Glucagon-like peptide-1, a new hormone of the entero-insular axis

Summary The post-translational processing of proglucagon in the small intestine gives rise to glucagon-like peptide-1 (PG 78–107 amide) which has profound effects on the endocrine pancreas, and in many species also on the stomach. Glucagon-like peptide-1 (PG 78–107 amide) is secreted in man in response to physiological stimuli e.g. a mixed meal. Glucagon-like peptide-1, in concentrations corresponding to those observed in response to meals, strongly stimulates insulin secretion, in all mammals studied, even more potently than the gastric inhibitory peptide. Thus, glucagon-like peptide-1 fulfills the classic criteria for being a hormone and is likely to be a new incretin. The glucagon inhibitory effect of glucagon-like peptide-1 (PG 78–107 amide) probably further potentiates the effect of glucagon-like peptide-1 on glucose metabolism and distinguished this peptide from other intestinal peptides which have been proposed as incretins. Glucagon-like peptide-1 also inhibits gastric acid secretion and gastric emptying in man. The latter delays nutrient entry to the intestine and thereby diminishes meal-induced glucose excursions. Elevated plasma concentrations of immunoreactive glucagon-like peptide-1 have been reported in Type 2 (noninsulin-dependent) diabetic patients, however, the consequences of the elevation are not yet known. However, elevated levels of glucagon-like peptide-1 in patients with increased gastric emptying rate (post-gastrectomy syndromes) may be responsible for the exaggerated insulin secretion seen in these patients.     

Glucagon-like peptide-1 stimulates insulin secretion by a Ca2+-independent mechanism in Zucker diabetic fatty rat islets of Langerhans

This study investigates the mechanisms responsible for glucagon-like peptide-1 (GLP-1)-induced insulin secretion in Zucker diabetic fatty (ZDF) rats and their lean control (ZLC) littermates. Glucose, and 100 nmol/L GLP-1 (7-37 hydroxide) in the presence of stimulatory glucose concentrations, induced insulin secretion in islets from ZLC animals. In contrast, ZDF islets hypersecreted insulin at low glucose (5 mmol/L) and were poorly responsive to 15 mmol/L glucose stimulation, but increased insulin secretion following exposure to GLP-1. The insulin secretory response to 100 nmol/L GLP-1 was reduced by 88% in ZLC islets exposed to exendin 9-39. The intracellular Ca2+ concentration ([Ca2+]i) increased in fura-2-loaded ZLC islets following stimulation with 12 mmol/L glucose alone or GLP-1 in the presence of 12 mmol/L glucose. The increases in [Ca2+]i and insulin secretion in ZLC islets induced by GLP-1 were attenuated by 1 micromol/L nitrendipine. In contrast, neither glucose nor GLP-1 substantially increased [Ca2+]i in ZDF islets. Furthermore, insulin secretory responses to GLP-1 were not significantly inhibited in ZDF islets by nitrendipine. However, the insulin secretory response to GLP-1 in both ZLC and ZDF islets was ablated by cholera toxin. Our findings indicate that in ZLC islets, GLP-1 induces insulin secretion by a mechanism that depends on Ca2+ influx through voltage-dependent Ca2+ channels, whereas in ZDF islets, the action of GLP-1 is mediated by Ca2+-independent signaling pathways.     

Effects of glucagon-like peptide-1 (7-36)amide on insulin stimulated rat skeletal muscle glucose transport

Glucagon-like peptide-1 binding sites have been reported in peripheral tissues including muscle. However, the potential extra-pancreatic effects of glucagon-like peptide-1(7-36)amide are controversial. To evaluate whether glucagon-like peptide-1(7-36)amide has any effects on skeletal muscle glucose transport, isolated rat soleus muscles were incubated in increasing concentrations of insulin (0–150 nmol/l) in the presence or absence of 1 nmol/l glucagon-like peptide-1(7-36)amide for 3 h. Subsequently glucose transport was measured as uptake of [³H]-O-methylglucose. It was found that glucagon-like peptide-(7-36)amide has a small but significant stimulating effect on skeletal muscle glucose transport independent of the insulin concentration (P<0.01). However, because of the magnitude of the observed effect, the physiological importance of glucagon-like peptide-1 (7-36)amide on skeletal muscle glucose metabolism is questionable. Received: 27 February 1998 / Accepted in revised form: 30 April 1998     

