Glucagon-Like Peptide-1 Inhibits Prandial Gastrointestinal Motility Through Myenteric Neuronal Mechanisms in Humans

Glucagon-Like Peptide-1 Inhibits Prandial Gastrointestinal Motility Through Myenteric Neuronal... Abstract Context Glucagon-like peptide-1 (GLP-1) secretion from l-cells and postprandial inhibition of gastrointestinal motility. Objective Investigate whether physiological plasma concentrations of GLP-1 inhibit human postprandial motility and determine mechanism of action of GLP-1 and analog ROSE-010 action. Design Single-blind parallel study. Setting University hospital laboratory. Participants Healthy volunteers investigated with antroduodenal manometry. Human gastric and intestinal muscle strips. Interventions Motility indices (MIs) obtained before and during GLP-1 or saline infusion. Plasma GLP-1 and glucagon-like peptide-2 (GLP-2) measured by radioimmunoassay. Gastrointestinal muscle strips investigated for GLP-1- and ROSE-010-induced relaxation employing GLP-1 and GLP-2 and their receptor localization, and blockers exendin(9-39)amide, Lω-nitro-monomethylarginine (L-NMMA), 2′,5′-dideoxyadenosine (DDA), and tetrodotoxin (TTX) to reveal target mechanism of GLP-1 action. Main Outcome Measures Postprandial gastrointestinal relaxation by GLP-1. Results In humans, food intake increased MI to 6.4 ± 0.3 (antrum), 5.7 ± 0.4 (duodenum), and 5.9 ± 0.2 (jejunum). GLP-1 administered intravenously raised plasma GLP-1, but not GLP-2. GLP-1 0.7 pmol/kg/min suppressed corresponding MI to 4.6 ± 0.2, 4.7 ± 0.4, and 5.0 ± 0.2, whereas 1.2 pmol/kg/min suppressed MI to 5.4 ± 0.2, 4.4 ± 0.3, and 5.4 ± 0.3 (P < 0.0001 to 0.005). In vitro, GLP-1 and ROSE-010 prevented contractions by bethanechol and electric field stimulation (P < 0.005 to 0.05). These effects were disinhibited by exendin(9-39)amide, L-NMMA, DDA, or TTX. GLP-1 and GLP-2 were localized to epithelial cells, GLP-1 also at myenteric neurons. GLP-1R and GLP-2R were localized at myenteric neurons but not muscle. Conclusions GLP-1 and ROSE-010 inhibit postprandial gastrointestinal motility through GLP-1R at myenteric neurons, involving nitrergic and cyclic adenosine monophosphate–dependent mechanisms. During a meal, gastrointestinal (GI) motility is stimulated with occurrence of irregular contractions, so-called segmentation, that move the food content back and forth, mixing it with digestive enzymes secreted into the intestine. The motility pattern governing transit through the small intestine is called the “fed” motility pattern, but has not been clearly defined in terms of GI muscle activity. This pattern is in contrast to the “fasting” motility pattern with the recurring migrating motility complex (MMC) propagating along the small intestine toward the colon. The physiological importance of these two different motor patterns differ. Whereas the “fed” motility pattern is assumed to enhance mixing and churning of the contents to optimize absorption, the “fasting” motility pattern has been ascribed a bacterial clearing effect to prevent bacterial overgrowth in the small bowel (1). Glucagon-like peptide-1 (GLP-1) is best known for its incretin effect, enhancing insulin secretion in response to a meal (2). GLP-1 also regulates several GI functions. It decreases gastric acid secretion (3), GI transit (4–7), motility (5, 8), and gastric wall tone (9). It is not clear how all these GI effects take place, but they are considered to be rapid and transient as GLP-1 has a plasma half-life of approximately 1 to 2 minutes (10). GLP-1 reduces contractions in human intestinal muscle (11). It seems that GLP-1 receptor (GLP-1R) agonists may be of value for treatment of certain motility disorders with a prominent overactivity in the gut, including irritable bowel syndrome (IBS) (12). Therefore, the stable GLP-1 analog ROSE-010 was investigated and found to reduce acute exacerbations of IBS (13). ROSE-010 stability is achieved by substitution of alanine with valine at amino acid position 8, which prolongs its plasma half-life after subcutaneous injection to about 60 minutes, however with some loss of intrinsic stimulating activity as compared with native GLP-1 (14). The mechanism of action of GLP-1 is enigmatic. GLP-1 inhibits gastric emptying in rats (5, 6, 15–17), pigs (18, 19), and humans (20). In animals, this inhibition was shown to be lost following vagal afferent denervation (6, 15), indicating that GLP-1 intervenes with the dorsal motor nucleus of the vagus in the central nervous system (CNS) and parasympathetic control of gastric emptying. Alternatively, circulating GLP-1 may gain access to the CNS through binding at the area postrema and subfornical organ where the GLP-1R is accessible via leaky capillaries (21, 22). Diffusion through the blood-brain barrier has also been suggested (23). However, the motility effects of GLP-1 can also be exerted peripherally. Research in animals indicates that locally produced nitric oxide (NO) is involved in the inhibitory effect of GLP-1 on fasting intestinal motility (5, 17, 24), and studies in humans show that gastric relaxation in response to a meal is dependent on NO (25). We have previously shown in rats that GLP-1 inhibits gastric emptying and small-bowel MMC myoelectric activity during fasting and after food intake concurrent with slowing of GI transit (5, 6). These findings are consistent with those of others showing inhibitory effects of GLP-1 on GI motility, including the MMC in humans (4, 8, 26). Furthermore, experimental work in the rat has shown that glucagon-like peptide-2 (GLP-2) acts additively with GLP-1 with respect to inhibition of fasting motility (27). Because GI motility is stimulated by food intake and released GLP-1 exerts an inhibitory effect on motility, the primary aim was to clarify whether GLP-1 at near physiological concentrations is able to inhibit digestive motility. Therefore, human manometry recordings were pursued with administration of low-dose GLP-1. As secondary aim, because the exact mechanism by which GLP-1 inhibits motility is not fully characterized, the actions of GLP-1 and its dipeptidyl peptidase-4-resistant analog ROSE-010 were studied in organ baths employing exendin(9-39)amide as GLP-1R antagonist tetrodotoxin (TTX) to block neuronal voltage-gated Na+ channels, 2´5′-dideoxyadenosine (DDA) as adenylate cyclase inhibitor, and Lω-nitro-monomethyl arginine (L-NMMA) as NO synthase inhibitor. In addition, the histological localization of GLP-1 and GLP-1R in the human stomach, small intestine, and colon were investigated. Because motility effects have also been ascribed to GLP-2 and this peptide is thought to coemerge in the circulation with GLP-1 during posttranslational processing of proglucagon (28), plasma concentrations of GLP-2 and localization of its receptor, GLP-2 receptor (GLP-2R), along the GI tract were also investigated. Materials and Methods Test subjects and patients Sixteen healthy male test subjects 18 to 55 years of age were studied. None of the subjects had GI symptoms or disease or earlier abdominal surgery. The subjects were screened for inclusion in the study by physical examination, body mass index of 20 to 25 m/kg2, normal blood counts, serum electrolytes, kidney function, and liver enzymes. On the day before and during the experiments, all test subjects abstained from smoking and intake of alcohol and caffeine. The in vivo experiments were approved by the Swedish Medical Products Agency (diary number: 151.2003/36261) and the the Ethics Committees of Karolinska Institutet and Uppsala University (01-313 updated version 2013/965-32) and carried out from 2008 to 2010. The in vitro experiments were approved by the Regional Ethics Committee at Uppsala University (2010/157 and 2010/184) and carried out from 2013 to 2016. Informed consent was obtained from all subjects. The study was registered at www.ClinicalTrials.gov (no. NTC02731664). Chemicals For in vivo experiments, GLP-1 [GLP-1-(7-36)-amide acetate] was purchased from the PolyPeptide Group (Wolfenbüttel, Germany) or Bachem (Bubendorf, Switzerland). The peptide was dissolved and diluted in sterile 0.9% NaCl and filtered (pore size, 0.22 µm; Millex-Micropore, Darmstadt, Germany). Vials of 10 mL were prepared, containing sterile GLP-1 (25 nmol/mL) with 0.25 mL albumin (200 mg/mL; Pharmacia, Uppsala, Sweden) added. Saline for intravenous (IV) infusion, 0.154 mol/L NaCl, was purchased from Pharmacia (Stockholm, Sweden). GLP-1 was diluted in saline in proportion to body weight. Saline or GLP-1 was infused (1 mL/min) using an infusion pump (Alaris IVAC 7100 Infusion Pump; P.M.S. Instruments Ltd, Maidenhead, United Kingdom). For in vitro experiments, GLP-1 (from PolyPeptide Group) and ROSE-010 (from Rose Pharma, Copenhagen, Denmark) were dissolved in sterile 0.9% saline to stock solutions of 100 μmol/L. Bethanechol and DDA were purchased from Sigma-Aldrich (St. Louis, MO). Exendin(9-39)amide was obtained from Mark Biosciences AG (Läufelfingen, Germany). TTX was purchased from Tocris Bioscience (Minneapolis, MN). For immunohistochemistry, rabbit polyclonal antibodies against GLP-1 (antiserum 2135-4) and mouse monoclonal against GLP-2 (antiserum 312-01) were obtained from JJ Holst. Goat polyclonal antibodies against GLP-1R (antiserum Y-12) and GLP-2R (H-57) were from Santa Cruz Biotechnology (Dallas, TX). Rabbit monoclonal antibody against neuronal enolase was purchased from Cell Signaling Technology [(Beverly, MA) (antiserum D20H2)]. Study design The study was conducted as an