Truncated glucagon-like peptide-1 interacts with exendin receptors on dispersed acini from guinea pig pancreas. Identification of a mammalian analogue of the reptilian peptide exendin-4.

To find mammalian analogues of exendin-4, a peptide from Helodermatidae venoms that interacts with newly discovered exendin receptors on dispersed acini from guinea pig pancreas, we examined the actions of recent additions to the vasoactive intestinal peptide/secretin/glucagon family of regulatory peptides. In every respect tested, the truncated form of glucagon-like peptide-1, GLP-1(7-36)NH2, mimicked the actions of exendin-4. Like exendin-4, GLP-1(7-36)NH2 caused an increase in acinar cAMP without stimulating amylase release. GLP-1(7-36)NH2-induced increases in cAMP were inhibited progressively by increasing concentrations of the specific exendin-receptor antagonist, exendin(9-39)NH2. In dispersed acini from guinea pig and rat pancreas, concentrations of GLP-1(7-36)NH2 that stimulated increases in cAMP caused potentiation of cholecystokinin-induced amylase release. Binding of 125I-[Y39]exendin-4 or 125I-GLP-1(7-36)NH2 to dispersed acini from guinea pig pancreas was inhibited by adding increasing concentrations of unlabeled exendin-4 or GLP-1(7-36)NH2. We conclude that the mammalian peptide GLP-1(7-36)NH2 interacts with exendin receptors on dispersed acini from guinea pig pancreas. Exendin(9-39)NH2, a competitive antagonist of the actions of GLP-1(7-36)NH2 in pancreatic acini, may be a useful tool for examining the physiological actions of this peptide.     

Metformin effects on dipeptidylpeptidase IV degradation of glucagon-like peptide-1

There is current interest in the use of inhibitors of dipeptidyl peptidase IV (DP IV) as therapeutic agents to normalize glycemic excursions in type 2 diabetic patients. Data indicating that metformin increases the circulating amount of active glucagon-like peptide-1 (GLP-1) in obese nondiabetic subjects have recently been presented, and it was proposed that metformin might act as a DP IV inhibitor. This possibility has been investigated directly using a number of in vitro methods. Studies were performed on DP IV enzyme from three sources: 20% human serum, purified porcine kidney DP IV, and recombinant human DP IV. Inhibition of DP IV hydrolysis of the substrate Gly-Pro-pNA by metformin was examined spectrophotometrically. Effects of metformin on GLP-1([7-36NH2]) degradation were assessed by mass spectrometry. In addition, surface plasmon resonance was used to establish whether or not metformin had any effect on GLP-1([7-36NH2]) or GLP-1([9-36NH2]) interaction with immobilized porcine or human DP IV. Metformin failed to alter the kinetics of Gly-Pro-pNA hydrolysis or GLP-1 degradation tested according to established methods. Surface plasmon resonance recordings indicated that both GLP-1([7-36NH2]) and GLP-1([9-36NH2]) show micromolar affinity (K(D)) for DP IV, but neither interaction was influenced by metformin. The results conclusively indicate that metformin does not act directly on DP IV, therefore alternative explanations for the purported effect of metformin on circulating active GLP-1 concentrations must be considered.     

The expression of GLP-1 receptor mRNA and protein allows the effect of GLP-1 on glucose metabolism in the human hypothalamus and brainstem

In the present work, several experimental approaches were used to determine the presence of the glucagon-like peptide-1 receptor (GLP-1R) and the biological actions of its ligand in the human brain. In situ hybridization histochemistry revealed specific labelling for GLP-1 receptor mRNA in several brain areas. In addition, GLP-1R, glucose transporter isoform (GLUT-2) and glucokinase (GK) mRNAs were identified in the same cells, especially in areas of the hypothalamus involved in feeding behaviour. GLP-1R gene expression in the human brain gave rise to a protein of 56 kDa as determined by affinity cross-linking assays. Specific binding of 125I-GLP-1(7-36) amide to the GLP-1R was detected in several brain areas and was inhibited by unlabelled GLP-1(7-36) amide, exendin-4 and exendin (9-39). A further aim of this work was to evaluate cerebral-glucose metabolism in control subjects by positron emission tomography (PET), using 2-[F-18] deoxy-D-glucose (FDG). Statistical analysis of the PET studies revealed that the administration of GLP-1(7-36) amide significantly reduced (p < 0.001) cerebral glucose metabolism in hypothalamus and brainstem. Because FDG-6-phosphate is not a substrate for subsequent metabolic reactions, the lower activity observed in these areas after peptide administration may be due to reduction of the glucose transport and/or glucose phosphorylation, which should modulate the glucose sensing process in the GLUT-2- and GK-containing cells.     

