HepG2 cells (kindly provided by T. Kieffer, University of British Columbia), an immortalised human liver cell line, were cultured in high-glucose (25 mmol/l) DMEM (DMEM-HG; #10-013-CV, Corning, Corning, NY, USA; or #D6429, Sigma-Aldrich, St Louis, MO, USA) with 10% (vol./vol.) heat-inactivated FBS (Sigma-Aldrich #F1051). INS-1 cells (kindly provided by C. Wollheim, University Medical Center, Geneva, Switzerland), an immortalised rat beta cell line, were cultured in RPMI 1640 (Corning #10-041-CV) with 10% (vol./vol.) FBS, 50 μmol/l 2-mercaptoethanol (Sigma-Aldrich), 10 mmol/l HEPES (#BP310, Thermo Fisher Scientific, Waltham, MA, USA) and 1 mmol/l sodium pyruvate (Sigma-Aldrich #S8636). MIN6 cells (kindly provided by J. Miyazaki, Osaka University Graduate School of Medicine, Osaka, Japan), an immortalised mouse beta cell line, were cultured in DMEM-HG with 10% (vol./vol.) FBS. βTC-6 cells (#CRL-11506, ATCC, Manassas, VA, USA), an immortalised mouse beta cell line, were cultured in DMEM-HG with 15% (vol./vol.) FBS. α-TC3 and α-TC1 clone nine cells (kindly provided by T. Kieffer), immortalised mouse alpha cell lines, were cultured in DMEM-HG (Sigma-Aldrich #D6429A) with 10% (vol./vol.) FBS. All cell lines were confirmed to be free of mycoplasma using the MycoAlert Mycoplasma Detection Kit (#CA11006554; Lonza, Basel, Switzerland).
Mouse islet isolation and culture
Islets were isolated from mice by pancreatic duct injection with collagenase (1000 U/ml; Sigma-Aldrich #C7657) dissolved in Hanks’ balanced salt solution (HBSS: 137 mmol/l NaCl, 5.4 mmol/l KCl, 4.2 mmol/l NaH2PO4, 4.1 mmol/l KH2PO4, 10 mmol/l HEPES, 1 mmol/l MgCl2, 5 mmol/l dextrose, pH 7.2). Pancreases were incubated at 37°C for 12 min, vigorously agitated and the collagenase reaction quenched by adding cold HBSS with 1 mmol/l CaCl2. The pancreas tissue was washed three times in HBSS+CaCl2 (centrifuging for 1 min at 1000 g in between washes) and resuspended in Ham’s F-10 (#SH30025.01, HyClone, GE Healthcare Bio-sciences, Pittsburgh, PA, USA; or Corning #10-070-CV) containing 0.5% (wt/vol.) BSA (Sigma-Aldrich #10775835001), 100 U/ml penicillin and 100 μg/ml streptomycin (Corning #30002CI). Pancreas tissue was filtered through a 70 μm cell strainer and islets were handpicked under a dissecting scope to >95% purity.
Human islet culture
Human islets for experiments in Figs. 3a, b, d, e (H216), 3c (H219), 3f–h (H210, H211, H220) and 4d (H215) were obtained from the Ike Barber Human Islet Transplant Laboratory (Vancouver, BC, Canada). Organ donor purity ranged from 60% to 90% islets. Human islets for experiments in Fig. 4a–c (R161) were obtained from the Alberta Diabetes Institute IsletCore (Edmonton, AB, Canada) and islet purity was 95%. All human islets were cultured in CMRL medium (Gibco #11530-037; Thermo Fisher Scientific) with 10% (vol/vol) FBS, 100 U/ml penicillin, 100 μg/ml streptomycin and 2 mmol/l l-glutamine (Sigma-Aldrich #59202C). Research with human islets was approved by the Research Ethics Boards at the University of British Columbia and Carleton University. Refer to the electronic supplementary material (ESM) for the Human Islets Checklist.
