The dual GCGR/GLP-1R agonist survodutide: Biomarkers and pharmacological profiling for clinical candidate selection.

AIM: To describe the biomarker strategy that was applied to select survodutide (BI 456906), BI 456908 and BI 456897 from 19 dual glucagon receptor (GCGR)/ glucagon-like peptide-1 receptor (GLP-1R) agonists for in-depth pharmacological profiling, which led to the qualification of survodutide as the clinical development candidate. MATERIALS AND METHODS: Potencies to increase cyclic adenosine monophosphate (cAMP) were determined in Chinese hamster ovary (CHO)-K1 cells stably expressing human GCGR and GLP-1R. Agonism for endogenously expressed receptors was investigated in insulinoma cells (MIN6) for mouse GLP-1R, and in rat primary hepatocytes for the GCGR. In vivo potencies to engage the GLP-1R or GCGR were determined, measuring improvement in oral glucose tolerance (30 nmol/kg) and increase in plasma fibroblast growth factor-21 (FGF21) and liver nicotinamide N-methyltransferase (NNMT) mRNA expression (100 nmol/kg), respectively. Body weight- and glucose-lowering efficacies were investigated in diet-induced obese (DIO) mice and diabetic db/db mice, respectively. RESULTS: Upon acute dosing in lean mice, target engagement biomarkers for the GCGR and GLP-1R demonstrated a significant correlation (Spearman correlation coefficient with p < 0.05) to the in vitro GCGR and GLP-1R potencies for the 19 dual agonists investigated. Survodutide, BI 456908 and BI 456897 were selected for in-depth pharmacological profiling based on the significant improvement in acute oral glucose tolerance achieved (area under the curve [AUC] of 54%, 57% and 60% vs. vehicle) that was comparable to semaglutide (AUC of 45% vs. vehicle), while showing different degrees of in vivo GCGR engagement, as determined by hepatic NNMT mRNA expression (increased by 15- to 17-fold vs. vehicle) and plasma FGF21 concentrations (increased by up to sevenfold vs. vehicle). In DIO mice, survodutide (30 nmol/kg/once daily), BI 456908 (30 nmol/kg/once daily) and BI 456897 (10 nmol/kg/once daily) achieved a body weight-lowering efficacy from baseline of 25%, 27% and 26%, respectively. In db/db mice, survodutide and BI 456908 (10 and 20 nmol/kg/once daily) significantly lowered glycated haemoglobin (0.4%-0.6%); no significant effect was observed for BI 456897 (3 and 7 nmol/kg/once daily). CONCLUSIONS: Survodutide was selected as the clinical candidate based on its balanced dual GCGR/GLP-1R pharmacology, engaging the GCGR for robust body weight-lowering efficacy exceeding that of selective GLP-1R agonists, while achieving antidiabetic efficacy that was comparable to selective GLP-1R agonism. Survodutide is currently being investigated in Phase 3 clinical trials in people living with obesity.

Evaluation of long acting GLP1R/GCGR agonist in a DIO and biopsy-confirmed mouse model of NASH suggest a beneficial role of GLP-1/glucagon agonism in NASH patients.

