Curcumin effect on human insulin amyloid aggregation and amyloid toxicity

Insulin has been widely used as model protein in the study of amyloid formation as, under specific experimental conditions, it is highly prone to form amyloid fibrils63,64,65. The rate of insulin amyloid fibril formation is affected by several factors, such as pH, temperature, protein concentration, ionic strength, and presence of denaturants66,67. Taking into account the importance of physiological pH and temperature, in this study the aggregation of insulin was performed incubating the protein at pH 7.0 under stirring with teflon ball at 37 °C.

To evaluate the effect of curcumin on insulin amyloid formation, we performed far-UV CD spectroscopy and Thioflavin T (ThT) fluorescence analysis at different times of incubation in aggregating conditions (Fig. 1). Curcumin concentrations were chosen in the 10−6 M range as this was suggested to be comparable with physiological concentration achieved by curcumin in the central nervous system by oral dosing68. Insulin amyloid formation occurs through an elongation process, the early prefibrillar aggregates (oligomers and protofibrils) are mainly characterized by an α-helical structure, while only amyloid fibrils show the typical cross-β-structure69,70. In order to monitor structural transitions, far-UV CD spectra of insulin were recorded in the absence and in the presence of curcumin at 12, 18 and 24 hours of incubation in aggregating conditions (Fig. 1A–C). As expected, the CD spectra recorded at the beginning of the aggregation process (time 0) showed no differences between the samples incubated in the absence and in the presence of curcumin (data not shown). After 12 hours of incubation, the spectrum of insulin in aggregating conditions almost resembled that of the native protein showing two minima at 222 nm and 208 nm, and a positive signal around 195 nm consistent with the presence of α-helical conformation likely associated to the early aggregating species. At 18 hours of incubation the spectrum displayed a decrease in the ellipticity at 208 nm suggesting that an α to β-transition was taking place. At 24 hours of incubation, the CD spectrum was characterized by a clear minimum at around 218 nm characteristic of extensive β-sheet structures. These data indicate that, after 24 hours of incubation in aggregating conditions, insulin undergoes a conformational transition from α-helix to β-sheet structure associated to the amyloid fibril formation. Differently, the α to β-transition was already observed at 18-hour incubation for samples incubated in the presence of curcumin indicating that it affects insulin aggregation kinetics accelerating the amyloid fibrils formation.

Figure 1 Effect of curcumin on insulin amyloid formation and cytotoxicity. The effect of curcumin on insulin amyloid formation was assayed by far-UV CD spectroscopy and ThT fluorescence at different times of incubation in aggregating conditions. In panels A–C are reported the CD spectra of insulin in the absence and in the presence of curcumin (10 and 100 µM) at 12 (A), 18 (B) and 24 hours (C) of incubation in comparison to the native protein. The ThT fluorescence emission was recorded at 482 nm upon excitation at 450 nm (D). Protein concentration were 0.3 mg/mL and 8 µM for CD and ThT measurements, respectively. (E) Cytotoxicity of insulin amyloid aggregates formed in the presence of curcumin. SH-SY5Ycells were exposed for 24 hours to insulin incubated for 0, 12, 18 and 24 hours in aggregating condition in the absence and in the presence of curcumin (10 and 100 µM) and cell viability was evaluated by MTT assay. Data are expressed as average percentage of MTT reduction ± SD relative to control cells from triplicate wells from 5 separate experiments (p < 0.01). CTR_Cur represents cell exposed to curcumin at the higher working concentration. Other experimental details are described in the Methods section. Full size image

The effect of curcumin on insulin amyloid fibril formation was also evaluated by ThT binding assay. ThT is an amyloid fibril-specific dye as, when bound to the cross-β amyloid structure, it shows a strong fluorescence intensity71. In Fig. 1D is reported the insulin aggregation kinetics in the presence and in the absence of curcumin monitored by ThT fluorescence. Samples incubated in the presence of curcumin showed a shorter lag phase and a higher ThT intensity at the end of the process at the tested concentrations thus indicating that curcumin accelerates insulin amyloid aggregation and favors fibril formation in aggregating conditions. Moreover, as additional control we monitored the protein concentration in the soluble fraction during the aggregation process by absorption spectroscopy and SDS-PAGE analysis (Supplementary Material Fig. S1). The results indicate that the amount of soluble protein decreases faster in the presence of curcumin. There are some concerns about the employment of ThT to detect fibril formation in the presence of curcumin, they are related to the superimposition of curcumin and ThT fluorescence emission spectra and to the ability of curcumin to compete with ThT for fibril binding72. This aspect should be considered when inhibitory effects are observed as they can yield false positive. However, in our case, ThT assay provides reliable information as we observe an increase in the intensity of fluorescence in the presence of curcumin. Moreover, no fluorescence emission was detected in samples incubated in the presence of curcumin before the addition of ThT.