In vitro cell treatments
To determine whether CYP1A1 could be induced in islets, we selected doses that are well established to maximally induce CYP1A1 in liver cells without causing toxicity. The upper doses reflect the half maximal inhibitory concentration (EC50) for each chemical. Since TCDD is 1000 times more potent than 3-MC [41,42,43], we used lower doses of TCDD (1–10 nmol/l) compared with 3-MC (100–1000 nmol/l). Adherent cell lines and isolated pancreatic islets (mouse and human) were treated for 48 h in vitro with 0.1 μmol/l or 1.0 μmol/l 3-MC (Sigma-Aldrich #46434-2ML-R, 100 ng/μl solution in acetonitrile), 1 nmol/l or 10 nmol/l TCDD (Sigma-Aldrich #48599, 10 μg/ml solution in toluene) or the appropriate vehicle control (acetonitrile, toluene or DMSO) in culture medium. Medium was refreshed after 24 h. Where indicated, human islets were also treated with 1 μmol/l thapsigargin (Sigma-Aldrich), a cytokine mixture (TNF-α, 50 ng/ml, #510-RT-010, R&D Systems, Minneapolis, MN, USA; IFNγ, 1000 U/ml, #213-10156-1AF, RayBiotech, Peachtree Corners, GA, USA; IL-1β, 10 ng/ml, #JM-4128-10, MBL International Corporation, Woburn, MA, USA) or 10 nmol/l exendin-4 (Sigma-Aldrich #E7144)
CYP enzyme activity assay
Enzyme activity was measured in various adherent cell lines (HepG2, MIN6, βTC-6, INS1, α-TC1, α-TC3) and isolated mouse and human islets using the P450-Glo CYP1A1 Assay (#V8752; Promega, Madison, WI, USA) and CYP1A2 Assay (#V8772, Promega). All assays were performed in 96-well white-walled plates with clear bottoms (#655098; Greiner Bio-One, Kremsmünster, Austria) using the lytic method, as described by the manufacturer. For adherent cell lines, the assay was performed on cells at 70–90% confluence. For islets, 50 mouse or human islets were handpicked into each well of the 96-well plate.
In vitro glucose-stimulated insulin secretion assays
To assess beta cell function, 25–50 mouse or human islets per condition were transferred to a pre-warmed (37°C) 24-well plate containing Krebs–Ringer bicarbonate buffer (KRBB) (115 mmol/l NaCl, 5 mmol/l KCl, 24 mmol/l NaHCO3, 2.5 mmol/l CaCl2, 1 mmol/l MgCl2, 10 mmol/l HEPES, 0.1% (wt/vol.) BSA, pH 7.4) with 2.9 mmol/l glucose (low glucose, LG) for a 40 min pre-incubation at 37°C. Islets were then transferred to 500 μl of LG KRBB for 1 h, followed by transfer to 500 μl of KRBB with 16.7 mmol/l glucose (high glucose, HG) for 1 h at 37°C. The LG KRBB and HG KRBB samples were centrifuged, and the supernatant stored at −30°C until use. To measure insulin content, islets were transferred to an acid-ethanol solution of 1.5% (vol./vol.) HCl in 70% (vol./vol.) ethanol at 4°C overnight and then neutralised with 1 mol/l Tris base (pH 7.5) before long-term storage at −30°C. Concentrations of human C-peptide (#10-1141-01; Mercodia, Uppsala, Sweden) and mouse insulin (#80-INSMS or 80-INSMSH; ALPCO, Salem, NH, USA) were measured by ELISA.
All mice received ad libitum access to a standard irradiated diet (Teklad Diet #2918; Harlan Laboratories, Madison, WI, USA) and were maintained on a 12 h light/dark cycle throughout the study. All experiments were approved by the University of British Columbia or Carleton University Animal Care Committees and carried out in accordance with the Canadian Council on Animal Care guidelines. Prior to beginning experimental protocols, animals were randomly assigned to treatment groups and matched for body weight and blood glucose levels (ensuring that these variables were not significantly different between groups).
As outlined in Fig. 5a, 8-week-old male C57Bl/6 mice (Jackson Laboratory, Bar Harbour, ME, USA) received a single i.p. injection of corn oil (25 ml/kg, vehicle control; n = 4), 20 μg/kg TCDD (n = 4), 100 μg/kg TCDD (n = 4) or 200 μg/kg TCDD (n = 4). Liver was flash frozen in liquid nitrogen and islets were isolated from all mice (as described above) 24 h after injection.
As outlined in Figs. 5h, 6a, 8-week-old male C57Bl/6 mice (Jackson Laboratory) received a single i.p. injection of corn oil (25 ml/kg, vehicle control; n = 24) or 200 μg/kg TCDD (n = 22). On days 7 (n = 9–10 per group) and 14 (n = 9–11 per group) following injection, islets were isolated from a subset of mice for ex vivo glucose-stimulated insulin secretion assays (25 islets per mouse), RNA isolation (~150–200 islets per mouse) and CYP1A enzyme activity assays (50 islets per mouse per assay). Liver was flash frozen in liquid nitrogen on days 7 (n = 5 per group) and 14 (n = 9–11 per group). Whole pancreas and liver were harvested from a different subset of mice on day 7 (n = 3 per group) and stored in 4% (vol./vol.) paraformaldehyde (PFA) for 24 h, followed by long-term storage in 70% (vol./vol.) ethanol.
