Glucagon Receptor Activation Research Articles

1017-P: Continuous Glucose Monitoring Reveals Comprehensive Glucose Control with MEDI0382, an Oxyntomodulin-Like Peptide with Targeted GLP-1/Glucagon Receptor Activity

Background: MEDI0382 is an oxyntomodulin-like peptide with GLP-1/glucagon receptor activity under development for type 2 diabetes mellitus (T2DM). Continuous glucose monitoring (CGM) is valuable for evaluating glucose-lowering efficacy. Methods: As an exploratory part of a double-blind phase 2a study, CGM was performed for 52 days with a Freestyle Libre. Patients with T2DM received daily SC MEDI0382 (n = 26) or placebo (n = 13) for 49 days; doses were up-titrated weekly from 50 to 300 µg. CGM assessed percent time spent in target range (70-140 mg/dL) and hypoglycemia (<54 mg/dL), as well as 7-day mean 24-h glucose and CV. Results: Over 52 days, 95% patients had >70% complete CGM data. Time spent in target range was significantly greater with MEDI0382 vs. placebo (64.8-78.0% vs. 42.5-53.0%; min-max; all P ≤ 0.05). Time spent at <54 mg/dL was <1% at all MEDI0382 doses and was not significantly different vs. placebo. A significant reduction in mean glucose with MEDI0382 (118.8-133.2 mg/dL) vs. placebo (153-154.8 mg/dL; all P ≤ 0.033) was observed, and CV was lower with MEDI0382 (0.19-0.21) vs. placebo (0.25-0.26; all P ≤ 0.030). Discussion: MEDI0382 rapidly stabilized glucose levels, improved glycemic variability, and delivered a consistent decrease in mean glucose. The benefits of more time spent in target range will need to be confirmed in larger trials. Disclosure V.E.R. Parker: Employee; Self; MedImmune. Stock/Shareholder; Self; AstraZeneca. D. Robertson: Employee; Self; AstraZeneca. Employee; Spouse/Partner; GlaxoSmithKline plc. Stock/Shareholder; Self; AstraZeneca. T. Wang: Employee; Self; MedImmune. D.C. Hornigold: Employee; Self; AstraZeneca. Stock/Shareholder; Self; AstraZeneca. M.G. Posch: None. T. Heise: Advisory Panel; Self; Mylan. Research Support; Self; ADOCIA, Boehringer Ingelheim International GmbH, Dance Biopharm Holdings Inc., Eli Lilly and Company, Gan & Lee Pharmaceuticals, Johnson & Johnson, MedImmune, Mylan, Nordic Bioscience, Novo Nordisk A/S, Pfizer Inc., Poxel, Saniona, Sanofi, Wockhardt, Zealand Pharma A/S. Speaker’s Bureau; Self; Eli Lilly and Company, Novo Nordisk A/S. L. Plum-Moerschel: None. J.J. Meier: Advisory Panel; Self; AstraZeneca, Boehringer Ingelheim International GmbH, Eli Lilly and Company, Merck Sharp & Dohme Corp., Novo Nordisk A/S, Sanofi. H. Schlichthaar: None. B.M. Klaus: None. P. Ambery: Employee; Self; AstraZeneca. B. Hirshberg: Employee; Self; AstraZeneca. Funding AstraZeneca

1010-P: Effects of MEDI0382, an Oxyntomodulin-Like Peptide with Targeted GLP-1/Glucagon Receptor Activity, on Amino Acids, Ketones, and Free Fatty Acids

Background: MEDI0382 is an oxyntomodulin-like peptide with targeted GLP-1/glucagon receptor activity under development for type 2 diabetes mellitus (T2DM). GLP-1 has been shown to promote glucose-dependent insulin release, delay gastric emptying, and suppress appetite. Glucagon has similar effects on appetite and gastric emptying and enhances energy expenditure by upregulating energy-expensive processes such as gluconeogenesis and fatty acid oxidation. Methods: This exploratory component of a phase 2a study measured fasting amino acid, β-hydroxybutyrate, and free fatty acid levels in overweight or obese T2DM patients randomized to once-daily SC MEDI0382 (n = 26) or placebo (n = 13) for 49 days. Doses were up-titrated weekly from 50 to 300 µg; measurements were taken at baseline and after 49 days of dosing. Results: A significant decrease from baseline to day 49 with MEDI0382 vs. placebo in alanine was observed (-0.5 vs. 0.4 mg/dL; P = 0.017). Numerical reductions in other amino acid levels, including glutamate, cystine, valine, lysine, and tyrosine, were also seen. There were no clinically or statistically significant changes from baseline to day 49 between treatment groups in β-hydroxybutyrate or free fatty acid levels. Conclusion: Despite the known effects of glucagon on fatty acid oxidation, MEDI0382 had no effect on ketogenesis or free fatty acids. Although the reason for this is unclear, enhanced insulin secretion may have counter-regulated this effect. MEDI0382 did promote reductions in key glucogenic amino acids, and this reduction could be related to increased gluconeogenesis and therefore glucagon receptor engagement. Similar patterns of amino acid reduction have been observed after bariatric surgery and have been attributed to excess circulating glucagon. We postulate that upregulation of an energy-consuming process such as gluconeogenesis could contribute to significant weight loss in subjects dosed with MEDI0382. Disclosure V.E. Parker: Employee; Self; MedImmune. Stock/Shareholder; Self; AstraZeneca. D. Robertson: Employee; Self; AstraZeneca. Employee; Spouse/Partner; GlaxoSmithKline plc. Stock/Shareholder; Self; AstraZeneca. T. Wang: Employee; Self; MedImmune. D.C. Hornigold: Employee; Self; AstraZeneca. Stock/Shareholder; Self; AstraZeneca. M.G. Posch: None. L. Plum-Moerschel: None. J.J. Meier: Advisory Panel; Self; AstraZeneca, Boehringer Ingelheim International GmbH, Eli Lilly and Company, Merck Sharp & Dohme Corp., Novo Nordisk A/S, Sanofi. H. Schlichthaar: None. B.M. Klaus: None. L. Jermutus: Employee; Self; AstraZeneca. Stock/Shareholder; Self; AstraZeneca. P. Ambery: Employee; Self; AstraZeneca. B. Hirshberg: Employee; Self; AstraZeneca. Funding AstraZeneca

