The use of perfused rat intestine to characterise the glucagon-like immunoreactivity released into serosal secretions following stimulation by glucose.

Isolated perfused intestine of rat was used to demonstrate the glucose-stimulated release of glucagon-like immunoreactivity (GLI) into serosal secretions. The released GLI was characterised using immunoaffinity chromatography on columns of immobilised antibodies specific for the N (residues 1 to 18) and for the C (residues 19-29) terminal portions of glucagon followed by gel-filtration. The immunoreactivity was present in a variety of molecular species. These include a large GLI which has a molecular weight about 12000 and binds to antibodies specific for the N-terminal portion of glucagon and two polypeptide fractions with molecular weight closer to that of glucagon. While one fraction of the small GLI boun both to antibodies specific for the C-terminal and N-terminal portions of glucagon the other bound only to the former antibodies. The relevance of these findings to the origins of circulating GLI and the possible precursor relationship between large and other forms of GLI is discussed.     

Dynorphin A immunoreactivity in human cerebrospinal fluid

Dynorphin A immunoreactivity in human cerebrospinal fluid has been characterized. Large quantities of the fluid were fractionated by molecular sieving on a Sephadex G-50 column and analyzed by radioimmunoassay. Active fractions were further analyzed by means of HPLC and an enzyme radioimmunoassay procedure for identification of Leu-enkephalin-Arg6 sequences. Several dynorphin A-active components of varying sizes were identified. However, most immunoreactive material derived from species of higher molecular weight (Mr 3000 and 5000) than authentic dynorphin A (Mr 2000). The Leu-enkephalin core was found to reside in both these large structures.     

Glucagon-like immunoreactivity in hypothalamic neurons of the rat

Summary Antisera specific for three different regions of pancreatic proglucagon were used to examine the distribution of such immunoreactivity in rat hypothalamus. Neurons in the supraoptic and paraventricular nuclei were immunoreactive with an antiserum against glucagon, but not with antisera directed towards the aminoterminal region of proglucagon (glicentin) or the glucagon-like peptide I sequence in the carboxyl-terminal region of proglucagon. These findings confirm a previous report of glucagon-like immunoreactivity in the supraoptic and paraventricular nuclei, but indicate that, while this material is immunochemically related to glucagon, it is not derived from a proglucagon-like precursor.     

The molecular forms of immunoreactive glucagon secreted by the isolated,perfused dog pancreas

The immunoreactive glucagon (IRG) in the plasma-free effluent of the arginine stimulated isolated dog pancreas was purified by immunoaffinity chromatography and characterized with respect to molecular weight. Only a 3500 dalton component was secreted from the pancreas during the first four minutes of stimulation but immunoreactive material having a molecular weight of between 150,000 and 200,000 was isolated from the secretions after prolonged stimulation. This component (which corresponds in size to the incompletely characterized “big plasma glucagon”) was dissociated to a 3500 dalton component and nonimmunoreactive material by 6 M guanadinium chloride. Components of molecular weight 9000 and 2000, which are found in plasma, and components with the immunological properties of gut GLI, were not identified in the pancreatic secretions.     

Effects of cooling and warming rates during vitrification on fertilization of in vitro-matured bovine oocytes

The purpose of this study was to clarify the relationship of cooling rates (CR) and warming rates (WR) during vitrification with postwarming viability of in vitro-matured bovine oocytes. In Experiment 1, oocytes were vitrified in a solution containing 7.2 M ethylene glycol and 1.0 M sucrose by use of open-pulled glass capillaries with five different outer diameters and were warmed by placement of the capillaries into 0.25 M sucrose solution. The capillaries of 2000-, 1400-, 1000-, 630-, and 440-mm diameters provided CR of 2000, 3000, 5000, 8000, and 12,000 degrees C/min and WR of 5000, 8000, 17,000, 33,000, and 62,000 degrees C/min, respectively. In oocytes vitrified in capillaries of 1400-mm diameter (CR, 3000 degrees C/min; WR, 8000 degrees C/min), the morphological survival rate (86% of vitrified), penetration rate (79% of inseminated), and normal fertilization rate (69% of penetrated) were higher or tended to be higher than those in the other vitrification groups. In Experiment 2, oocytes cooled at 2000, 3000, or 12,000 degrees C/min were warmed at 8000 degrees C/min, and oocytes cooled at 3000 degrees C/min were warmed at 5000, 8000, or 33,000 degrees C/min. Among these CR-WR combinations, cooling of oocytes at 3000 degrees C/min regardless of the WR resulted in higher postwarming survival. These results indicate that survival of in vitro-matured bovine oocytes after vitrification and subsequent warming is improved by a slightly rapid cooling rate in open-pulled glass capillaries compared to that obtained in conventional straws.     

