Abstract

The human islet amyloid polypeptide (hIAPP) or amylin is the major constituent of amyloidogenic aggregates found in pancreatic islets of type 2 diabetic patients that have been associated with β-cell dysfunction and/or death associated with type 2 diabetes mellitus (T2DM). Therefore, developing and/or identifying inhibitors of hIAPP aggregation pathway and/or compound that can mediate disaggregation of preformed aggregates holds promise as a medical intervention for T2DM management. In the current study, the anti-amyloidogenic potential of Azadirachtin (AZD)—a secondary metabolite isolated from traditional medicinal plant Neem (Azadirachta indica)—was investigated by using a combination of biophysical and cellular assays. Our results indicate that AZD supplementation not only inhibits hIAPP aggregation but also disaggregates pre-existing hIAPP fibrils by forming amorphous aggregates that are non-toxic to pancreatic β-cells. Furthermore, AZD supplementation in pancreatic β-cells (INS-1E) resulted in inhibition of oxidative stress; along with restoration of the DNA damage, lipid peroxidation and the associated membrane damage, endoplasmic reticulum stress and mitochondrial membrane potential. AZD treatment also restored glucose-stimulated insulin secretion from pancreatic islets exposed to hIAPP. All-atom molecular dynamics simulation studies on full-length hIAPP pentamer with AZD suggested that AZD interacted with four possible binding sites in the amyloidogenic region of hIAPP. In summary, our results suggest AZD to be a promising candidate for combating T2DM and related amyloidogenic disorders.

Introduction

Aberrant misfolding of proteins and peptides into insoluble fibrils and aggregates has been associated with many disorders, including Alzheimer's disease, Parkinson's disease and type 2 diabetes mellitus (T2DM) [1]. The human islet amyloid polypeptide (hIAPP) or amylin is a 37 amino acid long hormone (Supplementary Figure S1A) that is co-secreted with insulin from secretory granules of pancreatic β-cells. Under hyperglycemic conditions associated with T2DM, hIAPP is prone to form oligomeric species and amyloidogenic aggregates, which have been reported to mediate dysfunction and apoptosis in insulin-secreting pancreatic β-cells [25] and have been linked to insulin insufficiency in T2DM subjects. Longitudinal studies in non-human primates and cats (animal models that spontaneously develop T2DM) have shown that the formation of IAPP aggregates precedes β-cell dysfunction [3]. Increasing amyloid deposition has also been linked with decreased β-cell mass and hyperglycemia in hIAPP transgenic mice and rat models [6] and human subjects [7]. Previous studies indicate that these amyloid aggregates mediate cytotoxicity in a variety of ways, such as generation of inflammation, endoplasmic reticulum stress, mitochondrial dysfunction, creating reactive oxygen species (ROS) and overloading the misfolded protein response pathway [8,9]. Therefore, developing and/or identifying inhibitors of the hIAPP aggregation pathway and/or compounds that can mediate disaggregation of preformed aggregates holds promise as a medical intervention for T2DM management.

Natural products and their derivatives have received widespread attention for their medicinal properties against complex disorders including T2DM [10]. Indeed, the American Diabetes Association has included several spices, herbs and plant parts as food supplements for subjects affected with T2DM [11]. Along these lines, extracts of common fruits, like blackberry, blueberry, grapes and pineapple [12], vegetables, herbs and spices including curcumin, mint, red bell pepper and thyme have been shown to possess anti-amyloidogenic activity [13]. Among the different categories of natural compounds, polyphenolic compounds including terpenoids and flavonoids constitute the most actively investigated group for their potential as anti-diabetic agents by targeting hIAPP fibrillization [1422]. For example, polyphenolic compounds extracted from tea or coffee, including (−)-epigallocatechin 3-gallate (EGCG), caffeic acid, chlorogenic acid and morin hydrate, have been shown to strongly inhibit the hIAPP fibrillization, disaggregate amyloid fibrils and mediate protection of pancreatic β-cells from apoptosis [15,23]. In fact, preclinical trials with EGCG for different amyloidogenic proteins have been underway, wherein beneficial effects of green tea extracts have been reported on the progression of cardiac transthyretin amyloidosis [24]. However, the mechanism of action for these compounds needs to be investigated in details.

The leaf extract obtained from the wonder tree, Neem (Azadirachta indica A. Juss.; Meliaceae), well known for its medicinal properties (Table 1), has been documented to induce hypoglycemia in normal rats and lowering blood sugar levels in streptozotocin-induced diabetic rats [25,26]. Tetranortriterpenoids—meliacinolin, azadirachtolide [26,27] and limonoids—Azadiradione and Gedunin from A. indica leaves have been reported to inhibit pancreatic α-amylase activity [28]. Other limonoids such as Azadirachtin (AZD) (Supplementary Figure S1B) has also been documented in the literature for its insecticidal properties [29], radical scavenging properties [30] and anti-inflammatory activity [31]. Even though the anti-diabetic potential of extracts from Neem has been suggested (Table 1), detailed investigations on the effect of AZD supplementation on hIAPP fibrillization and preformed hIAPP fibrils need to be performed in order to gain insights into the mechanism by which hIAPP misfolding and aggregation promote T2DM. Therefore, in this study, we aimed to investigate the anti-diabetic and anti-amyloidogenic potential of AZD by targeting hIAPP fibrillization. To achieve the same, a battery of biophysical and cellular assays was performed to demonstrate that AZD inhibits the formation of hIAPP fibrils and mediates disaggregation of preformed hIAPP fibrils. Cellular studies on pancreatic β-cell line, INS-1E, showed that AZD provided protection against hIAPP-mediated cytotoxicity by preventing generation of cellular and mitochondrial oxidative stress, lipid peroxidation and the associated membrane damage. AZD supplementation was also associated with the restoration of mitochondrial membrane potential (MMP) in INS-1E cells exposed to hIAPP. In addition to this, AZD supplementation was associated with restoration of glucose-stimulated insulin secretion (GSIS) in pancreatic islets exposed to hIAPP. Insights from all-atom molecular dynamics (MD) simulation studies identified four possible binding sites for AZD on the hIAPP pentamer. Taken together, our results suggest AZD to be a promising candidate for the management of T2DM and related amyloidogenic disorders.

Table 1
Summary of previous studies performed to assess the anti-diabetic potential of compounds isolated from Neem
Compound isolated from Neem plantModel/systemRoleResultsRef
Azadirachtolide Adult male Wistar Rats α-Amylase and α-glucosidase Exhibited anti-diabetic and hypolipidemic effects in streptozotocin-induced diabetic rats [26
Meliacinolin Mice α-Glucosidase and α-Amylase Inhibited insulin resistance, improvement of renal function, lipid abnormalities and oxidative stress [27
Gedunin, Azadiradione, Epoxyazadiradione Rat pancreatic acinar-AR42J cell line Human pancreatic α-amylase (HPA) Exhibited porcine pancreatic α-amylase (PPA) inhibition [28
Nimbidiol In vitro α-Glucosidase Inhibited intestinal (mammalian) maltase-glucoamylase, sucrase-isomaltase, lactase, trehalase [77
Azadirachtin Pancreatic INS-1E cells hIAPP Exhibited anti-diabetic and anti-amyloidogenic effect in rat insulinoma cells Current study 
Compound isolated from Neem plantModel/systemRoleResultsRef
Azadirachtolide Adult male Wistar Rats α-Amylase and α-glucosidase Exhibited anti-diabetic and hypolipidemic effects in streptozotocin-induced diabetic rats [26
Meliacinolin Mice α-Glucosidase and α-Amylase Inhibited insulin resistance, improvement of renal function, lipid abnormalities and oxidative stress [27
Gedunin, Azadiradione, Epoxyazadiradione Rat pancreatic acinar-AR42J cell line Human pancreatic α-amylase (HPA) Exhibited porcine pancreatic α-amylase (PPA) inhibition [28
Nimbidiol In vitro α-Glucosidase Inhibited intestinal (mammalian) maltase-glucoamylase, sucrase-isomaltase, lactase, trehalase [77
Azadirachtin Pancreatic INS-1E cells hIAPP Exhibited anti-diabetic and anti-amyloidogenic effect in rat insulinoma cells Current study 

