Effects of histamine, of histidine and of anti-histaminic agents on the release of glucagon and insulin from the rat pancreas.

We have studied the effect of histamine (HA) and histidine (HIS) on the release of immunoreactive glucagon (IRG) and insulin (IRI) by isolated rat islets and pieces of pancreas. In isolated islets, HA and HIS stimulated IRG release at a glucose concentration of 3.0 mg/ml and IRI release at a glucose concentration of 0.5 mg/ml. In pieces of pancreas incubated in the presence of glucose (3.0 mg/ml), HA at a 2mM concentration stimulated IRG release and had no effect on the release of IRI; however, when the concentration of HA was doubled (4 mM), an inhibition of IRI release could also be demonstrated. HIS was ineffective. Perphenazine and dexchlorpheniramine, two anti-histaminic agents, inhibited IRG and stimulated IRI release. These results indicate that histamine, whether endogenous or exogenous, directly stimulated IRG and inhibits IRI release and suggest that some of the in vivo effects of histamine, such as hyperglycemia and lipid mobilization, may be mediated, at least in part, by these endocrinologic effects.     

Effects of serotonin, of its biosynthetic precursors and of the anti-serotonin agent metergoline on the release of glucagon and insulin from rat pancreas.

We have evaluated the effect of serotonin (5-HT) and of its biosynthetic precursors 5-Hydroxytryptophan (5-HTP) and tryptophan (TRP) on the release of immunoreactive glucagon (IRG) and insulin (IRI) from isolated islets and pieces of pancrease of the rat. In isolated islets, 5-HT inhibited the IRI response to a high glucose concentration (3.0 mg/ml), without affecting the IRG response to either a low (0.5 mg/ml) or a high glucose concentration; TRP stimulated the IRG and IRI response to the low glucose concentration, while 5-HTP was ineffective. When pieces of pancreas were used, 5-HT and 5-HTP inhibited IRG response to both glucose concentrations, while IRI release was inhibited only by 5-HT. The anti-5-HT agent metergoline enhanced the release of IRG and IRI by pieces of pancreas at both glucose concentrations. The results indicate that exogenous and endogenous 5-HT inhibit basal as well as glucose-mediated IRG and IRI release; that isolated islets are less sensitive than pieces of pancreas to the inhibitory effect of 5-HT and that TRP acts as an amino acid and not as a precursor of 5-HT.     

Insulin, glucagon and somatostatin localization in the pancreas of metamorphosed Xenopus laevis

The current study was designed to determine if insulin, glucagon and somatostatin-containing cells are present in the pancreas of adult Xenopus laevis. Localization methods utilized included cytochemical aldehyde fuchsin (AF) staining as well as the immunochemical peroxidase antiperoxidase (PAP) procedure for light microscopy. The results show numerous large clusters of AF-positive cells within a network of highly vascularized acinar tissue. PAP immunochemical localization with insulin antibody on adjacent sections demonstrates positive immunoreactivity to AF-positive cell groups and also the presence of immunoreactive insulin (IRI). Cells exhibiting this immunoreactivity are located in the central region of the islet-like structures. Serial sections not only show PAP immunoreactivity for IRI, but also for immunoreactive glucagon (IRG) and immunoreactive somatostatin (IRS) in the same islet-like structure. IRG and IRS-containing cells are situated around the periphery of the islet-like structures, surrounding the central core of IRI-containing cells. Antibody specificity was confirmed by homologous and heterologous antigen immuno-absorbance assays, as well as incubation of adjacent sections in preimmune sera. Based on this data we conclude that: the distribution of cells of the endocrine pancreas of metamorphosed Xenopus laevis is similar to that of many mammals and certain urodeles. Given the apparent specificity of the antigen-antibody reactions, it appears that Xenopus insulin, glucagon and somatostatin are structurally conserved.     

Response of plasma somatostatin-like immunoreactivity to the administration of alloxan in dogs.

The present study was designed to examine the effects of intravenously injected alloxan (75 mg/kg) upon plasma somatostatin-like immunoreactivity (SLI), glucagon (IRG), insulin (IRI) and glucose levels in 6 dogs. Within 2 hours of the injection of alloxan, SLI and IRI levels decreased significantly below their respective baselines, while IRG and plasma glucose concentrations increased. At 8 hours SLI levels had increased significantly by 55 pg/ml, together with a rise in IRI and a decrease in IRG and glucose concentrations. After 24 hours, marked hyperglycemia and hyperglucagonemia had developed whereas SLI levels were not different from preinjection values.     

