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Severe neonatal hypoglycemia with pathologic findings of diffuse nesidoblastosis of the pancreas is described in five children of both sexes from two families with unaffected parents. This appears to represent an autosomal recessive disorder of pancreatic development. Despite extensive testing, the diagnosis of hyperinsulinism was difficult in the index case of each family and delayed definitive treatment. Medical therapy with steroids and diazoxide was unsuccessful; pancreatectomy was required to treat persistent hypoglycemia. An abnormality of circulating glucagon found in one child with this disorder suggested that hyperinsulinism may not be the sole hormonal imbalance present, but rather that this disease is one of generalized disturbance of islet cell function. The history of severe, persistent neonatal hypoglycemia in an older sibling should lead the physician to investigate subsequent children for the presence of asymptomatic hypoglycemia.

After immunizations with glucagon or vasopressin, either conjugated to keyhole limpet hemocyanin or adsorbed to polyvinylpyrrolidone, both anti-hormone and anti-receptor activities were detectable in the serum of injected mice. Anti-hormone activity was identified by ELISA techniques; anti-receptor activity, by determining the ability of serum samples to compete with labeled hormone for glucagon or vasopressin receptors on rat liver plasma membranes. Anti-receptor activity appeared only after the peak anti-hormone response to each immunogen had been established, and required intensive immunizations (six to nine monthly injections). The presence of anti-idiotypic antibodies in serum samples containing glucagon or vasopressin anti-receptor activity was confirmed by demonstrating selective binding of such samples to corresponding rabbit idiotypic antibodies. Serum from mice immunized with insulin also contained anti-hormone activity, as determined by ELISA, and anti-receptor activity, as determined by noting insulin-mimicking properties in stimulating glucose transport in rat adipocytes. The anti-insulin receptor activity developed after only one boost with the hormone. These results are consistent with Jerne's network hypothesis in that the glucagon, vasopressin, and insulin anti-receptor activity may be attributed to antibodies produced in mice as part of an idiotypic-anti-idiotypic network.

Murine monoclonal antibodies against glucagon and insulin were generated by somatic cell hybridization and partially characterized. The monoclonal glucagon antibody K79bB10 exhibited no cross-reaction with gut glucagon. This antibody and the insulin antibody K36aC10 were found of very high concentration in ascites. An ascites dilution of 1:5,000 was used for immunohistochemical staining of insulin and glucagon on Bouin-fixed pancreatic tissue sections. By indirect immunofluorescence technique we could demonstrate a reduction of pancreatic insulin in Lewis rats after treatment with complete Freund's adjuvant. The glucagon staining was not altered. The results were confirmed by radioimmunoassay analysis of pancreatic insulin and glucagon content.

1. To further investigate the possible role of glucagon in appetite control, weaned rabbits were auto-immunized using a glucagon-bovine serum albumin conjugate (G-BSA). 2. At weekly intervals, the animals were weighed and blood samples collected and subsequently analysed for insulin, glucose and glucagon concentrations. Weekly food consumption was also recorded. 3. At the termination of the experimental period, each animal was subjected to a glucose tolerance test. Following this procedure, the animals were killed and the livers excised and frozen for subsequent glycogen determination. 4. No differences between the controls and auto-immunized group were found at any time for weekly weight gain, food intake, blood glucose or insulin concentrations. 5. Glucagon concentrations in the control group remain stable over the 7 week period; however, after the third week of the experiment, no glucagon could be detected in the blood of any of the auto-immunized animals. 6. The auto-immunized animals had significantly different glucose tolerance profiles and also had significantly more liver glycogen than the control group.

The ontogeny of different types of endocrine cells and nerves producing different hormonal peptides was studied by immunocytochemistry. The data indicate that several endocrine cell types are more frequent in the foetal than in the adult gastro-duodeno-pancreatic region. Some cell types were found to show an over-lapping distribution. Such distributional over-laps were more frequent in foetuses and neonates than in adults. Both parallel and non-parallel patterns of development of endocrine cells and peptide-producing nerves were detected. Together with available information on the trophic effects of the gastrointestinal hormonal peptides these findings indicate that foetal and neonatal endocrine cells may participate in the regulation of growth and morphogenesis in the gastrointestinal tract.