Somatostatin precursors: evidence for presence in and release from rat median eminence and neurohypophysis

Characterization of somatostatin-like immunoreactivity in rat median eminence and neurohypophysis by gel chromatography yielded two high molecular weight forms in addition to the tetradecapeptide somatostatin. The two larger molecules comprised 5% and 35% of the total tissue immunoreactivity, showed molecular weights of 25000 and 4000 dalton and were both releaseable in vitro in response to depolarizing stimuli. Their characteristic elution volumes remained unchanged after treatment with dithiothreitol or boiling in 8 M urea. Gentle trypsinization of the 25000 dalton molecule resulted in partial conversion into immunoreactive material coeluting with the tetradecapeptide somatostatin. Since the antibody employed in these studies is specific for the central and C-terminal portions of the tetradecapeptide somatostatin the present data suggest that both high molecular weight forms represent N-terminal extensions of somatostatin and that the 25000 molecular weight material might represent a prohormone for somatostatin.     

Oxyntomodulin (glicentin-(33-69)): pharmacokinetics, binding to liver cell membranes, effects on isolated perfused pig pancreas, and secretion from isolated perfused lower small intestine of pigs

The pharmacokinetics of purified synthetic oxyntomodulin were studied after infusing it into euglycaemic pigs at two rates. The elimination of the peptide from plasma was characterized by two components, a fast one (t1/2 7.2 +/- 0.6 min) and a slow one (t1/2 20.4 +/- 3.8 min) (mean +/- S.E.M., n = 7). The metabolic clearance rate was independent of infusion rate (6.96 +/- 0.99 vs 7.44 +/- 0.98 ml/kg . min (mean +/- S.E.M., n = 7). The synthetic peptide bound to pig hepatic glucagon receptors, but with approximately 2% of the affinity of glucagon, and showed insulinotropic and somatostatinotropic effects when infused into isolated perfused pig pancreases at concentrations higher than 10(-10) M. A dose-dependent increase was also shown for pancreatic glucagon output. A naturally occurring peptide, identified as oxyntomodulin by gel filtration and HPLC, was released into the circulation from the pig lower small intestinal mucosa upon intraluminal administration of glucose, and represented 25 +/- 3.8% of the secreted glucagon-like immunoreactivity. 11 +/- 2.3% of the secreted glucagon-like immunoreactivity was indistinguishable from glucagon itself upon gel filtration; thus at least 36% of the glucagon-like immunoreactivity secreted from the intestinal mucosa is already in an active form.     

A general procedure for analysis of proenkephalin B derived opioid peptides

Tryptic digestion followed by radioimmunoassay for (Leu)enkephalin-Arg6 has been used in this study as a general method to detect the presence of all possible products containing the enkephalin sequence from the opioid peptide prohormone, proenkephalin B. Tissue extracts of human hypothalamus and pituitary were examined. Gel filtration was used to separate the different precursor products according to molecular weight. The elution profile was also monitored with highly sensitive radioimmunoassays for dynorphin A and dynorphin B, respectively. Immunoreactive dynorphin A appeared in three peaks with the approximate molecular weight of 1000, 2000 and 5000. Immunoreactive dynorphin B partly occurred in other peaks, 1500, 5000 and 10 000 dalton. Profiles obtained by measuring immunoreactive (Leu)enkephalin-Arg6 in all fractions from gel filtration after trypsin digestion showed a more complex pattern compared to the profiles of immunoreactive dynorphin A and dynorphin B. The major peaks coincided with dynorphin A and dynorphin B but high levels of immunoreactive (Leu)enkephalin-Arg6 were also generated from higher molecular weight regions (MW greater than 5000).     

Species difference of glucagon-like materials in the brain

Glucagon-like materials were found in the canine, porcine, bovine, rat and human brain. Both glucagon immunoreactivity measured with an antibody against the C-terminal portion of glucagon and glucagon-like immunoreactivity measured with an antibody against the N-terminal portion of glucagon were detected in the thalamus-hypothalamus, brain stem and spinal cord, but not in the cerebrum, basal ganglia, pituitary gland or cerebellum. The distribution of glucagon-like material in the brain was common in all tested mammals.     

Half life of gastrointestinal glucagon-like immunoreactive materials.

