SCIENTIFIC REPSIRTS OPEN Pancreatic 3-Cell Membrane Fluidity and Toxicity Induced by Human Islet Amyloid Polypeptide “Cece Species Published: 16Febrvery 2016 ernity i, Pilkington", Esteban N. Gurzov"", Aleksandr Kakinen?, Sara A. Litwak?, William. Stanley®3, Thomas P. Davis & Pu Chun Ke? ‘Aggregation of human islet amyloid polypeptide (hlAPP) into fibrils and plaques is associated with pancreatic (3-cll loss in type 2 diabetes (T2D). However, due to the rapidness of hIAPP conversion in aqueous phase, exactly which hlAPP species is responsible for the observed toxicity and through what ‘mechanisms remains ambiguous. n light ofthe importance of understanding hIAPP toxicity forT2D_ here we show a biophysical scheme based on the use of a lipophilic Laurdan dye for examining MING cell membranes upon exposure to fresh and oligomeric hIAPP as well as mature amyloid. ithas been found that all three hlAPP species, especially fresh hiAPP, enhanced membrane fluidity and caused losses in cell viability. The cell generation of reactive oxygen species (ROS), however, was the most pronounced with mature amyloid hIAPP. The correlation between changes in membrane fluidity and cell viability and their lack of correlation with ROS production suggest hIAPP toxicity is elicited through both physical and biochemical means. This study offers anew insight into(3-cell toxicity induced by controlled hIAPP species, as well as new biophysical methodologies that may prove beneficial for the studies ofT20 as well as neurological disorders. Type 2 diabetes mellitus (T2D) isa metabolic disease currenty affecting 9% of the global adult population with prevalence expected to double by 2035, Though the major burden of the disease is largely preventable through healthy det and exercise, T2D is predicted to become the seventh leading cause of death by 2030°. key hallmark. during the onset of T2D is dysfunction and death of pancreatic 8-cells, located within the islets of Langerhans' Once cell mass decreases by 40-60%, development of T2D is irreversible. There is growing evidence that the 37-residue peptide human islet amyloid polypeptide (RIAPP) also known as amyl, directly contributes to 3-cell loss, and subsequently there is a need to establish the fundamental mechanisms of hLAPP-mediated toxicity IIAP is co-secreted with insulin by pancreatic }-cells and largely contributes to glycemic control”. Itis highly amyloidogenic,and aggregates kinetically in a concentration-dependent manner o form insoluble plaques and fibrils that ar present in 90% of T2D patients". The presence of f-cell granule components, including ins lin at a 1-250 molar ratio to hIAPP, prevents aggregation of the peptide at high concentrations within healthy 3-cels™", Consequently, a deviation in hIAPP secretion within a single cell can be capable of initiating amyloid fibrillation, and recent evidence has also suggested that amyloid could act to tigger amyloidosis in monomeric [IAP ina prion-like mechanism’. Ihas therefore been hypothesised that intracellular amyloidosis can trigger death of the J-cell and provide a ‘seed for larger plaque formation extracellularly", However, there has been considerable debate in the literature as tothe primary RIAPP conformation that induces -cell oxicty>™-namely, ‘monomeric, oligomeric, growing fibrils also referred to as protoibils) or mature amyloid hLAPP-and, addition ally, the mechanisms thereof. Within an aqueous environment, HLAPP fibillats rapidly, and multiple forms can *ARC Centre of Excellence in Convergent Bio-Nano Science and Technology, Monash Institute of Pharmaceutical Sciences, Monash University, 381 Royal Parade, Parkville, VIC 3052, Australia. St Vincent's Institute of Medical Research, 9 Princes Street, Fitzroy, VIC 3065, Australia, “Department of Medicine, t, Vincent's Hospital, The University of Melbourne, Melbourne, Australia. ‘Department of Chemistry, University of Warwick, Gibbet Mil, Coventry, CVA 7AL, United Kingdom. “These awthors contributed equally to this work. Correspondence and requests for materials should be addressed toT.PD. (email: thomas.p davis@monash.edu) or.CK. (email: pu-chun ke@ rmonash.edu) CCIENTIFIC REPORTS [621276|D01-10.1038/srp21274 2www.nature.com/scientificreports/ A Gel phase / quid