Evolutionarily conserved residues at glucagon-like peptide-1 (GLP-1) receptor core confer ligand-induced receptor activation

Glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) play important roles in insulin secretion through their receptors, GLP1R and GIPR. Although GLP-1 and GIP are attractive candidates for treatment of type 2 diabetes and obesity, little is known regarding the molecular interaction of these peptides with the heptahelical core domain of their receptors. These core domains are important not only for specific ligand binding but also for ligand-induced receptor activation. Here, using chimeric and point-mutated GLP1R/GIPR, we determined that evolutionarily conserved amino acid residues such as Ile(196) at transmembrane helix 2, Leu(232) and Met(233) at extracellular loop 1, and Asn(302) at extracellular loop 2 of GLP1R are responsible for interaction with ligand and receptor activation. Application of chimeric GLP-1/GIP peptides together with molecular modeling suggests that His(1) of GLP-1 interacts with Asn(302) of GLP1R and that Thr(7) of GLP-1 has close contact with a binding pocket formed by Ile(196), Leu(232), and Met(233) of GLP1R. This study may provide critical clues for the development of peptide and/or nonpeptide agonists acting at GLP1R.     

Conformational and molecular interaction studies of glucagon-like peptide-2 with its N-terminal extracellular receptor domain

Glucagon-like peptide-2 (GLP-2) is a therapeutic target used in the treatment of short bowel syndrome. In this paper, we present the three dimensional solution structure of GLP-2 peptide determined using nuclear magnetic resonance (NMR) and molecular modelling. The GLP-2 adopts an α-helical conformation similar to that of secretin family of hormones. In order to understand the molecular details governing the ligand binding and receptor activation, macromolecular docking studies were performed between the N-terminal extracellular domain of GLP-2 receptor and the GLP-2 hormone using a data driven docking program.     

Anti-inflammatory apoA-I-mimetic peptides bind oxidized lipids with much higher affinity than human apoA-I

4F is an anti-inflammatory, apolipoprotein A-I (apoA-I)-mimetic peptide that is active in vivo at nanomolar concentrations in the presence of a large molar excess of apoA-I. Physiologic concentrations ( approximately 35 microM) of human apoA-I did not inhibit the production of LDL-induced monocyte chemotactic activity by human aortic endothelial cell cultures, but adding nanomolar concentrations of 4F in the presence of approximately 35 microM apoA-I significantly reduced this inflammatory response. We analyzed lipid binding by surface plasmon resonance. The anti-inflammatory 4F peptide bound oxidized lipids with much higher affinity than did apoA-I. Initially, we examined the binding of PAPC (1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphatidylcholine) and observed that its oxidized products bound 4F with an affinity that was approximately 4-6 orders of magnitude higher than that of apoA-I. This high binding affinity was confirmed in studies with defined lipids and phospholipids. 3F-2 and 3F(14) are also amphipathic alpha-helical octadecapeptides, but 3F-2 inhibits atherosclerosis in mice and 3F(14) does not. Like 4F, 3F-2 also bound oxidized phospholipids with very high affinity, whereas 3F(14) resembled apoA-I. The extraordinary ability of 4F to bind pro-inflammatory oxidized lipids probably accounts for its remarkable anti-inflammatory properties.     

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.     

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.     

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.     

Differential structural properties of GLP-1 and exendin-4 determine their relative affinity for the GLP-1 receptor N-terminal extracellular domain