OBJECTIVE: The metabolic benefits of GLP-1 receptor (GLP-1R) agonists on glycemic and weight control are well established as therapy for type 2 diabetes and obesity. Glucagon's ability to increase energy expenditure is well described, and the combination of these mechanisms-of-actions has the potential to further lower hepatic steatosis in metabolic disorders and could therefore be attractive for the treatment for non-alcoholic steatohepatitis (NASH). Here, we have investigated the effects of a dual GLP-1/glucagon receptor agonist NN1177 on hepatic steatosis, fibrosis, and inflammation in a preclinical mouse model of NASH. Having observed strong effects on body weight loss in a pilot study with NN1177, we hypothesized that direct engagement of the hepatic glucagon receptor (GCGR) would result in a superior effect on steatosis and other liver related parameters as compared to the GLP-1R agonist semaglutide at equal body weight. METHODS: Male C57Bl/6 mice were fed a diet high in trans-fat, fructose, and cholesterol (Diet-Induced Obese (DIO)-NASH) for 36 weeks. Following randomization based on the degree of fibrosis at baseline, mice were treated once daily with subcutaneous administration of a vehicle or three different doses of NN1177 or semaglutide for 8 weeks. Hepatic steatosis, inflammation and fibrosis were assessed by immunohistochemistry and morphometric analyses. Plasma levels of lipids and liver enzymes were determined, and hepatic gene expression was analyzed by RNA sequencing. RESULTS: NN1177 dose-dependently reduced body weight up to 22% compared to vehicle treatment. Plasma levels of ALT, a measure of liver injury, were reduced in all treatment groups with body weight loss. The dual agonist reduced hepatic steatosis to a greater extent than semaglutide at equal body weight loss, as demonstrated by three independent methods. Both the co-agonist and semaglutide significantly decreased histological markers of inflammation such as CD11b and Galectin-3, in addition to markers of hepatic stellate activation (αSMA) and fibrosis (Collagen I). Interestingly, the maximal beneficial effects on above mentioned clinically relevant endpoints of NN1177 treatment on hepatic health appear to be achieved with the middle dose tested. Administering the highest dose resulted in a further reduction of liver fat and accompanied by a massive induction in genes involved in oxidative phosphorylation and resulted in exaggerated body weight loss and a downregulation of a module of co-expressed genes involved in steroid hormone biology, bile secretion, and retinol and linoleic acid metabolism that are also downregulated due to NASH itself. CONCLUSIONS: These results indicate that, in a setting of overnutrition, the liver health benefits of activating the fasting-related metabolic pathways controlled by the glucagon receptor displays a bell-shaped curve. This observation is of interest to the scientific community, due to the high number of ongoing clinical trials attempting to leverage the positive effects of glucagon biology to improve metabolic health.

Glucagon cell hyperplasia and neoplasia: a recently recognized endocrine receptor disease.

Glucagon cell hyperplasia and neoplasia (GCHN) is the name of an endocrine receptor disease, whose morphology was first described in 2006. Three years later, this rare disease was found to be to be caused by an inactivating mutation of the glucagon receptor (GCGR) gene. Functionally, the genetic defect mainly affects glucagon signaling in the liver with changes in the metabolism of glycogen, fatty acids and amino acids. Recent results of several studies in GCGR knockout mice suggested that elevated serum amino acid levels probably stimulate glucagon cell hyperplasia with subsequent transformation into glucagon cell neoplasia. This process leads over time to numerous small and some large pancreatic neuroendocrine tumors which are potentially malignant. Despite high glucagon serum levels, the patients develop no glucagonoma syndrome. In 2015, GCHN was identified as an autosomal recessive hereditary disorder.

The ubiquitination status of the glucagon receptor determines signal bias.

The pancreatic hormone glucagon activates the glucagon receptor (GCGR), a class B seven-transmembrane G protein-coupled receptor that couples to the stimulatory heterotrimeric G protein and provokes PKA-dependent signaling cascades vital to hepatic glucose metabolism and islet insulin secretion. Glucagon-stimulation also initiates recruitment of the endocytic adaptors, ßarrestin1 and ßarrestin2, which regulate desensitization and internalization of the GCGR. Unlike many other G protein-coupled receptors, the GCGR expressed at the plasma membrane is constitutively ubiquitinated and upon agonist-activation, internalized GCGRs are deubiquitinated at early endosomes and recycled via Rab4-containing vesicles. Herein we report a novel link between the ubiquitination status and signal transduction mechanism of the GCGR. In the deubiquitinated state, coupling of the GCGR to Gs is diminished, while binding to ßarrestin is enhanced with signaling biased to a ßarrestin1-dependent p38 mitogen activated protein kinase (MAPK) pathway. This ubiquitin-dependent signaling bias arises through the modification of lysine333 (K333) on the cytoplasmic face of transmembrane helix V. Compared with the GCGR-WT, the mutant GCGR-K333R has impaired ubiquitination, diminished G protein coupling, and PKA signaling but unimpaired potentiation of glucose-stimulated-insulin secretion in response to agonist-stimulation, which involves p38 MAPK signaling. Both WT and GCGR-K333R promote the formation of glucagon-induced ßarrestin1-dependent p38 signaling scaffold that requires canonical upstream MAPK-Kinase3, but is independent of Gs, Gi, and ßarrestin2. Thus, ubiquitination/deubiquitination at K333 in the GCGR defines the activation of distinct transducers with the potential to influence various facets of glucagon signaling in health and disease.