Excessive plasma levels of high molecular weight (MW) components of immunoreactive glucagon (IRG) have been found in three generations of a family in a pattern consistent with an autosomal dominant trait. Preliminary studies using gel filtration suggest the presence of two MW species of IRG greater than 3500 daltons in plasma of affected members who have normal oral glucose tolerance tests. The "hyperglucagonemia" in this pedigree is consonant with either a structural defect in biosynthetic precursors or an abnormality in enzyme-mediated cleavage of proglucagon.

In order to test the safety of glucagon (GG), the immunogenicity of GG was studied in rabbits and guinea pigs. Any sensitization, as revealed by anaphylactic shock, Arthus reaction, passive cutaneous anaphylaxis (PCA) or radioimmunoassay, was not demonstrated in animals immunized with GG alone. The anti-GG antibodies were detected by PCA and radioimmunoassay in two of the five animals immunized with GG mixed with Freund's complete adjuvant. Anti-GG IgE antibody production in mice was found in one of the ten mice given 570 micrograms and three of the six mice given 1,000 micrograms of GG with aluminum hydroxide gel (Alum), but other doses of GG with Alum produced no IgE antibody. These results indicate that the antigenicity of GG is very weak.

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.

Using glucagon-like peptide-1 N-terminus and C-terminus directed antisera, we investigated concentration and molecular forms of GLP-1 immunoreactivity (IR) in extracts of various tissues of the dog. GLP-1 IR measured with C-terminus-directed antiserum R2337 (GLP-1 IR-CT) was high in the ileum, appendix, jejunum, colon, and gastric fundus and body. GLP-1 IR measured with N-terminus-directed antiserum R1043 (GLP-1 IR-NT) was high only in the pancreas, and gastric fundus and body. Only GLP-1 IR-CT was found in the hypothalamus, thalamus and medulla oblongata. No immunoreactive materials were detected in the liver, spleen and kidney. Gel-filtration with Sephadex G-50 showed two peaks of both GLP-1 IR-CT and GLP-1 IR-NT, at 10kd and at the position of GLP-1 (1-36 amide) in the pancreatic extract, and one peak at 10kd in the stomach extract. Ileal extracts showed 3 peaks of GLP-1 IR-CT at 10kd, at the position of GLP-1(1-36 amide) and GLP-1(7-36 amide), respectively, but GLP-1 IR-NT was coeluted with GLP-1(1-36 amide). Hypothalamic extracts showed a single peak at the position of GLP-1(7-36 amide). These results suggest that processing of preproglucagon differs in different organs, and that the main GLP-1-related products are a large molecular form and GLP-1(1-36 amide) or GLP-1(1-37) in the pancreas, and GLP-1(7-36 amide) or GLP-1 (7-37) in the ileum and hypothalamus.

The C-terminal region-sepcific anti-glucagon sera were raised in rabbits using as immunogen, and conjugate of BSA and a C-terminal fragment of pancreatic glucagon. The hapten was prepared by trypsin digestion of the glucagon, which was proved to be a 1:3 mixture of glucagon (18--29) and (19--29). Six rabbits were immunized by subcutaneous injection of an emulsion of the conjugate with complete Freund's adjuvant and five of the rabbits produced antibodies to the glucagon (GC-1, GC-2, GC-3, GC-5 and GC-6). For comparison, rabbit antisera were also produced against glucagon polymer (GA-10) and syrupy glucagon fibrils (PGA-2). All these antisera as well as the pancreatic glucagon-specific antiserum 30 K were characterized with dog gut-extract (gut-GLI) and glucagon-related peptide fragments in the radioimmunoassay systems. The assay systems utilized 125 I-monosubstituted pancreatic glucagon as tracer and human mono-component glucagon as standard. All sera of the GC-series crossreacted with the dog gut-extract very weakly and antisera GC-5 and GC-6 exhibited the lowest crossreactivities with the extract, which were shown to be as low as that of 30k. Characterization of the antiserum GC-5 with purified glucagon related fragments indicated that the major antigenic determinant located exactly in the C-terminal region of glucagon. The present results clearly showed high efficiency of the use of the glucagon C-terminal fragment as hepatenic immunogen in obtaining the C-terminal region-specific, i.e., pancreatic glucagon-specific antisera.