Glucagon-like peptide-1 (GLP-1) and exendin-4 (Ex4) are homologous peptides with established potential for treatment of type 2 diabetes. They bind and activate the pancreatic GLP-1 receptor (GLP-1R) with similar affinity and potency and thereby promote insulin secretion in a glucose-dependent manner. GLP-1R belongs to family B of the seven transmembrane G-protein coupled receptors. The N-terminal extracellular domain (nGLP-1R) is a ligand binding domain with differential affinity for Ex4 and GLP-1: low affinity for GLP-1 and high affinity for exendin-4. The superior affinity of nGLP-1R for Ex4 was previously explained by an additional interaction between nGLP-1R and the C-terminal Trp-cage of Ex4. In this study we have combined biophysical and pharmacological approaches thus relating structural properties of the ligands in solution to their relative binding affinity for nGLP-1R. We used both a tracer competition assay and ligand-induced thermal stabilization of nGLP-1R to measure the relative affinity of full length, truncated, and chimeric ligands for soluble refolded nGLP-1R. The ligands in solution and the conformational consequences of ligand binding to nGLP-1R were characterized by circular dichroism and fluorescence spectroscopy. We found a correlation between the helical content of the free ligands and their relative binding affinity for nGLP-1R, supporting the hypothesis that the ligands are helical at least in the segment that binds to nGLP-1R. The Trp-cage of Ex4 was not necessary to maintain a superior helicity of Ex4 compared to GLP-1. The results suggest that the differential affinity of nGLP-1R is explained almost entirely by divergent residues in the central part of the ligands: Leu10-Gly30 of Ex4 and Val16-Arg36 of GLP-1. In view of our results it appears that the Trp-cage plays only a minor role for the interaction between Ex4 and nGLP-1R and for the differential affinity of nGLP-1R for GLP-1 and Ex4.     

Bean peptides have higher in silico binding affinities than ezetimibe for the N-terminal domain of cholesterol receptor Niemann-Pick C1 Like-1

Niemann-Pick C1 like-1 (NPC1L1) mediates cholesterol absorption at the apical membrane of enterocytes through a yet unknown mechanism. Bean, pea, and lentil proteins are naturally hydrolyzed during digestion to produce peptides. The potential for pulse peptides to have high binding affinities for NPC1L1 has not been determined. In this study , in silico binding affinities and interactions were determined between the N-terminal domain of NPC1L1 and 14 pulse peptides (5≥ amino acids) derived through pepsin-pancreatin digestion. Peptides were docked in triplicate to the N-terminal domain using docking program AutoDock Vina, and results were compared to those of ezetimibe, a prescribed NPC1L1 inhibitor. Three black bean peptides (−7.2 to −7.0 kcal/mol) and the cowpea bean dipeptide Lys-Asp (−7.0 kcal/mol) had higher binding affinities than ezetimibe (−6.6 kcal/mol) for the N-terminal domain of NPC1L1. Lentil and pea peptides studied did not have high binding affinities. The common bean peptide Tyr-Ala-Ala-Ala-Thr (−7.2 kcal/mol), which can be produced from black or navy bean proteins, had the highest binding affinity. Ezetimibe and peptides with high binding affinities for the N-terminal domain are expected to interact at different locations of the N-terminal domain. All high affinity black bean peptides are expected to have van der Waals interactions with SER130, PHE136, and LEU236 and a conventional hydrogen bond with GLU238 of NPC1L1. Due to their high affinity for the N-terminal domain of NPC1L1, black and cowpea bean peptides produced in the digestive track have the potential to disrupt interactions between NPC1L1 and membrane proteins that lead to cholesterol absorption.     

The positive charge at Lys-288 of the glucagon-like peptide-1 (GLP-1) receptor is important for binding the N-terminus of peptide agonists

Lysine-288 in the glucagon-like peptide-1 receptor was predicted to be ideally positioned to play a role in hormone binding. Subsequent mutation of Lys-288 to Ala or Leu greatly reduced hormone affinity, while substitution with Arg had minimal effect. Compared to wild type, the Lys288-Ala receptor had a reduced affinity for three peptide ligands with complete N-terminal sequences but not for their N-truncated analogues. Hence, the role of this positively charged residue, which is conserved at the equivalent position in all other Family B receptors, was determined to be important for receptor interaction with the N-terminal eight residues of peptide agonists.     

The N-terminal interferon-binding domain (IBiD) homology domain of p300 binds to peptides with homology to the p53 transactivation domain

