The aggregation potential of human amylin determines its cytotoxicity towards islet β-cells

The aggregation potential of human amylin determines itscytotoxicity towards islet b-cellsBarbara Konarkowska1,2, Jacqueline F. Aitken1,2, Joerg Kistler1, Shaoping Zhang1,2 andGarth J. S. Cooper1,2,3

1 School of Biological Sciences, Faculty of Science, University of Auckland, New Zealand

2 Centre for Research Excellence in Molecular Biodiscovery, Faculty of Science, University of Auckland, New Zealand

3 Department of Medicine, Faculty of Medical and Health Sciences, University of Auckland, New Zealand

Human amylin (hA) is a small protein cosecreted with

insulin from pancreatic islet b-cells upon stimulation

by glucose or other chemical signals [1,2]. Wild-type

hA, also known as insulinoma amyloid peptide, insuli-

noma amyloid polypeptide (IAPP) or islet amyloid

polypeptide [3], demonstrates a strong in vitro tendency

to aggregate into fibrils [4,5], which is dependent on

specific residues in different regions of the molecule,

notably, the amyloidogenic region of amino acids 20–

29 [6]. It is the main protein in the amyloid aggregates

that are frequently present in the islets of human sub-

jects with type 2 diabetes mellitus (T2DM) [7,8], as

well as in diabetic cats and primates [9]. It has been

reported that aggregated hA might contribute to the

loss of insulin-producing pancreatic b-cells cells in

T2DM [10,11]. The following lines of evidence have

associated hA with the pancreatic pathology of

T2DM: (a) amyloid is often concentrated near areas of

islet b-cell degeneration in humans or primates with

T2DM [10,12–14]; (b) synthetic hA is toxic to pancre-

atic b-cells in vitro [11,15–17]; (c) spontaneous loss of

b-cells has been reported in a line of hA-transgenic

mice [18]; and (d) toxicity of hA towards cultured cells

correlates with its ability to form fibrils - for example,

rat amylin does not form fibrils and nor does it evoke

b-cell apoptosis in vitro [11,17,19].

Keywords

amylin; amyloid formation; pancreatic islet

b-cells; protein aggregation; type 2 diabetes

Correspondence

G. J. S. Cooper, Level 4, Thomas Building,

School of Biological Sciences, University of

Auckland, Private Bag 92019, Auckland,

New Zealand

Fax: +64 9 373 7045

Tel: +64 9 373 7599, ext. 87394

E-mail: g.cooper@auckland.ac.nz

(Received 5 April 2006, revised 6 June

2006, accepted 8 June 2006)

doi:10.1111/j.1742-4658.2006.05367.x

Human amylin is a small fibrillogenic protein that is the major constituent

of pancreatic islet amyloid, which occurs in most subjects with type 2 dia-

betes. There is evidence that it can elicit in vitro apoptosis in islet b-cells,but the physical properties that underpin its cytotoxicity have not been

clearly elucidated. Here we employed electron microscopy, thioflavin T

fluorescence and CD spectroscopy to analyze amylin preparations whose

cytotoxic potential was established by live–dead assay in cultured b-cells.Highly toxic amylin contained few preformed fibrils and initially showed

little b-sheet content, but underwent marked time-dependent aggregation

and b-conformer formation following dissolution. By contrast, low-toxicity

amylin contained abundant preformed fibrils, and demonstrated high initial

b-sheet content but little propensity to aggregate further once dissolved.

Thus, mature amylin fibrils are not toxic to b-cells, and aggregates of fibrils

such as occur in pancreatic islet amyloid in vivo are unlikely to contribute

to b-cell loss. Rather, the toxic molecular species is likely to comprise sol-

uble oligomers with significant b-sheet content. Attempts to find ways of

protecting b-cells from amylin-mediated death might profitably focus on

preventing the conformational change from random coil to b-sheet.

Abbreviations

AM, acetomethoxyl ester; EthD, ethidium homodimer; hA, human amylin; HFIP, hexafluoroisopropanol; IAPP, insulinoma amyloid

polypeptide; JNK1, Jun NH2-terminal kinase 1; KRB, Krebs-Ringer bicarbonate buffer; RINm5F, rat insulinoma m5F; T2DM, type 2 diabetes

mellitus; TEM, transmission electron microscopy; ThT, thioflavin T.

3614 FEBS Journal 273 (2006) 3614–3624 ª 2006 The Authors Journal compilation ª 2006 FEBS

Preventing hA-induced loss of pancreatic b-cells has

been proposed as a route to attenuate the gradual

decline in endogenous production of insulin in T2DM

[20]. However, at this time our knowledge of the mech-

anism by which hA evokes pancreatic b-cell death is

incomplete. The small oligomeric structures formed

very early in the process of hA aggregation [4,21]

could be toxic to pancreatic b-cells, but whether such

structures form in vivo and whether mature fibrils are

cytotoxic remains unresolved.