Isolation and characterisation of GLP-1 7-36 amide from rat intestine. Elevated levels in diabetic rats

Glucagon-like peptide-1 (GLP-1) was purified to homogeneity by HPLC and anion-exchange chromatography. A molecular mass of 3297.4 Da was obtained by FAB mass spectrometry which corresponded exactly to GLP-1 7-36 NH2, providing evidence that amidation occurs at an arginine residue during the post-translational processing of GLP-1. The distribution of GLP-1 7-36 NH2-like immunoreactivity (GLP-1 7-36 NH2 IR) was determined in the rat gastrointestinal tract. Highest concentrations were found in terminal ileum and colon. Streptozocin-induced diabetic rats, who showed a significant increase in food intake, had a significant increase of GLP-1 7-36 NH2 IR in the colon.     

Second extracellular loop of human glucagon-like peptide-1 receptor (GLP-1R) has a critical role in GLP-1 peptide binding and receptor activation

The glucagon-like peptide-1 receptor (GLP-1R) is a therapeutically important family B G protein-coupled receptor (GPCR) that is pleiotropically coupled to multiple signaling effectors and, with actions including regulation of insulin biosynthesis and secretion, is one of the key targets in the management of type II diabetes mellitus. However, there is limited understanding of the role of the receptor core in orthosteric ligand binding and biological activity. To assess involvement of the extracellular loop (ECL) 2 in ligand-receptor interactions and receptor activation, we performed alanine scanning mutagenesis of loop residues and assessed the impact on receptor expression and GLP-1(1-36)-NH(2) or GLP-1(7-36)-NH(2) binding and activation of three physiologically relevant signaling pathways as follows: cAMP formation, intracellular Ca(2+) (Ca(2+)(i)) mobilization, and phosphorylation of extracellular signal-regulated kinases 1 and 2 (pERK1/2). Although antagonist peptide binding was unaltered, almost all mutations affected GLP-1 peptide agonist binding and/or coupling efficacy, indicating an important role in receptor activation. However, mutation of several residues displayed distinct pathway responses with respect to wild type receptor, including Arg-299 and Tyr-305, where mutation significantly enhanced both GLP-1(1-36)-NH(2)- and GLP-1(7-36)-NH(2)-mediated signaling bias for pERK1/2. In addition, mutation of Cys-296, Trp-297, Asn-300, Asn-302, and Leu-307 significantly increased GLP-1(7-36)-NH(2)-mediated signaling bias toward pERK1/2. Of all mutants studied, only mutation of Trp-306 to alanine abolished all biological activity. These data suggest a critical role of ECL2 of the GLP-1R in the activation transition(s) of the receptor and the importance of this region in the determination of both GLP-1 peptide- and pathway-specific effects.     

Characterization of high-affinity receptors for truncated glucagon-like peptide-1 in rat gastric glands

The truncated form of glucagon-like peptide-1 (TGLP-1, or proglucagon 78-108), secreted by the mammalian intestine, has potent pharmacological activities, stimulating insulin release and inhibiting gastric acid secretion. We have characterized high-affinity receptors for this peptide in rat isolated fundic glands. Scatchard analysis of binding studies using mono-125I-TGLP-1(7-36) amide as tracer showed a single class of binding site of Kd (4.4 +/- (SE) .08) x 10(-10) M, with a tissue concentration of 1.0 +/- 0.1 fmol sites/microgram DNA. Whole GLP-1 was approximately 700 times less potent in displacing tracer, while human GLP-2 and pancreatic glucagon produced no significant displacement at concentrations up to 10(-6) M. The data support a physiological role for TGLP-1 in the regulation of gastric acid secretion.     