Two high affinity Ser-20-phospho-LXXLL p53-binding domains of p300 map to the C-terminal interferon-binding domain (IBiD) and N-terminal IBiD homology domain (IHD) regions. Purified fractions of a recombinant IHD miniprotein are active in a set of in vitro assays highlighting its affinity to the N-terminal LXXLL domain of p53 including (i) dose-dependent binding to Ser-20-phosphorylated p53 tetramers; (ii) DNA-stimulated binding to p53 tetramers; and (iii) inhibition of MDM2-mediated p53 ubiquitination. The active component of the IHD miniprotein was localized to a 75-amino-acid fragment corresponding to amino acids 401-475 on human p300. This minimal IHD miniprotein can function in vivo as a p53-binding polypeptide in assays including: (i) complex formation with VP16-LXXLL peptide motifs in the two-hybrid assay; (ii) action as a dominant negative inhibitor of p53 from p21 luciferase templates; and (iii) attenuation of endogenous p21 protein levels. Further, we show here that the IRF-1-dependent stabilization and reactivation of p53DeltaPRO protein (LXXLL+/PXXP-) can be neutralized by the minimal IHD miniprotein, suggesting that IHD can bind to the p53 LXXLL domain in vivo. Phage-peptide display to the IHD miniprotein gave rise to an LSQXTFSXLXXLL consensus binding site that displays significant homology to the LXXLL transactivation domain of p53. These data validate the IHD scaffold as an independent LXXLL peptide-binding domain within the p300 protein, complementing the known peptide-binding domains including IBiD, C/H1, and C/H3.     

Nitrergic contribution to gastric relaxation induced by glucagon-like peptide-1 (GLP-1) in healthy adults

The incretin glucagon-like peptide-1 (GLP-1), which is used to treat diabetes mellitus, delays gastric emptying by inhibiting vagal activity. GLP-1 also increases fasting and postprandial gastric volume in humans. On the basis of animal studies, we hypothesized that nitric oxide mediates the effects of GLP-1 on gastric volumes. To assess the effects of nitrergic blockade on GLP-1-induced gastric accommodation in humans, in this double-blind study, 31 healthy volunteers were randomized to placebo (i.e., saline), GLP-1, or the nitric oxide synthase inhibitor N(G)-monomethyl-L-arginine acetate (L-NMMA; 4 mg.kg(-1) x h(-1)) alone or with GLP-1. Thereafter, 16 additional subjects were randomized to GLP-1 alone or together with a higher dose of L-NMMA (10 mg/kg bolus plus 8 mg.kg(-1).h(-1) infusion). Gastric volumes (fasting pre- and postdrug, postprandial postdrug) were measured by (99m)Tc-single-photon-emission computed tomography imaging. GLP-1 increased (P = 0.04) fasting gastric volume by 83 +/- 16 ml (vs. 17 +/- 11 ml for placebo) and augmented (P < or = 0.01) postprandial accommodation by 688 +/- 165 ml (vs. 542 +/- 29 ml for placebo). L-NMMA (low dose) alone did not affect fasting or postprandial gastric volume. L-NMMA (low dose) did not attenuate the effect of GLP-1 on gastric volumes. In contrast, L-NMMA (high dose) did not affect fasting volume but blunted GLP-1-mediated postprandial accommodation (postprandial change = 494 +/- 37 ml, P < or = 0.01 vs. GLP-1 alone). These data are consistent with the hypothesis that nitric oxide partly mediates the effects of GLP-1 on postprandial but not fasting gastric volumes in humans.     

The regulation of the lymphatic secretion of glucagon-like peptide-1 (GLP-1) by intestinal absorption of fat and carbohydrate

Glucagon-like peptide-1 (GLP-1) is an important incretin produced in the L cells of the intestine. It is essential in the regulation of insulin secretion and glucose homeostasis. Systemic GLP-1 concentrations are typically low in rodents, so it can be difficult to assay physiological levels or detect changes in response to nutrients. We have established a method of assaying GLP-1 in response to nutrients using the intestinal lymph fistula model. Intraduodenal infusion of Intralipid (4.43 kcal/3 ml) induced a significant increase of lymphatic GLP-1 concentration compared with saline control at the peak of 30 min. (P < 0.001). Isocaloric and isovolumetric treatment with dextrin, a glucose polymer, also caused a significant fourfold increase in peak concentration at 60 min (P = 0.001). These findings indicate that intestinal lymph contains high concentrations of postprandial GLP-1. Second, they reveal that GLP-1 secretion into lymph occurs in response to both enteral carbohydrate and fat, but the response to dextrin occurs later than to Intralipid with peak times at 60 and 30 min, respectively. Third, the combination of Intralipid plus dextrin demonstrated an additive effect in the stimulation of GLP-1 with peak at 30 min. These results indicate that assessment of levels in lymph is a novel and powerful means of studying the secretion of GLP-1 and potentially other gastrointestinal hormones in vivo. Furthermore, the lymph fistula rat model provides insight into the gut hormone concentrations to which the neurons and cells in the lamina propria of the gut are likely exposed.     

