Co-localisation and secretion of glucagon-like peptide 1 and peptide YY from primary cultured human L cells

The study was approved by the local research ethics committee. Fresh anonymised surgical specimens of human colon were obtained from Tissue Bank at Addenbrooke’s Hospital, Cambridge, stored at 4°C and processed within hours of surgery. Tissue samples (∼0.5 g in weight) were dissected free of muscle and connective tissue, rinsed, minced and digested four times (20 min each) with collagenase XI (0.5 mg/ml) in DMEM (4.5 g/l glucose) at 37°C. Supernatant fractions from each digest were centrifuged twice at 100 g for 3 min to pellet crypts, which were resuspended in 7 ml culture medium (DMEM supplemented with 10% (vol./vol.) fetal bovine serum, 2 mmol/l l-glutamine, 100 units/ml penicillin and 0.1 mg/ml streptomycin). Aliquots (250 μl) were plated into 24-well plates or glass coverslips coated thinly with Matrigel (BD Bioscience, Oxford, UK). During overnight incubation at 37°C in 5% CO₂, cells spread out to form a partial monolayer, similar in appearance to cultures from mouse colon that typically remain viable for up to 10 days [6].

After 16 h in culture, cells were fixed in 4% (wt./vol.) paraformaldehyde, washed three times with PBS, and blocked in a solution of 5% (vol./vol.) goat serum, 0.1% (vol./vol.) Triton X-100 in PBS for 30 min. Samples were incubated with guinea pig anti-PYY antibody (1:100; catalogue number 16066; Progen, London, UK;) and rabbit anti-proglucagon antibody (1:200; catalogue number sc-13091; Santa Cruz, CA, USA) for 3 h at room temperature, washed three times with 5% goat serum in PBS, and then incubated with Alexa-Fluor 633 anti-guinea pig and Alexa-Fluor 488 anti-rabbit antibodies (1:300 each; Invitrogen, Carlsbad, CA, USA) for a further hour. Control staining was performed by omitting proglucagon and/or PYY primary antibodies.

Colonic biopsies were stripped of connective tissue, minced and digested twice with 1.25 mg/ml collagenase (type XI) in Hanks’ buffered salt solution for 30 min at 37°C. After gentle trituration using a 10 ml pipette and filtering, the resultant single-cell suspension was fixed with 4% (wt./vol.) paraformaldehyde in PBS for 30 min at room temperature. Cell suspensions were incubated overnight in PBS–10% (vol./vol.) goat serum at 4°C and permeabilised with 10% goat serum, 0.3% Triton X-100 in PBS. Pellets were incubated in PBS–10% goat serum with/without proglucagon (1:200) and PYY (1:100) primary antibodies (as above) for 1 h. Cells were rinsed three times in PBS–10% goat serum and then incubated with secondary antibodies (as above; 1:1,000) for 30 min. After three further washes, cells were analysed using a BD LSRFortessa analyser (BD Biosciences, San Jose, CA, USA) equipped with 488 and 640 nm lasers. Data were analysed using FlowJo 7.6 software (Tree Star, Ashland, OR, USA).

Cultures were incubated with test reagents in saline buffer containing (in mmol/l) 4.5 KCl, 138 NaCl, 4.2 NaHCO₃, 1.2 NaH₂PO₄, 2.6 CaCl₂, 1.2 MgCl₂, 10 HEPES and 10 glucose (pH 7.4, NaOH) plus 0.1% fatty acid-free BSA, for 2 h at 37°C. At the end of the incubation, cells were lysed with buffer containing: 50 mmol/l Tris–HCl, 150 mmol/l NaCl, 1% (vol./vol.) IGEPAL CA-630, 0.5% (wt./vol.) deoxycholic acid and one tablet of protease inhibitor cocktail (Roche Diagnostics, Burgess Hill, UK), and mechanically disrupted to extract intracellular peptides. Supernatants and lysates were assayed for active GLP-1 and/or total PYY by ELISA (Millipore, USA). Hormone secretion was expressed as a fraction of the total hormone content per well, normalised to basal secretion measured in parallel control wells. Forskolin (FSK, 10 μmol), isobutylmethylxanthine (IBMX, 10 μmol/l), phorbol myristate acetate (PMA, 1 μmol/l), GW9508 (100 μmol/l), AR231453 (10 nmol/l) and GPBAR1-A (3 μmol/l; Roche, Basel), were added from 1,000x stocks in DMSO. GW9508 and AR231453 were synthesised by AstraZeneca (Macclesfield, UK) and supplied as a kind gift. Linoleic acid was applied at 100 μmol/l from a 5 mmol/l stock prepared in saline buffer + 5% (wt/vol.) BSA. All chemicals were obtained from Sigma (Poole, UK), unless otherwise stated.

One-sample Student’s t tests were performed using Microsoft Excel to test the null hypothesis that the data were from a population of mean = 1. A p value of less than 0.05 was considered significant.