Active metabolite of GLP-1 mediates myocardial glucose uptake and improves left ventricular performance in conscious dogs with dilated cardiomyopathy

We have shown previously that the glucagon-like peptide-1 (GLP-1)-(7-36) amide increases myocardial glucose uptake and improves left ventricular (LV) and systemic hemodynamics in both conscious dogs with pacing-induced dilated cardiomyopathy (DCM) and humans with LV systolic dysfunction after acute myocardial infarction. However, GLP-1-(7-36) is rapidly degraded in the plasma to GLP-1-(9-36) by dipeptidyl peptidase IV (DPP IV), raising the issue of which peptide is the active moiety. By way of methodology, we compared the efficacy of a 48-h continuous intravenous infusion of GLP-1-(7-36) (1.5 pmol.kg(-1).min(-1)) to GLP-1-(9-36) (1.5 pmol.kg(-1).min(-1)) in 28 conscious, chronically instrumented dogs with pacing-induced DCM by measuring LV function and transmyocardial substrate uptake under basal and insulin-stimulated conditions using hyperinsulinemic-euglycemic clamps. As a result, dogs with DCM demonstrated myocardial insulin resistance under basal and insulin-stimulated conditions. Both GLP-1-(7-36) and GLP-1-(9-36) significantly reduced (P < 0.01) LV end-diastolic pressure [GLP-1-(7-36), 28 +/- 1 to 15 +/- 2 mmHg; GLP-1-(9-36), 29 +/- 2 to 16 +/- 1 mmHg] and significantly increased (P < 0.01) the first derivative of LV pressure [GLP-1-(7-36), 1,315 +/- 81 to 2,195 +/- 102 mmHg/s; GLP-1-(9-36), 1,336 +/- 77 to 2,208 +/- 68 mmHg] and cardiac output [GLP-1-(7-36), 1.5 +/- 0.1 to 1.9 +/- 0.1 l/min; GLP-1-(9-36), 2.0 +/- 0.1 to 2.4 +/- 0.05 l/min], whereas an equivolume infusion of saline had no effect. Both peptides increased myocardial glucose uptake but without a significant increase in plasma insulin. During the GLP-1-(9-36) infusion, negligible active (NH2-terminal) peptide was measured in the plasma. In conclusion, in DCM, GLP-1-(9-36) mimics the effects of GLP-1-(7-36) in stimulating myocardial glucose uptake and improving LV and systemic hemodynamics through insulinomimetic as opposed to insulinotropic effects. These data suggest that GLP-1-(9-36) amide is an active peptide.     

Glucagon-like peptide-1 (7–36) amide as a novel neuropeptide

Although earlier studies indicated that GLP-1 (7-36) amide was an intestinal peptide with a potent effect on glucose-dependent insulin secretion, later on it was found that several biological effects of this peptide occur in the brain, rather than in peripheral tissues. Thus, proglucagon is expressed in pancreas, intestine, and brain, but post translational processing of the precursor yields different products in these organs, glucagon-like peptide-1 (7-36) amide being one of the forms produced in the brain. Also, GLP-1 receptor cDNA from human and rat brains has been cloned and sequenced, and the deduced amino acid sequences are the same as those found in pancreatic islets. Through these receptors, GLP-1 (7-36) amide from gut or brain sources induces its effects on the release of neurotransmitters from selective brain nuclei, the inhibition of gastric secretion and motility, the regulation of food and drink intake, thermoregulation, and arterial blood pressure. Central administration (icv) of GLP-1 (7-36) amide produces a marked reduction in food and water intake, and the colocalization of the GLP-1 receptor, GLUT-2, and glucokinase mRNAs in hypothalamic neurons involved in glucose sensing suggests that these cells may be involved in the transduction of signals needed to produce a state of fullness. In addition, GLP-1 (7-36) amide inhibits gastric acid secretion and gastric emptying, but these effects are not found in vagotomized subjects, suggesting a centrally mediated effect. Similar results have been found with the action of this peptide on arterial blood pressure and heart rate in rats. Synthesis of GLP-1 (7-36) amide and its own receptors in the brain together with its abovementioned central physiological effects imply that this peptide may be considered a neuropeptide. Also, the presence of GLP-1 (7-36) amide in the synaptosome fraction and its calcium-dependent release by potassium stimulation, suggest that the peptide may act as a neurotransmitter although further electrophysiological and ultrastructural studies are needed to confirm this possibility.     