It was initially assumed that mature amyloid fibrils

were the most likely toxic species, as these are the form

of aggregates that have been commonly detected adja-

cent to islet cells [11,22,23]. However, a growing body

of experimental data suggests that in the case of many

amyloid diseases, the most toxic form of protein to cells

are the prefibrillar aggregates [24–29]. Protofibrillar sol-

uble intermediates are known to exist in the early stages

of fibril formation of various amyloidogenic proteins,

including hA, b-amyloid and a-synuclein [21,30–32].

Prefibrillar aggregates of proteins not associated with

amyloid diseases have also been shown to cause cyto-

toxicity in a similar manner and extent to those of dis-

ease-associated proteins [33]. Production of an antibody

that recognizes micellar Ab, but not its soluble or fibril-lar forms, has been reported [31]. This antibody also

recognized and inhibited the cytotoxicity of soluble

oligomers of structurally unrelated proteins such as

a-synuclein, insulin, amylin and polyglutamine, suggest-

ing that these proteins may have a common structure

and may share a common pathogenic mechanism.

Clearly, the nature of the cytotoxic species of amylin has

implications that may be applicable to other amyloid

pathologies, making its investigation generally relevant.

Kayed et al. [34] and Tenidis et al. [35] previously

observed that a conformational transition of soluble

amylin into b-sheet precedes formation of insoluble

amylin aggregates, and in addition, the kinetics of this

amyloid formation are consistent with a nucleation-

dependent polymerization mechanism.

Wogulis et al. [36] suggested that the neuronal cell

death associated with b-amyloid and human amylin

required the presence of both fibrillar and soluble pep-

tide. They postulated that nucleation-dependent poly-

merization was a common feature of amyloid-mediated

cell death.

While studying their cytotoxicity in a b-cell culturemodel, we noted that different synthetic hA prepara-

tions vary markedly in their levels of intrinsic bioactivity

and hypothesized that this variation might be directly

related to differences in the rate of formation of soluble

cytotoxic intermediates, resulting from the initial aggre-

gation state of the synthetic hA preparation.

To test this hypothesis, we employed three independ-

ent techniques: transmission electron microscopy

(TEM), thioflavin T (ThT) fluoroscopy and CD spec-

troscopy, to characterize relevant physical properties

of hA preparations whose toxicity was known, as

determined by live–dead assay in rat insulinoma m5F

(RINm5F) islet b-cells. In particular, we characterized

their aggregation state with respect to the presence or

absence of preformed fibrils, aggregate content and

b-sheet content following initial dissolution. We also

measured time-dependent rates of aggregation and

b-sheet formation in solution.

We have previously described the RINm5F model of

amylin-mediated b-cell apoptosis [15–17,20]. Treatment

of these cells with fibrillogenic amylin preparations

(i.e. preparations that are able form fibrils from mono-

meric and oligomeric species) evokes apoptosis charac-

terized by typical changes in the cell membrane and

nucleus, formation of apoptotic bodies and internucle-

osomal cleavage of genomic DNA [37]. Fibrillogenic

amylin evokes cytotoxicity via a molecular signaling

pathway that incorporates sequential activation of Jun

NH2-terminal kinase 1 (JNK1), c-Jun, caspase 8, and

caspases 1 and 3 [16,17], as well as increased expres-

sion of p53 and p21WAF1 ⁄CIP1 [19]. Significantly, these

cellular and molecular events are only elicited by inter-

action between cells and fibrillogenic amylin variants,

whereas, by contrast, nonfibrillogenic amylin molecules

are nontoxic. We have also shown that responses

evoked by fibrillogenic amylin molecules in RINm5F

cells are echoed by those in the human islet b-cell line,CM [16,17].

Here we show that the cytotoxicity of preparations

of two fibrillogenic proteins, wild-type hA and 8)37hA,

correlates with their initial aggregation states.8)37hA is a fragment of hA that lacks the NH2-

terminal seven amino acids of the wild-type protein

(necessary for receptor binding and peripheral biologi-

cal activity [38]), but that retains its ability to elicit

cytotoxicity in b-cells [17,39]. Although the physiologic

function of 8)37hA is unknown, it is nevertheless a

second closely related amyloidogenic member of the

amylin family, and was therefore used to generate a

set of independent data to complement those obtained

with the wild-type molecule (Table 1).