Because in the dog, the gastric fundus contains the largest amount of glucagon immunoreactivity (IRG), the IRG of mucosal scrapes of 105 canine stomachs was extracted by acid-ethanol and then precipitated by ether-ethanol. The IRG recovered was measured by antisera 30K, specific for glucagon and K-4023, which cross-reacts with glucagon-like immunoreactivity. Extracts of mucosa of stomach fundus were further purified by gel filtration on Bio-Gel P-30 in 3M acetic acid. One pooled fraction corresponding to marker pancreatic glucagon in its elution volume was then gel-filtered on Bio-Gel P-30 in 0.05 M NH(4)HCO(3) and yielded one IRG peak, which, however, showed three immunoreactive components on polyacrylamide disc gel electrophoresis in urea. In addition, antiserum K-4023 reacted more strongly with that peak than antiserum 30K indicating the presence of glucagon-like immunoreactivity in this fraction. Subsequent ion-exchange column chromatography on DEAE-Sephadex A-25 and then CM-Bio-Gel A allowed purification to a single protein band on disc gel electrophoresis reacting equally to both antisera 30K and K-4023. 1.5 mug of purified gastric glucagon was obtained and its biological effects were compared to those of pancreatic glucagon in isolated rat hepatocytes. When immuno-equivalent amounts (300-2,500 pg/ml) of either type of glucagon were used, the same biological responses with respect to glycogenolysis and gluconeogenesis as well as urea, lactate, and pyruvate production were observed. Liver cyclic AMP was also raised to the same extent by either one of these hormones. We conclude that this moiety of gastric IRG is apparently identical to pancreatic glucagon because (a) their molecular weights, elution properties in ion exchange chromatography, and their electrophoretic mobility are indistinguishable and (b) both hormones elicited identical biological effects in isolated rat hepatocytes.

An intravenous glucose tolerance test was carried out to compare chronic pancreatitis patients (n = 17) who had undergone partial duodenopancreatectomy with (n = 9) and without (n = 8) occlusion of the residual pancreatic duct by Prolamin. The results obtained in 10 healthy volunteers were plotted as background information reflecting the normal metabolic response. Insulin- and C-peptide secretion were greatly decreased after both resection alone, and resection plus occlusion. However, the glucose tolerance (integrated glucose; K-values) appeared relatively well preserved in the two groups. The decrease in insulin appeared more marked after resection plus occlusion as compared with the non-occluded group. It is concluded that partial duodenopancreatectomy without or with ductal occlusion impairs insulin secretion, and leaves tolerance to an intravenous glucose load relatively stable. The mechanism underlying the latter observation is unknown at present.

The effects of tolbutamide infusion (1 gm. over forty minutes) on plasma pancreatic glucagon-like immunoreactivity (PGLI), serum insulin, and blood glucose were studied in six patients with chronic pancreatitis and six matched controls.asal PGLI levels were significantly higher in the patients, despite higher fasting glucose concentrations. Tolbutamide infusion had no significant effect on mean PGLI levels in controls but was associated with significant elevation in pancreatitis patients, despite higher circulating glucose levels in the latter. The data suggest that chronic calcific pancreatitis patients hypersecrete immunoreactive glucagon, possibly from a nonpancreatic source and that this immunocreactive material may be stimulated by sulfonylureas.

The production of a mouse monoclonal antibody from a hybrid myeloma and its use for the detection of glucagon in tissue sections is reported. The hybrid clone isolated after fusion of mouse myeloma cells with hyperimmune spleen cells from a mouse previously immunized with porcine glucagon allowed us a standardized and permanent source of monoclonal antibodies in a culture cell system. The monoclonal antibody (3 GL 31) specifically reacts with pancreatic A-cells in several species including pig, rabbit, tupaia belangeri and sheep. No immunoreactivity is observed against gut cells and neurons.