Amyloid aggregates are known to affect cell toxicity; it is well established that the amyloid cytotoxicity is associated to the early soluble oligomeric aggregates whereas amyloid fibrils are essentially harmless73. To assess the cytotoxicity of insulin aggregates formed in the presence of curcumin, we evaluated the cell viability by the MTT assay in SH-SY5Y neuronal cells (Fig. 1E). This cellular model was chosen as amyloid toxicity is generally associated to neurodegeneration and, also, this cell line is widely used for the evaluation of cytotoxicity in amyloid aggregates. To this aim, cultured human neuroblastoma SH-SY5Ycells were exposed to insulin incubated for 12, 18 and 24 hours in aggregating condition in the absence and in the presence of curcumin. As control, the cytotoxicity of insulin at the beginning of the aggregation process (time 0) was tested and no toxicity was observed both in the absence and in the presence of curcumin. As expected, in the absence of curcumin, early amyloid aggregates (12 hours) reduced cell viability by approximately 65% compared to untreated cells while amyloid fibrils (24 hours) were almost harmless31. Interestingly, samples aggregated in the presence of curcumin for 12 hours showed a reduced toxicity likely associated to a greater amount of harmless amyloid fibrils. These data suggest that curcumin, accelerating the amyloid fibrils formation, could have a protective effect on insulin amyloid toxicity.

Differently from data obtained for other amyloid proteins, in which curcumin showed anti-amyloid activity34,35,36,37,38,39,40,41,42, our data indicate that curcumin accelerates amyloid aggregation in human insulin at physiological pH thus favoring the formation of harmless fibrils. The effect of curcumin on the amyloid aggregation process could depend on the conformational organization of the model protein. Moreover, considering the high instability of curcumin under neutral pH, it is likely that the effect that we have observed is not caused by curcumin itself but mainly due to its degradation products49,74. For this reason, we performed the same experiments in the presence of vanillin, one of the main degradation products of curcumin at neutral pH.

Vanillin effect on human insulin amyloid aggregation

To evaluate the effect of vanillin in insulin amyloid formation, we tested the ability of insulin to form amyloid aggregates in physiological conditions in the presence of vanillin. To this aim, human insulin was incubated in the presence and in the absence of different concentrations of vanillin (10, 50,100, and 500 µM) and samples were analyzed by far-UV CD spectroscopy at 12, 18 and 24 hours of incubation in aggregating conditions (Fig. 2A–C). The CD spectra recorded at the beginning of the aggregation process (time 0) indicated no variations between the samples incubated in the absence and in the presence of vanillin. After 12 hours of incubation, while the sample incubated in the absence of vanillin was still mainly in a α-helical conformation, samples incubated in the presence of vanillin displayed a decrease in the ellipticity at 208 nm suggesting that a α to β-transition was taking place. At 18 hours of incubation, while the spectrum of insulin alone indicated that an α to β-transition was taking place, the spectra recorded in the presence of vanillin at all concentrations tested showed a clear shift to around 218 nm suggesting that the α to β-transition was completed. At 24 hours, all spectra were overlapping thus indicating that an extensive β-sheet structure was formed also for insulin in the absence of vanillin.

Figure 2 Effect of vanillin on insulin amyloid formation. CD spectra of insulin in aggregating conditions in the absence and in the presence of vanillin (10–500 µM) at 12 (A), 18 (B) and 24 hours (C) of incubation in comparison to the native protein. Protein concentration was 0.3 mg/mL. Absorption spectra of 5 µM CR bound to 8 µM insulin samples aggregated in the presence and in the absence of vanillin for 18 (D) and 24 hours (E) in comparison to the free CR. CR_Van represent the spectra of CR in the presence of vanillin at the higher working concentration. Other experimental details are described in the Methods section. Full size image