Two different preparations of each of these proteins

were studied: one containing a high proportion of

mature fibrils and the other containing an abundance

of small globular structures. These preparations

showed remarkable differences in cytotoxicity, differ-

ences in their secondary structure, and differences in

their potential to form new aggregates. Our results

demonstrate an inverse relationship between the extent

B. Konarkowska et al. Aggregation potential of human amylin

FEBS Journal 273 (2006) 3614–3624 ª 2006 The Authors Journal compilation ª 2006 FEBS 3615

of aggregation and the cytotoxic potential of hA. They

show that mature hA fibrils are not cytotoxic in vitro,

and instead implicate a much earlier precursor stage,

namely, the transition from random coil to b-sheetcoupled with de novo aggregation, as necessary proper-

ties for the induction of b-cell death.

Results and Discussion

Cytotoxic potential varies between different

hA preparations

We have previously noted major differences in cyto-

toxic potential among different commercial prepara-

tions of hA. Here, we employed a well-characterized

live–dead assay to measure the extent of cell death [37]

following 24 h incubations with various preparations

(Fig. 1). We employ the term ‘cytotoxic potential’ to

describe the (unrealized) cytotoxic activity present in

such preparations, as measured in the RINm5F-based

cytotoxicity assay.

The cytotoxic potential of different preparations of

hA (Fig. 1A) or 8)37hA (Fig. 1B) towards b-cells was

assessed and expressed as the number of dead cells as

a percentage of total cells counted. The degree of dif-

ference in cytotoxic potential was similar among prep-

arations of hA and 8)37hA, as illustrated (Fig. 1). The

six preparations tested fell into two categories, which

we named ‘high toxicity’ (Fig. 1A, preparations A and

B; Fig. 1B, preparation E), and ‘low toxicity’ (Fig. 1A,

preparations C and D; Fig. 1B, preparation F). There

was a definite dose–response effect of the ‘high-toxic-

ity’ peptides on cytotoxic potential (Fig. 1A, prepar-

ation B; Fig. 1B, preparation E) but no such effect

was seen with ‘low-toxicity’ preparations (Fig. 1A,

preparations C and D; Fig. 1B, preparation F).

Nonamyloidogenic rat amylin was used as a noncy-

totoxic control (data not shown). Amylin cytotoxicity

is correlated with the ability to form fibril intermedi-

ates, and the presence of three proline residues at posi-

tions 25, 28 and 29 of the amyloidogenic region in rat

amylin renders this protein unable to readily form

fibrils (Table 1) [6].

All preparations were shown to be substantially pure

as judged by MALDI-TOF MS. The mass spectra of

hA preparations B and C are shown as insets in

Table 1. Amino acid sequences of human amylin, 8)37human amylin and rat amylin. Differences in amino acid sequence between human

and rat amylin are shown in bold in the rat amylin sequence.

Human amylin KCNTATCATQRLANFLVHSSNNFGAILSSTNVGSNTY-NH28)37Human amylin TQRLANFLVHSSNNFGAILSSTNVGSNTY-NH2

Rat amylin KCNTATCATQRLANFLVRSSNNLGPVLPPTNVGSNTY-NH2

vehicle 10 µMA

10 µMB

25 µMB

10 µMC

40 µMC

10 µMD

30 µMD

vehicle 25 µME

40 µME

25 µMF

50 µMF

A B100

01000 2000 3000 4000 5000

ytisnetnI %

hA preparation B

1954.27

3904.47 10075

01000 2000 3000 4000 5000

ytisnetnI %

hA preparation C

1951.62

3904.33

Fig. 1. Marked variation in cytotoxic potential between different synthetic amylin preparations. Cytotoxic potential of different synthetic prep-

arations of (A) hA or (B) 8)37hA in cultured RINm5F islet b-cells, was measured using a live–dead assay at final protein concentrations as indi-

cated. Significance was determined by one-way ANOVA with Tukey’s post hoc test: P < 0.001, for both hA preparations A (n ¼ 3) and B

(25 lM; n ¼ 6) versus preparations C (n ¼ 6) and D (n ¼ 6). There was no statistically significant difference between preparations C and D.

Preparations A, B and D (30 lM only), but not preparation C, were statistically different from the vehicle control (*P < 0.001 for A and B;

**P < 0.01 for D). *P < 0.001 for 8)37hA preparations E (25 lM, n ¼ 23; 40 lM, n ¼ 2) versus both vehicle control and preparation F (25 lM,

n ¼ 20; 50 lM; n ¼ 12). There was no significant difference between preparations F and the vehicle control. Insets in (A) show mass spectra

of hA preparations B and C.