Liquid cisordered phase ld) ordered phase (lo) ; ee ee — eee ae ica Léa Figure 1. Laurdan dye as an indicator of cell membrane lipid order and ratiometric imaging. Laurdan (6-Dodecanosl-2-dimethylaminonaphthalene) sa ipophilie dye eapable of partitioning into cel phospholipid ‘membranes (A), When excited at the 405 nm laser line, Laurdan emits Muorescenceat 450mm when the cell ‘membranes in the geliquid ordered phase (1 ef panel, and redshifis to SOOnim when the cell membranes inthe liquid disordered phase (ight pane))-AIAPP monomers, oligomers and amyloid bis are predicted {6 causelipid disorder (A), Intent shits between th ordered and disordered channels can be qualified a, generalised polarisation (GP) values. A flowchart outlining the caleultion of GP values rom raw ratiometsc Confocal images inthe ordered and disordered channelsis represented by (B). co-exist at any given stage", When compared to the well-characterised amyloid polypeptide (A() implicated in Alzheimer’ disease, which has far slower aggregation rate of hours todays, isolation of diferent hIAPP species to examine their cytotoxic effects has thus far been dificult to accomplish®, further conteibuting to ambiguity svith regards to RIAPP toxicity. Early research ofthe past two decades favoured amylofd as a causative agent of )-cell failure, thought to be ‘mediated through physical association between the plagues and the cells, leading to membrane perturbation, production of reactive oxygen species (ROS), and/or apoptosis", Clinical studies in a Japanese population who produced « mutated form of HAPP with an increased aggregation propensity showed that they subsequently {developed T2D™"% Lorenzo etal. proposed that 3-cll viability is only reduced when hIAPP concentration i high enough to mediate fibrillation, and identified amyloid-membrane contact triggering apoptosis asthe primary ‘mechanism of toxicity’, Schubert eal, sereened a number of amyloid peptides in PC12 and B12 eells for pro- duction of ROS with 27-dichlorofluorescin diacetate, and determined that hIAPP was correlated with ROS pro- duction and subsequent loss of cell viahilty while no such species were measured with the non-amyloidogenic rat-derived LAPP". There is evidence that the hydrophobic amyloid can mediate formation of fon channels or pores in the J-cell membrane, through its high propensity for contacting and integrating phospholipids into {growing fibrils, leading to cell death by unregulated calcium ion influx or eytosol leakage’. Tn more recent yeas, however, the focus has shifted tothe soluble oligomeric form of hLAPP as the main toxic species, but considering similar mechanisms of toxicity as were postulated for hIAPP amyloid. In light of the biophysical nd biochemical connections between hIAPP and A fibrillation, the working paradigms concern: ing hIAPP toxicity have been influenced by the mechanistic studies of Alzheimer’s disease, an approach which remains to be Validated. Ritzel etal observed that hIAPP oligomers mediated a disruption in islet architecture x vivo, impairing cell coupling and insulin secretion and inducing apoptosis", Research from Kayed etal. and “Meier etal described cell membrane permeabilisation induced by MIAPP oligomers, while monomeric HAPP or amyloid fibrils showed no adverse effects’, and prevention of amyloid fibrillation did not mitigate toxicity". The latter study utilised rifampicin to prevent the formation of amyloid plaques, demonstrating its capacity to pre- vent fibrillation through ThioJlavin-T (TT) staining-an established method to detect amyloid protein. However, research by Meng cal. refuted ths, revealing that rifampicin had no effect on hIAPP fibrillation and demonstrat ing thatthe ligand can interfere with THT fluorescence”; thus illustrating limitations of current methodology to characterise HIAPP species, and proving that the exact science of hIAPP-mediated toxicity is till unclear. Inthe present study, we sought to further investigate fresh and oligomeric BIAPP, as wells mature amyloid as toxic agents to pancreatic B-cells (Fig. 1A), and attempted to establish theie respective mechanisms of ction, We CCIENTIFIC REPORTS [621276|D01-10.1038/srp21274 2hIAPP alone IN caer ash —n rsh Hydrodynamic size (nm) HIAPP + resveratrol