Glucagon-like Immunoreactive Material Research Articles

Plasma glicentin in diabetic and gastrectomized patients

Recent successful synthesis of human glicentin prompted us to establish an immunoassay method for determination of human glicentin in plasma. Human glicentin in plasma was measured using a newly developed sandwich ELISA. The mean fasting levels of human glicentin were 18.6±2.4 and 19.7±2.1 pM in normal subjects and diabetic patients, respectively. In diabetic patients with renal failure, plasma glicentin was elevated, exceeding 100 pM. In normal subjects, plasma glicentin increased to a peak level of about 130 pM at 60 min after an oral glucose load, and then decreased. In patients who underwent gastrectomy, plasma glicentin rapidly increased to a peak of about 300 pM at 30 min after oral glucose load. In a patient with short bowel syndrome plasma glicentin did not change following an oral glucose load. These results correspond with previous findings for gut glucagon-like immunoreactive materials (GLI) or enteroglucagon. We conclude that glicentin is secreted from the small intestine in response to intraluminal glucose stimulation in humans.

Regulatory peptides (glucagon, somatostatin, substance P, and VIP) in the brain and gastrointestinal tract of Ambystoma mexicanum

The concentrations of immunoreactive components of glucagon, somatostatin, substance P, and vasoactive intestinal polypeptide (VIP) in the brain, stomach, and gut of the neotenic Mexican axolotl ( Ambystoma mexicanum) were determined by radioimmunoassay using antibodies of defined regional specificity. The molecular forms of the immunoreactive components were analyzed by high-performance liquid chromatography (HPLC). The concentrations and molecular forms of somatostatin and VIP in axolotl brain were comparable to the concentrations in mammals but the substance P-like immunoreactivity was resolved by HPLC into components with the retention times of physalaemin and substance P together with their oxidized forms. No glucagon-like material was detected in the axolotl brain. The concentrations of substance P and VIP in the A. mexicanum digestive tract were appreciably lower than in the mammalian digestive tract and the VIP-like material did not coelute with porcine VIP. Somatostatin-14 represented the major molecular form in the axolotl stomach and gut. The distribution and molecular properties of the glucagon-like peptides in the axolotl digestive system were markedly different from these parameters in mammalian gut. Glucagon-like material is present only in low amounts in porcine and human stomach and, the concentration of enteroglucagon (N-GLI) in the gut is at least fiftyfold greater than pancreatic glucagon (C-GLI) concentrations. The axolotl stomach, in contrast, contains high levels of glucagon-like immunoreactive material and, in both stomach and gut, the levels of C-GLI and N-GLI were comparable. The glucagon-like material was heterogeneous on HPLC and was resolved into two major components but no component with the retention time of mammalian glucagon was present. The immunochemical properties of the axolotl glucagon-like peptides indicate that they possess strong homology with mammalian glucagon in the 10–18 and 25–29 regions of the molecule.

Inhibition of pancreatic exocrine secretion and augmentation of the release of gut glucagon-like immunoreactive materials by intraileal administration of bile in the dog.

The effect of intraileal instillation of bile, a stimulant of gut glucagon-like immunoreactive materials (gut GLI), on secretin-stimulated pancreatic secretion was examined in anesthetized dogs. Intraileal bile significantly inhibited the flow rate of secretin-stimulated pancreatic secretion. The inhibition of pancreatic secretion was accompanied by an elevation of plasma concentration of gut GLI. Taking the inhibitory effect of glucagon on pancreatic exocrine secretion into consideration, it could be reasonably postulated that gut GLI may be a mediator of bile-induced ileal inhibition of pancreatic exocrine function.

Effect of intraluminal bile or bile acids on release of gut glucagon-like immunoreactive materials in the dog.

The true biological role of gut glucagon-like immunoreactive materials (gut GLI) is still unknown, although the stimulatory effect of intraluminal nutrients on the secretion of gut GLI has been described. The present authors, using the canine intestinal loop prepared from the terminal portion of the ileum, investigated how gut GLI would respond to digestive juice or its components. When bladder bile collected from another dog and diluted to 10% in saline was instilled into canine ileal loop, gut GLI in a branch of regional mesenteric vein was elevated significantly. Cholic acid suspended in saline (0.25 g/50 ml) also stimulated gut GLI secretion in the similar pattern to that of bile administration. On the other hand, 154 mM NaHCO3 which is a major inorganic component of pancreatic juice did not affect the venous level of gut GLI.

Decrease in blood glucose and release of gut glucagon-like immunoreactive materials by bombesin infusion in the dog.

