Mammalian Glucagon Research Articles

Guinea pig glucagon differs from other mammalian glucagons

Guinea pig glucagon differs from other mammalian glucagons

Isolation and structures of coho salmon ( Oncorhynchus kisutch) glucagon and glucagon-like peptide

Glucagon and a glucagon-like peptide (GLP) containing 31 amino acids have been isolated from the principal islet of coho salmon ( Oncorhynchus kisutch) by gel filtration of acid alcohol extracts followed by HPLC, and the complete amino acid sequence of both peptides has been determined. Salmon glucagon is a simple 29 residue peptide differing at 3 positions when compared to catfish glucagon and at 8 positions when compared to porcine glucagon. Salmon GLP differs at 6 positions when compared with the N-terminal 31 amino acids of the 34 amino acid catfish GLP. Both coho salmon glucagon and GLP cross-react weakly in our mammalian glucagon radioimmunoassay and therefore this technique could not be used to determine tissue content. Glucagon and GLP isolated amounted to 156 μg/g and 350 μg/g wet tissue, respectively.

Primary structure of glucagon from an elasmobranchian fish, Torpedo marmorata

Glucagon has been isolated from the pancreas of Torpedo marmorata, an elasmobranchian cartilaginous fish, and purified to homogeneity using only reverse-phase high-performance liquid chromatography. Amino acid sequence analysis indicates that the molecule differs from mammalian glucagon at position 3 (glutamic acid for glutamine), position 16 (asparagine for serine), and position 20 (lysine for glutamine). Extracts of T. marmorata intestine and brain were associated with glucagon-like immunoreactivity determined by radioimmunoassay using antisera directed against the C-terminal and N-terminal to central regions of porcine glucagon. Although elasmobranchian and teleostean fish are believed to have diverged from the main line of vertebrate evolution at about the same time, the structure of two glucagons from the teleost, Lophius americanus (anglerfish) differ from mammalian glucagon by seven and nine residues. This study supports the assertion that the structure of glucagon has been highly conserved during evolution and suggests that the considerable morphological development of the pancreas is teleosts was associated with an accelerated rate of molecular evolution.

Amino acid sequence of Manduca sexta adipokinetic hormone elucidated by Combined Fast Atom Bombardment (FAB)/Tandem Mass Spectrometry

Combination of Fast Atom Bombardment Tandem Mass Spectrometry with Amino Acid Analysis assigns the amino acid sequence of the Manduca sexta adipokinetic hormone as pGlu-Leu-Thr-Phe-Thr-Ser-Ser-Trp-GlyNH 2. Similarities and differences with other invertebrate hormones and with mammalian glucagon are discussed.

Regulatory peptides (glucagon, somatostatin, substance P, and VIP) in the brain and gastrointestinal tract of Ambystoma mexicanum

The concentrations of immunoreactive components of glucagon, somatostatin, substance P, and vasoactive intestinal polypeptide (VIP) in the brain, stomach, and gut of the neotenic Mexican axolotl ( Ambystoma mexicanum) were determined by radioimmunoassay using antibodies of defined regional specificity. The molecular forms of the immunoreactive components were analyzed by high-performance liquid chromatography (HPLC). The concentrations and molecular forms of somatostatin and VIP in axolotl brain were comparable to the concentrations in mammals but the substance P-like immunoreactivity was resolved by HPLC into components with the retention times of physalaemin and substance P together with their oxidized forms. No glucagon-like material was detected in the axolotl brain. The concentrations of substance P and VIP in the A. mexicanum digestive tract were appreciably lower than in the mammalian digestive tract and the VIP-like material did not coelute with porcine VIP. Somatostatin-14 represented the major molecular form in the axolotl stomach and gut. The distribution and molecular properties of the glucagon-like peptides in the axolotl digestive system were markedly different from these parameters in mammalian gut. Glucagon-like material is present only in low amounts in porcine and human stomach and, the concentration of enteroglucagon (N-GLI) in the gut is at least fiftyfold greater than pancreatic glucagon (C-GLI) concentrations. The axolotl stomach, in contrast, contains high levels of glucagon-like immunoreactive material and, in both stomach and gut, the levels of C-GLI and N-GLI were comparable. The glucagon-like material was heterogeneous on HPLC and was resolved into two major components but no component with the retention time of mammalian glucagon was present. The immunochemical properties of the axolotl glucagon-like peptides indicate that they possess strong homology with mammalian glucagon in the 10–18 and 25–29 regions of the molecule.