inn ash —t sh Hydrodynamic size (am) Figure 2. Characterisation of BIAPP species. (A) Dynamic light scattering shows that monomeric hIAPP, (Onin) readily aggregated to form large fibrils in aqueous solution overtime (480 min), but was stabilised inoligomeric form by resveratrol. (B) TEM images of mature amyloid fibrils and plaques (2 weeks old), The characteristic helical structute ofthe fibrils can be seen in the lower image. additionally explored new methodologies to characterise and control AIAPP toxicity an fibrillation, respectively Resveratrol a polyphenol derived from red wine, proved a suitable candidate for prevention of amyloid fibrl- lation®-", and was employed in the current study to render hIAPP oligomers through off-pathway molecular self-assembly", Ratiometric imaging, a dual-channel confocal fluorescence technique, was used to visualise cll ‘membrane perturbation by each RIAPP species (Fig. 1A.B),This wasachieved via the employment of lipophilic aurdan dye utilised asa probe for membrane lipid order®™ and applied toa number of applications, inelud- ing nanoparticle exocytosis, vital budding", and yeast reproduction. Cell uptake and intracellular elects of the peptide were not a focus ofthis present study due tothe complexity of labelling the MIAPP species without altering their conformations. Th presence of ROS was also investigated to determine any correlation between ‘membrane perturbation, ROS production and 3-cell viability upon hIAPP exposure. We propose that oligomeric IAPR growing fibrils and mature amyloid are, though to different extents, toxic to pancreatic cells, but theit ‘modes of toxicity are not necessarily conserved between cach MIAPP species. In aaition to for research inT2D, this study demonstrates the use of ratiometric imaging as an effective tool for examining the biophysical an toxicological manifestations of hLAPP that remain a challenge due tothe kinetic nature ofthis "most aggregation prone polypeptide “Tis paper is arranged as follows. We ist presenta characterisation of MIAPP in aqueous and at micromo: lar concentrations, rendering in thre states s fresh and oligomeric AIAPP (stabilised by resveratrol a well as ‘mature amyloids, using high-throughput dynamic light scattering (DLS) We then show our ratiometric imag Of MENG pancreatic ells resulting rom their exposure to IAPP ofthe thre states, quand by generalised polarisation (GP) ofthe cell membranes partitioned with a Laurdan dye. Finally we compare our GP measure- ‘ment with the assays of MING cell viability and ROS generation induced by hIAPP ofthe three states and draw conclusions of the present study. Implications of understanding protein aggregation for research in neurological disorders and T2D are discussed a the end, Results BIAPP of three states. High-throughput DLS was utilised to observe hIAPP amyloid fibrillation in aqueous solution. In order to investigate the specific properties of oligomeric hIAPP, polyphenol resveratrol was utilised 28a fibrillation inhibitor. As shown in Fig. 2A, freshly dissolved monomeric RIAPP (approximately 2~311m) readily formed large amyloid fibrils within several hous (hydrodynamic rail of 0.1~4jum), When fresh APP. was incubated with resveratrol ata 2:1 molar ratio, the hydrodynamic radius of MIAPP remained consistent at SCIENTIFIC REPORTS [6:21274|D0L 10.1038)rep21274 3www.nature.com/scientificreports/ 43-Snm over 75h, demonstrating that peptide fibrillation was effectively inhibited and stabilised by the polyphe- ‘nol. Considering the established dynamic pracess of hIAPP fibrillation, including nucleation, elongation and saturation of amyloid formation, we term the peptides immediately dispersed in water as “Iresh’ stabilised by resveratrol a “oligomeric, and over 2 weeks as "mature Based on the DLS measurement, the “fresh” samples consisted intially of hIAPP monomers but evolved into oligomers and fibrils over a timescale of several hours" ‘nd the “oligomeric” samples were stable supramolecular astemblies of hLAPP-resveratrol formed on the spatial Scale ofa few nanometers over the timescale of nanoseconds (discrete molecular dynamics simulations, unpub- lished data). The “mature” amyloid samples s visualised by TEM in Fig. 2B, were characterised by bIAPP