Changes in plasma glucagon, pancreatic polypeptide and insulin during development of alloxan diabetes mellitus in dog

Changes in canine plasma glucose, immunoreactive glucagon (IRG), pancreatic polypeptide (PP) and insulin (IRI) were studied during the acute development of diabetes mellitus after iv alloxan injection. 100 mg or 75 mg/kg body weight of alloxan was injected iv and blood was taken successively till one or two days later. Plasma glucose showed four phases: first immediate and moderate decrease appeared 30 min after injection, second initial hyperglycemic phase, third hypoglycemic and fourth diabetic ones. Plasma IRI had already increased to 182 +/- 60 microU/ml 10 min after injection and again began to increase after about 6 h, peaking to 134 +/- 49 microU/ml at 18 h. Plasma IRG began increasing gradually soon after alloxan injection. The initial value was 196 +/- 26 pg/ml and it increased to 534 +/- 144 pg/ml at 4 h during the initial hyperglycemic phase, then reached a higher level through the hypoglycemic and diabetic phases. The change in plasma PP was similar to that in IRG. The initial value was 256 +/- 95 pg/ml at 12 h after injection, peaking to 840 +/- 100 pg/ml in the hypoglycemic phase. Similar blunted values were obtained following 75 mg/kg alloxan injection. Thus not only plasma IRI but also plasma IRG and PP varied greatly during the acute development of alloxan diabetes and some contribution of IRG to the initial hyperglycemic phase was suggested.     

Lack of effect of thyrotropin-releasing hormone (TRH) on the peripheral plasma levels of pancreatic glucagon in man

Based on the fact that human pancreas has thyrotropin-releasing hormone (TRH) immunoreactivity and bioactivity, we studied the effect of TRH on peripheral plasma levels of pancreatic glucagon (IRG) and insulin (IRI) in healthy subjects. During the infusion of 400 micrograms TRH for 120 min basal plasma IRI and IRG levels did not change significantly. In addition, intravenous infusion of 400 micrograms TRH did not affect the increments in the plasma IRG levels and the decrements in the blood glucose during insulin hypoglycemia.     

Important role of glucagon during exercise in diabetic dogs

To define the role of immunoreactive glucagon (IRG) during exercise in diabetes, 12 insulin-deprived alloxan-diabetic (A-D) dogs were run for 90 min (100 m/min, 12 degrees) with or without somatostatin (St 0.5 microgram . kg-1 . min-1). Compared with normal dogs, A-D dogs were characterized by similar hepatic glucose production (Ra), lower glucose metabolic clearance, and higher plasma glucose and free fatty acid levels during rest and exercise. In A-D dogs IRG was greater at rest and exhibited a threefold greater exercise increment than controls, whereas immunoreactive insulin (IRI) was reduced by 68% at rest but had similar values to controls during exercise. Basal norepinephrine, epinephrine, cortisol, and lactate levels were similar in normal and A-D dogs. However, exercise increments in norepinephrine, cortisol, and lactate were higher in A-D dogs. When St was infused during exercise in the A-D dogs, IRG was suppressed by 432 +/- 146 pg/ml below basal and far below the exercise response in A-D controls (delta = 645 +/- 153 pg/ml). IRI was reduced by 1.8 +/- 0.2 microU/ml with St. With IRG suppression the increase in Ra seen in exercising A-D controls (delta = 4.8 +/- 1.6 mg . kg-1 . min-1) was virtually abolished, and glycemia fell by 104 to 133 +/- 37 mg/dl. Owing to this decrease in glycemia, the increase in glucose disappearance was attenuated. Despite the large fall in glucose during IRG suppression, counterregulatory increases were not excessive compared with A-D controls. In fact, as glucose levels approached euglycemia, the increments in norepinephrine and cortisol were reduced to levels similar to those seen in normal exercising dogs. In conclusion, IRG suppression during exercise in A-D dogs almost completely obviated the increase in Ra, resulting in a large decrease in plasma glucose. Despite this large fall in glucose, there was no excess counterregulation, since glucose concentrations never reached the hypoglycemic range.     

