Metabolic manifestations of insulin deficiency do not occur without glucagon action

To determine unambiguously if suppression of glucagon action will eliminate manifestations of diabetes, we expressed glucagon receptors in livers of glucagon receptor-null (GcgR(-/-)) mice before and after β-cell destruction by high-dose streptozotocin. Wild type (WT) mice developed fatal diabetic ketoacidosis after streptozotocin, whereas GcgR(-/-) mice with similar β-cell destruction remained clinically normal without hyperglycemia, impaired glucose tolerance, or hepatic glycogen depletion. Restoration of receptor expression using adenovirus containing the GcgR cDNA restored hepatic GcgR, phospho-cAMP response element binding protein (P-CREB), and phosphoenol pyruvate carboxykinase, markers of glucagon action, rose dramatically and severe hyperglycemia appeared. When GcgR mRNA spontaneously disappeared 7 d later, P-CREB declined and hyperglycemia disappeared. In conclusion, the metabolic manifestations of diabetes cannot occur without glucagon action and, once present, disappear promptly when glucagon action is abolished. Glucagon suppression should be a major therapeutic goal in diabetes.     

Calcium,glucose and glucagon release

Summary Using the isolated, perfused canine pancreas the importance of calcium for the normal secretory function of the pancreatic alpha cell was investigated. It was found that 1. increases in perfusate Ca⁺⁺ from 1.3 to 4.8 mM and from 1.3 to 8.2 mM during perfusion with glucose concentrations of 25 and 150 mg/100 ml stimulate the release of both glucagon and insulin in a dose-related and a glucose-dependent fashion. The hormone responses to increases in calcium were, with few exceptions, biphasic 2. a Ca⁺⁺ free medium inhibited release of both hormones, and increases in perfusate glucose from 25 to 150 mg/100 ml were unable to suppress glucagon or to stimulate insulin. Addition of calcium (8.2 mM) resulted in re-establishment of the normal regulatory role of glucose upon release of both hormones, now being in a hyperactivated state by the high Ca⁺⁺ concentration; 3. sudden Ca⁺⁺ depletion of the perfusate from 2 mM at a glucose concentration of 200 mg/100 ml inhibited immediately the release of both hormones to very low levels, which remained low until the addition of Ca⁺⁺ (2 mM). Ca⁺⁺ is therefore an essential requirement for the normal secretory process of pancreatic glucagon, possibly involving uptake and accumulation within the A cell, as established for the B cell. It is suggested that Ca⁺⁺ exerts its effect on the microtubular microfilamentous system.Presented in part at the International Symposium on Glucagon, May 1976, Dallas, Texas and at the 12^th Annual Meeting of the European Association for the Study of Diabetes, September 3, 1976, Helsinki, Finland [Abstract 125, Diabetologia12, 398 (1976)]     

Postmaturity in the rat: Impairment of insulin,glucagon, and glycogen stores

Summary Prolonged gestation (2 extra days in utero) was obtained by daily subcutaneous injection of progesterone (2.5 mg) to pregnant rats from day 20.5 post coïtum (p. c.) through day 22.5 p. c. after reduction of the litter to 6 fetuses on day 14.5 p. c. Exogenous progesterone per se or litter reduction were without effect on fetal pancreas or fetal liver. Plasma insulin, insulin and glucagon in the pancreas, and liver glycogen stores have been systematically measured in postmature animals and in controls during the perinatal period. In 23.5 day-old postmature as compared to 21.5 day-old normal fetuses, the intrauterine mortality was increased (26%), the body weight was increased by 30%, the liver weight was decreased by 20%, the glycogen content of liver was dramatically depleted (1.1±0.2mg/g body weight on day 23.5 p.c. against 6.7±0.3 on day 21.5 p.c), the plasma insulin was lowered by 63% and the blood glucose level was normal. In postmature neonates during the first day of life the mortality rate was considerable (40%) and a dramatic fall of blood glucose was observed 6 hours after birth. The accumulation of insulin and glucagon in the pancreas, which normally occurs in the two first days after birth, was much lower in the postmature fetuses: in 23.5 day-old fetuses as compared to 2 day-old normal newborns of the same gestational age the insulin content was only 50% and the glucagon content 69%. The deficit of insulin accumulation in the postmature pancreas lasted at least five days. The ability of the endocrine pancreas to recover from this alteration was well shown by the lack of diabetes when the animals were examined three weeks later by a glucose tolerance test. These findings suggest that the drop of plasma insulin is a prime factor in causing the lack of glycogen stores in prolonged fetuses and the impairment of glycogen stores appears to be an important feature of postmaturity, since neonates exhibit, in these conditions, a lethal drop of blood glucose as glycogenolysis operates on very low glycogen stores.This work has previously been presented at the 11th Meeting of the European Association for the Study of Diabetes, Munich, September 1975 (1).     