Glucagon Acting at the GLP-1 Receptor Contributes to ß-Cell Regeneration Induced by Glucagon Receptor Antagonism in Diabetic Mice.

Dysfunction of glucagon-secreting α-cells participates in the progression of diabetes, and glucagon receptor (GCGR) antagonism is regarded as a novel strategy for diabetes therapy. GCGR antagonism upregulates glucagon and glucagon-like peptide 1 (GLP-1) secretion and, notably, promotes ß-cell regeneration in diabetic mice. Here, we aimed to clarify the role of GLP-1 receptor (GLP-1R) activated by glucagon and/or GLP-1 in the GCGR antagonism-induced ß-cell regeneration. We showed that in db/db mice and type 1 diabetic wild-type or Flox/cre mice, GCGR monoclonal antibody (mAb) improved glucose control, upregulated plasma insulin level, and increased ß-cell area. Notably, blockage of systemic or pancreatic GLP-1R signaling by exendin 9-39 (Ex9) or Glp1r knockout diminished the above effects of GCGR mAb. Furthermore, glucagon-neutralizing antibody (nAb), which prevents activation of GLP-1R by glucagon, also attenuated the GCGR mAb-induced insulinotropic effect and ß-cell regeneration. In cultured primary mouse islets isolated from normal mice and db/db mice, GCGR mAb action to increase insulin release and to upregulate ß-cell-specific marker expression was reduced by a glucagon nAb, by the GLP-1R antagonist Ex9, or by a pancreas-specific Glp1r knockout. These findings suggest that activation of GLP-1R by glucagon participates in ß-cell regeneration induced by GCGR antagonism in diabetic mice. ARTICLE HIGHLIGHTS: Glucagon receptor (GCGR) antagonism promotes ß-cell regeneration in type 1 and type 2 diabetic mice and in euglycemic nonhuman primates. Glucagon and glucagon-like peptide 1 (GLP-1) can activate the GLP-1 receptor (GLP-1R), and their levels are upregulated following GCGR antagonism. We investigated whether GLP-1R activated by glucagon and/or GLP-1 contributed to ß-cell regeneration induced by GCGR antagonism. We found that blockage of glucagon-GLP-1R signaling attenuated the GCGR monoclonal antibody-induced insulinotropic effect and ß-cell regeneration in diabetic mice. Our study reveals a novel mechanism of ß-cell regeneration and uncovers the communication between α-cells and ß-cells in regulating ß-cell mass.

Crispr-Cas9 mediated complete deletion of glucagon receptor in mice display hyperglucagonemia and α-cell hyperplasia.

Glucagon receptor plays an important role in the regulation of glucose metabolism. Studies have revealed that glucagon receptor antagonism is a potential effective treatment for diabetes. However, the functions of GCGR have not been fully illustrated. Although two Gcgr truncation knockout mice models have been widely used for GCGR function studies, truncated gene may remain neomorphic and/or dominant-negative function. In this study, we took the advantages of Crispr-Cas9 technique and generated a novel allele of GCGR in the mouse that yields complete loss of GCGR protein. Our studies reveal that complete deletion of Gcgr results in hyperglucagonemia, α-cell hyperplasia, improvement of glucose tolerance. These results are similar to the Gcgr-truncated mutation in mice. Hence, we provide a novel strain of GCGR knockout mice for the GCGR function studies.

Pancreatic alpha cell glucagon-liver FGF21 axis regulates beta cell regeneration in a mouse model of type 2 diabetes.

