Glucagon In Dogs Research Articles

Discovery of potent and orally bioavailable indazole-based glucagon receptor antagonists for the treatment of type 2 diabetes.

Type 2 diabetes mellitus (T2DM) is characterized by chronically elevated plasma glucose levels. The inhibition of glucagon-induced hepatic glucose output via antagonism of the glucagon receptor (GCGR) using a small-molecule antagonist is a promising mechanism for improving glycemic control in the diabetic state. The present work discloses the discovery of indazole-based β-alanine derivatives as potent GCGR antagonists through an efficient enantioselective synthesis and structure-activity relationship (SAR) exploration and optimization. Compounds within this class exhibited excellent pharmacokinetic properties in multiple preclinical species. In an acute dog glucagon challenge test, compound 13K significantly inhibited glucagon-mediated blood glucose increase when dosed orally at 10 mg/kg.

Comparison between emulsified isoflurane and propofol/isoflurane combination on plasma thyroid hormones, insulin, glucose, and glucagon in dogs

It is important to determine the varying effects of anaesthesia agents on the endocrine function. In this study, the effects of emulsified isoflurane and propofol-isoflurane on the endocrine function were compared in dogs. Sixteen dogs were randomly divided into two groups: one group (G1) of dogs were induced with intravenous 5 mg/kg propofol and maintained with inhaled 2–2.5% inspiratory isoflurane in 100% oxygen; second group (G2) of dogs were induced with intravenous 1 ml/kg emulsified isoflurane and maintained with intravenous 8 ml/kg/h emulsified isoflurane. Blood samples to determine the endocrine function were collected prior to induction of anaesthesia and at 15 min, 30 min, 60 min, 2 h, 6 h, and 24 h after drug administration. Serum triiodothyronine and tetraiodothyronine concentrations in G1 increased significantly (P < 0.05) at 15 min and were higher than in G2. Serum insulin at 15 min and 30 min were significantly (P < 0.05) higher in both G1 and G2. Serum insulin in G1 was higher after anaesthesia than in G2. Serum glucagon decreased at 15 min, 30 min, and 60 min, without significant changes (P > 0.05). Serum glucose increased at 15 min and 30 min after anesthesia, and was significantly higher in G1 than in G2 (P < 0.05) at 60 min. In conclusion, both emulsified isoflurane and propofol-isoflurane anaesthesia affected triiodothyronine, tetraiodothyronine, and insulin in dogs, but emulsified isoflurane anaesthesia had a minimal effect on triiodothyronine and tetraiodothyronine. With its less deleterious effect on the endocrine function, emulsified isoflurane may be more suitable for canine anaesthesia.

Insulin-induced hypoglycemia increases hepatic sensitivity to glucagon in dogs

In individuals with type 1 diabetes, hypoglycemia is a common consequence of overinsulinization. Under conditions of insulin-induced hypoglycemia, glucagon is the most important stimulus for hepatic glucose production. In contrast, during euglycemia, insulin potently inhibits glucagon's effect on the liver. The first aim of the present study was to determine whether low blood sugar augments glucagon's ability to increase glucose production. Using a conscious catheterized dog model, we found that hypoglycemia increased glucagon's ability to overcome the inhibitory effect of insulin on hepatic glucose production by almost 3-fold, an effect exclusively attributable to marked enhancement of the effect of glucagon on net glycogen breakdown. To investigate the molecular mechanism by which this effect comes about, we analyzed hepatic biopsies from the same animals, and found that hypoglycemia resulted in a decrease in insulin signaling. Furthermore, hypoglycemia and glucagon had an additive effect on the activation of AMPK, which was associated with altered activity of the enzymes of glycogen metabolism.

