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To date, type 2 diabetes is considered to be a "bi-hormonal disorder" rather than an "insulin-centric disorder," suggesting that glucagon is as important as insulin. Although glucagon increases hepatic glucose production and blood glucose levels, paradoxical glucagon hypersecretion is observed in diabetes. Recently, insulin resistance in pancreatic α cells has been proposed to be associated with glucagon dysregulation. Moreover, cell autonomous dysfunction of α cells is involved in the etiology of diabetes. In this review, we summarize the current knowledge about the physiological and pathological roles of glucagon.

In this work, we demonstrate the potential use of SPRi for secretion-monitoring of pancreatic islets, small micro-organs that regulate glucose homeostasis in the body. In the islets, somatostatin works as a paracrine inhibitor of insulin and glucagon secretion. However, this inhibitory effect is lost in diabetic individuals and little is known about its contribution to the pathology of diabetes. Thus, the simultaneous detection of insulin, glucagon and somatostatin could help understand such communications. Previously, the authors introduced an SPRi biosensor to simultaneously monitor insulin, glucagon and somatostatin using an indirect competitive immunoassay. However, our sensor achieved a relatively high LOD for somatostatin detection (246 nM), the smallest of the three hormones. For this reason, the present work aimed to improve the performance of our SPRi biosensor using gold nanoparticles (GNPs) as a means of ensuring somatostatin detection from a small group of islets. Although GNP amplification is frequently reported in the literature for individual detection of analytes using SPR, studies regarding the optimal strategy in a multiplexed SPR setup are missing. Therefore, with the aim of finding the optimal GNP amplification strategies for multiplex sensing we compared three architectures: (1) GNPs immobilized on the sensor surface, (2) GNPs conjugated with primary antibodies (GNP-Ab1) and (3) GNPs conjugated with a secondary antibody (GNP-Ab2). Among these strategies an immunoassay using GNP-Ab2 conjugates was able to achieve multiplex detection of the three hormones without cross-reactivity and with 9-fold LOD improvement for insulin, 10-fold for glucagon and 200-fold for somatostatin when compared to the SPRi biosensor without GNPs. The present work denotes the first report of the simultaneous detection of such hormones with a sensitivity level comparable to ELISA assays, particularly for somatostatin.

Diabetes arises from secretory defects in vascularized micro-organs known as the islets of Langerhans. Recent studies indicated that furthering our understanding of the paracrine effect of somatostatin on glucose-induced insulin secretion could represent a novel therapeutic avenue for diabetes. While many research groups are interested in insulin and glucagon secretion, few are particularly focused on studying the paracrine interaction in islets' cells, and none on monitoring a secretory fingerprint that contemplates more than two hormones. Surface plasmon resonance imaging can achieve high-throughput and multiplexed biomolecule quantification, making it an ideal candidate for detection of multiple islet's secretion products if arrays of hormones can be properly implemented on the sensing surface. In this study, we introduced a multiplex surface plasmon resonance imaging-based biosensor for simultaneous quantification of insulin, glucagon, and somatostatin. Performing this multiplex biosensing of hormones was mainly the result of the design of an antifouling sensing surface comprised by a mixed self-assembly monolayer of CH 3 O-PEG-SH and 16-mercaptohexadecanoic acid, which allowed it to operate in a complex matrix such as an islet secretome. The limit of detection in multiplex mode was 1 nM for insulin, 4 nM for glucagon, and 246 nM for somatostatin with a total analysis time of 21 min per point, making our approach the first reporting a label-free and multiplex measurement of such a combination of human hormones. This biosensor holds the promise of providing us with a mean for the further understanding of the paracrine effect of somatostatin on glucose-induced insulin secretion and consequently help develop novel therapeutic agents for diabetes.

Glucagon-like peptide-1 (GLP-1) receptor (GLP-1R), glucagon (GCG) receptor (GCGR), and glucose-dependent insulinotropic polypeptide (GIP, also known as gastric inhibitory polypeptide) receptor (GIPR), are three metabolically related peptide hormone receptors. A novel approach to the generation of multifunctional antibody agonists that activate these receptors has been developed. Native or engineered peptide agonists for GLP-1R, GCGR, and GIPR were fused to the N-terminus of the heavy chain or light chain of an antibody, either alone or in pairwise combinations. The fusion proteins have similar in vitro biological activities on the cognate receptors as the corresponding peptides, but circa 100-fold longer plasma half-lives. The GLP-1R mono agonist and GLP-1R/GCGR dual agonist antibodies both exhibit potent effects on glucose control and body weight reduction in mice, with the dual agonist antibody showing enhanced activity in the latter., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Aims/introduction: The aims of the present study were to investigate the performance of a novel sandwich enzyme-linked immunosorbent assay (ELISA) for measuring glucagon (1-29) with monoclonal antibodies against both the C- and N-terminal regions of glucagon (1-29), and to analyze the differences in plasma levels and responses of glucagon (1-29) to oral glucose loading in normal glucose tolerance (NGT) subjects and patients with type 2 diabetes mellitus., Materials and Methods: The cross-reactivity against proglucagon fragments using the ELISA kit and two types of conventional radioimmunoassay (RIA) kits was evaluated. A 75-g oral glucose tolerance test was carried out with NGT subjects and patients with type 2 diabetes mellitus, and the glucagon (1-29) concentration was measured using three types of kit., Results: The ELISA kit clearly had the lowest cross-reactivity against miniglucagon (19-29) and glicentin (1-61). The oral glucose tolerance test was carried out with 30 NGT and 17 patients with type 2 diabetes mellitus. The glucagon (1-29) levels measured by the ELISA kit after glucose loading were significantly higher at all time-points in the type 2 diabetes mellitus group than in the NGT group. However, the glucagon (1-29) levels measured by one RIA kit were significantly higher in the NGT group, and those measured with the other RIA kit were approximately the same among the groups., Conclusions: The novel sandwich ELISA accurately determines plasma glucagon (1-29) concentrations with much less cross-reactivity against other proglucagon fragments than conventional RIA kits.

Background: Proglucagon-derived peptides (PGDPs), which include glucagon-like peptide (GLP)-1, glucagon, and oxyntomodulin, are key regulators of glucose homeostasis and satiety. These peptide hormones are typically measured with immuno-based assays (e.g., ELISA, RIA), which often suffer from issues of selectivity., Methods: We developed a multiplexed assay for measuring PGDPs including GLP-1 (7-36) amide, GLP-1 (9-36) amide, glucagon, and oxyntomodulin by mass spectrometry and used this assay to examine the effect of a meal tolerance test on circulating concentrations of these hormones. Participants fasted overnight and were either given a meal (n = 8) or continued to fast (n = 4), with multiple blood collections over the course of 3 h. Plasma samples were analyzed by microflow immunoaffinity (IA)-LC-MS/MS with an isotope dilution strategy., Results: Assay performance characteristics were examined and established during analytical validation for all peptides. Intra- and interassay imprecision were found to be 2.2%-10.7% and 6.8%-22.5%, respectively. Spike recovery was >76%, and dilution linearity was established up to a 16-fold dilution. Immediately after the meal tolerance test, GLP-1 and oxyntomodulin concentrations increased and had an almost identical temporal relationship, and glucagon concentrations increased with a slight delay., Conclusions: IA-LC-MS/MS was used for the simultaneous and selective measurement of PGDPs. This work includes the first indication of the physiological concentrations and modulation of oxyntomodulin after a meal., (© 2015 American Association for Clinical Chemistry.)

