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 th
Trang 1of 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: liym@mail.tsinghua.edu.cn
(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.
Trang 2formation 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
Time (h)
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 lM in each group The concentration of
insulin in the control group was 100 lM The light scattering
intensity values are means ± SD, three replicate groups.
Amylin : Insulin = 1 : 1 Amylin : Insulin = 1 : 10 Insulin
40
30
20
10
0
Time (h)
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 lM in each group The ThT fluorescence intensity values are means ± SD, three replicate groups.
Trang 3intensity 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 amyamy-lin 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 lM in each group (A) Insulin
inhibits amylin aggregation after incubation
for 6 h (B) Insulin promotes amylin
aggrega-tion after incubaaggrega-tion 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 lM in each group The aggregation
of amylin was monitored by ThT
fluores-cence The fluorescence values are
means ± SD, three replicate groups.
Trang 4control 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 amyamy-lin 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
E
Fig 5 Insulin shows different effects on amylin fibril formation in
different time periods The concentration of amylin was 10 lM in
each sample The concentration of insulin was 10 lM in (B) and (D),
and 100 lM 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) reprerepre-sents 50 nm.
Trang 5Previous 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
0.06
0.04
0.02
0
Time (min)
Amylin Amylin : Insulin = 10 : 1 Amylin : Insulin = 1 : 1 Amylin : Insulin = 1 : 10 Insulin
Insulin oligomers
Amylin
Insulin
0 h
After 24 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 lL of
supernatant of the sample was loaded into an HPLC system for
SEC analysis The concentration of amylin was 10 lM, and the
concentration of insulin was 100 lM.
1000
Before addition of insulin
6 h after addition of insulin
24 h after addition of insulin
48 h after addition of insulin
800
600
400
200
0
Incubation time before addition of inuslin (h)
Fig 7 Insulin facilitates amylin aggregation by interacting with
amylin monomers or oligomers rather than preformed fibrils The
concentration of amylin was 10 lM 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.
Trang 6mately 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 amyinsu-lin 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 contribuinhibi-tor 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⁄ Pi (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 37C 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 solusolu-tion (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⁄ Pi 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⁄ Pi⁄ Tween-20 The the grid was
Trang 7then 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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