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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.)

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.

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.

Specific antibodies to pancreatic glucagon in a dose sufficient to neutralize 1.5 ng glucagon in vitro were intraportally infused during the first 2 min of spontaneous meals in ad libitum-fed male Sprague-Dawley rats. In separate tests, glucagon antibodies stimulated feeding during the first spontaneous meal of the dark phase (73% mean increase in meal size) and during spontaneous meals in the last quarter of the dark phase (58% increase). These results indicate that a glucagon-sensitive satiety mechanism has a physiological role in the control of nocturnal feeding in rats.

Everted intestinal rings from partially starved rats accumulate the nonmetabolized amino acid 1-amino-cyclopentane-5-carboxylic acid (ACPC) at an enhanced rate. Plasma glucagon concentrations were found to be markedly elevated in these partially starved rats as well as in rats with experimentally induced diabetes, a condition previously shown to be associated with augmented intestinal uptake of amino acid. Treatment of partially starved rats with repeated injections of glucagon-binding antiserum prevented increased ACPC uptake of intestinal rings. Chronically elevated plasma glucagon levels may participate in the mechanism of the functional changes in the intestine in partial starvation.

In some antisera raised against glucagon in rabbits a large fraction of the antibody binding sites are occupied by glucagon-like material which appears to be of endogenous origin. Six rabbits were immunized with a glucagon-albumin conjugate, boosted twice and bled 5 times during the course of 72 days. The antisera were found to contain between 110-750 nmol/l antibody-bound glucagon. The concentration of bound glucagon as well as the concentration of free antibody binding sites (titre) increased with time in 5 rabbits. By gel chromatography the bound material co-eluted with intact pancreatic glucagon (as opposed to the immunogen). Five different methods were examined for their ability to remove the ligand. Charcoal treatment of the antiserum at pH 2.5 for 48 h removed 91 +/- 5% of the bound ligand. After stripping, binding capacities (titres) and binding affinity (equilibrium constants) amounted to 63.2 +/- 19.4% and 105.0 +/- 25.4% of the respective pre-stripping values.

The need for chaotropic eluents in immunoaffinity chromatography is a consequence of the high affinities of antibodies towards their antigens. This affinity is decreased and elution of antiglucagon antibodies from a column of immobilized glucagon can be achieved under mild conditions when the steric complementarity to the antibody binding site is perturbed by selective chemical modification of the hormone. The effects of reaction with 2-hydroxy-5-nitrobenzyl bromide, tetranitromethane and hydrogen peroxide have been studied. Conversely, treatment of immobilized antibodies with 2-hydroxy-5-nitrobenzyl bromide facilitates the elution of glucagon during immunoaffinity chromatography. The general implications of these results are discussed.

The prevalence and titer of antibodies to pancreatic hormones in 21 diabetics treated with USP insulin preparations before 1964 (Group 1) and in another 21 diabetics treated with more highly purified insulin preparations after 1974 (Group 2) were determined. All subjects in Group 1 had antibodies to insulin, 11 had antibodies to pancreatic polypeptide (PP) and 3 to glucagon. In Group 2, 17 had antibodies to insulin, 5 to PP, and 1 to glucagon. Antibodies to somatostatin (SRIF) were not detectable in either group. Group 1 subjects had higher titers of antibodies to insulin, glucagon, and PP than Group 2 subjects. Of note, the Group 1 subjects with the highest insulin antibodies also had the greatest prevalence and highest titers of antibodies to glucagon and PP. In order to address the question of possible differences in immune responsivity between Group 1 and 2 subjects, antibody titers to 4 ubiquitous viral agents were determined. There was no significant difference in viral antibody titers between the groups.

The sera of 30 patients who had been treated with conventional beef insulin were tested for binding of insulin and other pancreatic hormones. All showed antibody binding of insulin, 29 binding of proinsulin, 29 binding of pancreatic polypeptide, two binding of glucagon but none of the sera bound vasoactive intestinal peptide or somatostatin. After changing therapy to highly purified pork insulin the binding capacity of sera for insulin and the other hormones was monitored for up to 35 months and a steady fall was found in nearly all cases. In eight of the patients conventional beef insulin treatment was resumed: in one month binding of insulin and of the other hormones increased back to the initial levels. In eighteen subjects who had only received highly purified pork insulin low levels of insulin binding were found with no binding of proinsulin or other hormones. The amounts of proinsulin and contaminating hormones in highly purified pork insulin are so low that they are not immunogenic; conventional beef insulin not only contains immunogenic amounts of proinsulin and the contaminating hormones pancreatic polypeptide and glucagon but also is more immunogenic than purified pork insulin.