Distribution and molecular forms of glucagon-like peptide in the dog

Using glucagon-like peptide-1 N-terminus and C-terminus directed antisera, we investigated concentration and molecular forms of GLP-1 immunoreactivity (IR) in extracts of various tissues of the dog. GLP-1 IR measured with C-terminus-directed antiserum R2337 (GLP-1 IR-CT) was high in the ileum, appendix, jejunum, colon, and gastric fundus and body. GLP-1 IR measured with N-terminus-directed antiserum R1043 (GLP-1 IR-NT) was high only in the pancreas, and gastric fundus and body. Only GLP-1 IR-CT was found in the hypothalamus, thalamus and medulla oblongata. No immunoreactive materials were detected in the liver, spleen and kidney. Gel-filtration with Sephadex G-50 showed two peaks of both GLP-1 IR-CT and GLP-1 IR-NT, at 10kd and at the position of GLP-1 (1-36 amide) in the pancreatic extract, and one peak at 10kd in the stomach extract. Ileal extracts showed 3 peaks of GLP-1 IR-CT at 10kd, at the position of GLP-1(1-36 amide) and GLP-1(7-36 amide), respectively, but GLP-1 IR-NT was coeluted with GLP-1(1-36 amide). Hypothalamic extracts showed a single peak at the position of GLP-1(7-36 amide). These results suggest that processing of preproglucagon differs in different organs, and that the main GLP-1-related products are a large molecular form and GLP-1(1-36 amide) or GLP-1(1-37) in the pancreas, and GLP-1(7-36 amide) or GLP-1 (7-37) in the ileum and hypothalamus.     

Glucagon-Like Peptide-1 Receptor Ligand Interactions: Structural Cross Talk between Ligands and the Extracellular Domain

Activation of the glucagon-like peptide-1 receptor (GLP-1R) in pancreatic β-cells potentiates insulin production and is a current therapeutic target for the treatment of type 2 diabetes mellitus (T2DM). Like other class B G protein-coupled receptors (GPCRs), the GLP-1R contains an N-terminal extracellular ligand binding domain. N-terminal truncations on the peptide agonist generate antagonists capable of binding to the extracellular domain, but not capable of activating full length receptor. The main objective of this study was to use Hydrogen/deuterium exchange (HDX) to identify how the amide hydrogen bonding network of peptide ligands and the extracellular domain of GLP-1R (nGLP-1R) were altered by binding interactions and to then use this platform to validate direct binding events for putative GLP-1R small molecule ligands. The HDX studies presented here for two glucagon-like peptide-1 receptor (GLP-1R) peptide ligands indicates that the antagonist exendin-4_[9-39] is significantly destabilized in the presence of nonionic detergents as compared to the agonist exendin-4. Furthermore, HDX can detect stabilization of exendin-4 and exendin-4_[9-39] hydrogen bonding networks at the N-terminal helix [Val19 to Lys27] upon binding to the N-terminal extracellular domain of GLP-1R (nGLP-1R). In addition we show hydrogen bonding network stabilization on nGLP-1R in response to ligand binding, and validate direct binding events with the extracellular domain of the receptor for putative GLP-1R small molecule ligands.     