Methods and materials

Materials

Synthetic hIAPP peptide (with end residue modification) was obtained from Peptide 2.0, Inc., U.S.A. AZD was obtained from Sigma–Aldrich, U.S.A., and hexafluoroisopropanol (HFIP), Thioflavin-T (ThT), and lactate dehydrogenase (LDH) release assay kit were obtained from HiMedia. JC-1 was procured from Thermo Fisher Scientific. Roswell Park Memorial Institute (RPMI) 1640 media, Dulbecco's modified Eagle's medium, ROMI1640, fetal bovine serum (FBS), phosphate-buffered saline (PBS) and penicillin–streptomycin solution were purchased from Life Technologies (Carlsbad, CA). Dimethyl sulfoxide (DMSO) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased from Sigma–Aldrich (St. Louis, MO, U.S.A.). Rabbit polyclonal anti-phospho-eIF2α (Ser51) and anti-eIF2α antibodies were purchased from Abcam (Cambridge, U.K.). The mouse monoclonal antibody for β-actin was purchased from Pierce. Mouse insulin ELISA kit was purchased from Mercodia (Sweden). Double-distilled water was filtered and deionized through a Millipore water purification system (Merck Millipore, U.S.A.). All other chemicals were of the highest grade available.

The stock solution of synthetic peptide (1 mM) was prepared in HFIP, centrifuged to avoid any pre-aggregation, filtered through 0.2 µm membrane and was diluted using 20 mM sodium phosphate saline buffer (pH 7.4) prior to use. Freshly prepared hIAPP was sonicated in a water bath-based sonicator prior to use for aggregation inhibition studies. For studies investigating disaggregation of preformed hIAPP fibrils, freshly prepared and sonicated hIAPP was incubated in a shaker incubator for 24 h at 37°C at 150 rpm to allow its proper mixing and aggregate formation. AZD stock (693.76 µM) solution was prepared by dissolving AZD in water–methanol (90 : 10) at room temperature filtered through a 0.2 µm membrane and stored in the refrigerator until further use.

ThT assay

The binding of the benzathiole dye — ThT — to the cross β-sheets of amyloid fibrils results in an increase in the fluorescence intensity [32]. The kinetics of fibril formation was monitored by the increase in fluorescence intensity of the peptide-bound ThT at room temperature for 12 h (Supplementary Material). Briefly, 15 µM peptide [diluted in buffer (20 mM NaH2PO4, 100 mM NaCl, 0.01% NaN3, pH 7.4)] was mixed with 3 µl of 1 mM ThT and incubated at room temperature for 15 min. For disaggregation experiments, 20 µM preformed hIAPP fibrils alone and in combination with different molar equivalents of AZD (hIAPP : AZD = 1 : 1 and 1 : 5) was mixed with 5 µl of 1 mM ThT and was further incubated for 15 min at room temperature. Post-incubation, fluorescence spectra were recorded on a spectrofluorophotometer (RF-5301PC, SHIMADZU, Japan) by exciting the samples at 450 nm and measuring the emission spectra in the range of 460–500 nm. The excitation and emission slit widths were kept at 5 nm.

Intrinsic tyrosine fluorescence measurements

The structural changes that accompany the fibrillization of proteins were monitored using intrinsic tyrosine fluorescence [33]. Briefly, the hIAPP peptide was diluted to 15 µM concentration in phosphate buffer (20 mM NaH2PO4, 100 mM NaCl, 0.01% NaN3, pH 7.4) and was incubated with different molar equivalents of AZD (mentioned above) for 12 h at 37°C. Post-incubation, the samples were placed in a 1 cm path length quartz micro-cuvette (Hellma, Forest Hills, NY, U.S.A.). For disaggregation experiments, 20 µM preformed hIAPP fibrils were incubated with different molar equivalents of AZD for 12 h at room temperature. Fluorescence spectra were recorded on a spectrofluorophotometer (RF-5301PC, SHIMADZU, Japan) by exciting the samples at 280 nm and measuring the emission spectra in the range of 290–400 nm. The excitation and emission slit widths were kept at 5 nm.

ANS fluorescence assay

ANS (8-anilinonaphthalene-1-sulfonic acid) fluorescence intensity measurements have been widely used for measuring changes in hydrophobicity in protein samples [34]. Briefly, peptide samples were diluted in phosphate buffer to a final concentration of 15 µM and were further incubated with different molar equivalents of AZD for increasing time durations (up to 12 h) at 37°C. The diluted samples were mixed with 60 µM ANS dye and were further incubated at RT for 10 min in dark. Similarly, for disaggregation experiments, 20 µM preformed hIAPP fibrils alone and in combination with different molar equivalents of AZD were incubated for 12 h at room temperature. Post-incubation, the samples were excited at 370 nm and emission spectra were recorded between 400 and 600 nm using a spectrofluorophotometer (RF-5301PC, SHIMADZU, Japan). The ANS fluorescence intensities at 490 nm were plotted against incubation time.

Transmission electron microscopy and atomic force microscopy

The morphology of hIAPP aggregates alone and/or in the presence of different molar concentrations of AZD was analyzed using transmission electron microscopy (TEM) and atomic force microscopy (AFM). For TEM imaging, samples prepared by diluting 60 µM of peptide in phosphate buffer (with and without AZD) were spotted on a carbon-coated grid (Electron Microscopy Sciences, Fort Washington, PA, U.S.A.), incubated for 3 min, washed thrice with distilled water and stained with 1% (w/v) aqueous uranyl acetate solution. The peptide-AZD solutions were incubated for 12 h prior to staining. Briefly, 1% uranyl acetate solution was freshly prepared and filtered through 0.22 µm sterile syringe filters (Millipore, U.S.A.). Electron microscopic analysis was performed using an FEI Tecnai G2 12 electron microscope at 120 kV with nominal magnifications of 43 000× and 60 000×. Images were recorded digitally using the SIS MegaView III imaging system (Olympus, U.S.A.). At least three independent experiments were carried out for each sample.

AFM imaging was performed using atomic force microscope (Asylum Research, CA, U.S.A.). Briefly, samples were prepared by diluting 40 µM peptide (with and without AZD) in phosphate buffer. The peptide-AZD solutions were incubated for 12 h prior to staining. The diluted samples were deposited on freshly cleaved mica sheets, air-dried, washed twice with double-distilled water and were kept for drying at room temperature for ∼40 min. For disaggregation, scanning was performed in tapping mode using Silicon nitride cantilever at a scan rate of 1 Hz. Random portions of the mica sheet were scanned to obtain desired images as well as to see the uniformity of the sample application. At least three independent experiments were carried out for each sample.

Circular dichroism spectroscopy

Far-UV circular dichroism (CD) measurements were performed on a JASCO J-810 spectropolarimeter (JASCO, Japan) using a 0.1 cm path length cuvette (Hellma, Forest Hills, NY, U.S.A.). Briefly, 30 µM peptide solutions and preformed hIAPP fibrils were incubated with different molar ratios of AZD and analyzed at 0, 6 and 12 h. All measurements were performed at 25°C. Raw data were smoothened and measurement from the buffer alone spectra was subtracted from that of the peptide samples. The CD spectra have been represented as a plot of molar ellipticity (θ) in deg cm2 dmol−1 versus the wavelength.

Fourier transform infrared spectroscopy

The secondary structural changes mediated by AZD supplementation in hIAPP fibrils were investigated using FTIR (Fourier transform infrared) spectroscopy. Briefly, 15 µM peptide alone and/or in the presence of different molar equivalents of AZD was spotted on KBr pellet and dried immediately under infrared lamp. The peptide-AZD solutions were incubated for 12 h prior to acquisition. Buffer (10 µl) alone spotted on another KBr pellet was used for determining the background spectra. Spectra were acquired in the region of 1600–1700 cm−1 corresponding to amide-I region [35] as an average spectrum of 32 scans using a Vertex-80 FTIR system (Bruker, Germany). To analyze the secondary structural components, the spectrum was further subjected to Fourier self-deconvolution of amide-I region (1600–1700 cm−1) followed by Lorentzian curve fitting using Opus 65 software (Bruker, Germany).