Effect of phenformin on the response of plasma intestinal glucagon-like immunoreactivity (GLI) to oral glucose in gastrectomized subjects.

The effect of phenformin (DBI) on the plasma intestinal glucagon-like immunoreactivity (GLI) and pancreatic glucagon (IRG) responses to oral and intravenous glucose loads were studied in 26 gastrectomized subjects, using a cross-reacting and an IRG-specific anti-serum. The drug produced no significant changes in fasting GLI and IRG levels. Thirty minutes after oral glucose alone, the total GLI level rose to a peak of 1.55 +/- 0.17 ng/ml in the untreated subjects and to a maximum level of 1.67 +/- 0.18 ng/ml in the DBI-pretreated subjects. However, the mean GLI levels obtained 120 and 180 min after oral glucose were significantly higher after treatment with DBI. The blood sugar and IRI responses to oral glucose were lowered significantly by DBI pretreatment. DBI did not alter the glucose, IRI, IRG and GLI response to intravenous glucose. These results suggest that the release of intestinal GLI is not related to the intestinal absorption of glucose.     

Pharmacokinetics and effects of intravenous infusion of somatostatin in normal subjects--a two-compartment open model

A direct radioimmunoassay of plasma somatostatin-like immunoreactivity (SRIF-LI) was developed and validated. The sensitivity was 16.0 pg/ml, and the specificity was good. The recovery of plasma SRIF-LI was 98.8 +/- 6.3%. The Scatchard plot of the antiserum binding data revealed a straight line, with a binding affinity of 3.52 X 10(-12) M and a binding capacity 4.06 X 10(-10) M. Synthetic SRIF (Stilamin), 250 micrograms, was infused intravenously over a 30-min period in 9 healthy volunteers. Plasma glucose, insulin (IRI), glucagon (IRG) and SRIF-LI were measured. A two-compartment open model was adopted to analyze the pharmacokinetic data of SRIF-LI. The results showed that plasma SRIF-LI rose from 192.2 +/- 16.2 pg/ml to a plateau of 2,129.8 +/- 288.2 pg/ml within 5-10 min after starting the infusion. The half disappearance time from plasma (Ta1/2) was 1.36 +/- 0.18 min, the half disappearance time from the 'remote' compartment (Tb1/2) was 49.6 +/- 10.9 min and the net half disappearance time from the two compartments together (Tn1/2) was 9.19 +/- 1.49 min. The metabolic clearance rate was 50.3 +/- 7.0 ml/kg/min. The plasma IRI, IRG and the IRI/IRG molar ratio were all suppressed during the infusion period. The recovery time of plasma IRG was mildly delayed in comparison to that of IRI. This indicates that there are dissociations between IRI and IRG in the extent and the duration of suppression caused by somatostatin infusion.     

Glucagon and insulin secretion by dispersed islet cells: possible paracrine relationships

The ability of dispersed islet cells in a perifusion system to secret glucagon and insulin in response to physiologic stimuli was investigated. Normal hamster islets were isolated by collagenase digestion and the cells dispersed by sequential digestion with collagenase and trypsin. Following a 50-min period of equilibrium in buffer with high glucose concentrations (5.0 mg/ml), glucagon secretion was stimulated by glucopenia and subsequently, inhibited by increasing the concentration of glucose. The responsiveness to glucose inhibition was significantly less in dispersed islet cells than in intact islets. However, the dispersed islet cells showed significantly greater response to arginine. Glucagon secretion by dispersed islet cells was stimulated to tolbutamide and epinephrine but somatostatin had no effect. Dispersed islet cell preparations did not augment insulin secretion in response to glucose but did secrete more insulin in response to arginine. Intact islets secreted insulin in response to glucose but not arginine. We conclude that A cells in cell suspension do not need direct contact or an intact intra-islet environment in order to respond to glucose, arginine, epinephrine, or tolbutamide but the extent of response may be influenced by paracrine effects. However, paracrine relationships may be important in determining the response of B cells to secretagogues.     