Coexpression of Glucagon-Like Peptide-1 (GLP-1) Receptor, Vasopressin, and Oxytocin mRNAs in Neurons of the Rat Hypothalamic Supraoptic and Paraventricular Nuclei

Abstract : This study was designed to gain better insight into the relationship between glucagon-like peptide-1 (GLP-1) (7-36) amide and vasopressin (AVP) and oxytocin (OX). In situ hybridization histochemistry revealed colocalization of the mRNAs for GLP-1 receptor, AVP, and OX in neurons of the hypothalamic supraoptic and paraventricular nuclei. To determine whether GLP-1(7-36)amide alters AVP and/or OX release, both in vivo and in vitro experimental study designs were used. In vivo, intravenous administration of 1 μg of GLP-1(7-36)amide into the jugular vein significantly decreased plasma AVP and OX concentrations. In vitro incubation of the neurohypophysis with either 0.1 or 1 μg of GLP-1(7-36)amide did not modify the release of AVP. However, addition of 1 μg of GLP-1(7-36)amide to the incubation medium increased slightly the secretion of OX. The coexpression of GLP-1 receptor and AVP mRNAs in hypothalamic supraoptic and paraventricular nuclei gives further support to the already reported central effects of GLP-1(7-36)amide on AVP. Our findings also suggest a dual secretory response of AVP and OX to the effect of GLP-1(7-36)amide, which most likely is related to the amount and/or the route of peptide administration.     

New screening strategy and analysis for identification of allosteric modulators for glucagon-like peptide-1 receptor using GLP-1 (9-36) amide

The glucagon-like peptide-1 receptor (GLP-1R) is an important physiologic regulator of insulin secretion and a major therapeutic target for diabetes mellitus. GLP-1 (7-36) amide (active form of GLP-1) is truncated to GLP-1 (9-36) amide, which has been described as a weak agonist of GLP-1R and the major form of GLP-1 in the circulation. New classes of positive allosteric modulators (PAMs) for GLP-1R may offer improved therapeutic profiles. To identify these new classes, we developed novel and robust primary and secondary high-throughput screening (HTS) systems in which PAMs were identified to enhance the GLP-1R signaling induced by GLP-1 (9-36) amide. Screening enabled identification of two compounds, HIT-465 and HIT-736, which possessed new patterns of modulation of GLP-1R. We investigated the ability of these compounds to modify GLP-1R signaling enhanced GLP-1 (9-36) amide- and/or GLP-1 (7-36) amide-mediated cyclic adenosine monophosphate (cAMP) accumulation. These compounds also had unique profiles with regard to allosteric modulation of multiple downstream signaling (PathHunter β-arrestin signaling, PathHunter internalization signaling, microscopy-based internalization assay). We found allosteric modulation patterns to be obviously different among HIT-465, HIT-736, and Novo Nordisk compound 2. This work may enable the design of new classes of drug candidates by targeting modulation of GLP-1 (7-36) amide and GLP-1 (9-36) amide.     

Structural Characterization by Affinity Cross-Linking of Glucagon-Like Peptide-1(7–36)Amide Receptor in Rat Brain

Abstract: Specific binding of glucagon-like peptide (GLP)-1(7–36)amide was detected in several rat brain areas, with the highest values being found in hypothalamic nuclei and the nucleus of the solitary tract. In hypothalamus and brainstem homogenate binding of ¹²⁵I-GLP-1(7–36)amide was time, temperature, and protein content dependent and was inhibited by unlabeled proglucagon-derived peptides. The rank order of potency was GLP-1(7–36)amide ? GLP-1(1–36)amide > GLP-1(1–37) ? GLP-2 > glucagon. Scatchard analysis of the steady-state binding data was consistent with the presence of both high- and low-affinity binding sites in hypothalamus and brainstem. Brain ¹²⁵I-GLP-1(7–36)amide-binding protein complexes were covalently cross-linked using disuccinimidyl suberate and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A single radiolabeled band of M_r 56,000 identified in both hypothalamus and brainstem homogenates was unaffected by reducing agents. An excess of unlabeled GLP-1(7–36)amide abolished the band labeling, whereas glucagon had no effect. Other unlabeled GLPs inhibited M_r 56,000 complex labeling with the following order of potency: GLP-1(1–36)amide > GLP-1(1–37) > GLP-2. The binding of ¹²⁵I-GLP-1(7–36)amide and the intensity of the cross-linked band were similarly inhibited in a dose-response manner by increasing concentrations of unlabeled GLP-1(7–36)amide. Covalent M_r 56,000 ¹²⁵I-GLP-1(7–36)amide-binding protein complexes solubilized by Triton X-100 were adsorbed onto wheat germ agglutinin. Our results suggest that the GLP-1(7–36)amide receptor in rat brain is a glycoprotein with a single binding subunit that has a greater molecular weight but binding features and ligand specificity similar to those of its peripheral tissue counterparts.     