Effect of gastrin-releasing peptide on the secretion of mouse islet hormones in vitro

Collagenase-isolated mouse islets were incubated with gastrin-releasing peptide (GRP). At 5.6 mmol glucose/l. 10 nmol GRP/l increased the release of insulin (by 50%) and glucagon (by twofold), decreased the release of pancreatic polypeptide (by 35%), but did not significantly affect the release of somatostatin. At 16.7 mmol glucose/l, 10 nmol GRP/l increased glucagon release (by fivefold) and decreased pancreatic polypeptide release (by 46%), without significantly altering insulin and somatostatin release. GRP (200 nmol/l) did not affect insulin release by perifused mouse islets at 2.8 mmol glucose/l, but increased both first and second phase insulin release after a square wave increase in the glucose concentration to 11.1 mmol/l. At 5.6 mmol glucose/l, GRP (100 pmol/1-100 nmol/l) increased (by 50-70%) insulin release by the RINm5F clonal cell line. GRP did not affect glucose oxidation or the cyclic adenosine monophosphate content of RINm5F cells. However, the intracellular free Ca2+ concentration of RINm5F cells was rapidly and transiently increased by GRP (maximum increase of 64% about 10 s after exposure to 1 mumol GRP/l). The rise of intracellular free Ca2+ was approximately halved in the absence of extracellular Ca2+. The results suggest that GRP may contribute to the normal regulation of the endocrine pancreas. The insulin-releasing effect of GRP is mediated via increased cytosolic free Ca2+, derived both from an increased net influx of extracellular Ca2+ and from mobilization of intracellular Ca2+ stores.     

Evidence that glucagon stimulates insulin secretion through its own receptor in rats

Summary Since glucagon-like peptide-1 (7–36) amide (7–37) (GLP-1) has been found to be a potent insulinotropic hormone, it has been postulated that glucagon stimulates insulin secretion from islet beta cells through the GLP-1 receptor. We therefore examined the effects of a GLP-1 receptor antagonist, exendin (9–39) amide, on glucagon- or GLP-1-stimulated insulin release from isolated perfused rat pancreas. When infusion of 100 nmol/l exendin (9–39) amide was started 5 min before that of 1 nmol/l glucagon, the stimulation of insulin release by glucagon was similar to that found in the control situation (preinfusion with vehicle alone). By contrast, when 0.3 nmol/l GLP-1 was used in the same experimental setting, exendin (9–39) amide clearly inhibited insulin release. These results indicate that glucagon stimulates insulin release mainly through glucagon receptors but not GLP-1 receptors on islet beta cells.Abbreviations GLP-1 Glucagon-like peptide-1 - BSA bovine serum albumin - IRI immunoreactive insulin     

Glucagon-like peptide-1 treatment delays the onset of diabetes in 8 week-old db/db mice

AIMS/HYPOTHESIS: Glucagon-like peptide-1 ameliorates the symptoms of diabetes through stimulation of insulin secretion and enhancement of beta-cell mass. We have therefore investigated the effects of glucagon-like peptide-1 on the development of diabetes, using db/db mice as a model of Type II diabetes. METHODS: The potent glucagon-like peptide-1 analogue Exendin-4 or vehicle (control) was administered (i.p.; 1 nmol/kg) to obese 6-week old db/db mice daily for 14 days ( n=10). RESULTS: By 8 weeks of age, control db/db mice developed hyperglycaemia (fasting: 10.4+/-0.5 mmol/l), hyperinsulinaemia and impaired glucose tolerance. However, Exendin-4 treatment prevented hyperglycaemia (fasting: 6.1+/-1.0 mmol/l, p<0.01), with reduced plasma insulin concentrations ( p<0.001) and improved glucose tolerance ( p<0.05). Peripheral insulin sensitivity was not affected. However, insulin release in vivo and in vitro from the perfused pancreas was improved by Exendin-4, as were pancreatic insulin concentrations (0.54+/-0.02 vs 0.32+/-0.01 micro g/mg protein, p<0.05). These changes occurred in conjunction with increased beta-cell mass (3.01+/-0.31 vs 2.22+/-0.22 mg, p<0.05) and proliferation (BrdU(+) beta-cells: 1.08+/-0.20 vs 0.47+/-0.11%, p<0.05), as well as decreased apoptosis (Tunel (+) beta-cells: 0.37+/-0.06 vs 1.20+/-0.21%). Western blot demonstrated increased expression of Akt1 (by fivefold, p<0.01) and p44 MAP kinase (by sixfold, p<0.01), and decreased activation of caspase-3 (by 30%, p<0.05). CONCLUSION/INTERPRETATION: Our results suggest that Ex4 treatment delays the onset of diabetes in 6-8 week old db/db mice, through a mechanism involving Akt1 and expansion of the functional beta-cell mass.     