Two-channel gastric pacing with a novel implantable gastric pacemaker accelerates glucagon-induced delayed gastric emptying in dogs

Background The aim of the current study was to investigate the efficacy of 2-channel gastric electrical stimulation (GES) with a custom-made implantable pacemaker on delayed gastric emptying and gastric dysryhthmia induced by glucagon in dogs. Methods Six dogs were studied in 4 randomized session (saline, glucagon, glucagon with single-channel or 2-channel GES). GES was applied via the first pair of electrodes for single-channel GES or the first and third pairs of electrodes for 2-channel GES. Gastric emptying was assessed for 90 minutes and gastric slow waves were recorded at the same time. Results Both single-channel and 2-channel GES improved gastric dysryhthmia ( P < .05 vs glucagon session). Two-channel GES but not single-channel GES improved glucagon-induced delayed gastric emptying at 30 minutes, 45 minutes, 60 minutes, 75 minutes, and 90 minutes. Conclusion Two-channel GES with a novel implantable pacemaker is more efficient and effective than single-channel GES in improving delayed gastric emptying induced by glucagon. This implantable multipoint pacemaker may provide a new option for treatment of gastric motility disorders.

Cloning and expression of canine glucagon receptor and its use to evaluate glucagon receptor antagonists in vitro and in vivo

Glucose homeostasis is maintained by the combined actions of insulin and glucagon. Hyperglucagonemia and/or elevation of glucagon/insulin ratio have been reported in diabetic patients and in animal models of diabetes. Therefore, antagonizing glucagon receptor function has long been considered a useful approach to lower hyperglycemia. Dogs serve as an excellent model for studying glycemic control and various aspects of glucagon biology in vivo; however, the amino acid sequence of the dog glucagon receptor has not been reported. To better understand the pharmacology of the dog glucagon receptor and to characterize glucagon receptor antagonists, we cloned a cDNA corresponding to the glucagon receptor from dog liver RNA. The dog glucagon receptor shares a significant (> 75%) homology at both nucleotide and amino acid levels with the glucagon receptor from human, monkey, mouse, and rat. The protein is highly conserved among all species in areas corresponding to the 7 trans-membrane domains. However, it shows significant divergence at the carboxy terminus such that the receptor from dog has the longest cytoplasmic tail among all species examined. When expressed in chinese hamster ovary cells, the dog glucagon receptor bound [ 125I]Glucagon with a K d of 477 ± 106 pM. Glucagon stimulated the rise of intracellular cAMP levels in these cells with an EC 50 of 9.6 ± 1.7 nM and such effects could be blocked by known peptidyl and non-peptidyl small molecule antagonists. In addition we show that a small molecule glucagon receptor antagonist with significant activity in cell based assays also blocked the ability of glucagon to induce elevation in blood glucose in beagle dogs. These data demonstrate that the cloned cDNA encodes a functional dog glucagon receptor. The availability of the dog cDNA will facilitate the understanding of glucagon pharmacology and aid in the characterization of novel glucagon antagonists that may serve as anti-hyperglycemic treatment for type 2 diabetes mellitus.

The action of glucagon and commonly used antispasmodics and analgesics on the canine ureter.

Ureteric peristalsis has been studied using extraluminal bipolar electrodes and metal foil strain gauges in both the unanaesthetized and anaesthetized dog. Electrical activity of the ureter was characterized by bipolar action potentials, which always preceded mechanical activity. In the acute studies glucagon 44 micrograms/kg i.v. was given during the unstimulated phase and again during a forced diuresis. Complete inhibition of ureteric activity was seen for 19 . 50 (+/- 3 . 76 s.e.) and 16 . 25 (+/- 1 . 59 s.e.) min respectively. During this period there was no change in the rate of urine flow. In the conscious dog glucagon was given as a bolus of 22 micrograms/kg followed by an infusion for 45 min. An infusion of 88 micrograms/kg h produced complete inhibition for 39 . 2 (+/- 2 . 41 s.e.) min. Propantheline, hyoscine, morphine, pethidine and buprenorphine were given in equivalent therapeutic human doses, but no consistent effect on ureteric peristalsis was seen. Glucagon may have a role to play in the management of ureteric colic.