Islet-activating protein (IAP)-induced adrenergic modulation of pancreatic A and B cell in dogs

Islet-activating protein (IAP) is a substance purified from the culture medium of Bordetella pertussis, and its main action is characterized by the enhancement of secretory response to glucose and other stimuli in pancreatic islet. In this experiment, the effect of IAP on epinephrine-induced secretion of immunoreactive insulin (IRI) and glucagon (IRG) was investigated in normal dogs. Epinephrine suppressed IRI secretion and it had a little increment to IRG secretion in control group, while IRI and IRG secretions were significantly increased by epinephrine in IAP pretreated group. Using beta-blocker (Propranolol) with epinephrine, these increments of IRI and IRG secretions in IAP pretreated group were abolished. However, using alpha-blocker (Phentolamine) with epinephrine, these secretions of IRI and IRG in IAP pretreated group were much more increased than epinephrine alone induced secretions. Blood glucose levels were lower in IAP pretreated group than in control group throughout the loading tests in all of the experiments. These findings suggest that (1) IAP decreases blood glucose level and (2) IAP enhances epinephrine-induced secretion of insulin and glucagon by acceleration of beta-adrenergic effect and by reduction of alpha-adrenergic suppression in dogs.     

The presence and actions of NPY in the canine endocrine pancreas

Immunofluorescent staining for neuropeptide Y (NPY) in canine pancreatic tissue was performed together with an evaluation of the effects of synthetic NPY on the release of insulin (IRI), glucagon (IRG) and somatostatin (SLI) from the duodenal lobe of the canine pancreas in situ. NPY-like immunoreactivity was localized in perivascular nerve fibers throughout the acinar tissue. NPY-immunoreactive fibers were also demonstrated in the islets, usually surrounding blood vessels but also occasionally in fibers associated with endocrine cells, primarily at the periphery of islets. In addition, the ganglia dispersed in the pancreatic parenchyma were densely innervated by NPY-immunoreactive fibers, and these ganglia regularly contained cell bodies staining for NPY. Direct infusion of NPY into the pancreatic artery (p.a.) produced a dose-dependent decrease of pancreatic SLI output and of pancreatic venous blood flow. Low-dose p.a. infusion of NPY (50 pmol/min) had no effect on basal IRI or IRG output or on the islet response to glucose (5-g bolus, i.v.). High-dose p.a. infusion of NPY (500 pmol/min) transiently stimulated IRI output and modestly increased IRG output. However, the comparatively sparse innervation of canine islets with NPY-like immunoreactive fibers and the relatively minor effects of large doses of synthetic NPY on pancreatic hormone release lead us to conclude that this peptide is not an important neuromodulator of islet function in the dog.     

Localization of insulin, glucagon and somatostatin in the pancreas of the adult newt, Notophthalmus viridescens

The current study is designed to demonstrate the presence of immunoreactive insulin (IRI), glucagon and somatostatin in the adult pancreas. Methods include aldehyde fuchsin (AF) staining and peroxidase anti-peroxidase (PAP) immunochemical localization for light microscopy as well as protein A gold (PAG) staining for scanning electron microscopy (SEM) in conjunction with backscattered electron imaging (BEI). Our results show the presence of large clusters of AF-positive cells within networks of highly vascularized pancreatic acinar tissue. PAP immunochemistry of pancreas serial sections exhibit positive immunoreactivity to the same AF-positive structure, thus demonstrating the presence of IRI. This immunoreactivity is found in a high percentage of cells in the islet-like structures. These cells tend to be centrally located within the cluster. Antibody specificity controls, including homologous antigen immunoabsorbance, as well as incubation of sections in normal guinea pig serum give negative immunoreactivity. Immunoreactive glucagon-containing cells and somatostatin-containing cells are distributed around the periphery of the central core of IRI-containing cells. SEM in conjunction with BEI confirm the presence of PAG within these cell clusters. We conclude that: (a) newt pancreatic IRI reacts in a specific manner with bovine antibody, suggesting a partial structural similarity to mammalian antigen; (b) IRI is localized within within pancreatic islet-like cell clusters and these IRI-containing cells form a central mass which is surrounded by glucagon and somatostatin-containing cells; this cellular distribution is similar to that found in many mammals. PAG conjugated insulin antibody is detectable by SEM in conjunction with BEI in islet cells of the newt pancreas.     

