Glucagon-like Peptide-1 Binding Research Articles

Physical exercise promotes memory function in diabetes mellitus rats: a look at glucagon like peptide-1 and glucagon like peptide-1 receptor

Diabetes mellitus (DM) is a metabolic disorder associated with declining of memory function. Glucagon like peptide-1 (GLP-1) has a role on memory function; binding of GLP-1 and GLP-1 receptor (GLP-1R) can enhance synaptic plasticity. Physical exercise has effect in increasing GLP-1 levels mediated by interleukin (IL)-6 in plasma. However, the effect of physical exercise on GLP-1 and GLP-1R in hippocampus is still unclear. Therefore, we investigated the effect of continuous and interval training on memory function through GLP-1/GLP-1R and its relation to hippocampal IL-6 of DM rats. This was an experimental study using 8-week-old Wistar rats, divided into four groups: normal control (Con); DM control (ConDM); DM with continuous training (DM-CT); and DM with interval training (DM-IT). DM-CT and DM-IT rats were trained six times a week for six weeks. All rats performed the forced alteration Y-maze test to verify spatial memory function. We analysed GLP-1 and IL-6 level by ELISA and GLP-1R by RT-PCR. We found decreased spatial memory function in DM rats accompanied by decreased hippocampal GLP-1 and GLP-1R. Physical exercise promote memory function in DM rats associated with restoration of hippocampal GLP-1 or GLP-1R level. The GLP-1 level is associated with hippocampal IL-6 level. Continuous training slightly increases GLP-1 level while interval training can maintain expression of hippocampal GLP-1R in DM rats. Our findings suggest that physical exercise may promote memory function by slightly increase the level of hippocampal GLP-1 and maintaining expression hippocampal GLP-1R.

Molecular insights into ago-allosteric modulation of the human glucagon-like peptide-1 receptor

The glucagon-like peptide-1 (GLP-1) receptor is a validated drug target for metabolic disorders. Ago-allosteric modulators are capable of acting both as agonists on their own and as efficacy enhancers of orthosteric ligands. However, the molecular details of ago-allosterism remain elusive. Here, we report three cryo-electron microscopy structures of GLP-1R bound to (i) compound 2 (an ago-allosteric modulator); (ii) compound 2 and GLP-1; and (iii) compound 2 and LY3502970 (a small molecule agonist), all in complex with heterotrimeric Gs. The structures reveal that compound 2 is covalently bonded to C347 at the cytoplasmic end of TM6 and triggers its outward movement in cooperation with the ECD whose N terminus penetrates into the GLP-1 binding site. This allows compound 2 to execute positive allosteric modulation through enhancement of both agonist binding and G protein coupling. Our findings offer insights into the structural basis of ago-allosterism at GLP-1R and may aid the design of better therapeutics.

Kisspeptin Amplifies GLP‐1 Potentiation of Insulin Secretion by a cAMP Independent Pathway

Glucagon Like Peptide‐1 (GLP‐1) and Kisspeptin (KP) receptors are co‐expressed on pancreatic b‐cells, and activation of both receptors potentiates glucose stimulated insulin secretion (GSIS). Unlike GLP‐1, KP has a low affinity for receptor activation (EC50 GLP‐1 ~20 nM vs KP >1 uM). The effects of GLP‐1 and a 10 amino acid analog of KP (KP10) on insulin secretion and cell signaling were studied in the INS 832/3 cell line. By itself KP10 has no effect on insulin secretion in the absence of glucose, but doubled GSIS (GSIS = 37.7 +/− 6.7; +KP10 = 69.2 +/− 1.9 ng/ml/80min) with an EC50 of ~5–10 mM. At saturating doses, KP10 (100 mm) and GLP‐1 (100 nM) had similar effects on GSIS, and in combination had an additive effect (GLP‐1 = 49.0 +/− 2.0, GLP‐1 + KP10 = 63.5 +/− 5.6 ng/ml/80min) consistent with activation of independent downstream signaling pathways. Neither GLP‐1 or KP‐10 altered intracellular Ca2+ levels at basal or activating glucose concentrations. GLP‐1 increased cAMP production (165.0 +/− 17.4 % over control), whereas KP‐10 was without effect. Conversely, pretreatment with KP‐10 enhanced glucose stimulated oxygen consumption and mitochondrial NADH fluorescence, although both responses were inconsistent. Therefore, the effects of KP on b‐cell metabolic activity needs to be investigated in more detail.We also developed a bivalent ligand composed of GLP‐17‐36 and KP10 binding domains, linked with a 3‐mer of proline/glycine repeats, to evaluate if linking KP10 to a high affinity ligand could promote KP10 activity at low concentrations by presumably increasing its local membrane concentration after GLP‐1 binding. The dimer also did not activate secretion in the absence of glucose, but doubled GSIS much like the individual monomers with an EC50 ~200nM; that is, an additive response was not obvious. Currently, we are determining the ability of GLP‐1/KP10 to initiate downstream signaling from both ligands to resolve the question: is KP active in the GLP‐1/KP at concentrations where monomeric KP10 is not.Support or Funding InformationFunded by the American Diabetes Association and Juvenile Diabetes Research Foundation