An improved synthesis of crystalline mammalian glucagon.

Mammalian glucagon was synthesized by the stepwise solid-phase method using several improvements developed in recent years. Peptide was assembled on a 4-(oxymethyl)phenylacetamidomethyl-copoly(styrene-divinyl benzene) resin support with N alpha-t-butoxycarbonyl and benzyl-based side-chain protection for most of the trifunctional amino acids. Crude synthetic glucagon was obtained in 75% yield by deprotection and cleavage from the resin with a new modified HF procedure. Pure material was isolated in 48% overall yield by a one-step purification on preparative C18 reverse-phase chromatography. It was crystallized from aqueous solution at pH 9.2. The synthetic glucagon activated adenylate cyclase in rat liver membranes in the same manner as natural glucagon, with both achieving half-maximum activation at a concentration of 7 nM.

Human preprovasoactive intestinal polypeptide contains a novel PHI-27-like peptide, PHM-27.

Vasoactive intestinal polypeptide (VIP), a 28-amino acid peptide originally isolated from porcine duodenum, is present not only in gastrointestinal tissues but also in neural tissues, possibly as a neurotransmitter, and exhibits a wide range of biological actions (for example, relaxation of smooth muscle, stimulation of intestinal water and electrolyte secretion and release of insulin, glucagon and several anterior pituitary hormones). As the structure of porcine and bovine VIP shows several similarities to those of mammalian glucagon, secretin and gastric inhibitory peptide (GIP), VIP is considered to be a member of the glucagon-secretin family. Recently, we have found that VIP is synthesized from a precursor, pro-VIP (molecular weight (Mr) 17,500), in human neuroblastoma cells and that the primary translation product of the mRNA encoding VIP is prepro-VIP (Mr 20,000). In an attempt to elucidate the primary structure of the precursor, we have now cloned the DNA sequence complementary to the mRNA coding for human VIP and analysed the nucleotide sequence. The entire amino acid sequence of the precursor, deduced from the nucleotide sequence, indicates that the precursor protein contains not only VIP but also a novel peptide of 27 amino acids. The peptide, designated PHM-27, differs by only 2 amino acids from PHI-27, a peptide recently isolated from porcine intestine, and is also closely related in sequence to VIP.

Pancreatic preproglucagon cDNA contains two glucagon-related coding sequences arranged in tandem.

We have constructed and cloned in bacteria recombinant plasmids containing DNA complementary to the mRNA encoding a pancreatic preproglucagon (Mr 14,500), a product of cell-free translation of angler fish islet mRNAs shown previously by immunoprecipitation analyses to be a precursor of glucagon. cDNAs of 630, 180, and 120 base pairs were isolated and correspond to most of the mRNA for the preproglucagon (650 bases). The cDNAs contain a protein coding sequence of 372 nucleotides and 5'- and 3'-untranslated regions of 58 and 206 nucleotides, respectively. From the coding sequence of the cDNAs, we find that the sequence of glucagon, identical to mammalian glucagon in 20 of 29 positions, resides in the preproglucagon of 124 amino acids flanked by NH2- and COOH-peptide extensions of 52 and 43 amino acids, respectively. The peptide extensions are linked to the glucagon by Lys-Arg sequences characteristic of the sites that are cleaved during the posttranslational processing of prohormones. Notable is the finding that, following the initial Lys-Arg sequence in the COOH-peptide extension is a pentapeptide. Ser-Gly-Val-Ala-Glu, followed by another Lys-Arg and a sequence of 34 residues that shows striking homology with glucagon and the other peptides of the glucagon family--gastric inhibitory peptide, vasoactive intestinal peptide, and secretin. Thus, the preproglucagon mRNA contains two glucagon-related coding sequences arranged in tandem. The finding of Lys-Arg sequences flanking the glucagon and glucagon-related sequences suggests that these two peptides and a pentapeptide are formed in vivo by posttranslational cleavages of a common precursor.

