Glucagon receptor antagonist upregulates circulating GLP-1 level by promoting intestinal L-cell proliferation and GLP-1 production in type 2 diabetes.

OBJECTIVE: Glucagon receptor (GCGR) blockage improves glycemic control and increases circulating glucagon-like peptide-1 (GLP-1) level in diabetic animals and humans. The elevated GLP-1 has been reported to be involved in the hypoglycemic effect of GCGR blockage. However, the source of this elevation remains to be clarified. RESEARCH DESIGN AND METHODS: REMD 2.59, a human GCGR monoclonal antibody (mAb), was administrated for 12 weeks in db/db mice and high-fat diet+streptozotocin (HFD/STZ)-induced type 2 diabetic (T2D) mice. Blood glucose, glucose tolerance and plasma GLP-1 were evaluated during the treatment. The gut length, epithelial area, and L-cell number and proliferation were detected after the mice were sacrificed. Cell proliferation and GLP-1 production were measured in mouse L-cell line GLUTag cells, and primary mouse and human enterocytes. Moreover, GLP-1 receptor (GLP-1R) antagonist or protein kinase A (PKA) inhibitor was used in GLUTag cells to determine the involved signaling pathways. RESULTS: Treatment with the GCGR mAb lowered blood glucose level, improved glucose tolerance and elevated plasma GLP-1 level in both db/db and HFD/STZ-induced T2D mice. Besides, the treatment promoted L-cell proliferation and LK-cell expansion, and increased the gut length, epithelial area and L-cell number in these two T2D mice. Similarly, our in vitro study showed that the GCGR mAb promoted L-cell proliferation and increased GLP-1 production in GLUTag cells, and primary mouse and human enterocytes. Furthermore, either GLP-1R antagonist or PKA inhibitor diminished the effects of GCGR mAb on L-cell proliferation and GLP-1 production. CONCLUSIONS: The elevated circulating GLP-1 level by GCGR mAb is mainly due to intestinal L-cell proliferation and GLP-1 production, which may be mediated via GLP-1R/PKA signaling pathways. Therefore, GCGR mAb represents a promising strategy to improve glycemic control and restore the impaired GLP-1 production in T2D.

Single and multiple ascending-dose study of glucagon-receptor antagonist RN909 in type 2 diabetes: a phase 1, randomized, double-blind, placebo-controlled trial.

PURPOSE: This first-in-human study assessed safety, immunogenicity, pharmacokinetics, and pharmacodynamics of RN909, a monoclonal antibody antagonist of the glucagon receptor, in type 2 diabetes (T2DM) subjects. METHODS: This study enrolled 84 T2DM subjects receiving stable metformin regimens. Forty-four subjects were randomized to receive single escalating doses of RN909 (0.3 to 6 mg/kg subcutaneously (SC), or 1 mg/kg intravenously (IV)), or placebo; 40 subjects were randomized to receive multiple escalating doses (50 to 150 mg SC) or placebo every 4 weeks for 12 weeks. RESULTS: RN909 was well tolerated; treatment-related elevated liver function tests (LFTs) were observed in 4/33 (12.1%) and 5/32 (15.6%) subjects treated with single and multiple doses, respectively, versus 1/10 (10%) and 0 in the respective placebo groups. RN909 dose-normalized AUCinf increased more than dose-proportionally following single SC doses, and after multiple doses, accumulation ratios ranged from 1.3 to 3.4. The incidence of antidrug antibodies (ADA) was 33% after single doses and 50% after multiple doses. RN909 produced dose-dependent, durable fasting plasma glucose (FPG)-lowering at day 29 (mean change -20.6 to -97.5 mg/dL) and day 85 (mean change; -27.2 to -43.5 mg/dL) after single and multiple doses, respectively. HbA1c also was reduced after single (mean change -0.30% to -1.44%), and multiple doses (-0.83% to -1.56%). CONCLUSION: RN909 was well tolerated after single and multiple doses in T2DM subjects, with diarrhea and elevated LFTs the most frequent adverse events. The appearance of ADA did not affect pharmacokinetics or efficacy. Robust lowering of FPG and HbA1c was observed.

