Animals and ethical considerations

Male Wistar rats ( ~ 250 g, 9 weeks) and male C57BL/6JRj mice (~25 g,12 weeks) were obtained from Janvier (Le Genest-Saint-Isle, France) and housed four rats per cage and eight mice/cage (allowing 102,5 cm2/mouse). Mice and rats were allowed one week of acclimatization and kept on a 12:12 h light/dark cycle with ad libitum access to water and standard chow.

Studies were conducted with permission from the Danish Animal Experiments Inspectorate (2023-15-0201-01408) and the local ethical committee (EMED P23-262 and P23-263) in accordance with the EU Directive 2010/63/EU and guidelines of Danish legislation governing animal experimentation (1987) and the National Institute of Health.

In vivo studies

Experiments were carried out on two occasions on mice (26.3 ± 1.9 g) fasted for 5 h. Mice were divided into weight-matched groups, (n = 8; l-valine and d-glucose group, n = 6; control group). At −30 min, mice received a cocktail consisting of the neprilysin (NEP) inhibitor, sacubitril (0.3 mg/kg, 5 uL/g, cat. no. 333-B1070, Nordic Biosite, Täby, Sweden) and the dipeptidyl peptidase (DPP)-4 inhibitor, sitagliptin (10 mg/kg, 5 uL/g, Xelevia; Merck, Sharp & Dohme) orally as described elsewhere [19]. At time 0 min, 75 uL tail blood was collected into ethylenediaminetetraacetic acid (EDTA) coated capillary tubes (cat. no. 167313, Micro Haematocrit Tubes, Vitrex Medical A/S, Herlev, Denmark) and instantly transferred onto ice. At time 0 min, mice were given orally either l-valine (1 g/kg, 20 uL/g), d-glucose (2 g/kg, 20 uL/g) or milliQ water (20 uL/g) mixed with acetaminophen (100 mg/kg). l-valine and d-glucose were prepared in milliQ water. Mice from the same cage received different treatments. Tail blood was collected at times; 0, 5, 10 and 30 min. Glucose concentration was measured prior to collection of blood samples. An extra blood sample for glucose measurement was taken at time 60 min. After blood collection, the mice were euthanized by cervical dislocation. Blood samples were centrifuged (6500 rpm, 4 °C, 10 min) within half an hour to obtain plasma. Plasma was transferred to pre-chilled PCR tubes (Thermowell, Gold PCR; Corning, NY) and placed on ice until storage at −20 °C.

Isolation and perfusion of the rat small and large intestine

Non-fasted rats were anaesthetized with a subcutaneous injection of Hypnorm/Midazolam (0.0158 mg fentanyl citrate+0.5 mg fluanisone+0.25 mg midazolam/100 g). When lack of reflexes was established, rats were placed on a heating plate (37 °C), and the abdominal cavity opened. The proximal small intestine (~37 cm) was isolated by ligating the vascular supply to the distal part and removing the colon. The colon was isolated by ligating the vascular supply to the cecum, the small intestine, the spleen, the stomach, the kidneys and the celiac artery, allowing isolation of the most proximal part of the colon to the part just proximal to the entry of inferior mesenteric artery (~10 cm). A plastic tube was placed in the lumen of the proximal small/large intestine, and the intestine was gently flushed with isotonic saline (room temperature) to remove luminal contents. Throughout the experimental protocol, a constant luminal flow of saline was applied via a syringe pump (0.25 ml/min; small intestine, 0.15 ml/min; colon). For perfusion of the small intestine, a catheter was inserted into the superior mesenteric artery, and the intestine was vascularly perfused with heated (37 °C), oxygenated (95% O2 and 5% CO2) perfusion buffer at a constant flow rate of 7.5 ml/min using a single pass perfusion system (UP100, Hugo Sachs Harvard Apparatus, Germany). For perfusion of the colon, a catheter was inserted into the abdominal aorta, which was ligated proximally to the superior mesenteric artery, tributaries to the small intestine were ligated and the colon was vascularly perfused at a constant flow rate of 3 ml/min. During both operations, a metal catheter was inserted into the vena portae to collect the venous effluent. As soon as proper flow was apparent, rats were euthanized by perforation of the diaphragm. To allow for equilibration of the system, the intestine/colon was perfused for 25 min before initiation of the experimental protocol.

Each protocol started with a baseline period followed by the addition of the test substance applied either through the luminal tube or the vascular route. The venous effluent was collected for 1 min periods using a fraction collector. Effluent samples were immediately placed on ice and stored at −20 °C until analysis. As an indicator of the health of the small/large intestine, perfusion pressure was monitored throughout the experiment. The procedure is described in more detail elsewhere [17, 18, 22].

Perfusion buffer and test substances

The perfusion buffer consisted of a modified Krebs-Ringer bicarbonate buffer supplemented with 3.5 mM glucose, 0.1% (w/v) bovine serum albumin (cat. no. 1.12018.0500, Merck, Denmark), 5% (w/v) dextran T-70 (to balance oncotic pressure; Pharmacosmos, Denmark), 5 mM of each fumarate, pyruvate and glutamate (Sigma Aldrich, Brøndby, Denmark) and 10 μM 3-isobutyl-1-methylxanthine (IBMX, cat no. 5879, Sigma Aldrich). The pH was adjusted to ~7.4 prior to experiments.