amy- loid fibrils of around 5-15 nm in diameter and tens of nanometers to micrometers in length" and mostly devoid. ‘of monomers and oligomers. Membrane disorders induced by hIAPP of three states. Given that « potential pathway of [IAPP-mediated cytotoxicity involves membrane perturbation, we investigated the interaction of fresh hIAPP, stabilised oligomeric LAPP and preformed amyloid fibrils with the cll membrane of pancreatic 8-cell using ratiometric imaging, Data was collected over 2h to reduce interference from laser mediated UV damage to cll “The lipophilic dye Laurdan (Fig. 1A) was utilised asa reporter for cell membrane lipid order, duc to its capability to undergo spectral redshilting upon an inerease in membrane ludity®. Changes in cell membrane lipid order, namely between the gelliquid ordered phase (|, predominantly fluorescent at 430-470 nm) andthe liquid dis- ordered phase (ip predominantly fluorescent at 480-550 nm), were determined by generalised polarisation (GP. see Fig, 1B & Methods) A negative GP shift, indicating decreased membrane lipid order, was observed inal cells, treated with hIAPP species, while membrane lipid order did not change in contro cells or cells incubated with resveratrol only (Fig. 3, right panel) The largest GP shift occurred withthe fees, or fibrillating, MIAPP (—1), followed by oligomeric (~0.08) and amyloid (0.05) species. Accordingly, at 2h the morphology of the cells exposed to BIAPP indicates they are unhealthy (Fig, 3, et panes)-shrinkage and cytoplasm leakage/blebbing twas observed in samples where the GP displays a net negative shift Pancreatic(3-cell viability upon exposure tohIAPP. Next, we determined what phase of MIAPP agare zation s responsible fr toxicity to pancreatic -ells For this purpose, MING cells were treated with fresh or aged !queous hIAPP (10).M) and/or resveratrol (20,M) to obtain the three stages ofthe polypeptide. After 24h trea ‘ment, cells were labeled with the DNA binding dyes Hoechst-33342 and propidium iodide to discriminate viable cell from dead cells. Hocchst-33342 labels dsDNA blue and is able to dilfuse freely through intact and damaged ‘membranes. Propidium iodide labels dsDNA as ed, however due tits molecular properties, i impermeable to cells wit intact membranes, thus staining only dead ces. Cell death was quantified by blue-red fluorescence or by fragmented nucki, tothe exclusion of viable cell identified by intact blue nucle We observed that treatment of MING cells with fesh NIAPP for 24h resulted in the greatest induction of cell death (6.2%, up from 3.7% of control) (Fig. 4). Treatment with either the oligomeric form of hIAPP or mature amo also induced el death toa minor extent (4.9% and 45%, respectively), while treatment with resveratrol alone als slightly affected cell viability (4.7%), ROS production in(-cells exposed to HIAPP. We then determined whether ROS production correlated with cell toxicity dependent on the aggregation state of hIAPP. To analyse this MIN6 cells were stained with 2,7-dichlorofluorescein diacetate (DCFDA) and subsequently treated with the three diferent forms of hIAPP. CFDA isa fluorogenic dye that diffuses readily into the cell and is then deacetylated by cellular esterases toa ‘non-fluorescent compound. After exposure to ROS, DCFDA is oxidised into 2,7-dichlorofluorescein (DCE). DCF fluorescence ean be measured by flow cytometry through excitation at 488 nm, and is detectable at 535mm, Aer 2h and 4h treatments with the three different forms of HIAPP we observed that treatment with mature amyloid fibrils resulted in the highest induction of ROS inside cells (up from 38 to 33% for 2h treatment, and up froma 7% 026% for 4h treatment, Fig. 4B. Treatment with fresh hIAPP had no effect on ROS production, while treatment with oligomeric BIAPP stabilised with resveratrol, or with resveratrol alone, tends to decrease ROS production in comparison tothe control cells. Importantly, fresh and mature hIAPP had no effect on DCEDA emission (Supplementary Figure 1) Discussion ‘The premise ofthis study was to investigate the impact of different stages in the hIAPP fibrillation process on ‘membrane fluidity, and the ways in which these diferent stages contribute to 3-cell toxicity. Determining the fundamental means