988-P: MEDI0382, an Oxyntomodulin-Like Peptide with Targeted GLP-1/Glucagon Receptor Activity, Promotes a Dose-Dependent Increase in Gastric Emptying Time

Background: MEDI0382 is an oxyntomodulin-like peptide with targeted GLP-1/glucagon receptor activity under development for type 2 diabetes mellitus (T2DM). GLP-1 and glucagon promote delayed gastric emptying, and GLP-1 tolerance has been described. Methods: This exploratory part of a double-blind phase 2a study measured gastric emptying time (GET) in T2DM patients randomized to daily SC MEDI0382 (n = 20) or placebo (n = 6) for 49 days. Doses were up-titrated fortnightly from 50 to 300 µg. GET was measured after 13C-octanoate ingestion via serial breath collection with plasma sampling for glucose, insulin, and MEDI0382 Ctrough levels. GET (t1/2 = time for 13C retention to decline 50%; tlag = time when percent excreted 13C dose peaks) was measured at baseline; 15, 29, 43, 50 days; and 28 days post-dose. Results: GET t1/2 was significantly prolonged from baseline, by 117.2 min (90% CI 56.2, 178.3) v placebo (-42.9 min; 90% CI -152.0, 66.2; P = 0.039), at 200 µg after 43 days of dosing. Also, at 43 days, tlag was significantly prolonged, by 46.5 min (90% CI, 16.9, 76) v placebo (-27.3 min; 90% CI, -80.0, 25.4; P = 0.048). Numerical increases in t1/2 and tlag were seen at all dose levels; tlag increased with exposure, as exposure increased with dose up to 200 µg. Lesser delay occurred in GET on day 50 at 300 µg with similar exposure, but alongside significant reduction in glucose AUC of -25.3% (90% CI, -28.2, -22.4; P < 0.0001). Postprandial insulin levels were unchanged on day 50 despite marked glucose reduction; delay in peak insulin levels was evident. Discussion: MEDI0382 promoted dose-dependent increase in GET. Although tolerance to this effect may be evident from day 50, reduced postprandial glucose was observed concurrently and may indicate an insulinotropic effect. This unique profile displays characteristics of both short- and long-acting GLP-1 agonism, suggesting that these effects may be mediated by both glucagon and GLP-1 receptor agonism. Disclosure D. Robertson: Employee; Self; AstraZeneca. Employee; Spouse/Partner; GlaxoSmithKline plc. Stock/Shareholder; Self; AstraZeneca. V.E. Parker: Employee; Self; MedImmune. Stock/Shareholder; Self; AstraZeneca. P. Ambery: Employee; Self; AstraZeneca. M. Petrone: Employee; Self; MedImmune. T. Wang: Employee; Self; MedImmune. T. Heise: Advisory Panel; Self; Mylan. Research Support; Self; ADOCIA, Boehringer Ingelheim International GmbH, Dance Biopharm Holdings Inc., Eli Lilly and Company, Gan & Lee Pharmaceuticals, Johnson & Johnson, MedImmune, Mylan, Nordic Bioscience, Novo Nordisk A/S, Pfizer Inc., Poxel, Saniona, Sanofi, Wockhardt, Zealand Pharma A/S. Speaker's Bureau; Self; Eli Lilly and Company, Novo Nordisk A/S. L. Plum-Moerschel: None. L. Jermutus: Employee; Self; AstraZeneca. Stock/Shareholder; Self; AstraZeneca. B. Hirshberg: Employee; Self; AstraZeneca. J.J. Meier: Advisory Panel; Self; AstraZeneca, Boehringer Ingelheim International GmbH, Eli Lilly and Company, Merck Sharp & Dohme Corp., Novo Nordisk A/S, Sanofi. Funding AstraZeneca

