Preparation of islets and tissues
All experiments with laboratory animals were approved by the committee for animal welfare at the Katholieke Universiteit Leuven. Islets of Langerhans from C57BL6/J (Janvier, Le Genest-Saint-Isle, France) female mice were collagenase-isolated as previously described . Other tissues were dissected from 10- to 12-week-old C57Bl6/J (Janvier) mice. Fetal tissue was isolated on pregnancy day (P) 15.5. Embryonic stem cells were isolated as previously described . Tissues were rinsed in PBS, frozen in liquid nitrogen and stored at −80°C. Mouse beta cells were purified from islets of Ripyy mice producing eYFP under the rat insulin promoter .
MIN6 cells were cultured in DMEM (Invitrogen-Gibco, Karlsruhe, Germany) 25 mmol/l glucose equilibrated with 5% CO2 and 95% air at 37°C and supplemented with 15% (vol./vol.) decomplemented FCS, 70 μmol/l β-mercaptoethanol, 4 mmol/l glutamax, 50 U/ml penicillin and 50 μg/ml streptomycin. MIN6 cells were used between passages 20 and 30. For immunocytochemistry, MIN6 cells were seeded on glass coverslips coated with Fibronectin-like engineered protein polymer plus (Sigma-Aldrich, St Louis, MO, USA) in a 12 well plate and incubated for 24 h in DMEM (25 mmol/l glucose, 2% [vol./vol.] decomplemented FCS, 4 mmol/l glutamax) with 0 or 500 ng/ml ovine PL (Prospec, Rehovot, Israel). For the inhibitor experiments, MIN6 cells were serum-starved for 24 h. Next they were pre-incubated for 15 to 30 min with one of the three inhibitors: tyrphostin AG490 (50 μmol/l; Sigma-Aldrich), mitogen-activated protein kinase kinase 1&2 (MEK1/2)-inhibitor-1 (10 μmol/l; Calbiochem-Merck Biochemicals, Darmstadt, Germany) or wortmannin (100 nmol/l; Sigma-Aldrich). Thereafter, they were incubated with or without 500 ng/ml PL, together with wortmannin for 7 h or together with tyrphostin AG490 or MEK1/2-inhibitor-1 for 24 h. For overexpression experiments, AMAXA technology (Lonza, Cologne, Germany) was used. Briefly, using the X13 program, 5×106 MIN6 cells were electroporated with 2 μg plasmid DNA and 100 μl nucleofector V (Lonza), and recovered for 7 h in RPMI (Invitrogen-Gibco; 11 mmol/1 glucose, 15% [vol./vol.] decomplemented FCS) medium.
Extracellular matrix-coated plates and coverslips were produced as previously described . In these coated plates, isolated islets were cultured in RPMI medium (10% [vol./vol.] decomplemented FCS, 100 U/ml penicillin, 100 μg/ml streptomycin, 4 mmol/l glutamax) for 7 days to form a monolayer. On day 7, islets were stimulated with 0 or 500 ng/ml PL.
Total RNA from mouse islets, pure beta cells and MIN6 cells was extracted using a kit (Absolutely RNA microprep; Stratagene, La Jolla, CA, USA). Total RNA was extracted from the other tissues as previously described . Total RNA quantity and quality were determined using a spectrophotometer (ND-1000; NanoDrop Technologies, Wilmington, DE, USA) and a bioanalyser (2100; Agilent, Waldbronn, Germany), respectively.
mRNA expression analysis via microarray
Microarray analysis was conducted on different tissues with MOE430-2.0 arrays (Affymetrix, Santa Clara, CA, USA) as described . To analyse the time series of pregnant islets, total RNA (100 ng) was used to hybridise the arrays (MoGene_1.0_ST; Affymetrix) according to manufacturer’s manual 701880Rev4 as described previously . The different conditions were compared with each other pairwise on the basis of p < 0.001 and a fold change of ≥1.5.
Following cDNA synthesis using a reverse transcriptase kit (RevertAid H Minus; Fermentas, St Leon-Rot, Germany), quantitative RT-PCR (Absolute QPCR mix; Abgene-Thermo Fisher Scientific, Waltham, MA, USA) was performed on a Rotorgene (Corbett Research, Mortlake, NSW, Australia) to estimate mRNA expression of different genes. The gene encoding RNA polymerase II (Polr2a) was used for normalisation. For primers and probes see ESM Table 1. For Tph2 we used a Taqman gene expression assay (Mm00557717_m1; Applied Biosystems, Carlsbad, CA, USA).