The glucagon-like peptide-1 receptor binding site for the N-terminus of GLP-1 requires polarity at Asp198 rather than negative charge

The mutation of Asp198 to Asn in the receptor for glucagon-like peptide-1(7–36)amide (GLP-1) had no effect upon GLP-1 affinity whereas substitution with Ala greatly reduced affinity, demonstrating the importance of polarity rather than negative charge at Asp198. However, the Asp198-Ala mutation had less effect upon the affinity of Exendin-4, a peptide agonist that has been shown previously not to require its N-terminus for high affinity. Moreover, the affinity of a truncated GLP-1 analogue lacking the first eight residues was not affected by the Asp198-Ala mutation, demonstrating that Asp198 is required for maintaining the binding site of the N-terminal region of GLP-1.     

GLP-1对人脐静脉内皮细胞释放NO的影响及机制的研究

目的:观察胰高糖素样肽-1(GLP-1)对脐静脉内皮细胞(HUVECs)释放一氧化氮(NO)的影响,并探讨GLP-1受体及GLP-1(9-36)在其中的作用。方法:分别以GLP-1、艾塞那肽、GLP-1(9-36)、GLP-1+exendin(9-39)、GLP-1+西格列汀、GLP-1+西格列汀+exendin(9-39)孵育HUVECs,取培养上清以硝酸还原酶法检测NO浓度。结果:GLP-1剂量依赖性的增加HUVECs中NO释放,艾塞那肽和GLP-1(9-36)均可刺激NO释放,exendin(9-39)和西格列汀均可部分阻断GLP-1引起的NO释放。结论:GLP-1可能通过GLP-1受体及GLP-1(9-36)相关的途径刺激HUVECs NO释放,发挥直接的血管保护作用。     

Crystal structure of the ligand-bound glucagon-like peptide-1 receptor extracellular domain

The glucagon-like peptide-1 receptor (GLP-1R) belongs to Family B1 of the seven-transmembrane G protein-coupled receptors, and its natural agonist ligand is the peptide hormone glucagon-like peptide-1 (GLP-1). GLP-1 is involved in glucose homeostasis, and activation of GLP-1R in the plasma membrane of pancreatic beta-cells potentiates glucose-dependent insulin secretion. The N-terminal extracellular domain (nGLP-1R) is an important ligand binding domain that binds GLP-1 and the homologous peptide Exendin-4 with differential affinity. Exendin-4 has a C-terminal extension of nine amino acid residues known as the "Trp cage", which is absent in GLP-1. The Trp cage was believed to interact with nGLP-1R and thereby explain the superior affinity of Exendin-4. However, the molecular details that govern ligand binding and specificity of nGLP-1R remain undefined. Here we report the crystal structure of human nGLP-1R in complex with the antagonist Exendin-4(9-39) solved by the multiwavelength anomalous dispersion method to 2.2A resolution. The structure reveals that Exendin-4(9-39) is an amphipathic alpha-helix forming both hydrophobic and hydrophilic interactions with nGLP-1R. The Trp cage of Exendin-4 is not involved in binding to nGLP-1R. The hydrophobic binding site of nGLP-1R is defined by discontinuous segments including primarily a well defined alpha-helix in the N terminus of nGLP-1R and a loop between two antiparallel beta-strands. The structure provides for the first time detailed molecular insight into ligand binding of the human GLP-1 receptor, an established target for treatment of type 2 diabetes.     

Microwave-assisted solid phase synthesis, PEGylation, and biological activity studies of glucagon-like peptide-1(7-36) amide

The insulinotropic hormone glucagon-like peptide-1 (GLP-1) is rapidly inactivated in the body. In order to improve its stability, we replaced the enzymatic hydrolyzation position Ala(8)with Gly and replaced Ala(30) with Cys firstly. Then the modified peptide was further PEGylated at thiol group of Cys(30). Biological activity studies showed that the resulting mPEG-MAL-Gly(8)-Cys(30)-GLP-1(7-36)-NH(2) exhibited long-lasting effect while maintaining moderate glucose-lowering activity.     