AIMS/HYPOTHESIS: Glucagon receptor (GCGR) antagonism ameliorates hyperglycaemia and promotes beta cell regeneration in mouse models of type 2 diabetes. However, the underlying mechanisms remain unclear. The present study aimed to investigate the mechanism of beta cell regeneration induced by GCGR antagonism in mice. METHODS: The db/db mice and high-fat diet (HFD)+streptozotocin (STZ)-induced mice with type 2 diabetes were treated with antagonistic GCGR monoclonal antibody (mAb), and the metabolic variables and islet cell quantification were evaluated. Plasma cytokine array and liver RNA sequencing data were used to screen possible mediators, including fibroblast growth factor 21 (FGF21). ELISA, quantitative RT-PCR and western blot were applied to verify FGF21 change. Blockage of FGF21 signalling by FGF21-neutralising antibody (nAb) was used to clarify whether FGF21 was involved in the effects of GCGR mAb on the expression of beta cell identity-related genes under plasma-conditional culture and hepatocyte co-culture conditions. FGF21 nAb-treated db/db mice, systemic Fgf21-knockout (Fgf21-/-) diabetic mice and hepatocyte-specific Fgf21-knockout (Fgf21Hep-/-) diabetic mice were used to reveal the involvement of FGF21 in beta cell regeneration. A BrdU tracing study was used to analyse beta cell proliferation in diabetic mice treated with GCGR mAb. RESULTS: GCGR mAb treatment improved blood glucose control, and increased islet number (db/db 1.6±0.1 vs 0.8±0.1 per mm2, p<0.001; HFD+STZ 1.2±0.1 vs 0.5±0.1 per mm2, p<0.01) and area (db/db 2.5±0.2 vs 1.2±0.2%, p<0.001; HFD+STZ 1.0±0.1 vs 0.3±0.1%, p<0.01) in diabetic mice. The plasma cytokine array and liver RNA sequencing data showed that FGF21 levels in plasma and liver were upregulated by GCGR antagonism. The GCGR mAb induced upregulation of plasma FGF21 levels (db/db 661.5±40.0 vs 466.2±55.7 pg/ml, p<0.05; HFD+STZ 877.0±106.8 vs 445.5±54.0 pg/ml, p<0.05) and the liver levels of Fgf21 mRNA (db/db 3.2±0.5 vs 1.8±0.1, p<0.05; HFD+STZ 2.0±0.3 vs 1.0±0.2, p<0.05) and protein (db/db 2.0±0.2 vs 1.4±0.1, p<0.05; HFD+STZ 1.6±0.1 vs 1.0±0.1, p<0.01). Exposure to plasma or hepatocytes from the GCGR mAb-treated mice upregulated the mRNA levels of characteristic genes associated with beta cell identity in cultured mouse islets and a beta cell line, and blockage of FGF21 activity by an FGF21 nAb diminished this upregulation. Notably, the effects of increased beta cell number induced by GCGR mAb were attenuated in FGF21 nAb-treated db/db mice, Fgf21-/- diabetic mice and Fgf21Hep-/- diabetic mice. Moreover, GCGR mAb treatment enhanced beta cell proliferation in the two groups of diabetic mice, and this effect was weakened in Fgf21-/- and Fgf21Hep-/- mice. CONCLUSIONS/INTERPRETATION: Our findings demonstrate that liver-derived FGF21 is involved in the GCGR antagonism-induced beta cell regeneration in a mouse model of type 2 diabetes.

Glucagon, from past to present: a century of intensive research and controversies.

2022 corresponds to the 100th anniversary of the discovery of glucagon. This TimeCapsule aims to recall the main steps leading to the discovery, characterisation, and clinical importance of the so-called second pancreatic hormone. We describe the early historical findings in basic research (ie, discovery, purification, structure, α-cell origin, radioimmunoassay, glucagon gene [GCG], and glucagon receptor [GLR]), in which three future Nobel Prize laureates were actively involved. Considered as an anti-insulin hormone, glucagon was rapidly used to treat insulin-induced hypoglycaemic coma episodes in people with type 1 diabetes. A key step in the story of glucagon was the discovery of its role and the role of α cells in the physiology and pathophysiology (ie, paracrinopathy) of type 2 diabetes. This concept led to the design of different strategies targeting glucagon, among which GLP-1 receptor (GLP1R) agonists were a major breakthrough, and combination of inhibition of glucagon secretion with stimulation of insulin secretion (both in a glucose-dependent manner). Taking advantage of the glucagon-induced increase in energy metabolism, biased coagonists were developed. Besides the GLP-1 receptor, these coagonists also target the glucagon receptor to further promote weight loss. Thus, the 100-year story of glucagon has most probably not come to an end.