Cell culture

INS-1E cells were obtained as a kind gift from Prof. Claes Wollheim and Prof. Pierre Machler, University of Geneva Medical Center. Cells between passages 56 and 65 were grown in monolayer cultures in a humidified 5% CO2 atmosphere at 37°C in RPMI 1640 media (Life Technologies) supplemented with 10 mM HEPES, 1 mM pyruvate, 50 µM 2-mercaptoethanol, 10% (v/v) heat-inactivated FBS, 100 units/ml penicillin and 100 µg/ml streptomycin [36].

Measurement of cell growth and loss in viability

Freshly prepared hIAPP was sonicated in a water bath-based sonicator, mixed with AZD in the indicated molar ratios and was applied to INS-1E cells for aggregation inhibition studies. On the other hand, for the disaggregation studies, the preformed fibrils (described above) were incubated AZD in the indicated molar ratios and incubated at RT for 8 h before application to INS-1E cells. hIAPP-mediated cytotoxicity was investigated at 24 h using the MTT assay and the LDH release assay (Himedia). For MTT assay, INS-1E cells were seeded onto 96-well plates (Corning) at an initial density of 1 × 104 cells/well and were allowed to adhere at 37°C for 24 h. Post-incubation, the medium was replaced with fresh culture medium (100 µl) containing hIAPP or the preformed hIAPP fibrils alone and in combination with different ratios of AZD. Media were removed after 24 h of incubation and the plates were incubated with 100 µl of MTT solution (0.5 mg/ml in PBS) for 4 h at 37°C. Untreated wells containing only cells in culture media and those treated with PBS were evaluated as controls. Post-incubation, formazan crystals were dissolved with 100 µl SDS/0.1 N HCl for 4 h at RT. Absorbance was measured at 540 nm using a plate reader (Thermo Scientific). The reduction in cell viability was expressed as a percentage for each treatment relative to the untreated (media only) control (set as 100%). The experiments were performed five times with three replicate wells for each treatment.

The LDH release assay was performed using the LDH release assay kit (HiMedia) as per the manufacturer's instructions. Briefly, 25 µl of lysis buffer was added to untreated (control) wells and incubated at 37°C for 30 min to obtain maximum LDH release. Subsequently, 25 µl of supernatant was taken from wells corresponding to different experimental conditions, and the wells were subjected to lysis and 25 µl of LDH reagents was added to it. The plate was incubated at 37°C for 15 min in dark. Post-incubation, 25 µl of stop solution was added and absorbance was measured at 450 and 620 nm using a plate reader (Thermo Scientific). Untreated wells containing only cells in culture media and those treated with PBS were evaluated as controls. The % cytotoxicity was calculated as per the formula below: 
formula
All experiments were performed four times with three replicate wells for each treatment.

ROS measurement

INS-1E cells were plated on 96-well plates and ROS levels were assessed by incubating the cells with either DCF-DA (10 µmol) or MitoSox (5 µmol) for 30 min at 37°C [37]. Simultaneous detection of cell viability was also carried out using the MTT assay (as described above). The data were normalized to the number of viable cells.

Measurement of cell growth and loss in viability

For relative quantification of cell death and apoptosis, flow cytometry was performed using the Annexin V-FITC apoptosis detection kit (Invitrogen, U.S.A.). Briefly, INS-1E cells (1 × 105) were seeded in six-well plates (Corning) and was allowed to recover for 24 h. The cells were treated with hIAPP in the presence of different molar equivalents of AZD for 24 h. Post-incubation, the cells were harvested, washed with 1× PBS and further resuspended in 1× binding buffer. Cells were stained with Annexin V-FITC and propidium iodide (PI) using the Cell Apoptosis Kit (Invitrogen) as per the manufacturer's instructions. Unstained cells (without Annexin V-FITC and PI) were used as a control and single stained cells (Annexin V-FITC or PI positive) were used for fluorescence compensation. Samples were acquired on an Attune NxT flow cytometer (Thermo Fisher Scientific) and analyzed using Attune NxT Flow Cytometer Software (Thermo Fisher Scientific). Ten thousand cells were analyzed for each sample.

Cell cycle analysis was carried out by flow cytometry according to an already established protocol [38]. Post-treatment, cells were harvested, washed with ice-cold PBS and fixed with 70% ice-cold methanol for 12 h (Supplementary Material). The fixed cells were stained with PBS containing 50 µg/ml PI and 20 µg/ml RNase A for 30 min in dark and acquired on an Attune NxT flow cytometer (Thermo Fisher Scientific) and analyzed using Attune NxT Flow Cytometer Software (Thermo Fisher Scientific).

Lipid peroxidation

Lipid peroxidation was determined in INS-1E cells using the protocol as described by Chen et al. [39]. INS-1E cells were seeded onto six-well plates at a density of 1 × 105 and were treated with hIAPP alone and/or in the presence of AZD for 24 h. Post-incubation, the cells were lysed and boiled with 500 µl of TBA reagent (0.8% TBA in 20% TCA) at 95°C for 20 min. Tetramethoxy propane was used as a standard. The resulting fluorescence was measured at excitation 485 nm and emission 535 nm. The amount of protein was quantitated using BCA (bicinchoninic acid) reagent and the results were expressed as nmol TBARS/mg protein.

MMP measurement

To determine the changes in mitochondrial membrane permeability, MMP was assessed using JC-1 dye (Thermo Fisher Scientific) as per the manufacturer's instructions. JC-1-stained cells were acquired using an Attune NxT flow cytometer (Thermo Fisher Scientific) and analyzed using Attune NxT Flow Cytometer Software (Thermo Fisher Scientific). JC-1 forms J-aggregates that emit red fluorescence at 590 nm in healthy mitochondria while J-monomers emit green fluorescence at 490 nm in depolarized mitochondria. An increased ratio of J-monomers and a shift in the green fluorescence indicate mitochondrial damage [37].

Immunofluorescence

INS-1E cells were grown on coverslips in six-well plates, treated with 5 µM hIAPP, 25 µM AZD (5 molar equivalent) and the 1 : 5 combination mixture of hIAPP : AZD (5 : 25 µM) for another 24 h. Post-incubation, the cells were fixed and stained with Thioflavin-S and γH2AX [37]. The coverslips were mounted onto slides with Vectashield antifade mountant containing DAPI (Vector Laboratories). Imaging was performed on a fluorescent microscope (Leica DM3000 LED) and the images were processed using Application Suite X (Leica).

Immunoblotting

INS-1E cells were seeded in six-well plates at a density of 1 × 105 and were treated with the peptide alone and/or in the presence of AZD for 24 h. Post-incubation, the cells were harvested and washed with 1× PBS. Total protein was extracted using protein extraction buffer (20 mM Tris–HCl, pH 7.4, 1 mM EDTA, 1 mM PMSF, 0.1% Triton X-100) and 1× cOmplete Mini Protease inhibitor cocktail (Roche) and quantitated using BCA reagent (Thermo Fischer Scientific). An equal amount of proteins were analyzed by SDS–PAGE and electrotransferred to the polyvinylidene fluoride membrane (Bio-Rad) at 70 mA constant current. β-actin, p-eIF-2α and total eIF-2α protein levels were determined by immunodetection.

Pancreatic islets isolation and GSIS assay

Pancreatic islets were isolated from 8- to 10-week-old Swiss Albino mice maintained under a standard 12 h light/dark cycle as described recently in ref. [37]. Animals were housed in polypropylene cages at 25 ± 2°C and were given animal feed and water ad libitum. The research was conducted in accordance with the guidelines of the Committee for the purpose of Control and Supervision of Experiments on Animals (CPCSEA), and the study protocol was approved by the Institutional Animal Ethics Committee, SPPU, Pune (Registration no. 538/02/c/CPCSEA).

The isolated islets were treated with freshly sonicated hIAPP fibrils in the presence/absence of different molar equivalents of AZD for 24 h. Following this incubation, the functionality of islets was checked by performing the GSIS assay as described recently [37] using the Mouse insulin ELISA kit (Mercodia, Sweden). Insulin release under basal (5 mM) and stimulated GSIS (11 mM) glucose conditions was measured.