Aggregation potential of human amylin B. Konarkowska et al.

Fig. 1A as representative examples. The peak in each

spectrum at 3904 Da ⁄ e corresponds to the molecular

weight of wild-type human amylin. The peaks at

1954 Da ⁄ e for preparation B and 1951 Da ⁄ e for pre-

paration C represent the doubly charged mass species

of human amylin. Thus differences in protein impurit-

ies did not explain the marked variability in cytotoxici-

ty between different amylin preparations. We selected

preparations B and E (high toxicity) and C and F (low

toxicity) as representatives of each category for further

investigation.

Low cytotoxicity correlates with high preformed

fibril content and low rates of de novo

aggregation

The cytotoxicity of hA has been associated with fibril

formation [11,19]. We sought to find structural corre-

lates to the observed differences in cytotoxicity by

characterizing the preformed fibril content and aggre-

gation potential of hA preparations with known cyto-

toxic potential. We employed TEM to examine fresh

aqueous solutions of the four selected preparations.

We found striking differences in the relative content of

mature fibrils [4], which were much more abundant in

preparations of low cytotoxic potential (Fig. 2).

Figure 2A illustrates a representative aspect of a fresh

(1 min) hA solution from preparation B containing rel-

atively few fibrils but many spheroidal structures,

which had previously been suggested to represent one

of the earliest forms of aggregate in hA preparations

[5,21]. Figure 2B illustrates an equivalent study of hA

preparation C, which, by contrast, contained many

fibrils and relatively few spheroidal structures. Similar

differences in the relative content of preformed fibrils

were observed in fresh 8)37hA solutions prepared from

material with high (preparation E) or low (preparation

F) cytotoxic potential. Figure 2C,D contrasts represen-

tative micrographs of 8)37hA preparations E and F,

respectively. It can be seen that their appearance mir-

rors those of the equivalent hA preparations.

We next employed ThT-binding assays to compare

aggregation potential between amylin preparations

(Fig. 3). Nonamyloidogenic rat amylin was used as a

control, and as expected, rat amylin solutions gener-

ated no ThT fluorescence above background (Fig. 3A).

However, major differences were present in the

Fig. 2. TEM of solutions containing amylin of high or low toxicity.

Fresh amylin solutions (500 lM) were studied by TEM. Aggregates

in (A) high-toxicity hA (preparation B) clearly differed from those in

(B) low-toxicity hA (preparation C). Similar contrasts were seen

between (C) high-toxicity 8)37hA (preparation E) and (D) low-toxicity8)37hA (preparation F). Scale bars, 0.2 lm.

ecnecseroulf evitaleR

Time [min]

Aecnecseroulf evitale

Time [min]

Fig. 3. ThT fluorescence of solutions containing amylin preparations

of high or low toxicity. ThT-binding assays were performed in solu-

tions containing 50 lM of (A) high-toxicity (n, preparation B) and

low-toxicity (m, preparation C) hA, or (B) high-toxicity (r, prepar-

ation E) and low-toxicity (., preparation F) 8)37hA. Rat amylin (d) is

shown as a nonfibril-forming control. All points shown are means ±

SEM, n ¼ 3.

ThT-binding profiles of hA preparations with differing

cytotoxic potential. First, the hA preparation of low

cytotoxic potential (preparation C) demonstrated high

levels of ThT-detectable aggregates immediately after

dissolution into ThT-containing buffer, and initial

fluorescence values comprised more than 80% of maxi-

mal values obtained during the time of the assay

(Fig. 3A). By contrast, the preparation of high cyto-

toxic potential (preparation B) contained few pre-

formed aggregates, and initial fluorescence values

comprised less than 5% of the maximal values that

developed during the time course (Fig. 3A). These data

are consistent with our TEM findings, both demonstra-

ting major differences in the aggregation state immedi-

ately after dissolution.

Prolonged incubation of these preparations with

ThT revealed very different time-dependent increases

in fluorescence. In the case of low-toxicity hA prepar-

ation C, this increase was small compared to that of

high-cytotoxicity preparation B (Fig. 3A). Moreover,

the ThT-binding profile of the highly toxic preparation

displayed a sigmoidal-shaped fluorescence time curve,

consistent with the presence of three phases: lag, elon-

gation and saturation. These findings are consistent

with those previously reported for hA [40]. By con-

trast, the best fit to the time-dependent ThT fluores-

cence data from the weakly toxic hA preparation was

a two-phase association curve with a relatively short

elongation phase followed by saturation. The presence

of ThT itself in the assay did not appear to affect the

kinetics of aggregation, as a control experiment where

samples were incubated in the absence of ThT and

aliquots removed at different times showed similar

results to those described above (data not shown).