A highly sensitive immunoenzymatic technic is presented. The method involves three sequential steps: (1) primary antibody, (2) biotin-labeled secondary antibody, and (3) avidin-biotin-peroxidase complex. Avidin, an egg white protein, has four binding sites for the low-molecular-weight vitamin biotin. Many moieties of biotin can be coupled to the peroxidase molecule. Thus, since a relatively large amount of avidin is incubated with biotin-labeled peroxidase, avidin serves as a link between biotin-peroxidase molecules; in turn, biotin-peroxidase serves as a link between avidin molecules. Consequently, this large lattice-like complex with biotin-binding capability can be attracted to the sites of biotin-labeled antibody, producing a superior staining sensitivity. Several commercially available radioimmunoassay antibodies (e.g., antiglucagon, prolactin, gastrin, growth hormone, and thyroid-stimulating hormone antibodies) were tested for immunohistochemical staining. The unlabeled antibody peroxidase-antiperoxidase method fails to stain gastrin or thyroid-stimulating secretory cells when using these antibodies, and a relatively high antibody concentration is required to produce a positive reaction for glucagon, prolactin, and growth hormone. In contrast, the avidin-biotin-peroxidase complex method successfully demonstrates polypeptide hormones even when antibodies are diluted 20 to 40 times.

Total pancreatectomy was performed in dogs; and arginine test was conducted at the first, second and third weeks after surgery, and measurement was made of blood glucose, glucagon and insulin on a time-course basis for a comparative study with normal dogs. 1) After total pancreatectomy, glucagon reacting to 30 K antibody decreased once but increased with the lapse of time when no insulin was administered. 2) In the arginine test, glucagon showed a biphasic reaction in normal dogs, but a monophasic reaction in totally depancreatized dogs. This reaction declined three weeks after surgery. 3) When blood samples were taken from the pancreaticoduodenal vein, the left-gastroepiploic and mesenteric veins, during and after arginine was infused for 30 minutes. Glucagon in the pancreaticoduodenal vein increased markedly, but there were no such prominent changes in the left-gastroepiploic or mesenteric veins, Meanwhile, the level of glucagon in the left-gastroepiploic vein increased remarkably in dogs one week after total pancreatectomy.

A substance present in the sera of diabetic children that interferes with the radioimmunoassay for glucagon was found in six of 66 children who were participating in an inpatient study of diabetic control. Detailed studies documented unequivocally that this glucagon-binding substance is a specific antibody to glucagon and is located in the immunoglobulins. In a survey of diabetic children in the outpatient diabetes clinic and in a diabetes summer camp, antibodies to glucagon were found in about 12% of those evaluated. However, no children who had had diabetes for less than three years were found to have antibodies, and there appeared to be an increase with increasing duration of disease of up to greater than 20% at eight years' duration. The presence of glucagon antibodies may be of pathologic significance in that the patients have a greater tendency to develop hypoglycemia than do diabetic children without glucagon antibodies.

Commercial sources provide good, though expensive antiinsulin antisera. We describe here a simple, fast and inexpensive method for the production of antiinsulin antisera. Purified pork insulin (Lente) was injected subcutaneously in oil/water/complete Freund adjuvant mixture. Three guinea pigs received 0.25 mg of insulin and three received 0.5 mg of insulin. Subsequent injections of the same dose were done 40 and 60 days later. Five animals developed antisera with titers superior to 1:10,000 40 days after the primary inoculation. Four out of five guinea pigs improved further their antibody titer after the 2nd and 3rd injection (p less than 0.0005). Good sensitivity was associated with titers superior to 1:50,000 and appeared only after the 2nd injection to improve further after the 3rd. Thus, four out of six animals developed antiinsulin antisera suitable for the radioimmunoassay (RIA). The antisera bound proinsulin on an equimolar basis with insulin while glucagon was not bound up to 100 ng/ml. The minimum detectable insulin concentration was about 12 pg (0.3 microU) at the optimum antiserum dilution. Six animals given a small dose of insulin (0.06 mg) developed antisera of a low titer and sensitivity, unsuitable for the RIA.

To assess the possible value of the use of high-purity pork insulin (HPPI) in the United States, the serum insulin (I), pancreatic polypeptide (PP), glucagon (G), and somatostatin (SRIF) antibody binding characteristics have been determined in 90 conventional insulin-treated diabetic subjects and related to their degree of metabolic control, as assessed by glycosylated hemoglobin (HbA1) concentration. All diabetic subjects had antibodies to insulin, but there was no relationship between any of the antibody binding characteristics and HbA1 level: 47% possessed PP antibodies; mean +/- SEM HbA1 in these patients was 14.5 +/- 0.3%, identical to those without PP antibodies (14.5 +/- 0.4%); 10% had G binding antibodies with HbA1 levels of 14.6 +/- 0.8%, similar to those without G antibodies. No subject possessed SRIF antibodies. This lack of correlation between antibody characteristics and metabolic control makes it unlikely that, in the majority of patients, treatment with a less immunogenic insulin (HPPI) versus conventional insulin will result in improved diabetic control.