exploratory, controlled, single-center, single-blind, parallel design study as illustrated in Fig. 1A. Randomization was done by envelope draw. After overnight fasting, 1-hour baseline recordings were performed with subjects in recumbent position receiving an IV saline infusion (n = 16). At the beginning of this hour, blood was drawn (baseline plasma samples). At the end of this hour, a 1-hour treatment period was started in which recordings were continued and subjects received infusions of saline as control (n = 6) or GLP-1 at doses of 0.7 (n = 4) or 1.2 (n = 6) pmol/kg/min in a randomized, single-blinded fashion. A 310-kcal mixed meal consisting of an egg omelet and a soft drink was consumed during the infusion. At the end of this hour, a prandial blood sample was drawn. Plasma was prepared for GLP-1 and GLP-2 measurements as detailed later. The lower dose of GLP-1 gives near-physiological fed-state plasma concentrations of the peptide in healthy subjects (22). Previous dose escalation studies (22, 29, 30) demonstrated that subjects tolerate doses up to 1.2 pmol/kg/min without adverse reactions, albeit slight queasiness. During experiments, the subjects were monitored for heart rate, arterial blood pressure, and peak expiratory flow. They were also questioned about adverse events during the infusion and the day after the experiment. Figure 1. View largeDownload slide Effects of infused GLP-1 on plasma GLP-1 and GLP-2 concentrations and upper GI motility. The protocol used is shown in (A). Baseline columns are combined data for the two GLP-1 doses used. Box-and-whisker plots showing steady-state concentrations of C-terminal (B) GLP-1 and (C) GLP-2 1 hour before and 1 hour after a mixed meal, during which time subjects were given IV infusions of saline or GLP-1 (0.7 or 1.2 pmol/kg/min). MIs of (D) gastric antrum, (E) duodenum, and (F) jejunum in healthy volunteers were quantified for a 1-hour interval of preprandial saline infusion (baseline) and then for the following prandial 1-hour infusion of continued saline or GLP-1 at doses of 0.7 or 1.2 pmol/kg/min. *P < 0.05, ***P < 0.001 (relative to baseline) and #P < 0.05, ##P < 0.01, P < 0.001 (relative to control). AUC, area under the curve. Figure 1. View largeDownload slide Effects of infused GLP-1 on plasma GLP-1 and GLP-2 concentrations and upper GI motility. The protocol used is shown in (A). Baseline columns are combined data for the two GLP-1 doses used. Box-and-whisker plots showing steady-state concentrations of C-terminal (B) GLP-1 and (C) GLP-2 1 hour before and 1 hour after a mixed meal, during which time subjects were given IV infusions of saline or GLP-1 (0.7 or 1.2 pmol/kg/min). MIs of (D) gastric antrum, (E) duodenum, and (F) jejunum in healthy volunteers were quantified for a 1-hour interval of preprandial saline infusion (baseline) and then for the following prandial 1-hour infusion of continued saline or GLP-1 at doses of 0.7 or 1.2 pmol/kg/min. *P < 0.05, ***P < 0.001 (relative to baseline) and #P < 0.05, ##P < 0.01, P < 0.001 (relative to control). AUC, area under the curve. Motility recordings The motility pattern of the proximal GI tract was monitored using a multichannel polyvinylchloride tube (length, 250 cm; diameter, 4.7 mm; eight channels 0.7 mm wide, ending as side holes, three located orally and spaced by 1.5 cm, another three side holes located 11.5 cm aborally and spaced by 2 cm, another separate side hole located 13 cm further aborally, and the last side hole 15 cm further beyond; William Cook, Bjaeverskov, Denmark). The tube was passed nasally and positioned by fluoroscopy with its distal recording sites beyond the angle of Treitz and tethered to the cheek. The proximal recording points were located in antrum, the middle group in duodenum, whereas the distal two were positioned in jejunum. Each channel was perfused with water at 0.1 mL/min from a low-compliance system (Arndorfer Medical Specialties, Greendale, WI) and connected to external pressure transducers (Synmed, Stockholm, Sweden). Pressure transducers were connected via a PC Polygraph HR (Synmed) to a computer (486D/66 MHz; Dell Corporation, Austin, TX). Polygram Lower GI 6.40 software (Synmed) recorded the data at 4-Hz sampling frequency. Pressure rise upon occlusion of each channel exceeded 200 mm Hg/s. Analysis of motility recordings Motility was computed for the 60-minute baseline period before food challenge and separately for the prandial 60 minutes of the saline or GLP-1 infusions. Polygram software (Synmed) was used to calculate the motility index (MI) from the frequency, duration, and amplitude of contractions, summarized as ln[Σ(mm Hg/s)/min] for area under the pressure curve per minute over each 60-minute recording period (20, 31). This calculation of MI has previously been evaluated by Holland et al. (32) Baseline threshold for contractions was set at 8 mm Hg. Infusions of saline or GLP-1 were carried out during a fed motility pattern (1), defined as a pattern of 2 to 10 phasic contractions per minute over 10-minute periods in the jejunum. Plasma concentrations of GLP-1 and GLP-2 Blood samples were drawn into cold 10-mL EDTA vacutainer tubes. Samples were immediately centrifuged (1500g, 4°C, 10 minutes), and the plasma supernatants were stored at –20°C until analysis. Before radioimmunoassay (RIA) of GLP-1 and GLP-2, the plasma samples were extracted in a final concentration of 75% ethanol to remove unspecific cross-reacting substances. The RIA for determination of plasma concentrations of GLP-1 was performed as previously described (33). The detection limit was <5 pmol/L, and the intra-assay coefficient of variation 7%. The RIA for GLP-2 was done as described elsewhere (33). The detection limit was <5 pmol/L and coefficient of variation 5%. Immunohistochemistry of GLP-1, GLP-2, and their receptors GLP-1R and GLP-2R Paraffin-embedded sections of human gastric corpus, jejunum, and colon were obtained from patients (three male, six female; age 37 to 71 years) undergoing surgeries as indicated previously. Primary antibodies were rabbit polyclonal against GLP-1 (1:120) and mouse monoclonal against GLP-2 (1:30), each resuspended from lyophilized powders to original volumes to prepare stocks, whereas antibodies against GLP-1R (1:50) and GLP-2R (1:100). Neuron-specific staining was confirmed with rabbit monoclonal primary antibody (Ab) against neuronal enolase (1:1000). The tissue was immunostained by horseradish peroxidase-diaminobenzidine [anti-mouse secondary antibody (Ab)] or alkaline phosphatase-Fast Red (anti-rabbit secondary Ab). Specific immunostaining was assessed in a blinded fashion by two independent researchers (M.A.H., D.-L.W.). Organ bath experiments of GI smooth muscle strips Tissue segments were obtained from gastric corpus and jejunum of patients undergoing gastric bypass surgery for morbid obesity. Colon tissues were noncancerous sections at the margin of resection obtained during surgery for colorectal cancer. Total number of patients were 47 (male n = 36, female n = 11; age 49 to 87 years). The tissue segments were placed in ice-cold Krebs solution (mmol/L: NaCl, 121.5; CaCl2, 2.5; KH2PO4, 1.2; KCl, 4.7; MgSO4, 1.2; NaHCO3, 25; d-glucose, 5.6) equilibrated with 5% CO2 and 95% O2, and within 5 to 10 minutes after resection were transported to the laboratory. The mucosa was removed and strips (2 to 3 mm wide, each 12 to 14 mm long) were cut along the circular axis. The strips (one to four strips from each patient) were mounted between two platinum ring electrodes in 5-mL organ bath chambers (PanLab, Barcelona, Spain) containing Krebs solution, continuously bubbled with 5% CO2 and 95% O2 and maintained at 37°C and pH 7.4. Tension was measured using isometric force transducers (MLT0201; PanLab). Data acquisition was performed using Powerlab hardware and LabChart 7 software (ADInstruments, Cambridge, United Kingdom). Muscle strips were equilibrated to a 2-g tension baseline for at least 60 minutes, during which the bathing medium was replaced every 15 minutes. After equilibration, muscle strips were stimulated with bethanechol 10 µmol/L for 8 minutes as control. This concentration of bethanechol showed submaximal contractile responses corresponding to the half maximal effective concentration (EC50) value. GLP-1 and ROSE-010 were given as pretreatments 2 minutes before bethanechol stimulation for 8 minutes. The response to GLP-1 and ROSE-010 was also tested in the presence of TTX (1 µmol/L), L-NMMA (100 µmol/L), exendin (9-39) amide (1 µmol/L), and DDA (10 µmol/L). These compounds were given 2 minutes before GLP-1 or ROSE-010 (i.e., 4 minutes before bethanechol stimulation). To test for possible prejunctional effects of GLP-1, tissue contraction was evoked by electric field stimulation (EFS) using biphasic square wave pulses of 0.6-ms duration (10 Hz, 50 V, 0.6 train/min) with a GRASS S88 stimulator (Grass Technologies, Astro-Med Inc., West Warwick, RI). For this purpose, GLP-1 was added to tissue preparations during continuous EFS. Statistics Results are presented as mean ± standard of the mean (SEM). For in vivo recordings, statistical comparisons of MIs between 60 minutes basal and 60 minutes prandial at each recording site were carried out employing the nonparametric Kruskal-Wallis test. Then, the motility responses to food intake were compared between basal and prandial, as well as between prandial with saline and the two doses of GLP-1 using the Kruskal-Wallis test, whereas comparison with the combined