This difference between the shapes of the fluores-

cence–time curves suggests that the initial aggregation

state differs markedly between high-toxicity and

low-toxicity hA preparations, consistent with the dif-

ferences observed by electron microscopy. In the

high-toxicity preparation, hA was initially mostly non-

aggregated, but underwent progressive aggregation

during the first 150 min in solution (Fig. 3A). By con-

trast, the low-toxicity hA was largely aggregated at the

beginning of the experiment, and aggregation was

essentially complete within 30 min.

The results obtained with hA were paralleled by sim-

ilar time-dependent fluorescence data for high-toxicity

and low-toxicity preparations of 8)37hA (Fig. 3B).

High-toxicity 8)37hA (preparation E) displayed a ThT-

binding profile similar to that of the high-toxicity pre-

paration of full-length protein, with the exception that

the lag phase was shorter. Initial fluorescence values

were low (they comprised less than 15% of maximal

values obtained during the time course of the assay),

indicating a low level of ThT-detectable aggregates at

the beginning. In the case of low-cytotoxicity 8)37hA

(preparation F), the initial fluorescence values were at

least three-fold higher than those observed for highly

toxic protein. Moreover, they remained within a narrow

range over the duration of the experiment, and there

was no statistically significant difference between the

initial and maximal values obtained during the time

course of the assay. The paucity of de novo formation of

ThT-detectable aggregates by the low-toxicity prepar-

ation is reflected in the observation that the data were

best fitted to a one-phase exponential decay model.

In summary, the results of the ThT-binding study

indicate that (a) the initial amount of ThT-detectable

aggregates in freshly prepared aqueous solutions of

hA, and (b) the extent of subsequent de novo forma-

tion of ThT-detectable aggregates, differed markedly

between the high-toxicity and low-toxicity preparations

of full-length or truncated amylin.

We next employed CD spectroscopy to probe the ini-

tial conformational status of hA from preparations

with different cytotoxic potential, and extended these

studies to characterize time-dependent conformational

changes of the proteins in solution. CD spectra from

the high-toxicity hA (preparation B) demonstrated a

time-dependent conversion from random coil to b-sheetconformation (Fig. 4A), as shown by a loss of signal at

205 nm and a subsequent appearance of a minimum at

217 nm (24 h), indicating the random coil to b-sheettransition. By contrast, low-toxicity hA already showed

predominantly b-sheet conformation immediately upon

dissolution in water (strong minimum at 217 nm), and

then underwent little conformational change (Fig. 4B;

preparation C).

Since the b-sheet conformer is reportedly character-

istic of hA in its fibrillar form [40], these CD data are

consistent with both the electron microscopy and ThT-

binding studies, which also showed an advanced aggre-

gation state immediately upon dissolution. Equivalent

differences of CD spectral behavior were found to exist

between preparations of 8)37hA of high (preparation

E) and low (preparation F) cytotoxic potential

(Fig. 4C,D).

The CD spectrum of rat amylin (a nonfibril-forming

control), although not shown, was similar to that of

hA (Fig. 4A) at 0 h.

Disaggregation restores cytotoxic potential to

low-toxicity hA

Based on the data presented so far, we observed

that the low cytotoxicity of an hA preparation was

associated with relatively high levels of pre-existing

fibrillar aggregates, a concomitant lack of propensity

to form new aggregates, and the absence of random

coil to b-sheet transition. On the other hand, a highly

cytotoxic preparation contained few fibrillar aggregates

at the time of dissolution and displayed a marked

potential to aggregate and change from a random coil

to a b-sheet conformation over time. We thus hypo-

thesized that dissociation of initial aggregates should

increase the cytotoxic potential. To test this hypothe-

sis, we employed a chaotropic solvent, hexafluoroiso-

propanol (HFIP), to dissociate pre-existing aggregates

into monomers. This treatment is similar to that with

guanidinium-HCl, which was employed to disaggregate

hA from isolated islet amyloid deposits during its ori-

ginal isolation from the diabetic pancreas [8]. Mature

fibrils were rarely seen in freshly prepared solutions of

low-toxicity hA following HFIP treatment (Fig. 5A,

preparation C). Instead, amorphous aggregates and

short flexible fibrils (possibly protofibrils [4,5,21]) were

abundant. Similar types of aggregates were also

observed in freshly dissolved, low-toxicity 8)37hA that

had been HFIP treated (Fig. 5B; preparation F).