Glucagon-like peptide-1 does not have a role in hepatic carbohydrate metabolism

Summary Glucagon-like peptide-1 does not have specific, high-affinity receptors on rat liver membranes, does not displace glucagon from glucagon receptors on these membranes and does not stimulate the production of cyclic AMP by isolated rat hepatocytes. In the presence of glucagon, high concentrations of glucagon-like peptide-1 do not significantly alter the production of cyclic AMP. Thus, glucagon-like peptide-1 appears unlikely to have a direct action on hepatic carbohydrate metabolism.     

Glucagon-like peptide-1 stimulates insulin secretion by a Ca-independent mechanism in Zucker diabetic fatty rat islets of Langerhans

This study investigates the mechanisms responsible for glucagon-like peptide-1 (GLP-1)-induced insulin secretion in Zucker diabetic fatty (ZDF) rats and their lean control (ZLC) littermates. Glucose, and 100 nmol/L GLP-1 (7-37 hydroxide) in the presence of stimulatory glucose concentrations, induced insulin secretion in islets from ZLC animals. In contrast, ZDF islets hypersecreted insulin at low glucose (5 mmol/L) and were poorly responsive to 15 mmol/L glucose stimulation, but increased insulin secretion following exposure to GLP-1. The insulin secretory response to 100 nmol/L GLP-1 was reduced by 88% in ZLC islets exposed to exendin 9-39. The intracellular Ca²⁺ concentration ([Ca²⁺]_i) increased in fura-2-loaded ZLC islets following stimulation with 12 mmol/L glucose alone or GLP-1 in the presence of 12 mmol/L glucose. The increases in [Ca²⁺]_i and insulin secretion in ZLC islets induced by GLP-1 were attenuated by 1 μmol/L nitrendipine. In contrast, neither glucose nor GLP-1 substantially increased [Ca²⁺]_i in ZDF islets. Furthermore, insulin secretory responses to GLP-1 were not significantly inhibited in ZDF islets by nitrendipine. However, the insulin secretory response to GLP-1 in both ZLC and ZDF islets was ablated by cholera toxin. Our findings indicate that in ZLC islets, GLP-1 induces insulin secretion by a mechanism that depends on Ca²⁺ influx through voltage-dependent Ca²⁺ channels, whereas in ZDF islets, the action of GLP-1 is mediated by Ca²⁺-independent signaling pathways.     

Effects of gastric inhibitory polypeptide, vasoactive intestinal polypeptide and peptide histidine isoleucine on the secretion of hormones by isolated mouse pancreatic islets