The role of the endocrine pancreas in glucose production (Ra), utilization (Rd), and metabolic clearance (R'd) was investigated during acute exposure to cold in normal normothermic dogs. Two ambient temperatures (TaN = +25 degrees C and TaC = -21 degrees C) were selected. At TaC, metabolic rate and glucose turnover of the shivering dogs were 4.3 and 2.4 times, respectively, higher than in dogs resting at TaN. As compared with the pre-experimental period, somatostatin infusion at TaN induced a 25% (arterial) and 34% (portal) glucagon deficiency, while insulin concentration dropped by 59% (arterial) and 74% (portal). Similar values were obtained at TaC for glucagon (39% arterial and 47% portal) and for insulin (52% arterial and 56% portal). At TaN, these simultaneous hormonal alterations provoked a slight reduction in plasma glucose concentration which levelled down to 4.4 mM. This reduction was due to a decrease in Ra, followed by a parallel decrease in Rd whereas R'd remained unchanged. At TaC, plasma glucose concentration dropped to the same level but quickly rose again during somatostatin infusion. This rise was due to a larger reduction in Rd than in Ra, accompanied by an abrupt fall in R'd. This reduction in R'd appears to be an important mechanism able to restore euglycemia during global pancreatic hormone deficiency in cold exposed dogs.

The requirement of using homologous antisera (primary antiserum and peroxidase-antiperoxidase (PAP) complex raised in the same species) in the unlabeled antibody enzyme method has been investigated at the light and electron microscopic level using the localization of insulin, glucagon and growth hormone as model systems. Optimum immunocytochemical staining for all three antigens was observed when sheep or goat antirabbit gamma-globulin (S-ARgammaG or G-ARgammaG) were used to couple rabbit peroxidase-antiperoxidase complex with either guinea pig antisera to insulin (GP-AIS) or glucagon (GP-AGS), or monkey antisera to rat growth hormone (M-ARGH). The cross-reactivity between S-ARgammaG or G-ARgammaG and immunoglobulins in these primary antisera were substantiated by immunoelectrophoresis and radioimmunoassay. S-ARgammaG was shown to produce precipitation arcs with GP-AIS and M-ARGH that were similar to those seen when the latter were reacted with rabbit antiguinea pig gamma-globulin antiserum and goat antimonkey gamma-globulin antiserum, respectively. Radioimmunoassay results revealed that immunoprecipitation of 6-10% as compared to homologous antisera controls yielded excellent staining localization when S-ARgammaG was used for immunocytochemistry. Thus, heterologous antisera (primary antiserum and PAP complex raised in different species) may be used in the unlabeled antibody enzyme method as long as the coupling antiserum shows cross-reactivity with immunoglobulins of the primary antiserum and the PAP complex.

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.

Glucagon immunoreactivity (IRG) was measured in plasma of 8 duodenopancreatectomized patients with antiserum 30-K. Arginine infusions failed to raise plasma IRG, whereas in control subjects IRG rose 3-fold. Column chromatography revealed that the basal IRG measured in these plasmas was not due to glucagon (molecular weight 3485) but to other plasma factors, mainly of high molecular weight. This suggests that diabetes mellitus does not require the presence of glucagon to produce the clinical picture, as suggested by other authors. Plasma levels of the amino acids alanine, serine, ornithine, and arginine were significantly (p less than 0.05) elevated, the former two being gluconeogenic substrates and the latter two constituents of the urea cycle. This amino acid abnormality may be a consequence of glucagon deficiency.

To investigate the possible discrepancy between immunoreactive glucagon (IRG) levels measured by two glucagon-specific antisera, OAL-123 and 30K, fasting plasma IRG levels were measured in 30 diabetics (DM group), 56 patients with impaired glucose tolerance (IGT group) and 96 normal subjects (normal group). The fasting plasma IRG measured by OAL-123 (OAL-IRG) in DM, IGT and normal groups were 121 +/- 53, 125 +/- 43 and 119 +/- 57 pg/ml, whereas the fasting plasma IRG measured by 30K (30K-IRG) were 109 +/- 92, 92 +/- 44 and 91 +/- 68 pg/ml, respectively. There was no significant difference in the distribution of the fasting IRG levels among DM, IGT and normal groups. Although there was a statistically significant correlation between OAL- and 30K-IRG, their correlation coefficient was as small as 0.29, and 31 of 182 cases showed a large discrepancy exceeding 100 pg/ml between the two IRG values. The OAL- and 30K-IRG measured again six months later did not show a significant change, where the first and second IRG levels showed a good correlation (0.72 and 0.83 for OAL- and 30K-IRG, both p less than 0.001). The gel chromatography of the 14 plasma with fasting OAL-IRG higher than 200 pg/ml showed that the main contributor to the fasting IRG was the component of big plasma glucagon (BPG). These data suggest that there is a significant discrepancy in the fasting plasma IRG levels measured by the different glucagon antisera and that the discrepancy is caused by the different immunoreactivity of BPG to the different glucagon antisera.