ThT binding of low-toxicity hA following HFIP

treatment resembled that of nontreated high-toxicity

preparations, with sigmoidal fluorescence time curves

demonstrating three phases: lag, elongation and sat-

uration. Representative data for HFIP-treated prepar-

ation C are shown in Fig. 6A. Similarly, following

Fig. 4. CD spectra of solutions made with amylin preparations of high or low toxicity. CD spectroscopy was used to characterize time-

dependent changes in protein conformation in 25 lM solutions containing (A) high-toxicity hA (preparation B) and (B) low-toxicity hA (prepar-

ation C), or (C) high-toxicity 8)37hA (preparation E) and (D) low-toxicity 8)37hA (preparation F).

Fig. 5. TEM of HFIP-treated, low-toxicity amylin preparations. Low-

toxicity (A) hA (preparation C) or (B) 8)37hA (preparation F) were

treated with HFIP. Aliquots were taken from freshly dissolved solu-

tions (500 lM), negatively stained and studied by TEM. Scale bars,

0.2 lm.

HFIP treatment, a weakly toxic batch of 8)37hA (pre-

paration F) generated a ThT-binding profile resem-

bling that of a high-toxicity preparation without HFIP

treatment (Fig. 6B). By contrast, HFIP treatment

failed to alter the ThT-binding characteristics of con-

trol rat amylin (Fig. 6A).

CD spectroscopy of HFIP-treated, low-toxicity hA

(preparation C) demonstrated a random coil structure

in solution (Fig. 7A), and time-dependent analysis

documented a gradual change in signal from predom-

inantly random coil to a flattening of the spectrum,

suggesting oligomerization to form insoluble amyloid

as visually observed by Congo red staining (24 h) [41].

HFIP treatment also generated a random coil structure

in low-toxicity 8)37hA (preparation F), which progres-

sively changed to b-sheet with time (Fig. 7B). These

spectra contrast with those from the corresponding

preparations before HFIP treatment, as shown in Fig.

4B (hA) and Fig. 4D (8)37hA), respectively.

Finally, the effect of HFIP treatment on the biologi-

cal activity of low-toxicity hA was assessed following

24 h of exposure of RINm5F b-cells to amylin solu-

tions. HFIP treatment caused marked increases in the

cytotoxicity of both hA and 8)37hA. Preparation C

(Fig. 8A) killed 82 ± 3% of cells versus its pretreat-

ment toxicity of 6 ± 2.5% (P < 0.001). A similar

effect was observed with treatment of low-toxicity8)37hA (preparation F; Fig. 8B), which killed 57.3 ±

4.1% of cells after treatment but only 2.3 ± 0.5%

before (P < 0.001).

The data shown above directly address the relation-

ship between mature hA fibrils and their cytotoxicity

towards islet b-cells. We have shown that the cytotoxic

potential of hA correlates inversely with the content of

mature fibrils in amylin preparations. Thus, the mature

fibril is clearly not the toxic form of hA. Preparations

with high proportions of fibrils show limited potential

to form new aggregates, with the remaining, soluble

portion of the protein predominantly in b-sheet con-

formation from the outset. By contrast, highly toxic

hA contains an abundance of spheroidal structures

and very few fibrils, and displays a high potential to

Time [min]

Fig. 6. ThT fluorescence of solutions containing HFIP-treated, low-

toxicity amylin preparations. ThT-binding assays were performed on

aqueous solutions containing 50 lM low-toxicity (A) hA (m, prepar-

ation C) or (B) 8)37hA (., preparation F), following HFIP treatment.

Rat amylin (d) is shown as a nonfibril-forming control. Data are

means ± SEM, n ¼ 3.

Fig. 7. CD spectra of solutions containing HFIP-treated, low-toxicity

amylin preparations. CD spectroscopy was used to characterize

time-dependent changes in protein conformation in 25 lM solutions

containing low-toxicity (A) hA (preparation C) or (B) 8)37hA (prepar-

ation F) that had been treated with HFIP.

form new aggregates, and initial CD spectra are indic-

ative of a mostly random coil conformation. These

highly toxic preparations undergo substantive time-

dependent changes in solution conformation, from ran-

dom coil to b-sheet. Furthermore, a preparation of

initially highly cytotoxic hA (preparation B) exhibited

significantly attenuated cytotoxicity after aging in

aqueous buffer for 7 days (17.85 ± 4.9% cell death

for 10 lm fresh amylin versus 1.2 ± 0.2% following

vehicle treatment, P < 0.01; and 2.75 ± 0.5% for

10 lm aged amylin, P nonsignificant versus vehicle

control). Visual inspection of Congo red-stained aged

amylin revealed the presence of insoluble aggregates.