The release of insulin, glucagon, somatostatin and pancreatic polypeptide (PP) by isolated mouse pancreatic islets was determined during 30-min incubations at 5.6 and 16.7 mmol glucose/l in the absence and presence of gastric inhibitory polypeptide (GIP), vasoactive intestinal polypeptide (VIP) and peptide histidine isoleucine (PHI) at concentrations of 1-1000 nmol/l. Insulin release was enhanced (greater than 50%) by GIP (100-1000 nmol/l) and VIP (1 mumol/l) at 5.6 mmol glucose/l, but not at 16.7 mmol glucose/l. Glucagon release was increased by GIP (100-1000 nmol/l), and by VIP and PHI (1-1000 nmol/l) at both glucose concentrations in a dose-related manner (maximum increases greater than tenfold). Somatostatin release was similarly increased by GIP (10-1000 nmol/l) at both glucose concentrations. Only the highest concentration (1 mumol/l) of PHI tested increased somatostatin release (twofold) at 5.6 mmol glucose/l, whereas PHI and VIP (1-1000 nmol/l) reduced (greater than 37%) somatostatin release at 16.7 mmol glucose/l. PP release was increased (49-58%) by 100-1000 nmol GIP/l, but was not significantly altered by VIP, and was reduced (39-56%) by PHI. The results indicate that GIP, VIP and PHI each stimulate glucagon release in a dose-related manner, but they exert discretely different effects on other islet hormones depending upon the dose and the prevailing glucose concentration.     

How glucagon-like is glucagon-like peptide-1?

Summary Although glucagon-like peptide-1 has the appearance of a glucagon-homologue that may be co-secreted with glucagon, synthetic glucagon-like peptide-1-(1–37) does not significantly affect plasma glucose and insulin concentrations when administered at high doses (100 and 400 g) to cortisone-pretreated rabbits. This synthetic preparation thus lacks the primary metabolic effect of glucagon at the doses tested. An intra- or extra-pancreatic role of glucagon-like peptide-1 has yet to be discovered.     

Once daily injection of exendin-4 to diabetic mice achieves long-term beneficial effects on blood glucose concentrations

Summary Glucagon-like peptide-1 is the main hormonal mediator of the enteroinsular axis. Recently, it has additionally received considerable attention as a possible new treatment for Type II (non-insulin-dependent) diabetes mellitus. Its major disadvantage is that its duration of action is too short to achieve good 24-h metabolic control. Exendin-4, which is produced in the salivary glands of Gila monster lizards, is structurally similar to glucagon-like peptide-1 and shares several useful biological properties with glucagon-like peptide-1. It binds the glucagon-like peptide-1 receptor, stimulates insulin release and increases the cAMP production in beta cells. We report that exendin-4 is a more potent insulinotropic agent when given intravenously to rats than is glucagon-like peptide-1 (ED₅₀ 0.19 nmol/kg for glucagon-like peptide-1 vs 0.0143 nmol/kg for exendin-4) and causes a greater elevation in cAMP concentrations in isolated islets. Of even greater interest we found that when given intraperitoneally only once daily to diabetic mice it had a prolonged effect of lowering blood glucose. After 1 week of treatment blood glucoses were 5.0 ± 2.6 mmol/l compared to diabetic concentrations of 13.2 ± 2.8 mmol/l. After 13 weeks of daily treatment HbA_{1 c} was 8.8 ± 0.4 % in non-treated diabetic animals compared with 4.7 ± 0.25 % in treated diabetic animals. Blood glucoses also were lower (p < 0.005) and insulin concentrations higher (p < 0.02) in the treated animals. Exendin-4 could therefore be preferable to glucagon-like peptide-1 as a long-term treatment of Type II diabetes. [Diabetologia (1999) 42: 45–50] Received: 30 March 1998 and in final revised form: 5 October 1998     

The Rap–B-Raf signalling pathway is activated by glucose and glucagon-like peptide-1 in human islet cells