Aims: To evaluate the performances of commercially available glucagon-like peptide-1 (GLP-1) assays and the implications for clinical studies., Methods: Known concentrations (5-300 pmol/l) of synthetic GLP-1 isoforms (GLP-1 1-36NH2, 7-36NH2, 9-36NH2, 1-37, 7-37 and 9-37) were added to the matrix (assay buffer) supplied with 10 different kits and to human plasma, and recoveries were determined. Assays yielding meaningful results were analysed for precision and sensitivity by repeated analysis and ability to discriminate low concentrations. Endogenous GLP-1 levels in clinical samples were assessed using three commercial kits., Results: The USCN LIFE assay detected none of the GLP-1 isoforms. The active GLP-1 enzyme-linked immunosorbent assays (ELISAs) from Millipore and DRG appeared identical and were specific for intact GLP-1 in buffer and plasma. The Meso Scale Discovery (MSD) total GLP-1 kit detected all six GLP-1 isoforms, although recovery of non-active forms was incomplete, especially in plasma. Millipore total GLP-1 ELISA kit detected all isoforms in buffer, but mainly amidated forms in plasma. The Alpco, Phoenix and Bio-Rad kits detected only amidated GLP-1, but the Alpco kit had a limited measurement range (30 pmol/l), the Phoenix kit had incomplete recovery in plasma and the Bio-Rad kit was insensitive (detection limit in plasma 40 pmol/l). The pattern of postprandial GLP-1 responses in clinical samples was similar between the kits tested, but the absolute concentrations measured varied., Conclusions: The specificity and sensitivity of commercially available kits for the analysis of GLP-1 levels vary considerably. This should be taken into account when selecting which assay to use and when comparing data from different studies., (© 2014 John Wiley & Sons Ltd.)

Objectives: To develop a novel, dual-monoclonal sandwich immunoassay with superior sensitivity that provides a rapid and convenient method for measuring glucagon. Glucagon is a 29-amino acid polypeptide hormone produced in the pancreas by the α-cells of the islets of Langerhans. Working in concert with insulin, glucagon is involved in regulating circulating glucose concentrations., Design and Methods: The immunoassay utilizes Meso Scale Discovery (MSD) electrochemiluminescence (ECL) technology and two affinity-optimized monoclonal antibodies. A series of experiments was performed to determine the linear range of the assay and to evaluate sensitivity, accuracy, recovery, precision, and linearity., Results: The sandwich assay was specific for glucagon and did not recognize the closely related peptide oxyntomodulin or other incretin peptides. The assay demonstrated excellent recovery, precision, and linearity, and a broad dynamic range of 0.14 pmol/L to 1950 pmol/L. In addition, assay results were highly correlated with those obtained using a previously described competitive RIA employing polyclonal antiserum., Conclusion: The use of affinity-optimized monoclonal antibodies in a sandwich immunoassay format provides a robust, sensitive, and convenient method for measuring concentrations of glucagon that is highly sensitive and specific. This immunoassay should help to improve our understanding of the role of glucagon in the regulation of glucose metabolism., (Copyright © 2012 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.)

The presence and distribution of glucose-dependent insulinotropic polypeptide or gastric inhibitory polypeptide (GIP), gastric-releasing peptide (GRP) and glucagon immunoreactivity were studied in the small intestine of the New Hampshire chicken using immunohistochemistry. This is the first report of the presence of GIP-immunoreactive (ir) cells in avian small intestine. GIP, GRP and glucagon immunoreactivity was localized in the epithelium of the villi and crypts of the duodenum, jejunum and ileum. In particular, both in the duodenum and in the jejunum immunoreactive endocrine cells to GIP, GRP and glucagon were observed. In the ileum, we noticed GIP-ir and glucagon-ir cells. GRP-ir was found in nerve fibres of all three segments of the small intestine. The distribution of these bioactive agents in the intestinal tract of the chicken suggests that GIP and glucagon may play a role in the enteropancreatic axis in which intestinal peptides modulate pancreas secretion., (© 2010 Blackwell Verlag GmbH.)

Resembling the main function of insect adipokinetic hormones (AKHs), the vertebrate hormone glucagon mobilizes energy reserves and participates in the control of glucose level in the blood. Considering the similarities, the effect of porcine glucagon was evaluated in an insect model species, the firebug Pyrrhocoris apterus. Using the mouse anti-glucagon antibody, presence of immunoreactive material was demonstrated for the first time in the firebug CNS and gut by ELISA. Mammalian (porcine) glucagon injected into the adult bugs showed no effect on hemolymph lipid level or on the level of AKH in CNS and hemolymph, however, it activated an antioxidant response when oxidative stress was elicited by paraquat, a diquaternary derivative of 4, 4'-bipyridyl. Glucagon elicited the antioxidant response by increasing glutathione and decreasing protein carbonyl levels in hemolymph, decreasing both protein carbonyl and protein nitrotyrosine levels in CNS. Additionally, when co-injected with paraquat, glucagon partially eliminated oxidative stress markers elicited by this redox cycling agent and oxidative stressor. This indicates that glucagon might induce an antioxidant defense in insects, as recently described for AKH. Failure of glucagon to alter AKH level in the bug's body indicates employment of an independent pathway without involving the native AKH.

We used atomic force microscopy (AFM) to measure the unbinding force between antigen coupled to an AFM tip and antibody coated on the substrate surface. Dynamic responses of glucagon/anti-glucagon pairs with multiple pull-off steps to pH and pulling velocity were studied by AFM. Force-distance curves of a specific glucagon-anti-glucagon interaction system with mono-, di-, and multi-unbinding events were recorded, which may be attributed to a single, sequential or multiple breaking of interacting bond(s) between glucagon and anti-glucagon. We studied the dynamic response of glucagon-anti-glucagon pairs to various pulling velocities (16.7-166.7 nm/s). It was found that the mean value of the unbinding force was shifted toward higher values with increasing pulling velocity at each pH. This indicates that the friction force between glucagon and anti-glucagon may contribute to the unbinding force. Moreover, the dynamic response of glucagon-anti-glucagon pairs to pH (4-10) with different pulling velocities was studied. Within the acid range, the bond strength between the glucagon/anti-glucagon complex showed a rapid increase from pH 4 to 7 and reached a maximum (256.4+/-48.9 pN at 166.7 nm/s) at neutrality, followed by a sharp decrease with increasing pH (pH 7-10). This could be attributed to the conformational change that occurred in glucagon when the pH value in solution was varied from the reference level at neutrality. This study demonstrated that the pH dependence of multiple antigen-antibody bond-rupture forces could be measured by a force-based AFM biosensor. Unraveling the relationship between inter-molecular force and intra-molecular conformational change in acid, neutral, and alkaline environments may provide new directions for future application of force measurements by AFM in proteomics or in the development of a clinical cantilever-based mechanical biosensor.

In type 2 diabetes, glucagon levels are elevated in relation to the prevailing insulin and glucose levels. The relative hyperglucagonemia is linked to increased hepatic glucose output (HGO) and hyperglycemia. Antagonizing the effects of glucagon is therefore considered an attractive target for treatment of type 2 diabetes. In the current study, effects of eliminating glucagon signaling with a glucagon monoclonal antibody (mAb) were investigated in the diabetic ob/ob mouse. Acute effects of inhibiting glucagon action were studied by an oral glucose tolerance test (OGTT) and by measurement of HGO. In addition, the effects of subchronic (5 and 14 days) glucagon mAb treatment on plasma glucose, insulin, triglycerides, and HbA1c (A1C) levels were investigated. Glucagon mAb treatment reduced the area under the curve for glucose after an OGTT, reduced HGO, and increased the rate of hepatic glycogen synthesis. Glucagon mAb treatment for 5 days lowered plasma glucose and triglyceride levels, whereas 14 days of glucagon mAb treatment reduced A1C. In conclusion, acute and subchronic neutralization of endogenous glucagon improves glycemic control, thus supporting the contention that glucagon antagonism may represent a beneficial treatment of diabetes.