Thus the cytotoxic potential of hA and 8)37hA prep-

arations depends on the presence of oligomeric nonfi-

brillar species in the solution at the time of addition to

the b-cells.

Our findings are consistent with the hypothesis that

hA toxicity is not mediated by mature fibrils, but by

oligomers representing intermediate assemblies in the

process of aggregation [4,5,21]. Our results indicate

that the fibrillar content, the potential to form new

ThT-detectable aggregates, and the initial secondary

structure of hA in aqueous solution, can predict the

level of cytotoxic activity in a particular preparation.

The chemical process by which the peptides are syn-

thesized may lead to the existence of preformed aggre-

gates that can affect the experimental outcomes of

studies involving synthetic amylin as well as other

amyloidogenic peptides. Thus it is critical that amy-

loidogenic peptides are prepared according to a defined

regime to produce a homogeneous monomeric sample

(e.g. through dissolution in HFIP [42]) and are charac-

terized prior to use in the study of hA-evoked b-cellapoptosis.

Our observations that hA in the form of mature

fibrils, similar to those present in pancreatic islet amy-

loid deposits in subjects with T2DM, is essentially inert

towards pancreatic b-cells, whereas hA preparations

that contain relatively few mature fibrils but undergo

time-dependent transformation into soluble b-conform-

ers are highly cytotoxic, are consistent with reported

results [43,44], where it was proposed that the forma-

tion of soluble oligomers of human amylin, rather than

islet amyloid per se, was related to the increased fre-

quency of b-cell apoptosis in the rodent human IAPP-

transgenic models. Hence, attempts at finding effective

ways of inhibiting hA-mediated b-cell apoptosis shouldconcentrate on either prevention of aggregation (inclu-

ding prevention of the conformational change of a sol-

uble protein from random coil to b-sheet) or, possibly,its acceleration to shorten the time of cell exposure to

toxic forms of the protein.

Experimental procedures

Proteins and other chemicals

All chemicals were of analytical grade or better, unless oth-

erwise stated. Human amylin [lots 0542559 (preparation A);

0538994 (preparation B); 0549416 (preparation C); 0551805

(preparation D)], 8)37hA [0523544 (preparation E); 0536090

(preparation F)] and rat amylin (0542554, 0516011) were

lyophilized preparations from Bachem California (Tor-

rance, CA, USA). Aqueous stock solutions were always

prepared freshly by dissolving proteins in purified water

(18 MW; MilliQ, Millipore, Bedford, MA) to an initial con-

centration of 500 lm. Purity of synthesized proteins was

verified by MALDI-TOF MS using a Voyager-DETM PRO

BioSpectrometryTM Workstation (Applied Biosystems,

laitnetop cixototyC

)sllec latot fo % sa sllec daed( vehicle non-treated

CHFIP-treated

laitnetop cixototyC

)sllec latot fo % sa sllec daed( non-treated

FHFIP-treated

vehicle

Fig. 8. Comparison of cytotoxicity of hA or 8)37hA from low-toxicity

preparations before and after HFIP treatment. Cytotoxic activity

was measured by live–dead assay in the control (vehicle) and in (A)

hA (preparation C) or (B) 8)37hA (preparation F) before and after

treatment with HFIP. Final peptide concentrations were 40 lM (hA)

or 50 lM (8)37hA). Significance was determined by one-way ANOVA

with Tukey’s post hoc test: *P < 0.001, for HFIP-treated hA and8)37hA versus respective nontreated peptides, n ¼ 4.

Foster City, CA, USA) in positive-ion mode with a matrix

of saturated a-cyano-4-hydroxycinnamic acid.

Cell culture and treatments

RINm5F cells were a gift from H. K. Oie (National Institutes

of Health, Bethesda, MD, USA). Cells were cultured in

RPMI 1640 medium (Roswell Park Memorial Institute

Media 1640; Invitrogen, Carlsbad, CA, USA) supplemented

with 10% (v ⁄ v) fetal bovine serum (Invitrogen), 290 lgÆmL)1

l-glutamine, 100 IUÆmL)1 penicillin, 100 lgÆmL)1 strepto-

mycin and 2.5 mgÆmL)1 NaHCO3 at 37 �C in a humidified

incubator with 5% CO2, as previously described [15,19]. All

experiments were performed using cells between passages 28

and 39, which were plated in 24-well culture plates at a

density of 1.2 · 105 per well.

