Human Glucagon Research Articles

Glucagon Clearance Is Decreased in Chronic Kidney Disease but Preserved in Liver Cirrhosis.

It is not completely clear which organs are responsible for glucagon elimination in humans, and disturbances in the elimination of glucagon could contribute to the hyperglucagonemia observed in chronic liver disease and chronic kidney disease (CKD). Here, we evaluated kinetics and metabolic effects of exogenous glucagon in individuals with stage 4 CKD (n =16), individuals with Child-Pugh A-C cirrhosis (n = 16) and matched control individuals (n = 16), before, during and after a 60-minute glucagon infusion (4 ng/kg/min). Individuals with CKD exhibited a significantly lower mean metabolic clearance rate of glucagon (14.0 [95% CI 12.2;15.7] mL/kg/min) both compared to individuals with cirrhosis (19.7 [18.1;21.3] mL/kg/min, P < 0.001) and to control individuals (20.4 [18.1;22.7] mL/kg/min, P < 0.001). Glucagon half-life was significantly prolonged in the CKD group (7.5 [6.9;8.2] minutes) compared to individuals with cirrhosis (5.7 [5.2;6.3] minutes, P = 0.002) and control individuals (5.7 [5.2;6.3] minutes, P < 0.001). No difference in the effects of exogenous glucagon on plasma glucose, amino acids, or triglycerides was observed between groups. In conclusion, chronic kidney disease, but not liver cirrhosis leads to a significant reduction in glucagon clearance, supporting the kidneys as a primary site for human glucagon elimination.

A Controversy Regarding the Identity of the Enzyme That Mediates Glucagon-Like Peptide 1 Synthesis in Human Alpha Cells.

Processing of proglucagon into glucagon-like peptide-1 (GLP-1) and GLP-2 in intestinal L cells is mediated by the prohormone convertase 1/3 (PC1/3) while PC2 is responsible for the synthesis of glucagon in pancreatic alpha cells. While GLP-1 is also produced by alpha cells, the identity of the convertase involved in its synthesis is still unsettled. It also remains to be determined whether all alpha cells produce the incretin. The aims of this study were first, to elucidate the identity of the proconvertase responsible for GLP-1 production in human alpha cells, and second, to ascertain whether the number of glucagon cells expressing GLP-1 increase during diabetes. To answer these questions, sections of pancreas from donors' non-diabetic controls, type 1 and type 2 diabetes were processed for double-labelled immunostaining of glucagon and GLP-1 and of each hormone and either PC1 or PC2. Stained sections were examined by confocal microscopy. It was found that all alpha cells of islets from those three groups expressed GLP-1 and PC2 but not PC1/3. This observation supports the view that PC2 is the convertase involved in GLP-1 synthesis in all human glucagon cells and suggests that the regulation of its activity may have important clinical application in diabetes.

Machine learning designs new GCGR/GLP-1R dual agonists with enhanced biological potency

Several peptide dual agonists of the human glucagon receptor (GCGR) and the glucagon-like peptide-1 receptor (GLP-1R) are in development for the treatment of type 2 diabetes, obesity and their associated complications. Candidates must have high potency at both receptors, but it is unclear whether the limited experimental data available can be used to train models that accurately predict the activity at both receptors of new peptide variants. Here we use peptide sequence data labelled with in vitro potency at human GCGR and GLP-1R to train several models, including a deep multi-task neural-network model using multiple loss optimization. Model-guided sequence optimization was used to design three groups of peptide variants, with distinct ranges of predicted dual activity. We found that three of the model-designed sequences are potent dual agonists with superior biological activity. With our designs we were able to achieve up to sevenfold potency improvement at both receptors simultaneously compared to the best dual-agonist in the training set.