Synthetic bombesin (100 ng/min) was infused under pentobarbital anesthesia in normal dogs and in insulin deprived depancreatized dogs 4 days after surgery. The release of gut glucagon-like immunoreactive materials (gut GLI) calculated as the difference between the values measured using cross-reacting antiserum and a so-called pancreatic glucagon specific antiserum was markedly stimulated by bombesin infusion. Plasma glucagon immunoreactivity (GI) measured by pancreatic glucagon specific antiserum also showed a small increase, whereas plasma glucose decreased significantly with a transient rise in insulin. The plasma glucose level did not decrease in depancreatized dogs. Gut GLI response in the regional mesenteric vein to 5% glucose administered into the loop of the ileum was strongly augmented by bombesin infusion. It is concluded that (1) bombesin infusion decreased blood glucose level in normal dogs but not in depancreatized dogs. (2) Bombesin infusion markedly augmented the release of GLI from the intestine. (3) Bombesin also stimulated the release of glucagon which was probably of gastrointestinal origin. (4) Insulin release was stimulated transiently by bombesin infusion. Thus, a competition of gut GLI with glucagon at the glucagon receptor site may be an explanation of the reduction in blood glucose.

Regional distribution of glucagon-like immunoreactive material in the brain of rats and sand rats. An immunohistochemical investigation

Glucagon-like immunoreactivity was demonstrated in different parts of rat and sand rat central nervous system by the indirect immunofluorescence method. In hyperglycemic animals the level of immunoreactive material was reduced to a great extent.

Production of a specific antiserum by synthetic C-terminal fragment of glucagon.

Seven rabbits were immunized with a synthetic C-terminal glucagon fragment [15--29] conjugated with bovine serum albumin by means of glutaraldehyde. Antisera for glucagon were produced in all the animals after six injections of the conjugate. One of them revealed a higher titer antiserum (G42), which did not cross react with gut glucagon-like immunoreactive material, secretin, insulin, gastric inhibitory polypeptide or vasoactive intestinal peptide. From the results of inhibition of 125 I-glucagon in binding with the antiserum by various glucagon-related fragments the immunogenic determinant of the antiserum was proved to be in the C-terminal residue of the glucagon molecule, although peptide [17--29] or [21--29] reacted weakly with the antiserum. The plasma glucagon levels measured by antiserum G 42 during an arginine test in five normal subjects were superposed on those obtained by other antiserum (G21), specific for pancreatic glucagon. Furthermore, a comparable standard curve for glucagon was obtained using antiserum G42, when a labelled p-hydroxyphenylacetylated glucagon fragment [15--29] was employed as a tracer. The present study clearly demonstrated that the C-terminal glucagon fragment could yield a specific antiserum for pancreatic glucagon, supporting the proposal that the C-terminal fragment of glucagon is responsible for such specific antisera. Furthermore, it is concluded that immunoassay for glucagon could be performed using the labelled glucagon fragment as a tracer.

Anomeric Specificity in the Response of Gut Glucagon-like Immunoreactive Materials to Glucose

An anomeric specificity of the glucose sensors of A cells and B cells of the pancreas has been reported. In this context the present authors investigated, using the canine intestinal loop prepared from the terminal portion of the ileum, how glucagon-like immunoreactive materials (GLI) of the gut would respond to glucose anomers in an attempt to explore a possible anomeric specificity of glucose-stimulated gut GLI secretion. As a result GLI was found to be more readily released into the blood stream after an intestinal alpha-glucose load than following beta-gluocse during a 15-minute observation period. It is thus suggested that gut GLI-secreting cells have glucose sensors similar to those of pancreatic A or B cells which are specific for the alpha-glucose anomer.

Half life of gastrointestinal glucagon-like immunoreactive materials.

The composition, half life and hyperglycemic action of the porcine gastrointestinal glucagon-like immunoreactive materials were examined. Glucagon immunoreactivity (GI) measured using specific antiglucagon serum was more abundunt in the extract from the gastric fundus than in the one from the small intestine. When the extract from the gastric fundus was injected in dogs, the half life (T1/2) of total glucagon-like immunoreactivity (total GLI) measured using nonspecific antiglucagon serum was 9.5 +/- 1.1 min (mean +/- SEM), which was longer than that of crystalline pancreatic glucagon, 3.4 +/- 0.2 min, but shorter than that of the extract from the small intestine, 15.9 +/- 1.3 min. On the other hand, T1/2 for GI from the gastric fundus was 5.1 +/- 0.9 min, which was not significantly different from that of crystalline pancreatic glucagon. Blood sugar levels were significantly increased from the basal by 25 +/- 4 mg/100 ml at 10 min and 19 +/- 4 mg/100 ml at 15 min following an injection of the extract from the gastric fundus, but such a change in blood sugar levels was not demonstrated when the extract from the small intestine was injected. These results suggest that GI of the gastric fundus is close to pancreatic glucagon in respect of its metabolism and hyperglycemic activity.