Assessment of cell viability

Cell viability following hA treatment was assessed by live–

dead assay [20,37]. After plating, cells were grown for

approximately 48 h. Following a single rinse with NaCl ⁄Pi,

fresh medium was added to each well, and aliquots of

aqueous amylin or vehicle added to a final volume of

200 lL. Following a 24 h incubation, cell viability was

determined by double-staining with calcein–acetomethoxyl

ester (AM) and ethidium homodimer-1 (EthD-1; Molecular

Probes, Eugene, OR, USA). Cells were gently rinsed with

Krebs-Ringer bicarbonate buffer (KRB) and incubated

with 1 lm calcein–AM and 4 lm EthD-1 in KRB in the

dark at 37 �C for 10 min. Green fluorescence of live cells

and red fluorescence marking dead cells were simulta-

neously visualized using a Zeiss Axiovert S100 microscope

(Zeiss filter set no. 09, Carl Zeiss International, Oberko-

chen, Germany). Live and dead cells were counted in two

to four fields per well (two wells per experiment) and pho-

tographed at 400· magnification using a digital camera

(Zeiss Axiocam). Statistical analysis of data was by one-

way ANOVA with Tukey’s post hoc test, and significance

was determined at P < 0.05.

HFIP treatment

Lyophilized amylin aliquots were weighed into microcentri-

fuge tubes, and HFIP added to a final protein concentra-

tion of 250 lm. Lids were sealed (Parafilm; American Can

Co., Greenwich, CT, USA) and protein dissolution was

aided by gentle agitation, with recovery of contents by

pulse-spinning. Tubes were stored at room temperature

(approximately 22 �C) in the dark for 5–24 h. Contents

were mixed and again pulse-spun. HFIP was removed by

evaporation under N2, leaving a thin transparent film of

peptide on the internal surface of the tube, which was

then dissolved in sterile water by trituration followed by

pulse-spinning. HFIP-treated amylin was dissolved in water

to a final concentration of 500 lm. Vehicle controls were

constructed exactly as described above, but with the omis-

sion of protein.

Aliquots (5 lL) of freshly prepared aqueous amylin

(500 lm) were adsorbed onto glow-discharged carbon-coa-

ted collodion film on 400-mesh copper grids for 30 s. Next,

grids were blotted, washed three times in water droplets,

negatively stained for 20 s with 2% (w ⁄ v) uranyl acetate,

reblotted and left to air-dry inside a glass Petri dish [40].

Grids were examined within 1–2 days following their pre-

paration, using a Tecnai Transmission Electron Microscope

(FEI, Hillsboro, OR, USA) operated at 120 kV. Images

were recorded at a nominal magnification of 6 · 104 using

a Bioscan digital camera (Gatan, Pleasanton, CA, USA).

All experiments were performed in duplicate or triplicate,

and the images shown are representative of at least six pho-

tomicrographs.

ThT-binding assay

The aggregation potential of amylin was assessed using the

ThT-binding assay. Experiments were performed in tripli-

cate using black plastic microtiter plates (Nunc, Roskilde,

Denmark). Assays were initiated by adding 89 lL of

10 mm Tris ⁄HCl, pH 7.2, buffer and 1 lL of a 1 mm aque-

ous solution of ThT per well. Next, an aliquot of a fresh

aqueous solution of amylin or vehicle was added to each

well to a final volume of 100 lL, giving final concentrations

of amylin and ThT of 50 lm and 10 lm, respectively.

Fluorescence was measured at 510 nm with an excitation of

450 nm and a cutoff filter at 495 nm using a Spectra MAX

Gemini XS fluorescence spectrophotometer (Molecular

Devices, Sunnyvale, CA, USA). Fluorescence measurements

were performed immediately after addition of amylin, and

subsequent measurements were made at intervals over the

next 2–6 h. Data analysis was performed by nonlinear

regression using graphpad prism v.4.00 for Windows

(GraphPad Software Inc., San Diego, CA, USA).

CD spectroscopy

Samples were prepared by diluting freshly prepared aque-

ous amylin (500 lm) into 10 mm Tris ⁄HCl, pH 7.2, buffer,

yielding a final protein concentration of 25 lm. Blanks wereprepared by adding water (vehicle) instead of amylin to the

buffer. Spectra were recorded at room temperature (p*-180Spectrometer; Applied Photophysics, Leatherhead, UK),

using a 1 mm pathlength quartz cuvette with 250 lLsample aliquots, at 1.0-nm intervals between k ¼ 200 and

250 nm, with automatic subtraction of buffer-blank spectra.

For presentation of the results, the data were expressed

as molar ellipticity (h). Interpretation was performed

according to generally accepted guidelines for the prediction

of protein secondary structure from CD spectra [40].

Acknowledgements

We thank J. D. Green, K. M. Loomes, J. Z. Bai, A.

Turner, M. S. Cameron-Cooper and C. A. Tse for dis-

cussions and criticism.

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