1027-P: A Phase 1, Open-Label, Single-Dose Study to Evaluate Pharmacokinetics of MEDI0382 in Patients with Renal Impairment

MEDI0382, an oxyntomodulin-like peptide with targeted GLP-1 and glucagon receptor activity, is in development for T2DM. This open-label, parallel-group study (NCT03235375) evaluated PK of SC MEDI0382 (100 µg; single dose) in subjects (N = 37) grouped by renal function: Group 1, ESRD or severe renal impairment (RI; CrCl < 20 mL/min); Group 2, severe RI (CrCl ≥ 20 mL/min to < 30 mL/min); Group 3, healthy controls (CrCl ≥ 90 mL/min); Group 4a, moderate RI (CrCl ≥ 30 mL/min to < 45 mL/min); and Group 4b, moderate RI (CrCl ≥ 45 mL/min to < 60 mL/min). Coprimary endpoints were geometric mean ratio AUC48h and Cmax of MEDI0382 (Table 1). MEDI0382 had similar PK profiles across all groups (Table 2). This suggests that PK of MEDI0382 is unaffected by renal function, allowing dosing in subjects with RI in future trials. Disclosure G.K. Klein: None. M. Petrone: Employee; Self; MedImmune. Y. Yang: Employee; Self; MedImmune. T. Hoang: Employee; Self; MedImmune. S. DiCostanza: Employee; Self; AstraZeneca. L. Hansen: Employee; Self; AstraZeneca, MedImmune. Stock/Shareholder; Self; Bristol-Myers Squibb Company, Novo Nordisk A/S. A. Flor: Employee; Self; AstraZeneca.

1030-P: Effect of MEDI0382 on the Pharmacodynamics and Pharmacokinetics of Warfarin or Esmolol in Healthy Volunteers

MEDI0382 is an oxyntomodulin-like peptide with targeted GLP-1 and glucagon receptor activity under development for T2DM treatment. Patients with T2DM have a high risk for cardiovascular disease. In an open-label, crossover, phase 1 study (NCT03347968), we evaluated the effect of MEDI0382 (SC; titrated up to 300 µg for 25 days) on the pharmacodynamics (PD) and PK of warfarin (PO; 25 mg on day [d] 2 [warfarin alone] and d 27 [warfarin + MEDI0382]) and PD of esmolol (IV; 200 µg/kg/min for 90 min on d 1 [esmolol alone] and d 26 [esmolol + MEDI0382]) in healthy volunteers (N = 22). Coprimary endpoints were prothrombin time (PT) maximum international normalized ratio (INRmax) and INRAUC of warfarin with MEDI0382, and change in heart rate from rest to after a 9-minute treadmill test (Bruce Protocol) with esmolol and MEDI0382. The coprimary endpoints were analyzed using paired t-tests. INRmax and INRAUC values were log transformed for analysis. MEDI0382 had no effect on PT INRmax, INRAUC (Table), or the PK profile of warfarin. Similar changes in heart rate with the treadmill test were observed with esmolol alone or with esmolol and MEDI0382 (Table). MEDI0382 resulted in an increased incidence of nausea and vomiting. There were no serious or severe adverse events. These results support co-administration of MEDI0382 and warfarin or esmolol and do not suggest safety concerns. Disclosure H.A. Coleman: None. M. Petrone: Employee; Self; MedImmune. T.M. Petrohoy: None. Y. Yang: Employee; Self; MedImmune. L.J. Sanford: None. L. Hansen: Employee; Self; AstraZeneca, MedImmune. Stock/Shareholder; Self; Bristol-Myers Squibb Company, Novo Nordisk A/S. A. Flor: Employee; Self; AstraZeneca.

1965-P: Glucagon Receptor Agonism Stimulates Body Weight Loss in Antibiotic Treatment in Obese Mice

