Báo cáo khoa học: Insulin is a kinetic but not a thermodynamic inhibitor of amylin aggregation pot

Insulin is a kinetic but not a thermodynamic inhibitor
of amylin aggregation
Wei Cui, Jing-wen Ma, Peng Lei, Wei-hui Wu, Ye-ping Yu, Yu Xiang, Ai-jun Tong, Yu-fen Zhao
and Yan-mei Li
Department of Chemistry, Key Laboratory of Bioorganic Phosphorus Chemistry and Chemical Biology (Ministry of Education),
Tsinghua University, Beijing, China
Amylin, or islet amyloid polypeptide, a 37 amino acid
peptide, is the major component of pancreatic islet
amyloid deposits in type 2 diabetes (T2D) [1–3]. Amy-
lin can readily form amyloid fibrils in vitro, but islet
amyloid deposits are rarely found in nondiabetic peo-
ple, even in obese individuals [4,5], whose amylin pro-
duction and secretion both surpass normal levels.
Although amylin has been shown to be cytotoxic in its
oligomeric forms as well as fibrillar forms, the mecha-
nism of amylin aggregation in vivo is still incompletely
understood [6]. The fact that no islet amyloid has been
observed in healthy individuals suggests the existence
of a natural mechanism for inhibition of amylin aggre-
gation [9,10]; understanding this mechanism could lead
to therapeutic benefits.
Insulin is cosecreted with amylin in secretory gran-
ules of pancreatic islet b-cells [6,7]. Previous studies
found that insulin significantly inhibited amylin aggre-
gation through binding with amylin in vitro [11,12,14],
so insulin was considered to be a major natural inhibi-
tor of amylin aggregation [9,13]. Nevertheless, other
results showed that insulin could promote amyloid
formation [10] and enhance the binding of amylin to
preformed fibrils[15], indicating that insulin might have
more than one effect on amylin aggregation.

In the present work, we investigated the effects of
insulin on amylin aggregation in vitro. We found that
insulin inhibited amylin fibril formation for only a lim-
ited time period, and amylin fibrillization was actually
promoted after long-term incubation. Both effects were
enhanced with a higher insulin ⁄ amylin ratio. We also
found that the promotional effect was caused by the
copolymerization of insulin and amylin. Furthermore,
we found that insulin significantly enhanced fibril
Keywords
aggregation; amylin; inhibition; insulin; type
2 diabetes
Correspondence
Y M. Li, Department of Chemistry, Key
Laboratory of Bioorganic Phosphorus
Chemistry and Chemical Biology (Ministry of
Education), Tsinghua University, Beijing
100084, China
Fax: +86 10 62781695
Tel: +86 10 62796197
E-mail:
(Received 8 January 2009, revised 3 April
2009, accepted 15 April 2009)
doi:10.1111/j.1742-4658.2009.07061.x
One of the most important pathological features of type 2 diabetes is the
formation of islet amyloid, of which the major component is amylin pep-
tide. However, the presence of a natural inhibitor such as insulin may keep
amylin stable and physiologically functional in healthy individuals. Some
previous studies demonstrated that insulin was a potent inhibitor of amylin
fibril formation in vitro, but others obtained contradictory results. Hence,

it is necessary to elucidate the effects of insulin on amylin aggregation.
Here we report that insulin is a kinetic inhibitor of amylin aggregation,
only keeping its inhibitory effect for a limited time period. Actually, insulin
promotes amylin aggregation after long-term incubation. Furthermore, we
found that this promotional effect could be attributed to the copolymeriza-
tion of insulin and amylin. We also found that insulin copolymerized with
amylin monomer or oligomer rather than preformed amylin fibrils. These
results suggest that the interaction between insulin and amylin may contri-
bute not only to the inhibition of amylin aggregation but also to the coag-
gregation of both peptides in type 2 diabetes.
Abbreviations
EM, electron microscopy; SD, standard deviation; SEC, size exclusion chromatography; T2D, type 2 diabetes; TEM, transmission electron
microscopy; ThT, thioflavin T.
FEBS Journal 276 (2009) 3365–3371 ª 2009 The Authors Journal compilation ª 2009 FEBS 3365
formation by interacting with amylin monomers or
oligomers rather than existing amylin fibrils. Our
results indicate that insulin plays different roles in
amylin aggregation, inhibiting amylin aggregation in
healthy individuals, but promoting aggregation during
T2D pathogenesis. In summary, amylin–insulin inter-
actions are most likely to play a complex and impor-
tant role in T2D.
Results
Insulin inhibits amylin aggregation for a limited
time period
We first performed a light scattering assay to deter-
mine the full kinetics of amylin aggregation in the
presence of insulin. Amylin was incubated with insulin
at different molar ratios. Figure 1 shows details of the
overall aggregation process. It is notable that amylin

