Reagents
Culture media, supplements and antibiotics were purchased from Gibco (Thermo Fisher Scientific, Carlsbad, CA, USA). Fetal Bovine Serum was from Natocor (Córdoba, Argentina). Compound A (2-(4-acetoxyphenyl)-2-chloro-N-methyl-ethylammonium chloride; CpdA) was synthesized as described [10, 11]. Dex and RU486 were purchased from Sigma-Aldrich. Recombinant cytokines were from R&D Systems (Minneapolis, MN, USA).
Animals
NOD, NODscid, and C57BL/6J mice (breeders from Jackson Laboratory, Bar Harbor, ME, USA) were housed under pathogen-free controlled environment (20–22°C, 12h light–dark cycle) in ventilated cages, and provided with food and water ad libitum. All procedures were conducted in accordance with the Guide for the care and use of laboratory animals, Eighth edition (2011). Studies were approved by the Animal Research and Care Committee (#0001 & #0069), FCEyN, University of Buenos Aires.
INS-1E cell line
The rat β- cell line INS-1E (Prof. Wollheim, University Medical Centre, Geneva, Switzerland) was used between passages 63 and 90, and cultured at 37°C in a humidified atmosphere containing 5% (vol./vol.) CO2 in complete RPMI 1640 medium [11 mM glucose, 10% (vol./vol.) heat-inactivated fetal bovine serum (FBS), penicillin (50 IU/ml), streptomycin (50 µg/ml), L-glutamine (2 mmol/l), 2-mercaptoethanol (50 µmol/l), HEPES (10 mmol/l) and sodium pyruvate (1 mmol/l)]. The possible presence of mycoplasma was periodically checked by PCR. INS-1E were seeded at 40 × 103 cells/cm2 in multiwell plates (Nunc, Thermo Scientific, Denmark) in complete medium with charcoal-treated FBS.
Mice islets isolation and culture
Islets (C57BL/6J) were isolated by collagenase digestion and handpicked after density gradient centrifugation [12]. For standardization, islets with 100–125 µm in diameter were considered as an islet equivalent (IEQ). Islets were cultured on ultra-low fixation plates (Corning Costar, Kennebunk, ME, USA), at 37°C in humidified atmosphere containing 5% (vol./vol.) CO2 in RPMI 1640 medium containing 5.5 mM glucose, 10% charcoal-treated FBS, penicillin (50 IU/ml), streptomycin (50 µg/ml), L-glutamine (2 mmol/l) and HEPES (10 mmol/l) for 16–24 h prior to performing experiments.
Human islets isolation and culture
Human pancreata were obtained from the Center for Organ Recovery and Education, Pittsburgh, PA. Islets were isolated using a semiautomated method following collagenase intraductal injection as previously described [13;14]. Islets were purified with a COBE 2991 cell separator using discontinuous Euro-Ficoll gradients; purity was assessed by dithizone staining, as described [15]. Available characteristics of the donors as well as islet preparations are summarized in the Table S1.
Islets were handpicked and cultured in ultra-low attachment plates (Corning Costar, Kennebunk, ME, USA), at 37°C in humidified atmosphere containing 5% (vol./vol.) CO2 in RPMI 1640 medium containing 5.5 mM glucose, 10% charcoal-treated FBS, penicillin (50 IU/ml), streptomycin (50 µg/ml), L-glutamine (2 mmol/l) and HEPES (10 mmol/l). For standardization, islets with a diameter of 150–200 µm were considered as an islet equivalent (IEQ). Islets were cultivated for 24–72 h before experimentation.
SDS-PAGE and Western blot
INS-1E cells were harvested on ice-cold PBS, washed and lysed in lysis buffer [50 mM Tris-HCl pH 7.4, 250 mM NaCl, 25 mM NaF, 2 mM EDTA, 0.1% Triton-X, protease inhibitors mix (Complete ULTRA, Roche)]. Protein concentration was determined using the BCA assay Kit (Pierce) and samples conserved at -20°C. Proteins were separated by 8–12% SDS-polyacrilamyde gel electrophoresis (SDS-PAGE), blotted onto nitrocellulose or PVDF membranes (Amersham GE-Healthcare, UK) and incubated with primary antibodies: IκBα (#sc-371; Santa Cruz Biotechnology, Santa Cruz, CA, USA); iNOS (#610332, BD Biosciences, San Jose, CA, USA); ORP150 (#ab124884), BIP (#ab21685, Abcam, Cambridge, MA, USA); phospho-IκBα (#9246), β-actina (#3700), phospho-eIF2α (#9721), eIF2α (#2103), ATF4 (#11815), CHOP (#2895); PDI (#3501) (Cell Signaling Technology, Danvers, MA, USA). Blots were incubated with HRP-conjugated secondary antibodies (Bio-Rad, Hercules, CA, USA) and visualized using ECL (Supersignal; Thermo Fisher Scientific, Carlsbad, CA, USA).
Confocal microscopy
INS-1E were cultured 72h onto fibronectin coated coverslips, treated as described in the figures, fixed by cold methanol and incubated with primary antibodies: NFκB p65 (RelA, #sc-8008) or GR (M-20) (#sc-1004, Santa Cruz Biotechnology). Secondary antibodies (1/200 dilution) were anti-goat or anti-rabbit Alexa Fluor 647 conjugated dye (Life Technology). The coverslips were mounted on slides with Mowiol and images were acquired on a Zeiss LSM 710 Confocal microscope (Carl Zeiss GmbH, Germany). Data acquisition was performed with ZEN Black 2011 software and quantification using Fiji software.
Nitric Oxide Production
Nitrite was measured as an indicator of nitric oxide (NO) production using Griess reagent (1% sulphanilamide and 0.1% naphthyl ethylene diamine dihydrochloride in 2.5% phosphoric acid) at 570 nm [16].
