Modulation of Islet Amyloid Polypeptide Induced β-Cell Toxicity and Amyloid Formation by Serum Albumin Proteins

Abstract

Human islet amyloid polypeptide (hIAPP, also known as amylin) is a 37-residue neuropancreatic hormone implicated in the progression of type 2 diabetes. hIAPP is soluble and partially structured under physiological conditions, but misfolds to form amyloid deposits in the islets of Langerhans in type 2 diabetes. Along the misfolding pathway, hIAPP forms species that are toxic to pancreatic β-cells, resulting in decreased β-cell function and mass. Serum albumin proteins are a key component of blood plasma and interstitial fluids and are omnipresent in mammalian cell culture media. Immortalized β-cell lines are widely used as model systems for mechanistic studies of hIAPP-induced cytotoxicity and for screening potential inhibitors of hIAPP toxicity. The effects of bovine serum albumin (BSA), human serum albumin (HSA) and fetal bovine serum (FBS) on hIAPP cytotoxicity are examined and the effects of BSA and HSA on hIAPP amyloid formation are explored. The time required for IAPP to form amyloid is lengthened by sub stoichiometric concentrations of BSA and HSA. Cell permeability and cell viability assays with cultured INS 832–13 pancreatic β−cells reveal that BSA, HSA, and FBS reduce hIAPP cytotoxicity. Protection against treatment with 40 μM hIAPP is observed for serum albumin concentrations that are only 1% (=3.75 μM) of the normal amount present in complete cell media. The implications for in vitro assays of hIAPP toxicity and studies of IAPP amyloid inhibitors are discussed.

1. Introduction

Islet amyloid polypeptide (IAPP, also known as amylin) is a neuropancreatic polypeptide hormone that plays a role in metabolic processes. hIAPP suppresses postprandial release of glucagon as well as regulating satiety and gastric emptying^1–4. IAPP is synthesized in the pancreatic β-cells, stored in the insulin secretary granule and co-secreted with insulin⁵. The aggregation of human IAPP (hIAPP) contributes to disease progression in type 2 diabetes^{6, 7}. Amyloid formation generates toxic species that reduce β-cell function and β-cell mass, by mechanisms that are not fully understood^{8, 9}. The process of islet amyloid formation by hIAPP is also believed to contribute to the failure of islet transplants^10–12. hIAPP remains soluble under physiological conditions, despite being stored in insulin granules at concentrations that would rapidly aggregate in vitro. The intragranular concentration of hIAPP is estimated to be on the order of 0.5 to 2.0 mM. The acidic local environment of the insulin granule, as well as hIAPP: insulin interactions and interactions with other granule components prevent irreversible aggregation of hIAPP monomers in the granule^13–16. Upon glucose stimulation, insulin granules are exocytosed and their cargo released and hIAPP ultimately enters the bloodstream, where it is exposed to a milieu of proteins and macromolecules^17,18. hIAPP is an intrinsically disordered protein and does not adopt a fixed conformational state as a monomer. During aggregation to form amyloid it populates a heterogeneous mixture of oligomeric states and these pre-fibril oligomeric species are the most toxic entities^1,3,9.

Serum albumin (SA) is the most abundant protein found in vertebrate blood plasma. Serum albumins fulfill many roles, including colloidal osmotic pressure regulation, endogenous ligand transport, drug binding and transport, and waste transportation^19–21. Synthesized in the liver, human serum albumin (HSA) is a 66.5 kDa multidomain protein with predominantly helical structure. Human blood plasma contains 35–50 mg/mL HSA, but over 60% of total HSA in the body is extravascular^{22, 23}. HSA is prevalent in interstitial fluids, extracellular fluids, and secretions^{21, 24, 25}. SA is an essential ingredient in the culture of mammalian cell lines as a component of fetal bovine serum (FBS). BSA is present in concentrations of 30–150 μM in a number of widely used culture media formulations for promoting the growth of cells. In FBS, typical BSA concentrations are on the order of 350 μM and sometimes higher.