by which hIAPP mediates cell dysunction and death is crucial not only forthe prevention and management of T2D, but also fora variety of other health concerns, as hIAPP toxicity can present an issue tothe body a large. Amyloid deposition has been observed to extend tothe vascular system, heart, lungs and. kidneys, and additionally hIAPP is capable of crossing the blood brain barrierco-loalisation with AS plaques in the central nervous system may cause increased morbidity in Alzheimer’s disease", Ratiometric imaging has proved to be a viable method for characterising membrane fluidity inthe presence and absence of hIAPP. We observed good correlations between hIAPP-cell membrane interaction, as determined by GP values (Fig 3), and decrease in 3-cel viability (Fig. 2, confocal images; Fig. 4A) within 24h after NIAPP. exposure. In accordance with the los in viability, observed in relation to a decrease in membrane lipid order ‘mediated by these forms of IAPR, and given the physical attributes ofeach HIAPP species, several hypotheses can bbe made for their modes of action, Structurally, oligomeric HIAPP-with or without the involvement of polyphenol resveratrol-could mediate H-H bonding with phospholipids to result in membrane disruption or damage, Mature amyloids can physically partition into the amphiphilic membrane, where the fatty acid tals bind to the fibrils through hydrophobic hydrophobic interactions. In terms of fibrillation of APP atthe cell membrane, a two-step SCIENTIFIC REPORTS [6:21274|DO4 10.1036/rep21274 4www.nature.com/scientificreports/ oh Og Cells only GP shift =0 Intensity per pixel oP hIAPP (fresh) AIAPP (oligomeric) <= 3 < a hi 2I h Intnstypepae h GP shift=-0.05 GP shift=o o or Figure 3. Ratiometric imaging of MIN6 cells treated for 2h with fresh and stabilised oligomeric hIAPP, as well as mature hIAPP amyloid. The shiftin GP values for MING cels labelled with Laurdan dye was recorded cover 2h (B). Blue channel =1, phase; green channel =|, phase; greyscale channel =lve/bright-field; blue-green channel = merge. Inset panel represent merged channels. Arrovts indicate signs of unhealthy cells including shrinkage, blebbing/cytoplasm leakage, and membrane disorder. Sale bars: 40m, process for insertion has recently been proposed utilising model membranes. Monomeric hIAPP and early fibrils «an partition into the membrane during the process of fibrillation as.a result of ther structural transformations and exposure of hydrophobic moieties, leading to changes in area per lipid molecule (and hence membrane fu igity) as well as release of calcium ions, further membrane damage is inhibited, but the fon release subsequently promotes fibril elongation at the membrane surface through incorporation of membrane lips into growing fibrils", This could provide an explanation forthe fresh’ NAP mediating the largest GP shit of the hIAPP species examined (Fig. 3). Overal, these data support published research? but also illustrate the differential capacity of LAPP of three states in physically disrupting ell membranes, which could ead to unregulated on exchange and cytosol leakage, and subsequently cell death Adltonally, interactions between the hIAPP species tnd cell membranes, through H-bonding andor hydrophobic interaction, could hinder the fibrillation process ofthe peptide via reduced availability ofthe peptide monomers and compromised cohesiveness of the peptide assembly architecture. Such reverse eect of LAPP. membrane interaction on peptide fibrillation isan interest. ing new topic to be explored in future studies, ait could also elicit consequences on ROS production and cel viability IFIC REPORTS [621274 D01-10.1038/erp21274ruckus ice kel A Contro! hIAPP hIAPP + Resveratrol a Amyloid Resveratrol Cell death (%) Bs . 3 -—————— 7 278 8 a zs : a —) g 2 6 hIAPP HIAPP+ = 11 Resveratrol 8 4 s 2a || 458 Control APP HAPP Amyloid Resy . 