Difference in calcium dependency of insulin,glucagon and somatostatin secretion in response to glibenclamide in perfused rat pancreas

Summary The extracellular calcium requirements for insulin, glucagon and somatostatin release induced by 1 g/ml of glibenclamide have been compared in the perfused, isolated rat pancreas. In the absence of glucose, the drug evoked insulin release equally well at physiological (2.6 mmol/l) and low (0.25 mmol/l) levels of total calcium. In contrast, glibenclamide evoked somatostatin release at 2.6 but not at 0.25 mmol/l of calcium. At 2.6 mmol/l of calcium, glibenclamide evoked bimodal effects (stimulation followed by inhibition) on glucagon secretion. At 0.25 mmol/l of calcium, basal secretory rates of glucagon were elevated and a small stimulatory effect of glibenclamide was seen. Addition of 0.5 mmol/l of EGTA to media with low calcium concentrations uniformly abolished the A, B and D cell secretory responses to glibenclamide. The possible modulation of calcium dependency by a non-stimulatory concentration of glucose was tested by its addition at 3.3 mmol/l to the perfusion media. Glucose enhanced glibenclamide-induced insulin secretion, both at 0.25 and 2.6 mmol/l of calcium. However, at 0.25 mmol/l of calcium, the enhancing effect of glucose was more pronounced than at 2.6 mmol/l. At 2.6 mmol/l of calcium, glucose diminished the somatostatin and abolished the glucagon response to glibenclamide. At 0.25 mmol/l of calcium, glucose did not influence somatostatin release while the presence of the sugar diminished basal and glibenclamide-induced glucagon secretion. The present data confirm the requirement of extracellular calcium for A, B and D cell secretion, demonstrating different calcium dependencies for the cell types and indicate that this dependency can, in part, be modulated by glucose.     

Importance of glucagon in the control of futile cycling as studied in alloxan-diabetic dogs

Summary In order to determine the role of glucagon in futile or substrate cycling in diabetes, we measured tracer determined glucose kinetics during a combined infusion of 2-³H-glucose (total glucose production) and 6-³H-glucose (glucose production) in six alloxan-diabetic dogs. The animals received either a 420 min infusion of (1) somatostatin alone (0.3 g·kg^–1· min^–1), (2) somatostatin with insulin replacement (100 U·kg^–1min^–1) or (3) glucagon (6 ng·kg^–1· min^–1) together with somatostatin and transient insulin replacement. When somatostatin was given alone, plasma glucagon (p<0.004) and insulin (p<0.0001) were suppressed. Glucose production and disappearance and plasma glucose concentrations fell (p<0.0001), but the metabolic clearance of glucose did not change significantly. In the basal state, futile cycling comprised 29±4%, 33±4% and 33±3% of total glucose production in the three goups of studies, which is high compared to normal dogs. The absolute rate of futile cycling fell slightly but significantly from 10.0±1.7 to 8.3±1.7 mol·kg·^–1min^–1 (p<0.0008). When insulin replacement was given during somatostatin infusion to correct for the small somatostatin-induced insulin suppression, there were similar changes in plasma glucagon, glucose concentrations and glucose kinetics as seen during the infusion of somatostatin alone. Futile cycling decreased to a slightly greater extent from 12.8±2.8 to 9.5±1.7mol·kg^–1·min.^–1 (p<0.02). When glucagon was infused together with somatostatin and insulin replacement, plasma glucagon (p<0.0002) increased and plasma glucose levels rose (p<0.001) due to a transient increase in glucose production. Metabolic clearance of glucose did not change significantly. There was a marked increase in futile cycling from 12.2±1.7 to 21.7±1.7mol· kg^–1·min^–1 (p<0.0001) in response to exogenous glucagon excess. There was a slight (p<0.01) drop in free fatty acid levels with somatostatin. Free fatty acid levels nearly doubled (p<0.025) with the infusion of glucagon together with somatostatin. In conclusion, (a) futile cycling was increased in alloxan-diabetic dogs; (b) glucagon suppression can suppress futile cycling only if total insulin deficiency is prevented; and (3) hyperglucagonaemia increases futile cycling, and this effect is more pronounced during insulin deficiency.     