doses of GLP-1 was done using the Mann-Whitney U test. In addition, statistical comparisons were made between the prandial motility response and the combined doses of GLP-1 using the nonparametric Mann-Whitney U test. For in vitro studies, the Student t test was used to compare two groups for all the treatment groups in the organ bath. One-way analysis of variance was applied to compare concentration-response relationships. The significance level was set at 0.05 employing a one-sided or two-sided test as appropriate. Results Vital signs and adverse events In all subjects, systemic arterial blood pressure, heart rate, and peak expiratory flow were unchanged during the infusion of GLP-1. No adverse reactions to GLP-1 were encountered, except for slight queasiness. Plasma concentrations of GLP-1 and GLP-2 At 1 hour prior to the meal, plasma concentrations of GLP-1 and GLP-2 were at baseline (Fig. 1B and 1C). After the meal, infusion of GLP-1 at 0.7 pmol/kg/min IV during the next hour increased plasma GLP-1 concentrations 2.7-fold and GLP-1 infusion at 1.2 pmol/kg/min IV increased plasma GLP-1 5.4-fold above the plasma levels in the saline-infused control group (Fig. 1B). Plasma GLP-2 did not rise measurably with this meal and was unaffected by the GLP-1 infusion (Fig. 1C). GLP-1 effects on prandial motility in vivo All subjects were in the fasted state as verified by a propagated MMC terminating at least 20 minutes before onset of the study. Motility of the gastric antrum, duodenum, and jejunum for 1 hour before and for 1 hour after the meal showed an increased MI during the prandial period. GLP-1 at 0.7 pmol/kg/min IV reduced the MI across all studied segments (antrum, P < 0.01; duodenum, P < 0.05; jejunum, P < 0.05; Fig. 1C–1E). The higher dose of GLP-1 at 1.2 pmol/kg/min IV gave a similar response (antrum, P < 0.05; duodenum, P < 0.05; Fig. 1D–1F). The combined effect of the two doses together reduced the MI in antrum as compared with saline by 1.3 ± 0.3 ln [Σ(mm Hg/s)/min] (P < 0.0001), in the duodenum by 1.2 ± 0.4 ln [Σ(mm Hg/s)/min] (P < 0.0002), and in the jejunum by 0.6 (P < 0.005) ln [Σ(mm Hg/s)/min]. Immunohistochemistry of GLP-1 and GLP-2 and their receptors As expected, immunoreactivity to GLP-1 and GLP-2 was localized in a subset of gut epithelial cells; stronger immunoreactivity was found in jejunum and colon compared with corpus (Fig. 2A). In the gastric corpus and jejunum, strong immunoreactivity to GLP-1 was also found in the nerve cell bodies of the myenteric plexus, whereas no GLP-2 immunoreactivity was found in nerves (Fig. 2A). In the colon, GLP-1 showed weaker immunoreactivity in nerve cell bodies. No immunostaining of either GLP-1 or GLP-2 was observed in smooth muscle. Figure 2. View largeDownload slide Immunostaining of (A) GLP-1 and GLP-2 and (B) their receptors GLP-1R and GLP-2R in the human gastric corpus, jejunum, and colon at myenteric plexus. Left panels are ×10 magnification, indicating low background throughout. Right panels are ×40 magnification centered at mucosa and myenteric plexus and ×20 magnification centered at myenteric plexus. Brown color is horseradish peroxidase-diaminobenzidine staining and pink-red color is alkaline phosphatase-Fast Red staining, each indicating immunoreactivity. Figure 2. View largeDownload slide Immunostaining of (A) GLP-1 and GLP-2 and (B) their receptors GLP-1R and GLP-2R in the human gastric corpus, jejunum, and colon at myenteric plexus. Left panels are ×10 magnification, indicating low background throughout. Right panels are ×40 magnification centered at mucosa and myenteric plexus and ×20 magnification centered at myenteric plexus. Brown color is horseradish peroxidase-diaminobenzidine staining and pink-red color is alkaline phosphatase-Fast Red staining, each indicating immunoreactivity. Strong GLP-1R and GLP-2R immunoreactivity was observed in nerve cell bodies of myenteric neurons (Fig. 2), whereas circular and longitudinal muscles were negative in all parts of the GI tract. Epithelial cells in all the tissues studied showed immunoreactivity to the GLP-1R, but little or none was found for the GLP-2R (data not shown). Mechanism of motility inhibition by GLP-1 and ROSE-010 To observe relaxation responses to GLP-1 and ROSE-010 in the organ bath, tissue contractions were stimulated with bethanechol or EFS. GLP-1 (1 to 100 nmol/L) caused concentration-dependent inhibition of bethanechol-induced contractions in the small bowel and colon muscle and reached statistical significance at and above 10 nmol/L of GLP-1 (P < 0.005 to 0.05; Fig. 3A). Data were reproduced with GLP-1-induced relaxation in the gastric corpus, but not enough for a complete set of experiments due to limited tissue availability. The EC50 was calculated to 40 nM. Similarly, ROSE-010 (1 to 1000 nmol/L) caused concentration-dependent reduction of bethanechol-induced contractions in the small bowel and colon (P < 0.005 to 0.05; Fig. 3B) with a slightly higher EC50, calculated to 50 nM. The responses obtained at the maximum intrinsic activity of GLP-1 (100 nmol/L) and ROSE-010 (1000 nmol/L) corresponded to ∼61% ± 13% reduction of the amplitude of bethanechol-stimulated contractions. At equal concentrations, GLP-1 was more potent to inhibit the motility response than ROSE-010, but ROSE-010 was able to reach the same degree of inhibition with higher concentrations. The smooth muscle relaxation effect of GLP-1 and ROSE-010 was completely reversible after washout with Krebs solution in between additions of the peptide. The response to EFS was inhibited by GLP-1 at and above concentrations of 10 nmol/L (Fig. 3C). Figure 3. View largeDownload slide Concentration-dependent inhibition of muscle contractions by GLP-1 and ROSE-010. (A) GLP-1 and (B) ROSE-010 concentration dependently inhibited bethanechol-induced contractions in human jejunum (n = 6) and colon (n = 6) tissue. Data are expressed as percentage of inhibition of 10 µM bethanechol-stimulated contractions. *P < 0.05 and **P < 0.005 compared with respective control. (C) GLP-1 inhibited EFS-induced contractions, reversibly with washouts between each GLP-1 treatment (n = 3). Data are mean ± standard error of the mean. Figure 3. View largeDownload slide Concentration-dependent inhibition of muscle contractions by GLP-1 and ROSE-010. (A) GLP-1 and (B) ROSE-010 concentration dependently inhibited bethanechol-induced contractions in human jejunum (n = 6) and colon (n = 6) tissue. Data are expressed as percentage of inhibition of 10 µM bethanechol-stimulated contractions. *P < 0.05 and **P < 0.005 compared with respective control. (C) GLP-1 inhibited EFS-induced contractions, reversibly with washouts between each GLP-1 treatment (n = 3). Data are mean ± standard error of the mean. Pretreatment with exendin(9-39)amide (1000 nmol/L) markedly disinhibited the smooth muscle relaxation effect of GLP-1 and ROSE-010 (Fig. 4A and 4B), as did pretreatment with DDA (10 µmol/L) (Fig. 4C and 4D). After addition of exendin(9-39)amide, the relaxation induced by GLP-1 was more effectively mitigated as compared with that of ROSE-010. Relaxation by GLP-1 and ROSE-010 was abolished by L-NMMA (10 mmol/L) (Fig. 5A and 5B) and by TTX (1 µmol/L) (Fig. 5C and 5D), indicating that responses are dependent on neural and nitrergic transmission. Neither TTX, L-NMMA, nor exendin(9-39)amide alone affected baseline tension of the muscle strips. Figure 4. View largeDownload slide Concentration-response curves for inhibitory effects induced by GLP-1 and ROSE-010 under control conditions and in presence of exendin(9-39)amide (Exen9-39) or DDA. Exendin(9-39)amide (1 µM) blocked (A) GLP-1-induced relaxation (n = 9) and (B) ROSE-010-induced relaxation (n = 6). Exendin(9-39)amide blocked GLP-1-induced relaxation more potently than that of ROSE-010. Relaxation by (C) GLP-1 (n = 9) and (D) ROSE-010 (n = 6) was blocked by the adenylate cyclase inhibitor DDA (10 µM). In all experiments, control contraction was induced by bethanechol in absence of GLP-1 or L-NMMA. In all experiments, control contraction was induced by bethanechol alone. *P < 0.05 and **P < 0.005 compared with respective control. Figure 4. View largeDownload slide Concentration-response curves for inhibitory effects induced by GLP-1 and ROSE-010 under control conditions and in presence of exendin(9-39)amide (Exen9-39) or DDA. Exendin(9-39)amide (1 µM) blocked (A) GLP-1-induced relaxation (n = 9) and (B) ROSE-010-induced relaxation (n = 6). Exendin(9-39)amide blocked GLP-1-induced relaxation more potently than that of ROSE-010. Relaxation by (C) GLP-1 (n = 9) and (D) ROSE-010 (n = 6) was blocked by the adenylate cyclase inhibitor DDA (10 µM). In all experiments, control contraction was induced by bethanechol in absence of GLP-1 or L-NMMA. In all experiments, control contraction was induced by bethanechol alone. *P < 0.05 and **P < 0.005 compared with respective control. Figure 5. View largeDownload slide Concentration-response curves for inhibitory effects induced by GLP-1 and ROSE-010 under control conditions and presence of L-NMMA or TTX. L-NMMA (100 µM) (A) abolished GLP-1-induced relaxation (n = 7) and (B) reduced ROSE-010-induced relaxation (n = 5). TTX (1 µM) abolished relaxation induced by (C) GLP-1 (n = 7) and (D) ROSE-010 (n = 6) in the colon. In all experiments, control contraction was induced by bethanechol alone. *P < 0.05 and **P < 0.005 compared with respective control. Figure 5. View largeDownload slide Concentration-response curves for inhibitory effects