Background/aims: Portal hypotensive effect of terlipressin is less effective when given during hemorrhage than in stable state. Blood retention in the stomach can induce splanchnic hyperemia which is mainly a consequence of an increased glucagon release. This study was undertaken to evaluate whether gastric blood retention contributes to the splanchnic hyporesponse to terlipressin., Methods: Plasma glucagon determination was performed under basal conditions and after intragastric blood gavage in sham-operated and cirrhotic rats. Additionally, splanchnic hemodynamic effects to terlipressin were measured in blood-gavaged cirrhotic rats with or without glucagon antiserum or octreotide infusion. Another set of air-gavaged cirrhotic rats was included for comparison., Results: Plasma glucagon level increased in both sham-operated and cirrhotic rats following blood gavage. Compared to air-gavaged cirrhotic rats, splanchnic hyporesponse to terlipressin was observed in cirrhotic rats receiving intragastric blood gavage. However, this splanchnic hyporesponse to terlipressin in blood-gavaged cirrhotic rats was overcome by glucagon antiserum or octreotide infusion., Conclusions: Intragastric blood gavage induced an elevation of plasma glucagon level and led to a splanchnic hyporesponse to terlipressin. Glucagon antiserum or octreotide administration overcame this hyporesponse. Excessive release of circulating glucagon may be an important factor for splanchnic hyporesponse to terlipressin in cirrhotic portal hypertension during hemorrhage.

New information regarding gastrointestinal mechanisms that participate in the control of food intake has extended our understanding of appetite control. Although each new signaling pathway discovered in the gut is a potential target for drug development in the treatment of obesity, the growing number of such signaling molecules indicates that a highly complex process controls food intake. The present summary focuses on the role of glucagon-like peptide 1 (GLP-1) in this regulatory process. The different biological effects of GLP-1 (glucose-lowering properties, inhibition of appetite and food intake) provide a powerful impetus for development of GLP-1-based new drugs.

Purpose: Some therapeutic peptides exhibit amyloidogenic properties that cause insolubility and cytotoxicity against neuronal cells in vitro. Here, we characterize the conformational change in monomeric therapeutic peptide to its fibrillar aggregate in order to prevent amyloidogenic formation during clinical application., Methods: Therapeutic peptides including glucagon, porcine secretin, and salmon calcitonin were dissolved in acidic solution at concentrations ranging from 1 mg/ml to 80 mg/ml and then aged at 37 degrees C. Amyloidogenic properties were assessed by circular dichroism (CD), electron microscopy (EM), staining with beta-sheet-specific dyes, and size-exclusion chromatography (SEC). Cytotoxic characteristics were determined concomitantly., Results: By aging at 2.5 mg/ml or higher for 24 h, monomeric glucagon was converted to fibrillar aggregates consisting of a beta-sheet-rich structure with multimeric states of glucagon. Although no aggregation was observed by aging at the clinical concentration of 1 mg/ml for 1 day, 30-day aging resulted in the generation of fibrillar aggregates. The addition of anti-glucagon serum significantly inhibited fibrillar conversion of monomeric glucagon. Glucagon fibrils induced significant cell death and activated an apoptotic enzyme, caspase-3, in PC12 cells and NIH-3T3 cells. Caspase inhibitors attenuated this toxicity in a dose-dependent manner, indicating the involvement of apoptotic signaling pathways in the fibrillar formation of glucagon. On the contrary to glucagon, salmon calcitonin exhibited aggregation at a much higher concentration of 40 mg/ml and secretin showed no aggregation at the concentration as high as 75 mg/ml., Conclusions: These results indicated that glucagon was self-associated by its beta-sheet-rich intermolecular structure during the aging process under concentrated conditions to induce fibrillar aggregates. Glucagon has the same amyloidogenic propensities as pathologically related peptides such as beta-amyloid (Abeta)1-42 and prion protein fragment (PrP)106-126 including conformational change to a beta-sheet-rich structure and cytotoxic effects by activating caspases. These findings suggest that inappropriate preparation and application of therapeutic glucagon may cause undesirable insoluble products and side effects such as amyloidosis in clinical application.

It is suggested that a T-helper cell 2 (Th2) shift and Th2 spreading of autoimmunity following immunization with beta-cell antigen causes diabetes protection. To address this, antibody titer and subclass to insulin, glutamic acid decarboxylase (GAD)65, IA-2, and IA-2beta proteins were measured by radiobinding assays in untreated or immunized female nonobese diabetic mice. Untreated nonobese diabetic mice developed autoantibodies to insulin (IAA), but not GAD or IA-2/IA-2beta, and IAA-positive mice had increased diabetes risk (P < 0.001). IAA were IgG1 and IgG2b. In immunized mice, IgG1 and lesser IgG2b insulin antibodies were promoted by subcutaneous injection of insulin plus incomplete Freund's adjuvant, insulin plus Montanide ISA 720, and glucagon plus incomplete Freund's adjuvant, but not by incomplete Freund's adjuvant plus GAD65, IA-2beta, or phenylethanolamine N-methyltransferase, or adjuvant alone. Diabetes incidence was significantly reduced in immunized groups with elevated insulin antibody (IA) responses. Spreading of antibody responses to GAD or IA-2/IA-2beta following immunization was rare, and antibody epitope spreading was only detected in IA-2beta immunized mice. Humoral autoimmunity in nonobese diabetic mice is, therefore, limited to IAA with Th2 subclass phenotype and is associated with increased diabetes risk. This contrasts the diabetes protection provided by immunization protocols that promote this response and suggests that Th2 immunity may not be the principal regulator of beta-cell destruction in autoimmune diabetes.

Background: Glucagon-like peptide 2 (GLP-2) is a newly discovered intestinotrophic hormone. We have recently reported that a 5-week GLP-2 treatment improved the intestinal absorptive capacity of short-bowel patients with no colon. Additionally, GLP-2 treatment was associated with changes in body composition that included a significant increase in total body bone mass. This article describes the effect of GLP-2 on spinal and hip bone mineral density (BMD) and biochemical markers of bone turnover in these patients., Methods: In an open-labelled pilot study, eight short-bowel patients (3M, 5F; mean age 49 years) with small-bowel resection and no colon received 400 microg s.c. of GLP-2 twice daily for 5 weeks. Four received home parenteral nutrition (mean length of residual jejunum 83 cm) and 4 did not (mean length of ileum resected 106 cm). The outcome measures were the mean percent change from baseline in spinal and hip BMD measured by dual-energy X-ray absorptiometry, changes in four biochemical markers of bone-turnover, PTH, 25-hydroxy vitamin-D, and the intestinal absorption of calcium., Results: Mean +/- s(x) (SEM) percent changes in spinal and hip BMD were 1.1+/-0.4% (P < 0.05) and 1.9+/-0.8% (P = 0.06), respectively. The intestinal calcium absorption increased by 2.7% (P = 0.87). Serum ionized calcium increased in 5/8 patients with a concomitant decrease in serum PTH values. Three of the four markers of bone turnover decreased., Conclusion: A 5-week GLP-2 administration significantly increased spinal BMD in short-bowel patients with no colon. The mechanism by which GLP-2 affects bone metabolism remains unclear, but may be related to an increased mineralization of bone resulting from an improved intestinal calcium absorption.

Miniglucagon, the COOH-terminal (19-29) fragment processed from glucagon, is a potent and efficient inhibitor of insulin secretion from the MIN 6 beta-cell line. Using the rat isolated-perfused pancreas, we investigated the inhibitory effect of miniglucagon on insulin secretion and evaluated the existence of an inhibitory tone exerted by this peptide inside the islet. Miniglucagon dose-dependently inhibited insulin secretion stimulated by 8.3 mol/l glucose, with no change in the perfusion flow rate. A concentration of 1 nmol/l miniglucagon had a significant inhibitory effect on a 1 nmol/l glucagon-like peptide 1 (7-36) amide-potentiated insulin secretion. A decrease in extracellular glucose concentration simultaneously stimulated glucagon and miniglucagon secretion from pancreatic alpha-cells. Using confocal and electron microscopy analysis, we observed that miniglucagon is colocalized with glucagon in mature secretory granules of alpha-cells. Perfusion of an anti-miniglucagon antiserum directed against the biologically active moiety of the peptide resulted in a more pronounced effect of a glucose challenge on insulin secretion, indicating that miniglucagon exerts a local inhibitory tone on beta-cells. We concluded that miniglucagon is a novel local regulator of the pancreatic islet physiology and that any abnormal inhibitory tone exerted by this peptide on the beta-cell would result in an impaired insulin secretion, as observed in type 2 diabetes.

