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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
Biochemistry. Author manuscript; available in PMC 2011 October 24.
Published in final edited form as:
PMCID: PMC3199968

The Flavanol (−)-Epigallocatechin 3-Gallate Inhibits Amyloid Formation by Islet Amyloid Polypeptide, Disaggregates Amyloid Fibrils and Protects Cultured Cells Against IAPP Induced Toxicity+


Islet amyloid polypeptide (IAPP, amylin) is the major protein component of 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.

Keywords: IAPP, Amylin, Amyloid Inhibitors, Islet Amyloid, Epigallocatechin 3-gallate, EGCG, Polyphenol, Flavanols

Amyloid formation plays a key role in a wide range of diseases including Alzheimer’s disease, Parkinson’s disease and Huntington’s disease (12). Human islet amyloid polypeptide (amylin, IAPP) is a 37 residue polypeptide which is the major component of the pancreatic islet amyloid associated with type 2 diabetes and is one of the most amyloidogenic polypeptides known (Figure 1) (311). IAPP has been identified in all mammalian species examined, and is a member of the calcitonin-like family of peptides which includes calcitonin, adrenomedullin, and calcitonin gene-related peptide (9). IAPP normally functions as an endocrine partner to insulin, is processed in parallel with insulin in the pancreatic β-cells, and is secreted in response to the same stimuli that lead to insulin secretion (8, 1213). Synthetic aggregates of human IAPP are toxic to pancreatic β-cells, arguing that the process of IAPP amyloid fibril formation contributes to islet cell death in type 2 diabetes (67, 1416). Longitudinal studies using animal models suggest a role for islet amyloid in type 2 diabetes, while autopsies indicate varying amounts of amyloid deposits in individuals diagnosed with type 2 diabetes (1718). Recent work has highlighted a potentially deleterious role for IAPP amyloid formation in islet transplantation (1922). Thus, there is considerable interest in the development of inhibitors of IAPP amyloid formation. There is a very large body of work on inhibitors of the Alzheimer beta amyloid peptide (Aβ), but much less attention has been paid to the development of IAPP amyloid inhibitors (2334).

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.

The ester of epigallocatechin and gallic acid, (−)-Epigallocatechin 3-Gallate [EGCG; (2R,3R)-5,7-dihydroxy-2-(3,4,5-trihydroxyphenyl)-3,4-dihydro-2H-1-benzopyran-3-yl 3,4,5-trihydroxybenzoate], is the most abundant biologically active compound in tea (3536). This tea derived flavanol has been reported to attenuate Aβ–induced neurotoxicity in cultured human neuronal cell lines, and to modulate both tau pathology and Aβ–mediated cognitive impairment in transgenic mouse models of Alzheimer disease (3739). The molecular basis of these effects is not well understood, but has been postulated to be related to EGCG’s radical scavenging properties, its potential to affect the processing of amyloid precursor protein, its ability to interfere with amyloid formation, or by its inhibition of c-Abl/FE65 nuclear translocation and GSK3 beta activation (3941).

Early studies using model homo-polymers of amino acids showed that catechins, a core component of the EGCG structure, interfered with and inhibited the coil to β-sheet transition (42). Recent work has shown that EGCG inhibits the in vitro amyloid formation of several natively unfolded polypeptides including Aβ, α-synuclein, polyglutamine peptides, and the model polypeptide κ-casein (41, 4344). The compound has also been shown to induce a transition of the cellular form of the prion protein into a detergent insoluble form, which differs from the pathological scrapie protein conformation, and to eradicate formation of a variety of prion structures (4546). It also inhibits in vitro amyloid formation by a malaria antigenic protein (47). However, its ability to inhibit amyloid formation by IAPP has not been tested, nor has its ability to protect cells against the toxic effects of IAPP amyloid formation been examined. These observations promoted us to examine the ability of EGCG to inhibit amyloid formation by IAPP and disaggregate amyloid fibrils, and to test its ability to protect cells against IAPP toxicity.