Effects of site-directed mutagenesis of GLP-1 and glucagon receptors on signal transduction activated by dual and triple agonists.

The paradigm of one drug against multiple targets, known as unimolecular polypharmacology, offers the potential to improve efficacy while overcoming some adverse events associated with the treatment. This approach is best exemplified by targeting two or three class B1 G protein-coupled receptors, namely, glucagon-like peptide-1 receptor (GLP-1R), glucagon receptor (GCGR) and glucose-dependent insulinotropic polypeptide receptor for treatment of type 2 diabetes and obesity. Some of the dual and triple agonists have already shown initial successes in clinical trials, although the molecular mechanisms underlying their multiplexed pharmacology remain elusive. In this study we employed structure-based site-directed mutagenesis together with pharmacological assays to compare agonist efficacy across two key signaling pathways, cAMP accumulation and ERK1/2 phosphorylation (pERK1/2). Three dual agonists (peptide 15, MEDI0382 and SAR425899) and one triple agonist (peptide 20) were evaluated at GLP-1R and GCGR, relative to the native peptidic ligands (GLP-1 and glucagon). Our results reveal the existence of residue networks crucial for unimolecular agonist-mediated receptor activation and their distinct signaling patterns, which might be useful to the rational design of biased drug leads.

Evaluation of commercially available glucagon receptor antibodies and glucagon receptor expression.

Glucagon is a major regulator of metabolism and drugs targeting the glucagon receptor (GCGR) are being developed. Insight into tissue and cell-specific expression of the GCGR is important to understand the biology of glucagon and to differentiate between direct and indirect actions of glucagon. However, it has been challenging to localize the GCGR in tissue due to low expression levels and lack of specific methods. Immunohistochemistry has frequently been used for GCGR localization, but antibodies targeting G-protein-coupled-receptors may be inaccurate. We evaluated all currently commercially available GCGR antibodies. The antibody, ab75240 (Antibody no. 11) was found to perform best among the twelve antibodies tested and using this antibody we found expression of the GCGR in the kidney, liver, preadipocytes, pancreas, and heart. Three antibody-independent approaches all confirmed the presence of the GCGR within the pancreas, liver and the kidneys. GCGR expression should be evaluated by both antibody and antibody-independent approaches.

Disruption of the glucagon receptor increases glucagon expression beyond α-cell hyperplasia in zebrafish.

The glucagon receptor (GCGR) is a potential target for diabetes therapy. Several emerging GCGR antagonism-based therapies are under preclinical and clinical development. However, GCGR antagonism, as well as genetically engineered GCGR deficiency in animal models, are accompanied by α-cell hyperplasia and hyperglucagonemia, which may limit the application of GCGR antagonism. To better understand the physiological changes in α cells following GCGR disruption, we performed single cell sequencing of α cells isolated from control and gcgr-/- (glucagon receptor deficient) zebrafish. Interestingly, beyond the α-cell hyperplasia, we also found that the expression of gcga, gcgb, pnoca, and several glucagon-regulatory transcription factors were dramatically increased in one cluster of gcgr-/- α cells. We further confirmed that glucagon mRNA was upregulated in gcgr-/- animals by in situ hybridization and that glucagon promoter activity was increased in gcgr-/-;Tg(gcga:GFP) reporter zebrafish. We also demonstrated that gcgr-/- α cells had increased glucagon protein levels and increased granules after GCGR disruption. Intriguingly, the increased mRNA and protein levels could be suppressed by treatment with high-level glucose or knockdown of the pnoca gene. In conclusion, these data demonstrated that GCGR deficiency not only induced α-cell hyperplasia but also increased glucagon expression in α cells, findings which provide more information about physiological changes in α-cells when the GCGR is disrupted.

Alpha-cells and therapy of diabetes: Inhibition, antagonism or death?