Aims/hypothesis Glucose and glucagon-like peptide-1 have been shown to activate extracellular signal-regulated kinase (ERK) and phosphoinositide 3-kinase in beta cells. We examined the contributions of the small GTPases Rap and Ras and the serine–threonine kinases B-Raf and Raf-1 to the activation of these kinases in human islet cells.Methods The expression of Rap, Ras, B-Raf and Raf-1 in human islets was examined by immunohistochemistry and immunoblotting. Human islets were incubated in glucose at concentrations of 2.5 and 15 mmol/l and were stimulated with 10 nmol/l glucagon-like peptide-1. The activation of ERK and Raf kinases was examined by phosphorylation-specific antibodies and immuno-complexed kinase assays. The activation of Rap and Ras was determined by pull-down assays. Stimulation of phosphoinositide 3-kinase was detected by immuno-complexed lipid kinase assays.Results Extracellular-regulated kinase and protein kinase B (a downstream target of phosphoinositide 3-kinase) were activated in islets stimulated with glucose and glucagon-like peptide-1. In these islets, the Rap–B-Raf signalling pathway was activated preferentially compared with Ras and Raf-1, and activated Rap and B-Raf mediated ERK stimulation in kinase assays in vitro. In addition, Rap rather than Ras mediated activation of phosphoinositide 3-kinase in islets stimulated with glucose and glucagon-like peptide-1.Conclusions/interpretation In human islet cells, glucose and glucagon-like peptide-1 activate the Rap and B-Raf signalling module, which mediates ERK activation in assays in vitro. Rap also activates phosphoinositide 3-kinase, delineating central roles for Rap and B-Raf as therapeutic targets for beta cell growth in diabetes mellitus.J. Trümper and D. Ross contributed equally to this article     

Interaction of glucagon-like peptide-1(7-36) amide and gastric inhibitory polypeptide or cholecystokinin on insulin and glucagon secretion from the isolated perfused rat pancreas.

The interaction of three incretin candidates, glucagon-like peptide-1(7-36)amide (t-GLP-1), gastric inhibitory polypeptide (GIP), and sulfated COOH-terminal octapeptide of cholecystokinin (CCK-8-S), on insulin and glucagon release from the isolated perfused rat pancreas was studied. Under the perfusate condition of 8.3 mmol/L glucose, coinfusion of 0.1 nmol/L t-GLP-1 and 0.1 nmol/L GIP resulted in an augmented insulin release greater than that obtained by the same dose of each peptide alone. The degree of stimulation elicited by t-GLP-1 and GIP reached a plateau at 0.3 nmol/L for both infusates, and no cooperative effect was observed by coinfusion at 0.3 nmol/L. Coinfusion of 0.1 nmol/L t-GLP-1 and and 0.1 nmol/L CCK-8-S also resulted in an augmented insulin release greater than that obtained by the same dose of each peptide alone. A similar cooperative effect was observed by coinfusion at 0.3 nmol/L, 1 nmol/L, and 3 nmol/L. With the same perfusion experiments, glucagon release was not significantly affected by any peptide at concentrations of 0.1, 0.3, 1, or 3 nmol/L. The coinfusion of 1 nmol/L t-GLP-1 and GIP elicited a transient, but significant, increase in glucagon release. A similar result was obtained by the coinfusion of 0.3 nmol/L and 3 nmol/L t-GLP-1 and GIP, respectively. The coinfusion of t-GLP-1 and CCK-8-S did not affect the glucagon release.(ABSTRACT TRUNCATED AT 250 WORDS)     

Insulin secretion defects in liver cirrhosis can be reversed by glucagon-like peptide-1

Liver cirrhosis is often accompanied by a disturbed carbohydrate metabolism similar to type 2 diabetes. To investigate the severity of the defect in insulin secretion in this form of diabetes, we measured insulin release from isolated pancreatic islets of rats with CCl(4)-phenobarbital-induced liver cirrhosis. Cirrhosis was confirmed by clinical signs, elevated liver enzymes and histology. Fasting venous plasma glucose concentrations were equal in rats with liver cirrhosis and in controls. Plasma insulin and glucagon concentrations were significantly greater (P<0.01) in cirrhotic rats than in control animals. Glucose (16.7 mM)-induced stimulation of insulin release from pancreatic islets revealed a twofold increase in control and cirrhotic rats. Basal and stimulated insulin secretion, however, were significantly lower in cirrhotic animals. The incretin hormone, glucagon-like peptide-1 (GLP-1), has therapeutic potential for the treatment of type 2 diabetes. Therefore, islets from control and cirrhotic animals were incubated with GLP-1 in concentrations from 10(-)(11) to 10(-)(6) M. GLP-1 stimulated insulin release in a concentration-dependent manner. In islets from cirrhotic rats, basal and stimulated insulin secretion was blunted compared with controls. These data show that the hyperinsulinemia observed in liver cirrhosis is not due to an increase of insulin secretion from islets, but could be explained by decreased hepatic clearance of insulin. GLP-1 may ameliorate diabetes in patients with liver cirrhosis.     