Corrination mitigates peptide aggregation as exemplified for Glucagon

Pharmaceutical development of glucagon for use in acute hypoglycemia has proved challenging, due in large part to poor solubility, poor stability and aggregate formation. Herein, we describe highly soluble, low aggregating, glucagon conjugates generated through use of the commercially available vitamin B12 precursor dicyanocobinamide (‘corrination’), which retain full stimulatory action at the human glucagon receptor. The modified glucagon analogs were tested in a chemical stability assay in 50 mM phosphate buffer and the percentage of original concentration retained was determined after two weeks of incubation at 37° C. Aggregate formation assays were also performed after 48 h of agitation at 37°C using a thioflavin (ThT) fluorescence-based assay. All corrinated compounds retained original concentration to a higher degree than glucagon controls and showed markedly decreased aggregation compared to their respective noncorrinated analogues. Based on the statistically significant increase in chemical stability coupled with the notably decreased tendency to form aggregates, analogues 2 and its corrinated conjugate 5 were used for a functional assay study performed after agitation at 37°C for 24-hr after which agonism was measured at the human glucagon receptor using a cAMP FRET assay. Corrinated 5 exhibited a 6.6-fold increased potency relative to glucagon, which was shown to have a 165-fold reduction in potency. The relative potency of 5 was also improved compared to that of 2 with EC50 values of 5.5 nM and 9.6 nM for 5 and 2, respectively. In conclusion, corrination of peptides mitigates aggregation, presenting a compound with prolonged stability and agonism as demonstrated for glucagon.

Abstract 14259: Contractile Effects of Glucagon in Atrial Preparations From Transgenic Mice and Men

Glucagon, a peptide composed of 29 amino acids, produced mainly in the pancreas can also stimulate cardiac function. Glucagon might contribute to hyperglycemia in diabetes mellitus type 2 and is thus of clinical relevance. There is controversy in the literature whether glucagon affects force and beating rate in the human atrium and which receptor is involved. Hence, we studied the effect of glucagon on atrial preparations of wild type mouse hearts (WT), from transgenic mice with cardiac overexpression of the human glucagon receptor (GR-TG) and for comparison in human right atrial preparations. We noted that glucagon exerted concentration- and time-dependent positive chronotropic effects in atrial preparations from WT and GR-TG starting at 0.1 nM glucagon and amounting to 25 % of pre-drug value at 3 nM glucagon (n=8, p&lt;0.05). These effects were antagonized by the glucagon receptor antagonist SC203973 (1 μM), the inhibitor of cAMP dependent protein kinase H89 (1 μM), by the M-cholinoceptor agonist 1 μM carbachol, by the A1-adenosine receptor antagonist R-phenylisopropyladenosine (1 μM) and the inhibitors of hyperpolarization-activated cyclic nucleotide gated channels namely ivabradine (1 μM) or cesium chloride (2 mM) (n=5-6, p&lt;0.05). In left atrial preparations from GR-TG but not WT, glucagon exerted starting at 1 nM a positive inotropic effect. Interestingly, in human atrial preparations glucagon at 1 μM in the additional presence of the phosphodiesterase III inhibitor cilostamide (1 μM) increased force of contraction by 34 % (n=5, p&lt;0.05). We conclude that glucagon can exert contractile effects in mammalian hearts via GR, but they exhibit considerable regional and species differences. The physiological and therapeutic relevance of glucagon in the human heart needs to be fully elucidated.

SAT149 In Vitro Characterization Of A Novel Long-acting Glucagon Analog (HM15136) And Its Potential Effect In Animal Models Of Chronic Hypoglycemic Diseases