Total protein extracts were obtained from freshly isolated islets or MIN6 cells, which were lysed in extraction buffer (50 mmol/l Tris, pH8; 1% [vol./vol.] NP-40; 150 mmol/l NaCl; 1 mmol/l EDTA). Protein extracts (100 μg for islets, 20 μg for MIN6 cells) were separated by SDS-PAGE (10% [vol./vol.] Tris/glycine gel; Invitrogen, Paisley, UK). The gels were blotted on polyvinylidene fluoride membranes and, after blocking with 4% (wt/vol.) milk, incubated with primary antibody (ESM Table 2). Thereafter, they were incubated with peroxidase-conjugated secondary antibody (ESM Table 2). Detection was by ECL-system (GE Healthcare-Amersham, Diegem, Belgium).
After stimulation with vehicle or PL, cells were washed with PBS and fixed with 4% (wt/vol.) paraformaldehyde. Next cells were washed with PBS, permeabilised with 0.2% (vol./vol.) Triton X-100 and washed again. For TPH1 staining, the cells were immersed in 0.1% (wt/vol.) SDS at 37°C to recover the antigen. The cells were then washed and immersed in Hoechst solution and thereafter washed and blocked with 5% (wt/vol.) BSA. After blocking, primary antibodies (ESM Table 2) were added for 1 h at room temperature in 1% (wt/vol.) BSA, after which cells were again washed. Finally, the cells were incubated with secondary antibodies (AlexaFluor; Invitrogen) (ESM Table 2) for 1 h at room temperature in 1% (wt/vol.) BSA and washed again. Images were obtained with a laser scanning confocal microscope (LSM510; Carl Zeiss Meditec, Jena, Germany) using a 63× oil objective or with an inverted microscope (Ti-E; Nikon, Brussels, Belgium) using 40× and 60× oil objectives.
The Tph1-knockout (KO) mice were obtained by knock-in of the lacZ gene and disruption of Tph1 in embryonic stem cells as previously described . The Tph1-null mutation was backcrossed on to a C57BL6/J background. Pancreases were dissected from pregnant wild-type and Tph1-KO mice, and fixed in 4% (wt/vol.) paraformaldehyde. Binding of primary serotonin antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA) (ESM Table 2) was detected with biotinylated anti-rat Ig in combination with streptavidin horseradish peroxidase; diaminobenzidine was used as substrate (Dako, Glostrup, Denmark). See also ESM Methods.
Insulin/serotonin release and content
To determine serotonin content, MIN6 cells and islets were homogenised in serotonin buffer (0.01 mol/l HCl, 1 mmol/l EDTA, 4 mmol/l sodium metabisulfite) by 3 min sonication. Other tissues were homogenised by douncing. After centrifugation (20,000 g) for 15 min at 4°C and addition of 0.1% (wt/vol.) ascorbic acid, lysates were stored at −20°C.
Freshly isolated islets from P15.5 pregnant female mice were incubated for 1 h in batches of 50 at 37°C. Incubation was in Krebs HEPES buffer (20 mmol/l HEPES, pH7.4; 119 mmol/l NaCl; 4.75 mmol/l KCl; 2.5 mmol/l CaCl2; 1.2 mmol/l MgSO4; 1.2 mmol/l KH2PO4; 5 mmol/l NaHCO3, 0.5% [wt/vol.] BSA) containing either 5 or 20 mmol/l glucose. After 1 h, medium was removed and stored at −20°C for measurement of insulin and serotonin release.
After 24 h incubation with PL, MIN6 cells were pre-incubated for 1 h in Earl’s HEPES buffer (0.5% [wt/vol.] BSA; 124 mmol/l NaCl; 5.3 mmol/l KCl; 0.8 mmol/l MgSO4; 1 mmol/l NaH2PO4; 10 mmol/l HEPES; 1.8 mmol/l CaCl2; 14.3 mmol/l NaHCO3; pH 7.35) containing 2 mmol/l glucose, 10 μmol/l clorgyline (Sigma-Aldrich), a monoamine oxidase A inhibitor and 0.1% (wt/vol.) ascorbic acid. Next, cells were incubated in 2 or 20 mmol/l glucose. After 1 h, medium was recovered for quantification of insulin and serotonin release using commercial ELISA kits (LDN, Nordhorn, Germany; Mercodia, Uppsala, Sweden, respectively).
All data are expressed as mean ± SEM, except the microarray analysis where data are mean ± SD. Statistical analysis was performed on experiments with n ≥ 3 animals/samples. Statistical significance was determined by a two-tailed Student’s t test or Welch’s t test, except for the monolayer experiments, where a Z test with value 1 (value of control sample) was used.