Isolation and structural characterization of insulin, glucagon and somatostatin from the turtle, Pseudemys scripta

The chelonians occupy an important position in phylogeny representing a very early branching from the ancestral reptile stock. Hormonal polypeptides in an extract of the pancreas of the red-eared turtle were purified to homogeneity by reversed phase HPLC and their primary structures were determined. Turtle insulin is identical to chicken insulin. Turtle glucagon differs from chicken glucagon by the substitution of a serine by a threonine residue at position 16 and from mammalian glucagon by an additional substitution of an asparagine by a serine residue at position 28. Turtle pancreatic somatostatin is identical to mammalian somatostatin-14. The crocodilians are phylogenetically much closer to the birds than are the chelonians. Alligator insulin, however, contains three amino acid substitutions relative to chicken insulin. Thus, caution is required when inferring phylogenetic relationships based upon a comparison of amino acid sequences of homologous peptides.     

Immunohistochemical colocalization of glucagon,serotonin, and aromatic L-amino acid decarboxylase in islet A cells of chicken pancreas

Summary The identity of monoamine-emitted, formaldehyde-induced fluorescence in some pancreatic islet cells was studied in pancreatic tissue of male chickens by fluorescence and immunohistochemistry either on the same tissue section or on serial tissue sections. Pancreatic islet cells emitting intense formaldehyde-induced fluorescence also react immunohistochemically with antisera directed against glucagon, serotonin and aromatic L-amino acid decarboxylase. These results show that chicken pancreatic islet A cells contain glucagon, serotonin, and aromatic L-amino acid decarboxylase, an enzyme involved in the synthesis of serotonin. The islet B cells identified with anti-insulin immunoreactivity, which displayed a very weak formaldehyde-induced fluorescence, did not react with anti-serotonin serum.     

The contribution of the pancreas and the intestine to the regulation of lipolysis in birds. I. Existence of highly potent lipolytic factors in ileum extracts.

Duck intestinal extracts were separated into three main peaks of proteins by chromatography on P6 BioGel columns. The first, which crossreacted with glucagon antibodies, gut "GLI", did not stimulate lipolysis in isolated chicken adipocytes. Both second and third peaks, corresponding to smaller molecular-size peptides were even more lipolytic than pancreatic glucagon.     

Interactions of glucagon and related peptides with chicken adipose tissue.

The effect of glucagon, Vasoactive Intestinal Polypeptide (VIP), secretin and gut glucagon on the cyclic adenosine 3'5' monophosphate (cAMP) level, and on the specific binding of these 125I-peptides to the adipocyte plasma membrane was measured in chicken adipocytes and compared to the results obtained in rat adipocytes. The displacement of 125I-glucagon from its specific sites was observed with about the same concentration of unlabeled hormone in fat cell plasma membranes of both species. However, the rise in cAMP induced by glucagon was much higher in chicken than in rat adipocytes. In chicken fat cells unlike rat fat cells, the cAMP accumulation elicited by glucagon was maintained during at least 60 min even in the absence of theophylline. Theophylline at 1-10 mM potentiated the glucagon-stimulated cAMP levels in rat fat cells, but had only a slight effect, if any, in chicken adiposyces. Porcine VIP, secretin or gut glucagon exerted no detectable action on the cAMP level of chicken adipocytes. The lack of cAMP accumulation was in good agreement with the absence of binding of 125I-VIP and 125I-secretin by chicken plasma membranes. These findings suggest that: 1) the difference of glucagon effect in rat and chicken fat cells results from variations in the rate of degradation of cAMP rather than from differences in the specific binding of glucagon between the two species; 2) the use of chicken fat cells is suitable to discriminate between glucagon and structurally related peptides from mammals.     

Purification and primary structure of ostrich pancreatic polypeptide

Pancreatic polypeptide has been isolated from ostrich pancreas by gel filtration, ion exchange chromatography and high pressure liquid chromatography. The ostrich peptide contains 36 amino acids and has an amino acid composition similar to pancreatic polypeptide of other avian species. The primary structure of ostrich pancreatic polypeptide differs from that of the chicken peptide only at residues 3 and 18 where the ostrich peptide contains an alanine and a valine residue compared to the serine and isoleucine residues found in those positions in the chicken peptide.     