Due to the ubiquity of SA in vivo, as well as in mammalian cell culture, the effects SA on amyloid formation are of particular interest. Previous work, primarily with the Aβ polypeptide of Alzheimer’s disease, has shown that SA interacts with amyloidogenic proteins, to prevent or slow fibrillization and HSA has been shown to prolong the time required for the in vitro aggregation of a range of amyloidogenic proteins in vitro^{26, 27}. SA is also able to bind amyloidogenic proteins in the bloodstream²⁸. For example, HSA is believed to bind 95% of Aβ in blood plasma^29–31 and is thought to inhibit the aggregation of Aβ in vivo, since binding prevents the self-association of Aβ monomers and facilitates Aβ clearance^{32, 33}. Furthermore, deposition of Aβ in the brain and spine have been reported to occur in areas with decreased HSA concentrations³⁴. HSA Aβ interactions have been targeted as a possible therapeutic approach via plasma exchange therapy^35–37.

In addition to the interactions of amyloidogenic proteins with HSA, it is also relevant to examine the effects of BSA in cell-based experiments as FBS is an essential component of cell media. BSA is also widely used as a crowding agent for in vitro biophysical studies and some crowding agents have been shown to reduce hIAPP cytotoxicity³⁸. However, it is not known if this is due to a direct effect of crowding or more specific interactions. In this study we examine the effects of BSA, HSA, and FBS on the toxicity of exogenously added hIAPP towards INS-1 832/13 β-cells and the effect of BSA and HSA on hIAPP amyloid formation in vitro. Concentrations are examined ranging from sub stoichiometric concentrations relative to hIAPP to those used in cell culture studies.

2. Materials and Methods

Peptide Synthesis and Purification

Human IAPP (UniProtKB id: P10997, residues 34–70) was synthesized on a 0.10 mmol scale using 9-Fluorenylmethyloxycarbonyl (Fmoc) chemistry with a CEM Liberty Blue peptide synthesizer. Fmoc-PAL-PEG-PS resin (0.18mmol/eq) was used to afford a C-terminal amide. Pseudoproline derivatives were used as previously described to prevent aggregation during synthesis^39,40. The first residue attached to the resin, beta branched amino acids, arginine, and all pseudoproline dipeptide derivatives were double coupled. A trifluoroacetic acid (TFA) based cleavage cocktail (92.5% TFA, 2.5% triisopropylsilane, 2.5% 3,6-Dioxa-1,8-Octanedithiol, and 2.5% H₂O) was used to cleave the synthesized peptide from the resin and scavenge side chain protecting groups. Crude peptides were dissolved in 15% acetic acid (4 mg/mL) and lyophilized. Peptides were oxidized to form the disulfide bond between residues Cys2 and Cys7 in 100% dimethyl sulfoxide (DMSO) at a concentration of 10.0 mg/ml, with 8.0 mM 2,2’-Dipyridyldisulfide (DPDS) added to facilitate disulfide bond formation. After one hour, a 30% molar excess (with respect to DPDS) of β-mercaptoethanol was added to quench excess DPDS. Peptides were purified using reverse-phase HPLC (RP-HPLC) (Higgins Analytical C18 preparative column, 25mm × 250mm), utilizing a gradient elution composed of buffer A (100% H₂O and 0.045% HCl) and buffer B (80% Acetonitrile, 20% H₂O, and 0.045% HCl). Purified peptides were lyophilized after purification. HCl was used as a counter ion for preparative purifications instead of TFA, as TFA can affect the rate of amyloid formation monitored by thioflavin-T kinetic assays and can affect cell toxicity experiments. A second HPLC purification was used to remove cleavage scavengers as well as residual TFA. 1,1,1,3,3,3-hexafluoroisopropanol (HFIP) was used to dissolve dry peptide for the second purification. Electrospray ionization / quadrupole time of flight (ESI / QTOF) mass spectrometry and analytical HPLC were performed to confirm the mass and the purity of hIAPP (WT), expected 3903.3, observed 3902.8.

Protein and Peptide Stock Solution Preparation

Purified peptides were dissolved in neat HFIP to a target concentration of 0.8 mg/mL and left to stand at room temperature for 4 hours. Peptide stocks were then filtered using a 0.22 μm Millex low protein binding durapore membrane filter. 10μL aliquots of stock were lyophilized for 24hr and reconstituted in PBS to determine the concentration of the HFIP stock solution.

Concentrations were determined by measuring the absorbance at 280 nm. Aliquots for Thioflavin T assays were lyophilized overnight to remove residual HFIP. Fatty acid free bovine serum albumin (UniProtKB id: P02769) and human serum albumin (UniProtKB id: P02768) were obtained from Sigma Aldrich (#A7030 and #A1887 correspondently). SA proteins stocks were prepared fresh the day of the experiment. 1.024 mM stock solutions of BSA and HSA were prepared in phosphate buffered saline (PBS, 10mM Phosphate buffer with 140mM KCl, pH 7.4) and filtered with a 0.22 μm membrane filter Millex low protein binding durapore membrane filter.