Glucagon, an essential regulator of glucose and lipid metabolism, also promotes weight loss in diet-induced obese (DIO) mice. Glucagon’s role as a primary counterregulatory hormone to insulin action has long received scientific attention, yet its broader therapeutic potential is an evolving research interest. We reported that chronic glucagon receptor (GCGR) activation increased energy expenditure and plasma bile acids (BA) in an farnesoid X receptor (FXR) dependent manner. Glucagon stimulates the conversion of cholesterol to BAs, with an increase in cholic and chenodeoxycholate species, critical regulators of FXR activity. Gut microbiota are known to alter the chemical structure of bile acids to produce secondary and tertiary BA. Thus, in these studies we tested the hypothesis that GCGR agonism regulates the gut microbiome to alter circulating BAs and stimulate FXR. GCGR agonism stimulated a profound change in the gut microbiome of DIO mice. These changes were largely associated with an increase in Actinobacteria and a decrease in Deferribacteria phylums. Assessment of functional microbiome composition via PICRUSt uncovered significant impacts on 130 out of 160 total KEGG pathways, including those involved in BA synthesis. To investigate the role of the gut microbiome on GCGR-stimulated weight loss we treated DIO mice with an antibiotic (AB) cocktail (ampicillin, vancomycin, metronidazole, neomycin, and amphotericin B) or vehicle. Mice were then treated with the GCGR agonist, IUB288, or vehicle for 14d to stimulate weight-loss. Surprisingly, AB-treated mice lost a similar amount of body-weight when treated with IUB288, suggesting that the gut microbiome may be dispensable for the weight loss effect. However, post hoc analysis of the cecal microbiome identified only a partial ablation of microbiota in AB-treated mice, with unexpected increases in Proteobacteria and Tenericutes. Thus, it is possible that GCGR-agonism acts via one of the resistant microbes to regulate weight loss. Disclosure T. Kim: None. J.P. Antipenko: None. S. Nason: None. N. Presedo: None. W.J. Van Der Pol: None. B. Finan: Employee; Self; Novo Nordisk A/S. R. DiMarchi: Employee; Self; Novo Nordisk Inc. C.D. Morrow: None. K.M. Habegger: Consultant; Self; Glyscend, Inc., Novo Nordisk Inc. Research Support; Self; Glyscend, Inc. Stock/Shareholder; Self; Glyscend, Inc. Funding National Institutes of Health

1011-P: Therapeutic Potential of Novel Combination of a Long-Acting Glucagon Analog (HM15136) and Antidiabetic Drugs for the Treatment of Obesity

The obesity epidemic represents a major challenge for public health. However, existing anti-obesity drugs have limited efficacy because they only suppress appetite, emphasizing the development of next generation therapeutic option. Recently, we showed that prolonged glucagon (GCG) receptor activation with HM15136, a long-acting GCG analog, could provide potent body weight loss (BWL) in obese animal models. Considering the stimulatory effect of GCG on glucose production, transient blood glucose (BG) elevation might be important when utilizing HM15136 as a novel anti-obesity drug. Antidiabetic drugs such as metformin, and DPP-4 inhibitor have been widely used for T2DM patients. Considering their safety profile and treatment adherence in addition to BG lowering nature, we hypothesized that combination of antidiabetic drugs might minimize the transient BG elevation by HM15136 and confer additional BWL. To investigate this hypothesis, the present study evaluated the combination effect of HM15136 and antidiabetic medication on BG and BW in vivo. In normal mice, HM15136-induced BG increase was effectively reversed by concomitant treatment of metformin, sitagliptin, or empagliflozin. To further confirm, DIO mice were chronically administered with HM15136 and/or antidiabetic drugs, and normal BG control was continuously maintained in COMBO groups. These results indicate that when combined, antidiabetic drugs could effectively counteract the transient BG elevation by HM15136. As to the BWL efficacy, combination of HM15136 either with metformin, sitagliptin, or empagliflozin provided additional BWL (-18% to -31% by metformin COMBO; -35% to -47% by sitagliptin COMBO; -35% to -40% by empagliflozin COMBO), highlighting the therapeutic benefit of this novel combination. In conclusion, our results demonstrate the prolonged GCG receptor stimulation with antidiabetic medication as a novel therapeutic option for obesity treatment. Disclosure Y. Park: None. J. Lee: None. J. Kim: None. D. Kim: None. Y. Kim: None. I. Choi: Employee; Self; Hanmi Pharm. Co., Ltd.

OR28-5 Bile Acid Sequestration Accelerates Glucagon Receptor-Mediated Body Weight Loss in Obese Mice

Glucagon, an essential regulator of glucose and lipid metabolism, also promotes weight loss. Previously, we reported that chronic glucagon receptor (GCGR) activation increased energy expenditure and plasma bile acid (BA) levels. Hepatic Farnesoid X Receptor (FXR) has been associated with this GCGR-BA pathway, and liver-specific FXR-deficient mice showed diminished response to the anti-obesity effects of GCGR agonism as compared to WT mice (Diabetes 2018 PMID: 29925501). In this study we hypothesized that BAs play an important role in GCGR-mediated weight loss. To test this, we utilized an anion-exchange BA-binding resin (BABR, cholestyramine or Questran, SANDOZ Inc.) known to reduce plasma total cholesterol, LDL, and BAs. Diet-induced obese C57BL6 mice were weight stable during the 10 d Cholestyramine pretreatment (1.5% in high fat diet 58 kcal%). Daily administration of GCGR agonist, IUB288 (10 nmol/kg, s.c.), for 14 d reduced body weight by 15%; whereas IUB288+Cholestyramine doubled this effect (30%, 2-way ANOVA Time p<0.0001; Drug p=0.019; Time x Drug interaction p<0.0001). As expected, plasma cholesterol was reduced by BABR. However, unexpectedly total BA levels were increased in plasma and feces. Thus, suggesting that cholesterol and BA excretion to feces stimulated BA synthesis and increased whole body energy expenditure. With cholestyramine treatment was increased to 3%, cholestyramine-dependent body weight loss was observed (p<0.05). Combined with daily IUB288 treatment, body weight was further reduced (Time p<0.0001; Drug p=0.0009; Time x Drug interaction p<0.0001). Together, these studies suggest a combination of BABR and GCGR agonism as a novel therapeutic approach for obesity. Unless otherwise noted, all abstracts presented at ENDO are embargoed until the date and time of presentation. For oral presentations, the abstracts are embargoed until the session begins. s presented at a news conference are embargoed until the date and time of the news conference. The Endocrine Society reserves the right to lift the embargo on specific abstracts that are selected for promotion prior to or during ENDO.