Inhibition of gastric acid secretion in humans by glucagon during euglycemia,hyperglycemia, and hypoglycemia

The effect of intravenous infusion of glucagon in a dose of 85 pmol/kg/hr on submaximal pentagastrin-stimulated gastric acid secretion was studied in eight healthy volunteers. The study was repeated four times in each subject. By a glucose-insulin clamp technique blood glucose levels were kept constant during the studies at 5.0 mmol/liter (euglycemic clamp), 2.5 mmol/liter (hypoglycemic clamp), or 7.0 mmol/liter (hyperglycemic clamp) on three different days. Glucose and insulin were not infused during one control day study. During glucagon infusion, plasma glucagon levels increased but the level reached was lower during the hyperglycemic condition when compared to euglycemic and hypoglycemic conditions. Glucagon infusion inhibited gastric acid secretion during hyper- and euglycemic conditions but not during hypoglycemic conditions. Hyperglycemia caused a modest but significant inhibition of acid secretion. Serum gastrin concentrations were unaltered during glucagon infusion regardless of the level of blood glucose. The present observations indicate that the inhibitory effect of glucagon is independent of the glucagon-induced hyperglycemia, but the effect is lost when blood glucose is below a certain limit, suggesting that blood glucose may have a modulating effect on gastric acid secretion.This study was supported by the Danish Hospital Foundation for Medical Research. Region of Copenhagen, The Faroe Islands and Greenland.     

Does glucagon play a role in the insulin resistance of patients with adult non-ketotic diabetes?

Summary Using a constant intravenous infusion technique we have measured in vivo insulin resistance in 17 normal subjects, five patients with chemical diabetes, and 13 non-ketotic diabetic patients with fasting hyperglycaemia (FBS>120 mg/ 100 ml). All of the diabetic patients were non-obese. The results demonstrated that the diabetic patients were insulin resistant compared to normals and that the degree of insulin resistance was greater the more severe the diabetes. No differences in plasma glucagon levels were found among the different groups during the infusion studies. These results demonstrate that non-obese, non-ketotic diabetic patients are insulin resistant and that abnormalities in plasma glucagon concentrations do not account for this insulin resistance.Dr. Olefsky is a Clinical Investigator with the Veterans AdministrationDr. Sperling is a recipient of a Research Career Development Award from the U. S. P. H. S., 1K04-HD 00029Dr. Reaven is a Medical Investigator with the Veterans Administration     

The effect of hGH deficiency on the insulin response to glucagon after oral glucose loading

Summary Six children and adolescents (aged from 2 6/12 to 16 years) with isolated hGH deficiency were subjected to a standard oral glucose tolerance test (OGTT) followed by the administration of IV glucagon at 180 mins. Three of them underwent a second test after several months of hGH therapy. Nine patients underwent a separate IV glucagon test and two of these patients had both tests. As controls served 14 endocrinologically normal children and adolescents, who underwent both tests. It was found that the patients with isolated hGH deficiency had lower basal plasma insulin and blood glucose levels and that their insulin response to IV glucagon even after oral glucose preloading was significantly lower than in the control group. This response was partially restored by several months of hGH treatment in the three patients tested. These findings are interpreted as further evidence for an insulinotrophic effect of hGH.Established Investigator of the Chief Scientist's Bureau, Ministry of Health     

Effect of insulin on glucagon enhanced lipolysis in vitro

Summary Glucagon in concentrations similar to those found in human plasma markedly stimulates lipolysis in rat adipose tissuein vitro. The effects of these physiological concentrations of glucagon are reduced or abolished by insulin at concentrations of 25 and 100U/ml. Considering the marked insulinogenic effect of glucagon these observations may provide an explanation for the delayed increase of blood FFA observed after glucagon injectionin vivo.This work was supported by the Fonds National de la Recherche Scientifique and the Fonds de la Recherche Scientifique Médicale, Belgium.     