Expression of glucagon-like peptide-1 (GLP-1) receptor and the effect of GLP-1-(7-36) amide on insulin release by pancreatic islets during rat ontogenic development.

The expression of glucagon-like peptide-1 (GLP-1) receptor and the effects of GLP-1-(7-36) amide (t-GLP-1) on glucose metabolism and insulin release by pancreatic islets during rat development were studied. GLP-1 receptor mRNA was found in significant amounts in pancreatic islets from all age groups studied, GLP-1 receptor expression being maximal when pancreatic islets were incubated at physiological glucose concentration (5.5 mM), but decreasing significantly when incubated with either 1.67 or 16.7 mM glucose. Glucose utilization and oxidation by pancreatic islets from fetal and adult rats rose as a function of glucose concentration, always being higher in fetal than in adult islets. The addition of t-GLP-1 to the incubation medium did not modify glucose metabolism but gastric inhibitory polypeptide and glucagon significantly increased glucose utilization by fetal and adult pancreatic islets at 16.7 mM glucose. At this concentration, glucose produced a significant increase in insulin release by the pancreatic islets from 10-day-old and 20-day-old suckling rats and adult rats, whereas those from fetuses showed only a significant increase when glucose was raised from 1.67 to 5.5 mM. t-GLP-1 elicited an increase in insulin release by pancreatic islets from all the experimental groups when the higher glucose concentrations were used. Our findings indicate that GLP-1 receptors and the effect of t-GLP-1 on insulin release are already present in the fetus, and they therefore exclude the possibility that alterations in the action of t-GLP-1 are responsible for the unresponsiveness of pancreatic beta cells to glucose in the fetus, but stimulation of t-GLP-1 release by food ingestion in newborns may partially confer glucose competence on beta cells.     

Small molecule ago-allosteric modulators of the human glucagon-like peptide-1 (hGLP-1) receptor

Following our previous publication describing the biological profiles, we herein describe the structure-activity relationships of a core set of quinoxalines as the hGLP-1 receptor agonists. The most potent and efficacious compounds are 6,7-dichloroquinoxalines bearing an alkyl sulfonyl group at the C-2 position and a secondary alkyl amino group at the C-3 position. These findings serve as a valuable starting point for the discovery of more drug-like small molecule agonists for the hGLP-1 receptor.     

Second extracellular loop of human glucagon-like peptide-1 receptor (GLP-1R) differentially regulates orthosteric but not allosteric agonist binding and function

The glucagon-like peptide-1 receptor (GLP-1R) is a prototypical family B G protein-coupled receptor that exhibits physiologically important pleiotropic coupling and ligand-dependent signal bias. In our accompanying article (Koole, C., Wootten, D., Simms, J., Miller, L. J., Christopoulos, A., and Sexton, P. M. (2012) J. Biol. Chem. 287, 3642-3658), we demonstrate, through alanine-scanning mutagenesis, a key role for extracellular loop (ECL) 2 of the receptor in propagating activation transition mediated by GLP-1 peptides that occurs in a peptide- and pathway-dependent manner for cAMP formation, intracellular (Ca(2+)(i)) mobilization, and phosphorylation of extracellular signal-regulated kinases 1 and 2 (pERK1/2). In this study, we examine the effect of ECL2 mutations on the binding and signaling of the peptide mimetics, exendin-4 and oxyntomodulin, as well as small molecule allosteric agonist 6,7-dichloro-2-methylsulfonyl-3-tert-butylaminoquinoxaline (compound 2). Lys-288, Cys-296, Trp-297, and Asn-300 were globally important for peptide signaling and also had critical roles in governing signal bias of the receptor. Peptide-specific effects on relative efficacy and signal bias were most commonly observed for residues 301-305, although R299A mutation also caused significantly different effects for individual peptides. Met-303 was more important for exendin-4 and oxyntomodulin action than those of GLP-1 peptides. Globally, ECL2 mutation was more detrimental to exendin-4-mediated Ca(2+)i release than GLP-1(7-36)-NH(2), providing additional evidence for subtle differences in receptor activation by these two peptides. Unlike peptide activation of the GLP-1R, ECL2 mutations had only limited impact on compound 2 mediated cAMP and pERK responses, consistent with this ligand having a distinct mechanism for receptor activation. These data suggest a critical role of ECL2 of the GLP-1R in the activation transition of the receptor by peptide agonists.     