Glucagon Switches between GLP-1 and Glucagon Receptors in Normal and Hyperglycemic Mice

Despite the well-known role of the GLP-1 receptor (GLP-1R) in potentiating glucose-stimulated insulin secretion (GSIS) and as a therapeutic drug target in diabetes treatment, how GLP-1R is activated under physiological and pathological conditions remains elusive. Against preconceptions in the field, we prove that glucagon released from pancreatic α-cells serves as the principal ligand for GLP-1R on β-cells and is essential for GSIS in normal mice. Activation of the cognate glucagon receptor (GCGR) by glucagon amplifies GSIS evoked by intermediate concentrations of glucose only, as high glucose bypasses GCGR in increasing cytosolic cAMP from the same downstream pool. As the endogenous dual-receptor agonist, glucagon switches from GLP-1R to GCGR in β-cells of mice fed a high-fat diet, which significantly enhances GSIS and reduces body weight. Therefore, developing dual-receptor agonists that target balanced activation of GCGR and GLP-1R in vivo may better preserve glycemic control than the activation of GLP-1R alone.

The brown adipose tissue glucagon receptor is functional but not essential for control of energy homeostasis in mice

ObjectiveAdministration of glucagon (GCG) or GCG-containing co-agonists reduces body weight and increases energy expenditure. These actions appear to be transduced by multiple direct and indirect GCG receptor (GCGR)-dependent mechanisms. Although the canonical GCGR is expressed in brown adipose tissue (BAT) the importance of BAT GCGR activity for the physiological control of body weight, or the response to GCG agonism, has not been defined. MethodsWe studied the mechanisms linking GCG action to acute increases in oxygen consumption using wildtype (WT), Ucp1−/− and Fgf21−/− mice. The importance of basal GCGR expression within the Myf5+ domain for control of body weight, adiposity, glucose and lipid metabolism, food intake, and energy expenditure was examined in GcgrBAT−/− mice housed at room temperature or 4 °C, fed a regular chow diet (RCD) or after a prolonged exposure to high fat diet (HFD). ResultsAcute GCG administration induced lipolysis and increased the expression of thermogenic genes in BAT cells, whereas knockdown of Gcgr reduced expression of genes related to thermogenesis. GCG increased energy expenditure (measured by oxygen consumption) both in vivo in WT mice and ex vivo in BAT and liver explants. GCG also increased acute energy expenditure in Ucp1−/− mice, but these actions were partially blunted in Ffg21−/− mice. However, acute GCG administration also robustly increased oxygen consumption in GcgrBAT−/− mice. Moreover, body weight, glycemia, lipid metabolism, body temperature, food intake, activity, energy expenditure and adipose tissue gene expression profiles were normal in GcgrBAT−/− mice, either on RCD or HFD, whether studied at room temperature, or chronically housed at 4 °C. ConclusionsExogenous GCG increases oxygen consumption in mice, also evident both in liver and BAT explants ex vivo, through UCP1-independent, FGF21-dependent pathways. Nevertheless, GCGR signaling within BAT is not physiologically essential for control of body weight, whole body energy expenditure, glucose homeostasis, or the adaptive metabolic response to cold or prolonged exposure to an energy dense diet.

Hepatic Glucagon Receptor Signaling Enhances Insulin-Stimulated Glucose Disposal in Rodents.

Glucagon receptor (GCGR) agonists cause hyperglycemia but also weight loss. However, GCG-like peptide 1 receptor (GLP1R)/GCGR mixed agonists do not exhibit the diabetogenic effects often attributed to GCGR activity. Thus, we sought to investigate the effect of glucagon agonism on insulin action and glucose homeostasis. Acute GCGR agonism induced immediate hyperglycemia, followed by improved glucose tolerance and enhanced glucose-stimulated insulin secretion. Moreover, acute GCGR agonism improved insulin tolerance in a dose-dependent manner in both lean and obese mice. Improved insulin tolerance was independent of GLP1R, FGF21, and hepatic glycogenolysis. Moreover, we observed increased glucose infusion rate, disposal, uptake, and suppressed endogenous glucose production during euglycemic clamps. Mice treated with insulin and GCGR agonist had enhanced phosphorylation of hepatic AKT at Ser473; this effect was reproduced in isolated mouse primary hepatocytes and resulted in increased AKT kinase activity. These data reveal that GCGR agonism enhances glucose tolerance, in part, by augmenting insulin action, with implications for the use of GCGR agonism in therapeutic strategies for diabetes.

