Somatostatin receptor 5 and cannabinoid receptor 1 activation inhibit secretion of glucose-dependent insulinotropic polypeptide from intestinal K cells in rodents

Animal procedures were approved by the local ethics committee and conformed to UK Home Office regulations. All mice used in the experiments were on a C57BL/6 background and housed in individually ventilated cages. Male Sprague Dawley rats were purchased from Charles River (Margate, UK) and housed in conventional caging.

K and L cells were isolated by FACS from GIP-Venus [11] and GLU-Venus [14] transgenic mice, respectively, as previously described. Briefly, small intestine (upper 10 cm) or colon was digested as described below for primary cultures, but using a higher collagenase concentration of 1 mg/ml in Hank’s buffered salt solution (HBSS), to obtain single cells. Cell suspensions were separated using a MoFlo Cytomation sorter (Beckman Coulter, High Wycombe, UK) (488 nm excitation) to obtain populations of Venus-positive or Venus-negative cells, which were collected directly into lysis buffer for mRNA extraction. Purified K and L cell populations were over 95% pure as assessed by fluorescence microscopy, and were enriched more than 2,000-fold for GIP and glucagon-like peptide-1 (GLP-1) concentrations, respectively, compared with non-fluorescent cells, as assessed by ELISA [11, 14].

RNA was extracted using a microscale RNA isolation kit (Applied Biosystems, Warrington, UK) as previously described [11, 14]. GLUTag cell RNA was isolated using TRI reagent. For hybridisation to mouse 430 2.0 arrays GeneChips (Affymetrix UK, High Wycombe, UK), RNA underwent two rounds of amplification based on in vitro translation from a T7 promoter introduced during oligo dT-priming (Two-cycles cDNA Synthesis Kit, Affymetrix UK; MEGAscript T7 kit, Ambion, Austin, TX, USA), and expression levels of each probe were determined by robust multichip average (RMA) analysis. Quantitative PCR was performed with cDNA reverse-transcribed from non-amplified RNA on a 7900 HT Fast Real-Time PCR system (Applied Biosystems, Foster City, CA, USA) using a QuantiTect SYBR Green PCR mix (Qiagen, Crawley, UK) and the primers given in Table 1.

In all cases, expression was compared with that of β-actin measured on the same sample in parallel on the same plate, giving a cycle threshold (C_t) difference (ΔC_t) for β-actin minus the test gene. C_ts for β-actin ranged from 20 to 25 in the different samples, and reactions in which the test gene was undetectable were assigned a C_t value of 40. A ΔC_t value of −12 to −15 is, therefore, close to the detection limit of the assay. Mean, SEM and statistics were performed on the ΔC_t data and only converted to relative expression levels (\( 2^{{\Delta {\text{C}}_{{\text{t}}} }} \)) for presentation in the figures.

Mice aged 2–6 months were killed by cervical dislocation and the guts collected into ice-cold Leibovitz-15 (L-15) medium (PAA, Yeovil, UK). Mixed intestinal cultures were produced, as previously described [11, 14]. Briefly, the intestine was opened longitudinally, rinsed in PBS and chopped into 1–2 mm pieces. Upper small intestinal cultures were derived from 10–15 cm of intestine immediately distal to the stomach, and colon cultures included tissue distal to the caecum. Tissue was digested three or four times with 0.4 mg/ml collagenase XI, centrifuged at 300 g, and resuspended in DMEM (25 mmol/l glucose) supplemented with 10% (vol/vol) FBS, 2 mmol/l l-glutamine, 100 units/ml penicillin and 0.1 mg/ml streptomycin. Cell/crypt suspensions were plated onto 24-well plates coated with a thin film of Matrigel (BD Bioscience, Oxford, UK) and incubated at 37°C in 5% CO₂.

GLUTag cells were cultured in DMEM (5.6 mmol/l glucose) supplemented with 10% (vol/vol) FBS, 2 mmol/l l-glutamine, 100 units/ml penicillin and 0.1 mg/ml streptomycin, at 37°C in 5% CO₂.

Around 20–24 h after cell plating, wells were washed thoroughly with bath solution before being incubated for 2 h at 37°C with test reagents in bath solution supplemented with 0.1% BSA. Five hundred KIU/ml aprotinin, 10 μmol/l amastatin and 0.1 mmol/l diprotin A were added to wells planned for GLP-1 assay. The supernatants were collected and briefly centrifuged before measuring the hormone content. Primary cultures were subsequently treated with lysis buffer containing 50 mmol/l Tris-HCl, 150 mmol/l NaCl, 1% IGEPAL-CA 630, 0.5% deoxycholic acid and one tablet of complete EDTA-free protease inhibitor cocktail (Roche Dignostics, Burgess Hill, UK). Cells were mechanically disrupted to extract intracellular peptides. GLP-1, GIP and/or somatostatin were assayed in supernatant fractions and cell extracts from primary intestinal cultures, and in supernatant fractions from GLUTag cells by ELISA (GLP-1 active ELISA kit, GIP Total ELISA kit, Millipore, USA; Somatostatin ELISA Kit, Phoenix Pharmaceuticals, Burlingame, CA, USA).