Abstract Disclosure: J. Kim: Employee; Self; Hanmi Pharm. Co., Ltd. Y. Kim: Employee; Self; Hanmi Pharm. Co., Ltd. B. Ye: Employee; Self; Hanmi Pharm. Co., Ltd. W. Kim: Employee; Self; Hanmi Pharm. Co., Ltd. J. Kim: Employee; Self; Hanmi Pharm. Co., Ltd. S. Bae: Employee; Self; Hanmi Pharm. Co., Ltd. D. Kim: Employee; Self; Hanmi Pharm. Co., Ltd. S. Lee: Employee; Self; Hanmi Pharm. Co., Ltd. I. Choi: Employee; Self; Hanmi Pharm. Co., Ltd. Glucagon plays an essential role in glucose homeostasis, and has been used to treat acute hypoglycemia in diabetic patients. However, due to poor solubility and stability at physiological pH as well as short duration of action, its utility was hampered when managing chronic hypoglycemic diseases. One such example is congenital hyperinsulinism (CHI). CHI is a rare genetic disorder characterized by unregulated insulin secretion which leads to severe hypoglycemia especially during fasting condition. Another example is post-bariatric hypoglycemia (PBH) which can also lead to postprandial neuroglycopenia. Despite this life-threatening condition, no drugs are approved for CHI and/or PBH, and available glucagon therapies are very inefficient and inconvenient due to abovementioned limitations. Thus, to provide more optimal therapy for these chronic hypoglycemic diseases, a novel long-acting glucagon analog (HM15136) was developed. Here, we evaluated HM15136’s 1) solubility and stability at physiologic pH, 2) in vitro properties, and 3) potential therapeutic effect in animal models of CHI or PBH. First, we demonstrated improved solubility of HM15136 (≥150 vs. 0.03 mg/mL) at pH 7.0 and stability at day 20, compared to those of native glucagon. Next, in vitro cAMP assay was performed in CHO cells expressing human glucagon receptor (hGCGR), and HM15136 selectively activated hGCGR as a full agonist (EC50 = 0.024 vs. 0.003 nM; relative activity = ca.12.7%). In line with this, HM15136 promoted glycogenolysis and gluconeogenesis in rat primary hepatocytes in a dose dependent manner (relative potency = ca.20%), confirming glucose producing nature of HM15136. To investigate in vivo therapeutic effect on CHI and PBH, chronic hypoglycemia rats induced by SC infusion of insulin (CHI rats) and postprandial hypoglycemia rats induced by vertical sleeve gastrectomy (VSG rats) were established, respectively. Notably, HM15136 treatment significantly restored FBG by 37.3 ∼ 69.7% (p&amp;lt;0.05 ∼ 0.001) in CHI rats. Consistently, HM15136 treatment significantly prevented a massive BG reduction during MMTT in VSG rats (ΔBG between 0 and 45 min = +21 vs. -30 mg/dL, p&amp;lt;0.001). Furthermore, weekly dosing potential and BG elevation effect of HM15136 were also demonstrated in healthy human subjects (1). Based on these results, HM15136 could be a potential therapeutic option for chronic hypoglycemic diseases including CHI and PBH. Phase 2 clinical study in CHI subjects is on-going to assess the clinical relevance of these findings. Reference: (1) Diabetes Obes Metab. 2022 Mar; 23(3):411-20 Presentation: Saturday, June 17, 2023

Glucagon and Its Receptors in the Mammalian Heart.

Glucagon exerts effects on the mammalian heart. These effects include alterations in the force of contraction, beating rate, and changes in the cardiac conduction system axis. The cardiac effects of glucagon vary according to species, region, age, and concomitant disease. Depending on the species and region studied, the contractile effects of glucagon can be robust, modest, or even absent. Glucagon is detected in the mammalian heart and might act with an autocrine or paracrine effect on the cardiac glucagon receptors. The glucagon levels in the blood and glucagon receptor levels in the heart can change with disease or simultaneous drug application. Glucagon might signal via the glucagon receptors but, albeit less potently, glucagon might also signal via glucagon-like-peptide-1-receptors (GLP1-receptors). Glucagon receptors signal in a species- and region-dependent fashion. Small molecules or antibodies act as antagonists to glucagon receptors, which may become an additional treatment option for diabetes mellitus. Hence, a novel review of the role of glucagon and the glucagon receptors in the mammalian heart, with an eye on the mouse and human heart, appears relevant. Mouse hearts are addressed here because they can be easily genetically modified to generate mice that may serve as models for better studying the human glucagon receptor.

1742-P: Loss of MAFB Compromises a-Cell Identity and Glucagon Secretion in Adult Human Islets