Absolute or relative hyperglucagonaemia is a characteristic of both Type 1 and Type 2 diabetes, resulting in fasting hyperglycaemia due in part to increased hepatic glucose production and lack of postprandial suppression of circulating glucagon concentrations. Consequently, therapeutics that target glucagon secretion or biological action may be effective antidiabetic agents. In this regard, specific glucagon receptor (GCGR) antagonists have been developed that exhibit impressive glucose-lowering actions, but unfortunately may cause off-target adverse effects in humans. Further to this, several currently approved antidiabetic agents, including GLP-1 mimetics, DPP-4 inhibitors, metformin, sulphonylureas and pramlintide likely exert part of their glucose homeostatic actions through direct or indirect inhibition of GCGR signalling. In addition to agents that inhibit the release of glucagon, compounds that enhance the transdifferentiation of glucagon secreting alpha-cells towards an insulin positive beta-cell phenotype could also help curb excess glucagon secretion in diabetes. Use of alpha-cell toxins represents another possible strategy to address hyperglucagonaemia in diabetes. In that respect, research from the 1920 s with diguanides such as synthalin A demonstrated effective glucose-lowering with alpha-cell ablation in both animal models and humans with diabetes. However, further clinical use of synthalin A was curtailed due its adverse effects and the increased availability of insulin. Overall, these observations with therapeutics that directly target alpha-cells, or GCGR signaling, highlight a largely untapped potential for diabetes therapy that merits further detailed consideration.

Glucagon receptor signaling at white adipose tissue does not regulate lipolysis.

Although the physiological role of glucagon receptor signaling in the liver is well defined, the impact of glucagon receptor (Gcgr) signaling on white adipose tissue (WAT) continues to be debated. Although numerous studies propose that glucagon stimulates WAT lipolysis, we lack evidence that physiological concentrations of glucagon regulate WAT lipolysis. In turn, we performed studies in both wild-type and WAT Gcgr knockout mice to determine if glucagon regulates lipolysis at WAT in the mouse. We assessed the effects of fasting and acute exogenous glucagon administration in wild-type C57BL/6J and GcgrAdipocyte+/+ versus GcgrAdipocyte-/- mice. Using an ex vivo lipolysis protocol, we further examined the direct effects of glucagon on physiologically (fasted) and pharmacologically stimulated lipolysis. We found that adipocyte Gcgr expression did not affect fasting-induced lipolysis or hepatic lipid accumulation in lean or diet-induced obese (DIO) mice. Acute glucagon administration did not affect serum nonesterified fatty acids (NEFA), leptin, or adiponectin concentration, but did increase serum glucose and FGF21, regardless of genotype. Glucagon did not affect ex vivo lipolysis in explants from either GcgrAdipocyte+/+ or GcgrAdipocyte-/- mice. Gcgr expression did not affect fasting-induced or isoproterenol-stimulated lipolysis from WAT explants. Moreover, glucagon receptor signaling at WAT did not affect body weight or glucose homeostasis in lean or DIO mice. Our studies have established that physiological levels of glucagon do not regulate WAT lipolysis, either directly or indirectly. Given that glucagon receptor agonism can improve dyslipidemia and decrease hepatic lipid accumulation, it is critical to understand the tissue-specific effects of glucagon receptor action. Unlike the crucial role of hepatic glucagon receptor signaling in maintaining glucose and lipid homeostasis, we observed no metabolic consequence of WAT glucagon receptor deletion.NEW & NOTEWORTHY It has been postulated that glucagon stimulates lipolysis and fatty acid release from white adipose tissue. We observed no metabolic effects of eliminating or activating glucagon receptor signaling at white adipose tissue.

Antagonistic Effect and In Vitro Activity of Dauricine on Glucagon Receptor.