Solid-phase synthesis of crystalline glucagon.

Mammalian glucagon was synthesized by a stepwise solid-phase method. The support was an alkoxybenzyl alcohol resin, and the biphenylylisopropyloxycarbonyl group was used for temporary alpha-amino protection. After purification by gel filtration and ion-exchange chromatography, the 29-residue hormone was readily crystallized from water at alkaline pH. The product was homogeneous and indistinguishable from natural bovine glucagon by gel electrophoresis, ion-exchange chromatography, reverse-phase high-pressure liquid chromatography, fluorescence spectroscopy, and amino acid analysis. The synthetic hormone was fully active in the rabbit hyperglycemia assay.

Synthesis of the nonacosapeptide corresponding to mammalian glucagon.

The nonacosapeptide corresponding to the entire amino acid sequence of mammalian glucagon was synthesized via the corresponding sulfoxide, [Met (O)27]-glucagon. For synthesis of the protected nonacosapeptide six subunits were prepared to serve for building blocks. The condensations of subunits were achieved by the HONB-DCC method. Finally, all the protecting groups of the protected peptide were removed by the treatment with methanesulfonic acid-anisole to give [Met (O)27]-glucagon. The sulfoxide was then treated with 3% aqueous thioglycolic acid to give mammalian glucagon. The product was successfully crystallized from dilute aqueous sodium chloride solution. The N to O acyl migration of serine and threonine residues with methanesulfonic acid treatment was studied in detail.

Synthesis of the nonacosapeptide corresponding to the proposed amino acid sequence of turkey glucagon.

The nonacosapeptide corresponding to the proposed amino acid sequence of turkey glucagon was synthesized via the corresponding sulfoxide, [Met (O)27]-turkey glucagon. For the synthesis of the protected nonacosapeptide six peptide fragments were prepared and the condensations of the fragments were achieved by the HONB-DCC method. Finally, all the protecting groups were removed by the treatment with anhydrous hydrogen fluoride-anisole to give [Met (O)27]-turkey glucagon. The sulfoxide was then treated with aqueous thioglycolic acid to give turkey glucagon, which was successfully crystallized from dilute aqueous sodium chloride solution. The biological activity, lipolysis on rat adipocytes, of the synthetic turkey glucagon was as potent as that of mammalian glucagon.

Species variations in the primary structure of glucagon

Porcine, 1 bovine, 2 and human 3 glucagons have identical sequences. The amino acid composition, crystal form, and electrophoretic behavior of rabbit, 4 camel, 5 and rat 6 glucagon strongly indicate their identity with porcine glucagon. Thus, the conservation of the primary structure of mammalian glucagon appeared to be complete. During attempts to isolate guinea pig glucagon it became clear that the glucagon from this mammal differed significantly from porcine glucagon. As guinea pig islets are used to study glucagon biosynthesis, we consider it relevant to present our experience with guinea pig glucagon. Solubility studies and ion-exchange chromatography showed that guinea pig glucagon had a much higher pI than porcine glucagon. When crude fractions of guinea pig glucagon were gel filtered the main peak of glucagon, as determined radioimmunologically with a nonspecific antiglucagon serum, eluted in front of insulin. After further purification the glucagon eluted between porcine insulin and porcine glucagon, indicating a molecular weight of 4000-5000. This was at variance with earlier observations where it eluted just in front of the insulin, but it may have been caused by protein-protein interaction which was eliminated during purification. Disc electrophoresis at pH 4.5 showed that the purified glucagon contained a main band and three minor components, all moving faster than porcine glucagon. These four bands were eluted and the immunoreactivity measured with antiglucagon sera K 68 (nonspecific) and K 814 (pancreas specific). All four components gave linear dilution curves with K 68, but did not react with K 814 at all. Histidine was the N-terminus of the main component , and its amino acid composition is shown in Table 1. The figures must be considered as tentatiVe because of the small amount of material analysed. The isolated component contains 40 residues in accordance with a molecular weight of 4000 5000 as found by gel filtration. The presence of two extra lysines and one extra arginine, but only one glutamic acid (glutamine) more than porcine glucagon accounts for the high pI observed during the isolation. The t ryptophan residue in the molecule justifies the use of tritium-labeled t ryptophan in the studies of glucagon biosynthesis in guinea pig islets. Because the isolated component does