Immunological properties and biological activity of chicken glucagon

A comparative study of avian (chicken) glucagon and commercial preparations of cattle glucagon was carried out with the view of studying the evolution of polypeptide hormones in some vertebrate species. A chromatographic procedure for obtaining crystalline hormone preparations from chicken tissues was developed. Study of immunological properties of chicken glucagon in radioimmune systems with highly specific antisera to mammalian glucagon revealed that the immunoreactivity of chicken hormone preparations purchased from CNR (USA) and RSL (USA) makes up to 40% and 60% of that of mammalian glucagon. Estimation of biological activity of the hormones by their ability to activate adenylate cyclase in a test system with plasma membranes of chicken and rat liver as well as to stimulate lipolysis in a test system with chicken adipocytes revealed that chicken glucagon possesses a biological activity within the same concentration range as its mammalian counterpart, i. e., 2.9 X 10(-10)-1 X 10(-5) M. In the majority of cases the effect of chicken glucagon taken in the above concentrations was less pronounced than that of the mammalian hormone. The data obtained suggest that even one amino acid substitution in the chicken glucagon molecule (in comparison with mammalian glucagon) affects the immunological properties of the hormone and its biological activity.     

Histological analysis of glucagon-like peptide-1 receptor expression in chicken pancreas

Glucagon-like peptide-1 (GLP-1) released from intestinal L cells in response to nutrient ingestion inhibits both gastrointestinal emptying and gastric acid secretion and promotes satiety. The main biological effect of GLP-1 is the stimulation of insulin secretion (thereby fulfilling the criterion for an incretin hormone) in order to reduce blood glucose levels in mammalian species. Chicken GLP-1 receptor (cGLP-1R) has also been identified in various tissues by gene expression analysis. Although certain effects of GLP-1 in mammals and birds are consistent, e.g., inhibition of food intake, whether GLP-1 has the same insulinotropic activity in chickens as in mammals is debated. Moreover, the expression of cGLP-1R in chicken pancreatic B cells has not been reported. The localization of cGLP-1R and its mRNA in pancreatic islets is studied by triple-immunofluorescence microscopy and in situ hybridization. Triple-immunofluorescence microscopy with antisera against cGLP-1R, somatostatin and insulin or glucagon revealed that cGLP-1R protein was exclusively localized in D cells producing somatostatin in chicken pancreatic islets. The D cells were localized in peripheral areas of the pancreatic islets and cGLP-1R mRNA was detected in the same areas, indicating that cGLP-1R mRNA was also expressed in D cells. This is the first report to demonstrate that cGLP-1R is expressed by D cells, not B cells as in mammals. Our study suggests that chicken GLP-1 performs its insulinotropic activity by a different mode of action from that of the mammalian hormone.     

Chicken glucagon: sequence and potency in receptor assay

Glucagon is a 29 amino acid peptide that is generally highly conserved. Among mammalian glucagons the only one that has been shown to differ significantly is that of the guinea pig which differs from the others in 5 of the 9 COOH-terminus amino acids. The amino acid content and partial sequencing of chicken glucagon had been reported earlier. This report describes the purification and complete amino acid sequencing of chicken glucagon and demonstrates that it differs from the usual mammalian glucagon by the replacement of asparagine at position 28 with serine. Chicken glucagon is indistinguishable from porcine glucagon in the rat liver receptor assay system.     

Effects of sulphydryl reagents on pancreatic glucagon secretion in vitro.

The effect of sulphydryl reagents on glucagon secretion of isolated Wistar rats islets was studied. Chloromercuribenzene-p-sulphonic acid (CMBS) is a strong stimulator of glucagon secretion, whereas 5,5'-dithiobis (2-nitrobenzoic acid) (DTNB) markedly inhibited the hormone release. The effect of CMBS could not be suppressed by 20 mM glucose, but in the presence of 1 mM 4-acetamido-4'-isothiocyano-stilbene-2,2'-disulphonic acid (SITS), and is independent of the glucagon content of islets. The results let us assume that sulphydryl-groups of pancreatic A-cells are involved in the regulation of glucagon secretion.     

Distribution of neurotransmitters and their effects on glucagon secretion from the in vitro normal and diabetic pancreatic tissues