DNA immunization combined with scFv phage display identifies antagonistic GCGR specific antibodies and reveals new epitopes on the small extracellular loops.

The identification of functional monoclonal antibodies directed against G-protein coupled receptors (GPCRs) is challenging because of the membrane-embedded topology of these molecules. Here, we report the successful combination of llama DNA immunization with scFv-phage display and selections using virus-like particles (VLP) and the recombinant extracellular domain of the GPCR glucagon receptor (GCGR), resulting in glucagon receptor-specific antagonistic antibodies. By immunizing outbred llamas with plasmid DNA containing the human GCGR gene, we sought to provoke their immune system, which generated a high IgG1 response. Phage selections on VLPs allowed the identification of mAbs against the extracellular loop regions (ECL) of GCGR, in addition to multiple VH families interacting with the extracellular domain (ECD) of GCGR. Identifying mAbs binding to the ECL regions of GCGR is challenging because the large ECD covers the small ECLs in the energetically most favorable 'closed conformation' of GCGR. Comparison of Fab with scFv-phage display demonstrated that the multivalent nature of scFv display is essential for the identification of GCGR specific clones by selections on VLPs because of avid interaction. Ten different VH families that bound 5 different epitopes on the ECD of GCGR were derived from only 2 DNA-immunized llamas. Seven VH families demonstrated interference with glucagon-mediated cAMP increase. This combination of technologies proved applicable in identifying multiple functional binders in the class B GPCR context, suggesting it is a robust approach for tackling difficult membrane proteins.

Glucagon Receptor Blockade With a Human Antibody Normalizes Blood Glucose in Diabetic Mice and Monkeys.

Antagonizing glucagon action represents an attractive therapeutic option for reducing hepatic glucose production in settings of hyperglycemia where glucagon excess plays a key pathophysiological role. We therefore generated REGN1193, a fully human monoclonal antibody that binds and inhibits glucagon receptor (GCGR) signaling in vitro. REGN1193 administration to diabetic ob/ob and diet-induced obese mice lowered blood glucose to levels observed in GCGR-deficient mice. In diet-induced obese mice, REGN1193 reduced food intake, adipose tissue mass, and body weight. REGN1193 increased circulating levels of glucagon and glucagon-like peptide 1 and was associated with reversible expansion of pancreatic α-cell area. Hyperglucagonemia and α-cell hyperplasia was observed in fibroblast growth factor 21-deficient mice treated with REGN1193. Single administration of REGN1193 to diabetic cynomolgus monkeys normalized fasting blood glucose and glucose tolerance and increased circulating levels of glucagon and amino acids. Finally, administration of REGN1193 for 8 weeks to normoglycemic cynomolgus monkeys did not cause hypoglycemia or increase pancreatic α-cell area. In summary, the GCGR-blocking antibody REGN1193 normalizes blood glucose in diabetic mice and monkeys but does not produce hypoglycemia in normoglycemic monkeys. Thus, REGN1193 provides a potential therapeutic modality for diabetes mellitus and acute hyperglycemic conditions.

Glucagon receptor antibody completely suppresses type 1 diabetes phenotype without insulin by disrupting a novel diabetogenic pathway.

Insulin monotherapy can neither maintain normoglycemia in type 1 diabetes (T1D) nor prevent the long-term damage indicated by elevated glycation products in blood, such as glycated hemoglobin (HbA1c). Here we find that hyperglycemia, when unaccompanied by an acute increase in insulin, enhances itself by paradoxically stimulating hyperglucagonemia. Raising glucose from 5 to 25 mM without insulin enhanced glucagon secretion ∼two- to fivefold in InR1-G9 α cells and ∼18-fold in perfused pancreata from insulin-deficient rats with T1D. Mice with T1D receiving insulin treatment paradoxically exhibited threefold higher plasma glucagon during hyperglycemic surges than during normoglycemic intervals. Blockade of glucagon action with mAb Ac, a glucagon receptor (GCGR) antagonizing antibody, maintained glucose below 100 mg/dL and HbA1c levels below 4% in insulin-deficient mice with T1D. In rodents with T1D, hyperglycemia stimulates glucagon secretion, up-regulating phosphoenolpyruvate carboxykinase and enhancing hyperglycemia. GCGR antagonism in mice with T1D normalizes glucose and HbA1c, even without insulin.