7 = 4n u 2 4 * 7 3 -————— Amyloid Resveratrol -& f\ 422] “i 148 | 8 : oi 8 2 | Bs S eee Control hIAPP hIAPP Amyloid Resv cFOR +Resv Figure 4. Viability and ROS production in MING cells treated with fresh, stabilised oligomeric and ‘mature amyloid LAPP. MIN6 cells were eft untreated (control) or incubated with fresh BIAPP, stable oligomeric MAPP, mature amyloid or resveratrol alone. (A) Cell viability after 24h of treatment was evaluated. by Hoechst-33342 (blue) alive stain, and propidium iodide (red) 2s dead stain. White arrows indicate propidium iodide positive cells. Data shown are means and SEM of independent experiments. “P< 0.05, Scale bar: 100y:m. (B) ROS positive cells were identified by DCFDA staining after treatment for 2and 4h. Representative FACS profiles after 2h treatment are shown, HQ, (1.96mM) was used as positive control. Data shown are means and SEM of 4-6 independent experiments, “P< 0.05, **P-< 0,001, REPORTS [621274 ]DOL-10.1038/5rep21274www.nature.com/scientificreports/ ‘Mechanisms of toxicity from other well-characterised amyloids, in particular AG, are frequently cross-referenced to characterise IAP toxicity. A} is proposed to have protective properties against ROS, ‘one mode of action being as a metal ion clearance agent, facilitated by three histidine residues”. Though, IIAPP-copper(Il) interactions do prevent amyloid fibrillation, any protective effect this provides against ROS, generation and subsequent ell death is disputed™.I'isknown that B-cells ae particularly vulnerable to ROS, and that it contributes to -cell dysfunction during development of T2D*. In our study significantly elevated. ROS production was associated with mature amyloid fibrils (Fig 4B), but levels of ROS did not change from the control when 3-cells were treated with fresh hIAPP Inthe presence of resveratrol, ROS were reduced in compar {son to the control cells illustrating the excellent antioxidant properties of the polyphenol. The contrast between negligible ROS in growing fibrils generated from rest HIAPP after 2h and 4h and increased ROS prodiicton in ‘mature amyloid suggests that properties of larger fibrils, including increased size and hydrophobicity determined the scale of ROS production. Given these data, itis ikely thatthe partitioning of amyloid into the cell membrane induces oxidative stress in the intracellular environment, similarly to what has been observed in other fibrous hhydrophobic materials, eg. carbon nanotubes. Consequent changes in the membrane fluidity could mediate further biochemical and biological processes-such as metal on concentrations and transport as well as metal on sssociation/chelation with the amyloid-and collectively result in 3-cell death. It has additionally been demon: strated in literature thatthe disassembly of amyloid fibrils, mediated by polyphenols, generates ROS through a ‘methionine-independent pathway, which may need to be a consideration when designing constructs for the purpose of disassembling amyloid deposits. Mitigation of ROS does not prevent cell death in all BIAPP species, however, as evidenced by the data from cells treated with fesh and oligomeric hIAPP (Fig. 4). Subsequently, ROS production likely plays significant part in hLAPP-mediated toxicity, but does not constitute the major mecha- nism of toxicity forthe species considered, In conclusion, we propose that oligomerkc ibrilating and amyloid BIAPP to different extents ae all toxic to pancreatic cells and that both membrane perturbation and ROS production contribute to hIAPP toxicity. ‘Membrane perturbation, through H-bonding, hydrophobic interaction and physical penetration, represents an overarching mechanism of toxicity for each ofthe thre respective hIAPP species examined. Amyloid hIAPP, additionally, induced marked ROS production in 3-cels, Collectively, the observed correlation between changes inmembrane fluidity and cell viability and their lck of correlation with ROS production suggest hLAPP toxicity Is licted through both physical and biochemical means. Lastly, this study has demonstrated new methodologies of ratiometric confocal luorescence imaging and high-throughput DLS for characterising hIAPP species and their toxicity in 3-cells, presenting biophysical means that have been recognised lacking” in investigating hIAPP {oxieity for T2D. Such methodologies, owing to their spatial and temporal resolution that are deemed sufiient for capturing peptide aggregation, should be extendable tothe studies of other amyloidogenic disorders associ- ated with ageing, Methods Materials. Human islet amyloid polypeptide (hIAPP, or amylin) (KCNTATCATQRLANFLVHSSNNFGAL