MD simulations

Co-ordinates of the hIAPP pentamer were a kind gift from the Tycko lab. The geometry of AZD structure was optimized using Gaussian 09 software package with B3LYP/6-31G** Basis set and using this structure CHARMM forcefield parameters were obtained from the CHARMMGUI server [40] (Supplementary Table S3). CHARMM36 [41] force field was used for hIAPP. Simulations were performed using GROMACS 4.6 [42] package. hIAPP was placed in a dodecahedron box with 1 nm distance between the protein and the box walls. Five AZD molecules were randomly added to the box. The box was solvated with TIP3P water and was neutralized using Na+Cl ions. The entire system was energy minimized using steepest descent algorithm until the largest force acting on the system was less than kJ mol−1 nm−1 followed by temperature (298 K) and pressure (1 atm) equilibration as per the protocol of Dantu et al. [43] using Berendsen thermostat and barostat [44]. Three independent production run simulations were performed using v-rescale thermostat [45] and Parrinello–Rahman barostat [46], with time constants of 1 ps and 2 ps, respectively. Electrostatics were treated using Particle Mesh Ewald algorithm [47], and atoms with bonds to hydrogen atoms were constrained with P-LINCS [48]. Each production run simulation was 300 ns long and data were collected at 40 ps intervals for analysis. All the simulations were performed using a time step of 2 fs. Contact frequencies were calculated using the g_contacts tool [49] and binding free energies were calculated using g_mmpbsa [50]. MD data were analyzed using PyMol and in-house python scripts.

Statistical analysis

All experiments were performed in triplicate and the results have been presented as mean ± SD, unless stated otherwise. Statistical analysis was performed by one-way ANOVA, Tukey HSD post-hoc test, and a value of P < 0.05 was considered to be a significant difference between groups.

Results

AZD inhibits hIAPP aggregation in vitro

The amyloidogenic fibrils/aggregates, rich in cross β-sheets, have been known to bind with ThT and show high ThT fluorescence intensities [51]. The effect of AZD supplementation on the kinetics of hIAPP aggregation/fibrillization was monitored using the ThT fluorescence assay over a period of 12 h. A significant increase in ThT fluorescence for hIAPP alone confirmed the formation of amyloidogenic aggregates with a lag time of 2 h (Figure 1A, cyan). On the other hand, a significant reduction in the formation of amyloidogenic fibril aggregates was observed by 4 h when 15 µM hIAPP was co-incubated with 7.5 µM AZD (1 : 0.5 ratio) (Figure 1A, green). No cross β-sheet aggregates were detected when hIAPP fibrillization was studied in the presence of 1 : 1 (Figure 1A, red) and 1 : 5 (Figure 1A, blue) molar equivalent of AZD to hIAPP as depicted by the remarkable decline in ThT fluorescence (P < 0.0001). Thus, concentration-dependent inhibition of hIAPP fibrillization was observed in the presence of AZD.

AZD supplementation alters the amyloidogenicity of hIAPP.

Figure 1.
AZD supplementation alters the amyloidogenicity of hIAPP.

(A) Kinetics of hIAPP aggregation monitored at different time intervals using the ThT fluorescence assay (N = 3); (B) ANS fluorescence intensity measurements at 490 nm of hIAPP with different molar equivalents of AZD (N = 3) to investigate the effect of AZD on hIAPP fibrillization. Disaggregation of preformed hIAPP fibrils investigated by performing (C) ThT fluorescence studies of hIAPP alone and in the presence of AZD in different concentrations (hIAPP : AZD = 1 : 1 and 1 : 5); and (D) ANS fluorescence measurements to assess the hydrophobic surface exposure of preformed hIAPP fibrils in the presence of different molar equivalents of AZD. The values of buffer intensities were subtracted in all cases. (E) Representative TEM and (F) AFM imaging of hIAPP alone; and in the presence of equimolar and 5 molar excess of AZD; (G) TEM and (H) AFM images of preformed hIAPP fibrils after incubation with AZD in different molar concentrations of AZD (hIAPP : AZD = 1 : 1 and 1 : 5). ***P < 0.001, ****P < 0.0001.

Figure 1.
AZD supplementation alters the amyloidogenicity of hIAPP.

(A) Kinetics of hIAPP aggregation monitored at different time intervals using the ThT fluorescence assay (N = 3); (B) ANS fluorescence intensity measurements at 490 nm of hIAPP with different molar equivalents of AZD (N = 3) to investigate the effect of AZD on hIAPP fibrillization. Disaggregation of preformed hIAPP fibrils investigated by performing (C) ThT fluorescence studies of hIAPP alone and in the presence of AZD in different concentrations (hIAPP : AZD = 1 : 1 and 1 : 5); and (D) ANS fluorescence measurements to assess the hydrophobic surface exposure of preformed hIAPP fibrils in the presence of different molar equivalents of AZD. The values of buffer intensities were subtracted in all cases. (E) Representative TEM and (F) AFM imaging of hIAPP alone; and in the presence of equimolar and 5 molar excess of AZD; (G) TEM and (H) AFM images of preformed hIAPP fibrils after incubation with AZD in different molar concentrations of AZD (hIAPP : AZD = 1 : 1 and 1 : 5). ***P < 0.001, ****P < 0.0001.

Further to eliminate the possibility of a competition between ThT and AZD for the same binding sites in hIAPP and/or due to quenching mediated by the interaction between ThT and AZD, intrinsic tyrosine fluorescence measurements and ANS-binding assays were performed. Intrinsic fluorescence from aromatic residues reveals information about the structure, local environment and binding interactions of proteins [52]. Previous studies have demonstrated that amyloid formation is closely associated with an increase in intrinsic fluorescence intensity at increasing time points [53,54]. It was observed that the intensity of tyrosine fluorescence was maximal for hIAPP peptide incubated at 37°C for 12 h (Supplementary Figure S2A). On the other hand, no or minimal fluorescence intensity was detected for buffer and AZD combination. However, the amyloidogenic potential of hIAPP (15 µM) was greatly compromised when hIAPP was co-incubated with AZD in concentrations as low as 7.5 µM (1 : 0.5 molar ratio), as can be seen by the reduction in fluorescence intensity (Supplementary Figure S2B). Similar to results obtained by ThT fluorescence assays, maximal inhibition was observed when hIAPP was incubated in 1 : 5 molar excess of AZD. These results hint towards a change in the secondary structure of hIAPP in the presence of the AZD.

To further probe the formation of hIAPP amyloidogenic aggregates, the change in the hydrophobic surface was monitored by using ANS—a dye which is regularly used to study the conformation changes in proteins [34]. The exposed hydrophobic surface should change as the free monomeric peptide proceeds towards forming amyloidogenic aggregates. Along these lines, hIAPP was allowed to form aggregates and the process was monitored over time. Indeed, a significant increase in ANS fluorescence in hIAPP was observed at 4 and 8 h, which remained constant at 12 h (Figure 1B, cyan). Similar to the results obtained from ThT measurements, a significant reduction in hIAPP aggregation and hence, hydrophobicity of the peptide was observed in the presence of different concentrations of AZD. Maximal inhibition in hIAPP aggregation was observed in the presence of 1 : 5 molar excess of AZD (Figure 1B, blue).

The morphology of hIAPP aggregates was visualized simultaneously using TEM (Figure 1E) and AFM microscopy (Figure 1F). hIAPP (30–40 µM) formed a long, unbranched fibrillar network after incubation at 37°C for 48 h (Figure 1E,F; Supplementary Figure S3A). On the other hand, loss in fibrillar morphology and formation of smaller amorphous aggregates of hIAPP were observed in samples when hIAPP was co-incubated with AZD in molar ratios of 1 : 1 and 1 : 5 (Figure 1E,F; Supplementary Figure S3B,C).