Islet-enriched transcription factors (TFs) are critical regulators of α and β cell development and function. One such TF, MAFB, exhibits a distinct expression pattern between species in that it is restricted to α cells postnatally in rodents, but produced in both developing and adult human α and β cells. While MAFB is essential for insulin production and secretion in human embryonic stem cell models, the impact of MAFB loss in mature α and β cells is unknown. Importantly, both T1D and T2D are associated with dramatic loss of islet MAFB expression. To test the hypothesis that MAFB is a critical regulator in adult human islets, dispersed islet cells were infected with lentiviruses expressing control or MAFB knockdown (MAFBKD) shRNAs, and reaggregated for 6 days to form pseudoislets (PIs; N=6 donors, ages 16-50 years). MAFBKD PIs exhibited a 56.4±4.2% reduction (P&amp;lt;0.001) in MAFB mRNA compared to control. Likewise, glucagon mRNA and hormone content were reduced by 42.5±6.9 and 28.6±7.6% (P&amp;lt;0.05), respectively, whereas insulin expression and content were unchanged. In static hormone secretion experiments, IBMX- and arginine-stimulated glucagon secretion was blunted in MAFBKD PIs (~33% lower than control; P&amp;lt;0.05), an observation that was replicated in α cell-enriched MAFBKD PIs. Insulin secretion, on the other hand, was largely unaffected. Bulk RNA-sequencing analysis of MAFBKD versus control PIs illustrated downregulation of α cell identity genes, upregulation of β, delta, and gamma cell identity genes, and misexpression of PYY, a gut peptide gene. Upregulated genes were also highly enriched in gene sets associated with epithelial mesenchymal transition and transforming growth factor β signaling. Intriguingly, marker genes in both pathways were shown recently to be upregulated in T2D α cells. Our findings suggest that MAFB is a molecular “gatekeeper” of α cell identity through gene activating and repressive functions, with its loss leading to dysregulation of human glucagon secretion. Disclosure K.C.Coate: None. R.Stein: None. X.Tong: None. J.Liu: None. R.Jenkins: None. R.Aramandla: None. N.Dey: None. S.Kim: None. M.Brissova: None. A.C.Powers: None.

Utilization of zebrafish larvae to monitor gastric motility in diabetes mellitus: targeting GLP‐1 and GIP receptors

BackgroundDiabetes mellitus (DM) is a prominent chronic disease associated with mortality and morbidity. The impact of DM is extensive, impacting with several major organs systems, including the gastrointestinal (GI) tract. For instance, the prevalence of DM in gastroparetic patients is approximately 30%. Glucagon‐like peptide‐1 (GLP‐1) and gastric inhibitory peptide (GIP) are hormones involved in glucose homeostasis but may also affect gastric motility. GLP‐1 and GIP receptors belong to class B G‐protein coupled receptors (GPCRs) and zebrafish GPCRs (zGPCRs) share structural similarities with the human GLP‐1 (hGLP‐1) and human glucagon receptors (hGCGR). Studies found that zebrafish GLP‐1 (zGLP‐1) receptors exhibit all the structural features of class B GPCRs and have ~50% homology with human GLP‐1 receptors. The aim of the studies is to establish experimental models of DM and investigate if GLP‐1 and GIP receptor agonists alter gastric function.MethodologyDiet‐induced DM zebrafish larvae models (from 5 days post fertilization (dpf) to 7 dpf) were established by (i) overfeeding, (ii) feeding with a high‐fat diet (egg yolk), (iii) exposure to high levels of glucose (30 mmol/L) or (iv) a knockdown of the pdx1 (insulin promoter factor 1) gene mutant. A body mass index (BMI) analyses and enzyme‐linked immunosorbent assays (ELISA) for insulin levels and glucose assay were used to grade DM. As obesity is associated with development of diabetes, adipose deposits in the abdominal area was also determined by an inverted microscope. Exendin‐4 and tirzepatide (100 µM – 1 nM) were administered via microgavage. Peristalsis was captured in anaesthetised animals using light microscopy and quantified in MATLAB using imaging velocimetry and pixel differences.ResultsExcept for the overfed and pdx1 mutated models, significant differences in BMI, insulin and glucose levels were observed relative to controls. The excess diet model showed high level of adipose deposits (2.4 ± 0.17 mm) and at 7 dpf an 11% increase in adipose area is seen but without DM properties (i.e. increase glucose levels and altered insulin levels). There were significant differences between DM models and control‐fed larvae in terms of mixing power, anterograde and retrograde peristalsis of several parameters including contractile intervals and velocity. Preliminary data indicated that larvae at7 dpf fed a standard diet, exendin‐4 (≥ 100 nM/larvae) and tirzepatide (≥100 µM/larvae) reduced motility in the anterior and mid‐intestine.ConclusionThree DM models and an obesity model using zebrafish larvae were successfully established. Advanced imaging and processing techniques revealed intricate subtypes of dysmotility in DM and obesity. Exendin‐4 and tirzepatide at high doses reduced motility in normal fish which may be consistent with their clinical side effect profile.