Abnormal increases in glucagon (GCG) are the primary cause of type II diabetes mellitus. When GCG interacts with a glucagon receptor (GCGR), GCG can increase the blood glucose level. In this paper, a compound that could interfere with the binding of GCG and GCGR to inhibit the increase of blood glucose was investigated. First, molecular docking was used to conduct preliminary screening of compounds whose active components could combine with GCGR by AutoDock Vina. The binding of the receptor-ligand complex was analyzed by PyMOL. Results showed that dauricine could tightly bind to the receptor pocket. Second, the plasmid pcDNA3.1(+)-GCGR containing the target gene was transfected into HEK293 cells for expression, which was the cell model established to screen GCGR antagonist. Dauricine, the lead compound of glucagon receptor antagonist (GRA), was screened using the GRA screening model in vitro. Finally, using [Des-His1, Glu9]-Glucagon amide as the positive control, flow cytometry was used to express the antagonistic effect of the compound. Consequently, dauricine can antagonize the GCGR.

Glucagon receptor-mediated regulation of gluconeogenic gene transcription is endocytosis-dependent in primary hepatocytes.

A number of G protein-coupled receptors (GPCRs) are now thought to use endocytosis to promote cellular cAMP signaling that drives downstream transcription of cAMP-dependent genes. We tested if this is true for the glucagon receptor (GCGR), which mediates physiological regulation of hepatic glucose metabolism via cAMP signaling. We show that epitope-tagged GCGRs undergo clathrin- and dynamin-dependent endocytosis in HEK293 and Huh-7-Lunet cells after activation by glucagon within 5 min and transit via EEA1-marked endosomes shown previously to be sites of GPCR/Gs-stimulated production of cAMP. We further show that endocytosis potentiates cytoplasmic cAMP elevation produced by GCGR activation and promotes expression of phosphoenolpyruvate carboxykinase 1 (PCK1), the enzyme catalyzing the rate-limiting step in gluconeogenesis. We verify endocytosis-dependent induction of PCK1 expression by endogenous GCGRs in primary hepatocytes and show similar control of two other gluconeogenic genes (PGC1α and G6PC). Together, these results implicate the endosomal signaling paradigm in metabolic regulation by glucagon.

Generation of an induced pluripotent stem cell (iPSC) line from a diabetic patient with glucagon receptor (GCGR) p.W83X mutation.

Induced pluripotent stem cell (iPSC) line, SJHi001-A, was generated from a diabetic patient carrying a heterozygous c.G248A mutation in GCGR gene that generated the p.W83X. The PBMCs from her father (no diabetes and no mutation site) were also prepared into iPSC (SJHi002-A) as a control. Both two cell lines had normal karyotype, expressed pluripotency markers and could differentiate into the three germ layers in vivo.

Molecular and in vivo phenotyping of missense variants of the human glucagon receptor.

Naturally occurring missense variants of G protein-coupled receptors with loss of function have been linked to metabolic disease in case studies and in animal experiments. The glucagon receptor, one such G protein-coupled receptor, is involved in maintaining blood glucose and amino acid homeostasis; however, loss-of-function mutations of this receptor have not been systematically characterized. Here, we observed fewer glucagon receptor missense variants than expected, as well as lower allele diversity and fewer variants with trait associations as compared with other class B1 receptors. We performed molecular pharmacological phenotyping of 38 missense variants located in the receptor extracellular domain, at the glucagon interface, or with previously suggested clinical implications. These variants were characterized in terms of cAMP accumulation to assess glucagon-induced Gαs coupling, and of recruitment of ß-arrestin-1/2. Fifteen variants were impaired in at least one of these downstream functions, with six variants affected in both cAMP accumulation and ß-arrestin-1/2 recruitment. For the eight variants with decreased Gαs signaling (D63ECDN, P86ECDS, V96ECDE, G125ECDC, R2253.30H, R3085.40W, V3686.59M, and R3787.35C) binding experiments revealed preserved glucagon affinity, although with significantly reduced binding capacity. Finally, using the UK Biobank, we found that variants with wildtype-like Gαs signaling did not associate with metabolic phenotypes, whereas carriers of cAMP accumulation-impairing variants displayed a tendency toward increased risk of obesity and increased body mass and blood pressure. These observations are in line with the essential role of the glucagon system in metabolism and support that Gαs is the main signaling pathway effecting the physiological roles of the glucagon receptor.

A glucagon analogue decreases body weight in mice via signalling in the liver.