Fluorescence Assays

Thioflavin-T kinetic assays were performed to monitor kinetics of IAPP amyloid formation inhibition by SAs using a Molecular Devices SpectraMax Gemini EM microplate reader (450 nm excitation and 485 nm emission). Experiments were conducted at 25°C without shaking. Peptides were resuspended in PBS. Final hIAPP concentration was 32μM, and the thioflavin-T concentration was 32μM. Inhibition experiments were conducted by mixing hIAPP with BSA or HSA to final SA concentrations of 1μM, 2μM, 4μM, 8μM, 16μM, 32μM, 64μM, and 256 μM in the presence of 32 μM hIAPP. The samples were loaded in a 96-well clear bottom plate with a non-binding coating and each sample was run in triplicate.

Transmission Electron Microscopy (TEM)

Transmission electron microscopy (TEM) was used to confirm the presence of amyloid fibrils. Images were taken at the Life Sciences Microscopy Center at Stony Brook University.

15 μl aliquots taken from the thioflavin-T kinetic assay at the end of the experiment were loaded on carbon-coated Formvar 300 mesh copper grid for one minute. After blotting the aliquot, one drop of 2% uranyl acetate was placed on the grid for one minute to negatively stain the sample.

Cell Viability Assays

INS-1 cells were purchased from AddexBio and cultured with optimized RPMI-1640 (AddexBio, #C0004–02) medium supplemented with 10% ultra-low IgG FBS (Gibco, #16250078) and 50 μM β-mercaptoethanol. CellTiter-Glo 2.0 (Promega, #G9242) and CellTox Green (Promega, # G8741) assays were used to evaluate the cytotoxicity of hIAPP towards INS-1 β-cells. SA stocks were prepared in RPMI-1640 supplemented with 50 μM β-mercaptoethanol from fatty acid free BSA (Sigma, #A7030) and HSA (Sigma, #A1887) and filtered through a 0.2 μm membrane before use. Ultra-low IgG FBS (Gibco, #16250078) was used as a 375 μM stock. hIAPP stocks were freshly prepared in ice-cold RPMI-1640 supplemented with 50 μM β-mercaptoethanol from lyophilized aliquots of hIAPP and used immediately in the experiments. The evaluation of the effect of the SAs on hIAPP cytotoxicity toward INS-1 β-cells was performed as follows. Cells were seeded at ~50% confluence on a 96-well half-area clear bottom white plates (Greiner, #675083) and incubated for 36 hrs in 5% CO₂ humidified incubator at 37°C. Freshly prepared 80 μM peptide in base media (i.e. media lacking FBS) was promptly mixed with an equal volume of 2X concentrated SA or FBS solutions prepared in base media. The culture medium on the assay plate was replaced with this mixture and the plate was then further incubated for six hours after which toxicity was monitored. This provides a six-hour exposure of cells to hIAPP in different solutions.

For the CellTox Green assay, the cells were exposed to the assay dye (1:5000 dilution) for the whole incubation period (6 hrs) before fluorescence intensity was measured using 480 nm excitation and 525 nm emission. For the CellTiter-Glo 2.0 assays, culture plates were cooled to RT and an equal volume of the assay reagent was added to the treated cells. The plates were vigorously (700 rpm) shaken for 1 min and luminescence intensity was measured using Clariostar plate reader.

3. Results and Discussion

3.1. Serum albumin proteins inhibit hIAPP aggregation at sub stoichiometric levels

hIAPP amyloid formation was monitored using a thioflavin-T based kinetic assay, and the presence of amyloid fibrils were confirmed using transmission electron microscopy (TEM) at the conclusion of the experiment. Thioflavin-T is an extrinsic dye that undergoes a significant increase in quantum yield when it binds to amyloid fibers, but the assay can yield false positives, as well as false negatives^41,42. Thus, we used TEM to confirm amyloid formation indicated by thioflavin-T. Background thioflavin-T fluorescence is observed in samples with high serum albumin concentrations. Over the range of serum albumin concentrations tested, this background fluorescence can reach as high as 50% of the final fluorescence intensity in an IAPP amyloid formation assay.