Glucoregulatory effects and prolonged duration of action of davalintide: a novel amylinomimetic peptide

Aims: Davalintide is a second‐generation amylinomimetic peptide possessing enhanced pharmacological properties over rat amylin to reduce food intake in preclinical models. The current experiments in rats describe additional glucoregulatory actions of davalintide consistent with amylin agonism, and explore the duration of action of these effects. Methods: Subcutaneous (SC) injection of davalintide slowed gastric emptying with equal potency to amylin (ED₅₀'s = 2.3 and 4.1 µg/kg). This effect was maintained for 8 h with davalintide, but not amylin. Intraperitoneal injection of davalintide also reduced food intake with a potency similar to amylin (ED₅₀'s = 5.0 and 11.3 µg/kg). Consistent with amylin agonism, davalintide (10 µg/kg, SC) suppressed the plasma glucagon response over 90 min following an intravenous arginine bolus in anaesthetized rats. The elimination t_1/2 of davalintide (200 µg/kg, SC) was 26 min, similar to the t_1/2 of amylin, suggesting that pharmacokinetic‐independent mechanisms contribute to davalintide's enhanced duration of action. Binding kinetic studies using ¹²⁵I davalintide revealed no appreciable dissociation from the amylin nucleus accumbens receptor after 7 h while ¹²⁵I rat amylin did dissociate from this receptor (K_off = 0.013/min). Sustained SC infusion of davalintide (275 µg/kg/day) or amylin (300) decreased plasma glucose after an oral glucose challenge at 2 weeks (by 27 and 31%) and suppressed gastric emptying at 3 weeks (by 29 and 47%), demonstrating durable glucoregulatory actions of both peptides. Conclusions: These data show glucoregulatory properties of davalintide consistent with amylin agonism and suggest that slowed receptor dissociation plays a role in davalintide's prolonged pharmacodynamic actions.     

Insulin and glucagon releasing activity of coleonol (forskolin) and its effect on blood glucose level in normal and alloxan diabetic rats

Summary Colenol, a diterpenoid isolated from the roots ofColeus forskohlii stimulates the release of insulin and glucagon from the islets bothin vitro andin vivo. Coleonol-stimulated release of glucagon from isletsin vitro is much more pronounced as compared to that of insulin. Glucose concentration of 5.6 mM in the medium is required for the coleonol stimulation of insulin release. Feeding of coleonol to alloxan diabetic rats cause 36.5% increase in blood glucose level as compared to alloxan diabetic control. Oral feeding of coleonol for 7 days to normal rats causes increase in blood glucose, serum insulin, glucagon and free fatty acid levels with corresponding increase in glucose-6-phosphatase activity and depletion of liver glycogen. Predominant stimulation of A-cells by coleonol is suggested for the above effects. C.D.R.I. Communication n ^o 4646.     

Divergent effect of glucagon antibodies on arginine and glucose-stimulated insulin secretion in the rat

Summary The effects of glucose and arginine on insulin secretion in the presence of glucagon antibodies were investigated in rats in vivo. In contrast to controls, animals given glucagon antibodies showed an inhibition of arginine-stimulated (p < 0.001), but not glucose-stimulated, insulin secretion. That these effects were not due to incomplete neutralisation of endogenous glucagon is evidenced by the presence of large antibody excess throughout the duration of the experiments. Both the glucagonotropic effect of arginine (319 ± 60ng/l, p < 0.01) and the insulinotropic effect of exogenous glucagon (8.3 ± 0.8 g/l, p < 0.001) were demonstrable under our experimental conditions in the absence of exogenous glucagon antibodies. These observations suggest that different mechanisms are involved in the stimulation of insulin release by arginine and by glucose, and that glucagon may play an important physiological role in the mediation and regulation of insulin secretion by secretogogues, such as arginine.     