AZD disaggregates the preformed hIAPP fibrils in vitro

High fibril concentration and formation of larger aggregates have been majorly associated with cytotoxicity of pancreatic β-cells. Therefore, reducing the hIAPP-mediated cytotoxicity could be an alternative strategy for alleviation of T2DM by dissolving pre-existing hIAPP fibrils. Along this direction, we investigated the effect of AZD supplementation on preformed hIAPP fibrils in vitro. Briefly, 20 µM of hIAPP was allowed to form fibrils by agitation at 37°C. After 24 h, the preformed fibrils of hIAPP were co-incubated with an equimolar concentration of AZD (hIAPP : AZD in molar ratios of 1 : 1) and the changes in ThT fluorescence were monitored over 12 h (Supplementary Figure S4). Indeed, there was a time-dependent decline in the intensity of ThT fluorescence and the maximum reduction was observed when the samples were co-incubated for a period of 12 h (Figure 1C, red triangles; Supplementary Figure S4A). A higher reduction in ThT fluorescence was observed when preformed hIAPP fibrils were co-incubated with 5-fold molar excess of AZD (1 : 5), thereby suggesting that AZD disassembled pre-existing hIAPP fibrils (Figure 1C, blue; Supplementary Figure S4B). We also observed that AZD itself did not interact with ThT dye and was associated with minimal ThT fluorescence (Figure 1C, open circles). A parallel reduction in ANS fluorescence measurements was also observed when preformed hIAPP fibrils were co-incubated with AZD in molar ratios of 1 : 1 (Figure 1D, red) and 1 : 5 (Figure 1D, blue), thereby suggesting a decline in the exposed hydrophobic surface. Indeed, the morphology of AZD supplemented preformed hIAPP fibrils, as analyzed by TEM (Figure 1G) and AFM imaging (Figure 1H; Supplementary Figure S5), revealed the disaggregation of the dense network of hIAPP fibrils in the presence of AZD. Co-incubation of hIAPP fibrils with AZD resulted in the formation of amorphous hIAPP aggregates (Figure 1G) with a globular morphology (Figure 1H).

AZD induces secondary structure changes in hIAPP

The monomeric form of hIAPP has random coil conformation, while an oligomeric intermediate formed during the lag phase has been associated with a helical and parallel β-sheet structure which ultimately changes into a partially disordered loop in the fibril [55], while mature amyloid fibril consists mostly of cross β-sheet structure [55]. To monitor the changes in the secondary structure of hIAPP in the presence of AZD, far-UV CD spectroscopy and FTIR spectroscopy studies were performed. As can be observed from Figure 2A, a structural transition in hIAPP was observed wherein the peptide acquired a typical β-sheet conformation (Cyan) characterized by a major negative peak at 220 nm after 12 h of incubation. On the other hand, AZD supplementation in equimolar concentrations of hIAPP induced a structural transition, wherein the peptide acquired an α-helical conformation at 12 h (Figure 2B, cyan; Supplementary Figure S6A). The structural changes were also observed at 6 h post-incubation (Figure 2B, red). Similarly, significant structural changes were also observed when preformed hIAPP fibrils were co-incubated with AZD in 1 : 1 (Figure 2B, green; Supplementary Figure S6B) and 1 : 5 (Figure 2B, pink; Supplementary Figure S6B) molar equivalents for 12 h. The peptide majorly acquired a helical conformation in the presence of AZD in equimolar concentration while random coil conformation was predominant when the concentration of AZD was increased to 1 : 5 molar equivalents.

Structural characterization of hIAPP in the presence of AZD.

Figure 2.
Structural characterization of hIAPP in the presence of AZD.

Far-UV CD spectra of (A) hIAPP alone and (B) hIAPP and preformed hIAPP fibrils supplemented with an equimolar concentration of AZD monitored at 0, 6 and 12 h. FTIR spectrum of (C) hIAPP alone and (D) supplemented with an equimolar concentration of AZD monitored after 12 h.

Figure 2.
Structural characterization of hIAPP in the presence of AZD.

Far-UV CD spectra of (A) hIAPP alone and (B) hIAPP and preformed hIAPP fibrils supplemented with an equimolar concentration of AZD monitored at 0, 6 and 12 h. FTIR spectrum of (C) hIAPP alone and (D) supplemented with an equimolar concentration of AZD monitored after 12 h.

FT-IR spectroscopy studies also confirmed the structural transitions in hIAPP supplemented with AZD. In FT-IR spectrum, hIAPP showed a predominant band at 1632 cm−1, suggesting β-sheet-rich structure with few unstructured regions (1645 cm−1) and β-turns (1671 cm−1) (Figure 2C). On the other hand, AZD supplementation in equimolar concentration induced structural changes in hIAPP wherein the majority of β-sheet was changed to α-helical conformation (Figure 2D).

AZD mediates protection of pancreatic β-cells from hIAPP-mediated cytotoxicity

The cytotoxic response of hIAPP on INS-1E cells was investigated using the MTT and the LDH release assays. These assays are commonly used to report the loss of cellular integrity. As previously documented in the literature [37], co-incubation of INS-1E cells with hIAPP for 24 h significantly reduced their viability when compared with the untreated control cells (Figure 3A). However, supplementation with AZD in molar ratios as low as 1 : 1 molar equivalent was able to mediate significant protection from cell death (P < 0.05) (Figure 3A). Control experiments suggested that AZD itself does not affect the cell viability at tested concentrations (Supplementary Figure S7). Indeed, co-incubation of hIAPP and preformed fibrils with AZD effectively reduced the cytotoxicity associated with hIAPP exposure and mediated protection in INS-1E cells (Figure 3C).

AZD mediates protection against hIAPP-induced cell death.

Figure 3.
AZD mediates protection against hIAPP-induced cell death.

Results from the MTT assay and LDH release assays used to measure cell viability in INS-1E cells exposed to (A) hIAPP in the presence of different molar ratios of AZD (inhibition of hIAPP aggregation experiments) and (C) preformed hIAPP aggregates in the presence of different molar ratios of AZD (disaggregation of preformed hIAPP fibrils). Intracellular and mitochondrial ROS levels as measured by DCF-DA and Mitosox, respectively, in INS-1E cells exposed to (B) hIAPP in the presence of different molar ratios of AZD (inhibition of hIAPP aggregation experiments) and (D) preformed hIAPP aggregates in the presence of different molar ratios of AZD (disaggregation of preformed hIAPP fibrils). *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 3.
AZD mediates protection against hIAPP-induced cell death.

Results from the MTT assay and LDH release assays used to measure cell viability in INS-1E cells exposed to (A) hIAPP in the presence of different molar ratios of AZD (inhibition of hIAPP aggregation experiments) and (C) preformed hIAPP aggregates in the presence of different molar ratios of AZD (disaggregation of preformed hIAPP fibrils). Intracellular and mitochondrial ROS levels as measured by DCF-DA and Mitosox, respectively, in INS-1E cells exposed to (B) hIAPP in the presence of different molar ratios of AZD (inhibition of hIAPP aggregation experiments) and (D) preformed hIAPP aggregates in the presence of different molar ratios of AZD (disaggregation of preformed hIAPP fibrils). *P < 0.05, **P < 0.01, ***P < 0.001.

To understand the mechanism of action of AZD in mediating protection from hIAPP-induced cytotoxicity, cellular and mitochondrial ROS levels were measured. As reported earlier [37], the cytotoxicity mediated by hIAPP in INS-1E cells was also associated with a significant increase in cellular and mitochondrial ROS levels (Figure 3B,D). AZD supplementation inhibited the formation of these species and mediated protection from hIAPP-mediated cytotoxicity in INS-1E cells (Figure 3B). Likewise, co-incubation of preformed hIAPP fibrils with AZD effectively reduced the cytotoxicity associated with hIAPP exposure, as evidenced by MTT and LDH release assays (Figure 3C), and hence, mediated protection in hIAPP-treated INS-1E cells by alleviation of cellular and mitochondrial ROS levels (Figure 3D).