Effects of S-Nitroso-N-acetyl-penicillamine Administration on Glucose Tolerance and Plasma Levels of Insulin and Glucagon in the Dog

It has been suggested that nitric oxide (NO, nitrogen monoxide) is a regulator of carbohydrate metabolism in skeletal muscle. The present study was undertaken to investigate the acute effects of the nitric oxide donor S-nitroso-N-acetylpenicillamine (SNAP) on blood glucose levels and on the gluco-regulatory hormones insulin and glucagon in healthy dogs. The acute effects of SNAP on mean arterial pressure and heart rate were also investigated. The drug was administered intravenously and the pre- and postprandial blood glucose, plasma insulin, and glucagon concentrations were determined at half-hour time intervals postadministration after a glucose challenge. The plasma nitrate and nitrite concentrations were measured and taken as the biochemical markers of in vivo NO formation. The oral glucose tolerance test revealed an impaired glucose tolerance in SNAP-treated dogs as reflected by the area under the glucose curve, 1150.50 ± 63.00 mmol × 150 min and 1355.25 ± 102.01 mmol/L × 150 min in dogs treated with 10 and 20 mg/kg of SNAP, respectively, compared with 860.25 ± 60.68 mmol/L × 150 min in captopril-treated controls (P < 0.05). The 2-h blood glucose concentration in dogs treated with 20 mg/kg body wt of SNAP was 9.17 ± 1.10 mmol/L compared with 5.59 ± 0.26 mmol/L for captopril-treated controls (P = 0.015). The oral glucose tolerance test also confirmed an impaired insulin secretion in the SNAP-treated dogs. While the plasma insulin concentration increased gradually in the captopril-treated controls to a peak value of 39.50 ± 2.55 μIU/ml, 1.5 h after a glucose challenge there was a decrease in the plasma insulin concentration in SNAP-treated dogs to a low value of 20.67 ± 0.88 μIU/ml (P = 0.006). In contrast, there were no significant differences in plasma glucagon concentration in SNAP-treated dogs and captopril-treated dogs at any time points. Using the Griess reaction, we found that there was a 27–95% increase in plasma nitrate/nitrite concentration on administration of SNAP. The sustained hyperglycemic effect observed in SNAP-treated dogs was accompanied by a marginal decrease in the mean arterial blood pressure and a significant increase in heart rate (P < 0.05). We conclude that acute administration of SNAP in the oral glucose tolerance test releases NO that modulates the parameters of carbohydrate metabolism.

Glucagon-like peptide 1 increases insulin sensitivity in depancreatized dogs.

To determine whether glucagon-like peptide (GLP)-1 increases insulin sensitivity in addition to stimulating insulin secretion, we studied totally depancreatized dogs to eliminate GLP-1's incretin effect. Somatostatin was infused (0.8 microg x kg(-1) x min(-1)) to inhibit extrapancreatic glucagon in dogs, and basal glucagon was restored by intraportal infusion (0.65 ng x kg(-1) x min(-1)). To simulate the residual intraportal insulin secretion in type 2 diabetes, basal intraportal insulin infusion was given to obtain plasma glucose concentrations of approximately 10 mmol/l. Glucose was clamped at this level for the remainder of the experiment, which included peripheral insulin infusion (high dose, 5.4 pmol x kg(-1) x min(-1), or low dose, 0.75 pmol x kg(-1) x min(-1)) with or without GLP-1(7-36) amide (1.5 pmol x kg(-1) x min(-1)). Glucose production and utilization were measured with 3-[3H]glucose, using radiolabeled glucose infusates. In 12 paired experiments with six dogs at the high insulin dose, GLP-1 infusion resulted in higher glucose requirements than saline (60.9+/-11.0 vs. 43.6+/-8.3 micromol x kg(-1) x min(-1), P< 0.001), because of greater glucose utilization (72.6+/-11.0 vs. 56.8+/-9.7 micromol x kg(-1) x min(-1), P<0.001), whereas the suppression of glucose production was not affected by GLP-1. Free fatty acids (FFAs) were significantly lower with GLP-1 than saline (375.3+/-103.0 vs. 524.4+/-101.1 micromol/l, P<0.01), as was glycerol (77.9+/-17.5 vs. 125.6+/-51.8 micromol/l, P<0.05). GLP-1 receptor gene expression was found using reverse transcriptase-polymerase chain reaction of poly(A)-selected RNA in muscle and adipose tissue, but not in liver. Low levels of GLP-1 receptor gene expression were also found in adipose tissue using Northern blotting. In 10 paired experiments with five dogs at the low insulin dose, GLP-1 infusion did not affect glucose utilization or FFA and glycerol suppression when compared with saline, suggesting that GLP-1's effect on insulin action was dependent on the insulin dose. In conclusion, in depancreatized dogs, GLP-1 potentiates insulin-stimulated glucose utilization, an effect that might be contributed in part by GLP-1 potentiation of insulin's antilipolytic action.