alone showed relatively higher light scattering intensity
after incubation for 6 h, corresponding to significant
amylin fibril formation. In contrast, amylin incubated
with insulin showed significantly lower light scattering
intensity, indicating the inhibitory effect of insulin on
amylin aggregation in this time period. This inhibitory
effect is also shown in Fig. 3A.
We also employed a thioflavin T (ThT) assay to
analyze the effects of insulin on amylin aggregation.
ThT can bind to amyloid fibrils, and its fluorescence
indicates the degree of fibril formation. A detailed view
of the early stage of aggregation was obtained with the
ThT assay (Fig. 2), which shows similar kinetic
features as the light scattering assay. The data show
that insulin obviously inhibited amylin aggregation
after short-term incubation (Figs 2 and 4A). This
result is consistent with the data from the light scatter-
ing assay. Transmission electron microscopy (TEM)
images (Fig. 5A,B) taken after 6 h of incubation also
demonstrate the inhibitory ability of insulin, by show-
ing reduced fibril formation in the sample that con-
tained insulin. It is notable that this inhibitory effect
could be achieved even with low concentrations of
insulin. Taking amylin alone as a reference, after incu-
bation for 6 h (Fig. 3A), approximately half of
the fibril formation was inhibited when amylin was
incubated with insulin.
Insulin promotes amylin aggregation after
long-term incubation
Previous studies found a continuous inhibitory effect

of insulin on amylin aggregation [9,13]. However,
our results show that this inhibitory effect is time-
dependent.
Our data show that amylin fibril formation was
facilitated when amylin was coincubated with insulin
for 72 h. Results from light scattering assays (Figs 1
and 3B) and ThT assays (Fig. 4B), and TEM images
(Fig. 5C,D), are all in accord with each other and sup-
port this conclusion. For amylin incubated alone, the
light scattering intensity reached a plateau after incu-
bation for 6 h and stayed almost the same for the
following 66 h. In contrast, the inhibitory effects of
insulin hardly remained after incubation for 12 h,
whereas the promotional effects began to appear
(Figs 1 and 2). Significantly enhanced light scattering
500
600
Amylin
Amylin : Insulin = 10 : 1
Amylin : Insulin = 1 : 1
Amylin : Insulin = 1 : 10
Insulin
400
300
200
100
0
0 10203040
Time (h)
50 60 70

Intensity of light scattering
Fig. 1. Light scattering assay, showing the full kinetics of amylin
aggregation in the presence of insulin. The inhibitory effect of
insulin on amylin aggregation is time-dependent, and long-term
incubation with insulin promotes amylin aggregation. The concen-
tration of amylin was 10 l
M in each group. The concentration of
insulin in the control group was 100 l
M. The light scattering
intensity values are means ± SD, three replicate groups.
50
Amylin
Amylin : Insulin = 10 : 1
Amylin : Insulin = 1 : 1
Amylin : Insulin = 1 : 10
Insulin
40
30
20
10
0
02468
Time (h)
10 12
Intensity of ThT fluorescence
Fig. 2. ThT assay, showing a detailed view of the early stage of
aggregation and the time-dependent inhibitory effect. The concen-
tration of amylin was 10 l
M in each group. The ThT fluorescence
intensity values are means ± SD, three replicate groups.