The pancreatic hormone glucagon hyperpolarizes the liver cell membrane under various conditions. Here we investigated the physiological relevance of this effect by testing the influence of infusions of glucagon antiserum on the liver cell membrane potential in vivo. Intracellular microelectrode recordings of liver cells (up to 60/rat over 2 h) were done in anesthetized male rats. Livers were fixed in place, and recordings were done 10-30 min after intraperitoneal injections of glucagon or hepatic portal vein infusions of glucagon or specific polyclonal glucagon antibodies raised in rabbits. The isotonic lactose vehicle was used as a control for glucagon, and equal amounts of nonimmunized rabbit IgG were used as a control for glucagon antibodies. Intraperitoneal glucagon (400 microg/kg) hyperpolarized the liver cell membrane up to 12 mV, and intraportal glucagon (10 or 60 microg/kg) dose dependently hyperpolarized the liver cell membrane by 3-7 mV. Intraportal infusion of glucagon antiserum (in vitro binding capacity of 4 ng glucagon/rat) significantly depolarized the liver cell membrane by approximately 2.5 mV. The effects of both glucagon and glucagon antiserum reversed after 60-90 min. We conclude that glucagon is a physiologically important modulator of the liver cell membrane potential.

Introduction: In this study, immunoneutralization of endogenous insulin, glucagon, and somatostatin with specific antibodies was used in an isolated perfused human pancreas (IPHP) model., Aims: To study intrapancreatic cellular interactions and pancreatic hormonal secretion., Methodology: Randomized, sequential 10-minute test intervals of single-pass perfusion with each antibody were performed at 3.9 mM or 11.5 mM steady-state glucose concentrations. Somatostatin, insulin, and glucagon levels were measured in the effluent during basal and immunoneutralization intervals., Results: At 3.9 mM glucose concentration, somatostatin antibody (SS-Ab) stimulated insulin and glucagon secretion, insulin antibody (IN-Ab) inhibited glucagon secretion, and glucagon antibody (GN-Ab) stimulated insulin secretion. At 11.5 mM glucose concentration, SS-Ab stimulated insulin secretion, IN-Ab stimulated glucagon and inhibited somatostatin secretion, and GN-Ab stimulated insulin secretion., Conclusion: The variation in hormonal responses to immunoneutralization during stimulated and nonstimulated glucose conditions suggests that a dynamic association exists between the pancreatic cells.

Background: Acute pancreatitis (AP) initiates a generalized inflammatory response that increases intestinal permeability and promotes bacterial translocation (BT). Impairment of the intestinal epithelial barrier is known to promote BT. Glucagon-like peptide 2 (GLP-2), a 33 residue peptide hormone, is a key regulator of the intestinal mucosa by stimulating epithelial growth. The purpose of this study was to determine whether GLP-2 decreases intestinal permeability and BT in AP., Methods: To examine whether GLP-2 can decrease intestinal permeability and thereby decrease BT in acute necrotizing pancreatitis, 34 male Sprague-Dawley rats (200 to 300 g) were studied. AP was induced in group I and group II by pressure injection of 3% taurocholate and trypsin into the common biliopancreatic duct (1 mg/kg of body weight). The potent analog to GLP-2 called ALX-0600 was utilized. Group I rats received GLP-2 analog (0.1 mg/kg, SQ, BID) and group II rats received a similar volume of normal saline as a placebo postoperatively for 3 days. Group III and group IV received GLP-2 analog and placebo, respectively. At 72 hours postoperatively, blood was drawn for culture of gram-negative organisms. Specimens from mesenteric lymph nodes (MLN), pancreas and peritoneum were harvested for culture of gram-negative bacteria. Intestinal resistance as defined by Ohm's law was determined using a modified Ussing chamber to measure transepithelial current at a fixed voltage. A point scoring system for five histologic features that include intestinal edema, inflammatory cellular infiltration, fat necrosis, parenchymal necrosis, and hemorrhage was used to evaluate the severity of pancreatitis. Specimens from MLN, pancreas, jejunum, and ileum were taken for pathology., Results: All group I and group II rats had AP. The average transepithelial resistance in group I was 82.8 Omega/cm(2) compared with 55.9 Omega/cm(2) in group II (P <0.01). Gram-negative BT to MLN, pancreas, and peritoneum was 80%, 0%, and 0%, respectively in group I compared with 100%, 30%, and 20% translocation in group II., Conclusion: GLP-2 treatment significantly decreases intestinal permeability in acute pancreatitis.

We demonstrate using physico-chemical techniques that insulin binds to glucagon with a Kd of 0.89 micromolar. While such binding is of little significance physiologically, it has important immunological consequences. Hormone binding is mirrored by specific binding between insulin antibody and glucagon antibody to form idiotype-antiidiotype complexes observable by Ouchterlony immunodiffusion and ELISA. These complexes may provide new insights into the formation of circulating immune complexes in diabetes. The insulin-glucagon complex is hyper-antigenic, inducing antibody production at concentrations that do not elicit immune responses from the individual hormones. The resulting immune response is not primarily against the individual hormones, but against the complex. In fact, all so-called insulin antibodies tested (rabbit, guinea pig, mouse and human) show substantially higher affinity for insulin-glucagon complex than for insulin alone, suggesting that this complex is the primary antigen in most, if not all, cases. These results lead to several testable predictions, including the possibility that glucagon antibody will bind to insulin receptors to cause type 2 (antibody mediated) insulin resistance.

Background: Glucagon-producing alpha cells play a crucial role during the perinatal period. Because of their peri-islet localization near the early dendritic and macrophage cell infiltration, we thought it pertinent to investigate alpha cells in greater depth in nonobese diabetic (NOD) mice, a well-recognized spontaneous model for human type I diabetes., Materials and Methods: We determined alpha-cell distribution (glucagon immunohistochemistry and image analysis) and activity (real-time reverse transcriptase polymerase chain reaction [RT-PCR] and glucagon radioimmunoassay [RIA]), in relationship to glycemia in NOD and lymphocyte-deficient NODscid mice as compared to control mice (C57BL/6) from birth onward., Results: NOD and NODscid mice, particularly at 1 day of age, had twice as many very small islets (<2,000 pixels) as C57BL/6 mice. During the postnatal period, the percentages of glucagon-positive areas in islets less than 2000 pixels were higher in NOD mice than C57BL/6; only a trend was found in NODscid. Pancreatic mRNA expression and glucagon content decreased in all strains at weaning. However, before weaning, pancreatic and blood glucagon levels were significantly lower in NOD and NODscid compared to C57BL/6 mice. Low basal nonfasting glycemia was observed in all strains before weaning with some strain differences: glycemia was significantly lower in NOD than C57BL/6, and higher in NODscid than NOD and C57BL/6., Conclusion: These data suggest that, before weaning, NOD and, to some extent NODscid pancreata contain more immature islets (as reflected by their small size and high percentages of glucagon-positive areas, concomitant with lower glucagon storage and basal secretion) than C57BL/6 pancreata.