Failure of somatostatin to decrease blood glucose by suppression of extrapancreatic glucagon in severely diabetic depancreatized dogs.

Effects of somatostatin on extrapancreatic glucagon secretion in totally depancreatized dogs were examined. Somatostatin infusion at a rate of 3 microgram/min showed a rapid decrease of total glucagon-like immunoreactive materials (total GLI) measured by nonspecific antiserum, AGS 10, and gut glucagon immunoreactivity (gut GI) measured by specific antiserum, AGS 18, in systemic blood. Gut GLI calculated as the difference between total GLI and GI did not decrease significantly within 30 min. No changes of blood glucose were noted. Significant decreases of all glucagon fractions were observed when the rate of somatostatin infusion was increased to 10 microgram/min and prolonged for 90 min, whereas again blood glucose did not change at all. It is concluded that somatostatin inhibits both gut GI and GLI secretion, although gut GLI remains in circulation longer than gut GI. Suppression of gut GI is not effective for the reduction of blood glucose once an extreme hyperglycemia is brought about by insulin deficiency.

Insulin releasing activity of gastrointestinal glucagon-like immunoreactive materials in perfused rat pancreas.

Insulin-releasing activity of porcine gastrointestinal glucagon-like immunoreactive materials purified by affinity chromatography was examined in the perfused rat pancreas. When glucose concentration of the perfusate was raised from 60 to 100 mg/dl, augmented insulin release was observed. The mean incremental area of immunoreactive insulin (sigma delta IRI) during the first 10 min thus observed was 19.07 +/- 3.76 ng/10 min. Pancreatic glucagon and the extract from the gastric fundus showed the enhancement of insulin release in this system when they were added to the perfusate at the rate of 100 ng/min for 5 min; delta IRI were 41.92 +/- 8.47 and 71.70 +/- 18.09 ng/10 min, respectively, which were significantly higher than that of 100 mg/dl of glucose alone. However, no significant difference in the insulinogenic activity was noticed between the extracts from the small intestine and the control. These results suggest that the extract from the gastric fundus has insulinogenic activity similar to that of pancreatic glucagon.

Elevated gut glucagon-like immunoreactive material in human and experimental diabetes and its suppression by somatostatin

Elevated gut glucagon-like immunoreactive material in human and experimental diabetes and its suppression by somatostatin

Persistent Pancreatic Glucagon but not Insulin Response to Arginine in Pancreatectomized Dogs

Effects of total pancreatectomy on plasma glucagon, insulin and glucose responses to arginine were determined in 5 dogs. Portal vein and femoral artery samples were obtained in response to an arginine infusion (10 g/30 min) prior to, 1 h, 1 day and 1 week after pancreatectomy. Glucagon was measured using pancreatic-specific antiserum 30K (Unger, Dallas). Before pancreatectomy arginine significantly increased portal vein glucagon from 373 plus or minus 36 to 595 plus or minus 31 pg/ml and femoral artery levels from 233 plus or minus 28 to 342 plus or minus 74 pg/ml. Portal vein and femoral artery insulin concentrations of 74 plus or minus 21 and 17 plus or minus 3 muU/ml increased significantly to 173 plus or minus 64 and 31 plus or minus 7 muU/ml. Glucose levels did not change. One h after pancreatectomy, portal vein glucagon decreased to 121 plus or minus 15 pg/ml but increased to 230 plus or minus 42 pg/ml after arginine. Elevated blood glucose and the necessity for insulin treatment established the adequacy of pancreatectomy. Furthermore portal vein insulin levels were undetectable and unresponsive to arginine or a combination of glucose, glucagon, and tolbutamide 1 week after pancreatectomy. One day after pancreatectomy arginine significantly increased portal vein glucagon from 343 plus or minus 42 to 776 plus or minus 152 pg/ml. One week after pancreatectomy basal glucagon values were 374 plus or minus 30 in the portal vein and 360 plus or minus 49 in the femoral artery and responded to 1226 plus or minus 641 and 825 plus or minus 270 pg/ml, respectively, with arginine. Chromatography of plasma from one pancreatectomized dog on Sephadex G-50 after arginine stimulation revealed that much of the material cross-reacting with antibody 30K was eluted from the column earlier than either 125I-insulin or 125I-glucagon. In contrast, peak glucagon activity in plasma obtained from a normal human given arginine eluted from the column between the peak of 125I-insulin and 125I-glucagon; glucagon added to human plasma also was recovered in this same area between the 125I-insulin and 125I-glucagon peaks. These results suggest that some of the material that reacted with 30K antibody and which increased after pancreatectomy in response to arginine has a molecular weight greater than pancreatic glucagon. At autopsy no pancreatic tissue could be identified. Thus, after pancreatectomy, validated by absent insulin responses, the glucagon response to arginine was normal or increased. Since arginine is not thought to increase intestinal glucagon-like immunoreactive material, the source and nature of the material measured as glucagon after pancreatectomy is unknown, but may be important to any understanding of plasma glucagon measurements.