Rational design of a humanized glucagon-like peptide-1 receptor agonist antibody.

Bovine antibody BLV1H12 possesses a unique "stalk-knob" architecture in its ultralong heavy chain CDR3, allowing substitutions of the "knob" domain with protein agonists to generate functional antibody chimeras. We have generated a humanized glucagon-like peptide-1 (GLP-1) receptor agonist antibody by first introducing a coiled-coil "stalk" into CDR3H of the antibody herceptin. Exendin-4 (Ex-4), a GLP-1 receptor agonist, was then fused to the engineered stalk with flexible linkers, and a Factor Xa cleavage site was inserted immediately in front of Ex-4 to allow release of the N-terminus of the fused peptide. The resulting clipped herceptin-Ex-4 fusion protein is more potent in vitro in activating GLP-1 receptors than the Ex-4 peptide. The clipped herceptin-Ex-4 has an extended plasma half-life of approximately four days and sustained control of blood glucose levels for more than a week in mice. This work provides a novel approach to the development of human or humanized agonist antibodies as therapeutics.

Inhibitory mechanism of an allosteric antibody targeting the glucagon receptor.

Elevated glucagon levels and increased hepatic glucagon receptor (GCGR) signaling contribute to hyperglycemia in type 2 diabetes. We have identified a monoclonal antibody that inhibits GCGR, a class B G-protein coupled receptor (GPCR), through a unique allosteric mechanism. Receptor inhibition is mediated by the binding of this antibody to two distinct sites that lie outside of the glucagon binding cleft. One site consists of a patch of residues that are surface-exposed on the face of the extracellular domain (ECD) opposite the ligand-binding cleft, whereas the second binding site consists of residues in the αA helix of the ECD. A docking model suggests that the antibody does not occlude the ligand-binding cleft. We solved the crystal structure of GCGR ECD containing a naturally occurring G40S mutation and found a shift in the register of the αA helix that prevents antibody binding. We also found that alterations in the αA helix impact the normal function of GCGR. We present a model for the allosteric inhibition of GCGR by a monoclonal antibody that may form the basis for the development of allosteric modulators for the treatment of diabetes and other class B GPCR-related diseases.

Human monoclonal antibodies against glucagon receptor improve glucose homeostasis by suppression of hepatic glucose output in diet-induced obese mice.

AIM: Glucagon is an essential regulator of hepatic glucose production (HGP), which provides an alternative therapeutic target for managing type 2 diabetes with glucagon antagonists. We studied the effect of a novel human monoclonal antibody against glucagon receptor (GCGR), NPB112, on glucose homeostasis in diet-induced obese (DIO) mice. METHODS: The glucose-lowering efficacy and safety of NPB112 were investigated in DIO mice with human GCGR for 11 weeks, and a hyperinsulinemic-euglycemic clamp study was conducted to measure HGP. RESULTS: Single intraperitoneal injection of NPB112 with 5 mg/kg effectively decreased blood glucose levels in DIO mice for 5 days. A significant reduction in blood glucose was observed in DIO mice treated with NPB112 at a dose ≥5 mg/kg for 6 weeks, and its glucose-lowering effect was dose-dependent. Long-term administration of NPB112 also caused a mild 29% elevation in glucagon level, which was returned to the normal range after discontinuation of treatment. The clamp study showed that DIO mice injected with NPB112 at 5 mg/kg were more insulin sensitive than control mice, indicating amelioration of insulin resistance by treatment with NPB112. DIO mice treated with NPB112 showed a significant improvement in the ability of insulin to suppress HGP, showing a 33% suppression (from 8.3 mg/kg/min to 5.6 mg/kg/min) compared to the 2% suppression (from 9.8 mg/kg/min to 9.6 mg/kg/min) in control mice. In addition, no hypoglycemia or adverse effect was observed during the treatment. CONCLUSIONS: A novel human monoclonal GCGR antibody, NPB112, effectively lowered the glucose level in diabetic animal models with mild and reversible hyperglucagonemia. Suppression of excess HGP with NPB112 may be a promising therapeutic modality for the treatment of type 2 diabetes.