Mechanistic insights into the protection mediated by AZD in hIAPP-treated cells

In line with our previous results, it was observed that the percentage of cells undergoing apoptosis, as assessed by Annexin V/PI staining, was increased in INS-1E cells exposed to hIAPP [37]. Apoptosis and cell death were significantly reduced upon AZD supplementation in 1 : 2 molar equivalents (Figure 4A; Supplementary Figure S8). In addition to this, the cell cycle analysis of hIAPP-treated INS-1E cells showed arrest in the S and G2/M phases of the cell cycle with a significant increase in the G0 population (Figure 4B). AZD supplementation in hIAPP-treated cells rescued these cells from the arrest and there was a significant reduction in the G0 population as compared with the hIAPP-treated cells (Figure 4C). AZD alone showed no significant differences in the cell cycle profile when compared with the control, suggesting that AZD was not toxic to the cell. The increased ROS levels in INS-1E cells exposed to hIAPP were also associated with a significant increase in lipid peroxidation (measured in terms of the amount of TBARS/mg protein), while significantly lesser membrane damage was observed in hIAPP-treated cells which were supplemented with AZD (Figure 4D).

AZD mediates protection from hIAPP-induced cytotoxicity in INS-1E cells.

Figure 4.
AZD mediates protection from hIAPP-induced cytotoxicity in INS-1E cells.

(A) Quantitation of Annexin V/PI-stained cells (N = 3). (B) Representative cell cycle profiles of INS-1E cells exposed to different experimental conditions. (C) Quantitation of cells in the different phases of cell cycle profile. (D) Lipid peroxidation in terms of nmol of TBARs/mg protein (N = 4). (E) Representative histograms showing flow cytometric analysis of JC-1 staining in untreated (control) and hIAPP-treated cells in the presence and/or absence of AZD. (F) Quantitation of JC-1-stained cells (N = 3). (G) Reduced γH2AX staining in AZD supplemented hIAPP-treated cells thereby indicating decreased DNA damage (N = 3). (H) Thioflavin-S staining in INS-1E cells exposed to the different experimental conditions to visualize amyloid aggregates. (I) Western blot analysis of phospho-eIF2α, total eIF2α and β-actin. (J) The ratio of phospho-eIF2α to total eIF2α in the western blots as determined by scanning densitometry and image analysis (ImageJ, NCBI). The data represent the mean (±standard error) of four separate experiments. (K) Pancreatic islets (isolated from Swiss albino mice) treated with hIAPP alone and/or in the presence of different molar ratios of AZD showed significantly different insulin secretion in the GSIS assay (N = 4). *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 4.
AZD mediates protection from hIAPP-induced cytotoxicity in INS-1E cells.

(A) Quantitation of Annexin V/PI-stained cells (N = 3). (B) Representative cell cycle profiles of INS-1E cells exposed to different experimental conditions. (C) Quantitation of cells in the different phases of cell cycle profile. (D) Lipid peroxidation in terms of nmol of TBARs/mg protein (N = 4). (E) Representative histograms showing flow cytometric analysis of JC-1 staining in untreated (control) and hIAPP-treated cells in the presence and/or absence of AZD. (F) Quantitation of JC-1-stained cells (N = 3). (G) Reduced γH2AX staining in AZD supplemented hIAPP-treated cells thereby indicating decreased DNA damage (N = 3). (H) Thioflavin-S staining in INS-1E cells exposed to the different experimental conditions to visualize amyloid aggregates. (I) Western blot analysis of phospho-eIF2α, total eIF2α and β-actin. (J) The ratio of phospho-eIF2α to total eIF2α in the western blots as determined by scanning densitometry and image analysis (ImageJ, NCBI). The data represent the mean (±standard error) of four separate experiments. (K) Pancreatic islets (isolated from Swiss albino mice) treated with hIAPP alone and/or in the presence of different molar ratios of AZD showed significantly different insulin secretion in the GSIS assay (N = 4). *P < 0.05, **P < 0.01, ***P < 0.001.

Depolarization of MMP has been associated with apoptosis. JC-1, an MMP probe, has a tendency to accumulate in the healthy mitochondria and form aggregates which yields red fluorescence. While damaged mitochondria with lowered MMP fail to accumulate JC-1, wherein it exists as a monomer and emits green fluorescence. As can be seen from Figure 4E, hIAPP exposure in INS-1E cells was associated with an increase in green fluorescence thereby pointing towards depolarization of MMP in these cells. In agreement with our other results, AZD supplementation in 1 : 2 molar equivalents restored the MMP in INS-1E cells exposed to hIAPP (Figure 4E,F).

The induction of double-strand breaks in DNA has been widely correlated with increased apoptosis [37,56]. γH2AX marks DNA double-strand breaks, which can be visualized by immunofluorescence [38]. As reported earlier [37], a significant increase in DNA double-strand breaks was observed when INS-1E cells were exposed to hIAPP (Figure 4G). Indeed, a reduction in the number of DNA double-strand breaks was observed in hIAPP-treated INS-1E cells supplemented with 5 molar excess of AZD, thereby pointing towards a protective role of AZD. In addition to this, the amyloid-specific probe, Thioflavin-S-stained positive in INS-1E cells exposed to hIAPP (Figure 4H). This staining was diminished in AZD supplemented hIAPP-treated INS-1E cells. Also, control cells and cells treated with AZD alone did not show any significant staining with Thioflavin-S.

The phosphorylation of eIF-2α leads to inhibition of protein synthesis and has been linked with different stresses, including apoptosis. As can be observed from Figure 4I, a significant increase in phosphorylation of eIF-2α was observed in hIAPP-treated INS-1E cells. A significant decrease in eIF-2α phosphorylation, consistent with an increase in cellular viability, was observed when hIAPP-treated INS-1E cells were supplemented with equivalent (1 :1) and 2-fold (1 : 2) molar excess of AZD (Figure 4J). These results indicate that AZD mediates protection in hIAPP-treated INS-1E cells by restoring translation machinery.

Taken together, our results so far indicate that AZD mediates protection from hIAPP-mediated cytotoxicity by preventing cellular and mitochondrial ROS generation, lipid peroxidation and formation of DNA double-strand breaks, and restoration of MMP and translation machinery in INS-1E cells.

AZD restores GSIS in hIAPP-treated mouse pancreatic islets

It is postulated that amyloid aggregates formed by hIAPP induce apoptosis and dysfunction in pancreatic β-cells [57]. This is reflected by defects in insulin secretion by these cells [37]. In this regard, islet culture experiments were performed to investigate the effect of hIAPP on the ability of pancreatic islets to secrete insulin in response to glucose stimulation. Pancreatic islets were isolated from Swiss Albino mice and treated with hIAPP alone or in combination with 1 : 2 molar excess of AZD. In the control islets, the amount of insulin release increased from 65.523 ± 5.234 µIU/ml under basal glucose conditions to 111.140 ± 1.568 µIU/ml under glucose-stimulated conditions (Figure 4K). On the other hand, in hIAPP-treated islets, insulin released under stimulated conditions significantly reduced when compared with islets exposed to basal glucose concentrations. AZD supplementation in hIAPP-treated islets restored the insulin secretion under basal and stimulated conditions thereby reflecting that AZD not only helps in promoting survival in these cells but also helps to maintain their functionality. AZD alone did not affect insulin secretion under basal and stimulated conditions, and it was similar to that of the untreated or control islets. Overall, the biophysical and cellular studies hint towards the fact that AZD promotes the inhibition of hIAPP aggregation and mediates disaggregation of pre-existing hIAPP fibrils that provides protection against cytotoxicity.

Binding modes and possible binding sites of AZD on hIAPP

To further understand the interaction between AZD and hIAPP fibril and gain insights into the residue-level details of disruption mechanism, three all-atom MD simulations were performed of the hIAPP pentamer without AZD (i.e. apo-hIAPP) and with AZD in equimolar concentration (hIAPP:AZD, 1 : 1) each. During the 300 ns simulation, randomly placed AZD molecules were shown (Supplementary Figure S9) to interact with hIAPP within the first 50 ns and some AZD molecules stayed bound with hIAPP pentamer for the rest of the 300 ns. We observed that a single hIAPP pentamer could interact with five AZD molecules at the same time (Figure 5A) and multiple binding sites were populated by AZD (Figure 5B). For all further analysis, only the last 50 ns (250–300 ns) of each simulation were used.

Binding of AZD to hIAPP pentamer in MD simulations.

Figure 5.
Binding of AZD to hIAPP pentamer in MD simulations.