Treatment of Verapamil Overdose With Glucagon in Dogs

Design: The study was performed in a nonblinded, controlled animal model.Interventions: Pentobarbital-anesthetized and instrumented dogs were maintained and observed for 60 minutes or until death. All animals were overdosed with 15 mg/kg IV verapamil over 30 minutes. Mean arterial pressure, heart rate, ECG, and cardiac output were monitored. The experimental group received a 2.5 mg glucagon IV bolus followed by a glucagon drip at 2.5 mg/hr.The control group received an equal volume of IV normal saline solution in the same fashion. Analysis was performed with the Dunnett and Tukey-Kramer methods, with a set at .05. Results: There were eight experimental and seven control animals, with mortality rates of 0% and 29%, respectively. The experimental group had increases in cardiac output and heart rate that were statistically significant at 45 and 60 minutes compared with those of the control group. In addition, there was a significant difference in heart rate at 30 minutes. No difference was noted between the groups for mean arterial pressure. Conclusion: Glucagon appears to reverse both the bradycardia and the depressed cardiac output associated with verapamil overdose in a canine model. [Stone CK, May WA, Carroll R: Treatment of verapamil overdose with glucagon in dogs. Ann Emerg Med March 1995;25:369-374.]

Insulin releasing action of N-terminal peptides of glucagon in dogs.

The relationship between the molecular structure and insulin releasing action of glucagon remains unknown. In order to investigate the direct action of N-terminal peptides of glucagon, glucagon (1-14), and glucagon (1-21) were studied using an in situ local circulation of the canine pancreas. These glucagon fragments as well as glucagon (1-29) were infused into the superior pancreaticoduodenal artery in a dose of 400 pmol for 10 min during the glucose or arginine infusion, and plasma insulin and glucagon in the superior pancreaticoduodenal vein were determined by radioimmunoassay. During the glucose infusion, glucagon (1-14) elicited a slight increase in plasma insulin, whereas glucagon (1-21) and (1-29) revealed significant changes in plasma insulin. In these experiments plasma glucagon did not change significantly following the administration of glucagon (1-14) or (1-21). During the arginine infusion all of the glucagon fragments studied enhanced insulin secretion markedly, whereas glucagon secretion was not affected. Furthermore, graded doses of glucagon (1-14) (50, 150, and 400 pmol) elicited an increase in plasma insulin in a dose-related manner. It is concluded from the present study that the N-terminal peptides of glucagon stimulate insulin release especially during the arginine infusion.

The Structure-Function Relationship of GLP-1 Related Peptides in the Endocrine Function of the Canine Pancreas.

In order to clarify the relationship between the structure and function of glucagon-like peptide (GLP) 1 in the endocrine function of the pancreas, the response of insulin and glucagon to various synthetic GLP-1-related peptides was investigated in anesthetized dogs. GLP-1-related peptides were administered in a dosage of 400 pmol within 10 min into the pancreatic artery during glucose or arginine infusion and the changes in plasma insulin and glucagon in the pancreatic vein were studied. GLP-1 (7-36) and (7-37), as well as glucagon enhanced insulin release during glucose infusion, whereas neither GLP-1 (1-37), (7-20), (6-37) nor (8-37) stimulated insulin release. The administration of GLP-1 (1-37), (7-36) and (7-37) reduced glucagon release during glucose infusion. When arginine was infused, GLP-1 (7-20), (7-36), (7-37), and glucagon enhanced insulin release. In contrast, glucagon release was increased by the administration of GLP-1 (7-20), (8-37), and (7-37). The present study indicates that histidine at the 7th position of GLP-1 is important in eliciting biological action and that only truncated GLP-1 (7-36), (7-37), and (7-20) showed an insulinotropic action as strong as glucagon in dogs. Furthermore, it is suggested that the response of insulin and glucagon to GLP-1-related peptides is dependent on a background condition.

Effect of pancreatic glucagon upon secretion of extrapancreatic glucagon in dogs.

Effect of pancreatic glucagon upon secretion of extrapancreatic glucagon in dogs.