When the effect of vanillin in insulin amyloid fibril formation was evaluated by ThT assay, the presence of vanillin was affecting the ThT emission, i.e. samples in the presence of vanillin were showing fluorescence at 482 nm (upon excitation at 350 nm) even before the addition of ThT. For this reason, the amyloid fibril formation was evaluated by Congo Red (CR) binding assay, a widely used method to detect amyloid fibrils. Indeed, while the free CR shows a maximum at 490 nm in the absorption spectrum, when it is bound to β-sheet-rich amyloid fibrils, a characteristic increase in absorbance accompanied by a red shift in the absorption maximum from 490 to 540 nm occurs75. Figure 2D–E shows the absorption spectra of CR bound to insulin samples aggregated in the absence and in the presence of vanillin for18 and 24 hours in comparison to that of free CR. The absorption spectra were first recorded at the beginning of the aggregation process (time 0) and no variations were observed both in the absence and in the presence of vanillin. As control, we recorded the CR spectrum in the presence of vanillin at the higher working concentration which was indistinguishable from that of CR alone. At 18 hours incubation time, the CR spectrum recorded for insulin sample in the absence of vanillin was almost superimposed to that of free CR, thus indicating that amyloid fibrils were not yet formed (Fig. 2D). Differently, the spectra recorded for insulin in the presence of vanillin showed a red shift at any concentration and a concentration-dependent increase in absorbance indicative of the presence of amyloid structures. At 24 hours, also the spectrum of insulin incubated in the absence of vanillin showed the typical red shift and absorption increase as expected. These results are in perfect agreement with the CD data and suggest that vanillin, as well as curcumin, strongly affects insulin aggregation kinetics accelerating the amyloid fibrils formation (Fig. 2E).

In order to confirm these data, samples of insulin incubated for 18 and 24 hours in the absence and in the presence of 100 µM vanillin, were analyzed by Transmission Electron Microscopy (TEM) (Fig. 3). At 18 hours of incubation, TEM images of insulin in the absence of vanillin showed predominantly small globular species, while in the presence of vanillin fibril formation was already observed. At 24 hours, also insulin sample incubated in the absence of vanillin showed the presence of amyloid fibrils. Moreover, TEM analysis revealed a similar morphology for fibrils formed in the absence and in the presence of vanillin with a greater amount of fibrils in the sample incubated with vanillin.

Figure 3 Effect of vanillin on insulin amyloid aggregation by TEM. TEM imaging of insulin samples incubated in aggregating conditions for 18 (A, B) and 24 hours (C, D) in the absence and in the presence of 100 µM vanillin. Panel E represents TEM imaging of vanillin. Scale bar represents 0.2 µm. Other experimental details are described in the Methods section. Full size image

Characterization of insulin-vanillin interaction

Vanillin-insulin interaction was investigated by fluorescence spectroscopy in native conditions (pH 7.0, 37 °C, without stirring). Insulin contains four tyrosyl residues as fluorescence emitters and its spectrum is characterized by the typical tyrosyl emission centered at 305 nm. The emission fluorescence spectra of insulin in the absence and in the presence of vanillin at different molar ratio are shown in Fig. 4A. The fluorescence intensity regularly decreased upon addition of increasing concentration of vanillin thus indicating that the insulin-vanillin interaction induces quenching of tyrosine emission. Interestingly, vanillin was able to strongly reduce the fluorescence intensity even at a very low molar ratio (1:0.25). The fluorescence quenching of a protein caused by small molecules may be collisional or due to the formation of a complex which has zero or small quantum yield. In order to exclude a collisional quenching by vanillin, we performed the same experiment on the monomeric tyrosyl residue (N-acetyl-L-tyrosine ethyl ester) (Fig. 4B).The F 0 /F values recorded for free tyrosine were significantly lower than those recorded for insulin at each molar ratio indicating the formation of an insulin-vanillin complex, as no collisional quenching is involved (Fig. 4C). In addition, the F 0 /F values recorded let us hypothesize a high binding affinity for the vanillin-insulin interaction in the low micromolar range. The emission spectra recorded in the presence of vanillin showed a blue-shift in the emission maximum around 301 nm indicative of a reduced polarity in the tyrosyl microenvironment. Moreover, the decrease of the fluorescence intensity at 305 nm was associated with the appearance of a new emission peak centred at 420 nm, indicative of an energy transfer between tyrosyl residues (donor) and vanillin (acceptor). Indeed, the emission spectrum of free vanillin upon excitation at 305 nm showed the appearance of an emission peak centred at 420 nm (data not shown). The formation of an insulin-vanillin complex is further supported by this evidence (Fig. 4D). A similar effect was reported for amyloid-β 1–42 upon interaction with vanillin76.