The ATP-sensitive potassium channel (KATP channel) is an essential ion channel involved in glucose-induced insulin secretion. The KATP channel is composed of an inwardly rectifying potassium channel, Kir6.2, and the sulfonylurea receptor (SUR 1); in the pancreas it is reported to be shared by all endocrine cell types. A previous study by our research group showed that Kir 6.2-knockout mice lacked KATP channel activities and failed to secrete insulin in response to glucose, but displayed normal blood glucose levels and only mild impairment in glucose tolerance at younger ages. In some aged knockout mice, however, obesity and hyperglycemia were recognizable. The present study aimed to reveal morphological changes in pancreatic islets of Kir 6.2-knockout mice throughout life. At birth, there were no significant differences in the islet cell arrangement between the knockout mice and controls. At 14 postnatal weeks glucagon cells appeared in the central parts of islets, and this image became more pronounced with aging. In animals older than 50 weeks insulin cells decreased in numbers and intensity of insulin immunoreactivity; most islets in 70- and 80-week-old mice were predominantly composed of glucagon cells and peptide YY (PYY)-containing cells. Staining of serial sections and double staining of single sections from these old mice demonstrated the frequent coexpression of glucagon and PYY, which is a phenotype for the earliest progenitor cells of pancreatic endocrine cells. These findings suggest that the KATP channel is important for insulin cell survival and also regulates the differentiation of islet cells.

Anti-rat CD45RA monoclonal antibody (MAb) has been used as a marker for rat B-lymphocytes (B-cells). When we applied the MAb to identify B-lymphocytes among immune cells infiltrating pancreatic islets of the BB rat, which is an animal model for human type 1 diabetes mellitus, a subpopulation of islet cells was immunostained as well. Our immunohistochemical investigations demonstrated that the stained islet cells are pancreatic alpha-cells and the MAb cross-reacts with glucagon.

Objective and Design: Co-localization of the four major pancreatic hormones, and also of islet amyloid polypeptide (IAPP), peptide tyrosine tyrosine (PYY), secretin and neurotensin, has been studied in the endocrine pancreas of human fetuses at 16, 18 and 22 weeks of gestation., Methods: Double and triple immunofluorescence stainings have been used., Results: All three fetal pancreata contained cells that showed insulin, glucagon, somatostatin, pancreatic polypeptide (PP), IAPP, secretin and PYY immunoreactivity. Neurotensin cells were found in the youngest fetus and gastric inhibitory polypeptide (GIP) in the two older fetuses. Co-localization of two hormones occurred in most of the endocrine cell types in the three fetuses examined, but three hormones occurred in only a few cells and especially in the youngest fetus. Somatostatin cells were the only cell type which was largely monohormonal. Our findings showed that there are two different co-localization patterns: insulin was co-localized mainly with IAPP and glucagon, while secretin and PYY occurred together with glucagon and PP., Conclusions: These data are the first to describe secretin and neurotensin in the fetal pancreas. Two different co-localization patterns could be distinguished: insulin, IAPP and glucagon, and glucagon, secretin, PP and PYY.

Combination therapy with enterotrophic agents may be useful in patients with the short bowel syndrome. The gut hormones neurotensin (NT) and glucagon-like peptide 2 (GLP-2) are potent enterotrophic factors when administered alone; however, their combined effects are not known. Using a GLP-2-producing tumor (STC-1), we determined whether administration of NT enhances the effect of GLP-2 on intestinal growth. Athymic mice were injected with STC-1 cells (6 x 10(6)) subcutaneously. Twenty-three days after STC-1 implantation, mice received either NT (300 microg/kg or 600 microg/kg) or saline solution (control) subcutaneously three times a day for 6 days. Two groups of tumor-free mice received either saline or NT for 6 days. At sacrifice, jejunum and ileum were collected, weighed, and analyzed for DNA and protein content. In the jejunum, NT combined with GLP-2 (from STC-1) increased weight, protein content (markers of mucosal hypertrophy), and DNA content (a marker of mucosal hyperplasia), compared to either NT or GLP-2 alone. In the ileum, the combination of NT and GLP-2 significantly increased weight and/or protein content compared to NT or GLP-2 alone. Administration of NT enhances the enterotrophic effects of GLP-2, augmenting hypertrophy of the entire small bowel and hyperplasia of the jejunum. The combination of NT and GLP-2 may be useful to enhance intestinal growth in patients with the short bowel syndrome.

The physiological action of endogenous active forms of glucagon-like peptide-1 (GLP-1) on the insulin response to intragastric glucose was studied in conscious male Wistar rats by immunoblockade with two monoclonal antibodies directed against different epitopes of GLP-1(7-36)amide. Plasma concentrations of intraperitoneally injected monoclonal antibodies were determined before and during each experiment by an enzyme-linked immunosorbent assay (ELISA) specific for GLP-1-binding antibodies. Three hours after injection of the two monoclonal antibodies, the plasma insulin response (area under the curve) following intragastric glucose 1 g/kg was reduced to a mean level (mean +/- SEM) of 60%+/-8% (n = 11) of control responses previously determined in the same rats, and the time course of the response showed almost no increase in insulin during the first 10 minutes, reaching a maximum of 45.1+/-4.6 microU/mL at 30 minutes, in contrast to the rapid increase of the control response to a maximum of 64.5+/-5.1 microU/mL at 15 minutes. Total C-terminally amidated GLP-1 measured by radioimmunoassay (RIA) of acid ethanol-extracted plasma increased from a mean basal level of 10+/-2 pmol/Lto a peak of 31+/-5 pmol/L at 15 minutes in the control experiments, while basal and response levels greater than 100 pmol/L were recorded after antibody treatment. The increase of plasma glucose was reduced in the presence of the antibodies, peaking at a mean of 9.7+/-0.3 mmol/L at 30 minutes, compared with 11.8+/-0.5 mmol/L at 30 minutes in the control experiments. The action of GLP-1 appears particularly important for the early insulin response to ingested glucose, and the unexpected effect of the antibodies on the glucose response may point to a net promoting effect of GLP-1 on intestinal glucose absorption.

Factors that stimulate gut mucosal proliferation may be beneficial during periods of gut disuse or atrophy. Recently glucagon-like peptide 2 (GLP-2) has been shown to stimulate small bowel growth. The purpose of our study was to compare the trophic effects of GLP-2 with those of neurotensin (NT), a potent gut trophic factor. Mice were randomized to receive either GLP-2, NT, or saline solution (control) for 10 days. The mice were killed on day 11, at which time the jejunum, ileum, and colon were removed, weighed, and DNA and protein content measured. Mice treated with GLP-2 showed a significant increase in the weight of the jejunum, ileum, and colon compared to both control and NT-treated mice. DNA content, a marker of cellular hyperplasia, was significantly increased in the small bowel and colon by treatment with GLP-2 and NT compared to control tissues. Small intestinal protein content, an indicator of cellular hypertrophy, was significantly increased by GLP-2 compared to both NT and control; protein content of the colon was greater in each of the treatment groups compared with control mice. We have demonstrated, for the first time, that GLP-2 stimulates colonic growth. In addition, GLP-2 is a potent trophic factor of normal small intestine with proliferative effects that are equal to or greater than those of NT. Administration of GLP-2 may be useful clinically to enhance small intestinal regeneration and adaptation during periods of disease and in the early phases of the short bowel syndrome.