Delineating the activation mechanism and conformational landscape of a class B G protein-coupled receptor glucagon receptor

Class B G protein-coupled receptors (GPCRs) are important targets in the treatment of metabolic syndrome and diabetes. Although multiple structures of class B GPCRs–G protein complexes have been elucidated, the detailed activation mechanism of the receptors remains unclear. Here, we combine Gaussian accelerated molecular dynamics simulations and Markov state models (MSM) to investigate the activation mechanism of a canonical class B GPCR, human glucagon receptor–GCGR, including the negative allosteric modulator-bound inactive state, the agonist glucagon-bound active state, and both glucagon- and Gs-bound fully active state. The free-energy landscapes of GCGR show the conformational ensemble consisting of three activation-associated states: inactive, active, and fully active. The structural analysis indicates the high dynamics of GCGR upon glucagon binding with both active and inactive conformations in the ensemble. Significantly, the H8 and TM6 exhibits distinct features from the inactive to the active states. The additional simulations demonstrate the role of H8 in the recruitment of Gs. Gs binding presents a crucial function of stabilizing the glucagon binding site and MSM highlights the absolute requirement of Gs to help the GCGR reach the fully active state. Together, our results reveal the detailed activation mechanism of GCGR from the view of conformational dynamics.

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 β-arrestin-1/2. Fifteen variants were impaired in at least one of these downstream functions, with six variants affected in both cAMP accumulation and β-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.

Dasiglucagon: an effective medicine for severe hypoglycemia.

Patients with type 1 diabetes mellitus (T1DM) receiving insulin therapy commonly suffer from insulin-mediated hypoglycemia and require glucagon for glycemic control to achieve normal plasma glucose (PG) levels. Severe hypoglycemia will endanger the life of patients and require intervention. Stable glucagon analog dasiglucagon was approved for the treatment of patients with severe hypoglycemia and is administered via Zegalogue autoinjector/Zegalogue prefilled syringe.The main purpose of this review article is to review the basic properties and clinical effects of dasiglucagon. We search related literature on CNKI, Web of Science and PubMed by keywords dasiglucagon, hypoglycemia, type 1 diabetes, glucagon. Carry out a careful review of the included literature. Dasiglucagon information on clinicaltrials.gov and https://www.fda.gov/ has been adopted. Dasiglucagon is a novel peptide analog of human glucagon, which can effectively rescue insulin-induced severe hypoglycemia in patients with T1DM and rapidly increase glycemic levels in a small dose under normal and hypoglycemic conditions. It has been proven that dasiglucagon has definite stability and solubility in aqueous formulations. Dasiglucagon has a higher absorption rate and longer plasma elimination half-life than traditional reconstituted glucagon. In three randomized, double-blind, placebo-controlled trials in children aged 6 to 17years and adults with T1DM the median time to glycemic recovery in 10min after dasiglucagon administration was significantly faster than placebo and 99% of patients recovered within 15min after subcutaneous injection of dasiglucagon in the key phase 3 clinical trial. The most common adverse reactions in these phase 3 trials were vomiting, nausea, diarrhea, headache, and injection site pain.

Deleterious mutation V369M in the mouse GCGR gene causes abnormal plasma amino acid levels indicative of a possible liver-α-cell axis.

Glucagon plays an important role in glucose homeostasis and amino acid metabolism. It regulates plasma amino acid levels which in turn modulate glucagon secretion from the pancreatic α-cell, thereby establishing a liver–α-cell axis described recently. We reported previously that the knock-in mice bearing homozygous V369M substitution (equivalent to a naturally occurring mutation V368M in the human glucagon receptor, GCGR) led to hypoglycemia with improved glucose tolerance. They also exhibited hyperglucagonemia, pancreas enlargement and α-cell hyperplasia. Here, we investigated the effect of V369M/V368M mutation on glucagon-mediated amino acid metabolism. It was found that GcgrV369M+/+ mice displayed increased plasma amino acid levels in general, but significant accumulation of the ketogenic/glucogenic amino acids was observed in animals fed with a high-fat diet (HFD), resulting in deleterious metabolic consequence characteristic of α-cell proliferation and hyperglucagonemia.