Glucose, insulin, pancreatic glucagon and glucagon-like immunoreactive materials in the plasma of normal and diabetic children. Effect of the initial insulin treatment.

Pancreatic glucagon (PG) and other glucagon-like immunoreactive materials (GLI) were measured in the plasma of normal and of newly diagnosed untreated diabetic children, using an antiglucagon serum (AGS) highly specific for pancreatic glucagon (AGS 18) and an AGS which crossreacts with extracts of intestinal mucosa (AGS 10). Gut GLI was considered to be the difference between "total" GLI (AGS 10) and PG (AGS 18). Glucose and immunoreactive insulin (IRI) were also measured. PG, total GLI and gut GLI were significantly elevated in children with severe insulin insufficiency and were reduced to normal by insulin treatment, even though a significant fasting hyperglycemia was still present. In three diabetic children who had high initial plasma IRI levels the three glucagon fractions were normal. We conclude that insulin insufficiency is characterized not only by high plasma levels of PG as previously reported, but also of gut GLI. These abnormalities can be corrected by the administration of insulin.

Plasma glucose, insulin, pancreatic, and enteroglucagon levels in normal and depancreatized dogs.

Summary and ConclusionPancreatic glucagon (IRG), total glucagon-like immunoreactive material (GLI), enteroglucagon (calculated from GLI and IRG values), glucose, and insulin (IRI) were measured in the systemic plasma of normal and depancreatized dog. IRG, GLI, and enteroglucagon began to increase 1 or 2 days after pancreatectomy, respectively, reaching values that were several times greater than those observed before the operation. Glucose, already elevated 1 hr after pancreatectomy, continued to increase thereafter; IRI showed a progressive decline. In other experiments, 50 ml of a 5% glucose solution were injected into 30-cm loops prepared from the upper portion of the jejunum or the lower portion of the ileum and blood samples were obtained from the regional mesenteric veins. Plasma enteroglucagon rose more and plasma glucose less when glucose was introduced into the ileum than when it was introduced into the jejunum. The enteroglucagon responses to glucose were much greater in depancreatized than in n...

An investigation of glucagon biosynthesis in isolated pancreatic islets of guinea pigs

It is well established that glucagon is stored within the AZ -cells of the pancreatic islets, although up till now little is known about the cellular mechanisms involved in the biosynthesis of this hormone. The possibility that glucagon, like insulin, may be initially synthesized as a larger precursor has been considered on the basis of the recent demonstration of glucagonlike immunoreactive material of at least twice the molecular size of glucagon in pancreatic extracts of dog and several other species including man [ 11. In addition, recent reports have indicated the occurrence of a possible precursor molecule of glucagon in fish [2] and in pigeon islets [3], but there appears to date to be little information available about the mechanisms of glucagon biosynthesis and its regulation in mammalian islets. We report here investigations of the biosynthesis of glucagon in isolated guinea-pig islets of Langerhans during incubation in vitro for 17 hr or in tissue culture for up to 6 days. Tryptophan, labelled with tritium, was used as a marker for newly synthesized glucagon in the guinea-pig islets since this amino acid is present in the glucagon molecule [4] but not in the proinsulin [5] or insulin [6] molecule of any species studied so far. After incubation labelled proteins were extracted from the islets and analysed by gel filtration. At each time examined the extracts contained labelled proteins eluting in the void volume and in addition a protein of molecular weight about 18,000 which could not be dissociated by gel filtration in the presence of 8 M urea or 3 M acetic acid. The elution volume of this protein corresponded with that of a peak of glucagon-

Effect of dog jejunum "glucagon-like immunoreactive material" on adipose tissue metabolism.

Effect of dog jejunum "glucagon-like immunoreactive material" on adipose tissue metabolism.