Peptide Synthesis and Purification

Human IAPP was synthesized on a 0.25 mmol scale using an applied Biosystems 433A peptide synthesizer, by 9-fluornylmethoxycarbonyl (Fmoc) chemistry as described (48). Pseudoprolines were incorporated to facilitate the synthesis. 5-(4′-fmoc-aminomethyl-3′,5-dimethoxyphenol) valeric acid (PAL-PEG) resin was used to afford an amidated C-terminal. The first residue attached to the resin, β-branched residues, residues directly following β-branched residues and pseudoprolines were double coupled. The peptide was cleaved from the resin using standard TFA protocols. Crude peptides were oxidized by dimethyl sulfoxide (DMSO) for 24 hours at room temperature (49). The peptides were purified by reverse-phase HPLC using a Vydac C18 preparative column. HCl was used as the counter-ion since the presence of TFA has been shown to affect amyloid formation by some IAPP derived peptides (50). After the initial purification, the peptide was washed with ether, centrifuged, dried and then redissolved in HFIP and subjected to a second round of HPLC purification. This procedure was necessary to remove residual scavengers that can interfere with toxicity assays. Analytical HPLC was used to check the purity of the peptide. The identity of the pure peptide was confirmed by mass spectrometry using a Bruker MALDI-TOF MS; IAPP observed 3904.6, expected 3904.8. An additional sample of human IAPP was purchased from Bachem.

Sample Preparation for in vitro Biophysical Assays of Amyloid Formation

Stock solutions (1.58 mM) of IAPP were prepared in 100% hexafluoroisopropanol (HFIP), and stored at 4°C. Aliquots of IAPP peptide in HFIP were filtered through a 0.45 μm filter and dried under vacuum. A Tris-HCl buffered (20 mM, pH 7.4) thioflavin-T solution was added to these samples to initiate amyloid formation. These conditions were chosen to match the method of sample preparation used for toxicity studies.

Thioflavin-T Fluorescence

Fluorescence measurements were performed using a Beckman model D880 plate reader. The samples were incubated at 25 °C in 96-well plates. An excitation filter of 430 nm and an emission filter of 485 nm were used. All solutions for these studies were prepared by adding a Tris-HCl buffered (20 mM, pH 7.4) thioflavin-T solution into IAPP peptide (in dry form) immediately before the measurement. The final concentration was 32 μM peptide and 25 μM thioflavin-T with or without 32 μM EGCG in 20 mM Tris-HCl. Seeding experiments were performed by adding IAPP to either preformed amyloid or to the final products of an IAPP plus EGCG kinetic experiment. The final concentration of seeds for the IAPP and IAPP: EGCG complex seeding experiments were 3.2 μM IAPP and 3.2 μM IAPP: 3.2 μM EGCG respectively, in monomeric units. EGCG was purchased from Sigma-Aldrich.

Transmission Electron Microscopy (TEM)

Peptide solution (5 μL) was blotted onto a carbon-coated Formvar 300 mesh copper grid for 1 min and then negatively stained with saturated uranyl acetate for 1 min. The same solutions that were employed for thioflavin-T fluorescence measurements were used for TEM studies so that samples could be compared under as similar conditions as possible.

