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: 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. 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). References 1 Cooper GJ, Willis AC, Clark A, Turner RC, Sim RB & Reid KB (1987) Purification and characterization of a peptide from amyloid-rich pancreases of type 2 diabetic patients. 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Insulin inhibits and promotes amylin aggregation FEBS Journal 276 (2009) 3365–3371 ª 2009 The Authors Journal compilation ª 2009 FEBS 3371 . but not a thermodynamic inhibitor of amylin aggregation, and insulin can eventually promote fibril formation. Insulin and amylin are cosecreted from granules. 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,