Cholesterol 7 Alpha–Hydroxylase (CYP7A1) Activity and 7–Alpha–Hydroxy–4–Cholesten–3–One (C4) Are Not Translatable Biomarkers of Glucagon Receptor (GCGR) Activation

Glucagon-like peptide 1 receptor/glucagon receptor (GLP–1R/GCGR) dual agonists are being pursued for obesity and type 2 diabetes mellitus (T2DM). A biomarker for GCGR would aid in optimizing receptor balance and in modeling dose selection to avoid potential adverse events from excess GCGR agonism. It has been reported that glucagon treatment decreases CYP7A1 transcription in primary rat and human hepatocytes, whereas GCGR antagonists increase CYP7A1 mRNA in mice and its product, 7–HCO (the precursor of C4), in diabetic patients. We tested glucagon, insulin, dulaglutide, and a GLP–1R/GCGR dual agonist, Cpd. A, in primary human and cynomolgus monkey hepatocytes. All treatments decreased CYP7A1 expression in monkey hepatocytes. C4 was undetectable in the culture media. Lean mice or rats treated with Cpd. A showed no changes in non–fasting C4. Diet–induced obese mice treated with Cpd. A or a GCGR–null analog had decreased non–fasting C4. To test whether GCGR (and/or GLP–1R) agonism affects plasma C4 in higher species, we infused saline or glucagon (at two different rates) on a background of somatostatin infusion in cynomolgus monkeys dosed with dulaglutide the day before. C4 levels did not differ between treatment groups. To determine whether this lack of an effect was specific to pre-clinical species, T2DM patients were dosed once a week for 4 weeks with a placebo or 2 doses of a second GLP-1R/GCGR dual agonist. There were no statistically significant differences in C4 levels between groups. Hence, in contrast to GCGR antagonists, GCGR and/or GLP–1R agonists did not modulate C4 levels in pre–clinical species or in T2DM patients. Disclosure R. Camacho: Employee; Self; Janssen Research &amp; Development. W. Li: None. T. Kirchner: None. R. Zhang: Employee; Self; Janssen Research &amp; Development. F. Bonilla: None. W. Jian: None. B. Gao: None. R. SinhaRoy: Employee; Self; Janssen Research &amp; Development. Employee; Spouse/Partner; Bristol–Myers Squibb Company. Stock/'Shareholder; Self; Merck &amp; Co., Inc.. Stock/Shareholder; Spouse/Partner; Merck &amp; Co., Inc. J. Leonard: Employee; Self; Janssen Research &amp; Development. T. Connolly: None.

Oxyntomodulin analogue increases energy expenditure via the glucagon receptor

The gut hormone oxyntomodulin (OXM) causes weight loss by reducing appetite and increasing energy expenditure. Several analogues are being developed to treat obesity. Exactly how oxyntomodulin works, however, remains controversial. OXM can activate both glucagon and GLP-1 receptors but no specific receptor has been identified. It is thought that the anorectic effect occurs predominantly through GLP-1 receptor activation but, to date, it has not been formally confirmed which receptor is responsible for the increased energy expenditure.We developed OX-SR, a sustained-release OXM analogue. It produces a significant and sustained increase in energy expenditure in rats as measured by indirect calorimetry. We now show that this increase in energy expenditure occurs via activation of the glucagon receptor. Blockade of the GLP-1 receptor with Exendin 9–39 does not block the increase in oxygen consumption caused by OX-SR. However, when activity at the glucagon receptor is lost, there is no increase in energy expenditure. Glucagon receptor activity therefore appears to be essential for OX-SR’s effects on energy expenditure. The development of future ‘dual agonist’ analogues will require careful balancing of GLP-1 and glucagon receptor activities to obtain optimal effects.

Central administration of coagonist of GLP-1 and glucagon receptors improves dyslipidemia