Pancreatic islet-cell antibodies (ICA) are important markers for two subtypes of insulin-dependent diabetes mellitus and stain the entire islet in the standard immunofluorescence test. This could indicate either a mixture of antibodies each directed against one cell type, or a population of antibodies reacting with a single antigen common to the endocrine pancreas. In the present experiments such a common antigen was demonstrated visually by application of animal antisera raised to each of the 4 pancreatic hormones, together with ICA-positive sera in a four-layer double immunofluorescent technique employing green and red anti-Ig conjugates. Double exposure photographs demonstrated that the patients' sera reacted equally with the different endocrine cells. The ICA antigen did not cross-react with gastric glucagon- or somatostatin-cells. By contrast, human antibodies against glucagon-cells (GCA) or somatostatin-cells (SCA) reacted with discrete antigens specific to each cell type and in 50% of cases the antibodies also stained the respective endocrine cells in the gastrointestinal tract. These refined discriminatory properties of human autoantibodies may lead to a better understanding of the intracellular membrane systems in these important endocrine organs.

In order to correlate the different cell types of the human endocrine pancreas to a specific secretion product, an immunoelectron microscopic localization of the hormones whose production had been attributed to pancreatic islets was conducted. Glucagon and insulin were respectively localized in the typical A- and B-cells, whereas no subclasses of A-cells could be identified. With antibodies that reacted with the gastrin cells in the human gastric mucosa, it was not possible to detect gastrin in any of the islet cell types. In confirmation of recent results obtained by light microscopy, somatostatin was found in all the typical D-cells containing large, weakly electron-dense secretory granules. The human pancreatic polypeptide (HPP), a newly postulated hormone, was clearly associated with a fourth cell type, which is characterized by the presence of small secretory granules (100-150 nm.). These results suggest that each of the four cell types that are easily identifiable by ultrastructural observations is responsible for the production of a specific secretory product.

Jejuno-ileal bypass (JIB) and resection are associated with hyperplastic and other changes in residual functional gut. In studies on endocrine changes, circulating hormone levels are usually measured. In this study with Sprague-Dawley rats, glucagon, secretin and vasoactive intestinal polypeptide (VIP), immunoreactivities were measured in extracts of gut tissues including formerly adjacent segments of functional and bypassed jejunum and ileum. Young adult (200-220 g) and mature (404-440 g) rats received greater than 90% JIB. Though slight differences were observed in the response of the young and old rats to the operations, and between the effects of JIB and resection, the results do not indicate major involvement of secretin, VIP or gut glucagon-like immunoreactivity in the effects of surgery. Changes include increased immunoreactivity of secretin in the upper and of VIP in the lower functional gut following JIB. N-terminal-to central glucagon immunoreactivity in the functional ileum did not change substantially after operations, but was significantly lower in the non-functional than functional ileum after bypass. Plasma levels of this immunoreactivity were higher following operations and highest following resection.

Insulin-like immunoreactivity can be localized to cells of the intestine in the area of the hepatopancreas of Mytilus edulis L. No cross-reactivity can be obtained with anti-glucagon, anti-gastrin, anti-pentagastrin or anti-caerulin. The cells containing the substance immunoreactive to mammalian anti-insulin, can be restrained with paraldehyde-fuchsin.

Effects of antisomatostatin and antiglucagon sera on insulin secretion were studied in isolated pancreatic islets of rats. Addition of antisomatostatin or antiglucagon serum to the incubation medium containing 5.5 mM glucose significantly increased insulin secretion from the islets. In contrast, these effect were not apparent when the glucose concentration was elevated to 20 mM. In the presence of antiglucagon serum the free glucagon concentration in the medium was decreased, while the total glucagon was markedly increased, suggesting that the antiglucagon serum caused an increase in glucagon secretion. These results suggest that A- and D-cells may play an important role in regulating insulin release at physiologic glucose concentration.