Figure 4 Insulin-vanillin interaction monitored by fluorescence. Tyrosine fluorescence emission was evaluated on both insulin (A) and free tyrosine (B) after addition of vanillin at different insulin:vanillin and tyrosine:vanillin molar ratio (1:0.25, 1:0.5, 1:0.75, 1:1). The dependence of F 0 /F on vanillin:insulin and vanillin:tyrosine molar ratio is shown in panel C. The fluorescence spectrum of insulin:vanillin (1:1) is shown in panel D. Working concentrations were 10 µM for insulin and 40 µM for free tyrosine. Other experimental details are described in the Methods section. Full size image

Analogous experiments were performed to check if also curcumin binds insulin in native conditions. The F 0 /F values recorded for insulin in the presence of increasing concentration of curcumin were comparable to those recorded for free tyrosine (Supplementary Material Fig. S2). These data indicate the occurrence of a collisional quenching thus suggesting that curcumin does not interact with insulin in native conditions.

To check if the insulin-vanillin interaction occurs through a covalent binding, we performed a mass spectrometry analysis on insulin incubated with vanillin in native conditions. No differences between insulin samples recorded in the absence and in the presence of vanillin were detected thus indicating that vanillin binds insulin through a non-covalent interaction (Supplementary Material Fig. S3). In conclusion, our data suggest that the insulin-vanillin interaction results in the formation of a non-covalent complex in which the hydrophobicity of the molecular regions surrounding tyrosyl residues is increased. This effect could involve other molecular regions affecting the overall hydrophobicity.

Vanillin effect on insulin amyloid cytotoxicity

Amyloid cytotoxicity is known to be associated to the early soluble oligomeric aggregates. To assess the cytotoxicity of insulin aggregates formed in the presence of vanillin, we performed both the MTT assay and cell cycle analysis. To this aim, SH-SY5Ycells were exposed for 24 hours to insulin incubated for 6, 12 and 24 hours in aggregating conditions in the presence of vanillin at different concentrations (0–10–50–100–500 µM) (Fig. 5A). As control, cell viability was assessed in the presence of insulin at the beginning of the aggregation process (time 0) and no toxicity was observed both in the absence and in the presence of vanillin at all tested concentrations. At 6 hours of incubation, while the sample incubated in the absence of vanillin did not affect cell viability, the ones in the presence of vanillin were able to induce cytotoxicity. In particular, cells exposed to insulin incubated with the higher vanillin concentration (500 µM) showed a 50% reduction of the cell viability compared to untreated cells. These data suggest that, at this time point, while insulin is mainly in a soluble not toxic conformation, samples incubated with vanillin are already in a highly toxic oligomeric state. At 12 hours of incubation also the insulin sample incubated in the absence of vanillin, induced a strong reduction of the cell viability (45%). At the same time, samples incubated with vanillin were still able to induce cell toxicity although with a minor extent respect to the 6-hour incubation. At 24 hours, all samples were not affecting cell viability indicating that the aggregates were mainly in the harmless fibril conformation. Similar indications were provided by the cell cycle analysis in which the cell toxicity is directly related to the pre-G1 content (Supplementary Material Fig. S4).

Figure 5 Cytotoxicity of insulin amyloid aggregates formed in the presence of vanillin. (A) SH-SY5Ycells were exposed for 24 hours to insulin incubated for 0, 6, 12 and 24 hours in aggregating conditions in the presence of vanillin at different concentrations (0–500 µM) and cell viability was evaluated by the MTT assay. Data are expressed as average percentage of MTT reduction ± SD relative to control cells from triplicate wells from 5 separate experiments (p < 0.01). CTR_Van represents cells exposed to vanillin at the higher working concentration. (B) Cell proliferation assay in cancer pancreatic cell line. Panc-1 cells were exposed for 24 hours to insulin incubated for 0, 6 and 12 hours in aggregating conditions in the presence of vanillin at different concentrations (0–500 µM) and cell proliferation was evaluated by MTT assay measuring the absorbance at 570 nm. Data are expressed as average ± SD from five independent experiments carried out in triplicate (p < 0.05). CTR: untreated cells; CTR_Van represents cells exposed to vanillin at the higher working concentration; Native: cells exposed to native insulin. Other experimental details are described in the Methods section. Full size image

Insulin, as well as glucose, is known to promote cell proliferation in pancreatic cancer cells contributing to chemoresistance77. This ability was used to monitor the presence of soluble insulin for the samples aggregated in the presence and in the absence of vanillin. To this aim, cell proliferation was assessed by MTT assay on Panc-1 cells, a human pancreatic cancer line. Cells were exposed for 24 hours to insulin incubated for 0, 6 and 12 hours in aggregating conditions in the presence of vanillin at different concentrations (0–10–50–100–500 µM) (Fig. 5B). At 6 hours of incubation, as expected, native insulin was able to induce cell proliferation as indicated by the increase of the absorbance at 570 nm. The same effect was observed for insulin incubated in the absence of vanillin under aggregating conditions suggesting that the protein is still in a native-like soluble state. Differently, samples incubated in the presence of vanillin were not able to promote cell proliferation. They rather induced a further reduction of cell growth respect to untreated cells, thus suggesting that insulin is in a toxic oligomeric state. At 12 hours, cells exposed to insulin sample incubated in the absence of vanillin, showed a strong reduction of the absorbance at 570 nm indicating both loss of the proliferative activity and gain of a toxic effect likely associated to the oligomeric state. Samples incubated in the presence of vanillin were still able to reduce cell proliferation although with a minor extent respect to the previous time point. These results are consistent with the cell viability data in SHSY5Y cells and indicate that vanillin accelerates the aggregation process in insulin promoting the formation of harmless amyloid fibrils.