Insulin and glucagon secretion by islets isolated from fetal and neonatal rats

Summary Islets were isolated by mild collagenase digestion and microdissection from rat fetuses 2 days before term and pups 1 or 2 days after birth and their insulin and glucagon secretion studied in vitro. Fetal B cells were stimulated by 16.7 mmol/l glucose, 20 mmol/l leucine or 20 mmol/l arginine. Fetal A cells were not affected by glucose or leucine, but were significantly stimulated by arginine. Somatostatin abolished the effect of arginine on both IRI and IRG output. Neonatal islets proportionally released more insulin and glucagon than their fetal counterparts, but reacted to the tested agents in a similar fashion. During the perinatal period, pancreatic insulin storage increased at a higher rate than that of glucagon. It is concluded that fetal B cells are equipped with sensors to a variety of agents and able to modulate their secretory rate according to the concentration of these agents. A cells are reactive to arginine 2 days before term but do not become glucose reactive until several days after birth.     

Beta endorphin modulation of the glucoregulatory effects of repeated epinephrine infusion in alloxan-diabetic and normal dogs

Summary When repeated epinephrine infusions are given to normal dogs as a partial stress model, there is exaggerated hyperglycaemia, associated with reduced plasma insulin levels and markedly decreased glucose clearance. In the present study, we have examined the hormonal and metabolic responses to two successive 60-min epinephrine (0.1 g-kg^–1·min^–1) infusions with or without concomitant infusion of beta endorphin (0.3 g·kg^–1·min^–1) in 6 alloxan-diabetic dogs. These studies have been compared to similar studies in 5 normal dogs.In the diabetic dogs, plasma glucose rose from 12.3±2.2 to 16.2±2.4 mmol/l (p<0.001) in response to the first epinephrine infusion and rose further to 18.1±2.5 mmol/l (p<0.001) during the second epinephrine infusion. The increases in plasma glucagon and glucose production were comparable with both infusions, but considerably greater than previously observed in normal dogs.In normal dogs, beta endorphin diminished the insulin response to the first epinephrine infusion (p<0.02), and abolished this response to the second (p<0.05). In addition beta endorphin also diminished the glucagon response to the second epinephrine infusion (p<0.01) and greatly potentiated epinephrine-induced suppression of glucose metabolic clearance during both infusions (p<0.001). However, beta endorphin did not appreciably alter the hyperglycaemic response to epinephrine due to a concomitant attenuation of the epinephrine-induced increase in hepatic glucose production.In contrast to normal dogs, beta endorphin did not modulate the effects of either the first or second epinephrine infusion on glucose kinetics in diabetic dogs. Also, beta endorphin failed to inhibit glucagon or insulin secretion in response to epinephrine in the diabetic animals. Since the alloxan-diabetic and normal dogs respond differently to the combined infusion of beta endorphin and epinephrine we conclude that the effects of beta endorphin observed in the normal dogs are dependent upon intact pancreatic endocrine function.     

Thyroid hormones and lipogenesis from glucose in rat fat cells

The effect of thyroidectomy on lipogenesis from glucose was investigated in rat fat cells. It was shown that thyroidectomy resulted in a clear increase in the oxidation of glucose to CO₂ and also in its conversion to fatty acids. Such an increase in lipogenesis from glucose after thyroidectomy was not due to a change in the cell size or to a modification of the cell surface. The diffusion of L-glucose and the facilitated diffusion transport of 3-0 methyl glucose were not increased; the increase in glucose oxidation observed after thyroidectomy is therefore not the result of an increased transfer of glucose. In contrast, the uptake of deoxyglucosc, a sugar which enters the cell and is phosphorylated by the hexokinase system, but is then not further metabolized, was markedly enhanced in cells from the thyroidectomized animals. A 4–5-fold increase over control in the V_max, but no change in the K_m, were observed. Since no effects were noted on sugar diffusion or transport, this result demonstrates that a major consequence of thyroidectomy is an increase in glucose phosphorylation. Previous observations (Corrèze et al., 1974; Van Inwegen et al., 1975) showed how fat cells from thyroidectomized rats lose their lipolytic capacity; in the present study we provide evidence for an increase in lipogenesis in these same cells. Thyroid hormones might therefore modulate a critical regulatory stcp(s) common to lipolysis and lipogenesis.     