induced by GLP-1 and ROSE-010 under control conditions and presence of L-NMMA or TTX. L-NMMA (100 µM) (A) abolished GLP-1-induced relaxation (n = 7) and (B) reduced ROSE-010-induced relaxation (n = 5). TTX (1 µM) abolished relaxation induced by (C) GLP-1 (n = 7) and (D) ROSE-010 (n = 6) in the colon. In all experiments, control contraction was induced by bethanechol alone. *P < 0.05 and **P < 0.005 compared with respective control. Discussion Unique approaches to stimulate the GLP-1R, such as with ROSE-010, may have a clinical application for functional GI disorders, including motility disorders and sensory mechanisms in the gut. This study with infusion of exogenous GLP-1 to only slightly more than physiological concentrations inhibited the fed motor activity in the antrum and reduced motility along the remaining intestinal segments. Based on the calculated MI, we can confirm in humans our previous animal findings where GLP-1 inhibited motility in the fasting as well as fed state (5, 6, 20, 33, 34). Even though the number of observations in our study is limited, the present results align with those of others studying different animal species (15, 16, 18, 19, 35) and man (4, 8, 26), in which also the metabolic impact of an inhibited antroduodenal motility has been shown. Indeed, the mechanism of GLP-1 action on gastric fasting volume and accommodation seems dependent on intact vagal functions (36). In our present study, plasma GLP-1 concentrations changed with dosing, and there was no consistent GLP-1 dose-response relationship for in vivo motility across the different intestinal segments. Possibly this reflects that even the lower doses result in saturation of all available receptors and therefore maximal effects were achieved. However, in smooth muscle experiments where a multistep graded concentration increase could be used, clear concentration-response relationships for GLP-1 and its analog ROSE-010 were observed. The GLP-1 concentrations required for in vitro responses were high relative to the receptor dissociation constant, Kd ∼28 pM (37). This could be due to instability of the GLP-1 peptide or to impaired permeability and diffusion conditions for the peptide in the incubated tissue sample. These findings in human tissue in vitro are in agreement with our previous study in man showing reduced small bowel fasting motility of GLP-1 in healthy subjects and patients with IBS (20), as well as other studies employing ROSE-010, shown to ameliorate abdominal pain in patients with IBS (13) and facilitate left colon emptying in diarrhea-dominant IBS by dampening excessive distal colonic contractions (38). Taken together, data from our group and others seem to converge on an inhibitory action of GLP-1, most markedly shown by the summarized data of both doses and its analog ROSE-010 on motility. These data are now further substantiated by our findings of the presence of GLP-1, possibly also with a contribution from GLP-2R, in neuronal tissue. We used bethanechol to selectively stimulate muscarinic receptors on GI smooth muscle strips. Then, GLP-1 was applied to induce a motility response. The GLP-1 concentrations used in vitro in this study are similar to those used in a previous ex vivo study (35). The inhibitory effect of GLP-1 on motility was blocked by exendin(9-39)amide as shown in human islets (39), indicating a GLP-1R-mediated mechanism for this action of the peptide. Furthermore, exendin(9-39)amide also inhibited the response of ROSE-010 on GI smooth muscle, consistent with GLP-1 and ROSE-010 acting on the same GLP-1R. The inhibitory effect of GLP-1 as studied in vitro appears to take place through neuronal signaling. Commensurate with our immunohistochemistry findings of GLP-1R on neuronal tissue, GLP-1 inhibited the contractile response to EFS. Also, TTX, which selectively blocks neuronal signal transmission, blocked the response to both GLP-1 and ROSE-010. Furthermore, L-NMMA blocked the inhibitory effect of GLP-1 through GLP-1R located on neurons, demonstrating that GLP-1 inhibition relies on nitrergic mechanisms. In different parts of the GI tract of various species, the inhibition of motility by exogenous GLP-1 has been shown to be dependent on endogenous NO (6, 17, 25, 35). Therefore, nitrergic transmission should be regarded as an important step in GLP-1-induced regulation of GI motility (40, 41). However, we cannot exclude the possibility that GLP-1 also has a direct inhibitory effect on cholinergic neurons in parallel to an excitation of nitrergic neurons. In our hands, immunohistochemistry revealed the presence of GLP-1R within the myenteric plexus, suggesting that GLP-1 acts on enteric neurons. Commensurate with this, our previous studies show presence of NO synthase in myenteric neurons (42, 43), with functional importance for motility (42), which supports a cross-talk between neuronal GLP-1 and NO. To this end, we found that the GLP-1R is coupled to the G-protein Gsα subunit that activates a cyclic adenosine monophosphate (cAMP)–dependent pathway through adenylyl cyclase. Therefore, agonist binding to the GLP-1R should result in adenylate cyclase activation with consequent cAMP production (44) and inhibition of motility. It is well recognized that cAMP is the main mediator of GLP-1R agonism. Our data using DDA supports that a similar mechanism underlies the motility responses to GLP-1 and ROSE-010. Furthermore, cAMP signaling can increase NO production (45, 46), so these two second messengers are suggested to act in concert and synchronize relaxatory mechanisms in the GI tract. To accomplish these effects, the GLP-1-induced motility response seems to involve both a neuronal and a smooth muscle step. In agreement with our findings, NO can be released from myenteric neurons (42, 43) as well as from interstitial cells of Cajal (47), resulting in an increase in cAMP at the level of the smooth muscle to induce relaxation. GLP-1 and GLP-2 are believed to be cosecreted from the l-cells of the mucosal lining in response to food intake. Because GLP-1R receptors are present on epithelial l-cells (48), as confirmed with immunohistochemistry in this study (not shown), it was unknown whether GLP-1 infusion would alter GLP-1 or GLP-2 secretion, which would theoretically influence the response to GLP-1. Unlike GLP-1, plasma GLP-2 was neither markedly increased after the meal, nor influenced by exogenous GLP-1 infusion. The fact that GLP-2 did not rise much following the meal suggests that GLP-1 is a more prominent signal for motility changes and insulin release during meals than GLP-2. A possible reason for disparities between GLP-1 and GLP-2 may also be explained by differences in the sensitivity to degradation by dipeptidyl peptidase-4 (49, 50) or differences in liver clearance. Moreover, baseline (fasted) GLP-2 was 17 times higher than baseline GLP-1 and 3 times higher than postprandial GLP-1 (saline group). Hence, cosecreted postprandial GLP-2 may also have been masked by the pre-existing high ambient GLP-2. To conclude, GLP-1 is able to reduce the digestive motility in the antroduodenojejunal region and slow gastric emptying in humans. The mechanism of this inhibitory response is likely exerted through the GLP-1R expressed in myenteric neurons as the primary functional target for GLP-1 and analogs, such as ROSE-010. Furthermore, both GLP-1 and ROSE-010 exercise receptor-mediated responses with NO and cAMP as second messengers, both of which are required for a regulatory action to take place. GLP-1 is suggested to primarily exert its effects on motility via direct local actions in the periphery, which may coexist with indirect actions mediated through the CNS, be it through vagal afferents originating in the GI tract or by binding to receptors within the CNS. Abbreviations: Ab antibody cAMP cyclic adenosine monophosphate CNS central nervous system DDA 2′,5′-dideoxyadenosine EC50 half maximal effective concentration EFS electric field stimulation GI gastrointestinal GLP-1 glucagon-like peptide-1 GLP-1R glucagon-like peptide-1 receptor GLP-2 glucagon-like peptide-2 GLP-2R glucagon-like peptide-2 receptor IBS irritable bowel syndrome IV intravenous L-NMMA Lω-nitro-monomethylarginine MI motility index MMC migrating motility complex NO nitric oxide NOS nitric oxide synthase RIA radioimmunoassay TTX tetrodotoxin. Acknowledgments Financial Support: This work was supported by the Swedish Research Council (7916) and the Karolinska Institutet (to P.M.H.), Bengt Ihre’s Foundation (SLS-411011, SLS-503061, SLS-506051, SLS-591271, SLS-594831), the Swedish Society of Medicine (SLS-411921, SLS-503131), the Gastroenterology Research Foundation (SLS-504191), Regionala forskningsrådet (RFR), Uppsala/Örebro region (RFR-476131), Björklund’s Foundation (SLS-589741), Sven Jerring’s Foundation and Avtal om läkarutbildning och forskning (ALF) funds, Sweden, and OE and Edla Johansson’s Science Foundation 2017. Clinical Trial Information: ClinicalTrials.gov no. NTC02731664 (registered 16 March 2016). Author Contributions: M.A.H., D.-L.W., and P.M.H. designed the study. J.J.H. performed RIA. P.M.H. enrolled healthy volunteers and performed randomization and motility experiments. M.A.H. performed immunostaining and organ bath experiments. M.S. and U.K. provided human tissue. 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Glucagon-Like Peptide-1 Inhibits Prandial Gastrointestinal Motility Through Myenteric Neuronal Mechanisms in Humans

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Endocrine Society