Effect of glucagon on the principal islets of a fresh-water fish, Channa punctatus (Bloch).

Intramuscular administration of crystalline mammalian glucagon (0.5-2.0 mg/kg body weight) evokes significant cytological alterations in the principal islets of Channa punctatus, a fresh-water fish. Initially there is degeneration of the alpha-cells but later the beta-cells are also atrophied. Regressive changes in some of the alpha-cells have been interpreted to reflect their possible role in glucagon secretion. The degeneration of beta-cells appears to be secondary to the fluctuations in the blood glucose level, induced by exogenous glucagon.

In vitro stimulation of hepatic glycogen phosphorylase activity by epinephrine and glucagon in the brown bullhead, Ictalurus nebulosus

In vitro studies with the brown bullhead, Ictalurus nebulosus, demonstrated that the addition of either epinephrine (20 μg/ml) or glucagon (20 μg/ml) to incubating liver pieces either prevented the decay of glycogen phosphorylase activity seen in controls after 30 min or, in some cases, increased the specific activity of phosphorylase above initial 0-min control levels. The stimulation of phosphorylase activity by hormones was effected both by an increase in total phosphorylase activity and by an increase in the percentage of this total phosphorylase in the active form. The stimulation of phosphorylase activity by hormones was accompanied by increased glucose liberation into the incubation medium, presumably as a result of glycogenolysis. Epinephrine was a more effective glycogenolytic agent than mammalian glucagon. All fish showed the same response regardless of acclimation temperature (20, 10, or 1°C).

The Avian Endocrine Pancreas

The avian endocrine pancreas is comprised of A-islets containing A1- and A2- cell types, and B-islets containing A1- and B-cell types. The function of the A2- and B-cells is the secretion of glucagon and insulin, respectively, while that of the A2-cells is uncertain. The avian pancreas contains small amounts of insulin, has poor insulinogenic potential, and releases the hormone “sluggishly” in response to high glucose load. Fasting, hormones, and/or vagal stimulation do not alter insulin release. Avian insulin is not anti-lipolytic and is poorly lipogenic in in vitro avian systems. Both avian pancreas and plasma contain 5-10 times more glucagon than observed in mammals; however, no studies have been reported employing the avian hormone. Birds are extremely sensitive to mammalian glucagon, exhibiting a rapid and marked hyperglycemia, hepatic glycogenolysis, hyperglycerolemia, and hypertriglyceridemia. The lipolytic effects of glucagon are intensified in ‘vitro’ by insulin. A pancreatic polypeptide (APP) containing 36 amino acid residues has been isolated from the avian pancreas, but not from gut, liver, proventriculus, or gizzard. APP circulates normally, fluctuates with nutritional manipulation, and is found in all avian species investigated. At high levels APP induces hepatic glycogenolysis and hypoglycerolemia. At low levels APP is a powerful “gastric” secretogogue, encouraging rapid proventricular volume, acid, pepsin, and protein release.