Coagonists of Glucagon-like peptide-1 (GLP-1) and glucagon receptors are under clinical investigation for treatment of obesity associated with diabetes. In addition to their role in glucose homeostasis, GLP-1 and glucagon modulate lipid metabolism. In this study, we have investigated the role of central GLP-1 receptor (GLP-1R) and glucagon receptor (GCGR) activation in regulation of lipid metabolism in cholesterol-fed hamsters. Hamsters were treated with coagonist alone (0.3 μg) or in combination with either GLP-1R antagonist (0.15 μg) or GCGR antagonist (0.3 μg) for 4 weeks by intracerebroventricular route (icv). A pair-fed control to coagonist was included in the experiment. In a separate experiment, vagotomized hamsters were treated with coagonist (0.3 μg) for four weeks. At the end of the treatment, plasma and hepatic lipids, bile homeostasis, and hepatic gene expression were determined. Coagonist treatment caused a reduction in plasma and liver lipids, and reduced triglyceride absorption from intestine. Also, hepatic triglyceride secretion, bile flow, and biliary cholesterol excretion were increased by the coagonist treatment. Coagonist treatment exhibited increased energy expenditure and reduced the expression of SREBP-1C, HMG-CoA reductase, SCD-1, FAS and ACC in liver. Increase in the expression of LDLR, ACOX1, CPT-1, PPAR-α, CYP7A1, ABCA1 and ABCB11 was also observed in liver. The effect of coagonist on lipids was partially blocked by either GLP-1R or GCGR antagonist. Coadministration of GLP-1R antagonist blocked the effect of coagonist on bile flow, while effect of coagonist on biliary cholesterol was blocked by co-administration of GCGR antagonist. Coagonist did not affect lipid metabolism in vagotomized hamsters. It appears that central administration of coagonist reduces dyslipidemia by activation of GLP-1R and GCGR, independent of its anorectic effect.

Selection and characterization of DNA aptamer against glucagon receptor by cell-SELEX

Excessive secretion of glucagon, a functional insulin antagonist, significantly contributes to hyperglycemia. Glucagon exerts its physiological functions through activation of the glucagon receptor (GCGR). Inhibition of GCGR activity represents a potential therapeutic approach for reducing excess glucose production in diabetes mellitus. Aptamers are short DNA or RNA oligonucleotides evolved from systematic evolution of ligands by exponential enrichment (SELEX). Here, we have successfully selected a DNA aptamer against GCGR by cell-SELEX, which can specifically bind membrane protein of CHO-GCGR cells with a Kd of 52.7 ± 5.1 nM. Aptamer-mediated pull-down and gcgr knockdown assay verified that GCGR was the target of aptamer GR-3. Binding analysis revealed that GR-3 could recognize other cells with different affinity according to the level of GCGR protein expressed in these cells. Hepatic tissue imaging suggested that GR-3 could bind the cell membrane of hepatic tissues. With the advantages of small size, high binding affinity, good stability, lack of immunogenicity, and easy synthesis, aptamer GR-3 against GCGR can be a promising tool with the potential to attenuate hyperglycemia in diabetes mellitus.

A novel glucagon-like peptide-1/glucagon receptor dual agonist exhibits weight-lowering and diabetes-protective effects

Glucagon has plenty of effects via a specific glucagon receptor(GCGR) like elevating the blood glucose, improving fatty acids metabolism, energy expenditure and increasing lipolysis in adipose tissue. The most important role of glucagon is to regulate the blood glucose, but the emergent possibilities of hyperglycaemia is exist. Glucagon could also slightly activate glucagon-like peptide-1 receptor(GLP-1R), which lead to blood glucose lowering effect. This study aims to erase the likelihood of hyperglycaemia and to remain the inherent catabolic effects through improving GLP-1R activation and deteriorating GCGR activation so as to lower the bodyweight and show diabetes-protective effects. Firstly, twelve cysteine modified GLP-1/GCGR dual agonists were synthesized (1–12). Then, the GLP-1R/GCGR mediated activation and biological activity in normal ICR mice were comprehensively performed. Compounds substituted by cysteine at positions 22, 23 and 25 in glucagon were observed to be better regulators of the body weight and blood glucose. To prolong the half-lives of derivatives, various fatty side chain maleimides were modified to optimal glucagon analogues. Laurate maleimide conjugate 4d was the most potent. Administration of 1000 nmol/kg 4d once every two days for a month normalized adiposity and glucose tolerance in diet-induced obese (DIO) mice. Improvements in plasma metabolic parameters including insulin, leptin, and adiponectin were observed. These studies suggest that compound 4d behaves well in lowering body weight and maintaining energy expenditure without a chance of hyperglycaemia, 4d has strong clinical potential as an efficient GLP-1/GCGR agonist in the prevention and treatment of obesity and dyslipidemia.

Rearrangement of a polar core provides a conserved mechanism for constitutive activation of class B G protein-coupled receptors

The glucagon receptor (GCGR) belongs to the secretin-like (class B) family of G protein-coupled receptors (GPCRs) and is activated by the peptide hormone glucagon. The structures of an activated class B GPCR have remained unsolved, preventing a mechanistic understanding of how these receptors are activated. Using a combination of structural modeling and mutagenesis studies, we present here two modes of ligand-independent activation of GCGR. First, we identified a GCGR-specific hydrophobic lock comprising Met-338 and Phe-345 within the IC3 loop and transmembrane helix 6 (TM6) and found that this lock stabilizes the TM6 helix in the inactive conformation. Disruption of this hydrophobic lock led to constitutive G protein and arrestin signaling. Second, we discovered a polar core comprising conserved residues in TM2, TM3, TM6, and TM7, and mutations that disrupt this polar core led to constitutive GCGR activity. On the basis of these results, we propose a mechanistic model of GCGR activation in which TM6 is held in an inactive conformation by the conserved polar core and the hydrophobic lock. Mutations that disrupt these inhibitory elements allow TM6 to swing outward to adopt an active TM6 conformation similar to that of the canonical β2-adrenergic receptor complexed with G protein and to that of rhodopsin complexed with arrestin. Importantly, mutations in the corresponding polar core of several other members of class B GPCRs, including PTH1R, PAC1R, VIP1R, and CRFR1, also induce constitutive G protein signaling, suggesting that the rearrangement of the polar core is a conserved mechanism for class B GPCR activation.