The safety and tolerability of GLP-1 receptor agonists in the treatment of type 2 diabetes: a review.

Although several classes of pharmacotherapy are available for type 2 diabetes, glycaemic control is often hampered by medication-related adverse effects and contraindications such as renal impairment. Glucagon-like peptide-1 (GLP-1) receptor agonists provide a new pharmacotherapeutic option based on the multiple glucose-lowering effects of the human hormone GLP-1. This mechanism of action not only provides therapeutic efficacy but also suggests that GLP-1 receptor agonists have distinct safety and tolerability concerns compared with other diabetes therapies. Stimulation of pancreatic insulin secretion by GLP-1 receptor agonists is glucose dependent, conferring a lesser risk of hypoglycaemia than that seen with sulfonylureas. Individual GLP-1 receptor agonists differ in their metabolism and excretion profiles, affecting the choice of agent for patients with renal impairment. As with other protein-based therapies, GLP-1 receptor agonists may induce the formation of antibodies that may attenuate therapeutic efficacy and affect safety. Conclusions on cardiovascular safety must await outcomes studies, but at present no signal of harm has been reported, and preclinical data and effects on risk markers suggest a potential for benefit. Current data on thyroid medullary cancer in humans and pancreatic malignancy in rodents do not suggest that there is any reason to restrict the clinical use of GLP-1 analogues in most people with diabetes. It is currently difficult to ascertain the possible contributory role of GLP-1 receptor agonists in increasing the risk of pancreatitis, and vigilance for signs and symptoms is prudent. Primary tolerability issues include transient gastrointestinal symptoms, common with GLP-1 receptor agonists, which can be reduced through dose titration.

Glucagon-like peptide-1 receptor signalling selectively regulates murine lymphocyte proliferation and maintenance of peripheral regulatory T cells.

AIMS/HYPOTHESIS: Glucagon-like peptide-1 receptor (GLP-1R) agonists improve glucose control in animals and humans with type 1 diabetes. However, there is little information on the role of the GLP-1R in the immune system. We studied the role of the GLP-1R in immune function in wild-type (WT) and nonobese diabetic (NOD) and Glp1r-/- mice. METHODS: Glp1r mRNA expression was examined in sorted immune subpopulations by RT-PCR. The effects of GLP-1R activation were assessed on cAMP production and proliferation, migration and survival of primary immune cells from WT and NOD mice. The ability of primary cells from Glp1r-/- mice to proliferate, migrate or survive apoptosis was determined. Immunophenotyping studies were performed to assess the frequency of immune subpopulations in Glp1r-/- mice. RESULTS: Ex vivo activation of the GLP-1R resulted in a modest but significant elevation of cAMP in primary thymocytes and splenocytes from both WT and NOD mice. GLP-1R activation did not increase proliferation of primary thymocytes, splenocytes or peripheral lymph node cells. In contrast, Glp1r-/- thymocytes exhibited a hypoproliferative response, whilst peripheral Glp1r-/- lymphocytes were hyperproliferative in response to mitogenic stimulation. Activation or loss of GLP-1R signalling did not modify apoptosis or chemotaxis in primary lymphocytes. Male Glp1r-/- mice exhibited a significantly lower percentage of peripheral regulatory T cells, although no differences were observed in the numbers of CD4+ and CD8+ T cells and B cells in the spleen and lymph nodes of Glp1r-/- mice. CONCLUSIONS/INTERPRETATION: These studies establish that GLP-1R signalling may regulate lymphocyte proliferation and maintenance of peripheral regulatory T cells.

Localization and activation of glucagon-like peptide-2 receptors on vagal afferents in the rat.