115-LB: Liver-Derived FGF21 Mediates the Promoting Effect of Glucagon Receptor Antagonism on Beta-Cell Regeneration in Type 2 Diabetic Mice

Objective: Based on the clear role for glucagon in the pathogenesis of diabetes, glucagon-blocking therapies have been pursued as a therapeutic strategy for this disease. Here, we used REMD 2.59, a human antagonistic glucagon receptor (GCGR) monoclonal antibody (mAb), to clarify the effects of GCGR mAb in promoting pancreatic β cell regeneration in type 2 diabetes (T2D), and its potential mechanism. Methods: GCGR mAb was administrated weekly for 4 weeks in db/db mice and HFD/STZ-induced T2D mice, including different lineage-tracing mice. The plasma insulin and glucagon were determined by using ELISA. The evaluation of islet morphology was performed with immunofluorescent analysis. GCGR mAb was also treated in the systemic Fgf21 gene knockout (Fgf21-/-) and hepatocyte-specific Fgf21 gene knockout (Fgf21Hep-/-) T2D mice. Results: Treatment with GCGR mAb lowered blood glucose, improved glucose tolerance, and elevated plasma glucagon and insulin levels in both db/db mice and HFD/STZ-induced T2D mice. The β cell dedifferentiation as indicated by β cell lineage-tracing was blocked by GCGR mAb. The β cell proliferation as indicated by insulin+ and BrdU+ cells was boosted by GCGR mAb. The α-to-β cell conversion, as evaluated by glucagon+ insulin+ cells and confirmed by α cell lineage-tracing, was facilitated by GCGR mAb. After GCGR mAb treatment, some insulin+ cells were located in the duct region or even colocalized with duct cell markers, suggestive of duct-derived β cell neogenesis that was also verified by Ngn3+ cell lineage-tracing. Notably, GCGR mAb could still improve glucose tolerance and increase β cell mass, but these effects were attenuated in Fgf21-/- and Fgf21Hep-/- T2D mice compared with WT T2D mice. Conclusions: GCGR mAb promotes β cell regeneration via inhibiting β cell dedifferentiation and promoting β cell self-replication, α-to-β cell conversion and duct-derived β cell neogenesis in T2D mice, which is partly mediated by liver-derived FGF21. Disclosure X. Cui: None. T. Wei: None. J. Feng: None. J. Yang: None. H. Yan: Board Member; Self; REMD Biotherapeutics, Other Relationship; Spouse/Partner; REMD Biotherapeutics. R. Wei: None. T. Hong: Speaker’s Bureau; Self; AstraZeneca, Boehringer Ingelheim International GmbH, Merck Sharp &amp; Dohme Corp., Novo Nordisk, Sanofi. Funding National Natural Science Foundation of China (81830022)

Scientific and Regulatory Considerations for the Approval of the First Generic Glucagon

Glucagon for Injection (NDA 020928) is a polypeptide hormone identical to human glucagon approved 20 years ago for severe hypoglycemia in patients with diabetes mellitus. On Dec 28, 2020, the U.S. FDA approved the first generic version of glucagon for injection USP, 1 mg/vial packaged in an emergency kit. The generic and the reference listed drug (RLD) version, i.e., the innovator version, of glucagon were each produced through different manufacturing processes. The RLD version of glucagon is produced via recombinant DNA in yeast while the generic version of glucagon is produced by peptide synthesis. The FDA published its current thinking on how to ensure sameness between the generic and innovator peptide products prepared with different manufacturing processes in a Draft Guidance for Industry: Submission of Abbreviated New Drug Applications for Certain Highly Purified Synthetic Peptide Drug Products, which applies to five peptide drug products, including glucagon. In this presentation, we aim to provide an overview of the regulatory recommendations for submitting generic glucagon drug products for approval, as outlined in the aforementioned draft guidance. Although glucagon may be produced using different manufacturing processes, the sameness in glucagon can be adequately demonstrated using analytical methods, which involve demonstrating physicochemical properties, as well as primary and secondary structures, oligomers and aggregation states. Biological assays may also be used as part of the demonstration of active pharmaceutical ingredient sameness. Synthetic glucagon may have different impurity profiles when compared to the RLD recombinant product. As part of the ANDA review, impurities in the synthetic drugs are analyzed and controlled, in addition, the potential immunogenicity of new impurities, which are not in the RLD products, are assessed and compared using non-clinical assays. In this work, we will discuss non-clinical assays for assessing the immunogenicity risk of these impurities, for both adaptive and innate immune responses. In conclusion, the sameness of an approved generic synthetic glucagon to an RLD can be adequately established through various analytical methods and biological assays.