As outlined in Fig. 5m and ESM Fig. 2a, 8-week-old male C57Bl/6 mice received multiple i.p. injections of corn oil (25 ml/kg, vehicle control) or a low-dose of TCDD (20 ng/kg per day) twice per week. This dose was previously shown to induce CYP1A1 in liver and lung [44, 45]. Furthermore, chronic administration of 46 ng/kg TCDD per day resulted in circulating TCDD concentrations of ~8.8 pg/g in rats , which is within the range of background dioxin levels reported in the USA (≤10 pg/g) and corresponds to individuals in the upper quartile (≥5.2 pg/g) with increased diabetes prevalence . In our study, the first group of mice (Fig. 5m–p; Jackson Laboratory) was treated at the University of Ottawa Roger Guindon Hall vivarium (n = 5 control, n = 8 TCDD); liver was flash frozen and islets were isolated after 2 weeks (i.e. five injections). A second group of mice (ESM Fig. 2) was generated by in-house breeding and then treated in the Modified Barrier Facility at the University of British Columbia (n = 4 per group) for metabolic assessment.
Cyp1a1/1a2 double knockout mice
Mice with a global deletion of both Cyp1a1 and Cyp1a2, originally generated and characterised by D. Nebert , were generously provided by F. Gonzalez (University of Cincinnati). Male mice were used for ex vivo experiments with isolated islets (Fig. 7) and as a negative control for CYP1A1 immunofluorescence staining of pancreas and liver tissues (ESM Fig. 1).
All metabolic analyses were performed in conscious, restrained mice and blood samples were collected via saphenous vein using heparinised microhematocrit tubes at the indicated time points. Blood glucose levels were measured using a handheld glucometer (Lifescan, Burnaby, BC, Canada).
Body weight and blood glucose levels were assessed weekly or bi-weekly following a 4 h morning fast. For all other metabolic tests, time 0 indicates the blood sample collected after fasting and prior to administration of glucose or insulin. For GTTs, mice received an i.p. bolus of glucose (2 g/kg; Vetoquinol, Lavaltrie, QC, Canada) following a 6 h morning fast. During the GTT, blood was collected at the indicated time points for measuring plasma insulin levels by ELISA (ALPCO mouse ultrasensitive insulin ELISA, #80-INSMSU-E01). For ITTs, mice received an i.p. bolus of insulin (0.7 U/kg, Novolin ge Toronto #02024233; Novo Nordisk Canada, Mississauga, ON, Canada) after a 4 h morning fast. For all tests, mice from different treatment groups were randomly distributed throughout the experiment to ensure that timing of blood collection was not a factor in our analysis.
Quantitative real-time PCR
RNA was isolated from cultured cell lines using the RNeasy Mini Kit (#74104; Qiagen, Hilden, Germany), human or mouse islets using the RNeasy Micro Kit (#74004; Qiagen) and liver using TRIzol reagent (#15596018; Invitrogen, Carlsbad, CA, USA) or the Qiagen RNeasy Mini Kit, according to the manufacturer instructions. DNase treatment was performed prior to cDNA synthesis with the iScript gDNA Clear cDNA Synthesis Kit (#1725035; Bio-Rad, Mississauga, ON, Canada). Quantitative real-time PCR (qPCR) was performed using the SsoFast EvaGreen Supermix (#1725200; Bio-Rad) or SsoAdvanced Universal SYBR Green Supermix (#1725271, Bio-Rad) and run on a CFX96 or CFX384 (Bio-Rad). Hprt/HPRT or Ppia/PPIA were used as the reference genes. Data were analysed using the ΔΔCt method. Primer sequences are listed in ESM Table 1.
Immunofluorescent staining and image quantification
Human islets were washed with PBS, resuspended in 4% (wt/vol.) PFA and stored overnight at 4°C. The following day, islets were washed twice with PBS, resuspended in 200 μl pre-warmed 2% (wt/vol.) agarose and cooled briefly at −20°C. The agarose pellet was resuspended in 4% (wt/vol.) PFA for 1 h at room temperature and then transferred to 70% (vol/vol.) ethanol for long-term storage at 4°C until paraffin embedding. Whole pancreas tissue was fixed in 4% (vol./vol.) PFA for 24 h and stored in 70% (vol./vol.) ethanol prior to paraffin embedding.