In an attempt to ascertain whether the sympathetic nervous system modulates glucagon release in man during starvation and hypoglycemia, the influence of alpha and beta adrenergic blockade on glucagon responses was studied in young, healthy men subjected to fasting and insulin-induced hypoglycemia. Six volunteers fasted for 84 h on three separate occasions. Plasma immunoreactive glucagon (IRG), measured initially at 12 h, climbed gradually from mean levels of 54 pg/ml to a zenith of 124 pg/ml at 48 h, with maintenance of these levels for the duration of the fast. The infusion of propranolol or phentolamine throughout the terminal 24 h of the second and third fasts failed to alter the pattern of IRG release. After an overnight fast, five volunteers received insulin intravenously, which evoked a mean rise in plasma IRG levels from 63 pg/ml to a maximum of 256 pg/ml at 30 min. The concurrent administration of propranolol or phentolamine did not modify the glucagon responses to insulin-induced hypoglycemia. These data suggest that the augmented glucagon release in man during starvation or after hypoglycemia is not significantly regulated by signals from the adrenergic nervous system.

Immunoreactive glucagon and immunoreactive vasoactive intestinal polypeptide (VIP) have been demonstrated in neuronal elements in the retina of a number of species by immunohistochemistry. In the present study, the concentrations of glucagon-like and VIP-like material in retinae from different species were determined by radioimmunoassay. The retinal concentration of glucagon-like immunoreactivity was 10-35 pg/mg in goldfish, chicken, pigeon, and frog, whereas retinae from cow, pig, rabbit, and rat contained very little. Retinae from the latter four species were on the other hand rich in VIP-like material whereas retinae from cat, guinea pig, and goldfish contained very little. The glucagon-like immunoreactive material in chicken and frog retina was subjected to gel chromatography and high performance liquid chromatography (HPLC). The results indicate that the extracted protein is of similar molecular size as porcine pancreatic glucagon, distinct from porcine glicentin. VIP immunoreactive material extracted from bovine retina was similar in molecular size as authentic porcine VIP by gel chromatography.

The comparative distribution of somatostatin- and glucagon-like-containing cells in the histomorphologically different gastric mucosae of the cartilaginous fishes Heptranchias perlo, Raja asterias, Scyliorhinus canicula, Squatina aculeata, and Torpedo marmorata was immunocytochemically studied to demonstrate a possible interrelationship between these endocrine cells and/or other endocrine or nonendocrine cells. In the gastric mucosa, these open-type glucagon and somatostatin immunoreactive cells show a double localization with different morphology and interrelationships. At the bottom of gastric pits, which corresponds to a proliferative zone, spindle or pear-shaped immunopositive cells appear rather numerously and are often in close proximity to each other. In gastric glands, triangular or oval immunopositive cells never in contact with each other were detected; their numeric ratio seems to be rather constant even if their numeric frequency and distribution vary according to the histomorphological aspect of selachian gastric glands. Glucagon immunoreactive cells seem to be more related to pepsinogenic cells, while somatostatin immunoreactive cells seem to be more ubiquitous. Both cell types can present basal cytoplasmic processes. From our results we can suggest a possible regulative role exerted by these two peptides on gastric secretion and cell proliferation.

Sixteen patients (aged 3.5-14.3 years) with normal jejunal mucosa, originally diagnosed as having coeliac disease at least 18 months before, were started on gluten challenge. The 'end point' of challenge was significant deterioration in jejunal mucosa morphologically and morphometrically. Studies carried out both before and after challenge included intestinal absorption of D-xylose and glucose, and release of insulin and N-terminal glucagon-like immunoreactivity (N-GLI). After gluten challenge, there were significant increases in plasma N-GLI at both 45 (P less than 0.05) and 120 minutes (P less than 0.03) after oral glucose. Significant reduction occurred in glucose absorption at 45 minutes (P less than 0.04), in one-hour D-xylose absorption (P less than 0.01) and fasting serum cholesterol (P less than 0.01). Plasma N-GLI showed significant negative correlations with D-xylose absorption (P less than 0.003) and serum cholesterol (P less than 0.004).