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Copyright © 2018 Endocrine Society
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0021-972X
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1945-7197
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10.1210/jc.2017-02006
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Abstract

Abstract Context Glucagon-like peptide-1 (GLP-1) secretion from l-cells and postprandial inhibition of gastrointestinal motility. Objective Investigate whether physiological plasma concentrations of GLP-1 inhibit human postprandial motility and determine mechanism of action of GLP-1 and analog ROSE-010 action. Design Single-blind parallel study. Setting University hospital laboratory. Participants Healthy volunteers investigated with antroduodenal manometry. Human gastric and intestinal muscle strips. Interventions Motility indices (MIs) obtained before and during GLP-1 or saline infusion. Plasma GLP-1 and glucagon-like peptide-2 (GLP-2) measured by radioimmunoassay. Gastrointestinal muscle strips investigated for GLP-1- and ROSE-010-induced relaxation employing GLP-1 and GLP-2 and their receptor localization, and blockers exendin(9-39)amide, Lω-nitro-monomethylarginine (L-NMMA), 2′,5′-dideoxyadenosine (DDA), and tetrodotoxin (TTX) to reveal target mechanism of GLP-1 action. Main Outcome Measures Postprandial gastrointestinal relaxation by GLP-1. Results In humans, food intake increased MI to 6.4 ± 0.3 (antrum), 5.7 ± 0.4 (duodenum), and 5.9 ± 0.2 (jejunum). GLP-1 administered intravenously raised plasma GLP-1, but not GLP-2. GLP-1 0.7 pmol/kg/min suppressed corresponding MI to 4.6 ± 0.2, 4.7 ± 0.4, and 5.0 ± 0.2, whereas 1.2 pmol/kg/min suppressed MI to 5.4 ± 0.2, 4.4 ± 0.3, and 5.4 ± 0.3 (P < 0.0001 to 0.005). In vitro, GLP-1 and ROSE-010 prevented contractions by bethanechol and electric field stimulation (P < 0.005 to 0.05). These effects were disinhibited by exendin(9-39)amide, L-NMMA, DDA, or TTX. GLP-1 and GLP-2 were localized to epithelial cells, GLP-1 also at myenteric neurons. GLP-1R and GLP-2R were localized at myenteric neurons but not muscle. Conclusions GLP-1 and ROSE-010 inhibit postprandial gastrointestinal motility through GLP-1R at myenteric neurons, involving nitrergic and cyclic adenosine monophosphate–dependent mechanisms. During a meal, gastrointestinal (GI) motility is stimulated with occurrence of irregular contractions, so-called segmentation, that move the food content back and forth, mixing it with digestive enzymes secreted into the intestine. The motility pattern governing transit through the small intestine is called the “fed” motility pattern, but has not been clearly defined in terms of GI muscle activity. This pattern is in contrast to the “fasting” motility pattern with the recurring migrating motility complex (MMC) propagating along the small intestine toward the colon. The physiological importance of these two different motor patterns differ. Whereas the “fed” motility pattern is assumed to enhance mixing and churning of the contents to optimize absorption, the “fasting” motility pattern has been ascribed a bacterial clearing effect to prevent bacterial overgrowth in the small bowel (1). Glucagon-like peptide-1 (GLP-1) is best known for its incretin effect, enhancing insulin secretion in response to a meal (2). GLP-1 also regulates several GI functions. It decreases gastric acid secretion (3), GI transit (4–7), motility (5, 8), and gastric wall tone (9). It is not clear how all these GI effects take place, but they are considered to be rapid and transient as GLP-1 has a plasma half-life of approximately 1 to 2 minutes (10). GLP-1 reduces contractions in human intestinal muscle (11). It seems that GLP-1 receptor (GLP-1R) agonists may be of value for treatment of certain motility disorders with a prominent overactivity in the gut, including irritable bowel syndrome (IBS) (12). Therefore, the stable GLP-1 analog ROSE-010 was investigated and found to reduce acute exacerbations of IBS (13). ROSE-010 stability is achieved by substitution of alanine with valine at amino acid position 8, which prolongs its plasma half-life after subcutaneous injection to about 60 minutes, however with some loss of intrinsic stimulating activity as compared with native GLP-1 (14). The mechanism of action of GLP-1 is enigmatic. GLP-1 inhibits gastric emptying in rats (5, 6, 15–17), pigs (18, 19), and humans (20). In animals, this inhibition was shown to be lost following vagal afferent denervation (6, 15), indicating that GLP-1 intervenes with the dorsal motor nucleus of the vagus in the central nervous system (CNS) and parasympathetic control of gastric emptying. Alternatively, circulating GLP-1 may gain access to the CNS through binding at the area postrema and subfornical organ where the GLP-1R is accessible via leaky capillaries (21, 22). Diffusion through the blood-brain barrier has also been suggested (23). However, the motility effects of GLP-1 can also be exerted peripherally. Research in animals indicates that locally produced nitric oxide (NO) is involved in the inhibitory effect of GLP-1 on fasting intestinal motility (5, 17, 24), and studies in humans show that gastric relaxation in response to a meal is dependent on NO (25). We have previously shown in rats that GLP-1 inhibits gastric emptying and small-bowel MMC myoelectric activity during fasting and after food intake concurrent with slowing of GI transit (5, 6). These findings are consistent with those of others showing inhibitory effects of GLP-1 on GI motility, including the MMC in humans (4, 8, 26). Furthermore, experimental work in the rat has shown that glucagon-like peptide-2 (GLP-2) acts additively with GLP-1 with respect to inhibition of fasting motility (27). Because GI motility is stimulated by food intake and released GLP-1 exerts an inhibitory effect on motility, the primary aim was to clarify whether GLP-1 at near physiological concentrations is able to inhibit digestive motility. Therefore, human manometry recordings were pursued with administration of low-dose GLP-1. As secondary aim, because the exact mechanism by which GLP-1 inhibits motility is not fully characterized, the actions of GLP-1 and its dipeptidyl peptidase-4-resistant analog ROSE-010 were studied in organ baths employing exendin(9-39)amide as GLP-1R antagonist tetrodotoxin (TTX) to block neuronal voltage-gated Na+ channels, 2´5′-dideoxyadenosine (DDA) as adenylate cyclase inhibitor, and Lω-nitro-monomethyl arginine (L-NMMA) as NO synthase inhibitor. In addition, the histological localization of GLP-1 and GLP-1R in the human stomach, small intestine, and colon were investigated. Because motility effects have also been ascribed to GLP-2 and this peptide is thought to coemerge in the circulation with GLP-1 during posttranslational processing of proglucagon (28), plasma concentrations of GLP-2 and localization of its receptor, GLP-2 receptor (GLP-2R), along the GI tract were also investigated. Materials and Methods Test subjects and patients Sixteen healthy male test subjects 18 to 55 years of age were studied. None of the subjects had GI symptoms or disease or earlier abdominal surgery. The subjects were screened for inclusion in the study by physical examination, body mass index of 20 to 25 m/kg2, normal blood counts, serum electrolytes, kidney function, and liver enzymes. On the day before and during the experiments, all test subjects abstained from smoking and intake of alcohol and caffeine. The in vivo experiments were approved by the Swedish Medical Products Agency (diary number: 151.2003/36261) and the the Ethics Committees of Karolinska Institutet and Uppsala University (01-313 updated version 2013/965-32) and carried out from 2008 to 2010. The in vitro experiments were approved by the Regional Ethics Committee at Uppsala University (2010/157 and 2010/184) and carried out from 2013 to 2016. Informed consent was obtained from all subjects. The study was registered at www.ClinicalTrials.gov (no. NTC02731664). Chemicals For in vivo experiments, GLP-1 [GLP-1-(7-36)-amide acetate] was purchased from the PolyPeptide Group (Wolfenbüttel, Germany) or Bachem (Bubendorf, Switzerland). The peptide was dissolved and diluted in sterile 0.9% NaCl and filtered (pore size, 0.22 µm; Millex-Micropore, Darmstadt, Germany). Vials of 10 mL were prepared, containing sterile GLP-1 (25 nmol/mL) with 0.25 mL albumin (200 mg/mL; Pharmacia, Uppsala, Sweden) added. Saline for intravenous (IV) infusion, 0.154 mol/L NaCl, was purchased from Pharmacia (Stockholm, Sweden). GLP-1 was diluted in saline in proportion to body weight. Saline or GLP-1 was infused (1 mL/min) using an infusion pump (Alaris IVAC 7100 Infusion Pump; P.M.S. Instruments Ltd, Maidenhead, United Kingdom). For in vitro experiments, GLP-1 (from PolyPeptide Group) and ROSE-010 (from Rose Pharma, Copenhagen, Denmark) were dissolved in sterile 0.9% saline to stock solutions of 100 μmol/L. Bethanechol and DDA were purchased from Sigma-Aldrich (St. Louis, MO). Exendin(9-39)amide was obtained from Mark Biosciences AG (Läufelfingen, Germany). TTX was purchased from Tocris Bioscience (Minneapolis, MN). For immunohistochemistry, rabbit polyclonal antibodies against GLP-1 (antiserum 2135-4) and mouse monoclonal against GLP-2 (antiserum 312-01) were obtained from JJ Holst. Goat polyclonal antibodies against GLP-1R (antiserum Y-12) and GLP-2R (H-57) were from Santa Cruz Biotechnology (Dallas, TX). Rabbit monoclonal