Ready-to-use Dasiglucagon Injection as a Rapid and Effective Treatment for Severe Hypoglycemia

Background: Severe hypoglycemia (SH) is an all too common and feared complication of insulin therapy and necessitates urgent medical intervention. Dasiglucagon, a novel peptide analog of human glucagon, is uniquely stable in aqueous solutions and is currently in development for treatment of SH via a ready-to-use auto-injector. Objective: To assess the efficacy and safety of dasiglucagon injection as a rapid and dependable treatment for SH. Methods: In the pivotal Phase 3 trial, NCT03378635, dasiglucagon (0.6 mg) was compared to placebo, and GlucaGen®, in adults with type 1 diabetes mellitus (N=168), randomized (2:1:1) after insulin-induced hypoglycemia. The primary endpoint was time to plasma glucose (PG) recovery, defined as first PG increase of 20 mg/dL after treatment initiation. Results: The median time to recovery was 10 min for dasiglucagon vs 12 and 40 min for GlucaGen® and placebo, respectively. In the dasiglucagon arm, 65% of patients recovered within 10 min vs 49% receiving GlucaGen®. After 15 min, 99% of dasiglucagon-treated patients recovered vs 95% treated with GlucaGen®. Dasiglucagon was generally well tolerated. Similar frequencies of nausea (55% and 53%), and vomiting (23% and 19%), were reported with dasiglucagon and GlucaGen®, respectively. Injection-site reactions 30 minutes post- dose were reported in 2%, 7%, and 5% of patients in the dasiglucagon, GlucaGen®, and placebo groups, respectively. Conclusions: Dasiglucagon 0.6 mg demonstrated rapid and efficacious treatment for SH. The stability of dasiglucagon in aqueous formulation supports its further development as a unique treatment option for the management of the entire spectrum of mild, moderate, and SH.

Human Glucagon Expression Is under the Control of miR-320a.

Increased glucagon is a hallmark of diabetes and leads to worsening of the hyperglycemia, but the molecular mechanisms causing it are still unknown. We therefore investigated the possibility that microRNAs might be involved in the regulation of glucagon. Indeed, analysis of the glucagon 3' untranslated region (UTR) revealed potential binding sites for miR-320a, and using luciferase reporter assays we found that miR-320a directly targets the 3' UTRs of human and rodent glucagon. In addition, endogenous glucagon mRNA and protein expression as well as glucagon secretion were reduced in response to miR-320a overexpression, whereas inhibition of miR-320a upregulated glucagon expression. Interestingly, miR-320a expression was decreased by high glucose, and this was associated with an increase in glucagon expression in human islets and mouse αTC1-6 cells. Moreover, miR-320a overexpression completely blunted these effects. Importantly, miR-320a was also significantly downregulated in human islets of subjects with type 2 diabetes and this was accompanied by increased glucagon expression. Thus, our data suggest that glucose-induced downregulation of miR-320a may contribute to the paradoxical increase in glucagon observed in type 2 diabetes and reveal for the first time that glucagon expression is under the control by a microRNA providing novel insight into the abnormal regulation of glucagon in diabetes.

Characterization of a naturally occurring mutation V368M in the human glucagon receptor and its association with metabolic disorders.