Although BSA and HSA are composed almost entirely of helical domains, they have been shown to bind thioflavin-T. Thioflavin-T is hypothesized to interact with a hydrophobic pocket which binds other small molecules⁴³. Other work has suggested a broader mechanism of enhanced thioflavin-T fluorescence with helical proteins involving π-π interactions with aromatic residues⁴⁴. Oligomers of BSA have been shown to exhibit enhanced binding of thioflavin-T at the subunit interface⁴⁵. These studies and the data presented here reinforce the view that thioflavin-T assays of amyloid formation should always be supported by orthogonal techniques. Thioflavin-T fluorescence can also be observed from amyloid fibrils formed by HSA and BSA, however the denaturing conditions required to form serum albumin fibrils were not used in this study, and TEM images of BSA and HSA alone do not show the presence of fibrils under the conditions used for in our experiments.^{46, 74}

The presence of BSA or HSA results in the inhibition of hIAPP amyloid formation, in the sense that the time to form amyloid is prolonged. All samples studied showed a thioflavin-T fluorescence vs time profile indicative of amyloid formation. The lengthening of the time to form amyloid was compared by measuring T₅₀, the time required for a given sample to reach half the maximum fluorescence intensity in a thioflavin-T assay. A sample of 32μM hIAPP in phosphate buffered saline (PBS) in the absence of SA has a T₅₀ value of 3.8 ± 0.2 hours under the conditions used (Table 1). The addition of BSA to a concentration of only 1.0 μM extends the T₅₀ of hIAPP to 7.9 ± 1.1 hour, i.e. two-fold larger than the untreated sample (Figure 1). Increasing the BSA concentration results in slower amyloid formation and larger T₅₀ values, however the effect appears to saturate at concentrations of BSA above 4 μM, with observed T₅₀ values ranging from 13.6 to 15.7 hours. At saturation, the T₅₀ is roughly four-fold larger than that of untreated hIAPP (Figure 1B). TEM imaging confirms the presence of fibrils in the untreated 32 μM sample, as well as in the presence of 1 μM and 32 μM SA. The relatively high concentration of BSA in the 256 μM sample results in amorphous protein deposits which may obscure any hIAPP fibrils. It is also possible that the hIAPP itself is forming amorphous material in this sample, thus we are cautious about interpreting the TEM images of this sample quantitatively (Figure 1C).

Table 1.

T₅₀ values for amyloid formation by 32 μM hIAPP measured in the presence of varying concentrations of BSA or HSA. Experiments were conducted in PBS, pH 7.4, 25°C

Serum Albumin Concentration	T50 (hours)
	BSA	HAS
0 μM	3.8 ± 0.2
1 μM	7.9 ± 1.1	4.4 ± 0.3
2 μM	10.5 ± 0.8	6.3 ± 0.6
4 μM	15.2 ± 2.0	8.0 ± 0.8
8 μM	14.1 ± 2.1	7.50 ± 0.4
32 μM	15.7 ± 0.6	10.4 ± 0.2
64 μM	13.6 ± 1.6	11.1 ± 0.2
256 μM	13.9 ± 0.8	14.7 ± 0.8

Figure 1.

Concentration dependent inhibition of IAPP amyloid formation by BSA. (A) Thioflavin-T monitored kinetics of amyloid formation by 32 μM IAPP alone (black) and in the presence of varying concentrations of BSA (red). (B) T_{50 values} of thioflavin-T curves normalized to the T₅₀ of hIAPP with no added SA. (C) TEM images of 32 μM hIAPP (black), 32 μM IAPP with 1 μM BSA (light red), 32 μM IAPP with 32 μM BSA (red), and 32 μM IAPP with 256 μM BSA (dark red). Scale bars represent 100 nm.

Similar trends were observed for the inhibition of hIAPP aggregation by HSA. Thioflavin-T background fluorescence is also observed at high HSA concentration (Figure 2A). HSA slows amyloid formation in a concentration dependent manner, but to a lesser extent than BSA except for the highest concentrations examined, which have similar T₅₀ values within experimental uncertainty (Table1). There is no indication that the inhibitory effects of HSA are saturated under the conditions studied here. HSA treated samples also show the formation of fibrils by TEM imaging. (Figure 2C) Additionally, large amounts of amorphous deposits are seen in the high concentration HSA samples. It is interesting and perhaps initially surprising that low concentrations of the bovine protein are more effective than low concentrations of the human protein at inhibiting hIAPP amyloid formation. However there appears to be very little, if any, evolutionary pressure to avoid amyloid formation by hIAPP^48–50. Thus it seems unlikely that the ability of albumin to inhibit hIAPP amyloid formation is selected for.

