Isolation and culture of mouse islets

Transgenic mice with hemizygous expression of hIAPP under the rat insulin promotor (B6D2-Tg(RIP-hIAPP)CStka; generated as described in [21]) were bred on an F1 C57BL/6 × DBA/2J background [10, 12]. Non-transgenic littermates were used as controls. Mice were housed and bred in a specific-pathogen-free vivarium at VA Puget Sound Health Care System, with ad libitum access to food and water. All animal studies described below were approved by the Institutional Animal Care and Use Committee at VA Puget Sound Health Care System, performed in an AAALAC-accredited animal research facility, and adhered to the Animal Research: Reporting of In Vivo Experiments guidelines.

Islets from 8–12 week-old male and female mice were isolated by collagenase digestion, as previously described [12]. Islets were handpicked and cultured overnight in complete RPMI-1640 medium containing 10% (vol./vol.) FBS, 1 mmol/l sodium pyruvate, 100 U/ml penicillin, 100 μg/ml streptomycin and 11.1 mmol/l glucose (complete medium). Thereafter, islets were distributed via a block randomisation method into islet pools and cultured for up to 144 h in complete RPMI medium containing either 11.1 mmol/l or 16.7 mmol/l glucose, the latter to induce amyloid deposition in hIAPP islets. The culture medium was renewed every 48 h. Subsets of islets were cultured for 48 h in the presence of Congo Red (200 μmol/l) or its vehicle control (DMSO), as done previously [22]. At the end of each experimental culture period, islets were collected for RNA extraction, plasmin activity measurement and/or histology, as described below.

For islet macrophage depletion, freshly isolated islets were cultured for 48 h in complete RPMI medium with 1 mg/ml clodronate-containing liposomes (Liposoma, Amsterdam, the Netherlands), or 1 mg/ml PBS-containing liposomes (Liposoma) or PBS alone as controls. Thereafter, they were transferred into complete RPMI medium containing 16.7 mmol/l glucose and cultured for 48 h, with the goal of inducing amyloid deposition in hIAPP transgenic islets. Subsequently, islets were collected for RNA extraction.

Islet plasmin activity assay

Plasmin enzymatic activity in islet protein lysates was assayed by measuring the release of para-nitroaniline from the chromogenic substrate of plasmin S-2251 (Molecular Innovation, MI, USA). Briefly, Glu-plasminogen (0.5 μmol/l in 50 mmol/l Tris-HCl+100 mmol/l NaCl, pH 7.4; Molecular Innovation) and then S-2251 (0.4 mmol/l) were added to islet lysate samples (10 μg protein/sample, in duplicate) and mixed. The subsequent colour change was quantified at 405 nm after 120 min of incubation at 37°C on a Beckman Coulter DTX880 plate reader (Beckman Coulter, CA, USA).

Histology and quantitative microscopy

Islets were formalin-fixed, paraffin-embedded and sectioned (10 µm). Sections were labelled with anti-insulin antibody (1:2000; Sigma-Aldrich, USA; catalogue no. I2018; RRID:AB_260137), followed by Cy3-conjugated goat anti-mouse IgG (1:250; Jackson ImmunoResearch Labs, West Grove, PA, USA; catalogue no. 115-165-146; RRID:AB_2338690, USA) and counterstaining with thioflavin-S (0.5% [wt/vol.] in aqueous solution; Sigma-Aldrich) to visualise beta cells and amyloid deposits [12]. The anti-insulin antibody was selected based on extensive validation in house. Sections were blocked for 1 h in buffer containing 0.05 mol/l PBS, 0.2% (wt/vol.) Triton X-100 (Sigma-Aldrich), 0.01% (wt/vol.) sodium azide (Sigma-Aldrich), 1% (wt/vol.) BSA (Sigma-Aldrich) and 2% normal goat serum (Vector Laboratories, USA). Antibodies were diluted in buffer containing 0.05 mol/l PBS, 0.2% (wt/vol.) Triton X-100, 0.01% (wt/vol.) sodium azide and 1% (wt/vol.) BSA. Islet images were acquired and analysed using a custom semi-automated workflow (Nikon TiE wide field microscope and Nikon NIS Elements AR v5.02.01 software; Nikon, USA). Briefly, islets were identified based on insulin immunofluorescence from a large area (×2) scan, and multichannel images were acquired at ×20. From these images, thioflavin-S-positive areas were computed based on pre-set pixel-density thresholds, using an automated method based on our previous manual approach [12] and image post-processing to compute islet cross-sectional areas. Amyloid prevalence was defined as the number of amyloid-positive islets/total number of islets×100, and amyloid severity as amyloid area/islet area×100 [3, 9, 13]. A mean of 18.5±1.8 islets per condition were analysed. The observer was blinded to genotype and culture conditions.