Paraffin sections (5 μm thickness) were prepared by Wax-it Histology Services (Vancouver, BC, Canada) or the University of Ottawa Heart Institute Histology Core Facility (Ottawa, ON, Canada). Briefly, slides were deparaffinised with sequential 5 min incubations in xylene (×3), 100% (vol./vol.) ethanol (×2), 95% (vol./vol.) ethanol (×1) and 70% (vol./vol.) ethanol (×1), and then transferred to PBS for 10 min on a shaker. Heat-induced epitope retrieval (HIER) was performed in citrate buffer (10 mmol/l sodium citrate, 0.05% (vol./vol.) Tween 20, pH 6.0) for 10 min at 95°C using the EZ Retriever microwave system (#MW014-MO; BioGenex, Fremont, CA, USA), unless indicated otherwise below. Slides were transferred to deionised water for a 5 min rinse and then to PBS for 5 min. Tissue sections were circumscribed with an ImmEdge Pen (#H-4000; Vector Laboratories, Burlingame, CA, USA) and then incubated at room temperature for a minimum of 30 min with Dako serum-free protein block (#X090930-2; Agilent, Santa Clara, CA, USA). Next, primary antibodies were diluted as indicated below for each antibody and added to each section; slides were stored in a humid chamber overnight at 4°C. The following day, slides were washed 3×10 min in PBS on a shaker and incubated in secondary antibodies for 1 h in a humid chamber at room temperature, protected from light. Finally, slides were washed 3×10 min in PBS on a shaker, and then coverslips were mounted with VECTASHIELD HardSet Mounting Medium with DAPI (#H-1500, Vector Laboratories) for counterstaining.
The following primary antibodies were used in this study: rabbit anti-MAFA (MAF bZIP transcription factor A) (15 min HIER, 1:1000, shared by T. Kieffer), rabbit anti-somatostatin (1:500, Sigma-Aldrich #HPA019472), rabbit anti-insulin (C27C9, 1:200, Cell Signaling Technology, Danvers, MA, USA; #3014), mouse anti-insulin (L6B10, 1:250, Cell Signaling Technology #8138BF) and mouse anti-glucagon (1:1000, Sigma-Aldrich #G2654). Numerous primary antibodies for CYP1A1 were tested, and these are outlined in ESM Table 2. The following secondary antibodies were used: goat anti-rabbit IgG (H+L) secondary antibody, Alexa Fluor 594 (1:1000, Invitrogen #A11037); and goat anti-mouse IgG (H+L) secondary antibody, Alexa Fluor 488 (1:1000, Invitrogen #A11029). All antibodies were diluted using Dako Background Reducing Antibody Diluent solution (#S302283-2, Agilent).
To measure apoptosis, pancreas and liver sections were stained with the Molecular Probes Click-iT Plus TUNEL Assay using Alexa Fluor 488 dye (Invitrogen #C10617), according to the manufacturers instructions. Pancreas sections were counterstained with rabbit anti-insulin (C27C9, 1:200, Cell Signaling #3014) and goat anti-rabbit IgG (H+L) secondary antibody, Alexa Fluor 594 (1:1000, Invitrogen #A11037). No antigen retrieval was used for counterstaining.
For islet morphology quantification, a minimum of eight islets were imaged with an EVOS FL Cell Imaging System (Invitrogen); the islet area, insulin-immuonoreactive area and glucagon-immunoreactive area were measured for each islet using ImageJ software (https://imagej.nih.gov/ij/index.html). Islet cell apoptosis was quantified as the percentage of insulin+ and insulin− islet cells that colocalised with TUNEL+ nuclei. Liver apoptosis was quantified in at least five images per mouse and expressed as the percentage of TUNEL+ cells in the field of view. Images were captured and quantified by a researcher blinded to the treatment groups.
H&E staining of pancreas and liver tissues was performed using standard protocols by Wax-it Histology Services. Colour images were taken with an Axio Observer 7 microscope (ZEISS, Oberkochen, Germany).
All statistics were performed using GraphPad Prism software (GraphPad Software, La Jolla, CA, USA). Specific statistical tests are indicated in the figure legends. For all analyses, p < 0.05 was considered statistically significant. Data are presented as mean ± SEM, unless indicated otherwise.