Effect of exogenous insulin and glucagon on exocrine pancreatic secretion in rats in vivo

Summary Background. The physiological roles of the islet hormones insulin and glucagon in the control of exocrine pancreatic secretion is not clear. It is still unknown whether these hormones have a stimulatory or an inhibitory effect on the basal exocrine pancreatic secretion. Methods. Thirty anesthetized rats were stimulated with doses of insulin and glucagon administered by continuous intravenous infusion. Doses varying from physiological to supraphysiological were used. Different groups of 5 rats were given each of these doses. The volume of pancreatic juice and amylase, lipase and trypsin activity, as well as enzyme output, were measured 0, 20, 40, and 60 min after starting infusion. The insulin, glucagon, and glucose levels were determined in serum at 0, 10, 30, and 60 min. Results. In the insulin group, the secreted volume of pancreatic juice increases with the maximum dose. All insulin doses results in amylase and lipase decreased activity. When submaximum and maximum insulin doses are administered, the trypsin activity also decreases. In the glucagon group, the activity of lipase and trypsin decreases regardless the dose, whereas the amylase activity decreases with submaximum and supramaximum doses. Conclusion. Both insulin and glucagon affect the basal exocrine pancreatic secretion in vivo when physiological doses are administered.     

Effect of furosemide on insulin and glucagon responses to arginine in normal subjects

Summary This study aimed at evaluating the influence of furosemide upon insulin and glucagon responses to arginine in healthy subjects. For this purpose, six normal subjects received two consecutive arginine pulses (3 g), 60 min apart, before and after the administration of furosemide (40 mg, IV). The acute insulin response (mean change from 3–10 min) to the second arginine pulse was significantly inhibited by furosemide (mean increase: 14.8 ±3.0 U/ml versus 11.7±2.5 U/ml, p<0.01). By contrast, the acute glucagon response was significantly increased (mean increase: 77±18 pg/ml versus 105±21 pg/ml, p<0.01). No significant changes in plasma glucose levels occurred. In control experiments, in which saline rather than furosemide was administered, the acute insulin and glucagon response to the first arginine pulse did not differ from that observed with the second pulse. The effect of furosemide on insulin and glucagon secretion might be mediated through enhanced release of endogenous prostaglandin E.     

A comparison of serum C-peptide response to intravenous glucagon, and urine C-peptide, as indexes of insulin dependence

Serum C-peptide (SCPR) at fasting and after intravenous injection of glucagon was evaluated in diabetic patients with various degrees of insulin dependence, and compared with 24 h urine C-peptide (UCPR). Fasting SCPR did not differ between healthy subjects and sulfonylurea-treated patients (SU) who were considered to have definite non-insulin-dependent diabetes (NIDDM); but was significantly lower in patients with insulin-dependent diabetes (IDDM) (0.24 +/- 0.10 ng/ml in IDDM vs. 1.43 +/- 0.61 ng/ml in SU, P less than 0.001). SCPR reached a peak at 6 min after glucagon injection, except for the IDDM group. The SCPR response at 6 min after 1 mg glucagon injection was significantly lower in the SU (NIDDM) group than in the normal group (2.86 +/- 1.21 v. 4.69 +/- 1.47 ng/ml, P less than 0.001). In the IDDM group, there was no increase of SCPR after glucagon injection. Among diabetic patients, SCPR response to glucagon correlated positively to the amounts of UCPR (P less than 0.001). By analysis of the distribution patterns of SCPR response to intravenous glucagon, SCPR of 1.0 ng/ml and the increment of SCPR of 0.5 ng/ml at 6 min are to be used as cut-off points to differentiate IDDM and NIDDM. These values correspond roughly to the UCPR values below 20 micrograms/day and above 30 micrograms/day, which we previously proposed as indexes to differentiate insulin-dependent and non-insulin-dependent diabetes.     

Lack of influence of arginine preloading on the insulin response to I.V. glucagon in children and adolescents

Summary Nine normal children (6 males and 3 females) aged from 7 1/2 to 14 1/2 years underwent a 30-min arginine infusion (0.5 g/kg) followed at 90 min by one bolus i.v. glucagon injection (0.03 mg/kg). On a separate occasion the same children underwent an i.v. glucagononly test. No significant difference was found when the glucose and insulin responses in the two glucagon tests were compared, in contrast to previous findings that preloading with glucose resulted in a significantly increased response of insulin to glucagon. Established Investigator of the Chief Scientist’s Bureau Ministry of Health.