All test substances were purchased from Sigma Aldrich (Brøndby, Denmark) unless otherwise stated. Test substances included the following compounds infused to reach the following final concentrations; 50 mM l-valine (cat. no. V0513), 10 µM nifedipine (cat. no. N7634), 250 µM diazoxide (cat no. D9035).

In vitro studies

GLUTag cells (generously provided by Professor Drucker from the University of Toronto in Canada) were cultured at 37 °C in an environment with 5% CO2 in Dulbecco’s modified Eagle’s medium (DMEM) with 5.6 mM glucose, supplemented with 10% fetal bovine serum, 1% penicillin (10,000 U/ml) and streptomycin (10,000 μg/ml), as well as 200 mM Glutamax (Thermofisher, Denmark). The cells were subcultured at a 1:5 dilution when they reached 80–85% confluence. For this study, we used cells with passage numbers ranging from 31 to 34.

In secretion studies, GLUTag cells were plated in 24-well culture plates at a density of 250,000 cells per well. Once the cells reached 80% confluence, which typically occurred within 24 to 48 h, the cells were subjected to a 30-minute period of starvation using freshly prepared Krebs-Ringer-HEPES (KRH) buffer. This KRH buffer contained 138 mM NaCl, 4.5 mM KCl, 4.2 mM NaHCO3, 1.2 mM NaH2PO4, 2.5 mM CaCl2, 1.2 mM MgCl2, and 10 mM HEPES, with a pH of 7.4, and it did not contain glucose or albumin. Following the starvation period, the cells were exposed to either KRH with 5.6 mM glucose (baseline secretion) or the same buffer containing varying concentrations of l-valine. We collected supernatants and processed them by centrifuging at 1500 × g, 4 °C, for 5 min to remove any debris. Supernatants were rapidly frozen and stored at −20 °C until further analysis. Experiments were conducted on three separate occasions, with each experiment having three technical replicates.

For calcium fluorimetry, we utilized a method employing Fluo-4 AM (Thermofisher). Cells were plated in a 96-well plate at a density of 50,000 cells per well and were cultured for 24–48 h. Before the experiment, the cells were loaded with 0.2% Fluo-4 AM, which was dissolved in HEPES-buffered Hank’s Balanced Salt Solution (HBSS, Thermofisher) containing 1 mM CaCl2, 1 mM MgCl2, and 0.5% probenecid (Thermofisher) for one hour. After a double-wash step with the same HBSS buffer without Fluo-4 AM, the plate was transferred to a FlexStation 3 from Molecular Devices. Fluorescence measurement was performed at excitation/emission λ of 494/506 nm. The background signal was registered for 20 s, followed by the addition of l-valine, using the instrument’s built-in automated pipetting system. Kinetic changes in intracellular calcium mobilization were registered for the total of 90 s. In some experiments, we introduced blockers to the wells after loading with Fluo-4 AM and waited for 15 min before taking readings. These blockers included nifedipine (2 mg/mL in DMSO), diazoxide (10 mg/mL in DMSO), and EDTA (0.5 M in NaOH), all purchased from VWR.

Biochemical measurements

Peptide hormones were measured using in-house radioimmunoassays: total GLP-1 (the sum of 7-36NH2, 9-36NH2 and potential mid-terminal cleaved fragments) was measured using a C-terminal specific antibody targeting amidated forms of GLP-1 (code no. 89390) [23]. Total PYY (PYY1–36 + PYY3–36) was measured with a porcine antiserum (cat. no. T-4093; Bachem) [24]. For the in vitro studies, GLP-1 was measured using an antibody targeting the mid-region of GLP-1 (code no. 2135). Blood glucose was measured using a glucometer (AccuChek Mobile, cat. no. 05874149001; Roche Diagnostics, Mannheim, Germany). Plasma concentrations of active GLP-1 (7–36) were measured by sandwich ELISA (cat. no. 80-GLP1A-CH01, Alpco, Salem, New Hampshire). Total L-amino acid concentrations in venous effluents were quantified using a commercial colorimetric L-amino acid assay kit from Abcam (cat. no. ab65347).

Calculations and statistical analysis

For analyses of GLP-1 secretion in perfusion experiments, hormone concentrations are presented in pM as mean values ± SEM. Mean concentrations were calculated for a 10-minutes baseline period before each stimulation/inhibition and for the last 10 min of each stimulation/inhibition period. For the baseline-subtracted concentrations, the individual baseline values were subtracted for each animal before calculating the mean value for the group. For within-group analyses, statistical significance was tested with a paired Student’s t-test or one-way ANOVA followed by Tukey’s post hoc analysis for multiple comparisons. For between-group analyses, statistical significance was tested with an unpaired Student’s t-test. The in vivo study was analyzed using two-way ANOVA followed by Šídák multiple comparisons test. Total areas under the curves were compared by one-way ANOVA followed by Tukey’s multiple comparison test. The in vitro studies were analyzed using one-way ANOVA. Post hoc analyses were performed using the Dunnett method.

Statistical testing and construction of graphs were done in GraphPad Prism version 10.1.2 (GraphPad Software, La Jolla, CA). P values < 0.05 were considered statistically significant.