Expression and distribution of pituitary adenylate cyclase-activating peptide in human prostate and prostate cancer tissues

The therapeutic potential of the intestinotrophic mediator glucagon-like peptide-2 (1-33) [GLP-2 (1-33)] has increased interest in the pharmacokinetics of the peptide. This study was undertaken to investigate whether the primary degradation product GLP-2 (3-33) interacts with the GLP-2 receptor. Functional (cAMP) and binding in vitro studies were carried out in cells expressing the transfected human GLP-2 receptor. Furthermore, a biologic response of GLP-2 (3-33) was tested in vivo. Mice were allocated to groups treated for 10 days (twice daily) with: (1) 5 microg GLP-2 (1-33), (2) 25 microg GLP-2 (3-33), (3) 5 microg GLP-2 (1-33)+100 microg GLP-2 (3-33), or (4) 5 microg GLP-2 (1-33)+500 microg GLP-2 (3-33). The intestine was investigated for growth changes. GLP-2 (3-33) bound to the GLP-2 receptor with a binding affinity of 7.5% of that of GLP-2 (1-33). cAMP accumulation was stimulated with an efficacy of 15% and a potency more than two orders of magnitude lower than that of GLP-2 (1-33). Increasing doses of GLP-2 (3-33) (10(-7)-10(-5) M) caused a shift to the right in the dose-response curve of GLP-2 (1-33). Treatment of mice with either GLP-2 (1-33) or (3-33) induced significant growth responses in both the small and large intestines, but the response induced by GLP-2 (3-33) was much smaller. Co-administration of 500 microg of GLP-2 (3-33) and 5 microg GLP-2 (1-33) resulted in a growth response that was smaller than that of 5 microg GLP-2 (1-33) alone. Consistent with the observed in vivo activities, our functional studies and binding data indicate that GLP-2 (3-33) acts as a partial agonist with potential competitive antagonistic properties on the GLP-2 receptor.     

GLP-1 and GLP-2 act in concert to inhibit fasted,but not fed,small bowel motility in the rat

Small bowel motility was studied in rats at increasing (1-20 pmol/kg/min) intravenous doses of either glucagon-like peptide-1 (GLP-1) or glucagon-like peptide-2 (GLP-2) alone, or in combination in the fasted and fed state. There was a dose-dependent inhibitory action of GLP-1 on the migrating myoelectric complex (MMC), where the dose of 5 pmol/kg/min induced an increased MMC cycle length. No effect was seen with GLP-2 alone, but the combination of GLP-1 and GLP-2 induced a more pronounced inhibitory effect, with significant increase of the MMC cycle length from a dose of 2 pmol/kg/min. During fed motility, infusion of GLP-1 resulted in an inhibition of spiking activity compared to control. In contrast, infusion of GLP-2 only numerically increased spiking activity compared to control, while the combination of GLP-1 and GLP-2 resulted in no change compared to control. In summary, this study demonstrates an additive effect of peripheral administration of GLP-1 and GLP-2 on fasted small bowel motility. In the fed state, GLP-1 and GLP-2 seem to display counter-balancing effects on motility of the small intestine.     

Carboxypeptidase-B-like processing of the C-terminus of glucagon-like peptide-2 in pig and human small intestine

We developed specific, C-terminal radioimmunoassays for three proglucagon (PG) fragments: PG 151-158, PG 151-160 and PG 126-159 (glucagon-like peptide-2 (GLP-2] in order to determine the exact C-terminal sequence of the newly isolated GLP-2 in man and pig. The antigens and the antisera showed no mutual cross-reactivity. By gel filtration of extracts of pig and human small intestine, the immunoreactivity eluting at the position of GLP-2 was identified by the radioimmunoassays for glucagon-like peptide-2 (PG 126-159) and for PG 151-158, whereas the assay for PG 151-160 was completely negative. We conclude that the C-terminal amino acid residue of pig and human ileal GLP-2 is PG 158. Thus the basic residues, PG 159 and 160 are removed during its processing in the small intestine.