Glucagon-like peptide-2 (GLP-2) is a nutrient-dependent proglucagon-derived hormone that stimulates intestinal growth through poorly understood paracrine and/or neural pathways. The relationship between GLP-2 action and a vagal pathway is unclear. Our aims were to determine whether 1) the GLP-2 receptor (GLP-2R) is expressed on vagal afferents by localizing it to the nodose ganglia; 2) exogenous GLP-2 stimulates the vagal afferent pathway by determining immunoreactivity for c-fos protein in the nucleus of the solitary tract (NTS); and 3) functional ablation of vagal afferents attenuates GLP-2-mediated intestinal growth in rats maintained with total parenteral nutrition (TPN). A polyclonal antibody against the N terminus of the rat GLP-2R was raised and characterized. The GLP-2R was localized to vagal afferents in the nodose ganglia and confirmed in enteroendocrine cells, enteric neurons, and nerve fibers in the myenteric plexus using immunohistochemistry. Activation of the vagal afferent pathway, as indicated by c-fos protein immunoreactivity in the NTS, was determined by immunohistochemistry after ip injection of 200 microg human GLP-2. GLP-2 induced a significant 5-fold increase in the number of c-fos protein immunoreactive neurons in the NTS compared with saline. Ablation of vagal afferent function by perivagal application of capsaicin, a specific afferent neurotoxin, abolished c-fos protein immunoreactivity, suggesting that activation of the NTS due to GLP-2 is dependent on vagal afferents. Exogenous GLP-2 prevented TPN-induced mucosal atrophy, but ablation of vagal afferent function with capsaicin did not attenuate this effect. This suggests that vagal-independent pathways are responsible for GLP-2 action in the absence of luminal nutrients during TPN, possibly involving enteric neurons or endocrine cells. This study shows for the first time that the GLP-2R is expressed by vagal afferents, and ip GLP-2 activates the vagal afferent pathway.

[Glucagon-like peptide 2, a neurotransmitter with a newly discovered role in the regulation of food ingestion]. / Glucagonlignende peptid-2, en neurotransmitter med en nyopdaget rolle i reguleringen af fødeindtagelsen.

We report here that glucagon-like peptide 2(GLP-2) and its receptor constitute a distinct projection system connecting the nucleus of the solitary tract with the dorsomedial hypothalamic nucleus (DMH). The DMH contains a dense plexus of GLP-2 immunoreactive fibres and is the only hypothalamic nucleus expressing GLP-2 receptor mRNA. Consistent with this, central application of GLP-2 activates the expression of neurones solely in the DMH. Furthermore, central administration of GLP-2 causes a dose-related, a pharmacologically and behaviourally specific inhibition of food intake in rats. Surprisingly, the alleged GLP-1 receptor antagonist, Exending (9-39), proved a functional antagonist of centrally applied GLP-2. These data implicate GLP-2 as an important neurotransmitter in the regulation of food intake and likely bodyweight. Our data therefore point to the DMH as a crossroad for endocrine and visceral information affecting feeding behaviour.

Structure of Fab hGR-2 F6, a competitive antagonist of the glucagon receptor.

The monoclonal antibody hGR-2 F6 has been raised against the human glucagon receptor and shown to act as a competitive antagonist. As a first step in the structural characterization of the receptor, the crystal structure of the Fab fragment from this antibody is reported at 2.1 A resolution. The hGR-2 F6 Fab crystallizes in the orthorhombic space group P2(1)2(1)2, with unit-cell parameters a = 76.14, b = 133.74, c = 37.46 A. A model generated by homology modelling was used as an aid in the chain-tracing and the Fab fragment structure was subsequently refined (final R factor = 21.7%). The structure obtained exhibits the typical immunoglobulin fold. Complementarity-determining regions (CDRs) L1, L2, L3, H1 and H2 could be superposed onto standard canonical CDR loops. The H3 loop could be classified according to recently published rules regarding loop length, sequence and conformation. This loop is 14 residues long, with an approximate beta-hairpin geometry, which is distorted somewhat by the presence of two trans proline residues at the beginning of the loop. It is expected that this H3 loop will facilitate the design of synthetic probes for the glucagon receptor that may be used to investigate receptor activity.

Insulinotropic glucagon-like peptide I receptor expression in glucagon-producing alpha-cells of the rat endocrine pancreas.