Design of Novel Exendin-Based Dual Glucagon-like Peptide 1 (GLP-1)/Glucagon Receptor Agonists

Dual activation of the glucagon-like peptide 1 (GLP-1) and glucagon receptor has the potential to lead to a novel therapy principle for the treatment of diabesity. Here, we report a series of novel peptides with dual activity on these receptors that were discovered by rational design. On the basis of sequence analysis and structure-based design, structural elements of glucagon were engineered into the selective GLP-1 receptor agonist exendin-4, resulting in hybrid peptides with potent dual GLP-1/glucagon receptor activity. Detailed structure-activity relationship data are shown. Further modifications with unnatural and modified amino acids resulted in novel metabolically stable peptides that demonstrated a significant dose-dependent decrease in blood glucose in chronic studies in diabetic db/db mice and reduced body weight in diet-induced obese (DIO) mice. Structural analysis by NMR spectroscopy confirmed that the peptides maintain an exendin-4-like structure with its characteristic tryptophan-cage fold motif that is responsible for favorable chemical and physical stability.

Defective insulin secretion by chronic glucagon receptor activation in glucose intolerant mice

Stimulation of insulin secretion by short-term glucagon receptor (GCGR) activation is well characterized; however, the effect of long-term GCGR activation on β-cell function is not known, but of interest, since hyperglucagonemia occurs early during development of type 2 diabetes. Therefore, we examined whether chronic GCGR activation affects insulin secretion in glucose intolerant mice. To induce chronic GCGR activation, high-fat diet fed mice were continuously (2 weeks) infused with the stable glucagon analog ZP-GA-1 and challenged with oral glucose and intravenous glucose±glucagon-like peptide 1 (GLP1). Islets were isolated to evaluate the insulin secretory response to glucose±GLP1 and their pancreas were collected for immunohistochemical analysis. Two weeks of ZP-GA-1 infusion reduced insulin secretion both after oral and intravenous glucose challenges in vivo and in isolated islets. These inhibitory effects were corrected for by GLP1. Also, we observed increased β-cell area and islet size. We conclude that induction of chronic ZP-GA-1 levels in glucose intolerant mice markedly reduces insulin secretion, and thus, we suggest that chronic activation of the GCGR may contribute to the failure of β-cell function during development of type 2 diabetes.

Investigating the Glucagon Receptor and Glucagon-Like Peptide 1 Receptor Activity of Oxyntomodulin-Like Analogues in Male Wistar Rats

AimsTo investigate the effect of Glu-3 OXM-like analogues on food intake and bodyweight in male rats. BackgroundOxyntomodulin (OXM) is a natural agonist at both the glucagon receptor (GCGr) and the glucagon-like peptide 1 receptor (GLP-1r), and peripheral administration reduces food intake and increases energy expenditure in rodents and humans. Substituting the native glutamine (Gln) at amino acid position 3 of OXM for glutamate (Glu) has previously been shown to diminish GCGr activity without affecting GLP-1r activity. The effects of Glu-3 OXM analogues have not been investigated in rats. MethodsThe effect of 2 Glu-3-substituted OXM-like analogues (eg, OXM14E3 and OXM15E3) on food intake and body weight was investigated in male Wistar rats during 6 days of daily subcutaneous (SC) administration. The effects of Glu-3 substitution on analogue binding and activity at the rat GCGr and rat GLP-1 receptor were investigated in vitro using Chinese hamster ovary or Chinese hamster lung cells. ResultsWe report the novel finding that 2 5-nmol/kg Glu-3 OXM-like analogues (OXM14E3 and OXM15E3) significantly increased rat body weight by up to 4% compared with the equivalent non-Glu-3 analogues (OXM14 and OXM15), without affecting food intake. The effect of OXM15E3 on body weight was dose–dependent. Glu-3 analogues, including Glu-3 OXM, decreased glucagon-mediated cyclic adenosine monophosphate accumulation in Chinese hamster ovary cells expressing the rat GCGr, suggesting they may be acting as antagonists. ConclusionsThe results indicate Glu-3 OXM-like analogues might not be suitable tools to investigate the mechanism of OXM analogue action in a rat model because they significantly increase body weight independent of food intake. Glu-3 OXM analogues are partial agonists at the rat GCGr and may also act as antagonists, possibly resulting in the observed increase in body weight.