Regulation of insulin synthesis in an insulin-producing cell line (RINm5F): Long-term experiments

Summary The insulin-producing cell line RINm5F, has been used in short-term experiments to evaluate insulin secretion. We sought to maintain the responsiveness of these cells to stimuli for up to 2 days. We examined the course of new insulin synthesis over this period by measuring at intervals immunoreactive insulin (IRI) in two parts: IRI in the medium (M) and IRI extracted from the cells (C). Control cells were incubated in RPMI 1640/2.8 mM glucose/10% fetal bovine serum/200 μg/ml bacitracin (to prevent insulin degradation). The addition of dibutyryl cAMP 10 mM to the experimental dishes significantly increased total (M+C) IRI at 48 hr to 37% above the insulin content of the control dishes (p<0.01). Theophylline 10 mM increased total (M+C) IRI by 24% over control (p<0.05) after 24 hrs. Glucose, glyceraldehyde, leucine, arginine, glucagon and tolbutamide, other stimulants of insulin production, had no effect. Under the experimental conditions reported here, including the use of bacitracin, IRI synthesis can be studied for up to 48 hr. Portions of this study have been published in abstract form for the 47th Annual Meeting of the American Diabetes Association, Indianapolis, Indiana, 1987. Supported in part by the American Diabetic Association, Maryland Affiliate.     

Glucagon and insulin relationships in genetically diabetic (db/db) and in streptozotocin-induced diabetic mice.

An inappropriate molar ratio of circulating insulin to glucagon is frequently associated with the metabolic alterations accompanying diabetes mellitus. Plasma immunoreactive insulin (IRI) and immunoreactive glucagon (IRG) levels were determined and the IRG:IRI ratio calculated at various intervals in overt diabetes in genetically diabetic (db/db) and in streptozotocin-treated mice. Plasma IRI levels in genetic mutants are elevated at nine weeks of age, but are comparable to values found in lean littermates by 21 weeks. The presence of a prevailing hyperglucagonemia is established for the first time in the intact db/db mice. Streptozotocin diabetics are found to have characteristically low plasma IRI and high plasma IRG values. The hormonal imbalance present in these two experimental animal models is accentuated when the data are expressed as the IRG:IRI ratio, which is seen to increase with the progression of diabetes.     

Changes in plasma glucose, insulin (IRI), glucagon (IRG) and free fatty acids (FFA) following alanine loading in hyperthyroid patients

The responses of plasma glucose, insulin (IRI), glucagon (IRG) and free fatty acids (FFA) following alanine loading (0.1 g/kg) were observed in 9 control subjects and 7 hyperthyroid patients, before and after restoration of thyroid function to normal. Despite the persistence of impaired glucose response to alanine, the blunted IRI and IRG responses in the hyperthyroid state were improved with a significant reduction in fasting IRI and IRG after treatment. Markedly increased FFA following alanine loading in hyperthyroid patients was reduced after treatment, but the FFA concentration remained greater than in the control subjects. We tentatively conclude that the impaired alpha and beta-cell responses to alanine were temporarily induced by the direct and/or indirect effects of thyroid hormone excess.     

The effect of long-distance running on plasma immunoreactive glucagon levels

Twelve highly conditioned long-distance runners were studied to determine the effects of marathon (42 km) and 10,000 m running on plasma immunoreactive glucagon (IRG), serum immunoreactive insulin (IRI), and serum glucose (G) levels. Blood samples were drawn just prior to and immediately upon completion of the run. Marathon running resulted in no significant change in G, IRI, or IRG levels. After running 10,000 m, plasma IRG levels did not change significantly, while IRI and G increased significantly. In evaluating the pooled data from both runs, a significant inverse correlation was observed between delta G and delta IRG. This relationship between delta G and delta IRG suggests that glucagon plays a role in maintaining normal blood glucose levels during strenuous exercise.     

Effect of subcutaneous pancreatic tissue transplants on streptozotocin-induced diabetes in rats. II. Endocrine and metabolic functions.