Figure 2.

Concentration dependent inhibition of IAPP amyloid formation by HSA. (A) Thioflavin-T monitored kinetics of amyloid formation by 32 μM IAPP alone (black) and in the presence of varying concentrations of HSA (blue). T_{50 values} of thioflavin-T curves normalized to the T₅₀ of hIAPP with no added SA. (C) TEM images of 32 μM hIAPP (black), 32 μM IAPP with 1 μM HSA (light blue), 32 μM IAPP with 32 μM HSA (blue), and 32 μM IAPP with 256 μM HSA (dark blue). Scale bars represent 100 nm.

3.2. Serum albumins partially mitigate hIAPP-induced cytotoxicity of cultured INS-1 832/13 β-cells

The effects of serum albumin on IAPP induced cytotoxicity of INS-1 832/13 β-cells were independently measured using two different assays, one monitoring cell permeability (CellTox Green) and the other the overall change of ATP levels in the cell (CellTiter-Glo). The INS-1 832/13 cell line is derived from a rat cell line (INS-1) that has been stably transfected to express human insulin and is widely used for studies of pancreatic β-cell function and insulin secretion.

Cells are stressed if grown for lengthy periods in media lacking FBS, so we designed a protocol in which the cells are first grown in normal rich media containing FBS and then the media exchanged for 40μM hIAPP in RPMI 1640 containing varying amounts of BSA, HSA, or FBS for six hours. At the end of this period cell viability was measured relative to cells that had been treated the same way, but without added HSA, BSA or FBS during the six-hour window. The six-hour incubation was long enough to see substantial hIAPP-related toxicity effects, but short enough to minimize reduction in cell viability directly caused by deprivation of FBS. The protocol helps to reduce background effects due to the exposure of the cells to serum free conditions during the 6-hour incubation period.

Cells were grown in an optimized RPMI 1640 medium supplemented with 10% FBS and 50 μM β-mercaptoethanol for 36 hours, before the medium was exchanged for 40μM hIAPP in RPMI 1640 containing varying amounts of BSA, HSA, or FBS. Assuming that FBS contains an average concentration of 375 μM of serum albumin, we expressed the FBS concentration used in our experiments in molar equivalents of BSA. For example, 50%, 10% and 1% FBS were labeled as 187.5 μM, 37.5 μM and 3.75 μM respectively. The CellTiter-Glo assays indicate that HSA, BSA and FBS protect cells from the toxic effects of hIAPP in a dose dependent manner, compared to samples treated with 40μM hIAPP in serum-free media. Measurable rescue was seen at the lowest concentration of the proteins or FBS (3.75μM), (Figure 3). Incubation of the cells with 37.5 μM and 187.5 μM SA showed a concentration dependent increase in viability. Complete FBS preserves cell viability somewhat more than BSA or HSA alone, and BSA shows a marginal improvement in viability compared to HSA. The cell permeability assay (CellTox Green) shows similar concentration-dependent trends in the inhibition of hIAPP induced effects. The data is plotted as normalized percent relative permeability which is defined as:

$$100\times \frac{\left(\text{Measured}\text{effect}\text{with}\text{hIAPP}\text{and}\text{HSA}/\text{BSA}\text{or}\text{FBS}\right)-\left(\text{Measured}\text{effect}\text{with}\text{HSA}/\text{BSA}/\text{FBS}\text{alone}\right)}{\left(\text{Measured}\text{effect}\text{with}\text{hIAPP}\text{no}\text{HSA}/\text{BSA}/\text{FBS}\right)-\left(\text{Measured}\text{effect}\text{without}\text{hIAPP}\text{no}\text{HSA}/\text{BSA}/\text{FBS}\right)}$$

This parameter is not a measure of absolute toxicity/permeability but rather examines the relative effect of inclusion of HSA, BSA or FBS. 100% relative permeability indicates that the effect of hIAPP is the same in the presence or absence of BSA/HSA/FBS and values below 100% indicate a protective effect. The treatment of the cells with 3.75 μM of SA reduced the relative cell permeability caused by 40 μM hIAPP and higher concentrations of SA (37.5 μM and 187.5 μM) resulted in a larger reduction (more rescue) of hIAPP-induced cell permeability. Overall, the data from both assays indicate SAs and FBS reduce β-cell toxicity induced by hIAPP.