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

Characterization of a heat stable, 12,000 Da, glucagon-like immunoreactive (GLI) peptide from porcine ileum that stimulates hepatic glucose production in vivo and in vitro

Glucagon-like immunoreactivity (GLI) was extracted from porcine ileal mucosa with boiling 2 M HAc followed by elution on DEAE A-50 and fractionation on Sephadex G-50 F. Intact GLI was isolated from mesenteric blood by fractionation of several plasma samples from a mesenteric vein of a dog on Sephadex G-50 M followed by refractionation of the pooled GLI from these columns on G-50 F. Analysis of the semipurified mucosal and plasma GLI on Sephadex G-50 SF, eluted with 0.1 M Tris/HCl + 8 M urea, pH 7.5, demonstrated a single, sharp peak of GLI with a relative molecular mass (Mr) between 12,000 and 13,000. Electrophoresis on PAGE gels at acid pH with 2 M urea demonstrated a single charged GLI species in both the plasma and mucosal fractions. Stimulation of the release of GLI from a loop of ileum isolated in situ in an anesthetized dog followed removal of the known sources of glucagon (stomach, pancreas, and duodenum) resulted in an immediate and sustained increase in hepatic glucose production. The isolated GLI from ileal mucosa (5 X 10(-8) M) stimulated gluconeogenesis from 10 mM [14C]alanine in hepatocytes isolated from fed rats. The stimulation was equal to 25% of the maximal stimulation observed with 10(-8) M glucagon. These experiments demonstrate that the ileum synthesizes and secretes a GLI peptide with a Mr of approximately 12,000 that stimulates hepatic glucose production in vivo and in vitro.     

Immunohistochemical localization of glucagon-like peptide 1. Use of poly- and monoclonal antibodies

We report the use of poly- and monoclonal antibodies to study the immunohistochemical distribution of glucagon-like peptide-1 immunoreactivity (GLP-1-IR) in various tissues. The polyclonal antibodies against GLP-1 reacted with pancreatic A cells, enteroglucagon (L) cells in the gut, and some neurons in the central nervous system of all species tested. In pancreas and gut the monoclonal antibodies against GLP-1 exhibited a similar, but species specific distribution, relative to the polyclonal antibodies. The colocalization of GLP-1 and glucagon immunoreactivity in pancreatic, intestinal, and nervous tissues is in agreement with previously reported findings that both peptides are part of a single precursor molecule (preproglucagon).     

Endogenous inhibitors of specific benzodiazepine binding in the cerebral cortex of the bull

Chromatography of an acidic extract from bovine brain cortex on a Sephadex G-10 column demonstrated 7 fractions capable of inhibiting specific binding of 3H-diazepam to brain cortex membranes. It was established by ultrafiltration that the molecular weights of the inhibitors were not higher than 5000 dalton. Scatchard analysis of 4 main fractions has revealed a type of inhibition similar to the competitive one. Activity of all the fractions remained unchanged after pronase treatment.     

Enrichment of Triadic and Terminal Cisternae Vesicles from Rabbit Skeletal Muscle

An enriched triad and terminal cisternae preparation was achieved from skeletal muscle through alterations of the differential centrifugation and muscle homogenization protocols. Both yield and specific activity (pmoles of radioligand binding per mg protein) were optimized for ³H-PN200-l10 (transverse tubule marker) and ³H-ryanodine (terminal cisternae marker) binding sites. By pelleting crude microsomes between 2,000 an 12,000 × g without any rehomogenizations, we improved both the yield and specific activity of transverse tubule and terminal cisternae markers in crude microsomes by approximately 4-fold to 1000–3000 pmoles binding sites (starting material: approximately 400 grams wet weight fast twitch skeletal muscle), with 10–15 pmoles/mg. Rehomogenization of the 1,000 × g pellet, which is typically discarded, allowed recovery of an additional 5000 pmoles PN200-110 binding sites and an additional 8000 pmoles ryanodine binding sites. Crude microsomes from the rehomogenized 1,000 × g pellets typically displayed specific activities of 20–25 pmoles binding/mg for both ³H-PN200-110 and ³H-ryanodine. Separation of crude microsomes on a sucrose gradient increased specific activity up to a maximum of 50 pmoles/mg in a specific fraction, a five- to ten-fold increase over standard triadic or terminal cisternae preparations. The mean specific activity for enriched triads was 30–40 pmoles/mg for both PN200-110 and ryanodine in pooled fractions, while pooled fractions of enriched terminal cisternae displayed low ³H-PN200-110 binding (3–5 pmoles/mg) and high ³H-ryanodine-specific activity (30–40 pmoles/mg).     