Enhancing β‐cell Sensitivity to Kisspeptin via Linkage to GLP‐1

Glucagon Like Peptide‐1 (GLP‐1) and Kisspeptin (KP) receptors are co‐expressed on pancreatic β‐cells, and activation of both receptors potentiate glucose stimulated insulin secretion (GSIS), although they appear to work via different downstream signaling pathways. Unlike GLP‐1, KP has a low affinity for receptor activation (EC50 20 nM vs >1 uM) making it difficult to use as a drug without significant side effects. We developed a bivalent ligand composed of GLP‐17–36 and KP‐10 binding domains linked together, to evaluate if linking KP to a high affinity ligand would promote its activity at low concentrations by presumably increasing its local membrane concentration after GLP‐1 binding. This feature may also provide some specificity for targeting to β‐cells which express both receptors. The effects of KP‐10, GLP‐1 and the KP‐10/GLP‐1 bivalent analog on insulin secretion from the INS 832/3 cell line were investigated. KP‐10 by itself had no effect on insulin secretion in the absence of glucose, but more than doubled GSIS (basal, 0.23 +/− 0.07; GSIS 0.60 +/− 0.02 ng/min) with an EC50 of ~25 μM. At saturating doses, KP‐10 (100 μm) and GLP‐1 (100 nM) had similar effects on GSIS, and together as monomers had an additive effect (0.79 +/− 0.07 (ng/min) consistent with activation of independent downstream signaling pathways. The dimer also did not activate secretion in the absence of glucose, but doubled GSIS much like the individual monomers (0.64 +/− 0.01 ng/min); that is, an additive response was not obvious. This finding suggests that either the KP‐10 moiety is not binding or is not active in the bivalent ligand, or that GLP‐1 activity is diminished in the bivalent construct such that the combined effect of GLP‐1/KP‐10 is diminished when compared to the additive effect or the monomers. We have evidence from previous studies that binding of the GLP‐1 moiety within a bivalent construct is decreased relative to monomeric GLP‐11, suggesting an additive response may be present. Currently, we are determining the ability of GLP‐1/KP‐10 to initiate downstream signaling from both ligands to resolve the question: is KP active in the GLP‐1/KP at concentrations where monomeric KP‐10 is not.Support or Funding InformationFunded by the American Diabetes Association and Juvenile Diabetes Research FoundationThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Glucagon-like Peptide 1 Conjugated to Recombinant Human Serum Albumin Variants with Modified Neonatal Fc Receptor Binding Properties. Impact on Molecular Structure and Half-Life.

Glucagon-like peptide 1 (GLP-1) is a small incretin hormone stimulated by food intake, resulting in an amplification of the insulin response. Though GLP-1 is interesting as a drug candidate for the treatment of type 2 diabetes mellitus, its short plasma half-life of <3 min limits its clinical use. A strategy for extending the half-life of GLP-1 utilizes the long half-life of human serum albumin (HSA) by combining the two via chemical conjugation or genetic fusion. HSA has a plasma half-life of around 21 days because of its interaction with the neonatal Fc receptor (FcRn) expressed in endothelial cells of blood vessels, which rescues circulating HSA from lysosomal degradation. We have conjugated GLP-1 to C34 of native sequence recombinant HSA (rHSA) and two rHSA variants, one with increased and one with decreased binding affinity for human FcRn. We have investigated the impact of conjugation on FcRn binding affinities, GLP-1 potency, and pharmacokinetics, combined with the solution structure of the rHSA variants and GLP-1-albumin conjugates. The solution structures, determined by small-angle X-ray scattering, show the GLP-1 pointing away from the surface of rHSA. Combining the solution structures with the available structural information about the FcRn and GLP-1 receptor obtained from X-ray crystallography, we can explain the observed in vitro and in vivo behavior. We conclude that the conjugation of GLP-1 to rHSA does not affect the interaction between rHSA and FcRn, while the observed decrease in the potency of GLP-1 can be explained by a steric hindrance of binding of GLP-1 to its receptor.

