest. There was no direct binding of COS and hIAPP, as revealed by surface plasmon resonance analysis. In addition, both chitin-oligosaccharide and the acetylated monosaccharide of COS and glucosamine had no inhibition effect on hIAPP amyloid formation. It is presumed that, mechanistically, COS regulate hIAPP amyloid formation relating to the positive charge and degree of polymerization. These findings highlight the potential role of COS as inhibitors of hIAPP amyloid formation and provide a new insight into the mechanism of COS against diabetes.

Keywords: amyloid; chitosan oligosaccharides; cytotoxicity; human islet amyloid polypeptide; type 2 diabetes.

Figure 1

Analysis of purified CsnA and chitosan oligosaccharides. (A) SDS-PAGE analysis of CsnA. Lane M, protein molecular mass marker; Lane 1, purified CsnA. (B) TLC analysis of the hydrolysis product of CsnA. (C) The chemical structure and MS analysis of chitosan oligosaccharides. ‘R’ can be either H or acetyl group depending on the DP. DP2, (GlcN)₂; DP3, (GlcN)₃ and (GlcN)₂(GlcNAc)₁; DP4, (GlcN)₄ and (GlcN)₃(GlcNAc)₁; DP5, (GlcN)₄(GlcNAc)₁; GlcN—2-amino-2-deoxy-d-glucose; GlcNAc—2-acetamido-2-deoxy-glucose; DP—degree of polymerization.

Figure 2

Thioflavin T (ThT) fluorescence analysis of chitosan oligosaccharides (COS) on human islet amyloid polypeptide (hIAPP) amyloid formation and disaggregation. (A) Effects of COS on hIAPP amyloid formation. (B) Effects of COS on the preformed hIAPP amyloid fibrils.

Figure 3

CD analysis of secondary structure transition of hIAPP. (A) Structural changes of hIAPP during fibrillation monitored by Far-UV CD. (B) Structural changes monitored at 217 nm. (C) COS affect hIAPP structure transition during aggregation. COS were used at the concentrations of 0, 2.5, 5.0 and 10.0 mg/mL. (D) COS disassemble of hIAPP fibrils at concentrations of 5.0 and 10.0 mg/mL.

Figure 4

Morphology changes of hIAPP aggregates. Representative TEM images of (A) monomeric hIAPP. (B) formed fibrils by monomeric hIAPP after 48 h. (C) monomeric hIAPP co-incubated with 2.5 mg/mL of COS for 48 h. (D) monomeric hIAPP co-incubated with 5.0 mg/mL of COS for 48 h. (E) 5.0 mg/mL of COS on preformed hIAPP fibrils for 48 h. (F) 10.0 mg/mL of COS on preformed hIAPP fibrils for 48 h. Scale bar represents 20 mm.

Figure 5

Binding affinity of COS with hIAPP and the effect of different oligosaccharides on hIAPP aggregation. (A) SPR detection of the binding between COS (red) and hIAPP. Mono-hIAPP was immobilized to the CM5 chip. Insulin (black) was used as the positive control. (B) ThT fluorescence detection of CHS (chitin oligosaccharides), GS (glucosamine sulfate), GH (glucosamine hydrochloride), GlcNAc (N-acetyl-d-glucosamine) on hIAPP amyloid fibrils formation.

Figure 6

Effect of COS on hIAPP induced cytotoxicity to β-TC-6 cells. (A) Lactate dehydrogenase release analysis. Cells were treated with 50 μM hIAPP alone or co-treated with 2.0 mg/mL of COS for 24 h. (B) Representative graphs obtained by flow cytometry using double staining with Annexin V-FITC (a marker for apoptosis) and PI (a marker for necrosis). (C) The apoptotic incidence of β-TC-6 cells exposed to 50 μM hIAPP in the presence or absence of 2.0 mg/mL COS for 24 h. Data are expressed as means ± SD of three independent experiments. ** p < 0.01 vs. Control, ## p < 0.01 vs. hIAPP group.

Figure 7

The effect of COS on hIAPP-induced cycle arrest in β-TC-6 cells. The G1, S and G2 phases in cell cycle are represented by green, yellow, and blue, respectively. The cells were exposed to 50 μM hIAPP in the presence or absence of 2.0 mg/mL COS for 24 h.