Quantitative real-time PCR
Total RNA was extracted from INS-1E cells with TRIzol reagent (Thermo Fisher Scientific, Carlsbad, CA, USA) according to manufacturer´s instructions. Nucleic acid quantification and quality control were performed with a NanoDrop One (Thermo Fisher Scientific, Carlsbad, CA, USA). For cDNA synthesis, 1 µg RNA was reverse-transcribed using RevertAid Reverse Transcriptase (Thermo Fisher Scientific, Carlsbad, CA, USA) in the presences of RiboLock RNase Inhibitor (Thermo Fisher Scientific, Carlsbad, CA, USA) and oligo(dT) primers. Real-time PCR was performed on a Bio-Rad CFX96 Touch Real-Time PCR Detection System, using SYBR Green mix (Thermo Fisher Scientific, Carlsbad, CA, USA). All reactions were performed in triplicate and HPRT or RPL19 were used as normalization controls. Relative expression was calculated with the 2−ΔΔCT method [17]. Primers are listed in the Table S2.
Transient transfections and luciferase reporter assays.
To determine ATF6 pathway activation we used a reporter plasmid containing the firefly luciferase gene under the control of five copies of ATF6 consensus binding site (5xATF6-LUC). To quantitatively measure XBP1 splicing, we used a splicing-specific reporter plasmid containing the coding sequence of firefly luciferase conjugated to the second ORF of XBP1u (XBP1u-LUC); luciferase is expressed only after IRE1-induced splicing removes the 26-nt intron.
Plasmids were transfected into INS-1E cells with Lipofectamine 3000 reagent (Thermo Fisher Scientific) in OptiMEM medium and cells were treated after 24h. LUC activity in cell lysates was measured using the Luciferase measure kit (Promega) with a Junior Portable luminometer (Berthold, Bad Wild- bad, Germany). Cells were co-transfected with RSV-β-galactosidase expression vector and β-gal activity was measured by ONPG assay as a normalization control for transfection efficiency.
Assessment of cell viability and apoptosis
For cell viability assays, INS-1E cells were seeded in 96-well plates. After treatment, medium was replaced by fresh medium containing 0.5 mg/mL MTT (Thermo Fisher Scientific, Carlsbad, CA, USA). After 3 h at 37°C, media was replaced for 100 µL of acidified isopropanol (40mM HCl) and incubated at room temperature 15 min. Absorbance was measured at 570 nm.
Apoptosis assessments were performed in isolated mouse islets. After treatment, islets were washed and stained with Hoechst 33342 (10 µg/mL) and propidium iodide (PI; 5 µg/mL) for 30 min at 37°C. Images were acquired under a Zeiss Axio Observer Z1 Inverted Phase Contrast Fluorescence Microscope (Carl Zeiss GmbH, Germany). The percentage of apoptotic cells was analyzed by two investigators blinded to the experiment using Fiji software.
Insulin quantification and Glucose-Stimulated Insulin Secretion (GSIS)
The quantification of insulin secreted by INS-1E cells and islets was performed by a sandwich ELISA [18]. For GSIS, cells were incubated in Krebs-Ringer phosphate buffer (KRB: 135 mmol/L NaCl, 0.5 mmol/L NaH2PO4, 3.6 mmol/L KCl, 0.5 mmol/L MgCl2, 1.5 mmol/L CaCl2, 5mM NaHCO3, pH 7.4), 10 mmol/L HEPES, 0.1% BSA, with 2 mmol/L glucose for a period of 2h. Cells/islets were incubated in KRB-HEPES-BSA 2 mmol/L glucose for 1 h; the solution was collected and the cells/islets were incubated in KRB-HEPES-BSA 20 mmol/L glucose for an additional 1 h before collecting the solution. Secreted insulin was measured by ELISA and stimulation index stimulation index (ratio between insulin released under high glucose versus low glucose conditions) was calculated.
Adoptive transfer of diabetes in mice and CpdA treatment.
Eight-week-old female NODscid mice were adoptively transferred with diabetogenic splenocytes (i.p. 5x106 cells/mice) isolated from diabetic NOD mice [16, 19] and injected i.p. with CpdA 100µg/200µL or vehicle (Control) three times a week from day − 1 to day 50. Body weight was registered weekly and animals monitored for appearance of treatment-related adverse effects. Tail-blood glucose was measured with a glucometer (Optium Xceed®, Abbott Laboratories, North Chicago, IL, USA); diabetes was diagnosed when glycemia reached ≥ 300 mg/dl in two consecutive days. The incidence of diabetes between groups was compared by Kaplan-Meier analysis and the log-rank test.
Histological examination
The pancreata were fixed in 10% formaldehyde and embedded in paraffin. Insulin immunolabelling was performed on 7 µm tissue sections with anti-insulin (clone HB125, #MU029-UC, Biogenex, Fremont, CA, USA) and HRP-conjugated donkey anti-mouse (#715-036–150, Jackson ImmunoResearch, Baltimore, PA, USA) and signal revealed with 3,3-diaminobenzidine (DAB) substrate; nuclei were counterstained with haematoxylin. Images were acquired under an optical microscope (Olympus CX31, Olympus, Tokyo, Japan). Two investigators blinded to the experiment scored at least 10 islets per mouse to calculate the infiltration percentage using the following criteria: 0, no insulitis; 1: <25%; 2: 25–50%; 3: 50–75%; and 4: >75%.
Statistical analysis
Results are presented as mean±SD. Comparison between groups were carried out using paired or unpaired Student´s t-test or ANOVA followed by Bonferroni´s multiple comparison test, as appropriate. A p < 0.05 was considered to indicate a statistically significant difference. All statistical analyses were performed using GraphPad Prism version 6.0 Software.