Truncated Glucagon-like Peptide-1 and Exendin-4 α-Conotoxin pl14a Peptide Chimeras Maintain Potency and α-Helicity and Reveal Interactions Vital for cAMP Signaling in Vitro

Glucagon-like peptide-1 (GLP-1) signaling through the glucagon-like peptide 1 receptor (GLP-1R) is a key regulator of normal glucose metabolism, and exogenous GLP-1R agonist therapy is a promising avenue for the treatment of type 2 diabetes mellitus. To date, the development of therapeutic GLP-1R agonists has focused on producing drugs with an extended serum half-life. This has been achieved by engineering synthetic analogs of GLP-1 or the more stable exogenous GLP-1R agonist exendin-4 (Ex-4). These synthetic peptide hormones share the overall structure of GLP-1 and Ex-4, with a C-terminal helical segment and a flexible N-terminal tail. Although numerous studies have investigated the molecular determinants underpinning GLP-1 and Ex-4 binding and signaling through the GLP-1R, these have primarily focused on the length and composition of the N-terminal tail or on how to modulate the helicity of the full-length peptides. Here, we investigate the effect of C-terminal truncation in GLP-1 and Ex-4 on the cAMP pathway. To ensure helical C-terminal regions in the truncated peptides, we produced a series of chimeric peptides combining the N-terminal portion of GLP-1 or Ex-4 and the C-terminal segment of the helix-promoting peptide α-conotoxin pl14a. The helicity and structures of the chimeric peptides were confirmed using circular dichroism and NMR, respectively. We found no direct correlation between the fractional helicity and potency in signaling via the cAMP pathway. Rather, the most important feature for efficient receptor binding and signaling was the C-terminal helical segment (residues 22-27) directing the binding of Phe(22) into a hydrophobic pocket on the GLP-1R.

A Synthetic Heterobivalent Ligand Composed of Glucagon-Like Peptide 1 and Yohimbine Specifically Targets β Cells Within the Pancreas.

β Cell specificity for a heterobivalent ligand composed of glucagon-like peptide-1 (GLP-1) linked to yohimbine (GLP-1/Yhb) was evaluated to determine its utility as a noninvasive imaging agent. Competition binding assays were performed on βTC3 cells and isolated rat islets. Immunostaining for insulin was used to co-localized intravenously injected Cy5-labeled GLP-1/Yhb in β cells of Sprague-Dawley rats. Rats were intravenously injected with In-111-labeled GLP-1/Yhb to determine clearance rates and tissue biodistribution. Tissue-specific binding was confirmed by competition with pre-administration of unlabeled GLP-1/Yhb and in Streptozotocin-induced diabetic rats. In βTC3 cells, high affinity binding of GLP-1/Yhb required interactions with both receptors because monovalent competition or receptor knockdown with RNAi lowered specificity and avidity of the heterobivalent ligand. Binding specificity for isolated islets was 2.6-fold greater than that of acinar tissue or islets pre-incubated with excess unlabeled GLP-1/Yhb. Immunofluorescent localization of Cy5-labeled GLP-1/Yhb was restricted to pancreatic islets. Within 30 min, ~90% of the In-111-labeled GLP-1/Yhb was cleared from blood. Tissue-specific accumulation of radiolabeled ligand was apparent in the pancreas, but not in other tissues within the abdominal imaging field. Pancreas specificity was lost in Streptozotocin-induced diabetic rats. The GLP-1/Yhb exhibits high specificity for β cells, rapid blood clearance rates, and low non-specific uptake by other tissues within the abdominal imaging field. These characteristics of GLP-1/Yhb are desirable for application to β cell imaging in vivo and provide a basis for developing additional multivalent β cell-specific targeting agents to aid in the management of type 1 diabetes.

Expression of the Glucagon Like Peptide-1 Receptor in Human Pancreas and Incretin Therapy