Peptide synthesis and purification

Full-length hIAPP, hIAPP 1–11 and hIAPP 12–37 were synthesised using 9-fluorenylmethylcarbonyl (Fmoc) chemistry on a 0.10 mmol scale with a CEM Liberty Blue peptide synthesiser (CEM, USA) [23]. Fmoc-PAL-PEG-PS resin (Agilent; 0.19 mmol/eq) was used for C-terminal amidation of full-length hIAPP and hIAPP 12–37. Peptides were cleaved from the resin using a trifluoroacetic acid (TFA)-based cleavage cocktail (92.5% [vol./vol.] TFA, 2.5% [vol./vol.] triisopropylsilane, 2.5% [vol./vol.] 3,6-dioxa-1,8-octanedithiol and 2.5% [vol./vol.] water). Crude peptides were dried and then dissolved in 20% acetic acid (4 mg/ml) followed by lyophilisation to improve their solubility. The Cys2 and Cys7 disulfide bridge was assembled by oxidising the crude peptide in 100% DMSO (10 mg/ml). Reverse-phase HPLC was used to purify the peptides. A Higgins Analytical C18 preparative column (Higgins Analytical, USA), 25 mm×250 mm, was employed with a binary A-B gradient of water and acetonitrile with 0.1% (vol./vol.) TFA. The purified peptides were lyophilised and redissolved in 1,1,1,3,3,3-hexafluoroisopropanol and subjected to a second round of reverse-phase HPLC purification. Analytical HPLC was used to confirm peptide purity and matrix-assisted laser desorption ionisation time-of-flight MS was used to verify the expected mass. mIAPP was purchased from Amyloid Peptide LLC (Danbury, CT, USA).

For cell treatment, islet amyloid polypeptide (IAPP) peptides were resuspended in Tris-HCl buffer (20 mmol/l, pH 7.4) to a final concentration of 250 μmol/l and then, immediately prior to use, diluted into complete media at final concentrations of 0–60 μmol/l.

Thioflavin-T fluorescence assays

Kinetics of amyloid formation were determined using solutions containing hIAPP 1–37 and/or hIAPP 1–11 and/or hIAPP 12–37, and thioflavin-T (32 μmol/l; catalogue no. T3516; Sigma), and Tris-HCl (20 mmol/l, pH 7.4) in the presence or absence of plasmin (0–4 μmol/l; Molecular Innovation) or tPA (8 nmol/l; Molecular Innovation). Samples were incubated in triplicate at 25°C or 37°C, with plate shaking every 10 min, for up to 72 h. Fluorescence was recorded every 10 min on a Beckman Coulter DTX880 plate reader using an excitation wavelength of 450 nm and an emission wavelength of 485 nm. Control reactions were carried out in the absence of hIAPP peptides.

Negative stain transmission electron microscopy

Samples (15 μl) of material collected at the end of the thioflavin-T assays were blotted onto carbon-coated formvar 300 mesh copper grids (Electron Microscopy Sciences, USA). The same volume of 1% (wt/vol.) depleted uranyl acetate was used to stain each sample. Images were recorded at the Central Microscopy Imaging Center facility at Stony Brook University (Stony Brook, NY, USA).