The present study examines the effect of subcutaneous pancreatic tissue grafts (SPTG) on endocrine and metabolic functions in streptozotocin (STZ)-induced diabetic rats using radioimmunoassay and biochemical techniques. SPTG survived even after 15 weeks of transplantation and significantly improved the weight of STZ-diabetic rats over a 15-week period. Although blood glucose-, cholesterol-, and glycosylated-haemoglobin (GHb) levels were not significantly lower in STZ-diabetic rats treated with SPTG, the values of these biochemical parameters were lower than those in untreated diabetic rats. Plasma and pancreatic immunoreactive C-peptide (IRCP) levels did not improve after SPTG (IRCP expressed as mean +/- standard deviation were 0.22 +/- 0.07, 0.072 +/- 0.02 and 0.08 +/- 0.03 pg ml-1 in the plasma non-diabetic diabetic and treated rats respectively, while IRCP levels in the pancreas of the non-diabetic, diabetic and treated rats were 433.8 +/- 0.1, 22.9 +/- 0.01 and 10.4 +/- 0.01 pg mg tissue-1 respectively). SPTG, however, improved plasma immunoreactive insulin (IRI) levels in both plasma and pancreas. IRI values in plasma were 54.7 +/- 13.6, 18.0 +/- 5.0 and 22.1 +/- 4.3 microUI ml-1 in non-diabetic, diabetic and treated rats respectively and were 277.3 +/- 37.1, 14.7 +/- 1.8 and 30.3 +/- 15.9 microIU micrograms tissue-1 in the pancreas of non-diabetic, diabetic and treated rats respectively. There was improvement in immunoreactive glucagon (IRG) levels after SPTG. IRG values in the plasma of non-diabetic, diabetic and treated rats were 147.0 +/- 10.7, 408.0 +/- 76.5 and 247.7 +/- 3 pg ml-1 respectively whereas, IRG measured in the pancreas was 1642.25 +/- 424.23, 1899.0 +/- 290.4 and 1714.1 +/- 301.98 pg micrograms tissue-1 in non-diabetic, diabetic and treated rats, respectively. The pancreas:plasma ratio of pancreatic hormones was deranged in untreated diabetes but improved after SPTG. In conclusion, SPTG significantly improved the weight gain, pancreatic insulin content, plasma IRG and pancreas: plasma ratio of IRCP, IRI and IRG. It also reduced blood glucose-, cholesterol-, and glycosylated-hemoglobin levels in STZ-diabetic rats.     

The ontogeny of insulin secretion mechanisms

Insulin secretion from pieces of pancreas of rabbits aged 6 weeks or 1 day, or of 24-day foetuses was studied in vitro in response to glucose, glucagon and theophylline. Glucose did not stimulate insulin release from foetal pancreas but was effective postnatally. Glucagon in medium containing 3.0 mg glucose/ml stimulated insulin secretion equally at each stage of development. Theophylline in medium containing 0.6 or 3.0 mg glucose/ml stimulated insulin secretion from foetal pancreas but was effective on postnatal pancreas only in the presence of 3.0 mg glucose/ml. Glucose potentiated the action of theophylline on the foetal β cell and theophylline potentiated the action of glucose on the adult β cell.     

A case with glucagonoma syndrome--heterogeneity of glucagon and insulin

The heterogeneity of glucagon and insulin in plasma and tissue extracts from a 57-year-old female with glucagonoma syndrome with surgically and autopsy verified islet-cell tumors was studied by Bio-Gel P-10 filtration. The preoperative plasma immunoreactive glucagon (IRG) level was 20.2 ng/ml, and plasma glucagon-like immunoreactivity(GLI) 25.8 ng/ml. The column chromatography of the preoperative plasma revealed three or four IRG components and four GLI components. Among these, peak II, the large glucagon immunoreactivity (LGI) peak, considered a candidate for proglucagon, was prominent, along with peak III. The resected metastatic liver tumor contained an enormous amount of IRG and an appreciable amount of immunoreactive insulin (IRI), indicating that the elevated plasma IRG was mainly of tumor origin. The IRG pattern of the tumor tissue extract revealed a small quantity of IRG in peaks I and II, and a large amount in peak III; control pancreatic tissue extract manifested a similar elution pattern. The IRI elution pattern of the tumor tissue extract revealed two major IRI peaks which migrated close to the elution volume of cytochrome C and insulin, respectively. This is a quite different pattern from the control pancreatic tissue extract in which the RI peak was localized in the elution volume of the insulin. We conclude that the present metastatic liver tumor produced not only enormous amounts of glucagon but heterogeneous peptides which contained immunological insulin determinants within their.