Response of extrapancreatic glucagon to arginine in dogs.

In order to investigate secretion of extrapancreatic glucagon in dogs, plasma glucagon and total immunoreactive glucagon (IRG) in response to arginine were determined with antisera specific and non-specific for pancreatic glucagon, respectively. Only a tiny rise of plasma glucagon was observed in the portal vein following arginine infusion performed immediately after total pancreatectomy in a group of 5 normal dogs. In contrast, total IRG in the portal vein increased significantly after arginine infusion even after pancreatectomy. In alloxan diabetic dogs, arginine infusion after total pancreatectomy caused a rise in plasma glucagon in the portal vein, although not significantly. The response of total IRG to arginine in the portal vein was exaggerated in alloxan diabetic dogs. In a group of one-week post-pancreatectomized animals, plasma glucagon and total IRG increased significantly in response to arginine infusion. Furthermore, in these dogs, response of plasma glucagon and plasma total IRG to arginine infusion was abolished after gastrectomy. From the present results it is concluded that secretion of extrapancreatic glucagon increased in response to arginine infusion in the diabetic state, both alloxan diabetic dogs and one-week post-pancreatectomized dogs. In addition, a rise in extrapancreatic glucagon following arginine infusion in the chronically pancreatectomized dogs almost certainly derived from the stomach, as the rise was abolished by gastrectomy.

Renal effects of glucagon in dogs with hemorrhagic hypotension

Previous studies in dogs with hemorrhagic hypotension, where the arterial blood pressure was maintained constant at 65 to 70 mm Hg, demonstrated that the intravenous infusion of glucagon at 5 μg/min may cause marked increments in the glomerular filtration rate and renal blood flow. To investigate the mechanism of this response we studied a similar model with micropuncture techniques. In 10 dogs, an acute hemorrhage raised intrarenal vascular resistance by 60%. By use of the Gomez equations ( Gomez, D. M. Evaluation of renal resistances with special reference to changes in essential hypertension. J. Clin. Invest. 30: 1143, 1951. ), it was demonstrated that the infusion of glucagon produced predominant preglomerular vasodilatation, though there was efferent dilatation as well. Glomerular permeability was not changed by the glucagon infusion. An increase in cardiac output or redistribution of glomerular flow did not contribute to the renal effects of glucagon in this model. Glucagon is able to raise glomerular filtration rate in dogs with hemorrhagic hypotension through its ability to dilate predominantly the afferent arteriole.

Somatotrophic diabetes: insulin release responses to arginine and glucagon in dogs.

Growth hormone injected daily in 6 dogs for 6 days caused a 20-fold elevation in fasting serum immunoreactive insulin (IRI) without appreciable change in serum glucose in 1 day. In the somatotrophic diabetes that occurred after 2 days, the hyperinsulinaemia was maintained and the serum IRI/glucose (I/G) ratio declined from the early high level but remained elevated. During this treatment, in response to glucose infusion, the rise in serum IRI above the initially high fasting level was 16 times the normal. In response to glucagon, the rise in IRI was twice the normal and the rise in glucose was more prolonged, resulting in a decline in the I/G ratio. In response to arginine infusion, the rise in serum IRI was 8 times the normal and the rise in the I/G ratio was twice normal. Following a meal, the rise in serum IRI was 8 times the normal. Thus, with growth hormone treatment the insulin secretory responses to these stimulating factors were magnified over the already elevated fasting level of secretion. The insulin content of the pancreas was reduced to less than 10% of normal by growth hormone treatment for 6 days, due apparently to elevation of the rate of secretion over the rae of formation of insulin.

Effect of intrapancreatic administration of vasoactive intestinal peptide upon the release of insulin and glucagon in dogs.