We have suggested that the order of cellular vascular perfusion within the islet is important in the regulation of islet hormone secretion. Anatomically, the A and D cells appear to be randomly dispersed throughout the mantle. Although islet capillary blood flow is known to be from the B-cell core to the A- and D-cell mantle, it has not yet been established whether the cells of the mantle may influence one another vascularly. Rat pancreata were perfused in vitro anterogradely and retrogradely with or without glucagon antibody in order to determine the order of cellular perfusion and interaction between the A and D cells in the islet mantle. Anterograde infusion of glucagon antibody did not affect insulin secretion, but rapidly decreased somatostatin secretion -46 +/- 8%, (p less than 0.005). Retrograde infusion of glucagon antibody decreased insulin secretion (-27 +/- 8%, p less than 0.005) but had no effect upon somatostatin secretion. This study not only confirms a core to mantle islet perfusion but also establishes that the A cell precedes the D cell in the terms of vascular perfusion. Thus within the islet, vascular borne insulin regulates the release of glucagon, which in turn, regulates the release of somatostatin. Somatostatin is vascularly neutral owing to its downstream position in the sequence (B to A to D) of cellular perfusion.

Sensitivity and detection efficiency of immunocytochemical methods were tested on cytochemical models and tissue material, respectively. Use of silver intensification procedures revealed that staining with immunogold reagents could be rendered equally or even more sensitive than the standard peroxidase-antiperoxidase (PAP) method. Further increases in sensitivity with both methods could be obtained by double application of the primary antiserum. Combined use of the immunogold techniques and the PAP method with development in diaminobenzidine and subsequent silver intensification resulted in the most sensitive procedures. The procedures were applied to a wide variety of tissue preparations, including whole mount preparations of the external longitudinal muscle layer of the gut wall and were found not to produce any unspecific staining in any tissue tested. Use of immunogold-silver and, particularly of the combined immunogold-silver-PAP methods may be valuable for analyzing tissues and tumours containing small amounts of antigen, for testing the quality of immunogold staining procedures intended for ultrastructural studies and for electroblotting techniques.

The passive transfer of rabbit anti-glucagon antibodies into mice induces periinsular and intrainsular inflammation and hyperplasia of alpha cells. Active immunization of rabbits with heterologous glucagon induces alpha cell hyperplasia.

High-titer (1:30,000) antiglucagon antibodies were detected in a 14-month-old patient with nesidioblastosis who had been treated by intramuscular long-acting glucagon since age 5 months. In this patient no correct equilibrium of blood glucose level could be obtained without maintaining glucagon treatment, even after subtotal pancreatectomy. Antiglucagon antibodies titer remained stable at age 18 months.

A method is described whereby commercially available radioimmunoassay-grade antibodies specific for the polypeptide hormones calcitonin, gastrin, glucagon, and somotastatin are used to detect these antigens on paraffin sections of routinely fixed tissue. The hormone antibodies are applied to deparaffinized tissue sections as the primary specific immune sera using the standard peroxidase technic. The use of these hormone antibodies to detect their respective antigens has proved valuable in demonstrating polypeptide forming tumor cells in pathologic specimens.

Two small peptide antigens, glucagon and enkephalin (5-L-leucine), were covalently immobilised using either glutaraldehyde or bis-(sulphosuccinimidyl) suberate to an adsorbed layer of phenylalanine-lysine copolymer (PL) or partially acetylated PL (APL) on polystyrene. Both copolymers formed stable layers, particularly APL after adsorption from solution in distilled water. Adsorption of the copolymers under these conditions and subsequent coupling of the antigens yielded solid phases with low non-specific immunoglobulin binding characteristics in an enzyme-linked immunosorbent assay (ELISA) to detect peptide-specific antibodies in rabbit serum. The signal-to-noise ratio in this ELISA was dependent on the combination of copolymer, antigen and coupling reagent employed. Removal from the solid-phase of weakly bound antigen by washing with solutions containing Tween 20 or sodium dodecyl sulphate (SDS) increased assay sensitivity, which was 2-4-fold greater than when simple antigen adsorption was utilised. In the ELISA, the coefficient of variation was lower when covalent antigen coupling was employed.