Analysis of the Effect of EGCG on IAPP-Induced Toxicity

Rat insulinoma (INS-1) beta cells were used to assess the ability of EGCG to protect against the toxic effects of human IAPP. INS-1 cells were grown in RPMI 1640 (Gibco-BRL) supplemented with 10% fetal bovine serum (FBS), 11 mM glucose, 10 mM Hepes, 2 mM L-glutamine, 1 mM sodium pyruvate, 50 μM β-mercaptoethanol, 100 U/ml penicillin (Gibco-BRL), and 100 U/ml streptomycin (Gibco-BRL). Cells were maintained at 37°C in a humidified environment supplemented with 5% CO2. Cells were grown for two passages prior to use and used in assays between passages 59 and 65. For toxicity experiments, cells were seeded at a density of 30,000 cells per well in 96-well plates and cultured for 24 hours prior to addition of solutions. Solutions of EGCG:IAPP at 1:1 molar ratio (30 μM IAPP and 30 μM EGCG) were prepared by adding aliquots of a 1.09 mM EGCG stock solution to dry IAPP (prepared as described in the sample preparation subsection) and diluting with Tris-HCl buffer (pH 7.4). Peptide samples and samples of peptide plus EGCG in Tris-HCl buffer (pH 7.4) were added directly to cells (30% final media concentration) after 11 hours of incubation at room temperature. The redox sensitive dye Alamar blue, (resazorin), (Biosource International, CA) was used to assess INS-1 cell viability (51). Alamar blue was diluted ten-fold in 30% culture media and cells were incubated for 5 hours at 37°C. Fluorescence (excitation 530; emission 590 nm) was measured with a Fluoroskan Ascent plate reader (Thermo Labsystems). Data are representative of a minimum of three independent experiments performed in triplicate. The experiments were repeated using human IAPP synthesized by two independent sources and similar results were obtained.

Light Microscopy

Changes in cell morphology were examined by light microscopy in order to provide a second method of evaluating cell viability. Transformed rat INS-1 beta cells were photographed immediately prior to assessment of toxicity by alamar blue cell viability assays. Images were captured using a Nikon Eclipse TS100 light microscope.


EGCG inhibits amyloid formation by IAPP in vitro

The primary structure of IAPP is shown in Figure 1, which also displays the structure of EGCG. The kinetics of in vitro amyloid formation are generally complex, and IAPP is no exception. Like other amyloidogenic polypeptides, it displays a lag phase during which no detectable amyloid fibrils are formed followed by a more rapid growth phase also called the elongation phase, which leads to a final state in which amyloid fibrils are in equilibrium with soluble peptide. The rate of amyloid formation by IAPP was measured in the presence and in the absence of EGCG using thioflavin-T binding assays. Thioflavin-T is a small molecule whose fluorescent quantum yield increases significantly when it binds to amyloid fibrils (52). The dye is thought to bind in grooves formed on the surface of the amyloid fibril which are generated by the in-register alignment of side chains in the regular cross β-sheet structure. Binding of the dye in a planar conformation eliminates rotation of the benzothiozole and benzaminic rings, and reduces self-quenching, resulting in an increase in fluorescence quantum yield.

Thioflavin-T monitored kinetic progress curves for IAPP in the presence and absence of EGCG are displayed in Figure 2. The sigmoidal curve observed in the absence of EGCG is typical of that observed for IAPP in vitro. The lag time is on the order of 20 hours for the IAPP sample in the absence of EGCG. The time course of amyloid formation observed here is comparable to other studies that have used similar sample preparation protocols, but is slower than has been observed for some biophysical studies that were conducted with constant stirring, and which initiate amyloid formation by dilution of stock solutions of IAPP in fluorinated alcohols, typically HFIP, into buffer. Stirring is well known to increase the rate of amyloid formation, most likely by inducing fiber fragmentation, and small amounts of residual HFIP have been shown to drastically accelerate amyloid formation by IAPP (5357).

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) ...

Experiments conducted in the presence of EGCG give strikingly different results than observed in the absence of the compound. No increase in thioflavin-T fluorescence is observed for the 1:1 mixture of IAPP with EGCG. The standard interpretation of curves such as those shown in Figure 2 is that the lack of thioflavin-T fluorescence indicates that no amyloid is formed. However, it is important to independently confirm the results of the thioflavin-T binding assays (58). Consequently, TEM images were recorded of aliquots removed at the end of the reaction. The TEM images of the sample without inhibitor revealed extensive amyloid fibrils with a morphology typical of that found for in vitro IAPP amyloid deposits (Figure 2B). In contrast, very few aggregates were observed on the grid when EGCG was present at the 1:1 ratio and the few fibrils detected had a different morphology (Figure 2C, Supporting Information). The TEM and thioflavin-T studies indicate that EGCG inhibits amyloid formation by IAPP in vitro.