The distribution of adrenergic, cholinergic and amino acid neurotransmitters and/or their enzymes were examined in both the normal and diabetic pancreatic tissues in rat using immunohistochemistry to determine whether changes in the pattern of distribution of nerves containing these neurotransmitters will occur as a result of diabetes mellitus. In addition to this, the effect of noradrenaline (NA), adrenaline (ADR), acetylcholine (ACh) and gamma-amino butyric acid (GABA) on glucagon secretion from the isolated normal and diabetic pancreatic tissues was also investigated. Pancreatic fragments from the tail end of normal and diabetic rats were removed and incubated with different concentrations (10(-8)-10(-4) M) of these neurotransmitters. Glucagon secretion into the supernatant was later determined by radioimmunoassay. NA at 10(-6) M evoked a three-fold increase in glucagon secretion from normal pancreatic tissue fragments. In diabetic pancreatic tissue, NA at 10(-6) M was able to increase glucagon secretion 1.5 times the value obtained from diabetic basal. ADR (10(-8) M) increased glucagon secretion slightly but not significantly in normal pancreatic tissue. ADR inhibited glucagon secretion from diabetic pancreas at all concentrations. ACh (10(-8) M) induced a five-fold increase in glucagon secretion from normal pancreatic tissue. In a similar way, ACh evoked a two-fold increase in glucagon secretion from diabetic pancreas at 10(-4) M. In normal pancreatic tissue, GABA produced a slight but not significant increase in glucagon secretion at 10(-4) M. In contrast to this it inhibited glucagon secretion from diabetic pancreatic tissue fragments at all concentrations. In summary, tyrosine hydroxylase- and choline acetyltransferase-positive nerves are equally well distributed in both normal and diabetic rat pancreas. There was an increase in the number of glucagon positive cells and a decrease in the number of GABA-positive cells in diabetic pancreas. NA and ACh have a potent stimulatory effect on glucagon secretion from normal pancreatic tissue fragments, whereas ADR and GABA produced a small but not significant increase in glucagon secretion from normal pancreas. NA and GABA stimulated glucagon secretion from diabetic pancreas. In contrast, ADR and ACh inhibited glucagon secretion from diabetic pancreas. Neurotransmitters vary in their ability to provoke glucagon secretion from either normal or diabetic pancreas.     

Cyclic-AMP-dependent phosphorylation of glicentin

Highly purified glicentin , a 69-amino-acid-residue peptide isolated from porcine intestine that contains the full sequence of glucagon and is probably biosynthetically related to glucagon, is a substrate for cyclic-AMP-dependent protein kinase in a cell-free system, Glicentin-related pancreatic peptide (residues 1–30 of glicentin) and glucagon were not phosphorylated under the same reaction conditions. It is postulated that the serine residue at position 34 of glicentin (position 2 of glucagon), t h a t is part of the sequence Lys.Arg. His.Ser., is the probable site of phosphorylation.     

Isolation and characterization of exendin-4, an exendin-3 analogue, from Heloderma suspectum venom. Further evidence for an exendin receptor on dispersed acini from guinea pig pancreas.

The recent identification in Heloderma horridum venom of exendin-3, a new member of the glucagon superfamily that acts as a pancreatic secretagogue, prompted a search for a similar peptide in Heloderma suspectum venom. An amino acid sequencing assay for peptides containing an amino-terminal histidine residue (His1) was used to isolate a 39-amino acid peptide, exendin-4, from H. suspectum venom. Exendin-4 differs from exendin-3 by two amino acid substitutions, Gly2-Glu3 in place of Ser2-Asp3, but is otherwise identical. The structural differences make exendin-4 distinct from exendin-3 in its bioactivity. In dispersed acini from guinea pig pancreas, natural and synthetic exendin-4 stimulate a monophasic increase in cAMP beginning at 100 pM that plateaus at 10 nM. The exendin-4-induced increase in cAMP is inhibited progressively by increasing concentrations of the exendin receptor antagonist, exendin-(9-39) amide. Unlike exendin-3, exendin-4 does not stimulate a second rise in acinar cAMP at concentrations greater than 100 nM, does not stimulate amylase release, and does not inhibit the binding of radiolabeled vasoactive intestinal peptide to acini. This indicates that in dispersed pancreatic acini, exendin-4 interacts only with the recently described exendin receptor.     

Lack of feedback regulation of cyclic 3':5'-AMP accumulation by free fatty acids in chicken fat cells.

Fat cells isolated from the mesenteric adipose tissue of chickens (pullets) responded to glucagon with an increase in lipolysis and a sustained rise in cyclic adenosine 3':5'-monophosphate (cyclic AMP) over a 30-min incubation. The prolonged accumulation of cyclic AMP due to glucagon in chicken fat cells was primarily intracellular. In addition, there was little increase in cyclic AMP accumulation due to theophylline alone or potentiation of the increase due to glucagon. These data indicate that chicken fat cells, unlike rat fat cells, are relatively insensitive to theophylline. Neither lipolysis nor cyclic AMP accumulation by chicken fat cells was inhibited by free fatty acid to albumin ratios (3 to 7) which markedly reduced both events in rat fat cells. However, in the absence of albumin from the medium, lipolysis in chicken fat cells was reduced, but not to the same extent as in rat fat cells. Chicken fat cells did accumulate more intracellular free fatty acids in response to lipolytic agents than did rat fat cells. The uptake of oleate by rat and chicken fat cells was identical. Glucagon-induced accumulation of cyclic AMP by chicken fat cell ghosts was unaffected by added oleate. Under identical conditions glucagon-induced adenylate cyclase activity of rat fat cell ghosts was markedly inhibited by added oleate. Triglyceride lipase activity of the pH 5.2 precipitate from a 40,000 x g infranatant of homogenized fat cells from chickens was less sensitive than that from rat fat cells to the ratio of oleate to albumin. These results suggest that the maintenance of cyclic AMP levels in chicken fat cells incubated with lipolytic agents results from the relative insensitivity of chicken fat cells to free fatty acid inhibition of cyclic AMP accumulation.     