LSSTNVGSNTY; disulfide bridge: 2-7; MW: 3,906) was obtained from Abcam in yophilised powder form. The [IAPP was weighed on a Cubis MSE balance (Sartorius, 0.01 mg resolution) and dissolved in Milli-Q water to forma 1 mg/ml (256}:M) stock solution immediatly prior to (freshly-dissolved monomers) or around two weeks. before (pre-formed mature” amyloid fibrils) commencing measurements. Resveratrol (purity > 99%; MW: 228) water to form 1204.M (0.027 mg/mL) stock solution dye (-Dodecanoyl-2-dimethylaminonaphthalene; MW: 354) was obtained from AnaSpec and main- 24M stock solution (1-4mg/ml,) in DMSO, with a 500M solution in Milli-Q made up immediately prior to addition to wells. Hoechst-33342 was obtained from Sigma Akirich and propidium iodide was obtained. from Life Technologies, bth were made to stack concentrations of 10mg/tL in PBS. The DCFDA celular ROS. detection kit was purchased from Abcam, High-throughput dynamic light scattering. The hydrodynamic sizes ofIAPP and bIAPP-resveratrol \wete acquired at room temperature over 430 min using an automated, high-throughput DLS device (DynaPro Plate Reader, Wyatt; instrument resolution: 0.5nim), with samples plated in quadruplicate in black 384-well plates (Thermo Fisher) Fach well contained a total volume of 20 of either 101M hIAPP or resveratrol 20M) mixed With hIAPP at a 1:2 molar ratio in aqueous solution, To ensure good mixing, samples were spun for 1 min at 1,000 rpm/164 RCE (Centrifuge 5804, Eppendorf) prior tothe stat of the experiment. 20 data points for each sample well were collected by an optical module while scanning through all samples continuously. Processing of the aequired data was automated through the Dynamics 7.1.7 software. Transmission electron microscopy. Mature amyloid fibrils were visualised by TEM as follows: a Su aliquot of amyloid BIABP (25u.M in Mill-C, ~2 weeks old) was pipetted onto a copper grid (300 mesh, carbon-coated) and allowed 6 of adsorption. Excess sample was then denen off using filter paper and the grids ‘washed using 101 ddH,0, with exces drawn off as previously described, The grids were stained with 5h 1% tranyl acetate for 30s, with exces stain drawn ofl. Grids were air-dried as needed. Electron microscopy was undertaken utilising a"Tecnai G? 130 Transmission Electron Microscope (FEL, Eindhoven, The Netherlands), operating at a voltage of 300KV. Images were recorded using a Gatan UltraScan 1000 (2k 2k) CCD camera (Gatan, California, USA) using Gatan Microscopy Suite contol software. Ratiometric imaging. _MIN6 pancreatic {}-cells were seeded in 300): DMEM (Invitrogen, UK), contain. ing 10% fetal calf seruni onto 8-ell slide plates (j-Slide,Tbidi, Germany) coated with poly-D-lysine. Cells were allowed to attach overnight in a humidified, 37°C, 5% CO, incubator, Prior to imaging, old media was removed. SCIENTIFIC REPORTS [6:21274|DO4 10.1036/rep21274 7www.nature.com/scientificreports/ and replaced with serum-free Opti-MEM (Life Technologies), with cells washed in between using Opti-MEM. Laurdan dye was added to each wel 1 a final concentration of 50}1M and allowed to equilibrate with the cel, ‘membranes for at least 30 min. Cells were transferred toa Leica SPS inverted confocal fluorescence microscope hhoused in a humiified, 37°C, 5% CO, environment. Appropriate areas ofeach well, containing at least 10 viable cells, were identified. Freshly dissolved hIAPP monomers (10M), pre-formed amyloid fibrils (10j1M), resver- trol (204M), and fresh BIAPP monomers + resveratrol in a 1:2 molar ratio were added to respective well. Imaging was undertaken at 1~30min timepoints for 2h with a 20 x 10.70 dry objective, Laurdan dye integrated. to cell membranes was excited along the 405m laser line and emission read at 430~470 nm (representing the lipid membrane at the gel/liqud ordered phase) or 480~550 nm (representing the lipid membrane a the liquid disordered phase) To calibrate dye background levels, well containing dye only was excited on the 405nim laser line using 0.5 and 2 x laser power. An additional contol of mixing individual hIAPP species with Laurdan abiot- ically didnot show any noticeable effect on the dye fluorescence in aqueous solution (Supplementary Figure S2). Postexperimental 3D images were taken ona 63 x /1 40 il immersion