(A) The structure at of hIAPP 300 ns from each simulation of hIAPP with an equimolar ratio of AZD. (B) Binding sites identified on hIAPP pentamer based on contact frequencies over the last 50 ns of the simulation between AZD and hIAPP. (C) Percentage of contact between AZD molecules and residues from hIAPP pentamer calculated from the last 50 ns of each hIAPP : AZD simulation with a distance cut-off of 0.3 nm. (D,E) Structure at 300 ns from simulation1-hIAPP : AZD showing the interaction of two AZD molecules (green) with hIAPP residues (cyan) at two patches (‘FLVHS’ of all five chains and ‘NVGSN’ from chains A, B and C). (F) Distribution of fiber twist angle calculated from apo-hIAPP and hIAPP : AZD. For each system, data from only the last 50 ns of the simulation were used.

Figure 5.
Binding of AZD to hIAPP pentamer in MD simulations.

(A) The structure at of hIAPP 300 ns from each simulation of hIAPP with an equimolar ratio of AZD. (B) Binding sites identified on hIAPP pentamer based on contact frequencies over the last 50 ns of the simulation between AZD and hIAPP. (C) Percentage of contact between AZD molecules and residues from hIAPP pentamer calculated from the last 50 ns of each hIAPP : AZD simulation with a distance cut-off of 0.3 nm. (D,E) Structure at 300 ns from simulation1-hIAPP : AZD showing the interaction of two AZD molecules (green) with hIAPP residues (cyan) at two patches (‘FLVHS’ of all five chains and ‘NVGSN’ from chains A, B and C). (F) Distribution of fiber twist angle calculated from apo-hIAPP and hIAPP : AZD. For each system, data from only the last 50 ns of the simulation were used.

To identify binding sites, contact frequencies were calculated between AZD and hIAPP. A protein residue was considered to be in contact with AZD if it was within 0.3 nm of AZD for >50% of the last 50 ns of the simulation (Figure 5D). Predominantly, ‘KCNTA’ at the N-terminus, ‘FLVHS’ and ‘FGAIL’ from the amyloidogenic core and ‘NVGSNT’ at the C-terminus were identified as the preferred binding sites in all the three simulations (Figure 5C; Supplementary Figure S10). Furthermore, a single AZD molecule was shown to interact with patches formed by residues from multiple hIAPP chains rather than a single chain (Figure 5D,E). In fact, the binding of AZD resulted in a shift in the fiber twist angle distribution in comparison with the apo-hIAPP (peak at 150°) in the hIAPP : AZD simulations (Figure 5F) with a minor peak at 60° (Figure 5F).

To gain further insights into the mechanism, the binding energies of AZD with hIAPP were calculated using MMPBSA for AZD molecules which were bound to the hIAPP pentamer for at least 45 ns out of the last 50 ns simulation time (Supplementary Table S1). The strongest binding was observed at the C-terminal of the peptide patch, i.e. site 4 followed by sites 1 and 2, suggesting that AZD showed a strong preference for amyloidogenic regions of hIAPP. Furthermore, the analysis of binding energies also suggested that the binding of multiple AZD molecules at adjacent sites could be more favorable (Supplementary Table S2).

Indeed, both electrostatic and van der Waal's energy were shown to contribute to the binding energy of AZD to the hIAPP fibril. Only in simulation 3 (designated as 3-hIAPP: AZD), a reduction in the number of inter-protein hydrogen bonds (from 114 in apo-hIAPP to 104 in hIAPP: AZD) and in the percentage of β-sheet content (from 45% in apo-hIAPP to 41% in 3-hIAPP: AZD) was observed. However, the complete disruption of the fibril or oligomer, as was observed in the biophysical assays performed at biological time-scales of hours, was not observed in any of these simulations. Therefore, longer simulations might be required to observe the complete disruption in the structural integrity of hIAPP oligomer/fibrils by AZD molecule in MD studies. However, with 300 ns simulation, we could show that upon interaction, AZD twisted fibril axis that may act as an initiation point for fibril disruption.

Discussion

The global prevalence of T2DM is increasing worldwide with estimates of ∼552 million people to suffer from diabetes by 2030 [58]. Among these, the percentage of children, adolescents and young adults among the affected subjects is alarming. The current therapeutic interventions for the management of T2DM have been developed with the aim to delay its progression. Therefore, the development of more effective drugs and therapeutic strategies has become imperative to deal with the worldwide increasing prevalence of T2DM. hIAPP is a major constituent of the amyloid deposits in the pancreas of most T2DM subjects and in kidneys, sensory neurons and heart of most T2DM subjects suffering from secondary complications [59]. The formation of such insoluble aggregates has been postulated as a major mechanism linked to pancreatic β-cell death [60]. It is now believed that designing inhibitors that can modulate hIAPP aggregation and membrane disruption by amyloidogenic species can emerge as an effective therapeutic strategy for T2DM treatment. In addition to this, since hIAPP aggregates form earlier in time, much before diabetes manifests in, these drug molecules might be able to delay onset of T2DM by inhibiting hIAPP aggregation process by reducing β-sheet extension and assembly. Therefore, designing/identifying potent inhibitors of amyloid aggregation and hence, targeting amyloid fibrils may be a useful strategy for the management of T2DM. As most protein aggregation diseases share a common mechanism for aggregation and toxicity, these molecules might work as generic inhibitors for aggregation-related diseases.

hIAPP is one of the most naturally occurring amyloidogenic peptide known till date [8]. Therefore, designing inhibitors against hIAPP aggregation can be extremely challenging. Previous studies have shown that IAPP displays ∼10% helical content in solution with predominantly unstructured conformation [61]. On the other hand, hIAPP undergoes a conformational transition from helix to cross β-sheet-rich conformation with the existence of long-lived partially folded intermediates as an oligomeric population [55,62]. Any points during this process could, therefore, serve as a target for the inhibition of hIAPP aggregation either by stabilizing monomers or by conversion of these intermediates to off-pathway oligomers.

Previous studies have reported that natural polyphenolic compounds including resveratrol, silibinin, EGCG, morin hydrate, curcumin and tanshinones (extracted from the dry roots of Salvia miltiorrhiza Bge), have the tendency to reduce amyloid-associated toxicity by either inhibiting self-association of hIAPP or by mediating disaggregation of preformed fibrils [15,18,23,63,64]. In addition to the natural polyphenols, various chemically synthesized compounds, short peptides, active metabolites, metallic ions and glycosidase inhibitors have also been investigating for their anti-amyloidogenic potential in T2DM [1422]. The aromatic ring-containing inorganic complexes such as ruthenium and copper complexes were found to effectively inhibit the fibril formation of hIAPP and have been shown to promote disaggregation of pre-existing IAPP fibrils [65,66]. Various chemotherapeutics, such as thiazolidinediones, meglitinide, sulfonylurea, non-aromatic and biguanide (metformin), have also been shown to improve β-cell function and mass in patients with islet amyloidosis [67,68]. Additionally, the non-phenolic dipeptidyl peptidase-4 inhibitors such as sitagliptin treatment prevented amyloid-induced β-cell loss in mice [69]. However, the exact mode of action of these inhibitors against T2DM needs to be investigated in detail.

The aromatic interactions in hIAPP peptide strongly promote the formation of the cross β-sheet fibril due to higher hydrophobicity or the formation of stabilizing pi–pi interactions [70]. In the current study, we speculated that the hydrophobic interactions of AZD with the proposed hIAPP pentamer might block the self-assembly of hIAPP and inhibit aggregation. In agreement with this, results from our biophysical experiments indeed suggest that indeed AZD inhibits hIAPP amyloid formation and mediates disaggregation of preformed hIAPP fibrils in vitro (Figure 1). This was also associated with secondary structure changes (as observed in by far-UV CD investigations; Figure 2) that involved conversion from a random coil to β-sheet structure (Figure 2). hIAPP-mediated toxicity in the pancreatic INS-1E cells was associated with the generation of ROS and the associated membrane and DNA damage that was restored upon AZD supplementation. In addition to this, AZD supplementation was also associated with restoration of GSIS from pancreatic islets cultured in the presence of hIAPP (Figure 6). It can be speculated that AZD treatment seems to have transformed the ordered fibrils into amorphous off-pathway aggregates that became non-toxic to pancreatic INS-1E cells.