Incretins are hormones released by gut enteroendocrine cells in response to nutrient ingestion that regulate circulating glucose levels. One member of the incretin family is the glucagon-like peptide-1 (GLP-1), which is secreted by intestinal L cells located mainly in the mucosa of ileum and colon. It is produced by posttranslational processing of the glucagon gene product. GLP-1 binds to a specific receptor (GLP-1R), a seven-transmembrane G proteincoupled receptor originally identified in islet -cells (1) with a broad range of tissue-specific functions (1–8). The binding of GLP-1 to the GLP-1R on pancreatic -cells augments glucose-stimulated insulin secretion, increases transcription of the insulin gene, and enhances both the stability of insulin mRNA and biosynthesis of insulin (reviewed in References 9 and 10). In addition to the glucoregulatory effects, GLP-1 has been reported to protect -cells from apoptosis (11) and to stimulate their neogenesis and proliferation (12, 13). After secretion, GLP-1 is rapidly degraded into GLP-1 (9–36) by the enzyme dipeptidyl-peptidase IV (DPP-4) (14). As a result, the biological half-life of GLP-1 is less than 5 minutes (15). DPP-4 enzymatic activity can be inhibited by DPP-4 inhibitors (DPP-4-i), which prevents the cleavage of GLP-1 and other DPP-4 targets. In doing so, the inhibitor maintains the intact levels of the molecules, improving glucose tolerance (reviewed in Reference 16). Because of their beneficial effect in controlling blood glucose levels, DPP-4is and GLP-1 agonists are current therapies for type 2 diabetes. Recently, however, the use of GLP-1-based therapies has been deemed dangerous by a publication indicating that they increase the risk of pancreatic cancer (17). Similarly, it was also suggested that incretin administration might be related to the development of thyroid cancer (18). One of the leading arguments that underpinned the proposed relationshipbetween incretins andcancersof the pancreas and thyroid was the reported localization of the GLP-1R to the pancreatic duct and acinar tissue (19) of rodents and to the human thyroid gland (18). The hypothesis raised by the authors of those studies proposed that long-term stimulation of cells expressing the GLP-1R resulted in cell hyperplasia and tumor formation. In the human thyroid, neoplastic and hyperplastic lesions of thyroid C cells were reported to contain GLP-1Rimmunopositive cells (18). The experiments analyzing the effect of GLP-1 stimulation in the pancreas were performed in rats receiving daily injections of the GLP-1 analog exendin-4 and in a mouse model of pancreatic cancer. These studies revealed the presence of immune-positive GLP-1R cells in lesions of the pancreatic duct [termed pancreatic duct glands (PDGs)] that appear during chronic injury and in pancreatic intraepithelial neoplasia (PanIN) (19). Because PDGs and PanIN were also found in the pancreas of diabetic patients who received DPP-4is, the conclusion was reached that the inhibitors caused tumor development (17). This report, suggesting a deleterious effect of DPP-4is in the pancreas of human diabetic patient, was highly controversial, mainly because the diabetic groups receiving either incretin therapy or other insulin secretagogues were not comparable with regard to age and duration of the disease. Several publications have already raised severe concerns regarding the clinical aspects of this publication (16, 20–23). A critical issue in the studies reporting a correlation between incretin therapy and cancer development is the

DNA methylation of the glucagon-like peptide 1 receptor (GLP1R) in human pancreatic islets

BackgroundInsulin secretion is enhanced upon the binding of Glucagon-like peptide-1 (GLP-1) to its receptor (GLP1R) in pancreatic β cells. Although a reduced expression of GLP1R in pancreatic islets from type 2 diabetic patients and hyperglycaemic rats has been established, it is still unknown if this is caused by differential DNA methylation of GLP1R in pancreatic islets of type 2 diabetic patients.MethodsIn this study, DNA methylation levels of 12 CpG sites close to the transcription start site of GLP1R were analysed in pancreatic islets from 55 non-diabetic and 10 type 2 diabetic human donors as well as in β and α cells isolated from human pancreatic islets. DNA methylation of GLP1R was related to GLP1R expression, HbA1c levels and BMI. Moreover, mRNA expression of MECP2, DNMT1, DNMT3A and DNMT3B was analysed in pancreatic islets of the non-diabetic and type 2 diabetic donors.ResultsOne CpG unit, at position +199 and +205 bp from the transcription start site, showed a small increase in DNA methylation in islets from donors with type 2 diabetes compared to non-diabetic donors (0.53%, p=0.02). Furthermore, DNA methylation levels of one CpG site located 376 bp upstream of the transcription start site of GLP1R correlated negatively with GLP1R expression (rho=−0.34, p=0.008) but positively with BMI and HbA1c (rho=0.30, p=0.02 and rho=0.30, p=0.03, respectively). This specific CpG site is located in an area with known SP1 and SP3 transcription factor binding sites. Moreover, when we compared the DNA methylation of the GLP1R promoter in isolated human β and α cells, we found that it was higher in α- compared with β-cells (p=0.009). Finally, there was a trend towards decreased DNMT3A expression (p=0.056) in type 2 diabetic compared with non-diabetic islets.ConclusionsTogether, our study shows that while BMI and HbA1c are positively associated with DNA methylation levels of GLP1R, its expression is negatively associated with DNA methylation of GLP1R in human pancreatic islets.