Glucagon receptor agonists show promise as components of next generation metabolic syndrome pharmacotherapies. However, the biology of glucagon action is complex, controversial, and likely context dependent. As such, a better understanding of chronic glucagon receptor (GCGR) agonism is essential to identify and mitigate potential clinical side-effects. Herein we present a novel, long-acting glucagon analogue (GCG104) with high receptor-specificity and potent in vivo action. It has allowed us to make two important observations about the biology of sustained GCGR agonism. First, it causes weight loss in mice by direct receptor signalling at the level of the liver. Second, subtle changes in GCG104-sensitivity, possibly due to interindividual variation, may be sufficient to alter its effects on metabolic parameters. Together, these findings confirm the liver as a principal target for glucagon-mediated weight loss and provide new insights into the biology of glucagon analogues.

Variation in the Evolution and Sequences of Proglucagon and the Receptors for Proglucagon-Derived Peptides in Mammals.

The mammalian proglucagon gene (Gcg) encodes three glucagon like sequences, glucagon, glucagon-like peptide-1 (GLP-1), and glucagon-like peptide-2 that are of similar length and share sequence similarity, with these hormones having cell surface receptors, glucagon receptor (Gcgr), GLP-1 receptor (Glp1r), and GLP-2 receptor (Glp2r), respectively. Gcgr, Glp1r, and Glp2r are all class B1 G protein-coupled receptors (GPCRs). Despite their sequence and structural similarity, analyses of sequences from rodents have found differences in patterns of sequence conservation and evolution. To determine whether these were rodent-specific traits or general features of these genes in mammals I analyzed coding and protein sequences for proglucagon and the receptors for proglucagon-derived peptides from the genomes of 168 mammalian species. Single copy genes for each gene were found in almost all genomes. In addition to glucagon sequences within Hystricognath rodents (e.g., guinea pig), glucagon sequences from a few other groups (e.g., pangolins and some bats) as well as changes in the proteolytic processing of GLP-1 in some bats are suggested to have functional effects. GLP-2 sequences display increased variability but accepted few substitutions that are predicted to have functional consequences. In parallel, Glp2r sequences display the most rapid protein sequence evolution, and show greater variability in amino acids at sites involved in ligand interaction, however most were not predicted to have a functional consequence. These observations suggest that a greater diversity in biological functions for proglucagon-derived peptides might exist in mammals.

Intestinal Growth in Glucagon Receptor Knockout Mice Is Not Associated With the Formation of AOM/DSS-Induced Tumors.

Treatment with exogenous GLP-2 has been shown to accelerate the growth of intestinal adenomas and adenocarcinomas in experimental models of colonic neoplasia, however, the role of endogenous GLP-2 in tumor promotion is less well known. Mice with a global deletion of the glucagon receptor (Gcgr-/-) display an increase in circulating GLP-1 and GLP-2. Due to the intestinotrophic nature of GLP-2, we hypothesized that Gcgr-/- mice would be more susceptible to colonic dysplasia in a model of inflammation-induced colonic carcinogenesis. Female Gcgr-/- mice were first characterized for GLP-2 secretion and in a subsequent study they were given a single injection with the carcinogen azoxymethane (7.5 mg/kg) and treated with dextran sodium sulfate (DSS) (3%) for six days (n=19 and 9). A cohort of animals (n=4) received a colonoscopy 12 days following DSS treatment and all animals were sacrificed after six weeks. Disruption of glucagon receptor signaling led to increased GLP-2 secretion (p<0.0001) and an increased concentration of GLP-2 in the pancreas of Gcgr-/- mice, coinciding with an increase in small intestinal (p<0.0001) and colonic (p<0.05) weight. Increased villus height was recorded in the duodenum (p<0.001) and crypt depth was increased in the duodenum and jejunum (p<0.05 and p<0.05). Disruption of glucagon receptor signaling did not affect body weight during AOM/DSS treatment, neither did it affect the inflammatory score assessed during colonoscopy or the number of large and small adenomas present at the end of the study period. In conclusion, despite the increased endogenous GLP-2 secretion Gcgr-/- mice were not more susceptible to AOM/DSS-induced tumors.