Hormone secretion from primary cultures was expressed as a fraction of the total hormone measured in each well and normalised to the basal secretion measured in parallel on the same day. Rates of hormone secretion from primary cultures under basal conditions were 5.1 ± 0.7 pg well⁻¹ 2 h⁻¹ (GIP, n = 47), 1.7 ± 0.1 fmol well⁻¹ 2 h⁻¹ (GLP-1 from small intestinal cultures, n = 37) and 3.9 ± 0.6 fmol well⁻¹ 2 h⁻¹ (GLP-1 from colonic cultures, n = 9), which was equivalent to 2.2 ± 0.2%, 6.3 ± 0.4% and 3.0 ± 0.3% of the content being released in 2 h, respectively. Secretion from GLUTag cells was normalised to control wells from the same plate. Each condition was tested in three or four wells per 24-well plate, so the number of individual experiments can be estimated by dividing the numbers above the bars by 3 or 4.

Single-cell measurements of cAMP levels were made using the Förster resonance energy transfer (FRET)-based sensor Epac2-camps [15]. GLUTag cells were seeded in 6 cm Matrigel-coated plastic dishes and, when 80–90% confluent, were transfected with 3 μg Epac2-camps DNA probe and 15 μl Lipofectamine 2000 (Invitrogen, Paisley, UK) for 24 h. Cells were trypsinised and re-seeded onto Matrigel-coated 35 mm glass-bottomed dishes (Mattek Corporation, Ashland, MA, USA) for experiments 24–48 h later.

Cells, continuously perfused with bath solution supplemented with 0.1% BSA with or without test reagents, were visualised with a ×40 oil immersion objective on an inverted microscope (Olympus IX71, Southend on Sea, UK). Excitation at 435 nm was achieved using a xenon arc lamp coupled to a monochromator (Cairn Research, Faversham, UK) controlled by MetaFluor software (Molecular Devices, Wokingham, UK). Cyan fluorescent protein (CFP) emission at 470 nm and YFP emission at 535 nm were monitored using an Optosplit II beam splitter (Cairn Research) and an Orca-ER digital camera (Hamamatsu, Welwyn Garden City, UK), and expressed as the CFP/YFP fluorescence ratio [16]. Maximum ratios were determined at baseline and following test reagent application, and calculated increments were normalised to that triggered by the first forskolin application. No significant differences in the response (by ANOVA) were observed with recurrent forskolin applications.

Male Sprague Dawley rats (Charles River, Margate, UK) underwent surgical placement of a single right jugular vein catheter as previously described [17], and were allowed to recover for 4–5 days. Only animals that had regained their preoperative body weight with no signs of infection were allowed to proceed to the study. On the day of study, overnight-fasted rats were acclimatised to the study room and caging for an hour prior to experimentation. A total of 30 min before the OGTT, rats in the methanandamide (mAEA) study were dosed intraperitoneally with either mAEA (at a dose of 10 mg/kg, as previously described [18]) suspended in Tocrisolve (1 ml/kg) or an equal volume of Tocrisolve alone. In the AM251 study, rats were dosed intraperitoneally with either vehicle control (lacking only AM251) or 4 mg/kg of the CB1 antagonist AM251 made up in DMSO, and dissolved in Tween 80 and normal saline in a 1:1:8 ratio, with an injection volume of 1 ml/kg [19]. An oral gavage of 2 g/kg glucose and 100 mg/kg acetaminophen dissolved in PBS (10 ml/kg) was administered at t = 0. Blood samples of 0.5 ml were taken at −30, 0, 5, 15 and 30 min after gavage. Blood was collected into tubes containing 4 μl EDTA and immediately spun for 90 s. Plasma glucose was analysed by Analox GM9 (Analox Instruments, London, UK); samples for GIP, GLP-1, insulin and acetaminophen were snap-frozen for later analysis. Plasma GIP was measured by ELISA (GIP Total ELISA Kit; Millipore, Watford, UK), total GLP-1 and insulin by immunoassay (MesoScale Discovery, Gaithersburg, MD, USA), and acetaminophen by colorimetric detection of p-aminophenol (Siemens, Camberley, UK). Rats were killed by overdose of anaesthesia immediately after the 30 min sample had been taken.

The bath solution contained (in mmol/l) 138 NaCl, 4.5 KCl, 4.2 NaHCO₃, 1.2 NaH₂PO₄, 2.6 CaCl₂, 1.2 MgCl₂, 10 HEPES, 10 glucose. Unless stated, all drugs and chemicals were obtained from Sigma (Poole, UK) and, where possible, drugs were made up as 1,000× stock. The mAEA (Tocris, Bristol, UK) was dissolved in ethanol for in vitro and Tocrisolve (Tocris) for in vivo studies. Somatostatin-14 was dissolved in water. AM251 (Tocris) and the SSTR5 antagonist, referred to as SSTR5A (Compound A described in [20]; a kind gift from F. Hoffmann-La Roche AG, Basel, Switzerland) were dissolved in DMSO.

Comparisons between conditions were made using Student’s t test (Microsoft Excel) or by one or two-way ANOVA with post hoc Bonferroni test (Prism5, GraphPad, La Jolla, CA, USA), with a threshold for significance of p < 0.05. All data are expressed as mean ± SEM.