Type 2 Diabetes Mellitus and Glucagon Like Peptide-1 Receptor Signalling

It has been estimated that approximately 8.4% of the world population currently live with diabetes mellitus and type 2 diabetes is the most common form. Type 2 diabetes increases the risk of complications such as heart attacks, blindness, amputations and kidney failure. Glucagon like Peptide-1 (GLP-1) is an effective insulinotropic agent and therefore its effects on insulin secretion have been greatly examined for more than two decades. It is a polypeptide hormone secreted by the intestinal L-cells into the blood in response to food uptake. GLP-1 has a very short half-life in vivo due to the rapid proteolytic degradation by Dipeptidyl Peptidase IV (DPP-IV). Therefore DPP-IV resistant GLP-1 analogues, Exenatide and Liraglutide, have been developed and are currently being used in the treatment of type 2 diabetes. GLP-1 agonist functions by binding to its receptor, GLP1R, on the cell surface. The GLP-1R belongs to the class B peptide receptor family based on its structure and function. The binding of GLP-1 to its receptor results in activation of Gαs coupled adenylyl cyclase and the production of cyclic Adenosine Monophosphate (cAMP), which enhances glucose-induced insulin secretion. Continuous GLP-1R activation also causes insulin secretion and pancreatic islet β-cell proliferation and neogenesis. The GLP-1R is internalised following its activation, which regulates the biological responsiveness of the receptor. Structurally the GLP-1R contains a large N-terminal extracellular domain (TM1-TM7) joined by three intracellular loops (ICL1, ICL2, ICL3) and three extracellular loops (ECL1, ECL2, ECL3), and an intracellular C-terminal domain. These domains play critical roles in GLP-1R trafficking to the cell surface, and also in agonist dependent activation and internalisation of the receptor. This review is focused on type 2 diabetes, its treatment with GLP-1, GLP-1R structure and function, and the physiological affects resulting from GLP-1R activation.

Microscopic Rates of Peptide–Phospholipid Bilayer Interactions from Single-Molecule Residence Times

The binding of glucagon-like peptide-1 (GLP-1) to a planar phospholipid bilayer is measured using single-molecule total internal reflection fluorescence microscopy. From several reports in the literature, GLP-1 has been shown to be a random coil in free solution, adopting a folded, α-helix conformation when intercalated into membrane environments. Single-molecule fluorescence measurements of GLP-1 binding to supported lipid bilayers show evidence of two populations of membrane-associated molecules having different residence times, suggesting weakly adsorbed peptides and strongly bound peptides in the lipid bilayer. The path to and from a strongly bound (folded, intercalated) state would likely include an adsorbed state as an intermediate, so that the resulting kinetics would correspond to a consecutive first-order reversible three-state model. In this work, the relationships between measured single-molecule residence times and the microscopic rates in a three-state kinetic model are derived and used to interpret the binding of GLP-1 to a supported lipid bilayer. The system of differential equations associated with the proposed consecutive-three state kinetics scheme is solved, and the solution is applied to interpret histograms of single-molecule, GLP-1 residence times in terms of the microscopic rates in the sequential two-step model. These microscopic rates are used to estimate the free energy barrier to adsorption, the fraction of peptides adsorbing to the membrane surface that successfully intercalate in the bilayer, the lifetime of inserted peptides in the membrane, and the free energy change of insertion into the lipid bilayer from the adsorbed state. The transition from a random coil in solution to a folded state in a membrane has been recognized as a common motif for insertion of membrane active peptides. Therefore, the relationships developed here could have wide application to the kinetic analysis of peptide-membrane interactions.

Lateral Allosterism in the Glucagon Receptor Family: Glucagon-Like Peptide 1 Induces G-Protein-Coupled Receptor Heteromer Formation

Activation of G-protein-coupled receptors (GPCRs) results in a variety of cellular responses, such as binding to the same receptor of different ligands that activate distinct downstream cascades. Additional signaling complexity is achieved when two or more receptors are integrated into one signaling unit. Lateral receptor interactions can allosterically modulate the receptor response to a ligand, which creates a mechanism for tissue-specific fine tuning, depending on the cellular receptor coexpression pattern. GPCR homomers or heteromers have been explored widely for GPCR classes A and C but to lesser extent for class B. In the present study, we used bioluminescence resonance energy transfer (BRET) techniques, calcium flux measurements, and microscopy to study receptor interactions within the glucagon receptor family. We found basal BRET interactions for some of the receptor combinations tested that decreased upon ligand binding. A BRET increase was observed exclusively for the gastric inhibitory peptide (GIP) receptor and the glucagon-like peptide 1 (GLP-1) receptor upon binding of GLP-1 that could be reversed with GIP addition. The interactions of GLP-1 receptor and GIP receptor were characterized with BRET donor saturation studies, shift experiments, and tests of glucagon-like ligands. The heteromer displayed specific pharmacological characteristics with respect to GLP-1-induced β-arrestin recruitment and calcium flux, which suggests a form of allosteric regulation between the receptors. This study provides the first example of ligand-induced heteromer formation in GPCR class B. In the body, the receptors are functionally related and coexpressed in the same cells. The physiological evidence for this heteromerization remains to be determined.