Insulin inhibits and promotes amylin aggregation W. Cui et al.
3366 FEBS Journal 276 (2009) 3365–3371 ª 2009 The Authors Journal compilation ª 2009 FEBS
intensity was observed in the samples of amylin coin-
cubated with insulin after incubation for 24 h. The
promotional effect of aggregation was enhanced with
increasing concentrations of insulin (Figs 1 and 3B).
Taking amylin alone as a reference, after incubation
for 72 h (Fig. 3B), there was approximately twice as
much fibril formation when amylin was incubated with
a low molar ratio of insulin (10 : 1), and approxi-
mately five to 10 times as much when amylin was incu-
bated with higher molar ratios of insulin (1 : 1, 1 : 10).
TEM images taken after 72 h also support this obser-
vation, by showing that insulin can stimulate amylin
to form more fibrils (Fig. 5C,D).
Insulin enhances amylin fibrillar aggregation
by copolymerization with amylin
Previous studies found that insulin could prevent amy-
lin aggregation through binding to amylin and forming
amylin–insulin complexes [9,11,14]. Here, we show the
possibility that amylin–insulin complexes can self-accu-
mulate and lead to enhanced aggregation [10]. In order
to determine how insulin facilitates amyloid deposit
formation, size exclusion chromatography (SEC) anal-
ysis and immunogold labeling electron microscopy
(EM) were performed (Figs 5E and 6). The SEC data
show that, after long-term incubation, the contents of
both amylin and insulin in the supernatant were signif-
icantly reduced. As the loaded sample was superna-
tant, the reduced amounts of amylin and insulin

indicated that insulin was copolymerized with amylin.
The immunogold EM image (Fig. 5E) also showed the
presence of insulin in amyloid fibrils.
It has been reported that insulin can exist as mono-
mers, dimers and hexamers in solution [18,19]. As
shown in Fig. 6, it seems that the peak at 30 min
contained insulin hexamers, and the peak at 40 min
contained insulin monomers and dimers. In another
Fig. 3. Both inhibitory and promotional
effects on amylin aggregation are observed
in a light scattering assay. These two
effects are enhanced with increasing con-
centrations of insulin. The concentration of
amylin was 10 l
M in each group. (A) Insulin
inhibits amylin aggregation after incubation
for 6 h. (B) Insulin promotes amylin aggrega-
tion after incubation for 72 h. The light scat-
tering intensity values are means ± SD,
three replicate groups.
Fig. 4. ThT assay, confirming both inhibitory
and promotional effects of insulin on amylin
aggregation. The concentration of amylin
was 10 l
M in each group. The aggregation
of amylin was monitored by ThT fluores-
cence. The fluorescence values are
means ± SD, three replicate groups.
W. Cui et al. Insulin inhibits and promotes amylin aggregation
FEBS Journal 276 (2009) 3365–3371 ª 2009 The Authors Journal compilation ª 2009 FEBS 3367

control experiment, using SEC analysis (data not
shown), the insulin itself (including monomers, dimers,
and hexamers) would not aggregate even after 72 h of
incubation. This result suggests that the enhanced
aggregation could be due to the interaction between
amylin and insulin rather than the self-assembly of
insulin. Considering the observation that insulin can
copolymerize with amylin after long-term incubation,
insulin may act as an important factor in amyloid
formation.
Insulin copolymerized with amylin monomers
or oligomers rather than amylin fibrils
As it was shown that insulin coaggregated with amylin
after long-term incubation, it was important to deter-
mine the details of how insulin facilitates amylin aggre-
gation. We performed a ThT assay in which insulin
was added at various time points of the incubation
process. The ratio of insulin to amylin was 10 : 1 in
each group. The results of this assay (Fig. 7) showed
that the time of insulin addition was critical for the
promotional effect of insulin on amylin aggregation.
Figure 7 shows that insulin could significantly enhance
aggregation if it was added at 3 h. However, if insulin
was added at 24 h or even later, the promotional effect
was shown to be greatly reduced. This result indicates
that insulin had little promotional effect on preformed
amylin fibrils, and that the promotional effect could
only be achieved by interaction between insulin and
amylin monomers or oligomers, which were the major
species in the early stage of aggregation.