MS

Full-length hIAPP (20 μmol/l) was incubated with or without human recombinant plasmin (0.4 μmol/l and 4 μmol/l; Molecular Innovation) at 37°C for up to 8 h. Samples were analysed by LC/MS after the indicated incubation time on an LTQ-Orbitrap XL mass spectrometer (ThermoFisher, USA).

Beta cell line experiments

The beta cell line INS-1 832/13 (RRID: CVCL_7226), originally provided by C. Wollheim (University of Geneva, Geneva, Switzerland) [24], which was negative for mycoplasma, was cultured in complete RPMI medium. In total, 15×103 cells/well were plated in triplicate into a gelatin-coated 96-well plate and incubated until confluency, after which the medium was replaced with complete RPMI medium containing freshly dissolved hIAPP peptides (0–60 μmol/l) or Tris-HCl buffer (20 mmol/l, pH 7.4), as a control. After a 24 h incubation, cell viability was assessed using the fluorescent CellTiter-Fluor viability assay (Promega, Madison, WI, USA). Absorbance of a blank sample (no cells) was used to determine assay background, which was subtracted from every experimental sample. Each sample was normalised to buffer-treated cells. In a subset of experiments, cells were collected for RNA extraction at the end of the 24 h incubation with hIAPP (20 μmol/l) or Tris-HCl buffer (20 mmol/l, pH 7.4), as control.

Bone marrow-derived macrophage experiments

Femur marrow from 6 month-old male Sprague Dawley rats (Charles River Laboratories, USA; www.criver.com/products-services/find-model/cd-sd-igs-rat?region=3611) was differentiated for 6 days in RPMI+10% (vol./vol.) FBS and 25 ng/ml recombinant human macrophage colony-stimulating factor (M-CSF; ThermoFisher) to make bone marrow-derived macrophages (BMDM). BMDM were then lifted from tissue culture plates using ice-cold PBS with 2 mmol/l EDTA, washed, plated and cultured overnight in RPMI+10% (vol./vol.) FBS and 25 ng/ml M-CSF. Subsequently, the medium was replaced with RPMI+10% (vol./vol.) FBS. After 48 h, the medium was replaced with complete RPMI containing freshly dissolved IAPP peptide (10 μmol/l) or Tris-HCl buffer (20 mmol/l, pH 7.4), as control. BMDM were incubated for 24 h and then collected for RNA extraction.

Human islets

Human islets from male and female donors with or without type 2 diabetes were obtained from the Integrated Islet Distribution Program. Characteristics of the donors are listed in the electronic supplementary material (ESM) Table 1 (human islets checklist). Sex of the donors was determined based on the medical history from the organ procurement organisation via the isolation centre. Freshly isolated shipped islets (25–50 per donor) were collected for RNA extraction. As the human islets were anonymised, their use was not considered human research by the institutional review board.

Gene expression analysis

Total RNA was extracted from islets or cells using the High Pure RNA Isolation Kit (Roche, Basel, Switzerland), reverse-transcribed using the High-Capacity cDNA Reverse Transcription Kit (ThermoFisher) and then subjected to quantitative RT-PCR (qRT-PCR).

cDNA samples from primary islet endothelial cells isolated from Sprague Dawley rats (Charles River Laboratories, USA; www.criver.com/products-services/find-model/cd-sd-igs-rat?region=3611) and treated for 24 h with IAPP peptides (20 μmol/l) or Tris-HCl buffer (20 mmol/l, pH 7.4) were made available from a previous study [25]. All cDNA samples were analysed in triplicate using pre-validated Taqman gene expression assays (Life Technology, Foster City, CA, USA), with specific probes listed in ESM Table 2. Expression of each gene was calculated using the ΔΔCt method, with Ppib or 18S as housekeeping genes.

Statistical analyses

Data are presented as mean±SEM. Numbers of experimental replications are represented by individual data points in figures. Mean data were compared among treatment groups by one-way ANOVA followed by Holm–Šidák’s multiple comparisons tests. A two-tailed Student t test was used when two groups were compared. A p value ≤0.05 was considered statistically significant. All statistical analyses were performed using Prism 9 (GraphPad Software, San Diego, CA, USA).