Refinement of Glucagon-like Peptide 1 Docking to Its Intact Receptor Using Mid-region Photolabile Probes and Molecular Modeling

The glucagon-like peptide 1 (GLP1) receptor is an important drug target within the B family of G protein-coupled receptors. Its natural agonist ligand, GLP1, has incretin-like actions and the receptor is a recognized target for management of type 2 diabetes mellitus. Despite recent solution of the structure of the amino terminus of the GLP1 receptor and several close family members, the molecular basis for GLP1 binding to and activation of the intact receptor remains unclear. We previously demonstrated molecular approximations between amino- and carboxyl-terminal residues of GLP1 and its receptor. In this work, we study spatial approximations with the mid-region of this peptide to gain insights into the orientation of the intact receptor and the ligand-receptor complex. We have prepared two new photolabile probes incorporating a p-benzoyl-l-phenylalanine into positions 16 and 20 of GLP1(7-36). Both probes bound to the GLP1 receptor specifically and with high affinity. These were each fully efficacious agonists, stimulating cAMP accumulation in receptor-bearing CHO cells in a concentration-dependent manner. Each probe specifically labeled a single receptor site. Protease cleavage and radiochemical sequencing identified receptor residue Leu(141) above transmembrane segment one as its site of labeling for the position 16 probe, whereas the position 20 probe labeled receptor residue Trp(297) within the second extracellular loop. Establishing ligand residue approximation with this loop region is unique among family members and may help to orient the receptor amino-terminal domain relative to its helical bundle region.

Role of glucagon-like peptide-1 and its agonists on early prevention of cardiac remodeling in type 1 diabetic rat hearts

Glucagon-like peptide-1 (GLP-1) is an incretin hormone secreted from intestinal L cells upon nutrients ingestion, and is currently used for treating diabetes mellitus. It plays an important role in receptor modulation and cross talk with insulin at the coronary endothelium (CE) and cardiomyocytes (CM) in diabetic type 1 rat heart model. We studied the effects of insulin, GLP-1 analogues (exendin-4), and dipeptidyl peptidase-IV (DPP-IV) inhibitor on GLP-1 cardiac receptor modulation. The binding affinity of GLP-1 to its receptor on CE and CM was calculated using a rat heart perfusion model with [(125)I]-GLP-1(7-36). Tissue samples from the heart were used for immunostaining and Western blot analyses. GLP-1 systemic blood levels were measured using ELISA. GLP-1 binding affinity (τ) increased on the CE (0.33 ± 0.01 vs. 0.25 ± 0.01 min; p < 0.001) and decreased on the CM (0.29 ± 0.02 vs. 0.43 ± 0.02 min; p < 0.001) in the diabetic non-treated rats when compared to normal. There was normalization of τ back to baseline on the CE and CM levels with insulin and DPP-IV inhibitor treatment, respectively. Histological sections and immunofluorescence showed receptor up-regulation in diabetic rats with significant decrease and even normalization with the different treatment strategies. Systemic GLP-1 levels increased after 14 days of diabetes induction (10 ± 3.7 vs. 103 ± 58 pM; p = 0.0005). In conclusion, there is a significant GLP-1 receptor affinity modulation on the CE and CM levels in rats with diabetes type 1, and a cross talk with GLP-1 analogues in early prevention of cardiac remodeling.