antibody against neuronal enolase was purchased from Cell Signaling Technology [(Beverly, MA) (antiserum D20H2)]. Study design The study was conducted as an exploratory, controlled, single-center, single-blind, parallel design study as illustrated in Fig. 1A. Randomization was done by envelope draw. After overnight fasting, 1-hour baseline recordings were performed with subjects in recumbent position receiving an IV saline infusion (n = 16). At the beginning of this hour, blood was drawn (baseline plasma samples). At the end of this hour, a 1-hour treatment period was started in which recordings were continued and subjects received infusions of saline as control (n = 6) or GLP-1 at doses of 0.7 (n = 4) or 1.2 (n = 6) pmol/kg/min in a randomized, single-blinded fashion. A 310-kcal mixed meal consisting of an egg omelet and a soft drink was consumed during the infusion. At the end of this hour, a prandial blood sample was drawn. Plasma was prepared for GLP-1 and GLP-2 measurements as detailed later. The lower dose of GLP-1 gives near-physiological fed-state plasma concentrations of the peptide in healthy subjects (22). Previous dose escalation studies (22, 29, 30) demonstrated that subjects tolerate doses up to 1.2 pmol/kg/min without adverse reactions, albeit slight queasiness. During experiments, the subjects were monitored for heart rate, arterial blood pressure, and peak expiratory flow. They were also questioned about adverse events during the infusion and the day after the experiment. Figure 1. View largeDownload slide Effects of infused GLP-1 on plasma GLP-1 and GLP-2 concentrations and upper GI motility. The protocol used is shown in (A). Baseline columns are combined data for the two GLP-1 doses used. Box-and-whisker plots showing steady-state concentrations of C-terminal (B) GLP-1 and (C) GLP-2 1 hour before and 1 hour after a mixed meal, during which time subjects were given IV infusions of saline or GLP-1 (0.7 or 1.2 pmol/kg/min). MIs of (D) gastric antrum, (E) duodenum, and (F) jejunum in healthy volunteers were quantified for a 1-hour interval of preprandial saline infusion (baseline) and then for the following prandial 1-hour infusion of continued saline or GLP-1 at doses of 0.7 or 1.2 pmol/kg/min. *P < 0.05, ***P < 0.001 (relative to baseline) and #P < 0.05, ##P < 0.01, P < 0.001 (relative to control). AUC, area under the curve. Figure 1. View largeDownload slide Effects of infused GLP-1 on plasma GLP-1 and GLP-2 concentrations and upper GI motility. The protocol used is shown in (A). Baseline columns are combined data for the two GLP-1 doses used. Box-and-whisker plots showing steady-state concentrations of C-terminal (B) GLP-1 and (C) GLP-2 1 hour before and 1 hour after a mixed meal, during which time subjects were given IV infusions of saline or GLP-1 (0.7 or 1.2 pmol/kg/min). MIs of (D) gastric antrum, (E) duodenum, and (F) jejunum in healthy volunteers were quantified for a 1-hour interval of preprandial saline infusion (baseline) and then for the following prandial 1-hour infusion of continued saline or GLP-1 at doses of 0.7 or 1.2 pmol/kg/min. *P < 0.05, ***P < 0.001 (relative to baseline) and #P < 0.05, ##P < 0.01, P < 0.001 (relative to control). AUC, area under the curve. Motility recordings The motility pattern of the proximal GI tract was monitored using a multichannel polyvinylchloride tube (length, 250 cm; diameter, 4.7 mm; eight channels 0.7 mm wide, ending as side holes, three located orally and spaced by 1.5 cm, another three side holes located 11.5 cm aborally and spaced by 2 cm, another separate side hole located 13 cm further aborally, and the last side hole 15 cm further beyond; William Cook, Bjaeverskov, Denmark). The tube was passed nasally and positioned by fluoroscopy with its distal recording sites beyond the angle of Treitz and tethered to the cheek. The proximal recording points were located in antrum, the middle group in duodenum, whereas the distal two were positioned in jejunum. Each channel was perfused with water at 0.1 mL/min from a low-compliance system (Arndorfer Medical Specialties, Greendale, WI) and connected to external pressure transducers (Synmed, Stockholm, Sweden). Pressure transducers were connected via a PC Polygraph HR (Synmed) to a computer (486D/66 MHz; Dell Corporation, Austin, TX). Polygram Lower GI 6.40 software (Synmed) recorded the data at 4-Hz sampling frequency. Pressure rise upon occlusion of each channel exceeded 200 mm Hg/s. Analysis of motility recordings Motility was computed for the 60-minute baseline period before food challenge and separately for the prandial 60 minutes of the saline or GLP-1 infusions. Polygram software (Synmed) was used to calculate the motility index (MI) from the frequency, duration, and amplitude of contractions, summarized as ln[Σ(mm Hg/s)/min] for area under the pressure curve per minute over each 60-minute recording period (20, 31). This calculation of MI has previously been evaluated by Holland et al. (32) Baseline threshold for contractions was set at 8 mm Hg. Infusions of saline or GLP-1 were carried out during a fed motility pattern (1), defined as a pattern of 2 to 10 phasic contractions per minute over 10-minute periods in the jejunum. Plasma concentrations of GLP-1 and GLP-2 Blood samples were drawn into cold 10-mL EDTA vacutainer tubes. Samples were immediately centrifuged (1500g, 4°C, 10 minutes), and the plasma supernatants were stored at –20°C until analysis. Before radioimmunoassay (RIA) of GLP-1 and GLP-2, the plasma samples were extracted in a final concentration of 75% ethanol to remove unspecific cross-reacting substances. The RIA for determination of plasma concentrations of GLP-1 was performed as previously described (33). The detection limit was <5 pmol/L, and the intra-assay coefficient of variation 7%. The RIA for GLP-2 was done as described elsewhere (33). The detection limit was <5 pmol/L and coefficient of variation 5%. Immunohistochemistry of GLP-1, GLP-2, and their receptors GLP-1R and GLP-2R Paraffin-embedded sections of human gastric corpus, jejunum, and colon were obtained from patients (three male, six female; age 37 to 71 years) undergoing surgeries as indicated previously. Primary antibodies were rabbit polyclonal against GLP-1 (1:120) and mouse monoclonal against GLP-2 (1:30), each resuspended from lyophilized powders to original volumes to prepare stocks, whereas antibodies against GLP-1R (1:50) and GLP-2R (1:100). Neuron-specific staining was confirmed with rabbit monoclonal primary antibody (Ab) against neuronal enolase (1:1000). The tissue was immunostained by horseradish peroxidase-diaminobenzidine [anti-mouse secondary antibody (Ab)] or alkaline phosphatase-Fast Red (anti-rabbit secondary Ab). Specific immunostaining was assessed in a blinded fashion by two independent researchers (M.A.H., D.-L.W.). Organ bath experiments of GI smooth muscle strips Tissue segments were obtained from gastric corpus and jejunum of patients undergoing gastric bypass surgery for morbid obesity. Colon tissues were noncancerous sections at the margin of resection obtained during surgery for colorectal cancer. Total number of patients were 47 (male n = 36, female n = 11; age 49 to 87 years). The tissue segments were placed in ice-cold Krebs solution (mmol/L: NaCl, 121.5; CaCl2, 2.5; KH2PO4, 1.2; KCl, 4.7; MgSO4, 1.2; NaHCO3, 25; d-glucose, 5.6) equilibrated with 5% CO2 and 95% O2, and within 5 to 10 minutes after resection were transported to the laboratory. The mucosa was removed and strips (2 to 3 mm wide, each 12 to 14 mm long) were cut along the circular axis. The strips (one to four strips from each patient) were mounted between two platinum ring electrodes in 5-mL organ bath chambers (PanLab, Barcelona, Spain) containing Krebs solution, continuously bubbled with 5% CO2 and 95% O2 and maintained at 37°C and pH 7.4. Tension was measured using isometric force transducers (MLT0201; PanLab). Data acquisition was performed using Powerlab hardware and LabChart 7 software (ADInstruments, Cambridge, United Kingdom). Muscle strips were equilibrated to a 2-g tension baseline for at least 60 minutes, during which the bathing medium was replaced every 15 minutes. After equilibration, muscle strips were stimulated with bethanechol 10 µmol/L for 8 minutes as control. This concentration of bethanechol showed submaximal contractile responses corresponding to the half maximal effective concentration (EC50) value. GLP-1 and ROSE-010 were given as pretreatments 2 minutes before bethanechol stimulation for 8 minutes. The response to GLP-1 and ROSE-010 was also tested in the presence of TTX (1 µmol/L), L-NMMA (100 µmol/L), exendin (9-39) amide (1 µmol/L), and DDA (10 µmol/L). These compounds were given 2 minutes before GLP-1 or ROSE-010 (i.e., 4 minutes before bethanechol stimulation). To test for possible prejunctional effects of GLP-1, tissue contraction was evoked by electric field stimulation (EFS) using biphasic square wave pulses of 0.6-ms duration (10 Hz, 50 V, 0.6 train/min) with a GRASS S88 stimulator (Grass Technologies, Astro-Med Inc., West Warwick, RI). For this purpose, GLP-1 was added to tissue preparations during continuous EFS. Statistics Results are presented as mean ± standard of the mean (SEM). For in vivo recordings, statistical comparisons of MIs between 60 minutes basal and 60 minutes prandial at each recording site were carried out employing the nonparametric Kruskal-Wallis test. Then, the motility responses to food intake were compared