Discussion
Insulin and amylin are two crucial peptides in pancre-
atic islets. The interaction between amylin and insulin
may contribute to the pathogenesis of T2D [2,3,7].
Several studies have investigated the effects of insulin
on amylin aggregation, and suggested that insulin
could prevent amylin aggregation through binding
with amylin [11,12,14]. However, other studies found
that insulin could promote amylin aggregation under
certain conditions [10], and enhance binding of amylin
to preformed fibrils [15]. Thus, the details of this inhi-
bitory effect are still incompletely understood.
Our work shows the dual effects of insulin on amy-
lin aggregation. A significant delay of amylin amyloid
fibrillogenesis induced by insulin was observed, sug-
gesting that amylin aggregation was inhibited by insu-
lin at various concentrations. However, we found that
this inhibitory effect was time-dependent, and insulin
eventually promoted amylin fibril formation after incu-
bation for a longer time. Moreover, our results show
that insulin facilitated amylin fibril formation by
copolymerization with amylin. It was also notable that
the promotional effect of insulin on amylin aggre-
gation was shown to be caused by interaction with
amylin monomers or oligomers rather than preformed
fibrils.
A
B
C
D

E
Fig. 5. Insulin shows different effects on amylin fibril formation in
different time periods. The concentration of amylin was 10 l
M in
each sample. The concentration of insulin was 10 l
M in (B) and (D),
and 100 l
M in (E). The identity of each sample is shown. (A,B)
Samples were prepared after incubation for 6 h. Insulin shows a
significant inhibitory effect on fibril formation. (C,D) Samples were
prepared after incubation for 72 h. Insulin shows a promotional
effect on fibril formation, and the sample has more amylin fibrils.
(E) Amylin and insulin copolymerize and form fibrils. Aggregates
were identified by immunogold labeling with insulin antibody and
immunogold goat anti-(rabbit IgG). The scale bars in (A–D) repre-
sent 100 nm. The scale bar in (E) represents 50 nm.
Insulin inhibits and promotes amylin aggregation W. Cui et al.
3368 FEBS Journal 276 (2009) 3365–3371 ª 2009 The Authors Journal compilation ª 2009 FEBS
Previous studies suggested that insulin could inhibit
amylin aggregation through the formation of amylin–
insulin complexes [9,11,14]. However, our results sug-
gest that amylin–insulin complexes only contribute to
the inhibition of the early stage of amylin aggregation.
As the incubation proceeds, increasing amounts of
amylin–insulin complex can accumulate and serve as a
nucleus for fibrillization of the remaining peptides [10].
A relatively higher concentration of insulin showed
more significant inhibitory and promotional effects
on amylin aggregation. In the SEC analysis (Fig. 6),
the peak of insulin supernatant almost disappeared

after 48 h of incubation, suggesting that amylin–insulin
complexes might also lead to insulin participating in
amyloid formation. Moreover, the ThT assay (Fig. 7)
showed that insulin could not depolymerize fibrils
which only contained amylin, indicating that the fibril
structure had been altered. This altered structure can
also be seen in Fig. 5C,D, and a recent study [20]
reported a similar phenomenon in the interaction
between various caseins during their fibrillation. Alto-
gether, these results indicate that insulin is a kinetic but
not a thermodynamic inhibitor of amylin aggregation,
and insulin can eventually promote fibril formation.
Insulin and amylin are cosecreted from granules in
pancreatic islet cells [7,8], where a relatively higher
concentration of amylin exists without amyloid forma-
tion [4,5]. It is believed that insulin serves as an impor-
tant biological factor that inhibits amylin aggregation
[10,11]. Early studies claimed that insulin might act as
a natural inhibitor under normal circumstances [9], so
that insulin deficiency in T2D might be crucial for islet
amyloid formation. However, our study demonstrates
that insulin itself does not act simply as a natural
inhibitor of amylin aggregation but has opposite influ-
ences on amylin aggregation during different time peri-
ods. Thus, a new mechanism is needed to explain the
different behaviors of amylin in healthy individuals
and T2D patients.
On the basis of our observations, we suggest a hypo-
thetical mechanism for the amylin–insulin interaction.
Early studies found that amylin and insulin degrada-