Glucagon is a peptide hormone secreted by islet α cells. It plays crucial roles in glucose homeostasis and metabolism by activating its cognate glucagon receptor (GCGR). A naturally occurring deleterious mutation V368M in the human GCGR leads to reduced ligand binding and down-regulation of glucagon signaling. To examine the association between this mutation and metabolic disorders, a knock-in mouse model bearing homozygous V369M substitution (equivalent to human V368M) in GCGR was made using CRISPR-Cas9 technology. These GcgrV369M+/+ mice displayed lower fasting blood glucose levels with improved glucose tolerance compared with wild-type controls. They also exhibited hyperglucagonemia, pancreas enlargement and α cell hyperplasia with a lean phenotype. Additionally, V369M mutation resulted in a reduction in adiposity with normal body weight and food intake. Our findings suggest a key role of V369M/V368M mutation in GCGR-mediated glucose homeostasis and pancreatic functions, thereby pointing to a possible interplay between GCGR defect and metabolic disorders.

Generation of a homozygous ARX nuclear CFP (ARXnCFP/nCFP) reporter human iPSC line (HMGUi001-A-4).

The aristaless related homeobox (ARX) transcription factor plays a crucial role in glucagon-producing α-cell differentiation. Here, we generate an ARX reporter iPSC line by 3' fusion of an intervening viral T2A sequence followed by a nuclear-localized histone 2B-cyan fluorescent protein (nCFP). The resulting cells have a normal karyotype and preserved pluripotency. In vitro differentiation of the ARXnCFP/nCFP reporter iPSCs towards the endocrine lineage confirmed the specific co-expression of the reporter protein in human glucagon+ α-like cells. Thus, ARXnCFP/nCFP iPSC line will provide a powerful tool to monitor human α-cell progenitor differentiation as well as ARX+ α-like cell function in vitro.

1910-P: Effects of Glucagon Concentrations on Splanchnic and Leg Glucagon Extraction in Healthy Humans

We have recently developed a novel isotope dilution method using nonradioactive, stable human Glucagon (Phe 6 13C9, 15N; Phe 22 13C9, 15N) tracer to measure glucagon turnover. In this study we combined this method with organ catheterization technique to estimate the effects of increasing circulating glucagon concentrations, spanning the physiological range, on splanchnic and leg glucagon extraction. We present data from the first 6 healthy subjects (age 25±3.6 yrs; BMI 26.7±4.5 kg/m2; fasting glucose 4.6±0.5 mM; HbA1c 5.1±0.3%) completed thus far. After IRB approval and informed consent, subjects were admitted in the morning after an overnight fast to Interventional Radiology, where catheters were placed in the right femoral artery and vein under local anesthesia with aseptic precautions. Under fluoroscopic guidance, a hepatic venous catheter was positioned through the femoral vein. Glucagon tracer was infused intravenously and Indocyanine Green infused via the femoral artery sheath to measure splanchnic and femoral plasma flows. After a 90 min period for basal sampling, exogenous glucagon was infused at 0.65 ng/kg/min for 60 minutes (low) followed by 1.5 ng/kg/min (high) for another 60 minutes. Plasma samples were collected periodically from femoral artery, femoral vein and hepatic vein for concentrations of Glucagon tracer (Tandem Mass Spectrometry), glucagon, glucose and Indocyanine Green (Spectrophotometry) concentrations. Arterial glucagon concentrations were 45.2±9.6 pg/ml (basal), 56.3±13.3 pg/ml (low) and 81.4±15.5 pg/ml (high). While fractional splanchnic glucagon extraction remained relatively unchanged (24.0±12.5% vs. 27.7±15.6% vs. 23.5±10.1%: basal vs. low vs. high), fractional leg glucagon extraction tended to increase (13.5±3.8% vs. 16.0±7.2% vs. 18.5±9.4%: basal vs. low vs. high) with increasing glucagon concentrations. These initial data suggest differing mechanisms of glucagon clearance across the splanchnic and leg tissues in humans. Disclosure F. Ruchi: None. Y.R. Yadav: None. D. Romeres: None. S. Sawleh: None. L.M. Benson: None. K.L. Johnson: None. C. Dalla Man: Research Support; Self; Sanofi-Aventis Deutschland GmbH. C. Cobelli: None. D.J. McCormick: None. L.R. Wilkins: None. R. Basu: Consultant; Self; GENFIT. Research Support; Self; AstraZeneca. A. Basu: Consultant; Spouse/Partner; GENFIT. Research Support; Spouse/Partner; AstraZeneca. Funding National Institutes of Health (R01DK029953 to R.B.), (R01DK085516 to A.B.), (DK059637, DK020593)