To investigate the effect of vasoactive intestinal peptide (VIP) on the endocrine function of the pancreatic islet, experiments with a local circulation of the pancreas were performed in anesthetized dogs. VIP was infused into the pancreatic artery in doses of 20, 100, 200 and 400 ng for 10 min. Blood glucose level in the femoral artery did not change during and after the infusion of VIP. Since VIP is known to increase blood flow, a circuit was made in the pancreatic vein and blood flow rate of the pancreatic vein was calculated from the time necessary to obtain a volume of blood. Blood flow rate of the pancreatic vein did not change throughout the experiment with graded doses of VIP. Plasma insulin in the pancreatic vein increased transiently following the infusion of 20 ng VIP but a constant and significant rise of plasma insulin was observed in the experiment with 400 ng of VIP. To the contrary, plasma glucagon in the pancreatic vein increased significantly after the infusion of VIP in doses of 20 to 400 ng for 10 min. The changes in the secretion of these hormones were similar to those in the plasma levels of these hormones in the pancreatic vein. From the present experiment it is concluded that VIP even in a small dose, which does not induce hyperglycemia or increase in blood flow, stimulates the secretion of insulin and glucagon from the pancreas.

Improved cardiovascular effects of glucagon in dogs with endotoxin shock.

From the Department of Surgery, University of Colorado, School of Medicine, and the Veterans Administration Hospital, Denver, Colorado

The effect of experimental insulin deficiency on glucagon secretion.

Suppression of pancreatic glucagon secretion by hyperglycemia is a characteristic of normal alpha cell function. However, in diabetic subjects, plasma glucagon is normal or high despite hyperglycemia. It seemed possible that the presence of glucose or its metabolites within the alpha cell might be essential for suppression of glucagon secretion, and that in diabetes an intracellular deficiency of glucose secondary to insulin lack might be responsible for the nonsuppressibility. The present study was designed to determine the effect upon glucagon secretion of blockade of glucose metabolism and of experimental insulin deficiency. Blockade of glucose metabolism was induced in dogs by administration of 2-deoxyglucose or mannoheptulose. A striking rise in glucagon was observed despite accompanying hyperglycemia and hyperinsulinemia, which, in the case of mannoheptulose, was induced by infusing crystalline insulin. To determine if insulin lack also causes paradoxical hyperglucagonemia, dogs were made severely diabetic by alloxan. Fasting glucagon levels ranged from 3 to 22 times normal despite severe hyperglycemia, and were quickly restored to normal by infusing insulin. Diabetes induced in rats by anti-insulin serum was also associated with significant elevation in plasma glucagon. However, diazoxide-induced insulin lack did not increase glucagon in dogs. It is concluded that normal suppression of glucagon secretion by hyperglycemia does not occur when glucose metabolism is blocked or when severe insulin deficiency is produced. It is suggested that normal glucose metabolism within the alpha cell may be an insulin-requiring process without which hyperglycemic suppression of glucagon release cannot occur.

Cardiovascular effects of glucagon in dogs with non-nodal pacemakers

In closed-chest dog preparations, glucagon (50 μg/kg) significantly increased ventricular heart rate up to 35% above control after surgical interruption of the His bundle. Under similar conditions, glucagon failed to alter ventricular rate in open-chest preparations when the study was conducted immediately after the surgery. Although ventricular rate was not increased in the latter group of animals, glucagon continued to exert a positive inotropic response equivalent to that observed in animals not subjected to heart block.

Alterations in glucose turnover following single intravenous injections of epinephrine and glucagon in dogs

The technique of priming injection-continuous infusion of tracer glucose-U-C 14 was employed in 7 anesthetized dogs, each serving as its own control, to determine the immediate changes in rates of appearance (Ra) and disappearance (Rd) of plasma glucose following single intravenous injections of epinephrine (10 μg. per Kg.) and glucagon (1 μg. per Kg.). Mean maximal hyperglycemia induced by the hormones was comparable in magnitude, but it was achieved in 4 minutes after epinephrine injection compared with 8 to 10 minutes after glucagon. Furthermore, Ra was increased 5- to 7-fold during the first 4 minutes following epinephrine injection and subsequently fell to below control levels for about 30 minutes. In contrast, following glucagon, Ra was increased 3- to 4-fold during the first 8 minutes after injection with subsequent return to control levels. After both epinephrine and glucagon, Rd increased 1.5- to 2-fold during the first 10 minutes; it returned to control levels 10 minutes after epinephrine and 30 minutes after glucagon. These data indicate that the increment in plasma glucose results from enhanced hepatic glycogenolysis. However, contrary to the suggestion of others, the effect of epinephrine on the liver does not appear to be mediated via glucagon release.