The flavanol (-)-epigallocatechin 3-gallate inhibits amyloid formation by islet amyloid polypeptide, disaggregates amyloid fibrils, and protects cultured cells against IAPP-induced toxicity

Abstract

Islet amyloid polypeptide (IAPP, amylin) is the major protein component of the islet amyloid deposits associated with type 2 diabetes. The polypeptide lacks a well-defined structure in its monomeric state but readily assembles to form amyloid. Amyloid fibrils formed from IAPP, intermediates generated in the assembly of IAPP amyloid, or both are toxic to β-cells, suggesting that islet amyloid formation may contribute to the pathology of type 2 diabetes. There are relatively few reported inhibitors of amyloid formation by IAPP. Here we show that the tea-derived flavanol, (-)-epigallocatechin 3-gallate [(2R,3R)-5,7-dihydroxy-2-(3,4,5-trihydroxyphenyl)-3,4-dihydro-2H-1-benzopyran-3-yl 3,4,5-trihydroxybenzoate] (EGCG), is an effective inhibitor of in vitro IAPP amyloid formation and disaggregates preformed amyloid fibrils derived from IAPP. The compound is thus one of a very small set of molecules which have been shown to disaggregate IAPP amyloid fibrils. Fluorescence-detected thioflavin-T binding assays and transmission electron microscopy confirm that the compound inhibits unseeded amyloid fibril formation as well as disaggregates IAPP amyloid. Seeding studies show that the complex formed by IAPP and EGCG does not seed amyloid formation by IAPP. In this regard, the behavior of IAPP is similar to the reported interactions of Aβ and α-synuclein with EGCG. Alamar blue assays and light microscopy indicate that the compound protects cultured rat INS-1 cells against IAPP-induced toxicity. Thus, EGCG offers an interesting lead structure for further development of inhibitors of IAPP amyloid formation and compounds that disaggregate IAPP amyloid.

Figure 1

(A) The primary sequence of human IAPP. The peptide contains a disulfide bridge between Cys-2 and Cys-7 and has an amidated C-terminus. (B) The structure of EGCG.

Figure 2

EGCG inhibits amyloid formation by IAPP in vitro. (A) Fluorescence detected thioflavin-T binding assays in the presence and absence of EGCG. IAPP alone (●); a 1:1 molar ratio mixture of EGCG and IAPP (○); (B) TEM image of IAPP alone. (C) TEM image of a 1:1 mixture of IAPP and EGCG. Aliquots were removed from the kinetic experiments depicted in panel A after 200 hours and blotted for TEM. Scale bars represent 100 nm. Samples contained 32 μM IAPP and experiments were performed in 20 mM Tris-HCl, pH 7.4, 25°C.

Figure 3

IAPP:EGCG complexes do not seed amyloid formation by IAPP. The results of thioflavin-T monitored kinetic experiments are shown. unseeded IAPP (●); IAPP seeded with IAPP amyloid fibrils (■); IAPP seeded with the IAPP:EGCG complex (○). Experiments were conducted in 20 mM Tris-HCl, pH 7.4, 25°C. The IAPP concentration was 32 μM. Seeds, when present, were added at a concentration of 3.2 μM (monomer units).

Figure 4

EGCG disaggregates IAPP amyloid fibrils. (A) thioflavin-T monitored kinetic experiments. Black, IAPP alone; Red, EGCG added at the point indicated by the arrow. (B) TEM image of IAPP before the addition of EGCG. The sample was removed at the time point indicated by the ( ). (C) TEM images of IAPP after the addition of EGCG. The sample was removed at the time point indicated by the ( ). (D) TEM image of IAPP after the addition of EGCG. The sample was removed at the time point indicated by the ( ). Scale bars represent 100 nm. Kinetic runs were conducted at pH 7.4, 25°C, 20 mM Tris-HCl with 32 μM IAPP.

Figure 5

EGCG protects rat INS-1 cells against the toxic effects of human IAPP. (A) Cell viability as determined via alamar blue assays, plotted as percent viability. Red, the effect of the addition of a 30μM solution of human IAPP. Blue, the effect of a 1:1 mixture of human IAPP (30 μM) and EGCG (30 μM). Green, the effect of a 30μM solution of EGCG. All solutions were incubated at room temperature for 11 hrs and then applied to rat INS-1 cells in 96-well plates. Values are relative to those of control cells treated with buffer only. All values represent means ± SEM (n=3). (B) Evaluation of apoptotic cell morphology by light microscopy. Transformed rat INS-1 beta cells were photographed immediate prior to assessment of cell viability by alamar blue. (B) The effect of the addition of 30 μM human IAPP. (C) The effect of a 1:1 mixture of human IAPP (30 μM) and EGCG (30 μM). (D) The effect of a 30 μM solution of EGCG.