Presence of a functional receptor for GLP‐1 in osteoblastic cells, independent of the cAMP‐linked GLP‐1 receptor

Glucagon-like peptide 1 (GLP-1) controls glucose metabolism in extrapancreatic tissues through receptors other than the pancreatic cAMP-linked GLP-1 receptor; also, GLP-1 induces an insulin- and PTH-independent bone anabolic action in insulin-resistant and type-2 diabetic rats. Here we searched for the presence and characteristics of GLP-1 receptors in osteoblastic MC3T3-E1 cells. [(125)I]-GLP-1 specific binding to MC3T3-E1 cells was time- and temperature-dependent, reaching maximal value at 30 min at 25 degrees C; in these conditions, [(125)I]-GLP-1 binding was dissociable, and displaced by GLP-1, partially by GLP-2, but not by exendin-4 (Ex-4), exendin-9 (Ex-9), glucagon or insulin; Scatchard analysis of the unlabeled GLP-1 data showed high and low affinity binding sites; cross-linking of GLP-1 binding revealed an estimated 70 kDa band, almost undetectable in the presence of 10(-6) M GLP-1. GLP-1, Ex-9, insulin or glucagon failed to modify cellular cAMP content, while GLP-2 and Ex-4 increased it. However, GLP-1 induced an immediate hydrolysis of glycosylphosphatidylinositols (GPIs) generating short-lived inositolphosphoglycans (IPGs), and an increase in phosphatidylinositol-3 kinase (PI3K) and mitogen activated protein kinase (MAPK) activities; Ex-4 also affected GPIs, but its action was delayed with respect to that of GLP-1. This incretin was found to decrease Runx2 but increased osteocalcin gene expression, without affecting that of osteoprotegerin or the canonical Wnt pathway activity in MC3T3-E1 cells which do not express the pancreatic GLP-1 receptor. Our data demonstrate for the first time that GLP-1 can directly and functionally interact with osteoblastic cells, possibly through a GPI/IPG-coupled receptor.

Role ofN-linked glycosylation in biosynthesis, trafficking, and function of the human glucagon-like peptide 1 receptor

The family B G protein-coupled glucagon-like peptide 1 (GLP-1) receptor is an important drug target for treatment of type 2 diabetes. Like other family members, the GLP-1 receptor is a glycosylated membrane protein that contains three potential sites for N-linked glycosylation within the functionally important extracellular amino-terminal domain. However, the roles for each potential site of glycosylation in receptor biosynthesis, trafficking, and function are not known. In this work, we demonstrated that tunicamycin inhibition of glycosylation of the GLP-1 receptor expressed in CHO cells interfered with biosynthesis and intracellular trafficking, thereby eliminating natural ligand binding. To further investigate the roles of each of the glycosylation sites, site-directed mutagenesis was performed to eliminate these sites individually and in aggregate. Our results showed that mutation of each of the glycosylation sites individually did not interfere with receptor expression on the cell surface, ligand binding, and biological activity. However, simultaneous mutation of two or three glycosylation sites resulted in almost complete loss of GLP-1 binding and severely impaired biological activity. Immunostaining studies demonstrated receptor biosynthesis but aberrant trafficking, with most of the receptor trapped in the endoplasmic reticulum and golgi compartments and little of the receptor expressed on the cell surface. Interestingly, surface expression, ligand binding, and biological activity of these mutants improved significantly when biosynthesis was slowed using low temperature (30 degrees C). These data suggest that N-linked glycosylation of the GLP-1 receptor is important for its normal folding and trafficking to the cell surface.

Molecular Basis of Glucagon-like Peptide 1 Docking to Its Intact Receptor Studied with Carboxyl-terminal Photolabile Probes

The glucagon-like peptide 1 (GLP1) receptor is a member of Family B G protein-coupled receptors and represents an important drug target for type 2 diabetes. Despite recent solution of the structure of the amino-terminal domain of this receptor and that of several close family members, understanding of the molecular basis of natural ligand GLP1 binding to its intact receptor remains limited. The goal of this study was to explore spatial approximations between specific receptor residues within the carboxyl terminus of GLP1 and its receptor as normally docked. Therefore, we developed and characterized two high affinity, full-agonist photolabile GLP1 probes having sites for covalent attachment in positions 24 and 35. Both probes labeled the receptor specifically and saturably. Subsequent peptide mapping using chemical and proteinase cleavages of purified wild-type and mutant GLP1 receptor identified that the Arg(131)-Lys(136) segment at the juxtamembrane region of the receptor amino terminus contained the site of labeling for the position 24 probe, and the specific receptor residue labeled by this probe was identified as Glu(133) by radiochemical sequencing. Similarly, nearby residue Glu(125) within the same region of the receptor amino-terminal domain was identified as the site of labeling by the position 35 probe. These data represent the first direct demonstration of spatial approximation between GLP1 and its intact receptor as docked, providing two important constraints for the modeling of this interaction. This should expand our understanding of the molecular basis of natural agonist ligand binding to the GLP1 receptor and may be relevant to other family members.