Mutations to the third cytoplasmic domain of the glucagon-like peptide 1 (GLP-1) receptor can functionally uncouple GLP-1-stimulated insulin secretion in HIT-T15 cells.

Glucagon-like peptide-1 (GLP-1) is an insulinotropic hormone with powerful antidiabetogenic effects that are thought to be mediated by adenylyl cyclase (AC). Recently, we generated two GLP-1 receptor mutant isoforms (IC3-1 and DM-1) that displayed efficient ligand binding and the ability to promote Ca2+ mobilization from intracellular stores but lacked the ability to couple to AC. In the present study, the wild-type rat GLP-1 receptor (WT-GLP-1 R) or the IC3-1 and DM-1 mutant forms were expressed for the first time in the insulin-producing HIT-T15 cells. Only cells expressing WT-GLP-1 R displayed dramatically elevated GLP-1-induced cAMP responses and elevated insulin secretion. The increase in GLP-1-stimulated secretion in cells expressing WT-GLP-1 R, however, was not accompanied by differences in glucose-stimulated insulin release. Prolonged exposure to GLP-1 (10 nM, 17 h), not only led to an increase in insulin secretion but also increased insulin mRNA levels, but only in cells expressing the WT-GLP-1 R and not the mutant isoforms. Electrophysiological analyses revealed that GLP-1 application enhanced L-type voltage-dependent Ca2+ channel (VDCC) currents > 2-fold and caused a positive shift in VDCC voltage-dependent inactivation in WT-GLP-1R cells only, not control or mutant (DM-1) cells. This action on the Ca2+ current was further enhanced by the VDCC agonist, BAYK8644, suggesting GLP-1 acts via a distinct mechanism dependent on cAMP. These studies demonstrate that the GLP-1 receptor efficiently couples to AC to stimulate insulin secretion and that receptors lacking critical residues in the proximal region of the third intracellular loop can effectively uncouple the receptor from cAMP production, VDCC activity, insulin secretion, and insulin biosynthesis.     

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.     

N-acetyl-GLP-1: a DPP IV-resistant analogue of glucagon-like peptide-1 (GLP-1) with improved effects on pancreatic beta-cell-associated gene expression

Glucagon-like peptide-1(7-36)amide (GLP-1) is a key insulinotropic hormone with the reported potential to differentiate non-insulin secreting cells into insulin-secreting cells. The short biological half-life of GLP-1 after cleavage by dipeptidylpeptidase IV (DPP IV) to GLP-1(9-36)amide is a major therapeutic drawback. Several GLP-1 analogues have been developed with improved stability and insulinotropic action. In this study, the N-terminally modified GLP-1 analogue, N-acetyl-GLP-1, was shown to be completely resistant to DPP IV, unlike native GLP-1, which was rapidly degraded. Furthermore, culture of pancreatic ductal ARIP cells for 72 h with N-acetyl-GLP-1 indicated a greater ability to induce pancreatic beta-cell-associated gene expression, including insulin and glucokinase. Further investigation of the effects of stable GLP-1 analogues on beta-cell differentiation is required to assess their potential in diabetic therapy.     

Plasma glucagon-like peptide-1 (GLP-1) responses to duodenal fat and glucose infusions in lean and obese men

It has been suggested that obesity is associated with a reduced glucagon-like peptide-1 (GLP-1) response to oral carbohydrate, but not fat. The latter may, however, be attributable to changes in gastric emptying. We have assessed plasma GLP-1 levels in response to these infusions in lean and obese subjects. Seven healthy lean (body mass index (BMI), 19.1-24.6 kg/m(2)) and seven obese (BMI, 31.3-40.8 kg/m(2)) young men received an intraduodenal infusion of glucose and fat for 120 min (2.86 kcal/min) on two separate days. Blood samples for plasma GLP-1 were obtained at baseline and every 20 min during the infusion. Plasma GLP-1 increased during infusion of glucose and fat (P = 0.001), but there were no differences between lean and obese subjects, nor the two nutrients. We conclude that GLP-1 secretion in response to duodenal infusion of glucose and fat is not altered in obese subjects.