Isolation of peptide hormones from the pancreas of the bullfrog (Rana catesbeiana). Amino acid sequences of pancreatic polypeptide, oxyntomodulin, and two glucagon-like peptides

Insulin, pancreatic polypeptide, glucagon, oxyntomodulin, and two distinct glucagon-like peptides were isolated from acidic ethanol extracts of bullfrog pancreas by gel filtration followed by high pressure liquid chromatography. The amino acid sequences of pancreatic polypeptide, oxyntomodulin, and both glucagon-like peptides were determined. Frog pancreatic polypeptide contains 36 amino acid residues and has a COOH-terminal phenylalaninamide. It is more homologous with human pancreatic polypeptide (61%) than other characterized members of this family of peptides. Frog glucagon has an amino acid composition identical to the NH2-terminal 29 residues of the larger, more abundant oxyntomodulin and was not sequenced. The finding of a single form of glucagon and oxyntomodulin, but two glucagon-like peptides in frog pancreas extract is similar to that found or deduced for mammals.     

Iodoglucagon. Preparation and characterization.

Iodinated derivatives of glucagon containing an average of 1 to 5 g-atoms of 127I per mol have been prepared by reacting the hormone with increasing amounts of iodine monochloride. Their iodoamino acid composition has been determined by ion-exchange chromatography and electrophoresis, following hydrolysis by pronase. Iodination of the two tyrosyl residues occurs first and is nearly complete after addition of a 4-fold molar excess of ICl. Iodination of the single histidyl residue is a later event and does not exceed an average of one atom per residue. Hydrolysis of iodoglucagon by trypsin and subsequent separation of the iodotyrosyl peptides shows that iodine is equally distributed between tyrosyl residues 10 and 13. Crude iodoglucagon containing an average of 1 g-atom of iodine per mol has been resolved into several components of differing iodine content and iodoamino acid composition by chromatography on DEAE-cellulose. Monoiodoglucagon isolated by this procedure shows a single band when analyzed by polyacrylamide gel electrophoresis. Iodoglucagons containing an average of 1 to 4 g-atoms of iodine per mol are more potent than native glucagon in their ability to stimulate adenylate cyclase activity and to bind to glucagon receptors of liver cell membranes of the rat. The maximal increase in biological potency occurring upon iodination is about 5-fold with respect to adenylate cyclase activity, and 2-fold with respect to binding to receptors; tetra and triiodinated derivatives show, respectively, the highest potency. Similar effects occur whether inactivation by liver membranes is inhibited or not, indicating an enhancement in the intrinsic affinity of iodoglucagon for the receptors. Iodination beyong 4 g-atoms per mol slightly decreases the affinity of the hormone for adenylate cyclase and for the receptors. Iodination causes a 2-20 fold decrease in the ability of liver plasma membranes and of blood plasma to inactivate glucagon in vitro; these effects correlate with the degree of iodination. With liver microsomal membranes, a decrease in glucagon inactivation occurs only at iodine contents exceeding 4 g-atoms per mol, and lower degrees of iodination result in opposite effects. Monoiodination causes a 4-6-fold increase in the plasma concentration of glucagon within the first 18 min following a single intrvenous injection of the hormone to rats. More extensive iodination results, in addition, in a marked decrease in the rate of dissappearance of glucagon from the blood. The immunological reactivity of glucagon is little affected by monoidination, but strongly depressed by higher degrees of iodination...     

Enzyme immunoassay of pancreatic glucagon at the picogram level using beta-D-galactosidase as a label.