Schematic representation of the effect of AZD on hIAPP fibrillization.

Figure 6.
Schematic representation of the effect of AZD on hIAPP fibrillization.

(Left) The representation of healthy pancreatic β-cells, wherein the mitochondrial and DNA integrity is maintained. These cells function by storing and releasing insulin in response to stimulants including glucose. (Middle) The nucleation pathway of hIAPP self-assembly in which conversion of monomer to amyloid fibrils occurs via oligomers as intermediates. The effect of AZD on inhibition of hIAPP aggregation and disaggregation of preformed aggregates has also been depicted. (Right) The effect of hIAPP oligomers on pancreatic β-cells which induces ROS production that is associated with changes in MMP and DNA damage; alteration in membrane permeability; and significantly alters the functionality of these cells by altering the insulin release in response to glucose.

Figure 6.
Schematic representation of the effect of AZD on hIAPP fibrillization.

(Left) The representation of healthy pancreatic β-cells, wherein the mitochondrial and DNA integrity is maintained. These cells function by storing and releasing insulin in response to stimulants including glucose. (Middle) The nucleation pathway of hIAPP self-assembly in which conversion of monomer to amyloid fibrils occurs via oligomers as intermediates. The effect of AZD on inhibition of hIAPP aggregation and disaggregation of preformed aggregates has also been depicted. (Right) The effect of hIAPP oligomers on pancreatic β-cells which induces ROS production that is associated with changes in MMP and DNA damage; alteration in membrane permeability; and significantly alters the functionality of these cells by altering the insulin release in response to glucose.

Earlier studies have pointed towards the amyloidogenic behavior of segments 8–20, 10–19, 20–29 and 30–37 [7173]. A smaller fragment comprising of amino acid 20–29 of hIAPP has also been associated with the fibrillization that forms the core region of hIAPP amyloid fibrils [55]. In the current study, results from all-atom MD simulations identified four possible sites of AZD interaction with hIAPP pentamer. The strongest binding was observed at the C-terminal of the peptide patch, i.e. site 4 followed by site 1 and site 2, suggesting that AZD showed a strong preference for amyloidogenic regions of hIAPP via hydrophobic interactions and hydrogen bonding. Indeed, mutation studies have shown that S20G (closer to site 2) substitution in hIAPP is more amyloidogenic and cytotoxic than wild-type hIAPP in mouse islets [74]. Both electrostatic and van der Waal's energy were shown to contribute to the binding energy of AZD to hIAPP. No pi–pi stacking interactions between AZD and hIAPP residues were observed possibly due to the absence of any unsaturated electronic rings in AZD. In addition, the aromatic amino acids such as F15 (site 2), F23 (site 3) and Y37 (site 4) have also been proposed to affect the hIAPP aggregation process. In fact, results from previous studies have shown that F23 and Y37 occur in proximal spaces in the fibrillar form of hIAPP [75]. H18 (site 2) has also shown to play an important role in determining specific intra- and intermolecular interactions that occur during fibrillogenesis [76].

In conclusion, we investigated the anti-diabetic and anti-amyloidogenic potential of AZD by targeting the hIAPP fibrillization pathway. AZD supplementation inhibits toxic aggregation of hIAPP by a reduction in oxidative stress, and restoration of the corresponding DNA damage, membrane damage, endoplasmic reticulum stress, and mitochondrial membrane damage, and restored GSIS from pancreatic islets exposed to hIAPP. In addition, AZD also disaggregates pre-existing hIAPP fibrils by forming amorphous aggregates that are non-toxic to pancreatic β-cells. AZD interacted with the amyloidogenic core region of hIAPP pentamer by hydrogen and hydrophobic interactions, thereby preventing hIAPP amyloidogenesis. In summary, our results suggest that AZD can be used for the management of T2DM and other protein-misfolding associated disorders.

Abbreviations

     
  • AFM

    atomic force microscopy

  •  
  • ANS

    8-anilinonaphthalene-1-sulfonic acid

  •  
  • AZD

    Azadirachtin

  •  
  • BCA

    bicinchoninic acid

  •  
  • CD

    circular dichroism

  •  
  • CPCSEA

    Control and Supervision of Experiments on Animals

  •  
  • DMSO

    dimethyl sulphoxide

  •  
  • EDTA

    ethylenediaminetetraacetic acid

  •  
  • EGCG

    (−)-epigallocatechin 3-gallate

  •  
  • FBS

    fetal bovine serum

  •  
  • FT-IR

    Fourier transform infrared

  •  
  • GSIS

    glucose-stimulated insulin secretion

  •  
  • HEPES

    4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid

  •  
  • HFIP

    hexafluoroisopropanol

  •  
  • hIAPP

    human islet amyloid polypeptide

  •  
  • LDH

    lactate dehydrogenase

  •  
  • MD

    molecular dynamics

  •  
  • MMP

    mitochondrial membrane potential

  •  
  • MTT

    3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide

  •  
  • PBS

    phosphate-buffered saline

  •  
  • PI

    propidium iodide

  •  
  • PMSF

    phenylmethylsulfonyl fluoride

  •  
  • ROS

    reactive oxygen species

  •  
  • RT

    room temperature

  •  
  • T2DM

    type 2 diabetes mellitus

  •  
  • TBA

    thiobarbituric acid

  •  
  • TBARS

    thiobarbituric acid reactive substances

  •  
  • TEM

    transmission electron microscopy

  •  
  • ThT

    Thioflavin-T

Author Contribution

A.K., R.D. and S.S. conceived and designed the experiments. R.D., K.P., N.M., P.B., J.D.A., D.M.S. and S.S. performed the experiments. R.D., K.P., J.D.A., S.S. and A.K. were involved in analysis and interpretation of data. S.C.D. performed and analyzed MD simulations data. R.C., S. Sarkar and S.G. did QM/MM calculations to parameterize AZD. S.S. and A.K. contributed to reagents/materials/analysis tools. R.D., S.C.D., K.P., J.D.A., S.S. and A.K. compiled the data. R.D., S.C.D., J.D.A., S.S. and A.K. wrote the manuscript.

Funding

This work is supported by Grant-in-Aid [37(1509)/11/EMR-II], CSIR-Government of India; BT/RLF/Re-entry/11/2012 (Department of Biotechnology-DBT, Government of India); F.4-5(18-FRP)(IV-Cycle)/2017(BSR) (University Grants Commission, Government of India); and SB/YS/LS-23/2014 (SERB-Science and Engineering Research Board, Government of India).

Acknowledgements

The authors acknowledge TEM, Bio-AFM facility funded by RFIC-IIT Bombay; Central Instrumentation Facility at Savitribai Phule Pune University (SPPU); and the flow cytometer facility at the Institute of Applied Biology Research and Development, Pune. R.D. is thankful for financial assistance from UGC-JRF, Government of India and A.K. acknowledges funding from CSIR, Government of India. S.C.D. is very grateful to CDAC PARAM Yuvall facility, Pune, India, for the computational time to perform the MD simulations. S.S. acknowledges funding from Ramalingaswami fellowship (Department of Biotechnology — DBT, Government of India) and University Grants Commission (MHRD, Government of India); Board of College and University Development (BCUD) grant (SPPU). S.S. laboratory has been generously funded by Research and Development grant and DST-PURSE grant to the Department of Biotechnology, SPPU; and UPE Phase II grant to SPPU. K.P. acknowledges DBT, GOI for her Master in Biotechnology fellowship. D.M.S. acknowledges funding from DBT- JRF programme, Government of India. J.D.A. acknowledges the funding from Start-up research grant by Science and Engineering Research Board, Government of India. INS-1E cells were obtained as a kind gift from Prof. Claes Wollheim and Prof. Pierre Machler, University of Geneva Medical Centre. We are also thankful to Prof. Robert Tycko, NIH for providing the pentameric co-ordinates of hIAPP for the molecular dynamics.

Competing Interests

The Authors declare that there are no competing interests associated with the manuscript.

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