between basal and prandial, as well as between prandial with saline and the two doses of GLP-1 using the Kruskal-Wallis test, whereas comparison with the combined doses of GLP-1 was done using the Mann-Whitney U test. In addition, statistical comparisons were made between the prandial motility response and the combined doses of GLP-1 using the nonparametric Mann-Whitney U test. For in vitro studies, the Student t test was used to compare two groups for all the treatment groups in the organ bath. One-way analysis of variance was applied to compare concentration-response relationships. The significance level was set at 0.05 employing a one-sided or two-sided test as appropriate. Results Vital signs and adverse events In all subjects, systemic arterial blood pressure, heart rate, and peak expiratory flow were unchanged during the infusion of GLP-1. No adverse reactions to GLP-1 were encountered, except for slight queasiness. Plasma concentrations of GLP-1 and GLP-2 At 1 hour prior to the meal, plasma concentrations of GLP-1 and GLP-2 were at baseline (Fig. 1B and 1C). After the meal, infusion of GLP-1 at 0.7 pmol/kg/min IV during the next hour increased plasma GLP-1 concentrations 2.7-fold and GLP-1 infusion at 1.2 pmol/kg/min IV increased plasma GLP-1 5.4-fold above the plasma levels in the saline-infused control group (Fig. 1B). Plasma GLP-2 did not rise measurably with this meal and was unaffected by the GLP-1 infusion (Fig. 1C). GLP-1 effects on prandial motility in vivo All subjects were in the fasted state as verified by a propagated MMC terminating at least 20 minutes before onset of the study. Motility of the gastric antrum, duodenum, and jejunum for 1 hour before and for 1 hour after the meal showed an increased MI during the prandial period. GLP-1 at 0.7 pmol/kg/min IV reduced the MI across all studied segments (antrum, P < 0.01; duodenum, P < 0.05; jejunum, P < 0.05; Fig. 1C–1E). The higher dose of GLP-1 at 1.2 pmol/kg/min IV gave a similar response (antrum, P < 0.05; duodenum, P < 0.05; Fig. 1D–1F). The combined effect of the two doses together reduced the MI in antrum as compared with saline by 1.3 ± 0.3 ln [Σ(mm Hg/s)/min] (P < 0.0001), in the duodenum by 1.2 ± 0.4 ln [Σ(mm Hg/s)/min] (P < 0.0002), and in the jejunum by 0.6 (P < 0.005) ln [Σ(mm Hg/s)/min]. Immunohistochemistry of GLP-1 and GLP-2 and their receptors As expected, immunoreactivity to GLP-1 and GLP-2 was localized in a subset of gut epithelial cells; stronger immunoreactivity was found in jejunum and colon compared with corpus (Fig. 2A). In the gastric corpus and jejunum, strong immunoreactivity to GLP-1 was also found in the nerve cell bodies of the myenteric plexus, whereas no GLP-2 immunoreactivity was found in nerves (Fig. 2A). In the colon, GLP-1 showed weaker immunoreactivity in nerve cell bodies. No immunostaining of either GLP-1 or GLP-2 was observed in smooth muscle. Figure 2. View largeDownload slide Immunostaining of (A) GLP-1 and GLP-2 and (B) their receptors GLP-1R and GLP-2R in the human gastric corpus, jejunum, and colon at myenteric plexus. Left panels are ×10 magnification, indicating low background throughout. Right panels are ×40 magnification centered at mucosa and myenteric plexus and ×20 magnification centered at myenteric plexus. Brown color is horseradish peroxidase-diaminobenzidine staining and pink-red color is alkaline phosphatase-Fast Red staining, each indicating immunoreactivity. Figure 2. View largeDownload slide Immunostaining of (A) GLP-1 and GLP-2 and (B) their receptors GLP-1R and GLP-2R in the human gastric corpus, jejunum, and colon at myenteric plexus. Left panels are ×10 magnification, indicating low background throughout. Right panels are ×40 magnification centered at mucosa and myenteric plexus and ×20 magnification centered at myenteric plexus. Brown color is horseradish peroxidase-diaminobenzidine staining and pink-red color is alkaline phosphatase-Fast Red staining, each indicating immunoreactivity. Strong GLP-1R and GLP-2R immunoreactivity was observed in nerve cell bodies of myenteric neurons (Fig. 2), whereas circular and longitudinal muscles were negative in all parts of the GI tract. Epithelial cells in all the tissues studied showed immunoreactivity to the GLP-1R, but little or none was found for the GLP-2R (data not shown). Mechanism of motility inhibition by GLP-1 and ROSE-010 To observe relaxation responses to GLP-1 and ROSE-010 in the organ bath, tissue contractions were stimulated with bethanechol or EFS. GLP-1 (1 to 100 nmol/L) caused concentration-dependent inhibition of bethanechol-induced contractions in the small bowel and colon muscle and reached statistical significance at and above 10 nmol/L of GLP-1 (P < 0.005 to 0.05; Fig. 3A). Data were reproduced with GLP-1-induced relaxation in the gastric corpus, but not enough for a complete set of experiments due to limited tissue availability. The EC50 was calculated to 40 nM. Similarly, ROSE-010 (1 to 1000 nmol/L) caused concentration-dependent reduction of bethanechol-induced contractions in the small bowel and colon (P < 0.005 to 0.05; Fig. 3B) with a slightly higher EC50, calculated to 50 nM. The responses obtained at the maximum intrinsic activity of GLP-1 (100 nmol/L) and ROSE-010 (1000 nmol/L) corresponded to ∼61% ± 13% reduction of the amplitude of bethanechol-stimulated contractions. At equal concentrations, GLP-1 was more potent to inhibit the motility response than ROSE-010, but ROSE-010 was able to reach the same degree of inhibition with higher concentrations. The smooth muscle relaxation effect of GLP-1 and ROSE-010 was completely reversible after washout with Krebs solution in between additions of the peptide. The response to EFS was inhibited by GLP-1 at and above concentrations of 10 nmol/L (Fig. 3C). Figure 3. View largeDownload slide Concentration-dependent inhibition of muscle contractions by GLP-1 and ROSE-010. (A) GLP-1 and (B) ROSE-010 concentration dependently inhibited bethanechol-induced contractions in human jejunum (n = 6) and colon (n = 6) tissue. Data are expressed as percentage of inhibition of 10 µM bethanechol-stimulated contractions. *P < 0.05 and **P < 0.005 compared with respective control. (C) GLP-1 inhibited EFS-induced contractions, reversibly with washouts between each GLP-1 treatment (n = 3). Data are mean ± standard error of the mean. Figure 3. View largeDownload slide Concentration-dependent inhibition of muscle contractions by GLP-1 and ROSE-010. (A) GLP-1 and (B) ROSE-010 concentration dependently inhibited bethanechol-induced contractions in human jejunum (n = 6) and colon (n = 6) tissue. Data are expressed as percentage of inhibition of 10 µM bethanechol-stimulated contractions. *P < 0.05 and **P < 0.005 compared with respective control. (C) GLP-1 inhibited EFS-induced contractions, reversibly with washouts between each GLP-1 treatment (n = 3). Data are mean ± standard error of the mean. Pretreatment with exendin(9-39)amide (1000 nmol/L) markedly disinhibited the smooth muscle relaxation effect of GLP-1 and ROSE-010 (Fig. 4A and 4B), as did pretreatment with DDA (10 µmol/L) (Fig. 4C and 4D). After addition of exendin(9-39)amide, the relaxation induced by GLP-1 was more effectively mitigated as compared with that of ROSE-010. Relaxation by GLP-1 and ROSE-010 was abolished by L-NMMA (10 mmol/L) (Fig. 5A and 5B) and by TTX (1 µmol/L) (Fig. 5C and 5D), indicating that responses are dependent on neural and nitrergic transmission. Neither TTX, L-NMMA, nor exendin(9-39)amide alone affected baseline tension of the muscle strips. Figure 4. View largeDownload slide Concentration-response curves for inhibitory effects induced by GLP-1 and ROSE-010 under control conditions and in presence of exendin(9-39)amide (Exen9-39) or DDA. Exendin(9-39)amide (1 µM) blocked (A) GLP-1-induced relaxation (n = 9) and (B) ROSE-010-induced relaxation (n = 6). Exendin(9-39)amide blocked GLP-1-induced relaxation more potently than that of ROSE-010. Relaxation by (C) GLP-1 (n = 9) and (D) ROSE-010 (n = 6) was blocked by the adenylate cyclase inhibitor DDA (10 µM). In all experiments, control contraction was induced by bethanechol in absence of GLP-1 or L-NMMA. In all experiments, control contraction was induced by bethanechol alone. *P < 0.05 and **P < 0.005 compared with respective control. Figure 4. View largeDownload slide Concentration-response curves for inhibitory effects induced by GLP-1 and ROSE-010 under control conditions and in presence of exendin(9-39)amide (Exen9-39) or DDA. Exendin(9-39)amide (1 µM) blocked (A) GLP-1-induced relaxation (n = 9) and (B) ROSE-010-induced relaxation (n = 6). Exendin(9-39)amide blocked GLP-1-induced relaxation more potently than that of ROSE-010. Relaxation by (C) GLP-1 (n = 9) and (D) ROSE-010 (n = 6) was blocked by the adenylate cyclase inhibitor DDA (10 µM). In all experiments, control contraction was induced by bethanechol in absence of GLP-1 or L-NMMA. In all experiments, control contraction was induced by bethanechol alone. *P < 0.05 and **P < 0.005 compared with respective control. Figure 5. View largeDownload slide Concentration-response curves for inhibitory effects induced by GLP-1 and ROSE-010 under control conditions and presence of L-NMMA or TTX. L-NMMA (100 µM) (A) abolished GLP-1-induced relaxation (n = 7) and (B) reduced ROSE-010-induced relaxation (n = 5). TTX (1 µM) abolished relaxation induced by (C) GLP-1 (n = 7) and (D) ROSE-010 (n = 6) in the colon. In all experiments, control contraction was induced by