tion were impaired in a rat model of T2D [17]. In
healthy individuals, the inhibitory effect of insulin on
amylin aggregation may be helpful for amylin degrada-
tion under normal circumstances, as amylin cannot be
degraded by enzymes such as insulin-degrading enzyme
after the formation of fibrils [16]. However, when amy-
lin cannot be degraded and cleared normally in T2D
patients, the inhibitory ability of insulin exists for only
a limited time period, and the promotional effect of
insulin on amylin aggregation may begin to appear.
This promotional effect will then lead to enhanced
amyloid formation and make amyloid degradation
more difficult. We showed that this promotional effect
was significantly enhanced with a relatively higher
ratio (1 : 10) of amylin to insulin (Fig. 3). Considering
that the molar ratio of amylin to insulin is approxi-
0.14
0.12
0.1
0.08
UV absorbation at 280 nm
0.06
0.04
0.02
0
20 25 30 35
Time (min)
40 45
Amylin
Amylin : Insulin = 10 : 1

Amylin : Insulin = 1 : 1
Amylin : Insulin = 1 : 10
Insulin
Insulin oligomers
Amylin
Insulin
0 h
After 24 h incubation
After 48 h incubation
Fig. 6. Insulin copolymerizes with amylin in the incubation process.
The sample was centrifuged at 10 600 g for 20 min, and 200 lLof
supernatant of the sample was loaded into an HPLC system for
SEC analysis. The concentration of amylin was 10 l
M, and the
concentration of insulin was 100 l
M.
1000
Before addition of insulin
6 h after addition of insulin
24 h after addition of insulin
48 h after addition of insulin
Intensity of ThT fluorescence
800
600
400
200
0
Incubation time before addition of inuslin (h)
0 3 24 72
Fig. 7. Insulin facilitates amylin aggregation by interacting with

amylin monomers or oligomers rather than preformed fibrils. The
concentration of amylin was 10 l
M in each group, and the ratio of
amylin to insulin was 1 : 10 in each group. Insulin was added after
incubation of amylin alone for different time periods. The fluores-
cence values are means ± SD, three replicate groups.
W. Cui et al. Insulin inhibits and promotes amylin aggregation
FEBS Journal 276 (2009) 3365–3371 ª 2009 The Authors Journal compilation ª 2009 FEBS 3369
mately 1 : 10 to 1 : 50 [10,15], it is possible that insulin
can promote amylin aggregation in vivo by similar
mechanisms as described above. As the intracellular
concentrations of both peptides are much higher than
those in extracellular spaces, the enhanced fibrillization
is more likely to occur intracellularly. It is noticeable
that insulin was not reported as the main component
of islet amyloid [15]. Thus, extracellular amyloid,
which is the major part of islet amyloid, may possibly
be formed by more complicated mechanisms. However,
insulin may still act as a contributor to amyloid forma-
tion in pancreatic islets and lead to a repetitive vicious
circle in the pathogenesis of T2D.
In conclusion, we have characterized the influences
of insulin on amylin aggregation. We found that insu-
lin could inhibit amylin aggregation for only a limited
time period, and that insulin promoted amylin fibril
formation after long-term incubation. These results
indicate that insulin may be not only a natural inhibi-
tor of amylin aggregation, but also a contributor to
the amyloid formation and pathogenesis of T2D. We
also found that the promotional effects were caused by

coaggregation of insulin and amylin after long-term
incubation. Furthermore, our results show that insulin
facilitates the aggregation by interaction of insulin with
amylin monomers or oligomers rather than preformed
fibrils. Considering the deficient amylin degradation
found in T2D, insulin may therefore act as an amyloid
inhibitor in healthy individuals and a promotional
agent of amyloid formation in T2D patients. Thus,
therapeutic strategies targeting the interaction between
insulin and amylin may need to be considered in
future.
Experimental procedures
Sample preparation
Synthesized human amylin(1–37) [KCNTATCATQRLAN-
FLVHSSNNFGAILSSTNVGSNTY(1–37), disulfide bridge:
C2 and C7] was obtained from American Peptide (Sunny-
vale, CA, USA). Recombined bovine insulin was obtained
from Sigma (St Louis, MO, USA). Amylin stock solution
was prepared by adding 1.0 mL of dimethylsulfoxide to
1.0 mg of dry purified peptide; the stock solution was then
sonicated at room temperature for 15 min, and shaken
overnight. Insulin stock solution was prepared by adding
2.18 mL of dimethylsulfoxide to 25 mg of dry, purified
peptide so that the final concentration was 2 mm; the
stock solution was then sonicated at room temperature for
15 min, and shaken overnight. All peptide stock solutions
were stored in 0.6 mL polypropylene Eppendorf tubes at
)20 °C.
Peptide aggregation
Amylin aggregation was initiated by adding amylin stock