We also examined the ability of EGCG to inhibit amyloid formation by IAPP when added at substoichiometric levels. Significant affects were observed at a 2:1 raio of IAPP to EGCG and at a 5:1 ratio of IAPP to EGCG. The final thioflavin-T fluorescence intensity was reduced by 94% for the 2:1 experiment, (IAPP in two fold excess), and was reduced by 80% even when IAPP was in five fold excess (Supporting Information). The lag phase was also much longer in the presence of substoichiometric amounts of EGCG and was increased approximately two fold for the 5:1, (IAPP to EGCG), sample. The observation that EGCG is a significant inhibitor at substoichiometric levels suggests that it binds to oligomeric species; of course it may also bind to monomers, or it may bind to monomers and form a structure which can associate with additional monomers to generate a complex that does not lead to amyloid.

The complex formed by IAPP and EGCG does not seed amyloid formation

Studies with Aβ and α-synuclein have shown that the EGCG peptide complexes formed are unable to seed amyloid formation by the parent protein (41). Seeding refers to the process of adding pre-aggregated species to a sample of unaggregated polypeptide. Seeding normally significantly accelerates amyloid formation by eliminating the lag phase. The inability of the EGCG: Aβ and the EGCG:α-synuclein complexes to seed aggregation of the parent proteins was taken as evidence that the species are off pathway (41). It is extremely difficult to determine if an intermediate is on or off pathway (59). Strictly speaking, the results with Aβ and α–synuclein indicate that the species formed are not capable of supporting growth of amyloid fibrils, but do not prove that they are off-pathway. Instead, EGCG may have trapped the respective polypeptides in an early intermediate state which is on pathway, but which has not yet reached the state where it is capable of promoting growth of cross β-structure. None-the-less, seeding experiments can provide important mechanistic insight. In particular, the observation of the ability to seed amyloid formation is consistent with the species being on pathway. Thus, a positive result in a seeding study is easily interpreted, while a negative result, although informative, is more ambiguous. Consequently, we investigated the ability of the material present at the end of the kinetic experiments to seed amyloid formation by IAPP. Figure 3 displays the results of IAPP seeding studies. Adding pure IAPP seeds eliminates the lag phase and leads to a thioflavin-T curve which is very similar to those reported in other seeding studies (55). In contrast, seeding by aliquots of the 1:1 EGCG: IAPP mixture collected at the end of the kinetic experiment displayed in Figure 2, had no detectable effect. This shows that the EGCG: IAPP complex does not seed amyloid formation by IAPP under these conditions (Figure 3).

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 (○). ...

EGCG disaggregates IAPP amyloid fibrils

We also tested the ability of EGCG to disaggregate IAPP amyloid fibrils. To the best of our knowledge, there are no small molecules which have been reported to disaggregate IAPP amyloid, although one large peptide-based inhibitor has been shown to do so (27). Figure 4 displays the results of a kinetic experiment for an IAPP control without EGCG; the second curve is from an experiment in which EGCG was added after the plateau region was reached. An initial rapid decrease in thioflavin-T fluorescence is observed after EGCG is added, followed by a slower decay of fluorescence. Samples of the solution were removed at various time points after addition of EGCG and used for TEM analysis. Images recorded from samples removed after the end of the initial, rapid, decay of thioflavin-T fluorescence revealed that the amyloid fibrils had converted to much shorter aggregates which, qualitatively appeared to have less tendency to clump together. Images were also recorded after the end of the second, slower decay phase. Most of the TEM grids were blank, while a few regions contained amorphous aggregates or very thin aggregates (Figure 4, Supporting Information).

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 ...