Vanillin effect on insulin glycation and AGE-induced toxicity

Insulin is susceptible to glycation by glucose, D-ribose and other highly reactive carbonyls, such as methylglyoxal, especially in diabetic conditions and the AGE products are considered the main cause of diabetes-related vascular complications29,31,78,79. Methylglyoxal (MG) is the most significant glycation agent in vivo and its plasma levels are found increased in diabetic patients80. Increasing evidence suggest that curcumin possesses a protective effect against MG-induced endothelial dysfunction attenuating oxidative stress and inflammatory response81,82. In order to assess if also vanillin is able to interfere with AGEs formation and related toxicity, we monitored both the kinetics of AGEs formation and their ability to affect cell viability in the presence and in the absence of vanillin (Fig. 6).

Figure 6 Effect of vanillin on insulin glycation kinetics and AGEs toxicity. (A) Insulin samples were incubated at 37 °C with 0.5 mM methylglyoxal at different concentrations of vanillin (0–500 μM) and AGE fluorescence (λ ex 320 nm/λ em 410 nm) was monitored at different time points. CTR: Insulin incubated in the absence of methylglyoxal. (B,C) SHSY5Y cells were exposed in the absence and in the presence of vanillin to glycated insulin and it was evaluated the cell viability after 48 h by the MTT assay (B) and ROS production after 72 h by the DCFH-DA assay (C). CTR_Van: cells treated with vanillin at the higher working concentration; CTR+: cells treated with 1.0 mM H 2 O 2 , Ins: cells treated with non-glycated insulin, AGE: cells treated with glycated insulin; AGE_Van: cells co-incubated with glycated insulin and vanillin 150 µM. For MTT experiments and DCFH-DA assay data are expressed as average percentage of MTT reduction ± SD relative to control cells from triplicate wells from 5 separate experiments (p < 0.01). Other experimental details are described in the Methods section. Full size image

Insulin glycation kinetics was monitored by fluorescence spectroscopy as AGEs are characterized by a typical fluorescence emission at 410 nm upon excitation at 320 nm. To this aim, insulin samples were incubated at 37 °C with 0.5 mM MG at different concentrations of vanillin (0, 10, 50, 100, 500 μM) and AGE fluorescence was monitored in time (Fig. 6A). In the absence of vanillin, the emission intensity at 410 nm increased markedly with incubation time and glycation reaction was completed in about 4 days. Differently, in the presence of vanillin, a drastic reduction of AGEs formation was detected at all incubation times as indicated by the decrease of fluorescence intensity. These data indicate that vanillin strongly restrains protein glycation by MG in a concentration-dependent manner. The same experiment was also performed using 0.5 M D-ribose as glycating agent but no significant effect of vanillin on protein glycation was detected (data not shown).

We have recently reported that insulin glycation by D-ribose produces AGEs adducts that strongly affect the cell viability31. In order to test the ability of vanillin to reduce the AGEs toxicity, we evaluated the cell viability in cells exposed to fully glycated species. In particular, we performed MTT assay on SHSY5Y cells co-incubated with ribosylated insulin and vanillin for 48 hours (Fig. 6B). As expected, glycated insulin induced a strong reduction of the cell viability (65%) while in the presence of vanillin only a 20% reduction was observed. Moreover, as AGE toxicity is generally associated to oxidative stress, the ability of vanillin to reduce ROS production was also tested31. To this aim, the intracellular ROS level in SHSY5Y cells co-incubated with ribosylated insulin and vanillin for 72 hours was measured by DCFH-DA fluorescence assay (Fig. 6C). Interestingly, while ribosylated insulin promotes ROS production as indicated by the increase in the DCF fluorescence, in the sample co-incubated with vanillin a strong reduction of ROS levels was observed. These data suggest that vanillin exerts a protective effect in AGE-induced cytotoxicity likely affecting death pathways mediated by intracellular ROS production.