An enzyme immunoassay of pancreatic glucagon was established by using E. coli beta-D-galactosidease [EC 3.2.1.23] as a marker. In order to increase the sensitivity of the immunoassay, different peptides obtained from glucagon fragments were used to produce the enzyme conjugate and the immunogen. Antiserum N6E raised against C-terminal fragment peptide (15-29) could be diluted to more than 1 : 100,000 in the assay and was highly specific for pancreatic glucagon. The antiserum reacted well with the C-terminal fragment peptide (21-29) as well as another fragment peptide (15-29) and pancreatic glucagon. The enzyme immunoassay using antiserum N6E and fragment peptide (21-29)-enzyme conjugate could detect as little as 1 to 2 pg of glucagon. The mean recovery of glucagon added to serum specimens was 104% and the coefficients of variation were 3.7-14.5% (within assay) and 9.0-18.5% (between assay).     

Isolation of a trypsin-like enzyme from Streptomyces paromomycinus (paromotrypsin) by affinity adsorption through Kunitz inhibitor-sepharose.

A trypsin-like enzyme has been isolated from the filtrate of a Streptomyces rimosus forma paromomycinus culture. Purification involves acetone fractionated precipitation, ultrafiltration on a Diaflo UM 10 membrane and affinity adsorption on to Kunitz pancreatic trypsin inhibitor linked to Sepharose. The trypsin-like enzyme (paromotrypsin) appears homogeneous by zone electrophoresis on gelatinized cellulose acetate. Specific activity toward Tos-Arg-OMe, calculated from amino acid analysis, is about 220 mu mg-1. The overall yield in activity is about 30%. The molecular weight of the trypsin-like enzyme, determined by gel filtration, is around 22,000-25,000 daltons. Electrophoretic migration on cellulose acetate strips indicates an isoelectric point around 8. Amino acid composition has been determined; the protein comprises about 210 residues on the basis of a single histidine residue per molecule. Paromotrypsin is unstable in acidic medium and is not stabilized by calcium ions. Enzymic activity towards Bz-Argo-OEt is not increased by the addition of calcium ion in contrast to the activating effect observed on bovine trypsin. Paromotrypsin is inhbited by TLCK and NPGB; it interacts with naturally occurring bovine trypsin inhibitors such as soya bean and Kunitz pancreatic inhibitors, but not with chicken ovomucoid. Proteolytic specificity, examined by hydrolysis of oxidized Kunitz pancreatic inhibitor and characterization of resulting peptides, seems similar to that of bovine trypsin.     

Effects of cysteamine administration on plasma concentration of metabolites, pancreatic glucagon and insulin in the chicken

1. The effects of subcutaneous injection of cysteamine (2-mercaptoethylamine, 300 mg/kg) were investigated in 5-6 week-old chickens. 2. In the short term (1 hr), cysteamine increased plasma levels of glucose, free fatty acids and insulin, and decreased that of alpha-amino non protein nitrogen. 3. In a longer term (17-24 hr), cysteamine increased the plasma level of glucose, did not modify those of alpha-amino non protein nitrogen, insulin and glucagon and decreased that of free fatty acids. 4. The disposal of an oral glucose load was impaired and the glucose-induced inhibition of pancreatic glucagon and stimulation of insulin release were blunted 17 hr after cysteamine administration. 5. Therefore, cysteamine exerts multiple effects on chicken pancreatic islet cells.     

Purification of peptide hormones from chinchilla pancreas by chemical assay

Glucagon was purified from chinchilla pancreas and its biological activity determined. It was isolated using a chemical assay to identify peptides with a histidyl residue at the N-terminus. Chinchilla glucagon has the amino acid sequence HSQGTFTSDYSKHLDSRYAQEFVQWLMNT. It differs from the usual mammalian glucagon by amino acid substitutions at positions 13, 18 and 21 from the N-terminus. Despite these sequence changes, its biological activity is conserved. Chinchilla glucagon has approximately the same potency as pig glucagon in stimulating liver membrane adenyl cyclase activity. Pancreatic polypeptide was also purified from chinchilla pancreas based on its Ala1 signal and has the sequence APLEPVYPGDNATPEQMAQYAAEMRRYINMLTRPRY#.     

Evidence for the existence of procolipase in chicken pancreas and pancreatic juice

Purified antibodies raised against chicken colipase were coupled to Sepharose 4B and colipase was isolated in a single step by immunoaffinity chromatography from an extract of chicken pancreas prepared under conditions where trypsin activation is avoided. The purified protein has a single amino terminal residue of alanine and its biochemical properties are similar to those of the precursor form of colipase (procolipase) previously isolated from porcine and equine pancreas or pancreatic juice. Further evidence for the existence of procolipase was obtained from kinetic studies of the hydrolysis of the Intralipid emulsion by untreated and trypsin-treated chicken pancreatic juice.