bethanechol alone. *P < 0.05 and **P < 0.005 compared with respective control. Figure 5. View largeDownload slide Concentration-response curves for inhibitory effects induced by GLP-1 and ROSE-010 under control conditions and presence of L-NMMA or TTX. L-NMMA (100 µM) (A) abolished GLP-1-induced relaxation (n = 7) and (B) reduced ROSE-010-induced relaxation (n = 5). TTX (1 µM) abolished relaxation induced by (C) GLP-1 (n = 7) and (D) ROSE-010 (n = 6) in the colon. In all experiments, control contraction was induced by bethanechol alone. *P < 0.05 and **P < 0.005 compared with respective control. Discussion Unique approaches to stimulate the GLP-1R, such as with ROSE-010, may have a clinical application for functional GI disorders, including motility disorders and sensory mechanisms in the gut. This study with infusion of exogenous GLP-1 to only slightly more than physiological concentrations inhibited the fed motor activity in the antrum and reduced motility along the remaining intestinal segments. Based on the calculated MI, we can confirm in humans our previous animal findings where GLP-1 inhibited motility in the fasting as well as fed state (5, 6, 20, 33, 34). Even though the number of observations in our study is limited, the present results align with those of others studying different animal species (15, 16, 18, 19, 35) and man (4, 8, 26), in which also the metabolic impact of an inhibited antroduodenal motility has been shown. Indeed, the mechanism of GLP-1 action on gastric fasting volume and accommodation seems dependent on intact vagal functions (36). In our present study, plasma GLP-1 concentrations changed with dosing, and there was no consistent GLP-1 dose-response relationship for in vivo motility across the different intestinal segments. Possibly this reflects that even the lower doses result in saturation of all available receptors and therefore maximal effects were achieved. However, in smooth muscle experiments where a multistep graded concentration increase could be used, clear concentration-response relationships for GLP-1 and its analog ROSE-010 were observed. The GLP-1 concentrations required for in vitro responses were high relative to the receptor dissociation constant, Kd ∼28 pM (37). This could be due to instability of the GLP-1 peptide or to impaired permeability and diffusion conditions for the peptide in the incubated tissue sample. These findings in human tissue in vitro are in agreement with our previous study in man showing reduced small bowel fasting motility of GLP-1 in healthy subjects and patients with IBS (20), as well as other studies employing ROSE-010, shown to ameliorate abdominal pain in patients with IBS (13) and facilitate left colon emptying in diarrhea-dominant IBS by dampening excessive distal colonic contractions (38). Taken together, data from our group and others seem to converge on an inhibitory action of GLP-1, most markedly shown by the summarized data of both doses and its analog ROSE-010 on motility. These data are now further substantiated by our findings of the presence of GLP-1, possibly also with a contribution from GLP-2R, in neuronal tissue. We used bethanechol to selectively stimulate muscarinic receptors on GI smooth muscle strips. Then, GLP-1 was applied to induce a motility response. The GLP-1 concentrations used in vitro in this study are similar to those used in a previous ex vivo study (35). The inhibitory effect of GLP-1 on motility was blocked by exendin(9-39)amide as shown in human islets (39), indicating a GLP-1R-mediated mechanism for this action of the peptide. Furthermore, exendin(9-39)amide also inhibited the response of ROSE-010 on GI smooth muscle, consistent with GLP-1 and ROSE-010 acting on the same GLP-1R. The inhibitory effect of GLP-1 as studied in vitro appears to take place through neuronal signaling. Commensurate with our immunohistochemistry findings of GLP-1R on neuronal tissue, GLP-1 inhibited the contractile response to EFS. Also, TTX, which selectively blocks neuronal signal transmission, blocked the response to both GLP-1 and ROSE-010. Furthermore, L-NMMA blocked the inhibitory effect of GLP-1 through GLP-1R located on neurons, demonstrating that GLP-1 inhibition relies on nitrergic mechanisms. In different parts of the GI tract of various species, the inhibition of motility by exogenous GLP-1 has been shown to be dependent on endogenous NO (6, 17, 25, 35). Therefore, nitrergic transmission should be regarded as an important step in GLP-1-induced regulation of GI motility (40, 41). However, we cannot exclude the possibility that GLP-1 also has a direct inhibitory effect on cholinergic neurons in parallel to an excitation of nitrergic neurons. In our hands, immunohistochemistry revealed the presence of GLP-1R within the myenteric plexus, suggesting that GLP-1 acts on enteric neurons. Commensurate with this, our previous studies show presence of NO synthase in myenteric neurons (42, 43), with functional importance for motility (42), which supports a cross-talk between neuronal GLP-1 and NO. To this end, we found that the GLP-1R is coupled to the G-protein Gsα subunit that activates a cyclic adenosine monophosphate (cAMP)–dependent pathway through adenylyl cyclase. Therefore, agonist binding to the GLP-1R should result in adenylate cyclase activation with consequent cAMP production (44) and inhibition of motility. It is well recognized that cAMP is the main mediator of GLP-1R agonism. Our data using DDA supports that a similar mechanism underlies the motility responses to GLP-1 and ROSE-010. Furthermore, cAMP signaling can increase NO production (45, 46), so these two second messengers are suggested to act in concert and synchronize relaxatory mechanisms in the GI tract. To accomplish these effects, the GLP-1-induced motility response seems to involve both a neuronal and a smooth muscle step. In agreement with our findings, NO can be released from myenteric neurons (42, 43) as well as from interstitial cells of Cajal (47), resulting in an increase in cAMP at the level of the smooth muscle to induce relaxation. GLP-1 and GLP-2 are believed to be cosecreted from the l-cells of the mucosal lining in response to food intake. Because GLP-1R receptors are present on epithelial l-cells (48), as confirmed with immunohistochemistry in this study (not shown), it was unknown whether GLP-1 infusion would alter GLP-1 or GLP-2 secretion, which would theoretically influence the response to GLP-1. Unlike GLP-1, plasma GLP-2 was neither markedly increased after the meal, nor influenced by exogenous GLP-1 infusion. The fact that GLP-2 did not rise much following the meal suggests that GLP-1 is a more prominent signal for motility changes and insulin release during meals than GLP-2. A possible reason for disparities between GLP-1 and GLP-2 may also be explained by differences in the sensitivity to degradation by dipeptidyl peptidase-4 (49, 50) or differences in liver clearance. Moreover, baseline (fasted) GLP-2 was 17 times higher than baseline GLP-1 and 3 times higher than postprandial GLP-1 (saline group). Hence, cosecreted postprandial GLP-2 may also have been masked by the pre-existing high ambient GLP-2. To conclude, GLP-1 is able to reduce the digestive motility in the antroduodenojejunal region and slow gastric emptying in humans. The mechanism of this inhibitory response is likely exerted through the GLP-1R expressed in myenteric neurons as the primary functional target for GLP-1 and analogs, such as ROSE-010. Furthermore, both GLP-1 and ROSE-010 exercise receptor-mediated responses with NO and cAMP as second messengers, both of which are required for a regulatory action to take place. GLP-1 is suggested to primarily exert its effects on motility via direct local actions in the periphery, which may coexist with indirect actions mediated through the CNS, be it through vagal afferents originating in the GI tract or by binding to receptors within the CNS. Abbreviations: Ab antibody cAMP cyclic adenosine monophosphate CNS central nervous system DDA 2′,5′-dideoxyadenosine EC50 half maximal effective concentration EFS electric field stimulation GI gastrointestinal GLP-1 glucagon-like peptide-1 GLP-1R glucagon-like peptide-1 receptor GLP-2 glucagon-like peptide-2 GLP-2R glucagon-like peptide-2 receptor IBS irritable bowel syndrome IV intravenous L-NMMA Lω-nitro-monomethylarginine MI motility index MMC migrating motility complex NO nitric oxide NOS nitric oxide synthase RIA radioimmunoassay TTX tetrodotoxin. Acknowledgments Financial Support: This work was supported by the Swedish Research Council (7916) and the Karolinska Institutet (to P.M.H.), Bengt Ihre’s Foundation (SLS-411011, SLS-503061, SLS-506051, SLS-591271, SLS-594831), the Swedish Society of Medicine (SLS-411921, SLS-503131), the Gastroenterology Research Foundation (SLS-504191), Regionala forskningsrådet (RFR), Uppsala/Örebro region (RFR-476131), Björklund’s Foundation (SLS-589741), Sven Jerring’s Foundation and Avtal om läkarutbildning och forskning (ALF) funds, Sweden, and OE and Edla Johansson’s Science Foundation 2017. Clinical Trial Information: ClinicalTrials.gov no. NTC02731664 (registered 16 March 2016). Author Contributions: M.A.H., D.-L.W., and P.M.H. designed the study. J.J.H. performed RIA. P.M.H. enrolled healthy volunteers and performed randomization and motility experiments. M.A.H. performed immunostaining and organ bath experiments. M.S. and U.K. provided human tissue. 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