To determine the character of the glucagon secretion and its modification by streptozotocin, were studied the plasma of a patient with recurrent pancreatic alpha-cell carcinoma. The plasma immunoreactive glucagon level before treatment of 4.80 ng per milliliter. Biogel column separation of the plasma immunoreactive glucagon revealed four components; the predominant component had a molecular weight of 9000 daltons and was designated as proglucagon-like. This fraction constituted 60 to 90 per cent of the total circulating immunoreactive glucagon, and had a biologic activity of 32 percent of that of an immunoequivalent amount of normal (porcine) pancreatic glucagon. After treatment with streptozocin (1.5 g per square meter) the plasma immunoreactive glucagon level decreased to 0.24 ng per milliliter. Treatment was accompanied by a marked reduction in the proglucagon-like component and the appearance of pancreatic glucagon (molecular weight of 3500 daltons) as the major post-therapy fraction. These findings support the use of streptozotocin in the management of unresectable glucagon-secreting tumors.

A 53 year old woman presented with diabetes mellitus, hyperglucagonemia (600 to 1,500 pg/ml), clinical hyperparathyroidism and an abdominal mass diagnosed on biopsy as an islet cell carcinoma. Glucagon content of the tumor was 0.78 mug/g wet weight. Hourly blood samples during a 24 hour period revealed a direct correlation between plasma glucose and glucagon. The oral administration of glucose paradoxically increased whereas the intravenous administration decreased plasma glucagon. Circulating glucagon levels were markedly increased with arginine and epinephrine infusion. Both short- and long-term administration of alpha adrenergic blockade depressed the glucagon response to epinephrine infusion. In contrast, long-term alpha adrenergic blockade increased glucagon secretion despite improved glucose tolerance during a second 24 hour study. Although the patient demonstrated overt clinical and chemical findings of hyperparathyroidism, parathyroid hormone (PTH) was not detected in her plasma. The pattern of tumor growth was consistent with an origin from pancreatic islets. We conclude that (1) the tumor was responsive to physiologic stimuli known to affect glucagon secretion; (2) elevations of plasma glucagon levels with oral and dietary glucose suggest regulation of secretion by intestinal factors; and (3) improvement of glucose tolerance with alpha adrenergic blockade may be related to increased insulin secretion.

Hybridoma technology has been successfully applied to the production of monoclonal antibodies against a variety of small soluble peptides. We report herein for the first time on the development of monoclonal antibodies to pancreatic glucagon. Twenty-three stable positive hybridomas were detected by radioimmunoassay from five separate fusions and cloned by the limiting dilution method. Four selected monoclonal antibodies were all of the IgG 2a subclass type kappa and bound to protein A. One monoclonal antibody (23.8B6) was shown to be directed toward the C-terminal region and another (23.6B4) toward the N-terminal to central region of the glucagon molecule. These antibodies did not cross-react with any of the other peptides tested. Two further monoclonal antibodies (23.4A1, 22.3A6) reacted with the C-terminal third of the glucagon molecule and showed a cross-reaction with the structurally related gastric inhibitory polypeptide of 0.7% and 9.1%, respectively. All but the C-terminal monoclonal antibody 23.8B6 showed a marked cross-reaction with ileal extracts. The N-terminally directed monoclonal antibody 23.6B4 was of sufficient avidity for use in the radioimmunoassay of pancreatic glucagon and gut glucagon-like immunoreactivity in tissue extracts, being sensitive to changes of pancreatic glucagon of 2.0 fmol/tube at a final titer of culture supernatant of 1:1.4 X 10(5). In gel permeation chromatography of intestinal extracts, two major peaks were detectable (Kav 0.27 and 0.54). The present findings show that monoclonal antibodies provide sensitive tools for detecting pancreatic glucagon and gut glucagon-like immunoreactivity. They will be valuable immunoreactants for the development of immunoradiometric assays as well as for large-scale immunoaffinity purification of gut glucagon-like immunoreactivity.

A primary ovarian carcinoid composed of both trabecular and strumal types was studied by histochemical, immunocytochemical, and biochemical techniques. High contents of glucagon, secretin, and calcitonin were demonstrated in the tumor homogenate. All of the tumor cells, irrespective of histologic type, showed properties of argyrophilia and neurosecretory granules on electron microscopy. Glucagon-producing cells were positive in trabecular carcinoid by immunoperoxidase techniques. Bio-Gel P10 gel filtration showed that the molecular weight of major immunoreactive glucagon in tumor was 20,000. It migrated faster than true glucagon after polyacrylamide gel electrophoresis. No clinical symptoms of glucagonoma developed.