Response of extrapancreatic immunoreactive glucagon to intraluminal nutrients in pancreatectomized dogs

To investigate the response of extrapancreatic glucagon to intraluminal stimuli, nutrients were administered to normal and pancreatectomized dogs through a stomach tube in a fully conscious state after an overnight fast. Plasma immunoreactive glucagon was determined with antisera specific and nonspecific to glucagon and was designated as IRG and total IRG, respectively. Oral glucose load elicited a decrease in plasma IRG and a remarkable rise of plasma total IRG in a group of 6 pancreatectomized dogs, as in the control dogs. When arginine was given, both plasma IRG and total IRG significantly increased in a group of 5 pancreatectomized dogs, while only total IRG rose significantly in the normal control dogs. Butter load did not reveal any changes in plasma IRG and total IRG in a group of 5 pancreatectomized dogs, whereas only total IRG increased in the normal control dogs. It is concluded that extrapancreatic glucagon responds to intraluminal administration of nutrients, as pancreatic glucagon does. In addition, gut glucagon-like immunoreactivity increased following glucose or arginine ingestion in pancreatectomized dogs. Furthermore, the failure in response of plasma IRG and total IRG to butter load in pancreatectomized animals suggests that its intraluminal hydrolysis is important in the secretion of extrapancreatic immunoreactive glucagon.     

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

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

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

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

Characterization of a chemically tritiated large molecular weight glucagon immunoreactive protein species by lectin-affinity column chromatography and reaction with anti-glucagon antibodies

High molecular weight glucagon immunoreactive material, obtained by gel-filtration (in the presence of 6 M guanidine hydrochloride) of fetal bovine pancreatic extracts, was tritiated by reductive methylation. Concanavalin-A-Sepharose column chromatography of the radiolabeled preparation yielded a discrete Concanavalin-A-reactive, α-methyl-mannoside-displaceable radioactive peak, coinciding with the glucagon immunoreactive peak. Submission of the Con-A-reactive material to wheat germ agglutinin-Sepharose column chromatography yielded a lectin-reactive, N-acetyl-glucosamine-displaceable radioactive peak, coninciding with the glucagon immunoreactive peak. The tritiated Con-A-reactive component interacted specifically with anti-glucagon antibodies. Sephacryl S-200 gel-filtration (in the presence of guanidine hydrochloride) dissociated a ～40 kDa radioactive species from the antibody-antigen complex. These data provide direct evidence for the existence of a large molecular weight glycosylated glucagon-related protein species from the fetal bovine pancreas.     

Immunochemical characterization of a human high molecular weight — melanoma associated antigen identified with monoclonal antibodies

Sodium dodecyl sulfate polyacrylamide gel analysis of a high molecular weight (HMW) human melanoma associated antigen (MAA) defined by murine monoclonal antibodies revealed a number of distinct polypeptides ranging from 80,000 up to 280,000 daltons, in addition to an extremely heterogeneous group of components distributed over a wide range in apparent molecular weight (300,000-700,000 daltons). The 280,000 dalton and the larger heterogeneous molecular weight material are glycosylated since they are labeled with 3H-sugars. The HMW-MAA is readily solubilized in the absence of detergents and the entire series of polypeptides fractionates together in the void volume of a Sephadex G200 column. Peptide maps of the various polypeptides of the HMW-MAA, generated by Staphylococcus aureus V-8 protease, are essentially the same except that some of the proteolytic fragments derived from the lower molecular weight polypeptides (80,000 daltons) are present in greater amounts than are similar fragments derived from the larger molecular weight polypeptides; the latter finding suggests that the complexity in molecular weight of the MAA may reflect combinations of several base subunits. Proteolytic cleavage of the HMW-MAA generates a number of peptides ranging in molecular weight from 77,000 daltons to less than 12,000 daltons, which still react with monoclonal antibodies and can distinguish monoclonal antibodies specific for different antigenic determinants of this MAA.     

Immunohistochemical localization of glucagon-like peptide 1

Summary We report the use of poly-and monoclonal antibodies to study the immunohistochemical distribution of glucagon-like peptide-1 immunoreactivity (GLP-1-IR) in various tissues. The polyclonal antibodies against GLP-1 reacted with pancreatic A cells, enteroglucagon (L) cells in the gut, and some neurons in the central nervous system of all species tested. In pancreas and gut the monoclonal antibodies against GLP-1 exhibited a similar, but species specific distribution, relative to the polyclonal antibodies. The colocalization of GLP-1 and glucagon immunoreactivity in pancreatic, intestinal, and nervous tissues is in agreement with previously reported findings that both peptides are part of a single precursor molecule (preproglucagon).Supported by the DFG, SFB 90