Glucagon-like peptide-1 inhibits LPS-induced IL-1β production in cultured rat astrocytes

Glia play an important role in neurotoxicity in neurodegenerative diseases. In this study, we investigated the expression of glucagon-like peptide-1 (GLP-1) and its receptor, and the effects of GLP-1 on lipopolysaccharide (LPS)-induced IL-1β mRNA expression and IL-1β production in glia. GLP-1-like immunoreactivity was observed in amoeboid microglia, but not ramified microglia or astrocytes. GLP-1 binding and GLP-1 receptor mRNA expression were observed in both astrocytes and microglia. GLP-1-induced morphological changes in microglia from the ramified type to the amoeboid type, suggesting an increase in production and release of endogenous GLP-1. GLP-1 prevented the LPS-induced IL-1β mRNA expression, which effect was, in turn, inhibited by pretreatment with SQ22536, an adenylate cyclase inhibitor. GLP-1 also increased cAMP concentration and cAMP response element-binding protein phosphorylation in astrocytes. These results suggest that GLP-1 may be a modulator of inflammation in the central nervous system.

In depth analysis of the N-terminal bioactive domain of gastric inhibitory polypeptide

Gastric inhibitory polypeptide/glucose-dependent insulinotropic polypeptide (GIP) is an important gastrointestinal regulator of insulin release and glucose homeostasis following a meal. Strategies have been undertaken to delineate the bioactive domains of GIP with the intention of developing small molecular weight GIP mimetics. The molecular cloning of receptors for GIP and the related hormone GLP-1 (glucagon-like peptide-1) has allowed examination of the characteristics of incretin analogs in transfected cell models. The current report examines the N-terminal bioactive domain of GIP residing in residues 1–14 by alanine scanning mutagenesis and N-terminal substitution/modification. Further studies examined peptide chimeras of GIP and GLP-1 designed to localize bioactive determinants of the two hormones. The alanine scan of the GIP 1–14 sequence established that the peptide was extremely sensitive to structural perturbations. Only replacement of amino acids 2 and 13 with those found in glucagon failed to dramatically reduce receptor binding and activation. Of four GIP 1–14 peptides modified by the introduction of DP IV-resistant groups, a peptide with a reduced bond between Ala 2 and Glu 3 demonstrated improved receptor potency compared to native GIP 1–14. The peptide chimera studies supported recent results on the importance of a mid-region helix for bioactivity of GIP, and confirmed existence of two separable regions with independent intrinsic receptor binding and activation properties. Furthermore, peptide chimeras showed that binding of GLP-1 also involves both N- and C-terminal domains, but that it apparently contains only a single bioactive domain in its N-terminus. Together, these results should facilitate development of incretin based therapies using rational drug design for potential treatment of diabetes.

Characterization of glucagon-like peptide-1 receptor-binding determinants.

Glucagon-like peptide 1 (GLP-1) is a potent insulinotropic hormone currently under study as a therapeutic agent for type 2 diabetes. Since an understanding of the molecular mechanisms leading to high-affinity receptor (R) binding and activation may facilitate the development of more potent GLP-1R agonists, we have localized specific regions of GLP-1R required for binding. The purified N-terminal fragment (hereafter referred to as NT) of the GLP-1R produced in either insect (Sf9) or mammalian (COS-7) cells was shown to bind GLP-1. The physical interaction of NT with GLP-1 was first demonstrated by cross-linking ((125)I-GLP-1/NT complex band at approximately 28 kDa) and secondly by attachment to Ni(2+)-NTA beads. The GLP-1R NT protein attached to beads bound GLP-1, but with lower affinity (inhibitory concentration (IC(50)): 4.5 x 10(-7) M) than wild-type (WT) GLP-1R (IC(50): 5.2 x 10(-9)M). The low affinity of GLP-1R NT suggested that other receptor domains may contribute to GLP-1 binding. This was supported by studies using chimeric glucose-dependent insulinotropic polypeptide (GIP)/GLP-1 receptors. GIP(1-151)/GLP-1R, but not GIP(1-222)/GLP-1R, exhibited specific GLP-1 binding and GLP-1-induced cAMP production, suggesting that the region encompassing transmembrane (TM) domain 1 through to TM3 was required for binding. Since it was hypothesized that certain charged or polar amino acids in this region might be involved in binding, these residues (TM2-TM3) were analyzed by substitution mutagenesis. Five mutants (K197A, D198A, K202A, D215A, R227A) displayed remarkably reduced binding affinity. These studies indicate that the NT domain of the GLP-1R is able to bind GLP-1, but charged residues concentrated at the distal TM2/extracellular loop-1 (EC1) interface (K197, D198, K202) and in EC1 (D215 and R227) probably contribute to the binding determinants of the GLP-1R.