The development of a sensitive radioimmunoassay (RIA) for C-terminally amidated forms of glucagon-like peptide-1 (GLP-1) is described. Rabbits immunized with GLP-1(7-36)amide conjugated to bovine serum albumin with glutaraldehyde produced antisera containing high-affinity antibodies directed against an epitope that included the free amidated C-terminus of the peptide. These antisera could be used in a sensitive RIA (detection limit 0.1 fmol/tube) that measured GLP-1(7-36)amide and GLP-1(1-36)amide equally. Total concentrations of amidated GLP-1 immunoreactivity in extracts of rat hypothalamus, pancreas and intestine were determined by RIA, and resolved into GLP-1(7-36)amide, GLP-1(1-36)amide and unidentified cross-reacting substances by HPLC. Whereas only GLP-1(7-36)amide could be identified in the hypothalamus, in amounts that represented 55-94% of total glucagon-like immunoreactivity (GLI), the pancreas produced chiefly GLP-1(1-36)amide, representing 0.8-3.4% of total GLI, and only trace or undetectable amounts of GLP-1(7-36)amide (0-0.36% of total GLI). This argues against any role of intrapancreatic GLP-1(7-36)amide in the secretion of insulin. In the terminal ileum total amidated GLP-1 immunoreactivity represented 27-73% of total GLI, and in five of six specimens only GLP-1(7-36)amide could be identified on HPLC, in amounts representing 13-17% of total GLI. Only one specimen of terminal ileum contained HPLC-identified GLP-1(1-36)amide (13% of total GLI) in addition to GLP-1(7-36)amide (31% of total GLI). Acid-ethanol extraction of peptide-free rat plasma with added GLP-1(7-36)amide gave recoveries of 91+/-SEM 2% in the range 20-200 pmol/l. Basal plasma amidated GLP-1 in six unanaesthetized rats was 4.1+/-1.1 pmol/l and rose to a maximum of 15.4+/-3.0 pmol/l 10 min after intragastric glucose 1 g/kg, illustrating the modest level of plasma responses of amidated forms of GLP-1.

Calretinin is a calcium-binding protein whose major functions are assumed to be buffering, transport of Ca2+, regulation of various enzyme systems and cellular protection. Moreover, calretinin does not seem to be a specific marker for any particular cell since it has been discovered in various mammalian and avian organs. In order to give clue to its function(s), we investigate its distribution in the rat pancreas using immunocytochemistry and transmission electron microscopy. We have found calretinin immunoreactive cells in rat pancreas only in the islets of Langerhans. These cells correspond to A cells producing glucagon. The colocalization of calretinin and glucagon was confirmed with double-labelling immunofluorescence. Nevertheless, the role of calretinin and its relationship with the production and/or the exocytosis of glucagon granules remains to be elucidated.

Glucagon-like peptide-1 (GLP-1), structurally similar to glucagon, is synthesized from a larger precursor, preproglucagon, and has been postulated to be a novel incretin. Recently, it was reported that central administration of GLP-1 decreased food intake in rats. The amino acid sequences of GLP-1 are identical in all mammals, and chicken GLP-1 exhibits a high homology with mammalian GLP-1. The aim of this study was to elucidate whether central injection of mammalian or chicken GLP-1 inhibits food intake in the chick, and to compare their effects. Intracerebroventricular administration of mammalian and chicken GLP-1 strongly inhibited food intake of chicks. However, the suppressive effect of both GLP-1 on food intake was similar. These results show that GLP-1 with a variety of amino acid sequences may be the most potent inhibitor of food intake in the chicken.

To elucidate the question of whether production of the insulinotropic gut hormones glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-1 (GLP-1) is altered by a diabetic metabolic state, their intestinal expression pattern was evaluated. Two rodent models for diabetes mellitus were used, non-obese diabetic (NOD) mice as a model for insulin-dependent diabetes and Zucker diabetic fatty (ZDF) rats for non-insulin-dependent diabetes mellitus (NIDDM). Expression of both incretin hormones followed typical patterns, which were similar in both animals and unaltered by the diabetic state. The GIP gene was greatly expressed in the duodenum, jejunum, and ileum, with a continuous decrease from the upper to lower intestines. This pattern was observed in both NOD mice and ZDF rats regardless of the diabetic state. This expression data was corroborated by radioimmunoassay (RIA) analysis of the gene product GIP. Expression of the proglucagon gene encoding GLP-1 had an opposite appearance. The highest expression was seen in the large bowel and the ileum. RIA analysis of the gene product GLP-1 mirrored these data. Although the distribution pattern was similar in both animal models, in contrast to diabetic NOD mice, a regulated expression was found in diabetic ZDF rats. Compared with lean nondiabetic controls, fatty hyperglycemic animals showed an increased expression of the proglucagon gene in the colon and a concomitant reduction in the small intestine. This was mirrored by the GLP-1 content of the colon and ileum. Overall, basal GLP-1 plasma levels were increased in ZDF rats (17.0 +/- 2.8 pmol) compared with lean Zucker rats (12.4 +/- 1.8 pmol). In conclusion, incretin hormone expression (GIP and GLP-1) follows specific patterns throughout the gut and is unaltered by the diabetic state. In ZDF rats, regulation of proglucagon expression occurs mainly in the large intestine.

Although a wide variety of neuropeptides have been localized in vertebrate retinas, many questions remain about the function of these peptides and the amacrine cells that contain them. This is because many of these peptidergic amacrine cells have been studied using only immunocylochemical techniques. To address this limitation, the present study used a combination of quantitative anatomy, biochemistry and electrophysiology to examine amacrine cells in the turtle retina that contain the neuropeptide glucagon. In the turtle retina, there is a small population of 2500 glucagonergic amacrine cells, which probably represents < 1% of the total number of amacrine cells. Circular distribution statistics indicated that many of these tristratified amacrine cells had asymmetric dendritic arborizations that were radially oriented toward the retinal periphery. The cells were found to have similar dendritic coverage factors, to be distributed in a non-random arrangement in all regions of the retina, and to peak in density in the visual streak region. Electron microscopic studies indicated that glucagonergic amacrine cells made synaptic contacts primarily with other amacrine cells, and small numbers of bipolar cells. The synaptic inputs and outputs were balanced in the inner strata of the inner plexiform layer, and were biased toward synaptic outputs in the outer strata of the inner plexiform layer. These contacts involved small unlabelled synaptic vesicles, and not the large labelled dense core vesicles also found in these neurons. The biochemical studies indicated that glucagon could be released from the retina in a calcium dependent manner by high potassium stimulation. The electrophysiology found no color opponency, and the glucagonergic amacrine cells gave sustained hyperpolarizing responses to small stimulation spots and had antagonistic surrounds. The results of these studies suggest that there are significant regional specializations of glucagonergic amacrine cells, and that they may provide OFF-modulation in interactions between the ON-and OFF-centre visual pathways in the turtle retina.

The role of glucagon in the regulation of blood glucose in fed and fasted anesthetized rats was studied by injecting intravenously 4 ml/kg of a high-capacity (40 nmol/ml) high-affinity (0.6 x 10(11) mol/l) monoclonal glucagon antibody. Blood glucose was lowered by the antibody by 2 mmol/l in fed rats but remained unchanged in 10- and 48-h-fasted rats. Antibody injection significantly reduced plasma insulin in both fed and 10-h-fasted rats. In 10-h-fasted rats, propranolol injection decreased blood glucose by 0.6 mmol/l, and combined with antibody administration, a decrease by 1.1 mmol/l was observed. Blood glucose was never < 3.3 mmol/l. Thus glucagon is partly responsible for maintenance of euglycemia in fed rats, whereas during fasting it plays a limited role. However, immunoneutralization of glucagon reduces insulin secretion irrespective of blood glucose. Additional mechanisms seem to be responsible for the maintenance of blood glucose in the fasting state when glucagon and the sympathoadrenergic system are blocked.

Pancreatic sections from diverse tetrapods, including various species of caviomorph rodents, were immunohistochemically investigated using two antisera which reacted with the N- and C-terminal portions of the glucagon molecule. While the antiserum against the N-terminal portion stained alpha cells in all the species studied, the antiserum against the C-portion failed to stain alpha cells in two caviomorphs of the Caviidae family (guinea pig and cuis) and in one of the Octodontidae family (degu). The observations in guinea pig and degu were expected, since their glucagons differ from those of many other tetrapods in the C-terminal portion of the molecule. In this paper, the cuis was added to these two species. It is noteworthy that among the caviomorphs studied herein (nine species), immunohistochemical differences were detected only in the three above-mentioned species and did not involve higher taxa, thus suggesting that these modifications are relatively recent in the evolution of this group of rodents.