Glucagon-like peptide I (GLP-I), an intestine-derived incretin hormone, is a potent stimulator of insulin and somatostatin secretion. In some studies, GLP-I is an inhibitor of glucagon secretion. It remains uncertain, however, whether the effect of GLP-I on the inhibition of glucagon secretion is direct, owing to interactions with GLP-I receptors on alpha-cells, or indirect, via paracrine suppression by insulin or somatostatin. The localization of the GLP-I receptor on insulin and somatostatin-producing cells in the islets is well established. Whether the GLP-I receptor also resides on the glucagon-producing alpha-cells remains controversial and is reported to be absent on rat alpha-cells. To investigate the distribution of the GLP-I receptor on islet cells, we examined the expression of GLP-I receptor mRNA in phenotypically distinct islet cell lines and islets, and the presence of immunoreactive GLP-I receptor in dispersed rat islet cells using a specific antiserum. GLP-I receptor mRNA was readily detected by reverse transcription-polymerase chain reaction (RT-PCR) in both rat islets and in established islet cell lines representing distinct alpha-, beta-, and delta-cell phenotypes. In addition, GLP-I receptor expression was detected in single rat alpha-cells by single-cell RT-PCR. In dispersed rat islet cells analyzed by double immunofluorescent staining, 90% of the insulin, 76% of the somatostatin, and 20% of the glucagon positive cells colocalized with the GLP-I receptor immunoreactivity. Thus, a substantial population of glucagon immunoreactive a-cells express the GLP-I receptor. These findings imply that GLP-I may have a direct receptor-mediated action in the regulation of the physiological functions on a substantial subpopulation of alpha-cells. We suggest that a possible role for GLP-I receptors on alpha-cells may be to provide positive autocrine feedback control on glucagon secretion during fasting and/or to dampen the potent paracrine suppression of glucagon secretion by insulin during feeding.

Human glucagon receptor monoclonal antibodies: antagonism of glucagon action and use in receptor characterization.

This paper describes the development and characterization of the first monoclonal antibody specific for the recently cloned human glucagon receptor (hGR), and its use in probing receptor structure and function. We demonstrate specificity of one of the antibodies, CIV395.7A, by immunofluorescence staining and immunoprecipitation analysis. In addition, CIV395.7A specifically competes with glucagon for the hormone binding site on the receptor, indicating that the antibody's specific recognition epitope overlaps with the receptor's hormone binding domain. As a consequence, the mAB antagonizes glucagon-stimulated signal transduction as assayed by in vitro cAMP accumulation. Binding inhibition studies further reveal that the antibody specifically recognizes the human and rat GR, but not mouse. Using hGR/glucagon-like peptide I receptor chimeras, we have localized the recognition epitope of the antibody to the membrane-proximal half of the amino-terminal extension of the receptor, thus defining a domain on the receptor which is involved in glucagon binding.

Antibodies against specific extracellular epitopes of the glucagon receptor block glucagon binding.

Polyclonal antibodies were prepared against synthetic peptides corresponding to four different extramembrane segments of the rat glucagon receptor. The antibodies bound specifically to native glucagon receptor as judged by immunofluorescence microscopy of cultured cells expressing a synthetic gene for the receptor. Antibodies to peptides designated PR-15 and DK-12 were directed against amino acid residues 103-117 and 126-137, respectively, of the extracellular N-terminal tail. Antibody to peptide KD-14 was directed against residues 206-219 of the first extracellular loop, and antibody to peptide ST-18, against the intracellular C-terminal tail, residues 468-485. The DK-12 and KD-14 antibodies, but not the PR-15 and ST-18 antibodies, could effectively block binding of 125I-labeled glucagon to its receptor in liver membranes. Incubation of these antibodies with rat liver membranes resulted in both a decrease in the maximal hormonal binding capacity and an apparent decrease in glucagon affinity for its receptor. These effects were abolished in the presence of excess specific peptide antigen. In addition, DK-12 and KD-14 antibodies, but not PR-15 and ST-18 antibodies, interfered with glucagon-induced adenylyl cyclase activation in rat liver membranes and behaved as functional glucagon antagonists. These results demonstrate that DK-12 and KD-14 antibodies are pharmacologically active glucagon antagonists and strongly suggest that residues 126-137 of the N-terminal tail and residues 206-219 of the first extracellular loop contain determinants of ligand binding and may comprise the primary ligand-binding site on the glucagon receptor.