Glucose induces glucagon release pulses antisynchronous with insulin and sensitive to purinoceptor inhibition

Both increase of the glucose concentration and activation of purinoceptors are known to affect pancreatic alpha-cells. Effects obtained with various purino derivatives at 2.8 and 8.3 mmol/liter glucose have been taken to indicate that external ATP is less potent than adenosine as a stimulator of glucagon release. However, when making a corresponding comparison at 20 mmol/liter glucose, we observed marked stimulation of glucagon release from isolated rat islets with 100 micromol/liter adenosine-5-O-2-thiodiphosphate but inhibition with 10 micromol/liter adenosine. Analyses of 30-sec samples of perfusate from rat pancreas indicated that a rise of the glucose concentration from 3 to 20 mmol/liter rapidly induces a glucagon peak followed by regular 4- to 5-min pulses. The glucagon pulses preceded those of insulin with a phase shift (1.8 +/- 0.1 min) near half the interpeak interval. Because of the antisynchrony, the maximal glucagon effect on liver cells will be manifested during periods with low concentrations of insulin. In support for the idea that neural P2Y(1) receptors are important for coordinating the secretory activity of the islets, both the insulin and glucagon pulses disappeared in the presence of the purinoceptor inhibitor MRS 2179 (10 micromol/liter). However, in contrast to what was observed for insulin, MRS 2179 lowered average glucagon release to the level of the oscillatory nadirs.     

Glucagon-like peptide-1(7-37)/(7-36)amide is a new incretin.

Glucagon-like peptide-1 (GLP-1) is the main product of the intestinal processing of proglucagon. It is released from the intestinal K-cells into the circulation in response to the oral ingestion of food. At the pancreatic beta cell GLP-1 is a potent insulin secretagogue in the presence of elevated glucose levels, defining glucagon-like peptide-1 as a new incretin. Its action is mediated by specific receptors coupled to the adenylate cyclase system by a stimulatory G-protein. Finally, glucagon-like peptide-1 stimulates proinsulin gene expression and it is thus involved at several levels in the regulation of insulin synthesis and secretion.     

Chronic exposure to glibenclamide impairs insulin secretion in isolated rat pancreatic islets

We investigated the effect of 24 h exposure to 100 nmol/l glibenclamide on insulin secretion in isolated rat pancreatic islets. The insulin content was similar in control islets and in islets preincubated with 100 nmol/l glibenclamide for 24 h. In islets preexposed to glibenclamide: 1) the subsequent response to a maximal glibenclamide stimulatory concentration (10 mumol/l, 1 h at 37 C) was greatly reduced in comparison to control islets (0.69 +/- 0.20% vs 2.16 +/- 0.41%; mean +/- SE; n = 14; p less than 0.001); 2) the response to 100 mumol/l tolbutamide stimulation was also reduced (0.55 +/- 0.15% vs 2.38 +/- 0.44%; n = 8; p less than 0.001); 3) the response to 16.7 mmo/l glucose, both in the presence or in the absence of 1 mmol/l IBMX, a phosphodiesterase inhibitor, was also diminished by about 50% (1.79 +/- 0.39% vs. 3.22 +/- 0.42%; n = 14, p less than 0.001). In glibenclamide pretreated islets, blunted responses to stimuli were confirmed also by dynamic studies using a perifusion system. The effect of glibenclamide preincubation was fully reversible: when islets cultured in the presence of glibenclamide were transferred to a glibenclamide-free medium for further 24 h, insulin release in response to glibenclamide stimulation returned to control values. We conclude that prolonged exposure of rat pancreatic islets to glibenclamide induces a reversible desensitization to a variety of metabolic stimuli. The inhibition by prolonged glibenclamide exposure of a common pathway in the mechanism of insulin release is one possible explanation for these results.