We had a patient with asymptomatic hyper-immunoreactive glucagonemia and with no evidence of pancreatic tumor detected by radiological examinations. The glucagon level was not decreased by the administration of glucose or somatosatin analogue (SMS 201-995). Gel filtration studies revealed that most glucagon immunoreactivity was eluted at the position of 150,000 daltons [big plasma glucagon (BPG)]. Binding studies with 125I-glucagon showed that glucagon autoantibody was negative. Acid treatment of plasma and reduction of immunoglobulin G (IgG) did not result in a shift of BPG to normal glucagon (3485 daltons). Glucagon immunoreactivity determined with anti-glucagon antiserum OAL 123 (C-terminal specific antiserum used in the present radioimmunoassay kit) did not dilute out in parallel to normal glucagon (3485 daltons), and the plasma glucagon level was normal with Unger's 30K (anther C-terminal specific antiserum) and OAL 196 (N-terminal specific antiserum). The patient's IgG dose-dependently reduced the binding of 125I-glucagon to anti-glucagon antiserum OAL-123. Glucagon degrading activity (GDA) was negative in the patient's plasma. These results suggest that the patient's IgG cross-reacted with the present anti-glucagon antiserum OAL 123, and caused a spuriously high plasma immunoreactive glucagon level.

Glucagon and glucagon-like peptide 1 (GLP-1) are homologous peptide hormones that are recognized by likewise homologous, but highly selective receptors. Analogs of glucagon and GLP-1, in which the divergent residues were systematically exchanged, were employed to identify the structural requirements for their selective receptor recognition. Substitutions in the NH2-terminal part of the glucagon molecule with the corresponding GLP-1 residues, as for example in [Ala2,Glu3]-glucagon and [Val10,Ser12]glucagon, reduced the binding affinity for the glucagon receptor several hundred-fold without increasing the affinity for the GLP-1 receptor. In contrast, introduction of GLP-1 residues into the far COOH-terminal part of the glucagon molecule, e.g. [Val27,Lys28,Gly29,Arg30]glucagon, had a minimal effect on recognition of the glucagon receptor, but improved the affinity of the analog for the GLP-1 receptor up to 200-fold. Similarly, substitutions in especially the far COOH-terminal part of the GLP-1 molecule with the corresponding glucagon residues, e.g. des-Arg30-[Met27,Asn28,Thr29]GLP-1, decreased the affinity for the GLP-1 receptor several hundred-fold (IC50 = 0.4-190 nM) without increasing the affinity for the glucagon receptor. Conversely, substitutions in the NH2-terminal part of the GLP-1 molecule impaired the affinity for the GLP-1 receptor only moderately. We conclude that the selective recognition of the glucagon and GLP-1 receptors is determined by residues located at opposite ends of the homologous peptide ligands. This conclusion is supported by the observation that a "chimeric" peptide consisting of the NH2-terminal part of the glucagon molecule joined to the COOH-terminal part of the GLP-1 molecule was recognized with high affinity by both receptors.

A mass spectrometric method is described for the rapid mapping of linear epitopes in proteins that are bound by monoclonal antibodies. The method consists of three steps. In the first step, an antigen protein is digested by a proteolytic enzyme to produce an appropriate set of peptide fragments. In the second step, peptide fragments containing the linear epitope are selected and separated from the pool of peptide fragments by immunoprecipitation with the monoclonal antibody. In the final step, the immunoprecipitated peptides are identified by matrix-assisted laser desorption mass spectrometry. The method allows the rapid determination of antigenic sites without tedious peptide synthesis or protein mutagenesis. The approach is demonstrated through the mapping of epitopes in two peptides (melittin and glucagon-like peptide-1 7-37) against which monoclonal antibodies were raised. In addition to epitope mapping, the successful coupling between matrix-assisted laser desorption mass spectrometry and immunoprecipitation provides a potentially powerful tool for determining binding sites between proteins.

The role of glucagon in diabetic hyperglycaemia has been a matter of controversy because of difficulties in the production of selective glucagon deficiency. We developed a high-capacity (40 nmol/ml), high-affinity (0.6 x 10(11) l/mol) monoclonal glucagon antibody (Glu-mAb) and gave i.v. injections (4 ml/kg) to rats in order to study the effect of selective glucagon deficiency on blood glucose. Controls received a mAb against trinitrophenyl. Glu-mAb completely abolished the hyperglycaemic effect of 2.86 nmol/kg glucagon in normal rats (p < 0.05, n = 6). In moderately hyperglycaemic rats injected with streptozotocin as neonates (N-STZ), Glu-mAb abolished a postprandial increase in blood glucose (from 11.2 +/- 0.7 mmol/l to 17.3 +/- 1.8 mmol/l in controls vs 10.5 +/- 0.9 mmol/l to 9.3 +/- 1.0 mmol/l; cross-over: n = 6, p < 0.05). No significant effect of Glu-mAb treatment was observed in more hyperglycaemic N-STZ rats (cross-over, n = 4) and in severely hyperglycaemic rats injected with STZ as adults (n = 6), but after insulin treatment of the latter, at doses partially restoring blood glucose levels (12.7 +/- 4.3 mmol/l), Glu-mAb administration almost normalized blood glucose (maximal difference: 6.0 +/- 3.8 mmol/l; cross-over: n = 5, p < 0.05). In conclusion, our results provide strong additional evidence for the hypothesis that glucagon is involved in the pathogenesis of diabetes. The hormone plays an important role in the development of STZ-diabetic hyperglycaemia, but glucagon neutralization only leads to normoglycaemia in the presence of insulin.

Intravenous glucagon inhibits exocrine pancreatic secretion in vivo, but exogenous glucagon does not affect exocrine secretion in vitro. Recent work, however, suggested that endogenous glucagon may be involved in the regulation of exocrine secretion even in vitro. We therefore investigated the effects of exogenous and endogenous glucagon on exocrine secretion by the isolated perfused rat pancreas in the presence of 1.8 mM glucose. Exogenous glucagon did not affect CCK-stimulated amylase output. 20 mM arginine stimulated glucagon release, but did not affect basal enzyme secretion. CCK-stimulated amylase output, however, was significantly inhibited in the presence of arginine. This inhibitory effect of arginine on exocrine pancreatic secretion could be blocked by glucagon antibodies, but not by nonspecific gammaglobulins. Thus exogenous glucagon failed to affect exocrine pancreatic secretion in vitro, but endogenously released glucagon or a glucagon-like peptide inhibited amylase release in the isolated perfused pancreas. We conclude that glucagon or a glucagon-like peptide may be a mediator in the islet-acinar axis.