objective, Images were false-coloured and. any adjustments made using LAS X software (Leia). Determination of generalised polarization (GP). ‘The acquisition of GP images was performed using the Image) (National Institute of Health) software and custom-written macro by Owen eal (2011), with mod: ifcations, GP values were then calculated for each piel ofa cell membrane according othe following equation: Gp = Lanto — Hoo s.0, Tygo-s00 ¥ Taras a ‘where I represents the intensity of pixels in the area of interest in the image acquired inthe ordered (430~470nm) and disordered (480-550 nm) spectral channels. The sample pool for GP analysis was an average value of 25 cells, (cach cell 960 + 330 pixels) from each image, totaling ~25,000 data points for each established histogram. GP. shit was observed by subtraction of the GP distribution peak maximum of each sample with 2h of incubation {ime from the GP value ofthe image taken at the beginning ofthe experiment (Oh) Cell culture and viability. ‘The insulin-producing MIN6 cell line was cultured in DMEM (Iavitrogen, UK) supplemented with 10% fetal caf serum. The percentage cell death of MIN6 was determined by labelling cells with the DNA dyes Hoechst-33342 (10j/mL) and propidium iodide (5j.g/mL.) and counting at last 600 cells, per experimental condition via inverted fluorescence microscopy. Cells were treated with 101M of fresh BIAPP, 10,.M of stabilised oligomeric hIAPP (with resveratrol 201M), 10M of preformed amyloid fibril, or 20j.M of resveratrol for 4h in Opti-MEM media (Life Technologies). Reactive Oxygen Species detection. ROS detection was performed using a DCEDA cellular ROS detec: tion kit (Abcam). Mixing individual hIAPP species with DCEDA abiotically did not show any effect on the dye fluorescence in aqucous solution (Supplementary Figure S1). Single cell suspensions of MING cells were stained. with 200nM of DCFDA for 30min and subsequently treated with fresh hIAPP, stabilised oligomeric hIAPP and ‘mature amyloid for 2h and 4h to avoid death of control cells in suspension. ROS levels were then measured. indirectly by the oxidation of nonfluorescent DCEDA to fluorescent DCF bya flow cytometer, exciting the dye at «488m and detection at 35mm. The percentage of ROS positive cells was then quantified using FlowJo Software, analysing the ROS (DCF*) population, Data are represented as means + SEM. Given the paired nature of the experimental design comparisons between treated groups were nade by analysis of varkance (ANOVA). A p value < 0.05 was considered statistically significant, report on noncommunicable ices 2014 (World Heath Organisation, Geneva, 201). Inet a Global estimates of diabetes prevalence for 2013 and projections for 2035. Dihtes Res. Ci, Pract, 13, (201. 1. Mathers C.D & Loner D. Projections of lb mortality and buen of disese fom 2002 to 2030 PLoS Med. 3201 (2006) 4. Let Pathogenesis of type 2 diabetes melts. Arch Me Res, 6 197-209 (2005). 5. Kahn SE, Zr, S, Uzachneder KM. & Hull RL The beta cellson inte? dabetes:there has tobe primary functional normaly Dialga 82, 1093-1012 (200). 6. Kamat, Ko et amyloid with macrophage mig ‘mpi 2, 191-201 201. 7s A al anda aman pe 2 daar sm destin Am Pat 4 Lorenz, A Razzabon, R, Weis, GC. Yank, B.A, Pancreatic set cll tonicity af amin assoiated with ype-2 diabetes smelt. Naar 368, 756-70 (1994). 9 Kaye Rot al Permeabliation of ip ayers i common conformation dependent activity of soluble amo oligomersin rote misoling diseases J Bl hem. 279, 46348 6846 (200, to, Neler |: Inhibstion of human APP ri ormation doesnot preven death: evidence fo tine ction oligomers nd fb offuman IAPP. Am. Pio. docrinol Metal 91, FLS17-E1324 (2008). 1H, ia RA Meter [Li C-¥, Vldhus, D8 Butler, PC-Human setae polypeptide oligomers disrupt cel coupling Snduce pops and impair inulin secretion in boat human iets Diseler 6,65-71 (207) 12, Schubert Deal Amy pptide oe otc ia common cutie roechaain, ro. NL Aca S- USA.92, 1989-1993 1995). Scherbaum, WA Teele of anya ia the piso glycemic conta. xp Cl. Endarial Dats 106 97-102 (1998 Kahn, SE The importance ofthe callin the pathogenesis ype 2 diabetes melts... Mel 108, 25-88 (200). 2n0 corsa wth augmented ill deficits in type 2 dabei pints SCIENTIFIC REPORTS [6:21274|DO4 10.1036/rep21274 8