EGCG protects INS-1 beta cells against the toxic effects of human IAPP

We compared the effects of 30 μM human IAPP and a 1:1 mixture of 30 μM human IAPP and 30 μM EGCG on rat INS-1 cells in order to determine whether EGCG was able to protect beta cells from the toxic effects of human IAPP. Rat INS-1 cells are transformed β-cell line which is widely used in studies of β-cell toxicity. Incubation of INS-1 cells with 30 μM human IAPP for 5 hours resulted in significant toxicity; cell viability was only 22 ± 0.3% relative to untreated control determined by alamar blue assays. The 1:1 mixture of EGCG and IAPP was significantly less toxic, increasing the percentage of viable cells to 77 ± 4% (Figure 5A). Changes in cell morphology were examined by light microscopy in order to provide a second method of evaluating cell viability. Cells were photographed immediately prior to assessment of toxicity by alamar blue assay. Analysis of 30 μM IAPP-treated INS-1 cells demonstrated induction of cell shrinkage and extensive detachment of cells from the cell culture substratum, indicative of cell death. In contrast, INS-1 cells treated with a 1:1 molar ratio of EGCG: IAPP, or with just EGCG demonstrated no observable signs of cell death (Figure 5B–D).

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 ...

The experiments demonstrate a significant level of EGCG-induced protection and indicate that EGCG is an effective inhibitor of human IAPP induced in vitro toxicity. Similar results were obtained using human IAPP from two independent sources, in-house prepared human IAPP and human IAPP purchased from Bachem. This is an important control since there have been reports of significant lot-to-lot variability in the toxicity of human IAPP from different sources (60).


The data reported here demonstrate that EGCG inhibits in vitro amyloid formation by IAPP and disaggregates IAPP amyloid. EGCG is the first small molecule that has been shown to disaggregate IAPP-derived amyloid fibrils. Studies of the interaction of EGCG with α–synuclein and Aβ lead to the proposal, based in part on seeding studies, that EGCG functions by a universal mechanism which involves diverting polypeptides from their normal amyloid formation pathway into non-productive off pathway states (41). It is worth noting, however, that it is extraordinarily difficult to prove if a species is on or off the pathway of amyloid formation (59, 61). Along these lines, studies with another polyphenol, exifone [3,4,5,2′,3′,4′-Hexahydroxybenzophenone;[2,3,4-Trihydroxyphenyl) (3,4,5-trihydroxyphenyl) methanon], has provided evidence that such compounds can function by an alternative mechanism in which they trap amyloidogenic proteins in an on-pathway intermediate state (62). Recent work with reduced carboxymethylated κ-casein showed that EGCG maintained the protein in a pre-amyloid state, but did not redirect the normal aggregation pathway (43). Thus EGCG can inhibit amyloid by a variety of ways. Under the conditions used here, EGCG appears to interact with IAPP in a fashion more similar to its interaction with Aβ and a-synuclein. Irrespective of the details, it is clear that EGCG has the ability to interact with a broad range of natively unfolded proteins and inhibit their in vitro aggregation while at the same time protecting cultured cells against toxicity.

Supplementary Material



We thank Ms. Ping Cao for her assistance with the synthesis and purification of human IAPP, and for helpful discussions. We also thank Prof. Martin T. Zanni and Dr. Chris T. Middleton for helpful discussions and for their continued interest in this work. We thank Dr. Mahiuddin Ahmed for helpful discussions.


the proteolytic fragment of amyloid precursor protein which is responsible for amyloid formation in Alzheimer’s disease
(−)-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
high performance liquid chromatography
human islet amyloid polypeptide
matrix assisted laser desorption ionization-time of flight mass spectrometry
5-(4′-Fmoc-aminomethyl-3′,5-dimethoxyphenyl) valeric acid
transmission electron microscopy
trifluoroacetic acid


+Grant Sponsor NIH GM078114 to D.P.R., Canadian Institute of Health Research grant MOP-14682 to C.B.V.


Additional TEM images of IAPP inhibited with EGCG and of the products of the disaggregation study. Thioflavin-T kinetic studies for 2:1 and 5:1 mixtures of IAPP and EGCG. This material is available free of charge via the internet at


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