The importance of changes in glucagon in the regulation of hepatic glucose production (Ra) during exercise has been questioned, as an increase in arterial immunoreactive glucagon (IRG) is not always detectable. However, IRG in the portal vein (PV) and not in the artery is most relevant, as flow through PV is approximately 80% of liver blood flow. To assess the extent that arterial IRG reflects the levels the liver is exposed to in PV, dogs (n = 5) were implanted with catheters in a carotid artery, hepatic vein (HV), and PV. Dogs were studied > or = 16 days later during rest and 150 min of moderate treadmill exercise, with indocyanine green and [3-3H]glucose infused to assess hepatic plasma flow (HPF) and hepatic Ra. IRG was 66 +/- 7, 73 +/- 8, and 81 +/- 7 pg/ml in the artery, HV, and PV at rest; it rose at 10 and 150 min of exercise to 89 +/- 9 and 127 +/- 13 pg/ml in the artery, 106 +/- 17 and 186 +/- 21 pg/ml in HV, and, by considerably more, to 153 +/- 20 and 261 +/- 25 pg/ml in PV. HPF fell by approximately 30% with exercise. The fall in HPF accounted for < 11% of the increased arterial-to-PV IRG gradient during exercise, with increased splanchnic IRG release comprising the remainder. Ra was linearly related to IRG levels in the three vessels.(ABSTRACT TRUNCATED AT 250 WORDS)

To study the effect of glucagon immunoneutralization on postoperative changes of urea synthesis, hysterectomized rats were given one injection of a specific high titre antibody against pancreatic glucagon 24 h before operation raising the plasma glucagon binding capacity to values 10-20 times higher than the plasma glucagon concentration in control animals. Earlier studies have shown that the spontaneous rate of urea-N synthesis (UNSR) doubles 3 h after operation, and that the Vmax of the process, the capacity of urea-N synthesis (CUNS) is 50% higher than normal values 24 h after operation. Therefore, the effect of glucagon on UNSR and CUNS were investigated 3 and 24 h postoperatively, respectively. Control animals were given non immune rabbit serum. Glucagon immunoneutralization partly normalized the early increase in UNSR 3 h postoperatively (control: 4.7 +/- 0.3, hysterectomy+serum: 6.7 +/- 0.4, hysterectomy+Gluc-Ab: 5.5 +/- 0.4 mumol (min.100 g BW)-1), but had no effect on the increase of CUNS 24 h postoperatively (control: 7.9 +/- 0.3, hysterectomy+serum: 9.5 +/- 0.3, hysterectomy+Gluc-Ab: 9.8 +/- 0.5 mumol (min.100 g BW)-1). This shows that glucagon is important for the early postoperative increase in the efficacy of urea synthesis, whereas the late increase in capacity seems not to depend on hyperglucagonemia.

The pharmacokinetics of intranasal, an intravenous infusion, and intramuscular glucagon has been studied in 5 healthy subjects and 11 patients with insulin-dependent diabetes mellitus. After infusion the elimination half-life was significantly longer in diabetics (11.9 vs 6.6 min) and the apparent volume of distribution was twice as high in diabetics (0.19 vs 0.37 l.kg-1). The metabolic clearance rates were the same in the two groups (18.9 and 21.3 ml.min-1.kg-1 in controls and in diabetics) and were about twice those previously reported. After 1 mg intranasally the Cmax of immunoreactive glucagon (IRG) was similar in diabetic and in healthy subjects. Administration of a higher dose (2 mg) to diabetic patients produced a higher plasma level, although not proportionately so. The AUC after 1 mg was also similar in controls and in diabetics. The elimination half-life in both groups was similar to the value found after i.v. infusion; it was significantly shorter in controls (5.5 min) than in diabetics (13.8 min). In both groups, mean Cmax was significantly lower than after IM glucagon, the relative bioavailability of 1 mg intranasally vs IM injection being less than 30%. After IM administration, the Cmax and AUC of IRG in controls and in diabetic patients, were identical. The apparent elimination half-life was also similar in the two groups, and was three- to four-times longer (28.6 and 31.4 min) than after infusion or intranasal administration, possibly because estimation of the t1/2 was affected by slow release of the hormone from the site of injection.

To determine the site of origin of pancreatic glucagon's inhibitory effect on spontaneous feeding in rats, glucagon was infused into either the hepatic portal vein or the inferior vena cava during spontaneous meals late in the dark phase. Hepatic portal infusion of 1.7-13.6 micrograms glucagon/meal reduced spontaneous meal size. In contrast, these doses did not significantly affect meal size when delivered via vena caval catheters that ended near the junction of the hepatic vein. This difference indicates that glucagon receptor sites in the liver initiate the satiating action of glucagon during spontaneous meals. The vagal dependency of glucagon satiety was also tested. Hepatic portal infusion of 13.6 micrograms glucagon/meal reduced the size of spontaneous meals both early and late in the dark in neurally intact rats, but not in hepatic-vagotomized rats. Finally, antagonism of endogenous glucagon with hepatic portal infusion of glucagon antibodies in a dose sufficient to neutralize 1 ng glucagon in vitro increased spontaneous meal size in intact rats, but not in hepatic-vagotomized rats. Thus the satiating effects of both exogenous and endogenous glucagon on spontaneous food intake appear to depend on the hepatic branch of the vagus. Taken together, these results are consistent with the hypothesis that glucagon acts in the liver to produce a satiety signal that is transmitted to the brain by the hepatic branch of the abdominal vagus.

Gut glucagon-like immunoreactivity (GLI), a supposed intestinal growth factor in the plasma and gastrointestinal tract, and weight of the gastrointestinal tract, an intestinal growth indicator, were examined in ventromedial hypothalamic (VMH)-lesioned rats 1 week after VMH lesions. Postprandial plasma gut GLI in VMH-lesioned rats was significantly higher than that in control rats. The content of gut GLI in all gastrointestinal sections except the duodenum in VMH-lesioned rats was significantly greater than that in control rats. Gel chromatography in the lower portion of the small intestine in which GLI content was the highest of all sections revealed the same pattern in both VMH-lesioned and control rats with two peaks of similar molecular size. Weight of all gastrointestinal sections except the cecum in VMH-lesioned rats significantly increased. These results demonstrated that both gut GLI secretion and production were enhanced in VMH-lesioned rats. The elevated gut GLI release may accelerate growth of the gastrointestinal tract in these animals.

To study the effect of glucagon neutralization on urea synthesis in diabetic rats, animals with newly induced (75 mg/kg streptozocin) experimental diabetes mellitus were divided into two groups. One group was given one weekly injection of nonimmune rabbit serum (n = 6), and the other group was given one weekly injection of a specific high-titer antibody against pancreatic glucagon (n = 6). Four weeks later, serum-treated diabetic rats had fasting glucagon concentrations 2-3 times higher than nondiabetic controls given one weekly injection of saline (control). Plasma glucagon binding capacity of diabetic rats given glucagon antibodies was 10-15 times higher than the glucagon concentration. A second group of nondiabetic controls were given nonimmune serum. Blood glucose concentration and urinary glucose output were identical in both groups of diabetic animals. Food intake doubled in both groups of diabetic rats. In control rats, the accumulated nitrogen balance, determined weekly for 4 wk, was positive at 81 +/- 3.1 mmol/96 h; in serum-treated diabetic rats, the accumulated nitrogen balance was negative, -8.3 +/- 2.4 mmol/96 h throughout the 4 wk, whereas it was higher at 4.7 +/- 2.3 mmol/96 h in the glucagon antibody-treated diabetic rats (P less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

An active or passive immunization against hormones and the subsequent neutralization of hormones by circulating antibodies is a valuable tool for the identification of hormonal action. To recognize presumed local (autocrine, paracrine) effects exerted by pancreatic hormones, the endocrine pancreas of rabbits was investigated electron-microscopically after long-term immunization against glucagon or somatostatin. Glucagon immunization resulted in hyperplasia and hypertrophy of glucagon- (A-) cells and in their increased metabolic activities: They showed prominent nucleoli, increased amounts of endoplasmic reticulum, Golgi areas, and mitochondria. These changes were paralleled by alterations in secretion granules (increased size, decreased hormonal content), increased numbers of lysosomes (crinophagic bodies), and an increment of the filamentous system. Basically, these findings point to an autocrine regulation of A-cells. Following somatostatin immunization, somatostatin- (D-) cells were hyperplastic but unchanged in their metabolic state. Instead, insulin-(B-) cells and A-cells exhibited equivalents of increased cellular activities (parameters, see above). This stimulation most probably is caused by cancelled paracrine (inhibitory) effects of somatostatin. The changes observed after both immunizations were differently expressed in morphologically heterogeneous islet types (size, angioarchitecture, cellular composition, microtopology of the various cell types). It is concluded, therefore, that the regulation of islets is not uniform. Autocrine and paracrine effects exerted by islet hormones are of different significance in individual islets, or they interfere differently with other regulatory signals.