Figure 3.

Serum albumin proteins and complete FBS improve the viability of INS-1 832/13 β-cells exposed to 40 μM hIAPP. (A) Changes in global ATP levels as determined by the CellTiter-Glo assay for “no FBS, BSA, or HSA” (hatched), 3.75μM (white), 37.5 μM (light gray), and 187.5 μM (dark gray) concentrations. The results are plotted as percentage of the viability of the cells not exposed to IAPP. (B) Changes in relative cell permeability (as defined in the text) caused by hIAPP treatment as determined by the CellTox Green assay for “no FBS, BSA or HSA” (hatched), 3.75μM (white), 37.5 μM (light gray), and 187.5 μM (dark gray) serum concentrations. Data in panel A and panel B are plotted as the mean ± SD, n=3.

4. Conclusions

Prior studies have provided evidence that BSA can bind Aβ monomers oligomers and protofibrils and exert effects at sub stoichiometric levels^29–31,51. The impact of low to moderate albumin concentrations on hIAPP amyloid formation argue that the effects are not due simply to sequestering hIAPP monomers since little effect would be expected when hIAPP is in large excess, yet a two-fold increase in T₅₀ was observed when hIAPP was present in a 32-fold excess. There are multiple examples of proteins which slow amyloid formation at sub stoichiometric levels including ones that impact hIAPP amyloid formation, possibly by binding to oligomers and modifying primary nucleation^52,53. Another, likely explanation is the inhibition of fibril surface catalyzed secondary nucleation. Secondary nucleation is believed to occur at specific sites on the fibril surface mediated by defects in the fibril structure and the concentration of such sites is noticeably less than the number of monomers in a fibril. Thus, proteins that target such sites can exert significant effects at sub stoichiometric levels^52–54. In addition, binding to the tips of fibrils will inhibit fibril growth. Multiple mechanisms likely come into play with the serum albumins. The effect of BSA and HSA on IAPP induced INS-1 832/13 β-cell death has implications for studies of hIAPP induced toxicity carried out in standard cell culture media as higher toxicity is observed in the absence of BSA. It appears that complete FBS has slightly more protective effects than BSA alone suggesting that either there are other factors in the complicated milieu of FBS which inhibit toxicity or which upregulate cell survival or induce BSA into a more potent inhibitory form. At first glance it may seem surprising that such a significant reduction in toxicity is observed since hIAPP is widely reported to be toxic to cultured cells with EC₅₀ values on the order of 20–40 μM. However, those studies involve longer incubation times of hIAPP on cells or preincubation of hIAPP to pre-populate toxic oligomers^9,10,28. In addition, the conditions required for the assays used here may induce a background stress when treating cells with hIAPP in the absence of SA or |FBS, even though the protocol seeks to minimize it.

The reduction in toxicity by HSA / BSA has potentially interesting implications for studies of inhibitors of hIAPP toxicity. One can envisage a molecule which inhibits hIAPP toxicity by interacting with the polypeptide, but which also competes for hIAPP binding to SA. In this case there will be two competing effects: the direct inhibition of hIAPP toxicity by inhibitor- hIAPP interactions coupled with a reduction of the protective SA-hIAPP interactions caused by the inhibitor interacting with SAs. Finally, the observations reported here also have implications for studies of the effect of molecular crowding on toxicity and amyloid formation by hIAPP. BSA is often used as a crowding agent, but the current work shows that it is not an inert crowder in the context of hIAPP studies

Highlights:

Human and bovine serum albumin (SA) inhibit in vitro amyloid formation by human IAPP.

Inhibitory effects are observed at sub stoichiometric concentrations.

Human and bovine SA reduce the toxicity of human IAPP to cultured beta-cells.

Fetal bovine serum reduces the toxicity of human IAPP to cultured beta-cells.

Abbreviations:

BSA

bovine serum albumin

EC₅₀

the concentration required to achieve 50% of the effective response in a hIAPP toxicity assay

FBS

fetal bovine serum

Fmoc

Fluorenylmethyloxycarbonyl

HPLC

high performance liquid chromatography

hIAPP

human islet amyloid polypeptide

HSA

human serum albumin

PBS

phosphate buffered saline (10mM Phosphate buffer with 140mM KCl, pH 7.4)

SA

serum albumin

TEM

transmission electron microscopy

T₅₀

the time required to reach half the maximum thioflavin-T fluorescent intensity in an amyloid formation assay