solution to NaCl ⁄ P
i
(pH 7.4) to a final concentration of
10 lm. Insulin at different concentrations (from 1 lm to
100 lm) was incubated with amylin to evaluate its effect on
amylin aggregation. Samples were incubated at 37 °C for
72 h with shaking, and were taken for ThT assays, light scat-
tering assays and HPLC analysis at selected time points.
ThT assay
To monitor peptide fibrillation, a ThT assay was performed
at selected time points by combining 20 lL of sample solu-
tion with 700 lL of ThT solution (10 lm, pH 7.4). ThT
was obtained from Sigma. Fluorescence measurements were
recorded on a Hitachi FP-4500 fluorescence spectrometer
(Hitachi High-Technologies Corp., Tokyo, Japan) at room
temperature using a 1 cm path length quartz cell. The ThT
signal was quantified by averaging the fluorescence emission
at 485 nm (slit width = 10 nm) over 30 s when the samples
were excited at 440 nm (slit width = 5 nm).
Light scattering assay
Light scattering was performed at selected time points to
monitor peptide aggregation during the incubation. The
intensity of light scattering was measured on a Hitachi
FP-4500 fluorescence spectrophotometer at room tempera-
ture, using a 1 cm path length quartz cell over 30 s. Both
the excitation and emission wavelengths were set to
405 nm, with a spectral bandwidth of 1 nm.
TEM and immunogold labeling
To observe the fibril growth at different time points, TEM
was employed. At selected time points, 8 lL of sample solu-

tion was placed on a 200 mesh copper grid coated with
formvar and carbon, and negatively stained with 1% (w ⁄ v)
fresh tungstophosphoric acid. The samples were then exam-
ined in a JEOL-1200EX electron microscope (JEOL, Tokyo,
Japan) at 100 kV.
To examine the content of amyloid fibrils, immunogold
labeling EM was used. The incubated sample solution was
centrifuged at 10 600 g for 20 min, and 10 lL of sample
solution containing precipitate was then placed on a 200
mesh nickel grid coated with formvar and carbon. Grids
were blocked in NaCl ⁄ P
i
with added egg albumin [0.2%
(v ⁄ v); Sigma] for 45 min, incubated with polyclonal anti-
body to bovine insulin (1 : 100 dilution; Beijing Biosyntheis
Biotech, Beijing, China) for 12 h at room temperature, and
then with immunogold goat anti-(rabbit IgG) (1 : 8 dilu-
tion; Beijing Biosyntheis Biotech.) for 1 h at room tempera-
ture, and washed in NaCl ⁄ P
i
⁄ Tween-20. The the grid was
Insulin inhibits and promotes amylin aggregation W. Cui et al.
3370 FEBS Journal 276 (2009) 3365–3371 ª 2009 The Authors Journal compilation ª 2009 FEBS
then negatively stained with 1% (w ⁄ v) fresh tungstophos-
phoric acid. The samples were examined in a JEOL-
1200EX electron microscope (JEOL) at 100 kV.
SEC assay
To examine the contents of sample solutions, an SEC (TSK-
G3000PWxl; Tosoh, Tokyo, Japan) assay was performed on
an HPLC system (Waters 600; Waters, Milford, MA, USA).

At selected time points, each sample was centrifuged at
10 600 g for 20 min, and 200 lL of supernatant of each
sample was loaded into the HPLC system. Dilution buffer
contained 30% acetonitrile and 0.006% trifluoroacetic acid.
Absorbance was measured at 280 nm, and the flow rate was
0.3 mL ⁄ min.
Statistical analysis
Data from three independent experimental groups are pre-
sented as mean values ± standard deviation (SD). Multiple
comparisons were performed with Student’s t-test. Differ-
ences with P < 0.05 were considered significant.
Acknowledgement
This work was supported by grants from the National
Natural Science Foundation of China (Nos. 20532020,
20672067, and 20825206).
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